Semen Analysis PDF

Semen Analysis Understanding Male Reproductive Health Prepared By T.A\ Ahmed Al-Aiashy  1 Introduction A semen analysis looks at the volume and quality of a man’s sperm. It is one of...

Semen Analysis Understanding Male Reproductive Health Prepared By T.A\ Ahmed Al-Aiashy  1 Introduction A semen analysis looks at the volume and quality of a man’s sperm. It is one of the first steps to detect male fertility issues. What is semen? This substance is released from a man’s penis when he has an orgasm (ejaculates). It contains: Sperm, male reproductive cells. The cells have a unique shape that contains: Head: includes genetic material (DNA) to fertilize a woman’s egg. Tail :that helps it travel (“swim”) through a woman’s reproductive system to reach the egg and fertilize it. Fluids which make it possible to deposit sperm toward the back of a woman’s vagina. Proteins, vitamins and minerals that fuel the sperm’s journey to the egg.  2 What is a semen analysis? A semen analysis is a lab test that examines a sample of semen under a microscope. It evaluates things such as sperm count, activity (motility) and shape (morphology). Importance of Semen analysis 1. Evaluate male reproductive function and fertility. 2. Choose infertility treatment for couples. 3. Assess the effectiveness of male contraception.  3 1. Patient instruction 1) The primary recommendation is ejaculating collection by masturbation. 2) Coitus interruptus is not recommended and should only be used in exceptional cases due to the risk of incomplete collection and contamination with vaginal fluid and cells. 3) Lubricants should be avoided, since they may contaminate the ejaculate and change its properties. 4) The ejaculate needs to be completely collected, and the man should report any loss of any fraction of the sample. 5) The ejaculate should be collected after a minimum of 2 days and a maximum of 7 days of ejaculatory abstinence. 6) To avoid exposure of the semen to temperature and to control the time between collection and analysis, it is recommended that the sample be collected in a private room close to the laboratory. Ideally, investigations should occur within 30 minutes after collection, but at least within 60 minutes.. 7) If not collected in the proximity of the laboratory, transport must not allow the sample temperature to go below 20 °C or above 37 °C.  4 2. Sample Collection & Labelling : the specimen container should be kept between 20 °C and 37 °C, to avoid large changes in temperature that may affect the spermatozoa. The specimen container should be a clean, wide-mouthed plastic container, and non-toxic for spermatozoa. Legal requirements for container identity markers may differ. It could be the man’s name and identification number, the date and time of collection, or unique sample identifying numbers. The following information should be recorded at sample reception and presented in the final report: o Identity of the man (e.g., Name, birth date, and personal code number) and ideally his confirmation that the sample is his own; o The period of prior ejaculatory abstinence; o The date and time of collection o The completeness of the sample and any difficulties in producing the sample (e.g., If collection was not done at the laboratory). o Ejaculate volume.  5 Examination and Post-Examination Procedures A. Assessment of Ejaculate Volume Precise measurement of volume is essential in any evaluation of ejaculate because it gives information on the secretory functions of the auxiliary sex glands. A reliable ejaculate volume assessment is also necessary for the calculation of the total number of spermatozoa, the total number of non-sperm cells in the ejaculate, and the total amounts of biochemical markers. The volume is best measured by weighing the sample in the container in which it has been collected. This can preferably be done at reception of the ejaculation container before incubation for liquefaction. Other methods introduce greater inaccuracy. i. Use a pre-weighed container for collection of the ejaculate, with the weight noted on the container and lid. ii. Weigh the vessel with the ejaculate in it. iii. Subtract the weight of the empty container. iv. Calculate the volume from the sample weight, assuming the density of semen to be 1 g/ml. (Semen density has been reported to vary between 1.03 and 1.04 g/ml, 1.00 and 1.01 g/ml, and an average of 1.01 g/ml).  6 B. Macroscopic evaluation Macroscopic evaluation comprises a number of important observations that may not be possible to assess in an exact numerical value – and thereby controlled by traditional quantitative methods – but can still be of great clinical importance. 1) Appearance A normal liquefied ejaculate has a macroscopically homogeneous, cream/grey-opalescent appearance. It may appear less opaque if the sperm concentration is very low; the color may also be different – i.e., slightly yellowish after longer abstinence times, red-brown when red blood cells are present (haemospermia), or clearer yellow in a patient with jaundice or taking certain vitamins or drugs. If the ejaculate appears viscous, totally clear, and colorless. 