Medicosnext
Understanding of human sperm and biology of human fertilization has improved over the years, after WHO started standardizing semen analysis and new research results became available. Objective data and information about important measures like mechanism of sperm capacitation, hyper activation of sperm, the ability of sperm to bind zona pellucid, and subsequent drilling, acrosome reaction, and zona digestion, the ability of sperm to fuse with oocyte, and finally, de-condensation of sperm nucleus to form male pronucleus started coming in. These parameters improved our knowledge of events leading to fertilization and formation of embryo.
This new information is helping us to diagnose new forms of male sub-fertility and predict success of attempts at natural or assisted conception, and to design in-vitro sperm function tests and treatments to overcome the diagnosed dysfunction. The basic semen analysis, followed by relevant functional tests, remains the most basic evaluation for clinical assessment of male infertility. Parameters of semen evaluation provide information on sperm production by the testes, potency and function of the male reproductive tract, activity of the accessory glands, and capability of ejaculation. Clinical usefulness of semen evaluation is improving rapidly as more objective, standardized methodology is introduced.
Fertilization of ovum requires only one sperm, which has to travel to the fallopian tube and mate with ovum. That one sperm comes from the testes, which produces more than 160 million sperms every day. Though semen analysis gives a general idea of the fertilizing ability of sperm, there is no specific test to find out which sperm has potential of mating with ovum, and which does not. Measurements made on the whole population of ejaculated spermatozoa cannot completely define the fertilizing ability of the few sperms that reach the site of fertilization. Semen analysis essentially provides information on the clinical status of the individual.
Clearly, the collection and analysis of semen must be undertaken by properly standardized procedures, if the results are to provide valid information. Normal semen is a mixture of spermatozoa suspended in secretions from the testes and epididymis, which at the time of ejaculation are combined with secretions from prostate, seminal vesicles, and bulbo-urethral glands. The final composition is a viscous fluid that comprises of ejaculate.
Variability in Semen Parameters
A single semen analysis is seldom adequate for even the most general assessment of a person’s fertility status. Multiple analyses are essential to establish quantitative parameters of semen quality. There is significant day-to-day variation of all the parameters, and coefficient of variation may exceed 50% for normal samples, and may be even more for abnormal samples. Therefore it is necessary to make repeated evaluations with at least 2 -3 analyses spaced 15 days apart to come to average quality parameters.
The period of abstinence from previous ejaculation has significant effect on semen parameters. At least two days, and maximum of five days of abstinence, should be observed to get consistent quality. The patient must be given proper detailed written or oral instructions before collection of sample and note should be made of strict adherence to the instructions.
Sample Collection and Delivery
Reliable results require proper collection. The semen collection for some patients is a stressful and unpleasant act. It may even be distasteful, difficult, and embarrassing. For these reasons, it is very important to relax and reassure the patient and allow him to collect sample in surroundings that are most comfortable, both physically and emotionally, to him. Collection in situations of stress can, at times, produce inadequate sample, and can lead to misdiagnosis/missed cycle for treatment. The sample should be obtained by masturbation, and ejaculated into a clean, wide-mouth, plastic, disposable container.
When circumstances prevent collection by masturbation, special condoms are available for semen collection. Ordinary latex condoms should never be used, as they contain spermicidal coating. Similarly, coitus interruptus is also not acceptable, as many a time the first rich portion of semen is lost before collection. It is very important to emphasize that semen sample should be complete, as first portion is rich in sperms.
As far as possible, the sample should be collected in a room near the lab. It this is not possible, the sample should be protected from extremes of temperature and brought to lab within an hour of collection. The container should be labeled with patient’s name, date of collection, and other identifying information.
Physical Characteristics of Semen
The semen should be evaluated macroscopically as soon as possible after receiving in the laboratory, pending detailed examination at the uniform time of one hour after collection. Semen sample can store be stored at room temperature, which should be around 24-27 °C. But, if extremes of temperature are expected, it is better to store at
37 °C in an incubator. It is good to make uniform practice of storing samples at 37 °C, so as to achieve standardization.
