PREGNANCY TESTS

Written by Wednesday, 16 August 2017 12:56
Pregnancy tests detect human chorionic gonadotropin (hCG) in serum or urine. Although pregnancy is the most common reason for ordering the test for hCG, measurement of hCG is also indicated in other conditions as shown in Box 836.1.
 
Box 836.1 Indications for measurement of β human chorionic gonadotropin

• Early diagnosis of pregnancy
• Diagnosis and management of gestational trophoblastic disease
• As a part of maternal triple test screen
• Follow-up of malignant tumors that produce β human chorionic gonadotropin.
Human chorionic gonadotropin is a glycoprotein hormone produced by placenta that circulates in maternal blood and excreted intact by the kidneys. It consists of two polypeptide subunits: α (92 amino acids) and β (145 amino acids) which are non-covalently bound to each other. Structurally, hCG is closely related to three other glycoprotein hormones, namely, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyroid-stimulating hormone (TSH). The α subunits of hCG, LH, FSH, and TSH are similar, while β subunits differ and confer specific biologic and immunologic properties. Immunological tests use antibodies directed against β-subunit of hCG to avoid cross-reactivity against LH, FSH, and TSH.
 
Syncytiotrophoblastic cells of conceptus and later of placenta synthesize hCG. Human chorionic gonadotropin supports the corpus luteum of ovary during early pregnancy. Progesterone, produced by corpus luteum, prevents ovulation and thus maintains pregnancy. After 7-10 weeks of gestation, sufficient amounts of progesterone are synthesized by placenta, and hCG is no longer needed and its level declines.
 
CLINICAL APPLICATIONS OF TESTS FOR HUMAN CHORIONIC GONADOTROPIN
 
  1. Early diagnosis of pregnancy: Qualitative serum hCG test becomes positive 3 weeks after last menstrual period (LMP), while urine hCG test becomes positive 5 weeks after LMP.
  2. Exclusion of pregnancy before prescribing certain medications (like oral contraceptives, steroids, some antibiotics), and before ordering radiological studies, radiotherapy, or chemotherapy. This is necessary to prevent any teratogenic effect on the fetus.
  3. Early diagnosis of ectopic pregnancy: Trans-vaginal ultrasonography (USG) and quantitative estimation of hCG are helpful in early diagnosis of ectopic pregnancy (before rupture).
  4. Evaluation of threatened abortion: Serial quantitative estimation of hCG is helpful in following the course of threatened abortion.
  5. Diagnosis and follow-up of gestational trophoblastic disease (GTD).
  6. Maternal triple test screen: This consists of measurement of hCG, α-fetoprotein, and unconjugated estriol in maternal serum at 14-19 weeks of gestation. The maternal triple screen identifies pregnant women with increased risk of Down syndrome and major congenital anomalies like neural tube defects.
  7. Follow-up of ovarian or testicular germ cell tumors, which produce hCG.
 
Normal Pregnancy
 
In women with normal menstrual cycle, conception (fertilization of ovum to form a zygote) occurs on day 14 in the fallopian tube. Zygote travels down the fallopian tube into the uterus. Division of zygote produces a morula. At 50-60-cell stage, morula develops a primitive yolk sac and is then called as a blastocyst. About 5 days after fertilization, implantation of blastocyst occurs in the uterine wall. Trophoblastic cells (on the outer surface of the blastocyst) penetrate the endometrium and develop into chorionic villi. There are two main forms of trophoblasts—syncytiotrophoblast and cytotrophoblast. Placental development occurs from chorionic villi. After formation of placenta, the conceptus is called as an embryo. When embryo develops most major organs, it is called as fetus (after 10 weeks of gestation).
 
Figure 836.1 Level of human chorionic gonadotropin during pregnancy
Figure 836.1 Level of human chorionic gonadotropin during pregnancy
 
Box 836.2 Diagnosis of early pregnancy

• Positive serum hCG test: 8 days after conception or 3 weeks after last menstrual period (LMP)
• Positive urine hCG test: 21 days after conception or 5 weeks after LMP
• Ultrasonography for visualization of gestational sac:
– Transvaginal: 21 days after conception or 5 weeks after LMP
– Transabdominal: 28 days after conception or 6 weeks after LMP
Human chorionic gonadotropin is synthesized by syncytiotrophoblasts (of placenta) and detectable amounts (~5 mIU/ml) appear in maternal serum about 8 days after conception (3 weeks after LMP). In the first trimester (first 12 weeks, calculated from day 1 of LMP) of pregnancy, hCG levels rapidly rise with a doubling time of about 2 days. Highest or peak level is reached at 8-10 weeks (about 100,000 mIU/ml). This is followed by a gradual fall, and from 15-16 weeks onwards, a steady level of 10,000-20,000 mIU/ml is maintained for the rest of the pregnancy (Figure 836.1). After delivery, hCG becomes non-detectable by about 2 weeks.
 
Box 836.2 shows minimum time required for the earliest diagnosis of pregnancy by hCG test and ultrasonography (USG).
 
Two types of pregnancy tests are available:
 
  • Qualitative tests: These are positive/negative result types that are done on urine sample.
  • Quantitative tests: These give numerical result and are done on serum or urine. They are also used for evaluation of ectopic pregnancy, failing pregnancy, and for follow-up of gestational trophoblastic disease.
 
