Infertility Tests Guide

1 Infertility Investigation Tests
Written by . DR. Abdulrazzaq Othman Alagbare
LECTURE NOTES
For Medical Laboratory Students
Infertility Investigation Tests
Dr.Abdulrazzaq Othman Alagbare
MD. Clinical pathologist
Yemeni – Jordanian University
Yemen- Sana'a
2016
Infertility Tests
1. Reproductive System Hormones
2. Gonadotropin- hormone
3. LH and FSH
4. Female Reproductive system
5. Function of female Reproductive system
6. Progesterone
7. Estrogen (General)
8. Estradiol (E2)
9. Estriol Total
10. testing
11. Estriol, Free (F-E3)
12. Prolactin
13. Beta Human Chronic Gonadotropin (Beta
hCG) Pregnancy Tests
14. Testosterone free
15. Testosterone Total
16. Semen analysis
17. Anti- Sperm Antibodies (ASA)
18. Seminal fluid Sperm Wash (Swim-Up Test)
19. Fructose (semen)
20. DHEA, Non-Sulfate (Dehydroepiandrosterone)
21. DHEA-S (Dehydroepiandrosterone sulfate)
22. Dihydrotestosterone (DHT)
23. Karyotyping (chromosome analysis) or genetic

Female Reproductive system
Female Reproductive system includes
a) the female gonads (ovaries),
b) the genital ducts (uterus, fallopian tubes, and vagina),
c) and the supporting structures (mammary glands, or breasts, and vulva or genitalia).
Ovary function:
a) It is site of ova production and maturation
b) Produce female sex hormones
Ovary hormones
· Produced from no pregnant women and pregnant women
· Called steroid hormones
They are
a) Esterogen
b) Progesterone
Function of female Reproductive system
a) The female tract manufactures the ova (egg),
b) accepts sperm,
c) fosters fertilization,
d) and allows development,
e) birth
f) and nourishment of the offspring.
Progesterone
What is Progesterone?
Progesterone is a female hormone produced by the ovaries during ovulation.
Main action
Progesterone helps prepare the lining of the uterus (endometrium) to receive the egg if it becomes
fertilized by a sperm. If the egg is not fertilized, progesterone levels drop and menstrual bleeding
Reproductive System Hormones
a) Normal reproductive function depends on complex hormonal communication between endocrine
and target organs.
b) Normal function is essential to sexual development at puberty and to the cyclic processes of
ovulation and menstruation

Gonadotropin- hormone
The hypothalamus secretes a small peptide, gonadotropin-releasing hormone (GnRH), also known as
luteinizing hormone-releasing hormone, which regulates TO release of the gonadotropins
a) luteinizing hormone (LH)
b) and follicle-stimulating hormone (FSH) from the anterior pituitary gland
LH and FSH promote maturation of ova and stimulate secretion of estrogen and progesterone from the
ovaries.
FSH and LH
 FSH and LH are necessary for the male and female reproductive system
 The FSH and LH present in the plasma of both male and female at all ages
FSH-LH plasma Level
FSH and LH have:
1. A Small increase at puberty
2. A great increase in in women after menopause
3. In the ovulating women rises sharply from basal level just prior to ovulation and then rapidly falls
Infertility Testing
Infertility is the inability of a couple to achieve pregnancy after a year of unprotected sex (without using
any form of birth control). Between 10% and 25% of couples have infertility problems sometime during
their lives. Infertility problems are more common in people over the age of 35.
Steps of investigation
1. full history
2. sexual practices (including birth control),
3. sexually transmitted diseases (STDs)
4. the use of caffeine, tobacco, alcohol, and recreational drugs (such as marijuana). Kat
5. The woman's menstrual history
Blood Tests
The levels of
a) luteinizing hormone (LH) AND FSH
b) progesterone
c) Prolactin hormone
d) Thyroid function tests
And some tests for a man such as
a) Seminal fluid
b) Testosterone etc

Infertility Tests
The Hormones LH and FSH
Follicle Stimulating Hormone (FSH)
FSH secreted by the anterior pituitary gland, under the
influence of gonadotropin realizing hormones
The FSH needs for study cases
such as:
a) Infertility.
b) Low sperm count.
c) Delayed puberty,
d) pituitary gland tumor.
e) Early puberty an irregulars (amenorrhea
FSH increases in:
a) at puberty,
b) after menopause.
c) Prior to ovulation and then rapidly falls.
Decrease in:
a) primary pituitary dysfunction
b) useful in investigating in Infertility,
c) amenorrhea,
d) And testicular dysfunction.
High FSH levels in a man may indicate
a) Klinefelter's syndrome,
b) testicles are absent,
c) are not Functioning properly, or have been
damaged.
d) In prepubescent children, high FSH levels may
indicate the impending start of puberty
Sample Serum
Unit mIU/ml
Normal Female
Follicular 4-13
Luteal 5-22
Post menopause 2-13
Male 1-8

