Presented

by: Yasmine Amr Assistant lecturer in Medical Biochemistry department

Female sex hormones

Progesterone

(21 C) It is secreted from:

Corpus luteum. Placenta (after 10 weeks of pregnancy).

It

is also formed in the adrenal cortex as a precursor of C19 and C21 corticosteroids hormones .

Estrogens (18 C):

They are secreted from mature graffian follicle in ovaries. They contain 18C, the first ring is completely unsaturated (aromatic) with no CH3 group at C10. There are 3 types: Estrone (E1), Estradiol (E2) and Estriol (E3) The most circulating one is E1 while the most active one is E 2

Male sex hormones (Androgens) 19C

Testosterone

is the most potent one. It is synthesized in the testis.

Diagnosis of pregnancy

Pregnancy can be diagnosed by measuring Human chorionic gonadotropin(HCG) in blood or urine

Detectable amounts ~ 5 IU/L Appears 8-11 days after conception and reach the peak(~ 100,000 IU/L) at 8-10 weeks pregnancy. In case of twin pregnancy, the amount of HCG is doubled.

Serial HCG can be used to determine abortion and ectopic pregnancy:

In normal pregnancy, HCG doubles in 1.5 days in the first 5 weeks then every 2-3 days after 5 months In ectopic pregnancy or abortion HCG rises more slowly or even decreases

Diagnosis of fetal anomalies

Down

syndrome: is usually due to an extra copy of chromosome 21. It is commonly associated with increased maternal age. Neural tube defects: e.g. anencephaly, meningomyocele,encephalocele. Usually associated with folic acid deficiency during pregnancy

 These

two serious anomalies can be diagnosed in second trimester (between 16-18 weeks) using triplet test, Measuring:

-fetoprotein (FP), unconjugated estriol 3, and HCG.

Down

syndrome: FP, 3, HCG Neural tube defects: FP, 3, HCG

Infertility
Infertility

is defined as inability of a couple to conceive after at least 1 year of unprotected, well timed intercourse. This may be due to male, female or a combination of both causes.

Laboratory tests to determine female infertility:

Blood tests that measure the levels of various hormones aid greatly in determining the cause of infertility. Some examples include: Luteinizing hormone (LH) Follicle-stimulating hormone (FSH) Prolactin (PRL) Estradiol Progesterone

 Because

changes in pituitary or thyroid function can also affect the menstrual cycle and ovulation, blood tests that measure thyroid function (TSH and/or T4) and steroids, such as testosterone and DHEA-S ( dehydroepiandrosterone sulfate is used in producing androgens and estrogens), are also informative.

Laboratory tests to determine male infertility:

Those

include blood tests and more important semen analysis Blood samples can be used to measure:

Free and total testosterone Luteinizing hormone (LH) Follicle-stimulating hormone (FSH) Prolactin (PRL)

Semen analysis

Semen is made up of the secretion of all the accessory glands of the male genital tract:

Testes 5% Seminal vesicle 46-48% Prostate 13-33% Bulbourethral gland 2-5%.

Semen is a grey opalescent fluid which is formed at ejaculation. It is composed of suspension of spermatozoa in seminal plasma.

Semen analysis

Physical properties Volume Colour pH Vicosity Specific gravity

Microscopic examination count Morphology Motility Viability Non sperm cells

Biochemical tests Fructose Acid phosphatase ASA Acrosin Zinc L-carnitine Alpha glucosidase

PHYSICAL PROPERTIES OF SEMEN

Volume: Average volume is from 2-5 ml/ejaculation. Abnormalities: 1. Aspermia: Total absence of ejaculation (rare). 2. Hypospermia: the seminal fluid volume is less than 2 ml. 3. Hyperspermia: Increased volume of semen above 10 ml (rare).

Colour Greyish white. It is opalescent due to its high content of protein and the presence of more than 60 million sperms /ml. abnormalities: 1. Urine produces pale yellow discoloration easily detected by the consistency of the semen and the urineferous odor. 2. Jaundice: bilirubin will also cause coloration of semen in deep jaundice. The semen may be a very bright yellow. 3. Blood (haematospermia) traces of fresh blood will color semen pink, while large amounts of blood give bright red color.

pH:
Between

7.3-8.1 only recorded on fresh semen by using pH paper with a range of 7-9. Inflammatory conditions of the prostate or seminal vesicle may alter the pH of semen. Viscosity: Normal viscosity is that which allows semen to be poured drop by drop out of the container. It is measured the time taken by one drop to leave the standard pipette. Specific gravity: 1.028

MICROSCOPICAL EXAMINATION:
This includes: Sperm

count motility morphology

Non-sperm

cells

Sperm count
Total

sperm count is the number of sperms in an ejaculation. Normally, it is 20 million/ml, i.e. about 60 millions/ejaculation. It is obtained by multiplying the sperm concentration by the volume.

How to conduct a sperm count

Hemacytometers

were developed for counting blood cells, but can also be used to count spermatozoa. A hemacytometer has two chambers and each chamber has a microscopic grid etched on the glass surface. The chambers are overlaid with a glass coverslip that rests on pillars exactly 0.1 mm above the chamber floor. Thus, the volume of fluid above each square of the grid is known with precision.

Procedure The semen must be killed to prevent movement and diluted before loading into the hemacytometer. This can be done by diluting the semen into a buffer containing a small quantity of formaldehyde. The dilution factor must be recorded to allow calculating the concentration. When there are 20-25 cells per large square, the sample is at the proper dilution.

The example at right shows red lines where cells on the line would be counted. If red dots represent cells, one would count 3 cells in the top middle large square.

