" SEMEN ANALYSIS PA 23.3a (CC): Describe and interpret the abnormalities in semen analysis. Q. Write a short note on semen analysis. Semen (seminal fluid) consists of spermatozoa (sperms) and the fluid part. About 40% of cases of infertility are due to abnormalities in semen and therefore, semen analysis is the first test to be performed while investigating for infertility. The defect of sperms may be quantitative (absence of sperms, lack of enough sperms) or qualitative. Indications for semen analysis are listed in Box 15.24. BOX 15.24: Indications for semen analysis. Assessment of fertility/infertility To monitor the success of surgical procedures, such as varicocelectomy and vasectomy Determine the suitability of donor semen for artificial insemination Medicolegal purpose: In alleged rape cases, vaginal pool smears are examined to detect sperms For selection of assisted reproductive technology (e.g., in vitro fertilization, gamete intrafallopian transfer technique) Collection of the Sample Q. Write a short note on a collection of semen for analysis. The patient is asked to collect the semen by masturbation after a minimum of 2 days and a maximum of 7 days of sexual abstinence (2-7 days of ejaculatory abstinence). The specimen should be collected in a clean, dry, wide-mouthed plastic/glass container. Collection of condom samples is not advisable because they often contain spermicidal agents which impair sperm motility. Examination of Semen Q. Write a short notes on various tests/parameters of the semen with normal range. Physical Examination Liquefaction: Immediately after ejaculation, the semen is normally a semisolid coagulated mass. At room temperature, the semen usually begins to liquefy (become thinner) within a few minutes and completely liquefles within 15 minutes Within 30 minutes it becomes more homogeneous and watery. Fallure to liquefy may be due to inadequate prostate secretion. Semen viscosity: Fresh semen is viscid and the viscosity can be estimated by gently aspirating semen into a wide-bore (approximately 1.5 mm diameter) plastic disposable pipette, allowing the semen to drop by gravity. Normal semen falls drop by drop and if viscosity is abnormal, the drop will form a thread more than 2 cm long. Normal viscosity is important since the increase in viscosity affects sperm motility. Appearance: Freshly ejaculated semen is an opaque, white-gray, and viscid fluid. After liquefaction, it has a homogeneous gray-opalescent appearance. Semen may have red-brown color when red blood cells are present (hemospermia) or yellow in patients with jaundice or ingestion of certain vitamins or drugs. The yellow hue appearance is associated with pyospermia; a rust color is due to small bleeding in the seminal vesicle. Semen volume: Normal volume >1.4 mL. First sample is weighed and then volume is calculated by assuming the density of semen to be 1 g/mL.. Low semen volume may be due to obstruction of the ejaculatory duct, congenital bilateral absence of the vas deferens, or can also be due to difficulty in collection. High semen volume may be due to active exudation in cases of inflammatory lesions of the accessory organs. Semen pH: Alkaline and ranges from 7.2-8 (>7.2). The pH should be measured after liquefaction, preferably after 30 minutes. Microscopic Examination Q. Write a short note on the microscopic examination of semen. Sperm aggregation or agglutination: The adherence either of immotile spermatozoa to each other or of motile spermatozoa to mucus strands, nonsperm cells. or debris is considered to be nonspecific aggregation and should be noted. Agglutination refers to motile spermatozoa sticking to each other, head-to-head, tail to tail, or in a mixed way. Any motile spermatozoa that stick to each other by their heads, tails, or midpieces should be recorded. QW Cla 15. ofs US in Cellular elements other than spermatozoa: During microscopic examination, a search should be made for 1. the presence of cells other than spermatozoa. Some of these cells may be clinically relevant, which includes epithelial cells from the genitourinary tract and "round cells" (leukocytes and immature germ cells). Assessment of sperm motility: Motility of the sperms helps in the penetration of cervical mucus and migra-tion of the sperms into the fallopian tube. In normal 2. semen, 42 (40-43)% of sperms should be motile (rapidly progressive and slowly progressive). This is assessed by placing a drop