Luteinizing Hormone (LH)
LH secreted by the anterior pituitary gland, under the
influence of gonadotropin realizing hormone with FSH
hormone
Is necessary for the male and female reproductive system
Along with other hormones (follicle-stimulating hormone
and estrogen),
LH helps to regulate, the menstrual cycle in women
And occur ovulation .
The FSH and LH present in the plasma of both male
(stimulate testosterone production) and female at all ages
LH increase in:
a) hypogonadism in male,
b) after menopause,
c) primary ovarian hypo function
d) polycystic ovarian disease,
Decrease in:
a) primary pituitary dysfunction useful in
investigating in infertility,
b) amenorrhea,
c) and testicular dysfunction.
Sample Serum
Unit mIU/ml
Normal Male 2-12
Female
Follicular 1.0-18
Luteal 0.4-20
Mid cycle 24-105
Post menopause 15-62
*The amounts of hormones measured in both
a man and a woman to determine why the
couple cannot achieve pregnancy (infertility).
*Infertility Tests: LH, FSH, Estrogen,
Progesterone, Testosterone

Progesterone (P)
Progesterone is a female hormone produced by the
ovaries during ovulation.
It is useful for, to study fertility problems (see below)
Increase of progesterone indicates:
a. Pregnancy.
b. A tumor of the ovaries or adrenal glands,
c. a molar pregnancy,
d. overproduction of hormones by the adrenal glands
Decrease of progesterone indicates:
a. Problems with ovulation.
b. In a pregnant woman, low progesterone levels may
indicate a threatened miscarriage
Sample Serum
Unit ng/ml nmolL
Normal
follicular 0.27-2.61 0.9-8.3
luteal 3.28-38.63 10.4-122.8
post -
menopause
< 0.2-0.82 < 0.7-2.7
Pregnant female
1st
Trim 12.26-81.8 40.9-272.7
2nd
Trim 11.11-81.4 37.0-271.3
3rd
Trim 39.3-387.8 131.0-1292.7
Male 0.2-3.37 0.7-11.2
Estrogen (General)
Originate in the ovarian follicles
And in the placenta (pregnant case) types of estrogens
are:
Estradiol.
Estriol.
Estrone in a blood sample.
Action: Participate in the menstrual cycle
Essential for development and maintenance
of the reproductive organs and secondary
sex characteristics

Estradiol (E2)
Is the most potent natural estrogen
derived almost exclusively from the
ovaries?
The test used for study:
A. Hypoestrogenism in cases
delayed puberty.
B. primary and secondary
amenorrhea, and menopause.
Increased level are found in:
a) Tumors producing estrogen.
Gyneomastia.
b) Post-menopause bleeding.
c) Hepatic cirrhosis and uses of oral
contraceptives.
Low in:
low level indicate pituitary or ovarian
malfunction and menopause
Sample Serum
Unit pg/ml Pmol/L
Normal
Follicular 39-189 144-696
Luteal 48-309 177-1137
Mid cycle 94-508 346-1869
Post -menopause <20-41 <74-151
Male <20-77 <74-283
State day of menstrual cycle, steroid therapy affect the
result
Estradiol is the most commonly measured type of
estrogen for
No pregnant women. Menstrual cycle. After
menopause, estradiol production drops to a very low
but constant level. The amount of estradiol in a
woman's blood varies widely
Estriol Total
In pregnancy produced in the feto-placental unit,
exist in blood as a mixture of the unconjugated
form and as a number of conjugates.
Low estriol level indicate:
a) Diabetic mother.
b) Post-date gestation.
c) Intrauterine growth retardation and
placental sulfatase deficiency.
Sample Serum
Unit ng/ml
Normal
28-31 wks 5.1-23.0
28-31 wks 6.1-26.2
28-31 wks 7.8-30.4
28-31 wks 9.1-33.5

