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Transcript
The National Ribat University
Faculty of Graduate Studies and Scientific Research
Hormonal Profile among Amenorrheic Sudanese
Women under 04 years
A thesis submitted for M.Sc. in medical laboratory science
(Chemical pathology)
By:
Rasha Moh amed Musa
B.Sc. medical laboratory Sciences
Omdurman Ahlia University (2001)
Supervisor:
Dr. Eltayeb Mohamed Ahmed Tayrab
Assistant professor of chemical pathology
June 2014
Dedication
To my …..
……. Husband,
……. Parents,
……. Friends &
……. Teachers;
I dedicate this work.
I
Acknowledgements
I would like to express my profound thanks to my supervisor,
Dr. Eltayeb Mohamed Ahmed Tayrabfor his fruitful guidance, unlimited
assistance, encouragement and sustained interest throughout the course of
this work. I wish to extend my warmest thanks to the staff of the clinical
chemistry department especially Gaafar Mahmoud Gaafar, Ribat
University Hospital, Maternity Hospital, Reproductive Health Care center
and Soba Teaching Hospital, for their support. I’m also grateful to all people
from whom samples were taken.
II
Abstract
This cross- sectional study was conducted in National Ribat University
Hospital, Omdurman Maternity Hospital, Reproductive Health Care Center
and Soba University Hospital. This study including 200 amenorrheic
Sudanese women and 106 ages matched apparently healthy volunteer
women with proven fertility as control. The study was done from May 2012
to June 2014 to assess the role of endocrine hormones including luteinizing
hormone (LH), follicle stimulating hormone (FSH), thyroid stimulating
hormone(TSH) and prolactin in infertile amenorrheic women under 40
years, attending infertility centers.
Materials and methods: ELISA method was used to measure serum levels
of FSH, LH, TSH and prolactin. Data were analyzed by Statistical Package
for Social Studies (IBM, SPSS) version 20.
Result: The mean age of the amenorrheic women was (33.5±6.0 years),
while in the control group was (29.2±5.9 years). The prevalence of primary
amenorrhea among the study group was (4%), while in the secondary
amenorrhea was (96%). The mean age at menarche in the secondary
amenorrheic women was (13.8±3.2 years), while in the control group was
(14.2± 1year). There was significant decrease in mean age at menarche in
the amenorrheic women compared to the normal controls.
Among the amenorrheic women 126 (63%) were vaccinated against
meningitis 80 (40%), tetanus 39 (19.5%), hepatitis 2 (1%), tetanus &
meningitis 3 (1.5%) tetanus & hepatitis 1 (0.5%) and meningitis & hepatitis
1 (0.5%). The study also showed that mean of the prolactin in the
amenorrheic women was (121.5 ± 265 ng/ml) versus (15.0±6.0 ng/ml) in the
III
control group; with P. value (0.000). The mean of the FSH in the
amenorrheic women was (50.3 ±34.0 IU/ml), versus (8.7 ± 31.3 IU/ml) in
the control group; with P.value (0.000). The mean of LH in the amenorrheic
women was (34.89±22.7 IU/ml), versus (6.27± 2.55 IU/ml) in the control
women with P.value (0.000). The mean of TSH in the amenorrheic women
was (1.7±2.0 IU/ml) versus (1.2±0.7 IU/ml) in the control group with
P.value (0.004)
Conclusion: The age at menarche in secondary amenorrheic Sudanese
women significantly decreases, 40% of vaccinated amenorrheic women
immunize against meningitis. Serum FSH, LH, prolactin and TSH
significantly increase in the amenorrheic Sudanese women.
IV
‫مستخلص الدراسة‬
‫أجشٗج ُزٍ الذساست الومطؼَ٘ بوسخشفٔ الشببؽ الجبهؼٖ‪ ٬‬هسخشفٔ الْالدٍ ام دسهبى‪ ٬‬الوشكض‬
‫االسخشبسٓ الهشاع الٌسبء ّالخْل٘ذ ّالؼمن ببلخشؽْم ّهسخشفٔ سْبب الجبهؼٔ‪ ٬‬شولج ُزٍ الذساست‬
‫‪ 200‬اهشأة سْداًَ٘ هٌمطغ هٌِي دم الح٘غ فٔ ػوش الل هي ‪ 40‬سٌت‪ ٬‬بجبًب ‪ 106‬اهشأة هي‬
‫االطحبء ّهوبثالث لِي فٔ الؼوش كؼٌ٘بث ػببطت‪ُ .‬زٍ الذساست اجشٗج فٔ الفخشٍ هب ب٘ي هبْٗ‬
‫‪ 2012‬الٔ ًْْٗ٘ ‪ّ 2014‬رلك لخم٘ن دّس ُشهًْبث الغذد الظوبء فٔ ُزٍ الظبُشٍ ّلذ شولج‬
‫الِشهْى الوٌبَ للجشٗب‪ُ ٬‬شهْى اللْ٘حٌبصٗي‪ُ ٬‬شهْى اللبي ّالِشهْى الوحفض للغذٍ الذسلَ٘‪.‬‬
‫الطرق والقياس‪ :‬اسخخذم جِبص االل٘ضا لم٘بط ُزٍ الِشهًْبث كوب اسخخذم البشًبهج االحظبئٔ‬
‫الخبص ببلذساسبث االجخوبػَ٘ للخحل٘ل االحظبئٔ‪.‬‬
‫النتائج ‪ :‬اظِشث الذساست اى هخْسؾ أػوبس الٌسبء الوظبببث ببًمطبع دم الح٘غ لبل اّاًَ ُْ‬
‫(‪ )6.0±33.5‬سٌت‪ّ ٬‬كبى رلك الوخْسؾ ػٌذ الؼٌ٘بث الؼببطَ ُْ (‪ )5.9±29.2‬سٌت‪ً .‬سبت الٌسبء‬
‫الوظبببث بؼذم الح٘غ االّلٔ آ لن ٗظِش دم الح٘غ ػٌذُي هطلمب (‪ًّ ٬)4%‬سبت الٌسبء‬
‫الوظبببث بؼذم الح٘غ الثبًْٓ (‪ ٬)96%‬كوب كبى هخْسؾ ػوش البلْؽ ػٌذُي (‪ )3.2±13.8‬سٌت‬
‫ّػٌذ الٌسبء الؼببطبث (‪ )1.0±14.2‬سٌت‪ .‬ػذد الٌسبء الوطؼوبث فٔ هجوْػَ البحث ‪)63%(126‬‬
‫‪ )40%( 80‬اهشأة بوظل السحبئٔ‪39 ٬‬‬
‫اهشأة‪ّ ٬‬كبى رلك الخطؼ٘ن ػلٔ الٌحْ الخبلٔ‪:‬‬
‫(‪ )19.5%‬بوظل الخ٘خبًْط‪ ٬‬اهشاح٘ي (‪ )1%‬بوظل الكبذ الْببئٔ‪ ٬‬ثالثَ ًسبء (‪ )1.5%‬بوظل‬
‫الخ٘خبًْط ّالسحبئٔ‪ ٬‬اهشأة ّاحذٍ (‪ )0.5%‬بوظل الخ٘خبًْط ّ الكبذ الْببئٔ‪ّ ٬‬اهشأة ّاحذٍ اخشٓ‬
‫(‪ )0.5%‬بوظل السحبئٔ ّالكبذ الْببئٔ هؼب‪ .‬هخْسؾ حشاك٘ض الِشهًْبث ّسؾ الٌسبء الوظبببث‬
‫ببًمطبع الح٘غ لبل االسبؼ٘ي كبى ػلٔ الٌحْ الخبلٔ‪ :‬الِشهْى الوٌبَ للجشٗب‪ُ ٬‬شهْى اللْ٘حٌبصٗي‪٬‬‬
‫ُشهْى اللبي ّالِشهْى الوحفض للغذٍ الذسلَ٘ (‪121.5( ٬)22.7±34.8( ٬)34.0 ±50.3‬‬
‫‪ ٬)2.0± 1.7( ٬)265.1±‬ػلٔ الخْالٔ‪ ٬‬اهب هخْسطِزٍ الِشهًْبث ػٌذ الؼٌ٘بث الؼببطَ فكبى ػلٔ‬
‫الٌحْ الخبلٖ (‪ )0.7± 1.2( ٬)2.5± 6.2( ٬)31.2± 8.7‬ػلٔ الخْالٔ اٗؼب‪ّ ٬‬رلك ببسحفبع رّ داللت‬
‫هؼٌْٗت ػلٔ الٌحْ الخبلٖ‪.)0.004 ّ 0.000 ٬0.0000 ٬0.000( :‬‬
‫الخالصة‪:‬الٌسبء السْداً٘بث الوظبببث ببًمطبع دم الح٘غ الثبًْٕ ٗبلغي ببكشا‪.‬‬
‫‪ %40‬هي الٌسبء ّالوظبببث ببًمطبع دم الح٘غ الوبكش ٗخن حطؼ٘وِي بلمبح السحبئٖ‪ ٬‬كوب‬
‫ٗظِشػٌذُي اسحفبع رّ داللت هؼٌْٗت فٔ هخْسؾ هسخْٓ كل هي الِشهْى الوٌبَ للجشٗب‪ُ ٬‬شهْى‬
‫اللْ٘حٌبصٗي‪ُ ٬‬شهْى اللبي ّالِشهْى الوحفض للغذٍ الذسلَ٘‪.‬‬
‫‪V‬‬
Contents
Pages
Subject
‫االٗت‬
Dedication
I
Acknowledgments
II
Abstract
III
‫هسخخلض البحث‬
V
List of contents
VII
List of tables
XII
Abbreviations
XIII
Chapter one
NO
1.
1.1
1.1.2
1.1.2.1
1.1.2.2
1.1.3
1.1.4
1.1.5
Subject
Introduction and Literature review
Infertility
Gonadotropins
Luteinizing hormone
Follicle-stimulation hormone
Gonadotropin-releasinghormone:
Control of FSH and LH
Activity of gonadotropin-releasing hormone
1
1
2
3
5
7
7
8
1.2
1.3
1.3.1
1.3.2
1.3.2.1
1.3.2.2
1.3.2.3
1.3.2.4
1.3.2.5
Human prolactin
Causes of female infertility
Hormonal disorder
Hormonal imbalance
Glandular problems
Ovulatory disorders
Polycystic ovarian Syndrome
Premature menopause
Abnormal cervical Mucous
8
11
11
11
11
12
12
12
13
VI
pages
1.4
1.4.1
1.4.1.1
1.4.1.2
1.4.1.3
1.4.1.4
1.4.2
1.4.3
1.4.4
1.4.5
Menstrual cycle
Menstrual cycle Phases
Menstruation
Follicular phase
Ovulation
Luteal phase
Pathophysiology of menstrual cycle
Behavioral change
Length of menstrual cycle
Effect on other systems
14
16
18
19
20
22
24
24
25
25
1.4.6
1.5
Cycle abnormalities and disorders
Amenorrhea
26
27
1.5.1
28
1.5.1.3
1.5.1.4
1.6.
1.7
1.8
1.8.1
1.8.2
1.9
1.10
1.11
1.12.
1.12.1
1.12.2
1.12.3
1.12.4
1.12.5
Types of amenorrhea based on
hypothalamic (HPO) axis etiology
Hypothalamic amenorrhea
Gonadotropin-releasing hormone deficiency
in adults
Pituitary amenorrhea
Ovarian causes of primary amenorrhea
Congenital and anatomical abnormalities
Receptor and enzyme defects
Etiology of amenorrhea