• Immediate assessment should be made for presence of coagulation. Semen normally gets liquefied within 20 minutes. Specimens that do not liquefy within one hour of collection exhibit high viscosity and should be recorded as abnormal liquefaction and viscosity. The sample should be thoroughly mixed by swirling or repeated pipetting with transfer pipettes. Care should be taken to avoid frothing.
• Semen color and odor should be evaluated. Normal color is grayish white opalescent. Translucent color is associated with low or absent sperm counts. Yellow, pink, or red coloration should be noted and denote disease condition. Yellow color is due to jaundice, red and pink color is due to blood, contamination.
• Any abnormal smell of purification or urine should be noted.
• Semen volume is measured by transferring the entire content to conical-bottom test tubes, and volume is recorded to nearest half milliliter. Better and accurate measurement of volume can be done by using gravimetric method using small pocket weighing machines, which are accurate to milligrams.
• High or low volumes are associated with infertility. Normal volume is 2-6 ml per ejaculate.
• Viscosity is noted while transferring to test tube. If the sample leaves pipette in clear drops, the viscosity is normal. If it leaves with a thread of more than 2 cm, it is high viscosity.
• After noting physical parameters, pH is measured with the use of narrow range pH papers with range of 6-9 pH points.
• The pH strip is moistened with the seminal plasma and the color is compared with stabilization time of 30 seconds. pH is noted to nearest 0.1 pH points.
• Normal pH is alkaline, between 7.2 to 8.0. If pH is less than 7.0 with azospermia, there may be obstruction of the ejaculatory ducts or bilateral congenital absence of the vasa.
• pH of more than 8.0 is mostly associated with infections and high viscosity.
Visual Assessment of Sperm Motility
After physical parameters, sperm motility assessment is carried out with simple wet mount. Standardization can be achieved by using measured quantity of semen with same size of cover slip used. 10 microlitre drop of semen with 22 x 22 mm cover slip is good enough. This preparation achieves approximately 20 micron depth and gives almost single layer of sperms to make evaluation easy. Phase contrast microscope is most useful for detailed evaluation, though ordinary binocular microscope also can be used to evaluate semen samples. At least 5-10 randomly selected fields under high power should be evaluated, and motility should be evaluated based on these criteria.
Several scoring systems exist for sperm motility assessment, based on speed of sperm movement, quality of progression, etc.
The motility is judged on semi-subjective scale.
Class a: Rapid progressive motility
Class b: Slow sluggish progressive motility
Class c: Non-progressive motility
Class d: Immotile
The latest WHO manual recognizes only three categories, namely, progressive motile, non progressive motile, and non-motile, which is sufficient enough for evaluation and interpretation. In each selected small area of analysis, rapidly progressive sperms are counted first at the same time. New sperms entering into the area are excluded and only the ones present in the area at the determined time are counted. Then, slow, sluggish, non-progressive, and immotile are scored. The scoring is continued till at least total of 200 sperms are counted. Normal semen contains more than 50 % motile sperms with at least 25% rapidly progressive motile sperms. These observations can be simultaneously carried out along with total count in Cryocell Sperm Counting Chamber or Makler Chamber or disposable counting slides.
Other Microscopic Observations
After assessment of motility, sperm aggregation or agglutination can be observed. If sperms show aggregation, observation should be made whether they are head to head, tail to tail, or tail to head agglutinates. Estimation of percentage of sperms involved in agglutinates should be made. Agglutinates denote possible presence of anti-sperm antibodies.
Other Cellular Components and Debris
Even normal semen samples from fertile donors often contain substantial numbers of other cells and debris contamination.
