INTRODUCTION
Semen (seminal fluid) consists of spermatozoa (sperms) and the fluid part. About 40% cases of infertility are due to abnormalities in semen and therefore semen analysis is the first test to be performed while investigating for infertility. Defect of sperms may be quantitative (absence of sperms, lack of enough sperms) or a qualitative.
Indications for Semen Analysis
•• Assessment of fertility/infertility.
•• Determine the effectiveness of vasectomy.
•• Determine the suitability of 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
Patient is asked to collect the semen by masturbation after a minimum of 2 days and a maximum of 7 days of sexual abstinence. Specimen should be collected in a clean, dry, widemouthed plastic/glass container. Collection of condom sample is not advisable because they often contain spermicidal agents which impair the sperm motility.
DIFFERENT PROCEDURES OF SEMEN ANALYSIS
They can be mainly divided into three types—standard, optional and research procedures.
Standard Procedures
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 liquefies within 15 minutes. Within 30 minutes it becomes more homogeneous and watery.
•• Semen viscosity: Fresh semen is fairly 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 increase in viscosity affects sperm motility.
•• Appearance: Freshly ejaculated semen is an opaque, white gray and viscid fluid. After liquefaction it has homogeneous grey-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.
•• Semen volume: Normal volume ranges from 1.4 to 1.7 mL per ejaculate. Low semen volume may be due to obstruction of the ejaculatory duct, congenital bilateral absence of the vasdeferens 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 to 8 (>7.2). The pH should be measured after liquefaction, preferably after 30 minutes.
Microscopic Examination
•• Sperm aggregation or agglutination
–– Aggregation of spermatozoa: 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 of spermatozoa: 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. The grading of agglutination of spermatozoa is shown in Table 53.1.
•• Cellular elements other than spermatozoa: During microscopic examination search should be made for the presence of cells other than spermatozoa. Some of these cells may be clinically relevant, which includes epithelial cells from the genitourinary tract and “roundcells” (leukocytes and immature germ cells).
•• Assessment of sperm motility: Motility of the sperms helps in penetration of cervical mucus and migration of the sperms into the fallopian tube. In normal semen, 38–42% of sperms should be motile (progressively motile and non-progressively motile). This is assessed by placing a drop of liquefied semen on a clean glass slide with a coverslip placed over it and examining under microscope. According to the WHO (2010), the motility of each spermatozoon (plural is spermatozoa) is categorized as progressively motile (PR), nonprogressively motile (NP) and immotile (IM). This grading system (Table 53.2) has replaced the older grading system of a, b, c or d (I to IV) grades.
•• Sperm vitality: It is important to know whether immotile spermatozoa are alive or dead. Normally 55 to 63 % live forms are observed. 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.
–– Dye exclusion method: It is based on the principle that damaged plasma membranes, such as those found in nonvital (dead) cells, allow entry of membrane-impermeant stains.
- ◆◆ Vitality test using eosin–nigrosin: The nigrosin helps to increases the contrast between the background and the sperm heads, thereby makes them easier to identify. The dead sperms are stained by eosin.
- ◆◆ Vitality test using eosin alone: This is a simple and rapid method, but the wet preparations cannot be stored for quality control purposes.
–– Hypo-osmotic swelling test: The principle is based on the fact that only cells with intact membranes (live cells) will swell in hypotonic solutions.
•• 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). The composition of semen diluting fluid is shown in Table 53.3. Normal range of sperm count: 33–46 millions/ejaculate.
•• Sperm morphology (Fig. 53.1): 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 end piece is difficult to see under light microscope, the spermatozoa can be considered to consists of a head (and neck) and tail (midpiece and principal piece). Normally more than 30% of sperms have normal morphology. For a spermatozoon to be considered as normal, both its head and tail must be morphologically normal. All other forms should be considered as abnormal.
–– Head: It is oval in shape with smooth outline. It has an acrosomal region which constitutes 40–70% of the head area. The acrosomal region does not contain large vacuoles and not more than two small vacuoles which do not occupy more than 20% of the sperm head. The post-acrosomal region of the head does not contain any vacuoles.
–– Neck and midpiece: It is slender, regular and has the same length as the sperm head. The major axis of the midpiece is in line with the major axis of the sperm head.
–– Tail (principal) piece: It has a uniform caliber and is thinner than the midpiece. It is about 10 times the head length and may be looped back on itself. considered as normal, both its head and tail must be morphologically normal. All otherforms should be considered as abnormal.
Classification of abnormal sperm morphology (Fig. 53.1):
Abnormal spermatozoa usually have a lower fertilizing capacity. The abnormalities in morphology can be classified as;
–– Head defects:
- ◆◆ Abnormal size: Large or small head
- ◆◆ Abnormal shape: Pointed/tapered, round, pyriform, amorphous
- ◆◆ Acrosomal defects
◊ Vacuolated acrosomal region: More than two vacuoles or >20% of the head area occupied by vacuoles. Presence of vacuoles in the post-acrosomal region.
◊ Abnormality of acrosomal areas: Small or large acrosomal areas (<40% or >70% of the head area).
- ◆◆ Abnormal number: Double-headed.