Ectopic Pregnancy
 
Ectopic pregnancy refers to the implantation of blastocyst at a site other than the cavity of uterus. The most common of such sites (>95% cases) is fallopian tube. Early diagnosis and treatment of tubal ectopic pregnancy is essential since it can lead to maternal mortality (from rupture and hemorrhage) and future infertility. Ectopic pregnancy is a leading cause of maternal death during first trimester. Diagnosis of ectopic pregnancy can be readily made in most cases by ultrasonography and estimation of β-subunit of human chorionic gonadotropin.
 
Early diagnosis of unruptured tubal pregnancy can be made by quantitative estimation of serum hCG and ultrasonography. In normal intrauterine pregnancy, hCG titer doubles every 2 days until first 40 days of gestation. If hCG rise is abnormally slow, then an unviable pregnancy (either ectopic or abnormal intrauterine pregnancy) should be suspected.
 
Transabdominal USG can detect gestational sac in intrauterine pregnancy 6 weeks after LMP. The level of hCG in serum at this stage is >6500 mIU/ml. If gestational sac is not visualized at this level of hCG, then there is a possibility of ectopic pregnancy. Transvaginal ultrasonography can detect ectopic pregnancy average 1 week earlier than abdominal ultrasonography; it can detect gestational sac if β-hCG level is 1000-1500 mIU/ml. Therefore, if gestational sac is not visualized in the presence of >1500 mIU/ml of β-hCG level, an ectopic pregnancy can be suspected.
 
Early diagnosis of ectopic pregnancy provides the option of administration of intramuscular methotrexate (rather than surgery), which causes dissolution of conceptus. This improves the chances of patient’s future fertility. Serial measurements of hCG after surgical removal of ectopic pregnancy can help in detecting persistence of trophoblastic tissue.
 
Abortion
 
Termination of pregnancy before fetus becomes viable (i.e. before 20 weeks) is called as abortion.
 
In threatened abortion, vaginal bleeding is present but internal os is closed and process of abortion, though started, is still reversible. It is possible that pregnancy will continue.
 
Serial quantitative titers of hCG showing lack of expected doubling of hCG level and USG are helpful in diagnosis and management of abortion.
 
Gestational Trophoblastic Disease (GTD)
 
It is characterized by proliferation of pregnancyassociated trophoblastic tissue. The two main forms of GTD are hydatidiform (vesicular) mole (benign) and choriocarcinoma (malignant). Clinical features of GTD are as follows:
 
  • Short history of amenorrhea followed by vaginal bleeding.
  • Size of uterus larger than gestational age; uterus is soft and doughy on palpation with no fetal parts and no fetal heart sounds.
  • Excessive nausea and vomiting due to high hCG.
  • Characteristic snowstorm appearance on pelvic USG.
 
Quantitative estimation of hCG is helpful in diagnosis and management of GTD.
 
Trophoblastic cells of GTD produce more hCG as compared to the trophoblasts of normal pregnancy for the same gestational age. Concentration of hCG parallels tumor load. Also, hCG continues to rise beyond 10 weeks of gestation without reaching plateau (as expected at the end of first trimester).
 
After evacuation of uterus, weekly estimation of hCG is advised till subsequent three (weekly) results are negative; following evacuation of vesicular mole, hCG becomes undetectable (after 2-3 months) on follow-up in 80% of cases. Plateau or rising hCG indicates persistent GTD. In such cases, chemotherapy is indicated.
 
Negative results for hCG after therapy should be regularly followed up every 3 months for 1-2 years.
 
LABORATORY TESTS FOR HUMAN CHORIONIC GONADOTROPIN
 
These are classified into two main groups:
 
  • Biological assays or bioassays
  • Immunological assays
 
Bioassays
 
In bioassay, effect of hCG is tested on laboratory animals under standardized conditions. There are several limitations of bioassays like need for animal facilities, need for standardization of animals, long time required for the test results, low sensitivity, and high cost. Therefore, bioassays have been replaced by immunological assays.
 
In Ascheim-Zondek test, urine from pregnant woman is injected into immature female mice. Formation of hemorrhagic corpora lutea in ovaries (after 4 days) is a positive test. Friedman test is similar except that urine is injected into female rabbit. In rapid rat test, injection of urine containing hCG into female rats is followed by hyperaemia and hemorrhage in ovaries. Yet another test measures release of spermatozoa from male frog after injection of urine containing hCG.
 
Immunological Assays
 
These are rapid and sensitive tests for detection and quantitation of hCG. Variable results are obtained by different immunological tests with the same serum sample; this is due to differences in specificity of different immunoassays to complete hCG, β-subunit, and β-core fragment. A number of immunological tests are commercially available based on different principles like agglutination inhibition assay, enzyme immunoassay including enzyme linked immunosorbent assay or ELISA, radioimmunoassay (RIA), and immunoradiometric assay.
 
A commonly used qualitative urine test is agglutination inhibition assay. Early morning urine specimen is preferred because it contains the highest concentration of hCG. Causes of false-positive test include red cells, leukocytes, bacteria, some drugs, proteins, and excess luteinizing hormone (menopause, midcycle LH surge) in urine. Some patients have anti-mouse antibodies (that are used in the test), while others have hCG-like material in circulation, producing false-positive test. Anti-mouse antibodies also interfere with other antibody-based tests and are known as ‘heterophil’ antibodies. Fetal death, abortion, dilute urine, and low sensitivity of a particular test are causes of false-negative test. Renal failure leads to accumulation of interfering substances causing incorrect results.
 