At least two chambers should be counted, including at least 100 cells within each central counting area of each chamber.

 count/ml

= (Dilution Factor)(Count in 5 squares)(0.05 X 106) convention, sperm concentration is usually expressed in terms of sperm X 106/ml.

By

 Abnormalities: 1. 2. 3.

Azoospermia means no spermatocytes (male sterility). Oligozoospermia mean less than 20 million/ml less than 50 millions/ejaculation Polyzoospermia may reach 350 millions/ejaculation

Motility: Percentage motility (the percentage of sperms in the seminal fluid which are highly active) is performed soon after the production of the sample and is repeated after 1,2,3 and 6 hours after semen production. Normally, after one hour there must be over 80% active sperms.

W.H.O divided grades of motility into: ARapid forward progress motility BSlow or sluggish progressive motility CNon progressive motility DImmotility. The cutoff value for normal is 50% grade A+B or 25% grade A motility. Asthenospermia: sperm motility less than the WHO cutoff levels

Morphology Normally, the sperm count contains fewer than 20% abnormal forms e.g. bitailed, short tailed, 2 heads....etc.

Examples of abnormal sperm morphology

Non-sperm cells

RBCs: Normally, there are no RBCs. If present, this indicates haematospermia. WBCs: Normally there are very little number of WBCs which increase in cases of inflammation. Epithelial cells: always present in semen. Pus cells: 0- few number. Presence of large numbers of these cells indicates inflammation. Spermatocytes (germinal cells) : usually present in normal semen, but few in number.

Other tests in semen analysis

Viability When the motility is reported as less than 5% to 10%, viability testing is recommended because profoundly low motility may indicate dead sperm (necrospermia) . The most common viability assessment involves staining with Eosin Y followed by counter staining with Nigrosin. The viable sperm with its intact cell membrane will not take up the dye and will remain unstained.

Hypo-osmotic swelling test (HOST) an alternative method to assess sperm viability. It is based on the principle that viable sperm have intact cell membranes. Exposure of the sperm to hypo-osmotic fluid will cause water to flow into the viable cells seen as swelling of the cytoplasmic space and curling of the sperm tail. Nonviable sperm with nonfunctional cell membranes will not exhibit this effect because they cannot maintain an osmotic gradient

BIOCHEMICAL TESTS
Fructose in semen: Secreted from the seminal vesicle (150-650 mg%). It is secreted for nutrition of sperm cells. It disappears in cases of:
1. 2. 3.

absence of seminal vesicle; obstruction of ejaculatory duct; inflammation of seminal vesicle.

It is decreased in case of testosterone deficiency. fructose is used as fertility test. The used test is Seliwanoffs.

Acid phosphatase: Secreted from the prostate. The test is used as: 1. A marker of prostatic functions 2. In forensic laboratories as a test for the presence of semen.

Antisperm Antibody Testing: Approximately 10% of infertile men will present with antisperm antibodies (ASA). Hence it has been suggested to be tested routinely in all men undergoing infertility work-ups. Acrosin: Low acrosin activity has been associated with low sperm density, motility, and poor normal morphology.

Zinc: It is necessary for chromatin stability and decondensation, as well as for headtail detachment during fertilization. L- carnitine: It is secreted by the epididymis and is concentrated in the seminal plasma at up to 10 times the serum levels. It has a role in sperm maturation. Low L- carnitine levels are found in oligoasthenozoospermic men.

Alpha glucosidase: This has been used to distinguish non obstructive from obstructive azoospermia. It is used as a specific marker for epididymal function and is believed to play a role in sperm maturation in the epididymis. A cutoff value of 12 mIU/mL distinguishes ductal obstruction from primary testicular failure.

Normal semen parameters

Test
Liquefaction Morphology Motility pH Sperm count Volume White blood cell

Normal values
Within 20 minutes >70%normal,mature spermatozoa >60% >7.0 (average 7.7) >20 million sperm/ml 1.5-5.0 ml < 1 million cell/ ml

Precautions and steps of semen sample collection

There

should be 2 to 7 days of sexual abstinence before collection. The duration of abstinence should be constant, if possible.. Two separate samples at least 7 days apart should be analyzed. It is best to collect the specimen in a clean (not necessarily sterile), wide-mouthed jar.

 It

is important that the entire specimen be collected, because the initial fraction contains the greatest density of sperm. Ideally, collection should take place in the location where the analysis will be performed.

 The

degree of sperm motility should be determined as soon as possible after liquefaction, which usually occurs 15 to 20 minutes after ejaculation Semen should not be exposed to marked changes in temperature, and if collected at home during cold weather, the specimen should be kept warm during transport to the laboratory.

 In

order to allow liquefaction and mixing, semen is placed in a 370 C gently shaking incubator for 30 minutes. The semen sample should be examined within 1 hour of production and receipt in the laboratory.

Clinical cases

Case 1 : A 51 year old male with a history of 3 children in a prior marriage, an unremarkable medical history, and several (four) semen analyses that have revealed considerable variability in terms of sperm concentration (12 million per mL, 26 million per mL, 31 million per mL, and 94 million per mL). The semen collections were all thought to be complete and the other variables assessed in the semen analysis (including motility and morphology) were entirely normal. Question: What should be considered given this information?

Answer: There is a normal variability in sperm concentration for a normal fertile man. The sperm concentration occasionally is decreased even in the normal fertile male population. Therefore, the fact that most of the semen analyses report a normal concentration is encouraging. Also, the fact that this gentleman has proven fertility in the past is encouraging.

 Case

2: A 38 year old male with a history of 2 children, an unremarkable medical history, and a semen analysis that has revealed persistent pyospermia (an excess number of WBCs in the semen) Question: What should be considered given this information?

 Answer

Course

of (broad spectrum) antibiotic treatment should be given.