Semen Analysis

Objective: Physiology of semen formation Indication of semen analysis Macroscopic examinationof semen Microscopic examination Antibody testing in semen Reference ranges

Physiology of Seminal fluid Semen is grey opalescent fluid , which consists of suspension of spermatozoa in seminal plasma. It is made up of the secretions of all accessory glands of the male genital tract.

The Percentage contribution of Each of the Secretions in Seminal Fluid % Ejaculate Source of Secretion 5 Testis 40-80 Seminal vesicles 13-33 Prostate 2-5 Bulbo-uretheral & uretheral glands

Fructose: imp energy for sperms especially needed for sperms motility Prostaglandins: play a role in controlling sperm movement & sperm penetration of cervical mucus Fibrinogen-like substance : acted upon by enzyme vesiculase to induce clotting that occurs in semen The prostatic fluid pH =6.5 Enzymes: vesiculase Acid phosphatase

Bulbo-uretheral & uretheral glands The fluid rich in mucoprotein , so it lubricates the urethra Occasionally in the secretions contain antisperm antibodies( may be of importance in infertility cases)

During ejaculation , each of the components that make up semen may be discharged from the urethra in a sequence The first part is made up of testicular component of semen followed by secretion of the prostate , lastly the secretion of the vesicles are expelled.

Indication of Semen Analysis Assessment of fertility/infertility ( most common) Determination the effectiveness of vasectomy Determination of suitability of semen for artificial insemination Follow up of fertility after cancer treatment by radio- or chemotherapy Forensic purpose

Semen analysis Specimens: 2 samples should be collected for initial evaluation, the interval between the two collections should not be less than 7 days or more than 3 weeks , if the results of these assessments are markedly different , additional samples should be examined

Examination of Semen Macroscopic Examination Microscopic Examination Volume Liquefaction time Appearance pH Viscosity Microscopic Examination Sperm count Sperm motility Sperm viability Sperm morphology Agglutination Antibodies coating of sperms Biochemical Tests Fructose test Acid phosphatase

Macroscopic Examination

Volume : ( 2-6ml/ ejaculation) Aspermia: total absence of ejaculation ( rare) Hypospermia or oligospermia: the seminal fluid is < 2ml Hyperspermia: volume > 10 ml ( spermia denotes seminal fluid not spermatozoa)

Liquefaction time: ( forms gel-like clot immediately after ejaculation) a normal semen sample liquefies with 15 -60 mint . ( prolongation must be recorded).

Appearance: homogeneous , grey-opalescent appearance The color is due to high content of protein &presence of > 60 million sperms/ml May be less opaque if sperm concentration is very low Dense white turbid in inflammation and high WBCs Haematospermia :when red blood cells are present (blood) Yellow in patient with jaundice (very bright yellow due to bilirubin) or taking some vitamins Or contaminated with urine ( uriniferous odour)

pH : between7.2-8.0 recorded in fresh semen by using pH paper . A patient exceeding pH 8.0 may suggest acute disease of the seminal vesicles Lowering of pH may be due chronic inflammatory of seminal vesicles Or contamination with urine

Viscosity : Normal viscosity is that which allows semen to be poured drop by drop out of the container. In case of abnormal viscosity drop will form a thread more than 2 cm long High viscosity : can interfere determination of sperm motility

Microscopical Examination

Sperm Count: Total number of sperms in an ejaculation. It is 20 million/ml i.e. 60 million / ejac It is obtained by multiplying the sperm concentration by the volume Azoospermia: means no spermatocytes ( male sterility) Oligozoospermia: < 20 million/ml (less than 50 million/ejaculation Polyzoospermia : may reach 350millions/ejaculation

Sperm Count Decreased: vasectomy varicocele primary testicular failure (Klinefelters) secondary testicular failure congenital vas deferens obstruction endocrine causes (prolactinemia, low testosterone)

Assessment of sperm motility: the motility of each spermatozoon is graded : ‘a’ =rapid progressive motility ‘b’= slow or sluggish motility ‘c’= non progressive motility ‘d’= immotility

Viability should be determined if the % of immotile spermatozoa exceeds 50 % Supravital stain: Eosin /Nigrosin Viable do not take up the stain

Assessment of sperm morphology

Normally the sperm count contains fewer than 20 % abnormal forms e. g Normally the sperm count contains fewer than 20 % abnormal forms e.g. bitailed, short tailed , 2 heads …..etc.

Other cellular elements: Leukocytes: leukocytes predominantly neutrophils are present in most human ejaculate. Normal <1x106 /ml or < 5/HPF 5-20 WBC/HPF =increased 20-40 WBCs/HPF =+ >40 WBCs/ HPF = ++ If increased leukospermia or pyospermia may be associated with infection and poor sperm quality When the number is increased, microbiological tests should be performed to investigate the presence of of infection in any of the accessory glands

Agglutination Reported when motile sperm stick to each other in a definite pattern. Head-head Tail-tail Head-tail Immunological cause of infertility

Biochemical Tests Fructose Test: (a) absence of seminal vesicle; It is secreted for sperm nutrition from seminal vesicle Impairment of seminal vesicular secretion will result in reduced fructose secretion in semen and the motility of the sperms will be reduced. Fructose disappears in cases of : (a) absence of seminal vesicle; (b) obstruction of ejaculatory duct; and (c) inflammation of seminal vesicle. It is decreased in case of testosterone deficiency. So, fructose is used as fertility test.

Secreted from the prostate. The test is used as: Acid phosphatase: Secreted from the prostate. The test is used as: 1)A marker of prostatic functions; and 2)In forensic laboratories as a test for the presence of semen.

Testing for Antibody coating of spermatozoa Sperms can induce immune response not only between species but also within one species and within an individuals Sperm antibodies may : Cause agglutination of sperm Cause reduction in motility Inhibit the ability of sperm to penetrate cervical mucus Impede binding of the sperm to the oocyte Antibodies may be IgA & IgG It must be remembered that sperm antibodies may be present in the cervical mucus itself , may inhibit the entry of sperm in the female genital system

Reference Ranges

Total count > 40 million/ejaculate Volume 2.0-6.0 ml pH 7.2-8.0 Color: greyish white Liquefaction: 15-60 min. Count >20 million/ml Total count > 40 million/ejaculate Motility: > 50 % motile( grades a+b) or > 25% with progressive motility ( grade a) within 60 minutes of ejaculaton Morphology > 30% normal form Viability > 75% viable WBC< 1million/ml RBC none Quoted from the WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction ,1999