Estriol, Free (F-E3)
Estriol is the main estrogen hormone in the blood during
pregnancy.
a) Synthesis by the fetus and placenta,
b) secreted into maternal circulation,
c) excreted in urine and
d) This test used as a sensitive index of:
e) Fetoplacental status, in combination with other tests
calls Triple screen Tests (AFP/hCG/uE3).Which
include also.Alpha Fetoprotein., Beta Human Chronic
Gonadotropin
Increased in:
Caused by a pregnancy with more than one fetus (such as
twins or triplets).
Decreased in:
a) Down's syndrome.
b) Single test for one time don’t give meaningful
information about the fetus condition.
Sample Serum
Unit ng/ml
Normal in gestation case
at 18 wks 2.5-7.0
at 22 wks 3.8-11.0
at 26 wks 4.6-15.0
at 30 wks 5.5-18.0
at 34 wks 7.8-25.0
State last menstrual period, During
pregnancy, levels of estriol that begin
to decrease or that do not rise may
indicate a problem with the fetus or
placenta.
Prolactin (PRL) Infertility Tests
Prolactin is a hormone produced by the pituitary gland;
Pregnant women have high levels of prolactin.
Action: Prepares their breasts to produce milk.
After the baby is born, maintains the mother's milk
production (lactation).
The pituitary glands of men and no pregnant women also
produce prolactin, but its function in these people is not
understood.
Increase during:
a) times of physical stress,
b) emotional stress,
c) during sleep,
d) pituitary tumors,
e) ectopic tumor,
f) hypothyroidism,
g) renal failure and pregnancy,
Sample Serum
Unit ng/ml mIU/L
Normal
male up to 19 up to 621
female up to 24 up to 529
patient must be fasting avoid taking sample
after, Emotional stress or strenuous exercise
or pelvic examination, nipples Stimulation
In male high level of prolactin may cause
infertility and impotence, very high level
may indicate presence of tumor

Pregnancy Tests
a) The test for pregnancy is done by measuring the amount of human chorionic gonadotropin (hCG)
in blood or urine.
b) HCG is a hormone produced by the placenta during pregnancy
Beta human chorionic gonadotropin
HCG is a glycoprotein similar in structure to follicle-stimulating hormone,
luteinizing hormone (LH),and thyroid-stimulating hormone (TSH).
Positive pregnancy test
a) Detected in maternal serum and urine is evident only after implantation and vascular
communication has been established
b) 8-10 days after conception.
Time of detection is related to the sensitivity of the assay being used. Most current pregnancy tests
c) have a sensitivity of 25 mIU/mL
Equally, finding serum Progesterone levels of less than 5 ng/mL (<15.9 nmol/L) can aid in the
diagnosis of a nonviable pregnancy with 100% sensitivity
HCG is detectable in the serum of PREGNANT WOMAN approximately
a) 5% of patients 8 days after conception
b) 98% of patients by day 11.
c) Levels peak at 10-12 weeks
and then begin to decline rapidly until another more gradual rise begins at 22 weeks, which continues until
term.
Abnormal hCG
a) Beta-hCG is produced by all patients with choriocarcinomas (TYPE OF CANCER)
and 40-60% of patients with embryonal carcinomas.
b) patients with pure seminomas, 5-10% have elevated beta-hCG.
c) In 60% of patients with testicular cancer
d) 50-70% have an elevated AFP

Beta Human Chronic Gonadotropin (Beta hCG) Pregnancy Tests
Beta hCG is a glycoprotein produced by trophoblastic
cells, resemble
pituitary LH, they have similar
hormonal action in stimulating the
corpus luteum to produce progesterone, progesterone
maintain the
pregnancy by preventing menstruation,
The presence of beta hCG in serum of males with a
testicular nodule serves as a tumor marker
The test is useful in the following cases:
a) detecting early pregnancy, (if the
serum concentration is over 30ng/ml).
b) Diagnosis of ectopic pregnancy or threatened abortion,
c) screening test for Down's syndrome (triple
Screen, Maternal Serum)
High Level in:
Hydatid form, mole and gonadal tumors (reach up to one
million ng/ml)
Sample Serum
Unit ng/ml
Normal
Non-pregnant individual < 5.0
Wks of Gestation
1-2 9-130
2-3 75-2.600
3-4 850-20.800
4-5 4000-100,000
5-10 11,500-298,000
10-14 18,300-137,000
14-27 1400-53,000
27-39 940-60,000
State LMP (last menstrual period).
*Avoid hemolytic sample, the test of
serum Beta hCG dose not react either
affect with the

Semen analysis
A semen analysis measures
1. the volume of the semen,
2. the number of sperm (sperm count),
3. the percentage of sperm that appear normal,
4. the percentage of sperm that can move forward normally,
5. and the number of white blood cells in the semen
Indications / Reasons for Testing
1. Infertility
2. Post-vasectomy
3. Identification of a fluid as semen (forensic medicine)
4. Artificial insemination programs – sperm donors