Figure 836.2 Principle of agglutination inhibition test for diagnosis of pregnancy
Figure 836.2 Principle of agglutination inhibition test for diagnosis of pregnancy
 
In latex particle agglutination inhibition test (Figure 836.2), anti-hCG antibodies are incubated with patient’s urine. This is followed by addition of hCGcoated latex particles. If hCG is present in urine, anti-hCG serum is neutralized, and no agglutination of latex particles occurs (positive test). If there is no hCG in urine, there is agglutination of latex particles (negative test). This is commonly used as a slide test and requires only a few minutes.
 
Sensitivity of agglutination inhibition test is >200 units/liter of hCG.
 
Radioimmunoassay, enzyme immunoassay, and radioimmunometric assay are more sensitive and reliable than agglutination inhibition assay.
 
Quantitative tests are employed for detection of very early pregnancy, estimation of gestational age, diagnosis of ectopic pregnancy, evaluation of threatened abortion, and management of GTD.
 
REFERENCE RANGES
 
  • Serum human chorionic gonadotropin:
    – Non-pregnant females: <5.0 mIU/ml
    – Pregnancy: 4 weeks after LMP: 5-100 mIU/ml
    – 5 weeks after LMP: 200-3000 mIU/ml
    – 6 weeks after LMP: 10,000-80,000 mIU/ml
    – 7-14 weeks: 90,000-500,000 mIU/ml
    – 15-26 weeks: 5000-80000 mIU/ml
    – 27-40 weks: 3000-15000 mIU/ml
 
 Further Reading:
 

SEMEN ANALYSIS FOR INVESTIGATION OF INFERTILITY

Written by Tuesday, 15 August 2017 23:54
 Box 835.1 Contributions to semen volume
 
• Testes and epididymis: 10%
• Seminal vesicles: 50%
• Prostate: 40%
• Cowper’s glands: Small volume
Semen (or seminal fluid) is a fluid that is emitted from the male genital tract and contains sperms that are capable of fertilizing female ova. Structures involved in production of semen are (Box 835.1):
 
  • Testes: Male gametes or spermatozoa (sperms) are produced by testes; constitute 2-5% of semen volume.
  • Epididymis: After emerging from the testes, sperms are stored in the epididymis where they mature; potassium, sodium, and glycerylphosphorylcholine (an energy source for sperms) are secreted by epididymis.
  • Vas deferens: Sperms travel through the vas deferens to the ampulla which is another storage area. Ampulla secretes ergothioneine (a yellowish fluid that reduces chemicals) and fructose (source of nutrition for sperms).
  • Seminal vesicles: During ejaculation, nutritive and lubricating fluids secreted by seminal vesicles and prostate are added. Fluid secreted by seminal vesicles consists of fructose (energy source for sperms), amino acids, citric acid, phosphorous, potassium, and prostaglandins. Seminal vesicles contribute 50% to semen volume.
  • Prostate: Prostatic secretions comprise about 40% of semen volume and consist of citric acid, acid phosphatase, calcium, sodium, zinc, potassium, proteolytic enzymes, and fibrolysin.
  • Bulbourethral glands of Cowper secrete mucus.
 
Normal values for semen analysis are shown in Tables 835.1 and 835.2.
 
Table 835.1 Normal values of semen analysis (World Health Organization, 1999)
Test Result
1. Volume ≥2 ml
2. pH 7.2 to 8.0
3. Sperm concentration ≥20 million/ml
4. Total sperm count per ejaculate ≥40 million
5. Morphology ≥30% sperms with normal morphology
6. Vitality ≥75% live
7. White blood cells <1 million/ml
8. Motility within 1 hour of ejaculation  
    • Class A ≥25% rapidly progressive
    • Class A and B ≥50% progressive
9. Mixed antiglobuiln reaction (MAR) test <50% motile sperms with adherent particles
10. Immunobead test <50% motile sperms with adherent particles
 
Table 835.2 Biochemical variables of semen analysis (World Helath Organization, 1992)
 1. Total fructose (seminal vesicle marker) ≥13 μmol/ejaculate 
 2. Total zinc (Prostate marker)  ≥2.4 μmol/ejaculate
 3. Total acid phosphatase (Prostate marker)  ≥200U/ejaculate
 4. Total citric acid (Prostate marker)  ≥52 μmol/ejaculate
 5. α-glucosidase (Epididymis marker)  ≥20 mU/ejaculate
 6. Carnitine (Epididymis marker)  0.8-2.9 μmol/ejaculate
 
INDICATIONS FOR SEMEN ANALYSIS
 
Box 835.2 Tests done on seminal fluid
 
• Physical examination: Time to liquefaction, viscosity, volume, pH, color
• Microscopic examination: Sperm count, vitality, motility, morphology, and proportion of white cells
• Immunologic analysis: Antisperm antibodies (SpermMAR test, Immunobead test)
• Bacteriologic analysis: Detection of infection
• Biochemical analysis: Fructose, zinc, acid phosphatase, carnitine.
• Sperm function tests: Postcoital test, cervical mucus penetration test, Hamster egg penetration assay, hypoosmotic swelling of flagella, and computer-assisted semen analysis
Availability of semen for examination allows direct examination of male germ cells that is not possible with female germ cells. Semen analysis requires skill and should preferably be done in a specialized andrology laboratory.
 
  1. Investigation of infertility: Semen analysis is the first step in the investigation of infertility. About 30% cases of infertility are due to problem with males.
  2. To check the effectiveness of vasectomy by confirming absence of sperm.
  3. To support or disprove a denial of paternity on the grounds of sterility.
  4. To examine vaginal secretions or clothing stains for the presence of semen in medicolegal cases.
  5. For selection of donors for artificial insemination.
  6. For selection of assisted reproductive technology, e.g. in vitro fertilization, gamete intrafallopian transfer technique.
 
