Semen analysis & Sperm processing Jalal Ghasemzadeh Andrology lab

What is Andrology? “Andro” from the Greek “andros”, man. Andrology is the study of male fertility and laboratory aspects of infertility diagnosis and treatments. Science of diseases of males, including infertility, spermatogenesis and sexual dysfunction.

Semen analysis Alternative names : sperm test male infertility test Semen analysis (SA) is a gateway test and primary test for evaluation of male infertility and treatment planning for helping couples achieve pregnancy. Safe, inexpensive, non-invasive procedure Alternative names : sperm count sperm test male infertility test spermogeram

Indications of semen analysis 1) Assessment of fertility (2 sample,7days to 3weeks) Phosphatase acid 2) Forensic purposes PSA Sperm 3) Suitability for artificial insemination (IUI, IVF) 4) Post vasectomy (2 sample, 3 months and 6 months )

Semen source Characteristics Volume Source of secretion alkaline & gelatinous fructose, prostaglandins seminogelin 1-3 cc (46-80%) Seminal vesicles acidic & watery contain zinc, citric acid, acid phosphatase & PSA 0.5-1 cc (13-33%) Prostate contain spermatozoa , carnitine & inhibin 0.1-0.2 cc (5%) Testis,epididymids & vasa deferens viscous & clear contain IgA & Mucoproteins 0.1-0.2 cc (2-5%) Bulbourethral glands

Male reproductive system Erectile tissue Prostate gland Urinary bladder Bulbourethral gland Vas deferens Epididymis Testis Seminal vesicle (behind bladder ) Urethra Scrotum Glans penis Erectile tissue Prostate gland Urinary bladder Bulbourethral gland Vas deferens Epididymis Testis Seminal vesicle (behind bladder ) Urethra Scrotum Glans penis

Accessory glands dysfunction 1) Seminal vesicles dysfunction : Low volume Decreased PH Decreased fructose concentration 2) Prostate dysfunction : Delayed liquefaction Increased viscosity Increased PH Decreased zinc concentration

Spermatogenesis Begins at puberty and continues throughout adult life of a male. Sperm are produced within the seminiferous tubules. Sertoli cells (nurse cells) feed and protect from a blood-testis barrier developing sperms. Primordial germ cells differentiate into spermatogonia (diploid cells). Spermatogonia during mitosis division become two primary spermatocyts.

Spermatogenesis Each primary spermatocytes (diploid cells ) through meiosis I produce two secondary spermatocytes (haploid cells). Each secondary spermatocytes during meiosis II divides to produced two spermatid (haploid cells with 23 chromosomes). As a result of the two meiotic divisions, each primary spermatocyte produces four spermatids.

Spermiogenesis Spermatogenesis takes 64 days in the human. Changes that transform spermatids into sperm : 1) Chromatin condensation 2) Flagellum formation 3) Acrosome cap development 4) Discarding excess cytoplasm Sperms transported to the epididymis (caput corpus caudal ) here they are stored. Spermatogenesis takes 64 days in the human.

Examinations of human semen 1) Standard procedures Sample collection & delivery Initial microscopic & macroscopic examinations Anti-sperm antibody test (IBT & MAR test) 2) Optional tests Indices of multiple defect (TZI & SDI ) HOS test Semen culture CASA Biochemical assay (fructose, zinc) 3) Research tests Sperm function tests ROS Acrosome reaction CASA morphology

Sample collection & Delivery 1) Two samples 7days……3weeks 2) Sexual abstinence 2days……7days 3) Adequate collection masturbation non toxic glass or plastic container no condoms no lubricants complete (important) 4) Adequate lab delivery ≤1 hour post collection 5) Adequate temperature 20 0C – 40 0C

Long sexual abstinence (Levitas et al. 2005) & (Pellestor et al.1994) Sample collection Long sexual abstinence (Levitas et al. 2005) & (Pellestor et al.1994) Increase sperm concentration Increase volume Decrease motility Decrease normal morphology Loss of the first part of the ejaculate : Increase PH Decrease sperm concentration Delay liquefy Loss of the second part of the ejaculate : Decrease PH Decrease volume Lack of coagulum formation

Sample collection at clinic or at home, which‼? Elzanaty & Malm, 2007 OBJECTIVE : Comparison of sperm parameters in samples collected by masturbation at clinic and at home. at a clinic (n = 273) PATIENTS 379 men at a home (n = 106) RESULTS : 1) Sperm concentration, total sperm count & rapid progressive motility were statistically significantly higher at home-collected samples. 2) Semen volume, normal morphology, PSA, Zinc & fructose did not differ significantly between groups. CONCLUSION : Superior semen quality in samples collected by masturbation at home compared with at a clinic. this should be taken into consideration in infertility investigation. Home Clinic oligozoospermia 19/106 (18%) 81/273 (30%) asthenozoospermia 68/106 (64%) 205/273 (75%)

Semen collection methods 1) Masturbation 2) Coitus interruptus : Loss of first portion of the ejaculate Cellular & bacteriological contamination The acid PH of the vaginal fluid 3) Semen collection in sexual dysfunction Vibrator Electro ejaculator (E.E) Sperm retrieval methods : PESA (Precutaneous Epididymal Sperm Aspiration) MESA (Microsurgical Epididymal Sperm Aspiration) TESE (TEsticular Sperm Extraction) MESA PESA