Causes of primary amenorrhea
Causes of secondary amenorrhea
Epidemiology of amenorrhea
Prognosis of amenorrhea
Patient education
Amenorrhea clinical presentation
Primary amenorrhea
Secondary amenorrhea
Disorders of the outflow tract
Ovarian disorders
Hypothalamic/pituitary disorders
1.12.6
Functional hypothalamic impairment
42
1.12.7
1.13
Chronic diseases
Hormonal studies
44
45
1.5.1.1
1.5.1.2
VII
28
29
29
30
32
32
33
34
35
36
37
38
38
39
39
40
41
41
1.13.1
1.13.2
1.14
1.13.3
1.13.4
1.14.5
1.15
1.15.1
1.15.2
Prolactin
FSH, LH, and estradiol
Thyroid hormones
Thyroid gland
Thyroid physiology
Type of thyroid hormones
Rationale and objectives
Rationale
Objectives
45
46
47
47
48
48
51
51
52
Chapter two
Materials and methods
2.1
2.2
2.3
2.4
2.5
2.6
2.7
Study duration:
Study design and sample size
Study setting
Study population
Inclusion criteria
Exclusion criteria
Sampling technique
53
53
53
53
53
53
54
2.8
2.9
2.10
2.11
2.12
2.13
2.14
2.15
2.15.1
2.15.2
2.15.3
2.15.4
2.15.5
Tools of data collection
Ethical consideration
Organization
Excepted out comes
Specimen collection and preparation
Statistical analysis
Reagents
Assay procedure
General remarks
Test procedure
Calculations
Quality control
Data analysis
54
54
54
54
54
55
55
56
56
56
57
58
58
Chapter three
Results
1.
Results
59
VIII
Chapter four
Discussion, conclusion and recommendations
4.1.
4.2.
4.3.
1.
Discussion
Conclusion
Recommendations
References
70
72
73
74
1.
2.
Appendixs
Questionnaire
Agreement consent
90
91
IX
List of Tables
NO
Subject
Pages
1.1
17
Menstrual cycle phases
3.1
Descriptive study of mean age of amenorrheic Sudanese
61
women and their control.
3.2
62
Descriptive study of type of amenorrhea among
amenorrheic Sudanese women.
3.3
Descriptive study of experience of cycle (menarche)
63
among amenorrheic Sudanese women under study.
3.4
Descriptive study of experience of cycle (menarche)
64
among control group.
3.5
Comparison of mean of menarche in the test group and
65
their control.
3.6
Descriptive study of vaccination among amenorrheic
66
Sudanese women.
3.7
Descriptive study of the type of vaccination among
67
amenorrheic Sudanese women.
3.8
Descriptive study of the type of vaccination among
68
control group.
3.9
69
Comparison
study
of
hormonal
amenorrheic women and their controls.
X
profile
among
ABBREVIATIONS
AIDS
Acquired immune deficiency syndrome
AMP
Adenosine monophosphate
CAUV
Congenital absence of the uterus and vagina
CG
Chorionic gonadotropin
CNS
Central nervous system
CYP17
17 Alpha-hydroxylase deficiency
DIT
Diiodotyrosine
DNA
Dioxynecluic acid
E2
Estradiol
F T4
Free thyroxin
FSH
Follicle stimulating hormone
GnRF
Gonadotropin-releasing factor
GnRH
Gonadotropin-releasing hormone
HIV
Human immuno-deficiency Virus
HPO
Hypothalamus, pituitary and ovary
ICSH
Interstitial cell-stimulating hormone
LH
Luteinizing hormone
MIT
Monoiodotyrosine
XI
MRKH
Mayer-Rokitansky-Kuster-Hauster
NWHIC
National Women's Health Information Center
PCOS
Polycystic ovarian syndrome
PMS
Premenstrual syndrome
POD
Polycystic ovarian disease
POF
Premature ovarian failure
POI
Primary ovarian insufficiency
SRV
Sex-determining region of y chromosome
T3
Triiodothyronin
T4
Thyroxin
TRH
Thyrotropin – releasing hormone
TSH
UK
Thyroid stimulating hormone
United King
XII
CHAPTER ONE
Introduction & Literature review
1.Introduction and literature review
1.1. Infertility
Fertility is important to all societies. The inability to have children has
traditionally been a source of pain, anxiety and shame, flagging the worse
consequences to couples that are infertile. In Africa,couples who are unable
to rear as many children as they wish may feel anguish or emotional panic.
Hence several reports have focused on the causes, prevention and treatment
of infertility in the continent (Brunham, et al., 1992). Primary infertility is
the infertility affecting individuals who have had no previous successful
pregnancies while secondary infertility is infertility affecting individuals
who have previously had a successful pregnancy, but are currently unable to
conceive. Technically; secondary infertility is not present if there has been a
change of partners (Bamigbowu and Adegoke, 2005). Many causes of
infertility have been established. Endocrinology studies, which have
revealed so much about female infertility, brought to limelight problems of
anovulatory cycle and hyper prolactinemia. In the female, ovulation
depends on a number of factors, including normal egg development during
fetal life and complex interactions among hormones secreted from the brain,
the pituitary gland, and the ovary after reproductive maturity. During the
menstrual cycle, the concentrations of hormones, including estrogen and
progesterone change dramatically, resulting in ovulation and preparation of
the uterus for implantation of the fertilized egg. If this highly orchestrated
and tightly controlled sequence of events is interrupted, infertility or reduced
fertility may result (Speroff, et al., 1994).
1
In women, failure to ovulate normally can be caused by genetic or
environmental factors, including toxic exposures. Endocrine problems such
as thyroid disease can also interfere with normal ovulation (Speroff, et al.,
1994). Ovulation disorders generally arise from disruption of the
hypothalamic-pituitary ovarian axis. Causes of ovulation disorders include
diminished ovarian reserve, gonadal dysgenesis (Turner syndrome), ovarian
tumor, premature ovarian failure (POF), hypothalamic dysfunction due to
environmental, physical stress, emotional stress, pituitary adenoma with or
without hyperprolactinemia, Sheehan syndrome or pituitary hypofunction,
hypogonadism and or corpus luteum insufficiency, premature menopause,
oral or injectable contraceptives and polycystic ovarian disease (POD) –
)leventhal syndrome( characterized by enlarged ovaries with follicular cysts,
amenorrhea, abnormal hair growth and obesity(Ladipo, 1980).
1.1.2. Gonadotropins
Gonadotropins are glycoprotein hormones secreted by gonadotrope cells of
the pituitary of vertebrates(Parhar,et al.,2002(.This is a family of proteins,
which include the mammalian hormones follicle-stimulating hormone
(FSH), luteinizing hormone (LH) and placental chorionic gonadotropin
(hCG) (Goodwin ,et al.,1983). These hormones are central to the complex
endocrine system that regulates normal growth, sexual development, and
reproductive function .The hormones LH and FSH are secreted by the
anterior pituitary gland, while placental chorionic gonadotropin (hCG) is
secreted by the placenta )Golos,et al.,1991).
The two principal gonadotropins in vertebrates pituitary are luteinizing
hormone (LH) and follicle-stimulating hormone (FSH), although primates
2
produce a third gonadotropin called chorionic gonadotropin (CG). LH and
FSH are heterodimers consisting of two peptide chains, an alpha chain and a
beta chain. LH and FSH share nearly identical alpha chains (about 100
amino acids long), whereas the beta chain provides specificity for receptor
interactions. These subunits are heavily modified by glycosylation
(Goodwin, et al., 1983).
The alpha subunit is common to each protein dimer (well conserved within