COLLECTION OF SEMEN FOR INVESTIGATION OF INFERTILITY
 
Semen specimen is collected after about 3 days of sexual abstinence. Longer period of abstinence reduces motility of sperms. If the period of abstinence is shorter than 3 days, sperm count is lower. The sample is obtained by masturbation, collected in a clean, dry, sterile, and leakproof wide-mouthed plastic container, and brought to the laboratory within 1 hour of collection. The entire ejaculate is collected, as the first portion is the most concentrated and contains the highest number of sperms. During transport to the laboratory, the specimen should be kept as close to body temperature as possible (i.e. by carrying it in an inside pocket). Ideally, the specimen should be obtained near the testing site in an adjoining room. Condom collection is not recommended as it contains spermicidal agent. Ejaculation after coitus interruptus leads to the loss of the first portion of the ejaculate that is most concentrated; therefore this method should not be used for collection. Two semen specimens should be examined that are collected 2-3 weeks apart; if results are significantly different additional samples are required.
 
Box 835.3 Semen analysis for initial investigation of infertility
 
• Volume
• pH
• Microscopic examination for (i) percentage of motile spermatozoa, (ii) sperm count, and (iii) sperm morphology
EXAMINATION OF SEMINAL FLUID
 
The tests that can be done on seminal fluid are shown in Box 835.2. Tests commonly done in infertility are shown in Box 835.3. The usual analysis consists of measurement of semen volume, sperm count, sperm motility, and sperm morphology.
 
Terminology in semen analysis is shown in Box 835.4.
 
EXAMINATION OF SEMEN TO CHECK THEEFFECTIVENESS OF VASECTOMY
 
 Box 835.4 Terminology in semen analysis

• Normozoospermia: All semen parameters normal
• Oligozoospermia: Sperm concentration <20 million/ml (mild to moderate: 5-20 million/ml; severe: <5 million/ml)
• Azoospermia: Absence of sperms in seminal fluid
• Aspermia: Absence of ejaculate
• Asthenozoospermia: Reduced sperm motility; <50% of sperms showing class (a) and class (b) type of motility OR <25% sperms showing class (a) type of motility.
• Teratozoospermia: Spermatozoa with reduced proportion of normal morphology (or increased proportion of abnormal forms)
• Leukocytospermia: >1 million white blood cells/ml of semen
• Oligoasthenoteratozoospermia: All sperm variables are abnormal
• Necrozoospermia: All sperms are non-motile or non-viable
The aim of post-vasectomy semen analysis is to detect the presence or absence of spermatozoa. The routine follow-up consists of semen analysis starting 12 weeks (or 15 ejaculations) after surgery. If two successive semen samples are negative for sperms, the semen is considered as free of sperm. A follow-up semen examination at 6 months is advocated by some to rule out spontaneous reconnection.
 
Further Reading:
 

SPERM FUNCTION TESTS OR FUNCTIONAL ASSAYS

Written by Tuesday, 15 August 2017 01:44
These tests are available only in specialized andrology laboratories. The tests are not standardized thus making interpretation difficult. If used singly, a sperm function test may not be helpful in fertility assessment. They are more predictive if used in combination.
 
Postcoital (Sims-Huhner) Test
 
This is the examination of the cervical mucus after coitus and assesses the ability of the sperm to penetrate the cervical mucus. The quality of the cervical mucus varies during the menstrual cycle, becoming more abundant and fluid at the time of ovulation (due to effect of estrogen); this facilitates penetration of the mucus by the spermatozoa. Progesterone in the secretory phase increases viscosity of the mucus. Therefore cervical mucus testing is scheduled just before ovulation (determined by basal body temperature records or follicular sizing by ultrasonography). Postcoital test is the traditional method to detect the cervical factor in infertility. Cervical mucus is aspirated with a syringe shortly before the expected time of ovulation and 2-12 hours after intercourse. Gross and microscopic examinations are carried out to assess the quality of cervical mucus (elasticity and drying pattern) and to evaluate the number and motility of sperms (Box 834.1). If ≥ 10 motile sperms are observed the test is considered as normal. An abnormal test may result from: (a) poor quality of cervical mucus due to wrong judgment of ovulation, cervicitis or treatment with antioestrogens (e.g. Clomid), and (b) absence of motile sperms due to ineffective technique of coitus, lack of ejaculation, poor semen quality, use of coital lubricants that damage the sperm, or presence of antisperm antibodies. Antisperm antibodies cause immotile sperms, or agglutination or clumping of sperms; they may be present in either partner. If cervical factor is present, intrauterine insemination is the popular treatment. The value of the postcoital test is disputed in the medical literature.
 
Box 834.1 Interpretation of postcoital test
  • Normal: Sperms are normal in amount and moving forward in the mucus; mucus stretches atleast 2 inches (5 cm) and dries in a fern-like manner.
  • Abnormal: Absence of sperms or large number of sperms are dead or sperms are clumped; cervical mucus cannot stretch 2 inches (5 cm) or does not dry in a fern-like manner.
 
This test can be carried out if semen analysis is normal, and the female partner is ovulating and fallopian tubes are not blocked. It is also done if antisperm antibodies are suspected and male partner refuses semen analysis.
 
Cervical Mucus Penetration Test
 
In this test, greatest distance traveled by the sperm in seminal fluid placed and incubated in a capillary tube containing bovine mucus is measured. Majority of fertile men show score >30 mm, while most infertile men show scores <20 mm.
 