• Round cells: It is important to differentiate round cells into leukocytes or immature germ cells. If more than one round cell per high power field is seen, it is necessary to do differential test for presence of leukocytes. Specific staining procedure can be carried out where leukocytes show presence of brown granules, with germ cells remaining unstained. Detailed evaluation can be carried out using stained smears. Presence of high number of leukocytes is indicative of infection. High number of round cells is seen in oligospermia, with rapid rate of spermatogenesis leading to sloughing of germinal cells in various stages of development.
• Epithelial cells: Epithelial cells from the reproductive tract normally contaminate semen. High number is associated with infection.
• Erythrocytes: RBCs are not normal contaminants of semen. Presence of large number of RBCs with pink/red coloration of semen is definitely abnormal.
• Particulate debris: Particulate debris matter, which also includes anucleate cytoplasmic masses smaller than sperm head, may be plentiful in normal semen.
Measurement of Sperm Concentration
Accurate determination of sperm concentration and total sperm count are important quantitative parameters for evaluating spermatogenesis. Due to importance of these parameters, various devices are designed to give accurate counts.
Various chambers
Traditional methods rest on accurate volumetric dilutions using positive displacement pipettes and improved Neubauer Chamber for counting. Neubauer Chamber is world standard method for accurate sperm counts. Method rests on dilution of semen 1:20 in sperm diluting fluid consisting of sodium bicarbonate and formaldehyde. After loading the Neubauer Chamber and allowing to settle for five minutes in moist chamber, five squares in RBC counting area are counted. The count will give million/ml count.
Sperm Counting Chamber or other such chambers are designed to give counts in undiluted samples. Added advantage is that you can assess motility and count at the same time with assessment of morphology if phase contrast microscope is used. This special sperm counting chamber has grid of 0.1 mm square with depth of l0 microns. Sperm count of more than 20 million/ml is considered normal.
Assessment of Sperm Morphology
An assessment of the morphological characteristics is as important for complete evaluation of the semen sample as count and motility. Ideally, morphology should be assessed on unfixed, unstained, wet preparations, using phase contrast microscopy. But, this requires great skill, as the size of sperm head is very small. Therefore, it is necessary to make air-dried smears from fresh semen sample. These smears are made on thoroughly cleaned slides, fixed, and then stained with suitable stains. Frequently used stains are Papanicolaou, Geimsa, Leishmann Shorr, etc. All are suitable, and choice is by personal preference. For strict morphology assessment, stained smears are must. Stained smears also can be stored for record purpose for a long time.
These criteria are considered normal by WHO:
Volume 1.5 ml or more
pH 7.2 or more
Sperm concentration 15 million/ ml
Motility 50 % motile
Vitality 50 % or more
Round cells less than 1 million/ml
Immunobead test < 50 % with bead bound
Sperm Morphology Classification
The major problem of morphological assessment is the pleomorphism of human spermatozoa. Studies on the selection of spermatozoa in-vitro and within the female genital tract in-vivo have helped to define the limits of normal human spermatozoa. The most widely-accepted criteria for normal sperm are endorsed by WHO, which originally provided the metric standards for normal sperm in stained smears. More recently, Kruger et al. have suggested strict criteria that have been useful in predicting the success in in-vitro fertilization. Though both are based on similar metric criteria of head length and width and tail length, strict criteria recognize many more subtle abnormalities that are considered as normal by WHO standards.
Normal sperm head is considered to be 3-5 micron in length and 2-3 microns in width with perfect oval shape. Mid-piece is about 1 micron in diameter with straight and regular outline. It must be aligned to the longitudinal axis of head and should be 7-8 microns in length. The tail must be slender, uncoiled, and at least 45 microns in length. Any sperm not meeting these criteria is considered abnormal in strict criteria.
Interpretation of Semen Parameters
A number of clinical approaches have been used to identify the minimum standards for fertile semen. One approach is to compare the results of semen evaluations in groups of fertile and infertile males. Second approach is to compare infertile couples with those conceiving during the treatment cycles. Results are inconsistent in some of the criteria used for evaluations, and common shortcoming is the reliance on a single semen evaluation for prediction. It is difficult to depend on single evaluation or single group of tests for predicting fertility, as the fertility depends on sum total of all the functional parameters of the sperm, and reliance on any one will be an ‘elephant and seven blind men’ story.