Semen Analysis
Semen fluid, is the fluid, which
produced from the male
reproductive system it is useful
to study of man sub fertility
and infertility
Sample Semen fluid
SP: 3-days or 4 days from sexual intercourse, no more and no lower
than that. Avoid long standing of the sample over 30 min -Urine test
before the semen analysis is important -in cases with azospermia,
urine analysis must be post ejaculation
Semen Color (for more details see Table: 6)
Yellow (infection, high blood bilirubin, drugs)
Abnormal: Bloody (bleeding, trauma)
Sample Semen fluid
Normal Gray-White
Seminal fluid Sperm Wash (Swim-Up Test)
sperm selection is performed to improve the quality of seminal fluid spacemen required for intra uterine
insemination (IUI), selection is based on either sperm motility (swim-up technique) or sperm density
Sample fresh seminal fluid
Sp :
collected in special container
3-days or 4 days from sexual
intercourse, no more and, no lower than that.
Avoid old sample than 1 hour proper patient required

Antibody tests (anti-sperm antibodies)
Antibodies are substances produced by the body's immune system to detect and destroy foreign substances
(such as bacteria and viruses). Sometimes the body makes antibodies
against normal cells, such as sperm cells (anti-sperm antibodies).
Antibody tests may be done to determine whether antibodies against the man's sperm are present in his
or the woman's reproductive tract.
Doctors disagree about the value of antibody tests for determining the cause of infertility.
Anti- Sperm Antibodies (ASA)
The test used in investigation of infertility,
antibodies may be detected in serum of
both sexes, seminal and cervical mucus.
Sample:
Serum and seminal plasma
Unit Titer
Normal Negative
Avoid hemolytic, lipemic and contaminated samples
Testosterone – Total
Testosterone is a male hormone (androgen), affects
sexual features and development, produced in large
amounts by the testicles.
Before puberty, the testosterone level in boys is low.
The level of testosterone continues to rise during
adulthood until it peaks around age 40, then it
gradually decreases.
Increase in:
a) a tumor in the testicles or adrenal glands.
b) In boys younger than age 10, indicate early
(precocious) puberty.
In women.
c) A high level of testosterone may indicate a tumor
of the ovaries.Adrenal glands or polycystic ovary
syndrome
Sample Serum
Unit ng/ml nmol/L
Normal
<1yr 0.12-0.21 0.42-0.72
1-6yrs 0.03-.032 0.10-1.12
7-12yrs 0.03-0.680 0.10-2.37
13-17yrs 28-11.1 0.98-38.5
>17yrs 2.8-8.0 9.9-27.8
Female 0.06-0.82 0.22-2.9
Low serum testosterone:
a) Certain inherited diseases (such as
Klinefelter's syndrome).
b) Liver disease (cirrhosis).
c) Treatment for cancer of the prostate gland

Testosterone free
It is useful investigation tests for patients with hirsutism,
loss of lipdo and male hypogonadism,
High level may indicate:
Adrenocortical tumor,
Ovarian tumor (secreting androgen), Pituitary tumor
Decreased in:
Hypopituitarism,
Estrogen therapy.
Kleinfelter`s syndrome.
Sample Serum
Unit Pg/ml Pmol/L
Normal Male:
20-39yrs 8.8-27.0 31-94
40-50yrs 7.2-23.0 25-80
60-80yrs 5.6- 19.0 19-66
Female:
20-39yrs 0.06-2.57 0.19-8.9
40-59yrs 0.04-2.03 0.14-7.0
60-80yrs 0.03-1.35 0.09-5.4
Others infertility tests
Fructose (semen)
Increased in
 fructosuria,
 hepatic failure and fructose intolerance.
Absent in
 congenital absence of vas deferens and seminal vesicles,
 or obstruction of ejaculatory ducts.
Sample: Fresh Semen
Normal: 120-500 mg/dl or 6.7-
27.8mmol/L
DHEA, Non-Sulfate (Dehydroepiandrosterone)
 Dehydroepiandrosterone (DHEA)
produced by both the adrenal cortex
and Testis.
 This test done for evaluates adrenal
cortex carcinomas, which secrete
large amount of DHEA,
 and estimation of delayed,
puberty.
Sample Serum or EDTA plasma
Unit nmol/L
Normal
Age/yrs Male Female
2-4 < 0.5 < 2.4
5-7 < 4.1 < 2.6
8-10 < 6.9 0.3-10.4
11-13 1.7-13.8 0.3-10.4
13-20 3.4-31.2 5.2-27.7
>20 5.0-31.0 4.1-28.0
Avoid hemolytic, lipemic and contaminated samples