species, but differing between them, and a unique beta subunit, which
confers biological specificity. The alpha chains are highly conserved
proteins of about 100 amino acid residues which contain ten conserved
cysteines all involved in disulfide bonds (Isaacs, et al., 1994). Intracellular
levels of free alpha subunits are greater than those of the mature
glycoprotein, implying that hormone assembly is limited by the appearance
of the specific beta subunits and hence that synthesis of alpha and beta is
independently regulated. Another human gonadotropin is human chorionic
gonadotropin (hCG), produced by the placenta during pregnancy (Goodwin,
et al., 1983).
1.1.2.1. Luteinizing hormone (LH)
Luteinizing hormone is produced in both men and women from the anterior
pituitary gland in response to luteinizing hormone-releasing hormone (LHRH or Gn-RH), which is released by the hypothalamus (Harris, et al., 1970).
Luteinizing hormone also called interstitial cell-stimulating hormone (ICSH)
in men; is glycoprotein with a molecular weight of approximately 30.000
daltons (Whitely, et al.,1978).
It is composed of two non- covalently
associated dissimilar amino acid chains, alpha and beta (Pierce and Parsons,
3
1981). The alpha chain is similar to that found in human thyroid-stimulating
hormone (TSH), follicle stimulating hormone (FSH), and human chorionic
gonadotropin (hCG).The difference between these hormones lie in amino
acid composition of their beta subunits, which account for their
immunological differentiation (Bardin, et al., 1981).
The basal secretion of LH in men is episodic and has the primary function
of stimulating the interstitial cells to produce testosterone. The variation in
LH concentration in women is subject to the complex ovulatory cycle of
healthy menstruating women, and depends upon a sequence of hormonal
events along the gonado-hypothalamic-pituitary axis. The decrease in
progesterone and estradiol levels from the preceding ovulation initiates each
menstrual cycle (Ross, et al., 1981). As result of decreased in hormone level,
the hypothalamus increases the secretion of gonadotropin-releasing factor
(GnRF), which in turn stimulates the pituitary to increase FSH production
and secretion (Whitely, et al., 1978). The rising FSH levels stimulate several
follicles during the follicular phase; one of these will mature to contain the
egg. As the follicle develops, estradiol is secreted, slowly at first, but by day
12 or 13 of normal cycle increasing rapidly. LH is released as result of this
rapid estradiol rise because of direct stimulation of the pituitary and
increasing GnRF and FSH levels. These events constitute the pre-ovulatory
phase (Acosta and Wright, 1983).
Ovulation occurs approximately 12 to 18 hours after the egg is released,
corpus luteum is formed which secrets progesterone and estrogen to
feedback regulators of LH (Jeffcoat and Marshall, 1975).
4
The luteal phase rapidly follows this ovulatory phase, and is characterize by
high progesterone levels.
A decreased in steroid hormone production in females is result of immature
ovaries. Primary ovarian failure, polycystic ovary disease, or menopause: in
these cases, LH secretion is not regulated (yen. et al., 1970). A similar loss
of regulatory hormones occurs in males when the testes develop abnormally
or anorchia exists. High concentrations of LH may also be found in primary
testicular failure and Klinefelter syndrome, although LH levels will not
necessarily be elevated if the secretion of androgens continues. Increased
concentrations of LH are also present during renal failure, cirrhosis,
hyperthyroidism, and severe starvation (Cohen, 1977).
A lack of secretion by the anterior pituitary may cause lower LH levels. As
may be expected, low levels may result in infertility in both males and
females. Low levels of LH may also be due to the decreased secretion of
GnRH by the hypothalamus.
Althoughthesameeffect
maybeseenbyafailureof
theanterior
pituitaryto
respondtoGnRH stimulation, low LH values may therefore indicate some
dysfunction of the pituitary or hypothalamus, but the actual source of the
problem must be confirmed by other test. In the differential diagnosis of
hypothalamic, pituitary, or gonadal dysfunction, assays of LH concentration
are routinely performed in conjunction with FSH assays since their roles are
closely interrelated. Furthermore, the hormone levels are used to determine
menopause and monitor endocrine therapy (Marshall, 1975).
5
1.1.2.2. Follicle-stimulation hormone
Follicle-stimulation hormone and luteinizing hormone are intimately
involved in the control of the growth and reproductive an activities of the
gonadal tissue, which synthesize and secrete male and female sex hormones
through a negative feedback relationship (Marshalland Jeffcoate, 1975).
FSH is glycoprotein secreted by the basophiles cells of the anterior pituitary.
Gonadotropin-releasing hormone (GnRH), produced in the hypothalamus,
controls the release of FSH from the anterior pituitary. Like other
glycoprotein, such as LH, TSH, HCG, FSH consists of subunits designated
as alpha and beta. Hormones of this type have alpha subunits that are very
similar structurally; therefore the biological and immunological properties of
each are dependent on the unique beta subunit (Marshalland Cohen, 1977).
In the female, FSH stimulates the growth and maturation of ovarian follicles
by acting directly on the receptors located on the granulose cells, follicular
steroidgenesis is promoted and LH production is stimulated. The LH
produced then binds to the theca cells and stimulates steroid genesis.
Increased intra-ovarian estradiol production occurs as follicular maturation
advances, there upon stimulating increased FSH receptor activity and FSH
follicular binding. FSH, LH, and estradiol are therefore intimately related in
supporting ovarian recruitment and maturation in women (Ross, et al., 1981)
FSH levels are elevated after menopause, castration, and in premature
ovarian failure. The levels of FSH may be normalized through the
administration of estrogens, which demonstrate a negative feedback
mechanism. Abnormal relationships between FSH and LH, between FSH
and estrogen have been linked to anorexia nervosa and polycystic ovarian
6
disease. Although there are significant exceptions ovarian failure is indicated
when random FSH concentrations exceed 40mlu/ml (Rebar, 1982).
High level of FSH in men may be found in primary testicular failure and
khlinefelter Syndrome. Elevated concentrations are also present in cases of
starvation; renal failure, hyperthyroidism, and cirrhosis. The growth of the
seminiferous tubules and maintenance of spermatogenesis in men are
regulated by FSH (Marshalland Cohen, 1977).
1.1.3. Gonadotropin-releasinghormone (GnRH)
Gonadotropin receptors are embedded in the surface of the target cell
membranes and coupled to the G-protein system. Signals triggered by
binding to the receptor are relayed within the cells by the cyclic AMP
second messenger system. Gonadotropins are released under the control of
gonadotropin-releasing hormone (GnRH) from the arcuate nucleus and