Hamster Egg Penetration Assay
 
Hamster oocytes are enzymatically treated to remove the outer layers (that inhibit cross-species fertilization). They are then incubated with sperms and observed for penetration rate. It can be reported as (a) Number of eggs penetrated (penetration rate <15% indicates low fertility), or as (b) Number of sperm penetrations per egg (Normal >5). This test detects sperm motility, binding to oocyte, and penetration of oocyte. There is a high incidence of false-negative results.
 
Hypo-osmotic Swelling of Flagella
 
This test assesses the functional integrity of the plasma membrane of the sperm by observing curling of flagella in hypo-osmotic conditions.
 
Computer-assisted Semen Analysis
 
Computer software measures various characteristics of the spermatozoa; however, its role in predicting fertility potential is not confirmed.
ANTISPERM ANTIBODIES
 
The role of antisperm antibodies in causation of male infertility is controversial. The immunological tests done on seminal fluid include mixed antiglobulin reaction (MAR test) and immunobead test.
 
The antibodies against sperms immobilize or kill them, thus preventing their passage through the cervix to the ovum. The antibodies can be tested in the serum, seminal fluid, or cervical mucus. If the antibodies are present bound to the head of the sperm, they will prevent the penetration of the egg by the sperm. If antibodies are bound to the tail of the sperm, they will retard motility.
 
a. SpermMAR™ test: This test can detect IgG and IgA antibodies against sperm surface in semen sample. In direct SpermMAR™ IgG test, a drop each of semen (fresh and unwashed), IgG-coated latex particles, and anti-human immunoglobulin are mixed together on a glass slide. At least 200 motile spermatozoa are examined. If the spermatozoa have antibodies on their surface, antihuman immunoglobulin will bind IgG-coated latex particles to IgG on the surface of the spermatozoa; this will cause attachment of latex particles to spermatozoa, and motile, swimming sperms with attached particles will be seen. If the spermatozoa do not have antibodies on their surface, they will be seen swimming without attached particles; the latex particles will show clumping due to binding of their IgG to antihuman immunoglobulin.
 
In direct SpermMAR™ IgA test, a drop each of fresh unwashed semen and of IgA-coated latex particles, are mixed on a glass slide. The latex particles will bind to spermatozoa if spermatozoa are coated with IgA antibodies.
 
In indirect SpermMAR™ tests, fluid without spermatozoa (e.g. serum) is tested for the presence of antisperm antibodies. First, antibodies are bound to donor spermatozoa which are then mixed with the fluid to be analyzed. These antibodies are then detected as described above for direct tests.

Atleast 200 motile spermatozoa should be counted. If >50% of spermatozoa show attached latex particles, immunological problem is likely.
 
b. Immunobead test: Antibodies bound to the surface of the spermatozoa can be detected by antibodies attached to immunobeads (plastic particles with attached anti-human immunoglobulin that may be either IgG, IgA, or IgM). Percentage of motile spermatozoa with attached two or more immunobeads are counted amongst 200 motile spermatozoa. Finding of >50% spermatozoa with attached beads is abnormal.
The most important test in semen analysis for infertility is microscopic examination of the semen.
 
SPERM MOTILITY
 
The first laboratory assessment of sperm function in a wet preparation is sperm motility (ability of the sperms to move). Sperm motility is essential for penetration of cervical mucus, traveling through the fallopian tube, and penetrating the ovum. Only those sperms having rapidly progressive motility are capable of penetrating ovum and fertilizing it.
 
Principle: All motile and non-motile sperms are counted in randomly chosen fields in a wet preparation under 40× objective. Result is expressed as a percentage of motile spermatozoa observed.
 
Method: A drop of semen is placed on a glass slide, covered with a coverslip that is then ringed with petroleum jelly to prevent dehydration, and examined under 40× objective. Atleast 200 spermatozoa are counted in several different microscopic fields. Result is expressed as a percentage of (a) rapidly progressive spermatozoa (moving fast forward in a straight line), (b) slowly progressive spermatozoa (slow linear or non-linear, i.e. crooked or curved movement), (c) non-progressive spermatozoa (movement of tails, but with no forward progress), and (d) immotile spermatozoa (no movement at all) (WHO critera). Sperms of grades (c) and (d) are considered to be poorly motile (asthenospermia). Normally, ≥ 25% of sperms show rapid progressive motility, or ≥ 50% of sperms show rapid progressive and slow progressive motility.
 
If the proportion of motile spermatozoa is < 50%, then proportion of viable sperms should be determined by examining an eosin preparation.
 
SPERM VIABILITY OR VITALITY
 
Principle: A cell with intact cell membrane (a vital or viable cell) will not take up the eosin Y and will not be stained, while a non-viable or dead cell will have damaged cell membrane, will take up the dye, and will be stained pink-red (Figure 832.1). Another stain (e.g. nigrosin) may  be used to stain the background material. The test is performed if motility is abnormal.
 
Figure 832.1 Eosin nigrosin stain
Figure 832.1 Eosin-nigrosin stain. Dead sperms are stained pink-red, while live sperms are stained white
 
Method
 
  1. Mix one drop of semen with 1 drop of eosin-nigrosin solution and incubate for 30 seconds.
  2. A smear is made from a drop placed on a glass slide.
  3. The smear is air-dried and examined under oilimmersion objective. White sperms are classified as live or viable, and red sperms are classified as dead or non-viable. At least 200 spermatozoa are examined.
  4. The result is expressed as a proportion of viable sperms against non-viable as an integer percentage.
 
Seventy-five percent or more of sperms are normally live or viable.
 