2) Liquefaction Immediately after ejaculation into the collection vessel, the ejaculate is typically a semi-solid coagulated mass or a gel-like clump. Usually, the ejaculate begins to liquefy (become thinner) within a few minutes at room temperature, at which time a heterogeneous mixture of semi-solid lumps will be seen in the fluid. As liquefaction continues, the ejaculate becomes more homogeneous and more watery but still with a higher viscosity than water. In the final stages of liquefaction, only small areas of coagulation remain. A temperature of 37 °C will facilitate liquefaction. Complete ejaculate liquefaction is normally achieved within 15–30 minutes at room temperature. If liquefaction is not complete within 30 minutes, this should be recorded and noted in the final report. The ejaculate could then be left at 37 °C for another 30 minutes.  7 3) Viscosity After liquefaction, the viscosity of the ejaculate can be estimated by gently aspirating it into a wide-bore (approximately 1.5 mm diameter) plastic disposable pipette (verified as non-toxic to sperm and, if needed, sterile), allowing the semen to drop by gravity and observing the length of any thread. A normal liquefied ejaculate falls as small discrete drops. If viscosity is abnormal, the drop will form a thread more than 2 cm long. 4) Odor There is considerable variability in the ability of different individuals to perceive the normal smell of a human ejaculate. Information of a strong odor of urine or putrefaction can be of clinical importance; it is therefore important to note this in the report. 5) pH The pH at ejaculation depends on the relative contribution of acidic prostatic secretion and alkaline seminal vesicular secretion. it should be done at a uniform time, preferably 30 minutes after collection, but in any case, within 1 hour of ejaculation. For normal samples, pH test strips in the range 6.0–10.0 should be used. 1. Mix the semen sample well. 2. Spread a drop of semen evenly onto the pH strip. 3. Wait for the color of the impregnated zone to become uniform (< 30 seconds). 4. Compare the color with the calibration strip to read the pH. A pH value under 7.2 may be indicative of a lack of alkaline seminal vesicular fluid. It can also be due to urine contamination.  8 C. Microscopic Examination 1. Preparation of sample for initial microscopic examination Thoroughly mix the semen specimen to ensure as much as possible that the sample you are examining is representative of the whole specimen. One recommended mixing method is to gently aspirate the sample 10 times into a wide-bore disposable plastic pipette so as not to create air bubbles. Obtain the sample to be examined immediately after mixing the contents in the original container. Initial microscopic examination of semen is first done using 40X Then 100X magnification. Observe mucous strand formation, sperm aggregation, and/or sperm agglutination. 2. Sperm Aggregation Non-specific aggregation of spermatozoa should be recorded but is usually not clinically significant. Aggregations include immotile sperm adhering to each other in a random fashion and motile sperm adhering to mucous strands, epithelial cells, or other debris in the specimen.  9 3. Sperm agglutination Agglutination specifically refers to motile spermatozoa sticking to each other, head-to-head, tail-to-tail, or in a mixed way. The motility is often vigorous, with a frantic shaking motion, but sometimes the spermatozoa are so agglutinated that their motion is limited. Any motile spermatozoa that stick to each other by their heads, tails or mid-pieces should be noted.  10 4. Non-sperm Cells The ejaculate contains cells other than spermatozoa, some of which may be clinically relevant. These include epithelial cells from the genitourinary tract, as well as leukocytes and immature germ cells, the latter two collectively referred to as “round cells Immature Germ Cell  11 5. Sperm motility Assessing sperm motility in semen analysis is critical for understanding fertility potential. The total number of progressively motile sperm is calculated by multiplying the sperm count by the percentage of motile cells. To standardize this assessment, it's essential to maintain a consistent temperature, ideally 37°C, throughout the process. Categories of sperm movement A four-category system for grading motility is recommended. Clinical data from both manual assessments of sperm motility as well as computer-aided sperm analysis demonstrate that the identification of rapidly progressive spermatozoa is important. Therefore, the recommended categories are (with approximate velocity limits): Rapidly progressive (25 µm/s) – spermatozoa moving actively, either linearly or in a large circle, covering a distance, from the starting point to the end point, of at least 25 µm (or ½ tail length) in one second; Slowly progressive (5 to < 25 µm/s) – spermatozoa moving actively, either linearly or in a large circle, covering a distance, from the starting point to the end point, of 5 to < 25 µm (or at least one head length to less than ½ tail length) in one second;  12 Non-progressive (< 5 µm/s) – all other patterns of active tail movements with an absence of progression – i.e., swimming in small circles, the flagellar force displacing the head less than 5 µm (one head length), from the starting point to the end point; and Immotile – no active