preoptic area of the hypothalamus. The gonads — testes and ovaries — are
the primary target organs for LH and FSH. The gonadotropins affect
multiple cell types and elicit multiple responses from the target organs. As a
simplified generalization, LH stimulates the Leydig cells of the testes and
the theca cells of the ovaries to produce testosterone (and indirectly
estradiol), whereas FSH stimulates the spermatogenic tissue of the testes and
the granulosa cells of ovarian follicles(Parhar and Ishwar, 2002).
1.1.4. Control of FSH and LH
At the pituitary, GnRH stimulates the synthesis and secretion of the
gonadotropin, follicle-stimulating hormone (FSH), and luteinizing hormone
(LH). These processes are controlled by the size and frequency of GnRH
pulses, as well as by feedback from androgens and estrogens. Low7
frequency GnRH pulses lead to FSH release, whereas high-frequency GnRH
pulses stimulate LH release. There are differences in GnRH secretion
between females and males. In males, GnRH is secreted in pulses at a
constant frequency, but, in females, the frequency of the pulses varies during
the menstrual cycle, and there is a large surge of GnRH just before ovulation
(Mahmood, et al., 2013).
GnRH secretion is pulsatile in all vertebrates, and is necessary for correct
reproductive function. Thus, a single hormone, GnRH1, controls a complex
process of follicular growth, ovulation, and corpus luteum maintenance in
the female, and spermatogenesis in the male (Mahmood, et al., 2013).
1.1.5. Activity of gonadotropin-releasing hormone(GnRH)
GnRH activity is very low during childhood, and is activated at puberty.
During the reproductive years, pulse activity is critical for successful
reproductive function as controlled by feedback loops. However, once a
pregnancy is established, GnRH activity is not required. Pulsatile activity
can be disrupted by hypothalamic-pituitary disease, either dysfunction (i.e.,
hypothalamic suppression) or organic lesions (trauma, tumor). Elevated
prolactin levels decrease GnRH activity. In contrast, hyperinsulinemia
increases pulse activity leading to disorderly LH and FSH activity, as seen in
polycystic ovary syndrome (PCOS). GnRH formation is congenitally absent
in Kallmann syndrome (Schwanzel,et al.,1989).
The GnRH neurons are regulated by many different afferent neurons, using
several different transmitters (including norepinephrine, GABA, glutamate).
For instance, dopamine appears to stimulate LH release (through GnRH) in
estrogen-progesterone-primed females; dopamine may inhibit LH release in
8
ovariectomized females(Jayes,et al.,1997).Kisspeptin appears to be an
important regulator of GnRH release )Dungan, et al., 2006). GnRH release
can also be regulated by estrogen (Franceschini, et al., 2006).
1.2. Human prolactin (lactogenic hormone)
Prolactin is secreted from the anterior pituitary gland in both men and
women (Shome and Parlow, et al.,1977). Human prolactin is a single chain
polypeptide hormone. The release and synthesis of prolactin is under neuroendocrinal control, primary through prolactin releasing factor and prolactin
inhibiting factor (Friesen, et al., 1972). Women normally have slightly
higher basal prolactin levels than men: apparently, there is an estrogen –
related rise at puberty and a corresponding decrease at menopause. The
primary functions of prolactin are to initiate breast development and to
maintain lactation. Prolactin also suppresses gonadal function. During
pregnancy, prolactin levels increase progressively to between 10 and 20
times normal values. Declining to non-pregnant levels by 3-4 weeks postpartum (Cowden, et al., 1979).
Breast feeding mothers maintain high levels of prolactin, and it may take
several months for serum concentrations to return to non-pregnant levels.
The demonstration of prolactin concentration is helpful in diagnosing
hypothalamic-pituitary disorders (Friesen, et al., 1979). Micro adenomas
(small pituitary tumors) may cause hyperprolactinemia, which is sometimes
associated with male impotence (Thomer, et al., 1977). High prolactin levels
are commonly associated with galactorrhea and amenorrhea. Prolactin
concentrations have been shown to be increased by estrogens, thyrotropin releasing hormone (TRH). Prolactin levels are elevated in renal disease and
hypothyroidism, and some situation of stress, exercise and hypoglycemia.
9
Additionally, the release of prolactin is episodic and demonstrates diurnal
variation .Prolactin concentrations may also increase by drugs such as
chloropromazine and reserpin, and may be lowered by bromocyptine and Ldopa(Daughday, et al., 1981).
Female infertility is often caused by a woman’s inability to ovulate or
release an egg. Prolactin concentrations may also be increase by drugs such
as chlorpromazine (Failure to ovulate is usually rooted in hormonal
problems). In many infertility cases, a woman may be producing too little of
one hormone or too much of another (Cowden, et al., 1979).
Puberty:at birth, female infants have a predetermined number of primordial
follicles that are arrested during meiosis 1 at the diplotene stage of prophase
until stimulation at puberty. Until puberty, the hypothalamus is in a
quiescent state. At approximately age 8 years, GnRH is synthesized in the
hypothalamus and released. The adrenal cortex begins to produce
dehydroepiandrostenedione to initiate the start of adrenarche (ie, the
development of sexual hair),(Pletcher and Slap, 1999).
The orderly progression of puberty begins with breast budding (thelarche),
accelerated growth, and menses (menarche). Pubarche, which is independent
from GnRH function, typically occurs between breast budding and
accelerated growth but may occur anywhere along the puberty timeline. In
the United States, the average age of girls at menarche is 12.6 years, with a
range of 9-15 years. (Age 15 years is 2 standard deviations above the mean,
while age 16 years is 3 standard deviations above), (Reindollar, et al., 1979).
Menarche and sustained menstrual cycles requires normal function of the
endocrine axis comprising the hypothalamus, pituitary, and ovaries. Any
10
disruption in this axis may result in amenorrhea. Defining the level of
primary dysfunction is critical in determining the pathophysiology of
amenorrhea(Pletcher and Slap, 1999).
1.3. Causes of female infertility
1.3.1. Hormonal disorder:
Gonadotropin deficiency due to pituitary disease results in hypogonadism,
which can lead to infertility. Treatment includes administered gonadotropin,
which, therefore, work as fertility medication. Such can either be produced
by extraction and purification from urine or be produced by recombinant
DNA.Failure or loss of the gonads usually results in elevated levels of LH
and FSH in the blood (Golos, et al., 1979).
1.3.2. Hormonal imbalance
1.3.2.1. Glandular problems
Hormonal imbalances can sometimes be traced back to the primary glands
(hypothalamus, thyroid and pituitary) that produce reproductive hormones.
The hypothalamus, pituitary and ovaries send signals back and during the
process that cause changes in hormone production (Adegoke, et al., 2011(.