SPERM COUNT
 
Principle: The sperm count is done after liquefaction in a counting chamber following dilution and the total number of spermatozoa is reported in millions/ml (106/ml).
 
Method
 
  1. Semen is diluted 1:20 with sodium bicarbonateformalin diluting fluid (Take 1 ml liquefied semen in a graduated tube and fill with diluting fluid to 20 ml mark. Mix well).
  2. A coverslip is placed over the improved Neubauer counting chamber and the counting chamber is filled with the well-mixed diluted semen sample using a Pasteur pipette. The chamber is then placed in a humid box for 10-15 minutes for spermatozoa to settle.
  3. The chamber is placed on the microscope stage. Using the 20× or 40× objective and iris diaphragm lowered sufficiently to give sufficient contrast, number of spermatozoa is counted in 4 large corner squares. Spermatozoa whose heads are touching left and upper lines of the square should be considered as ‘belonging’ to that square.
  4. Sperm count per ml is calculated as follows:

    Sperm count =                Sperms counted × correction factor             × 1000
                              Number of squares counted × Volume of 1 square
                           = Sperms counted × 20 1000
                                        4 × 0.1
                           = Sperms counted × 50, 000

  5. Normal sperm count is ≥ 20 million/ml (i.e. ≥ 20 × 106/ml). Sperm count < 20 million/ml may be associated with infertility in males.
 
SPERM MORPHOLOGY
 
A smear is prepared by spreading a drop of seminal fluid on a glass slide, stained, and percentages of normal and abnormal forms of spermatozoa are counted. The staining techniques used are Papanicolaou, eosinnigrosin, hematoxylin-eosin, and Rose Bengal-toluidine blue stain. Atleast 200 spermatozoa should be counted under oil immersion. Percentages of normal and abnormal spermatozoa should be recorded.
 
Normal morphology: A spermatozoon consists of three main components: head, neck, and tail. Tail is further subdivided into midpiece, main (principle) piece, and end piece (Figure 832.2 and Box 832.1).
 
Figure 832.2 Morphology of spermatozoa
Figure 832.2 Morphology of spermatozoa
 
Head is pear-shaped. Most of the head is occupied by the nucleus which has condensed chromatin and few areas of dispersed chromatin (called nuclear vacuoles). The anterior 2/3rds of the nucleus is surrounded by acrosomal cap. Acrosomal cap is a flattened membranebound vesicle containing glycoproteins and enzymes. These enzymes are required for separation of cells of corona radiata and dissolution of zona pellucida of ovum during fertilization.
 
Neck is a very short segment that connects the head and the tail. Centriole in the neck gives rise to axoneme of the flagellum. Axoneme consists of 20 microtubules (arranged as a central pair surrounded by 9 peripheral doublets) and is surrounded by condensed fibrous rings.
 
Middle piece is the first part of the tail and consists of central axoneme surrounded by coarse longitudinal fibers. These are surrounded by elongated mitochondria that provide energy for movement of tail.
 
Principle or main piece constitutes most of the tail and is composed of axoneme that is surrounded by 9 coarse fibers. This central core is surrounded by many circularly arranged fibrous ribs.
 
Endpiece is the short tapering part composed of only axoneme.
 
Normally, > 30% of spermatozoa should show normal morphology (WHO, 1999). The defects in morphology that are associated with infertility in males include defective mid-piece (causes reduced motility), an incomplete or absent acrosome (causes inability to penetrate the ovum), and giant head (defective DNA condensation).
 
Box 832.1 Normal sperm morphology
• Total length of sperm: About 60 μ
• Total length of sperm: About 60 μ
• Head:
   – Length: 3-5 μ
   – Width: 2-3 μ
   – Thickness: 1.5 μ
• Neck: Length: 0.3 μ
• Middle piece:
   – Length: 3-5 μ
   – Width: 1.0 μ
• Principal piece:
   – Length: 40-50 μ
   – Width: 0.5 μ
• End piece: 4-6 μ
 
Abnormal morphology (Figure 832.3): WHO morphological classification of human spermatozoa (1999) is given below:
 
  1. Normal sperm
  2. Defects in head:
    • Large heads
    • Small heads
    • Tapered heads
    • Pyriform heads
    • Round heads
    • Amorphous heads
    • Vacuolated heads (> 20% of the head area occupied by vacuoles)
    • Small acrosomes (occupying < 40% of head area)
    • Double heads
  3. Defects in neck:
    • Bent neck and tail forming an angle >90° to the long axis of head
  4. Defects in middle piece:
    • Asymmetric insertion of midpiece into head
    • Thick or irregular midpiece
    • Abnormally thin midpiece
  5. Defects in tail:
    • Bent tails
    • Short tails
    • Coiled tails
    • Irregular tails
    • Multiple tails
    • Tails with irregular width
  6. Pin heads: Not to be counted
  7. Cytoplasmic droplets
    • > 1/3rd the size of the sperm head
  8. Precursor cells: Considered abnormal
 
Figure 832.3 Abnormal morphological sperm forms
Figure 832.3 Abnormal morphological sperm forms: (1) Normal sperm, (2) Large head, (3) Small head, (4) Tapered head, (5) Pyriform head, (6) Round head, (7) Amorphous head, (8) Vacuoles in head, (9) Round head without acrosome, (10) Double head, (11) Pin head, (12) Round head without acrosome and thick midpiece, (13) Coiled tail, and (14) Double tail

ROUND CELLS
 
Round cells on microscopic examination may be white blood cells or immature sperm cells. Special stain (peroxidase or Papanicolaou) is required to differentiate between them. White blood cells >1 million/ml indicate presence of infection. Presence of large number of immature sperm cells indicates spermatogenesis dysfunction at the testicular level.
Biochemical markers (Table 831.1) can be measured in semen to test the secretions of accessory structures. These include fructose (seminal vesicles), zinc, citric acid or acid phosphatase (prostate), and α-glucosidase or carnitine (epididymis).
 