–– Neck and midpiece defects:
◆◆ Sharply bent neck
◆◆ Asymmetrical insertion of the midpiece into the head
◆◆ Thick or thin or irregular midpiece
–– Tail (principal) piece defects: These abnormalities include short or bent or coiled or double tail.
Tests for Antibody Coating of Spermatozoa
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 it 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 anti-sperm antibodies in sperm-free fluids (“indirect” tests).
Optional Procedures
These tests are not necessary for routine semen analysis, but may be useful in certain circumstances.
Indices of Multiple Sperm Defects
Spermatozoa which morphologically appear abnormal often have multiple defects (of the head, midpiece or principal piece, or combinations of these defects). A detailed examination of the incidence of morphological abnormalities is more useful than a simple evaluation of the percentage of morphologically normal spermatozoa. Three useful indices of multiple sperm defects are:
•• Multiple anomalies index (MAI): It is the mean number of anomalies per abnormal spermatozoon. It includes all the head, midpiece and principal piece anomalies.
•• Teratozoospermia index (TZI): In this, a maximum of four defects (one from each component of spermatozoon such as midpiece, tail piece) counted, irrespective of real number of anomalies in the abnormal spermatozoon.
•• Sperm deformity index (SDI): It is the number of defects divided by the total number of spermatozoa. These indices have been correlated with fertility in vivo (MAI and TZI) an in vitro (SDI).
Panleukocyte (CD45) Immunocytochemical Staining
All leukocytes express a CD45 which can be detected by immunocytochemical staining. This is useful for distinguishing between leukocytes and germ cells.
Interaction Between Spermatozoa and Cervical Mucus
Cervical mucus favors sperm penetration during the mid-cycle of the menstrual cycle.
•• In vivo (postcoital) test: The postcoital test is used to determine the number of active spermatozoa in the cervical mucus and to evaluate sperm survival and sperm behavior few hours after coitus.
•• In vitro tests: Assessment of sperm–cervical mucus interaction may also be undertaken using in vitro penetration tests.
Biochemical Assays for Accessory Sex Organ Function
Qualitative defects in semen may be due to testicular production of abnormal spermatozoa or from post-testicular damage to spermatozoa in the epididymis or the ejaculate from abnormal accessory gland secretions. Secretions from accessory glands can be measured to assess gland function, e.g. citric acid, zinc, γ-glutamyl transpeptidase and acid phosphatase for the prostate; fructose and prostaglandins for the seminal vesicles; free L-carnitine, glycerophosphocholine(GPC) and neutral α-glucosidase for the epididymis.
•• Measurement of zinc in seminal plasma: The amount of zinc in semen is a reliable measure of prostate gland secretion. The lower reference limit for zinc is 2.4 μmol per ejaculate.
•• Measurement of fructose in seminal plasma: Fructose is the main sugar in the semen reflects the secretory function of the seminal vesicles. It is reduced when there is androgen deficiency or obstruction to the ejaculatory duct. There is inverse relationship between sperm count and fructose level. The lower reference limit for fructose is 13 μmol per ejaculate.
•• Measurement of neutral α-glucosidase in seminal plasma: Seminal plasma contains both a neutral α-glucosidase isoenzyme, which originates in the epididymis, and an acid isoenzyme contributed by the prostate. The lower reference limit for neutral α-glucosidase is 20 mU per ejaculate.
•• Acid phosphatase test: It is used in alleged rape cases and is performed on vaginal aspirates. Normal level of acid phosphatase in semen is less than or equal to 200 U/ejaculate.
Computer-aided Sperm Analysis
Computer aided sperm analysis (CASA) can be used to measure sperm concentration. By the use of fluorescent DNA stains with CASA, the concentration of motile sperm and percentage motility can be determined.
Research Procedures
Several diagnostic tests have been developed which are capable of detecting the precise nature of the processes that are defective in the spermatozoa of infertile men. These are mentioned in Table 53.4.
CRYOPRESERVATION OF SPERMATOZOA
One of the methods of storing spermatozoa is by cryopreservation.
Indications for Cryopreservation
•• Donor semen: Donor spermatozoa can be used for artificial insemination (AI), intrauterine insemination (IUI), in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI).
•• Fertility preservation: Semen may be stored before a man undergoes a procedure or exposure that might prevent or impair his fertility. These include vasectomy, treatment with cytotoxic agents or radiotherapy.
•• Infertility treatment: Spermatozoa may be stored for artificial insemination by husband’s semen.
•• Minimizing infectious disease transmission: For men with HIV controlled by antiretroviral therapy, semen samples with an undetectable viral load may be stored for IUI, IVF or ICSI, to attempt conception which reduces the risk of HIV transmission to the female partner.
Terminologies used in semen analysis (Table 53.5).
Points to note:
•• Semen analysis is used for assessment of fertility, effectiveness of vasectomy, to determine its suitability for artificial insemination and medicolegal purpose.
•• Semen analysis is mainly divided into three types: standard, optional and research procedures.
•• Standard procedure includes physical examination, microscopic examinations and tests for antibody coating spermatozoa.
•• Normal sperm count ranges from 33 to 46 millions/ejaculate.
•• Morphologically, spermatozoa have a head, neck, middle piece (midpiece), principal piece and endpiece and abnormality may involve any one or more of these components.
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