Hypothalamus: this gland can be affected by injury, stress, starvation
and some

medications.
Thyroid: An underactive thyroid gland causes hypothyroidism and can
be characterized by excessive levels of the hormone prolactin, which
interferes with ovulation.
11

Pituitary: Microscopic tumors (prolactinomas) on the pituitary gland
can secrete the hormone prolactin, which interferes with ovulation
(Bayrak, et al., 2005(.

Glandular problems, ovulatory disorders, polycystic ovarian syndrome
(PCOS), premature menopause and stress. This can be classified and
detailed as fallowed (Adam and Balen, 2008).
1.3.2.2. Ovulatory Disorders
Problems with ovulation are one of the leading causes of female infertility,
accounting for about 20% of cases. Some women ovulate irregularly or do
not ovulate at all (this is called anovulation). When women have problems
with ovulation it’s usually because they have hormonal imbalances such as
too much prolactin (a milk-producing hormone that suppresses ovulation) or
an overproduction of male hormones androgens, such as testosterone (Oriel
and Schrager, 1999).
1.3.2.3. Polycystic Ovarian Syndrome (PCOS)
PCOS is a condition in which hormonal imbalances interfere with ovulation.
The adrenal glands and ovaries produce excessive amounts of male
hormone, which leads to an abnormally high production of luteinizing
hormone (LH) and an abnormally low production of follicle-stimulating
hormone (FSH). As a result, the ovary fills with cysts of immature follicles
that are unable to generate eggs. Women with this condition may experience
Irregular periods, enlarged ovaries, excessive facial and body hair, oily skin,
acne and obesity(Bauer and cooper, 2008).
12
1.3.2.4. Premature menopause
Women under 40 years of age whose ovaries are not producing sufficient
hormones to sustain ovulation and menstruation are deemed prematurely
menopausal. Premature menopause, also known as premature ovarian failure
(POF), occurs when a woman has prematurely depleted her supply of eggs
(Bardoni, et al., 2000).
1.3.2.5. Abnormal cervical mucous
Normal cervical secretions are a vital component to successful impregnation.
Many women may notice a change in the consistency of their cervical
mucous throughout the menstrual cycle. Cervical mucous is thin and watery
near the time of ovulation, making it easy for sperm to enter the uterus.
Abnormal cervical mucous may involve unusually thick mucous that blocks
the movement of sperm. The presence of anti-sperm IgA antibodies in
cervical mucous may also provide a hostile environment for sperm.
Hormonal imbalances are sometimes traced back to the primary glands in
the body that produce reproductive hormone, specifically the hypothalamus,
thyroid and pituitary glands. These glands are constantly sending signals
back and forth in order to keep hormones in check by making adjustments in
production of certain hormones (Burry, 2007).
1.4. Menstrual cycle
The menstrual cycle, under the control of the endocrine system, is necessary
for reproduction. It is commonly divided into three phases: the follicular
phase, ovulation, and the luteal phase. It is also occasionally misclassified
using the uterine cycle: menstruation, proliferative phase, and secretory
13
phase. Menstrual cycles are counted from the first day of menstrual
bleeding. Hormonal contraception interferes with the normal hormonal
changes with the aim of preventing reproduction (Greenberg,et al.,
2012),and stimulated by gradually increasing amounts of estrogen in the
follicular phase, discharges of blood (menses) slow then stop, and the lining
of the uterus thickens. Follicles in the ovary begin developing under the
influence of a complex interplay of hormones, and after several days one or
occasionally two become dominant (non-dominant follicles atrophy and die).
Approximately mid-cycle, 24–36 hours after the luteinizing hormone (LH)
surges, the dominant follicle releases an ovum, or egg in an event called
ovulation. After ovulation, the egg only lives for 24 hours or less without
fertilization while the remains of the dominant follicle in the ovary become a
corpus luteum; this body has a primary function of producing large amounts
of progesterone, under the influence of progesterone, the endometrium
(uterine lining) changes to prepare for potential implantation of an embryo to
establish a pregnancy. If implantation does not occur within approximately
two weeks, the corpus luteum will involute, causing sharp drops in levels of
both progesterone and estrogen. These hormone drops cause the uterus to
shed its lining and egg in a process termed menstruation (Greenberg,et al.,
2012).
In the menstrual cycle, changes occur in the female reproductive system as
well as other systems (which lead to breast tenderness or mood changes, for
example). A woman's first menstruation is termed menarche, and occurs
typically around age 12-13. The average age of menarche is about 12.5 years
in the United States(Anderson, et al., 2003) 12.72 in Canada, 12.9 in the UK
and 13.06 ± 0.10 years in Iceland. Factors such as hereditary, diet and
14
overall health can accelerate or delay menarche. The end of woman’s
reproductive phase is called the menopause, which commonly occurs
somewhere between the age of 45 to 55 years (Magnússon, 1978).
The average age of menarche in humans is 12–13 years, but is normal
anywhere between ages 8 and 16. Factors such as heredity, diet and overall
health can accelerate or delay menarche(Beyene andYewoubdar, 1989). The
cessation of menstrual cycles at the end of a woman's reproductive period is
termed menopause. The average age of menopause in women is 52 years,
with anywhere between 45 and 55 being common. Menopause before age 45
is considered premature in industrialized countries. Like the age of
menarche, the age of menopause is largely a result of cultural and biological
factors. However, illnesses, certain surgeries, or medical treatments may
cause menopause to occur earlier than it might have otherwise.The length of
a woman's menstrual cycle will typically vary, with some shorter cycles and
some longer cycles. A woman who experiences variations of less than eight
days between her longest cycles and shortest cycles is considered to have
regular menstrual cycles. It is unusual for a woman to experience cycle
length variations of less than four days. Length variation between eight and
20 days is considered as moderately irregular cycles. Variation of 21 days or
more between a woman's shortest and longest cycle lengths is considered
very irregular(Greenfield and Marjorie, 2001).
15
1.4.1 Menstrual cycle phases
The menstrual cycle can be divided into several different phases. The
average length of each phase is shown below; the first three are related to
changes in the lining of the uterus whereas the final three are related to
processes occurring in the ovary (Greenfield,etal.,2001).
16
Table (1.1) Menstrual cycle Phases(Greenfield,etal.,2001).
Name of phase
Average
start
Average
Average
day end day
duration
assuming a
number
28-day
of days
cycle
Menstrual phase (menstruation)
1
4
4
13
9
13
16
4
Luteal phase (also known as secretary 16
28
13
28
2
Proliferative phase (some sources 5
include menstruation in this phase)
Ovulatory phase (ovulation)
phase)
Ischemic phase
27
17
1.4.1.2. Follicular phase
This phase is also called the proliferative phase because a hormone causes
the lining of the uterus to grow, or proliferate, during this time. Through the
influence of a rise in follicle stimulating hormone (FSH) during the first
days of the cycle, a few ovarian follicles are stimulated. These follicles
which were present at birth have been developing for the better part of a year
in a process known as folliculogenesis, compete with each other for
dominance. Under the influence of several hormones, all but one of these
follicles will stop growing, while one dominant follicle in the ovary will
continue to maturity. The follicle that reaches maturity is called a tertiary, or
Graafian, follicle, and it contains the ovum (Losos, et al., 2002).
In physiologic terms, the first day of menses is considered the first day of the
menstrual cycle. The following 13 days of the cycle are designated the
follicular phase. As levels of progesterone, estradiol, and inhibin decline 2-3
days before menses, the pituitary begins to release higher levels of folliclestimulating hormone (FSH), which recruits oocytes for the next menstrual
cycle. The hypothalamus is the initiator of the follicular phase via
gonadotropin-releasing hormone (GnRH) (Doufas and Mastorakos, 2000).
The GnRH pump in the hypothalamus releases GnRH in a pulsatile fashion
into the portal vessel system surrounding the anterior pituitary gland. GnRH
interacts with the anterior pituitary gland to stimulate release of FSH in the
follicular phase. FSH is secreted into the circulation and communicates with
the granulosa cells surrounding the developing oocytes (Hu, et al., 2008).
18
As FSH increases during the early portion of the follicular phase, it meshes
with granulosa cells to stimulate the aromatization of androgens into
estradiol. The increase in estradiol and FSH leads to an increase in FSHreceptor content in the many developing follicles.Over the next several days,
the steady increase of estradiol (E2) levels exerts a progressively greater
suppressive influence on pituitary FSH release. Only one selected lead
follicle, with the largest reservoir of estrogen, can withstand the declining
FSH environment. The remaining oocytes that were initially recruited with
the lead follicle undergo atresia. Immediately prior to ovulation, the
combination of E2 and FSH leads to the production of luteinizing-hormone
(LH) receptors on the granulosa cells surrounding the lead follicle (Jone,
2007).
During the late follicular phase, estrogen has a positive influence on LH
secretion, instead of suppressing pituitary LH secretion as it does early in the
follicular phase. To have this positive effect, the estradiol (E2) level must
achieve a sustained elevation for several days. The LH surge promotes
maturation of the dominant oocyte, the release of the oocyte and then the
luteinization of the granulosa cells and the surrounding theca cells of the
dominant follicle resulting in progesterone production.The appropriate level
of progesterone arising from the maturing dominant follicle contributes to
the precise timing of the midcycle surge of LH. Estradiol (E 2) promotes
uterine
endometrial
gland
growth,
implantation(Pletcher and Slap, 1999).
19
which
allows
for
future
1.4.1.3. Ovulation
During the follicular phase, estradiol suppresses production of luteinizing
hormone (LH) from the anterior pituitary gland. When the egg has nearly
matured, levels of estradiol reach a threshold above which this effect is
reversed and estrogen actually stimulates the production of a large amount of
LH. This process, known as the LH surge, starts around day 12 of the
average cycle and may last 48 hours. The exact mechanism of these opposite