Table 831.1 Biochemical variables of semen analysis (World Helath Organization, 1992)
1. Total fructose (seminal vesicle marker) ≥13 μmol/ejaculate
2. Total zinc (Prostate marker) ≥2.4 μmol/ejaculate
3. Total acid phosphatase (Prostate marker) ≥200U/ejaculate
4. Total citric acid (Prostate marker) ≥52 μmol/ejaculate
5. α-glucosidase (Epididymis marker) ≥20 mU/ejaculate
6. Carnitine (Epididymis marker) 0.8-2.9 μmol/ejaculate
 
TEST FOR FRUCTOSE
 
Resorcinol method is used for detection of fructose. In this test, 5 ml of resorcinol reagent (50 mg resorcinol dissolved in 33 ml concentrated hydrochloric acid; dilute up to 100 ml with distilled water) is added to 0.5 ml of seminal fluid. The mixture is heated and brought to boil. If fructose is present, a red-colored precipitate is formed within 30 seconds.
 
Absence of fructose indicates obstruction proximal to seminal vesicles (obstructed or absent vas deferens) or a lack of seminal vesicles. In a case of azoospermia, if fructose is absent, it is due to the obstruction of ejaculatory ducts or absence of vas deferens, and if present, azoospermia is due to failure of testes to produce sperm.
Examination is carried out after liquefaction of semen that occurs usually within 20-30 minutes of ejaculation.
 
1. VISUAL APPEARANCE
 
Normal semen is viscous and opaque gray-white in appearance. After prolonged abstinence, it appears slightly yellow.
 
 
Immediately following ejaculation, normal semen is thick and viscous. It becomes liquefied within 30 minutes by the action of proteolytic enzymes secreted by prostate. If liquefaction does not occur within 60 minutes, it is abnormal. The viscosity of the sample is assessed by filling a pipette with semen and allowing it to flow back into the container. Normal semen will fall drop by drop. If droplets form ‘threads’ more than 2 cm long, then viscosity is increased. Increased semen viscosity affects sperm motility and leads to poor invasion of cervical mucus; it results from infection of seminal vesicles or prostate.
 
3. VOLUME
 
Volume of ejaculated semen sample should normally be > 2 ml. It is measured after the sample has liquefied. Volume < 2.0 ml is abnormal, and is associated with low sperm count.
 
4. pH
 
A drop of liquefied semen is spread on pH paper (of pH range 6.4-8.0) and pH is recorded after 30 seconds. Normal pH is 7.2 to 8.0 after 1 hour of ejaculation. The portion of semen contributed by seminal vesicles is basic, while portion from prostate is acidic. Low pH (< 7.0) with absence of sperms (azoospermia) suggests obstruction of ejaculatory ducts or absence of vas deferens. Low pH is usually associated with low semen volume (as most of the volume is supplied by seminal vesicles).
This includes examination of material obtained from vagina, stains from clothing, skin, hair, or other body parts for semen. This is carried out in cases of alleged rape or sexual assault.
 
Collection of Sample
 
  • Vagina: Direct aspiration or saline lavage
  • Clothing: When scanned with ultraviolet light, semen produces green white fluorescence. A small piece (1 m2) of clothing from stained portion is soaked in 1-2 ml of physiologic saline for 1 hour. A similar piece of clothing distant from the stain is also soaked in saline as a control.

LABORATORY PROCEDURES
 
1. MICROSCOPIC EXAMINATION FOR SPERMS
 
Presence of motile sperms in vaginal fluid indicates interval of < 8 hours. Smears prepared from collected samples are stained and examined for the presence of sperms.
 
2. ACID PHOSPHATASE
 
Acid phosphatase is determined on vaginal or clothing samples. Due to the high level of acid phosphatase in semen, its presence indicates recent sexual intercourse. Level of ≥50 U/sample is considered as positive evidence of semen.
 
3. DETERMINATION OF BLOOD GROUP SUBSTANCES
 
When semen is positively identified in vaginal fluid or other sample, test can be carried out for the presence of blood group substances in the same sample. The ‘secretor’ individuals (80% individuals are secretors) will secrete the blood group substances in body fluids, including semen.
 
4. FLORENCE TEST
 
This test detects the presence of choline found in high concentration in semen. To several drops of sample, add equal volume of reagent (iodine 2.54 g, potassium iodide 1.65 g, distilled water 30 ml); in positive test rhombic or needle-like crystals of periodide of choline form. False-positive tests can occur due to high choline content of some other body fluids.

TESTS FOR DETECTION OF BLOOD IN URINE

Written by Sunday, 13 August 2017 02:34
1. MICROSCOPIC EXAMINATION OF URINARY SEDIMENT
 
Definition of microscopic hematuria is presence of 3 or more number of red blood cells per high power field on microscopic examination of urinary sediment in two out of three properly collected samples. A small number of red blood cells in urine of low specific gravity may undergo lysis, and therefore hematuria may be missed if only microscopic examination is done. Therefore, microscopic examination of urine should be combined with a chemical test.
 