Sperm Function Tests
Various sperm function tests are proposed and endorsed by different researchers, in addition to routine evaluation just discussed, to evaluate fertility. These tests detect function of a certain part of spermatozoon and give insight on the events in fertilization of oocyte.
• Sperm membrane function: Sperm membrane structure and function can be determined by evaluating sperm viability and hypo-osmotic swelling test.
• Sperm viability test: Viable sperms with an intact sperm membrane do not allow eosin stain to pass into sperm head, therefore, sperm appears white against a dark background. Sperms that have a structural membrane defect allow eosin to leak into the sperm, and these appear pink. Therefore, membrane structural integrity can be tested with sperm viability test.
• Hypo-osmotic swelling test: HOS test gives functional integrity of the sperm membrane. Although sperm viability and HOS test both show a good correlation in results, they give different information about sperm membrane function. Spermatozoa are exposed to hypo-osmotic solution, which results in imbibing of water intracellular when the sperm membrane is functionally intact. This results in coiling of tails to various extents. The cells with defective sperm membrane do not show coiling of tails.
Tests of Sperm Membrane Binding, Capacitation, and Penetration
•• Hyper-activated sperm motility: hyper-activation of sperm is associated with a particular type of sperm motility and is probably related to capacitation of sperms. It is defined as increased flagellar movement and beat frequency with reduced progression. Observation of such motility is correlated with capability for capacitation.
• Hemi-zona Assay: This test measures capability of spermatozoa ability to bind with empty hemi-zona, and correlates well with the in-vitro fertilization results.
• Zona free Hamster Oocyte Test or Sperm Penetration Assay detects the ultimate fertilization capability. This is a highly complex test and can be performed in very specialized research labs only.
• Acrosome detection and Reaction: Sperms undergo biochemical changes over a several-hour period in-vivo in female genital tract that result in capacitation of sperm. This is a prerequisite for acrosome reaction, and for sperm to penetrate zona. Freshly ejaculated sperms are not able to fertilize eggs without undergoing changes either in-vitro or in the female genital tract. The process involves complex biochemical as well as physical changes in the sperm membrane.
• Acrosome area of sperm head contains acrosin in inactive form, which is first activated and then released at Zona, resulting in penetration of sperm. Capacitated sperms can release the acrosin, therefore, counting of acrosome reacted sperms and sperms with ability to acrosome react is a very important sperm function test. These are fairly complex tests beyond purview of most fertility centers.
• Triple Staining Triple staining technique evaluates the intact acrosome with special staining involving Rose Bengal. Rose Bengal selectively stains acrosome, and absence of stain indicates lack or loss of acrosome. This staining can be used as challenge test to evaluate both the initial and post-challenge percentage of acrosome reacted spenns.
• Fluorescent techniques : Various fluorescent ligands attach to various binding sites in acrosome line outer membrane, inner membrane, acrosin, activated acrosin, etc, and give various functional parameters. The fluorescent ligands used are various lectins, chlortetracycline, polyclonal and monoclonal sera to various proteins, etc.
• Acrosin Measurement: Acrosin is a proteolytic enzyme that can be measured with specific assay.
• Gelatinfilm lysis test: This is one of the simplest acrosome assays, and detects intactness of acrosome. The spermatozoa are thinly spread over a gelatin film and incubated in moist atmosphere. The intact acrosin spermatozoa will show proteolytic halo around the sperms due to digestion of gelatin film.
• Antisperm Antibodies: Antibodies present on the sperm surface can be detected by direct immunobead test. Presence of IgG and IgA type of antibodies can be detected by using IgG or IgA-coated beads, respectively. Similarly, indirect test using anti-globulin can be used for detecting antibodies in semen.