DHEA-S (Dehydroepiandrosterone sulfate)
 DHEA sulfate is the principle adrenal androgen
and is secreted together with cortisol under the
control of ACTH and prolactin hormones,
This test used to evaluate and diagnosis cases such as:
 Heurism,
 amenorrhea,
 polycyctic ovarian syndrome
 and Cushing's syndrome.
Congenital adrenal hyperplasia due to the rare
deficiency of 3-beta-hydroxydehydrogenase causes
decrease secretion of androgens and estrogens,
but increase secretion of DHEA and DHEA-S,
reflected in increase plasma level and increase urinary
17-oxosteroids.
Sample Serum
Unit µg/dl
Normal
Age/yrs Female Male
10-14 54.4-255 49.5-245
15-19 66.6-306 133-553
20-24 158-412 210-496
25-34 98-315 163-425
35-44 60.8-338 110-424
45-54 35.4-256 44.3-321
55-64 18.9-205 51.7-305
65-74 9.4-246 33.6-140
Dihydrotestosterone (DHT)
DHT is responsible for development of the male
external genitalia and prostate, a deficiency of
enzyme 5a-reductase will lead to deficiency of
DHT and hypogonadism,
This test done for evaluate and diagnose
hypogonadism in male
(development of external genitalia
and prostate)
High level of DHT result in hirsutism
Sample Serum
Unit pg/ml
Normal
Age Ad. Male Ad. Female
20-49 yrs 155-5553 5.3-170
>50yrs 36-573 4.0-71
Avoid hemolytic, lipemic and contaminated
samples

Karyotype
In order for chromosomes to be seen with a microscope, they need to be stained. Once stained, the chromosomes look like strings with
light and dark "bands" and their picture can be taken. A picture, or chromosome map, of all 46 chromosomes is called a karyotype.
Karyotyping (chromosome analysis) or genetic testing
Karyotyping is a test that examines the genetic material (chromosomes) in a person's cells for abnormalities.
Some genetic abnormalities interfere with the processes necessary to achieve pregnancy. Genetic testing may be done to help
determine the cause of infertility.
Karyotyping refers to use of a microscope to examine
a) the size,
b) shape,
c) and number of chromosomes in a sample of body cells.
d) Extra, missing,or abnormal positions of chromosome pieces can cause problems with a person's growth, development, and
body functions.
Why Karyotyping Done
a) Determine whether the chromosomes of an adult have an abnormality that can be passed on to a child.
b) Determine whether a chromosome defect is preventing a woman from becoming pregnant or causing a pregnant woman to
miscarry.
c) Determine whether a chromosome defect is present in a fetus or infant. Karyotyping also may be done to determine whether
chromosomal problems may have caused a fetus to be stillborn.
d) Determine the cause of a child's birth defects or disability.
e) Help determine the appropriate treatment for some types of cancer.
f) Identify the sex of a person by determining the presence of the Y chromosome. This may be done when a newborn's sex is not
clear.
Samples for Karyotyping test
a) a sample of bone marrow
b) amniocentesis or chorionic villus sampling (CVS)
c) a blood sample taken from a vein.
Karyotyping Test Results
Results of karyotyping are usually available within 1 to 2 weeks.
Normal Result
a) There are 46 chromosomes that can be grouped as 22 matching pairs and 1 pair of sex chromosomes (XX for a female and XY
for a male).
b) The size, shape, and structure are normal for each chromosome.
Abnormal:
1. There are more than or fewer than 46 chromosomes.
2. The shape or size of one or more chromosomes is abnormal.
3. A chromosome pair may be broken or incorrectly separated.
4. Down syndrome results from having an extra (number 21) chromosome.
5. In Turner syndrome, a defect that results in abnormal female sexual development, only one X chromosome is present.
6. In Klinefelter's syndrome, a disorder that affects males, at least one extra X chromosome is present.

Chromosomal Karyotyping, Blood
Chromosomal karyotyping is recommended for the
detection and evaluation of possible
 congenital malformations,
 mental retardation,
 growth retardation,
 infertility,
 cryptorchidism,
 hypogonadism,
 primary amenorrhea,
 abnormal/ambiguous genitalia,
 recurrent miscarriage,
 Turner syndrome,
 Klinefelter syndrome,
 Down syndrome, or other suspected chromosomal
disorders.
Fluorescence in situ hybridization (FISH) ordered as
needed
Sample Heparinized Blood
Unit
Normal Negative
Must be sterile, do not refrigerate or
freeze.
Maintain sample at room temperature.
Clinical details required.
Avoid hemolyzed and clotted specimens
Normal karyotyping

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