2. CHEMICAL TESTS
 
These detect both intracellular and extracellular hemoglobin (i.e. intact and lysed red cells) as well as myoglobin. Heme proteins in hemoglobin act as peroxidase, which reduces hydrogen peroxide to water. This process needs a hydrogen donor (benzidine, orthotoluidine, or guaiac). Oxidation of hydrogen donor leads to development of a color (Figure 828.1). Intensity of color produced is proportional to the amount of hemoglobin present.
 
Chemical tests are positive in hematuria, hemoglobinuria, and myoglobinuria.
 
Figure 828.1 Principle of chemical test for red cells
Figure 828.1 Principle of chemical test for red cells, hemoglobin, or myoglobin in urine
 
Benzidine Test
 
Make saturated solution of benzidine in glacial acetic acid. Mix 1 ml of this solution with 1 ml of hydrogen peroxide in a test tube. Add 2 ml of urine. If green or blue color develops within 5 minutes, the test is positive.
 
Orthotoluidine Test
 
In this test, instead of benzidine, orthotoluidine is used. It is more sensitive than benzidine test.
 
Reagent Strip Test
 
Various reagent strips are commercially available which use different chromogens (o-toluidine, tetramethylbenzidine).
 
Causes of false-positive tests:
 
  • Contamination of urine by menstrual blood in females
  • Contamination of urine by oxidizing agent (e.g. hypochlorite or bleach used to clean urine containers), or microbial peroxidase in urinary tract infection.
 
Causes of false-negative tests:
 
  • Presence of a reducing agent like ascorbic acid in high concentration: Microscopic examination for red cells is positive but chemical test is negative.
  • Use of formalin as a preservative for urine
 
Evaluation of positive chemical test for blood is shown in Figure 828.2.
 
Figure 828.2 Evaluation of positive chemical test for blood in urine
Figure 828.2 Evaluation of positive chemical test for blood in urine

Telomere Indicator of Physiological Age

Written by Saturday, 12 August 2017 13:06
Have you ever wondered, what is your physiological age? Is it more or less than your chronological age? Physiological age determines a person’s health condition. Are we able to determine physiological age? You would think the answer is NO. but it can be done by determining telomere’s length. “Telomere is a repetitive nucleotide sequence (having no meaningful information) at each end of chromosome to protect DNA from deterioration and or from fusion with other chromosomes.” This sequence is about 3000-15000 base pairs in length. In vertebrates this repeated sequence is TTAGGG.
 
Significance of Telomeres
 
Cells divide and increase their number, DNA duplication also occurs. Enzymes involved in this duplication process, can’t continue duplication all the way to the end so some part of DNA is lost and chromosome is shortened. This lost part is some base pairs of telomere. Somatic cells lose about 50-100 nucleotides on each cell division. In this way, telomeres, having no meaningful information, act as CAPS preventing the important information (DNA) from deterioration and preserve the critical information. Telomeres are never tied to each other which allows chromosomes to remain segregate. Without telomeres, chromosomes would fuse with each other. Telomere Shortening Telomeres shorten because of the two major factors:
 
  1. End replication problem in eukaryotes accounts for loss of 20 base pairs per cell division.
  2. Oxidative stress accounts for loss of 50-100 base pairs per cell division.
 
Figure 827.1
 
Oxidative stress in the body depends on lifestyle factors. Smoking, poor diet and stress can cause increase in oxidative stress. With each cell division telomeres shorten, so there are limited number of divisions that a cell can undergo, this limit is called Hayflick Limit. This is to prevent the loss of vital DNA information and to prevent production of abnormal cells. When a cell reaches this limit it undergoes apoptosis that is a programmed cell death. Telomere Lengthening to reverse telomere shortening, there is an enzyme named Telomerase that adds telomere sequence nucleotides and replenish the lost telomere nucleotides. Telomerase activity is not present in all cells. It is almost absent in somatic cells including; lung, liver, kidney cells, adult tissues, cardiac and skeletal muscles etc. In the presence of telomerase enzyme, a cell can divide to unlimited extent without ageing giving rise to tumors. That’s why it is found only in some cells in considerable concentration including germline cells and stem cells. These cells don’t show signs of ageing.
 
Figure 827.3
 
Relation between Telomere’s Shortening and Ageing
 
Figure 827.2It is still controversial that whether telomere shortening is a reason of ageing or is a sign of ageing just like grey hair. Whatever it is, the thing is, it determines your physiological age because ageing cells mean an ageing body. Telomere shortening is related with poor lifestyle. People who are active and have a healthy lifestyle have the same telomere length as someone 10 years younger than them has. Depression causes increase in oxidative stress in the body so the higher the stress, the shorter the telomere is Link between Telomeres and Cancer “Cancer in general is defined as an uncontrollable rapid growth of cells.”
 
What causes these cells to grow uncontrollably?
 
These cells have active telomerase enzyme, which doesn’t let the telomere to shorten, so no Hayflick limit reaches and cell continues to divide. This is the reason why telomerase is not used as an anti-ageing medicine because it has potential to turn normal body cells into cancerous cells. Without telomerase activity cancer cells activity would stop, which is an under research treatment for cancer. However, drugs inhibiting telomerase activity, can interfere with normal functioning of cells that require telomerase. In healthy female breast there is a portion of cells named, luminal progenitors, with critically short telomere length. In these cells telomerase becomes active causing these cells to turn into cancer cells on higher activity. To tackle breast cancer, use of telomerase inhibiting drugs should be practiced. Telomere biology is very important for understanding cancer biology and scientists are working hard on it.
 
 
Reviewed by Dr. Nida Hayat Khan
Editor @ BioScience.pk 
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