responses of LH levels to estradiol is not well understood )Lentz,et al.,
2013). In animals, a Gonadotropin-releasing hormone (GnRH) surge has
been shown to precede the LH surge, suggesting that estrogen's main effect
is on the hypothalamus, which controls GnRH secretion .This may be
enabled by the presence of two different estrogen receptors in the
hypothalamus: estrogen receptor alpha, which is responsible for the negative
feedback estradiol-LH loop, and estrogen receptor beta, which is responsible
for the positive estradiol-LH relationship )Hu,etal., 2008). However in
humans it has been shown that high levels of estradiol can provoke abrupt
increases in LH, even when GnRH levels and pulse frequencies are held
constant.Suggesting that estrogen acts directly on the pituitary to provoke
the LH surge(Lentz,et al., 2013).The release of LH matures the egg and
weakens the wall of the follicle in the ovary, causing the fully developed
follicle to release its secondary oocyte (Losos,et al., 2002).
The secondary oocyte promptly matures into an ootid and then becomes a
mature ovum. The mature ovum has a diameter of about 0.2 mm(Gray and
Henry, 2000). Ovaries—left or right—ovulates appear essentially random;
no known left and right co-ordination exists.Occasionally, both ovaries will
20
release an egg; if both eggs are fertilized, the result is fraternal
twins(Ecochard and Gougeon, 2000).
After being released from the ovary, the egg is swept into the fallopian tube
by the fimbria, which is a fringe of tissue at the end of each fallopian tube.
After about a day, an unfertilized egg will disintegrate or dissolve in the
fallopian tube.Fertilization by a spermatozoon, when it occurs, usually takes
place in the ampulla, the widest section of the fallopian tubes. A fertilized
egg immediately begins the process of embryogenesis, or development. The
developing embryo takes about three days to reach the uterus and another
three days to implant into the endometrium. It has usually reached the
blastocyst stage at the time of implantation (Losos,et al., 2002).
In some women, ovulation features a characteristic pain called
mittelschmerz (German term meaning middle pain), (John, 2007). The
sudden change in hormones at the time of ovulation sometimes also causes
light mid-cycle blood flow(Wechsler, 2002).
Ovulation occurs approximately 34-36 hours after the onset of the LH surge
or 10-12 hours after the LH peak and 24-36 hours after peak estradiol (E2)
levels. The rise in progesterone increases the distensibility of the follicular
wall and enhances proteolytic enzymatic activity, which eventually breaks
down the collagenous follicular wall. After the ovum is released, the
granulosa cells increase in size and take on a yellowish pigmentation
characteristic of lutein. The corpus luteum then produces estrogen,
progesterone, and androgens and becomes increasingly vascularized (Gray
and Henry, 2000).
21
1.4.1.4. Luteal phase
The luteal phase is also called the secretory phase. An important role is
played by the corpus luteum, the solid body formed in an ovary after the egg
has been released from the ovary into the fallopian tube. This body continues
to grow for some time after ovulation and produces significant amounts of
hormones, particularly progesterone (Losos,et al., 2002). Progesterone plays
a vital role in making the endometrium receptive to implantation of the
blastocyst and supportive of the early pregnancy; it also has the side effect of
raising the woman's basal body temperature(Wechsler, 2002).
After ovulation, the pituitary hormones FSH and LH cause the remaining
parts of the dominant follicle to transform into the corpus luteum, which
produces progesterone. The increased progesterone in the adrenals starts to
induce the production of estrogen. The hormones produced by the corpus
luteum also suppress production of the FSH and LH that the corpus luteum
needs to maintain itself. Consequently, the level of FSH and LH fall quickly
over time, and the corpus luteum subsequently atrophies (Losos,et al., 2002).
Falling levels of progesterone trigger menstruation and the beginning of the
next cycle. From the time of ovulation until progesterone withdrawal has
caused menstruation to begin, the process typically takes about two weeks,
with 14 days considered normal. For an individual woman, the follicular
phase often varies in length from cycle to cycle; by contrast, the length of
her luteal phase will be fairly consistent from cycle to cycle (Wechsler,
2002).
The loss of the corpus luteum can be prevented by fertilization of the egg;
the resulting embryo produces placental chorionic gonadotropin (hCG),
22
which is very similar to LH and which can preserve the corpus luteum.
Because the hormone is unique to the embryo, most pregnancy tests look for
the presence of placental chorionic gonadotropin (hCG), (Losos,et al.,
2002).
The life span and steroidogenic capacity of the corpus luteum depends on
continued LH secretion from the pituitary gland. The corpus luteum secretes
progesterone that interacts with the endometrium of the uterus to prepare it
for implantation. This process is termed endometrial decidualization. In the
normal ovulatory menstrual cycle, the corpus luteum declines in function 911 days after ovulation. If the corpus luteum is not rescued by placental
chorionic gonadotropin (hCG) hormone from the developing placenta,
menstruation reliably occurs 14 days after ovulation. If conception occurs,
placental hCG interacts with the LH receptor to maintain luteal function
until placental production of progesterone is well established(Pletcher and
Slap, 1999).
1.4.2. Pathophysiology of menstrual cycle
The menstrual cycle is an orderly progression of coordinated hormonal
events in the female body that stimulates growth of a follicle to release an
egg and prepare a site for implantation if fertilization should occur.
Menstruation occurs when an egg released by the ovary remains unfertilized;
subsequently, the soggy decidua of the endometrium (which was primed to
receive a fertilized egg) is sloughed in a flow of menses in preparation for
another cycle.The menstrual cycle can be divided into 3 physiologic phases:
follicular, ovulatory, and luteal. Each phase has a distinct hormonal
secretory milieu. Consideration of the target organs of these reproductive
23
hormones (hypothalamus, pituitary, ovary, and uterus) is helpful for
identifying the disease process responsible for a patient’s amenorrhea
(Speroff,et al., 1999).
1.4.3. Behavioral change
In some cases, hormones released during the menstrual cycle can cause
behavioral changes in females; mild to severe mood swings can
occur(Schmidt,et al.,1998).
1.4.4. Length of menstrual cycle
Although many people believe the average menstrual cycle takes 28 days, a
large study of more than 30,000 cycles from more than 2300 women showed
that the mean cycle length was 29.1 with a standard deviation of 7.5 days
and a 95% prediction interval of between 15 and 45 days.In that study, the
subset of data with cycle lengths between 15 and 45 days had an average
length of 28.1 days with a standard deviation of 4 days. A smaller study of
140 women performed in 2006 found a mean cycle length of 28.9 days. The
variability of menstrual cycle lengths are highest for women under 25 years
of age and are lowest, that is, most regular, for ages 35 to 39 (Chiazze,et
al.,1968).Subsequently, the variability increases slightly for women aged 40
to 44. Usually, length variation between eight and 20 days in a woman is
considered as moderately irregular menstrual cyclesVariation of 21 days or
more is considered very irregular(Greenfield and Marjorie, 2001).
24
1.4.5. Effect on other systems
Some women with neurological conditions experience increased activity of
their conditions at about the same time during each menstrual cycle. For
example, drops in estrogen levels have been known to trigger migraines,
especially when the woman who suffers migraines is also taking the birth
control pill. Many women with epilepsy have more seizures in a pattern
linked to the menstrual cycle; this is called catamenial epilepsy(Herzog,
2008). Different patterns seem to exist (such as seizures coinciding with the
time of menstruation, or coinciding with the time of ovulation), and the
frequency with which they occur has not been firmly established. Using one
particular definition, one group of scientists found that around one-third of
women with intractable partial epilepsy have catamenial epilepsy(Herzog,
2008). An effect of hormones has been proposed, in which progesterone
declines and estrogen increases would trigger seizures (Scharfman and
Maclusky, 2006).Recently, studies have shown that high doses of estrogen
can cause or worsen seizures, whereas high doses of progesterone can act
like an antiepileptic drug. Studies by medical journals have found that
women experiencing menses are 1.68 times more likely to commit suicide
(García, et al., 2003).
Estrogen levels may affect thyroid behavior (Doufas and Mastorakos, 2000).
For example, during the luteal phase (when estrogen levels are lower), the
velocity of blood flow in the thyroid is lower than during the follicular
phase(Krejza,et al., 2004).
25
1.4.6. Cycle abnormalities and disorders
Infrequent or irregular ovulation is called oligoovulation (Galan and Nicole,
2008). The absence of ovulation is called an ovulation. Normal menstrual
flow can occur without ovulation preceding it: an anovulatory cycle. In some
cycles, follicular development may start but not be completed; nevertheless,
estrogens will form and will stimulate the uterine lining. Anovulatory flow
resulting from a very thick endometrium caused by prolonged, continued
high estrogen levels is called estrogen breakthrough bleeding. Anovulatory
bleeding triggered by a sudden drop in estrogen levels is called changes.
Anovulatory cycles commonly occur before menopause (perimenopause)
and in women with polycystic ovary syndrome (Weschler, 2002).
Very little flow (less than 10 ml) is called hypomenorrhea. Regular cycles
with intervals of 21 days or fewer are polymenorrhea; frequent but irregular
menstruation is known as metrorrhagia. Sudden heavy flows or amounts
greater than 80 ml are termed menorrhagia. Heavy menstruation that occurs
frequently and irregularly is menometrorrhagia. The term for cycles with
intervals exceeding 35 days is oligomenorrhea(Oriel and Schrager, 1999).
Amenorrhea refers to more than three to six months without menses (while
not being pregnant) during a woman's reproductive years (Oriel and
Schrager, 1999).
1.5. Amenorrhea
Amenorrhea is the absence of menstrual bleeding.Amenorrhea is a normal
feature in prepubertal, pregnant, and postmenopausal females, in females of
reproductive age. Diagnosing amenorrhea is a matter of first determining
26
whether pregnancy is the etiology. In the absence of pregnancy, the
challenge is to determine the exact cause of absent menses (Hunter,et
al.,2006).