of liquefied semen on a clean glass slide with a coverslip placed over it and examining it under a microscope. According to the 3. WHO (2021), the motility of each spermatozoon (plural is spermatozoa) is categorized as rapidly progressive (29-31%), and slow-progressive (1%). Te A rapidly progressive spermatozoon moves >5 head lengths per second. Immotile sperms may be 54 (50-56)%. Th th ar pl Te di te T Sperm vitality: It is important to know whether immotile spermatozoa are alive or dead. Normally, 54 (50-56)% of live forms are observed. The vitality of the spermatozoa is estimated by identifying those with an intact cell membrane and is especially important for samples with less than about 40% progressively motile spermatozoa. The percentage of live spermatozoa is assessed either (" by dye exclusion or by hypotonic swelling. Q. Write a short note on sperm count. Total sperm count: Sperm count is carried out in an improved Neubauer chamber using a Thoma pipette in a dilution of 1 in 20 (as for total leukocyte count) using semen diluting fluid. T Normal range of sperm count: >39 million/ejaculate. Aspermia: No semen. Azoospermia: No spermatozoa in the ejaculate. Oligospermia: Total number of spermatozoa below the lower reference limit. Sperm morphology (Fig. 15.18): Smear prepared from semen is fixed and stained with Papanicolaou stain to identify the morphological features. Normal spermatozoa have a head, neck, middle piece (midpiece), principal piece, and endpiece. Since the endpiece is difficult to see under a light microscope, the spermatozoa can be considered to consist of a head (and neck) and a tail (midpiece and principal piece). Normally, >4% of sperms have normal morphology. For a spermatozoon to be considered normal, both its head and tail must be morphologically normal. All other forms should be considered as abnormal. Q. Write a short answer on abnormal forms of sperm. Classification of abnormal sperm morphology (Fig. 15.18): During morphological examination all major parts of spermatozoa should be assessed, abnormal spermatozoa usually have a lower fertilizing capacity. The abnormalities in morphology can be classified as: 1. Head defects: Abnormal size: Large or small head. Abnormal shape: Pointed/tapered, round, pyriform, amorphous. Acrosomal defects: Vacuolated acrosomal region, abnormality of acrosomal areas. Abnormal number: Double-headed. Neck and midpiece defects: Sharply bent neck. a-al 2. e S a d e n y Asymmetrical insertion of the midpiece into the head. Thick or thin or irregular midpiece. 3. Tail (principal) piece defects: These abnormalities include short or bent or coiled or double tail. Tests for Antibody Coating of Spermatozoa -e The agglutination of spermatozoa may be the result of the anti-sperm antibodies (ASAs) or other factors present in the semen. ASAs in the semen belong to IgA and IgG types and they may also be found in sperm-free fluids (seminal plasma, blood serum, and cervical mucus). Tests for antibodies on spermatozoa ("direct tests"): Two direct tests include the mixed antiglobulin reaction (MAR) - test and the immunobead (IB) test. Tests for antisperm antibodies in sperm-free fluids ("indirect" tests). " Make this text of dr. Priyanka’s pathology prof buster book. Removal of unnecessary words like a, an, the, is, are, it is, there is wherever possible. And then make masterchart of semen analysis including everything.

Create masterchart of semen analysis.

### **SEMEN ANALYSIS** ## Definition * Semen = **spermatozoa (sperms) + seminal fluid** * ~40% infertility cases due to semen abnormalities * First investigation in infertility workup * Defects: * **Quantitative** → absent/low sperm number * **Qualitative** → abnormal sperm function/morphology --- ## Indications * Assessment of fertility/infertility * Monitor success of: * Varicocelectomy * Vasectomy * Assess donor semen for artificial insemination * Medicolegal purpose: * Alleged rape cases → vaginal pool smear for sperm detection * Selection of assisted reproductive techniques: * IVF * GIFT --- # Collection of Sample ### Requirements * Masturbation collection * Ejaculatory abstinence: **2–7 days** * Collect in: * Clean * Dry * Wide-mouthed plastic/glass container ### Avoid Condom Sample * May contain spermicidal agents * Impair sperm motility --- # Examination of Semen # I. Physical Examination ## 1. Liquefaction ### Normal * Immediately after ejaculation → semisolid coagulated