tail movement 6. Sperm Vitality Sperm vitality, determined by assessing membrane integrity, is routinely measured in semen analysis. However, it's unnecessary when at least 40% of sperm are motile. In cases with poor motility, the vitality test helps distinguish between immotile dead and immotile live sperm. A high proportion of live but immotile cells might indicate structural defects, while a high percentage of immotile and dead cells could suggest epididymal issues or an immune reaction due to infection. Vitality is assessed by identifying sperm with intact cell membranes, typically using the eosin-nigrosin test. It's crucial to conduct this assessment as soon as possible after liquefaction, ideally within 30 minutes to 1 hour, to minimize the impact of dehydration or temperature  13 changes on vitality. Vitality test using eosin–nigrosin This one-step staining technique uses nigrosin to increase the contrast between the background and the sperm heads, which makes them easier to discern. It also permits slides to be stored for re-evaluation, training and quality control purposes.  14 7. Counting spermatozoa and other cells The total number of spermatozoa per ejaculate and the sperm concentration are related to both time to pregnancy and pregnancy rates and are predictors of conception. The number of spermatozoa in the ejaculate is calculated from the concentration of spermatozoa and the ejaculate volume. For normal ejaculates, when the male tract is unobstructed and the abstinence time short, the number of spermatozoa is correlated with testicular volume and thus is a measure of the capacity of the testes to produce spermatozoa, the patency of the male tract and, potentially, the number of spermatozoa transferred to the female during coitus. The concentration of spermatozoa in the ejaculate, while related to fertilization and pregnancy rates, is influenced by the volume of the secretions from the seminal vesicles and prostate and is not a good measure of testicular function. Sperm concentration is the number of sperm/mL in a semen sample. The number of sperm/mL in a sample is determined by counting sperm in a counting chamber. According to the WHO 5th edition, the lower reference limit for sperm concentration is 15 x 106 spermatozoa/mL. Sperm count is the total number of sperm in the entire ejaculation. To determine the sperm count, sperm concentration is multiplied by the total sample volume submitted. The lower reference limit for sperm count is 39 x 106 spermatozoa per ejaculate.  15 8. Sperm Morphology Human sperm morphology assessment is essential for both predicting fertility and providing diagnostic insights into the functional status of male reproductive organs. Beyond measuring the proportion of "normal" sperm, this evaluation involves assessing specific features such as head, neck/midpiece, tail morphology, and the presence of abnormal cytoplasmic residues. It helps determine the overall health and functionality of sperm, including their capacity for fertilization. Spermatozoa consists of a head and a tail, with the tail further divided into segments like the midpiece and principal piece. Normal characteristics in the head, midpiece, tail, and cytoplasmic residue are all necessary for a spermatozoon to be considered free from abnormalities. Recognizing subtle variations in the shape of the entire spermatozoon, including the head and tail, is crucial in evaluating sperm morphology, with an emphasis on the head's shape rather than its exact size. Abnormally large heads are not considered normal in this assessment.  16 Terminology of Human Semen The motility and morphology are percentages and the total number of motile or normal sperm, respectively, should be taken into account before a diagnosis of male factor fertility is made. For example, 15 m/ml is the lower limit of concentration and 4% is the lower limit of morphology. If a semen analysis reveals 3% morphology, and the concentration is 15 m/ml that is more concerning than a semen analysis that shows a sample with 3% morphology, but a concentration of 100 m/ml.  17 There are some common terms utilized when discussing the interpretation of results : Sperm / semen analysis – abnormal results Abnormalities Definition Aspermia Complete absence of seminal fluid azoospermia Absence of sperm in the semen Hypospermia Low semen volume Hyperspermia high semen volume Oligozoospermia Very low sperm count Polyzoospermia Abnormally high sperm count in the ejaculate Asthenozoospermia Poor sperm motility teratozoospermia Sperms that have morphological defects Necrozoospermia All the sperms in the ejaculate are dead Leucospermia A high level of white blood cells presents in the semen Hematospermia Presence of red blood cells in the ejaculate  18 Computer-Assisted Semen Analysis (CASA) Computer-Assisted Sperm Analysis or refers to a system where a complex electronic imaging system is used to visualize the sperm, while advanced software programs help measure the numerous individual parameters of said sperm. In this method, the semen specimen is examined with a microscope’s help, which has a high-resolution video camera attached to it. The data is fed via the camera to a computer, which processes it using the required software. 19 20 21