Initial examinations 1) Macroscopic : 2) Microscopic : Liquefaction Appearance Viscosity PH Volume 2) Microscopic : Sperm motility Sperm viability Sperm concentration Sperm morphology Cellular elements other than sperm

Macroscopic examinations 1) Liquefaction time Definition : the change from the coagulated to liquid. Principl : proteolysis semenogelin by PSA Normal : within 60 minutes bromelain(1g/l) Abnormal : >60 minutes after ejaculation alpha amylase chymotripsin(150 USP/ml) 2) Appearance Normal: homogenous & grey-opalescent Abnormal: Opaque (debris or WBC) Red −Brown (RBC) Yellow (jaundice or taking vitamins) Clear (poor sperm quantity)

Macroscopic examinations- continued 3) Viscosity (consistency) Semen viscosity refers to the fluid nature ↑ Viscosity = ↓ sperm motility Normal: ≤ 2 cm thread 4) PH Normal = 7.2 or more PH is important because sperm die at PH < 6 Routine measurement of PH is not necessary.

Macroscopic examinations- continued 5) Volume  Low volume (hypospermia) : < 0.5ml Seminal vesicle dysfunction or agenesis Ejaculatory ducts obstruction Retrograde ejaculation Incomplete collection High volume (hyperspermia) : > 6ml Long periods of sexual abstinence Over production of accessory glands No ejaculate (Aspermia) : Surgery

Assessment of motility Microscopic examinations CASA ( Computer- Aided/Assisted Sperm Analysis )  Systematic (manual method) : Grade a → rapid progressive motility ( ≥25 µm/s at 37 0C ) Grade b → slow progressive motility ( 5-25µm/s at 37 0C) Grade c → non progressive motility (<5 µm/s at 37 0C) Grade d → immotility Asthenozoospermia: Sperm structure defects Prolonged abstinence periods Varicocele Anti-sperm antibody Cartagena's syndrome Necrozoospermia : all sperms are dead Cilia immotile syndrome

Microscopic examinations- continued Viability Microscopic examinations- continued Distinguish live non motile sperm from dead. Indication > 50% immotile spermatozoa Determined by :  Eosin staining  Eosin- nigrosine staining  Hypo-osmotic swelling test (HOS test ) Hoechst staining Live Dead Live Dead

Microscopic examinations- continued Sperm concentration Sperm concentration = number of sperm per ml of semen. Azoospermia (no sperm in the ejaculate) Obstruction Hormonal insufficiency Congenital Klinefelter's syndrome Oligozoospermia ( <20 million sperm/ml ) Procedural causes Varicocele Retrograd ejaculation Polyzoospermai ( >250 million sperm/ml ) Long sexual abstinence Improved neubauer Makler counting chamber

Assessment of morphology Microscopic examinations- continued Most confusing & time-consuming area of semen analysis Smearing, air-drying, fixation, staining Staining method :  Papanicolaou stain→ method recommended  Pap-quick stain  Diff-quick stain→ rapid staining method  Shorr stain  Wright-gimsa stain

Structure of normal spermatozoa 1) Head nuclear region Oval in shape Length: 4-5 µm & Width: 2.5-3.5 µm Length-to-width ratio: 1.5 to 1.75 µm Acrosomal region: 40-70% head area Contain nucleus (DNA) 2) Midpiece metabolic region Slender & Width <1 µm Length: about 1.5 times the length of the head Contain axoneme & mitochondria Cytoplasmic droplets less than half the size of the normal head 3) Tail locomotor region Straight, uniform, thinner than the midpiece, uncoiled Length: approximately 45 µm

Classification of sperm morphology 1) Head defects Small Large Tapered Round Amorphous Vacuolated Pyriform 2) Neck & midpiece defects Bent Thin Thick Asymmetrical insertion 3) Tail defects Short Coiled Double 4) cytoplasmic droplet or excess residual cytoplasmic ( >1/3rd normal head )

Diff- quick staining (x100)

Scanning Electron Microscope of spermatozoa

Sperm morphology classification systems Normal reference range 1) Macleod >60% 2) WHO manual 2nd edition >50% 3) WHO manual 3rd edition >30% 4) Strict (menkveld & kruger ) / WHO manual 4th edition >14% 5) WHO manual 5th edition >4% 6) ASCP (American society clinical pathology ) >80%

Cellular elements other than spermatozoa Microscopic examinations- continued Cellular elements other than spermatozoa Round cells : < 5x106/ml Immature germ cells : Spermatogenic cells : fever, radiation, cytotoxic drug Prostatic cells Epithelial cells Leukocytes : Leukocytospermia ( >1x106/ml ) Pyospermia (high amount of WBC) Produce ROS, motility, aggregation Antibiotic treatment

Reference values of semen variables (WHO 1999) 2.0 ml or more Volume 7.2 or more pH 20x106 spermatozoa/ml or more Sperm concentration 40x106 spermatozoa per ejaculate or more Total sperm count 50% or more motile (grades a+b ) or 25% or more with progressive motility (grade a ) Motility 30% or more with normal morphology Morphology 50% or more live i.e. excluding dye Viability Fewer than 1x106/ml White blood cells Fewer than 50% motile spermatozoa with adherent particles MAR test

Lower reference limits for semen (WHO 2010)