mass * Begins liquefying within few minutes * Complete liquefaction within **15 min** * By **30 min** → homogeneous, watery ### Abnormal * Failure to liquefy → inadequate prostate secretion --- ## 2. Semen Viscosity ### Method * Aspirate semen into wide-bore pipette (~1.5 mm) * Allow drop by gravity ### Normal * Falls drop by drop ### Increased Viscosity * Thread >2 cm long ### Significance * Increased viscosity ↓ sperm motility --- ## 3. Appearance ### Normal * Fresh semen: * Opaque * White-gray * Viscid ### After Liquefaction * Homogeneous gray-opalescent ### Abnormal Colors | Color | Cause | | ------------ | --------------------------------- | | Red-brown | RBCs (hemospermia) | | Yellow | Jaundice, certain vitamins/drugs | | Yellow hue | Pyospermia | | Rust colored | Small bleeding in seminal vesicle | --- ## 4. Semen Volume ### Normal * > 1.4 mL ### Measurement * Weigh sample * Density assumed = **1 g/mL** ### Low Volume * Ejaculatory duct obstruction * Congenital bilateral absence of vas deferens * Collection difficulty ### High Volume * Active exudation due to inflammatory lesions of accessory organs --- ## 5. Semen pH ### Normal * Alkaline * **7.2–8** * > 7.2 ### Measurement * After liquefaction * Preferably after 30 min --- # II. Microscopic Examination ## 1. Sperm Aggregation vs Agglutination ### Aggregation * Nonspecific * Immotile sperms stick together * Motile sperms attach to: * Mucus strands * Debris * Nonsperm cells ### Agglutination * Motile sperms stick to each other: * Head-to-head * Tail-to-tail * Mixed pattern --- ## 2. Cellular Elements Other Than Sperms Look for: * Epithelial cells from genitourinary tract * Round cells: * Leukocytes * Immature germ cells --- ## 3. Sperm Motility ### Importance * Cervical mucus penetration * Migration into fallopian tube ### Normal * **42% (40–43%) motile sperms** Includes: * Rapidly progressive * Slowly progressive ### Method * Drop of liquefied semen on slide * Coverslip * Microscopic examination ### WHO 2021 Categories #### Rapidly Progressive * 29–31% * Movement >5 head lengths/sec #### Slow Progressive * ~1% #### Immotile * 54% (50–56%) --- ## 4. Sperm Vitality ### Purpose * Determine whether immotile sperms alive or dead ### Normal * **54% (50–56%) live forms** ### Based On * Intact cell membrane ### Especially Important * Progressive motility <40% ### Methods * Dye exclusion test * Hypotonic swelling test --- ## 5. Total Sperm Count ### Method * Improved Neubauer chamber * Thoma pipette * Dilution 1:20 * Same as TLC method ### Normal * > 39 million/ejaculate ### Terms #### Aspermia * No semen #### Azoospermia * No spermatozoa in ejaculate #### Oligospermia * Sperm count below lower reference limit --- # 6. Sperm Morphology ### Stain * Papanicolaou stain ### Normal Sperm Parts * Head * Neck * Midpiece * Principal piece * Endpiece ### Practical View * Head + neck * Tail (midpiece + principal piece) ### Normal Morphology * > 4% normal forms ### Requirement * Both head and tail normal ### Otherwise * Abnormal sperm --- # Classification of Abnormal Sperm Morphology ## A. Head Defects ### Abnormal Size * Large head * Small head ### Abnormal Shape * Pointed * Tapered * Round * Pyriform * Amorphous ### Acrosomal Defects * Vacuolated acrosomal region * Abnormal acrosomal area ### Abnormal Number * Double-headed sperm --- ## B. Neck & Midpiece Defects * Sharply bent neck * Asymmetrical midpiece insertion * Thick midpiece * Thin midpiece * Irregular midpiece --- ## C. Tail (Principal Piece) Defects * Short tail * Bent tail * Coiled tail * Double tail --- # Tests for Antibody Coating of Spermatozoa ## Antisperm Antibodies (ASA) ### Cause * May produce sperm agglutination ### Types * IgA * IgG ### Present In * Semen * Seminal plasma * Blood serum * Cervical mucus --- ## Direct Tests (Detect antibodies on sperm) ### 1. MAR Test * Mixed Antiglobulin Reaction test ### 2. Immunobead (IB) Test --- ## Indirect Tests (Detect antisperm antibodies in sperm-free fluids) * Performed on: * Seminal plasma * Serum * Cervical mucus --- ### Ultra-Short Revision Box **Normal Values** * Volume → **>1.4 mL** * pH → **7.2–8** * Motility → **42%** * Vitality → **54%** * Sperm count → **>39 million/ejaculate** * Normal morphology → **>4%** **Definitions** * Aspermia = No semen * Azoospermia = No sperms * Oligospermia = Low sperm count * Hemospermia = Blood in semen * Pyospermia = Pus cells in semen * Agglutination = Motile sperms stick to each other "