Primary amenorrhea is the failure of menses to occur by age 16 years, in the
presence of normal growth and secondary sexual characteristics. If by age 13
menses has not occurred and the onset of puberty, such as breast
development, is absent, a workup for primary amenorrhea should start
(Speroff and Fritz, 2005).
Secondary amenorrhea is defined as the cessation of menses sometime after
menarche has occurred. Oligomenorrhea is defined as menses occurring at
intervals longer than 35 days ) Speroff, et al., 1999(.
No consensus has been reached regarding the point at which oligomenorrhea
becomes amenorrhea. Some authors suggest the absence of menses for 6
months constitutes amenorrhea, but the basis for this recommendation is
unclear. For a postmenarchal girl or a reproductive-aged woman to
experience a menstrual cycle interval of more than 90 days is statistically
unusual. Practically speaking, this should be an indication for an evaluation
to seek the cause (Tarannum,et al.,2006).
1.5.1. Types of amenorrhea based on hypothalamic (HPO) axis etiology
1.5.1.1. Hypothalamic amenorrhea
Hypothalamic dysfunction results in decreased or inhibited GnRH secretion,
which affects the pulsatile release of LH and FSH, causing an ovulation. A
common
cause
of
amenorrhea
27
is
functional
hypothalamic
amenorrhea(Greenspan, 1991).It is characterized by abnormal hypothalamic
GnRH secretion, decreased gonadotropin pulsations, low or normal LH
concentrations, absent LH surges, abnormal follicular development, and low
serum estradiol. Serum FSH concentrations are usually in the normal range,
with high FSH-to-LH ratio) Santoro, et al., 1986(.
Functional hypothalamic amenorrhea can be caused by eating disorders,
exercise, or high levels of prolonged physical or mental stress. This can also
include major psychiatric disorders such as depression. Hypothyroidism,
hyperthyroidism, sarcoidosis, galactosemia or any severe chronic medical
condition may result in amenorrhea.
Congenital GnRH deficiency leads to low gonadotropin levels. When this
occurs with anosmia, it is diagnosed as Kallman syndrome. Kallman
syndrome may be associated with midline facial defect, renal agenesis, and
neurologic deficiency. Most often, it is an X-linked recessive disorder.
Autosomal dominant and autosomal recessive inheritances are possible but
less common (Kallman and Schoenfeld, 1944).
1.5.1.2. Gonadotropin-releasing hormone deficiency in adults
Evidence suggests a negative correlation between body fat levels and
menstrual abnormalities. A critical body fat level must be present for the
reproductive system to function normally (Greenspan, 2001).
In some female athletes, the synergistic effects of excessive exercise and
disordered eating cause severe suppression of GnRH, leading to low
estradiol levels. The female athletic triad, as defined by the American
College of Sports Medicine, is characterized by disordered eating,
28
amenorrhea, and osteoporosis. Study conducted found that about half of
exercising women could be amenorrhric)De Souza٬et al., 2009(.
Anorexia nervosa is a serious psychiatric disease with severe medical
complications including primary amenorrhea (15%), osteopenia (52%), and
osteoporosis (35%), Functional causes of amenorrhea include severe chronic
disease, rapid weight loss, malnutrition, depression or other psychiatric
disorders, recreational drug abuse, and psychotropic drug use (Miller,et al.,
2006(.
1.5.1.3. Pituitary amenorrhea
A deficiency in FSH and LH may be a result of GnRH receptor gene
mutations. Mutations in the FSH beta gene have been associated with
amenorrhea. These women have low FSH and estradiol levels and high LH
levels. Primary amenorrhea caused by hyperprolactinemia is a rare condition
characterized by the onset of thelarche and pubarche at appropriate ages but
arrest of pubertal development before menarche (Kawano, et al., 2000(.
Hyperprolactinemia is associated with suppression of the GnRH from the
hypothalamus and subsequent inhibition of LH and FSH, suppressed
gonadal function and galactorrhea. Prolactinomas are the most common
cause of persistent hyperprolactinemia, accounting for 40-50% of pituitary
tumors.Pituitary tumors may suppress gonadotropin secretion, such as in
Cushing
disease
or
hypothalamic
tumors,
Craniopharyngioma,
or
germinoma. Brain injury or cranial irradiation may also result in
amenorrhea. Other pituitary causes include empty sella syndrome, pituitary
infarct, hemachromatoses, and sarcoidosis (Crosignani, et al., 2006(.
29
1.5.1.0. Ovarian causes of primary amenorrhea
Gonadal dysgenesis most commonly occurs in Turner syndrome (45, X).
Accelerated loss of the germ cells in the gonads occurs. The gonads usually
contain only fibrous tissue and are called streak gonads. Gonadotropin
levels, especially the FSH levels, are high during early childhood and after
age 9-10 years.
Additional anomalies associated with Turner syndrome include short stature,
webbed neck, coarctation of the aorta (10%), renal abnormalities (50%), and
hypertension, pigmented nevi, short forth metacarpal and metatarsals,
Hashimoto thyroiditis, obesity, and osteoporosis. Depletion of ovarian
follicles causes amenorrhea (Greenspan, 1991).
Spontaneous 46,XX primary ovarian insufficiency (POI), (also known as
premature ovarian failure [POF] and premature menopause) affects 1 in
10,000 women by age 20 years, 1 in 1,000 women by age 30 years, 1 in 250
women by age 35 years, and 1 in 100 women by age 40 years. POI is
hypergonadotropic hypogonadism, characterized by oligomenorrhea,
estrogen deficiency, and its associated symptoms such as hot flashes, vaginal
dryness, dyspareunia, and insomnia (Bardoni, et al., 2000).
The fragile X permutation accounts for approximately 6% of cases of overt
POI. It is caused by an increased number of CGG repeats in the FMR1 gene
located on the long arm of the X chromosome, in the permutation, the
number of CGG repeats ranges from 55-200. Approximately 21% of
permutation carriers have POF/POI compared with 1% in the general
30
population. Autoimmune oophoritis occurs in 3-4% of POI cases (Sherman,
et al., 2002(.
Amenorrhea is also seen in pure 46, XX gonadal dysgenesis and in 46, XY
gonadal dysgenesis. These women have significantly elevated FSH levels
due to the absence of ovarian follicles and reduction in negative feedback on
FSH from estradiol and inhibin A and B (Bakalov, et al., 2002(.
The early stages of testicular formation require the action of several genes,
of which one of the earliest and most important is the sex-determining
region of the Y chromosome (SRY). In Swyer syndrome, the fetus has a 46,
XY karyotype but with mutations of the SRY gene such that the testes never
form and antimüllerian hormone is not produced. As a result, these
individuals have a vagina, uterus, and fallopian tubes. Germ cells in the
ovaries are lost before birth. The streak gonads must be surgically removed
because of the increased risk for developing germ cell tumor. Pure gonadal
dysgenesis occurs when the syndrome affects the gonads only and no other
dysmorphic features are noted )Coulam, et al., 1986).
Polycystic ovarian syndrome (PCOS) usually presents as secondary
amenorrhea, but in some cases may present as primary amenorrhea )Azziz,
et al., 1986).
1.6. Congenital and anatomical abnormalities
A uterus and patent vaginal tract are needed for normal menstrual flow to
occur. Female reproductive tract abnormalities account for about one fifth of
primary amenorrhea cases. Cyclic pelvic pain is common in girls with
disorders of the reproductive tract involving outflow obstruction.
31
Imperforate hymen causes an outflow obstruction. These patients can have
blood in the vagina that collects and can result in a perirectal mass.
Transverse vaginal septum can be anywhere along the tract between the
hymenal ring and cervix. Vaginal agenesis or müllerian dysgenesis, also
known as Mayer-Rokitansky-Kuster-Hauser [MRKH] syndrome is caused
by agenesis or partial agenesis of the müllerian duct system. It is
characterized by congenital aplasia of the uterus and upper two thirds of the
vagina in women showing normal development of the secondary sexual
characteristics and a normal 46, XX karyotype. The first sign is primary
amenorrhea. It affects 1 of 4500 women. It can be associated with renal,
vertebral, and, to a lesser extent, auditory and cardiac defects(Morcel, et al.,
2002).
1.7. Receptor and enzyme defects:
Congenital adrenal hyperplasia as a result of 17 alpha-hydroxylase
deficiency (CYP17) causes an excess of deoxycortisone to be produced and
deficiency of cortisol and adrenal and gonadal sex steroids. Patients with this
disorder who experience primary amenorrhea can be either genotypic males
(XY) or females (XX) (Greenspan, 1991).
Vanishing testes syndrome is characterized by genotypic males 46, XY
whose gonads do not develop completely. As a result, no testosterone,
estrogen, or müllerian-inhibiting substance is produced. These patients
appear phenotypically female. The diagnosis is made from the findings of
gonadal failure with lack of pubertal progression, high serum FSH and LH
concentrations, and male karyotype. Androgen insensitivity syndrome
occurs when patients are resistant to testosterone. It is an X-linked disease.
32
Patients appear as phenotypically normal females. The testes, located
internally and sometimes in the labia or inguinal area, do make müllerianinhibiting hormone, so all müllerian structures, fallopian tubes, uterus, and
upper third of the vagina are absent)Morcel, et al., 2002).
Gonadotropin resistance is rare, but inactivating mutations of the receptors
for LH and FSH can cause anovulatory amenorrhea (Huhtaniemi, et al.,
2006).
Aromatase deficiency is also a rare disorder. Aromatase catalyzes the
conversion of androgen to estrogen,when estrogen synthesis cannot occur.
Often, girls have cystic ovaries and resultant amenorrhea (Jones, et al.,
2007).
1.8. Etiology of amenorrhea
Amenorrhea can be divided into 2 groups: (1) amenorrhea without evidence
of associated androgen excess and (2) amenorrhea with evidence of
androgen excess (e.g., hirsutism, fertilization, sexual ambiguity) (Pletcher
and Slap, 1999).
1.8.1. Causes of primary amenorrhea
First and for most, it is imperative to rule out pregnancy. Additional
diagnoses of primary amenorrhea usually result from a genetic or anatomic
abnormality. The cause of primary amenorrhea includes hypergonadotropic
hypogonadism (48.5% of cases). Hypogonadotropic hypogonadism (27.8%)
and Eugonadism (pubertal delay with normal gonadotropin; 23.7%)(Barber
and Franks, 2010(.
33
(a) The hypergonadotropic hypogonadism category includes patients with
abnormal sex chromosomes (ie, Turner syndrome), who make up
29.7% of all primary amenorrhea cases, and those with normal sex
chromosomes. The latter group includes both patients who are 46, XX
(15.4%) and those who are 46, XY (3.4%) )Reindollar, et al., 1989(.
(b) Hypogonadotropic hypogonadism includes the following:

Congenital abnormalities

Endocrine disorders

Tumor

Systemic illness (2.6%)

Eating disorder (2.3%)
Congenital abnormalities that can cause hypogonadotropic hypogonadism
include the following:

Isolated GnRH deficiency (8.3%)

Forms of hypopituitarism (2.3%)

Congenital central nervous system (CNS) defects (0.8%)

Constitutional delay (6%)
Endocrine disorders that can cause hypogonadotropic hypogonadism include
the following:

Congenital adrenal hyperplasia (CAH) (0.8%)

Cushing syndrome (0.4%)

Pseudohypoparathyroidism (0.4%)

Hyperprolactinemia (1.9%)
34
Tumors that can cause hypogonadotropic hypogonadism include the
following:

Unclassified pituitary adenoma (0.8%)

Craniopharyngioma (1.1%)

Unclassified malignant tumor (0.4%)
(c) Eugonadism may result from anatomic abnormalities or intersex
disorders. Anatomic abnormalities include congenital absence of the
uterus and vagina (CAUV; 16.2%) and cervical atresia (0.4%).
Intersex disorders include androgen insensitivity (1.5%), 17ketoreductase deficiency (0.4%), and inappropriate feedback (5.3%)
)Reindollar, et al., 1989(.
1.8.2. Causes of secondary amenorrhea
Disorders associated with a low or normal FSH, which account for 66% of
cases of secondary amenorrhea, include the following (De Souza and
Toombs, 2010).

Weight loss/anorexia

Nonspecific hypothalamic

Chronic anovulation including PCOS

Hypothyroidism

Cushing syndrome

Pituitary tumor, empty sella, Sheehan syndrome
Disorders in which the FSH is high (12%) include the following:

46XX, spontaneous POI
35

Premature ovarian failure due to abnormal karyotype (45,XO)

Pure gonadal dysgenesis
Disorders associated with a high prolactin level comprise 13% of cases.
Anatomic disorders (ie, Asherman syndrome) account for 7%.
Hyperandrogenic states as a cause of secondary amenorrhea (2%) include
the following:

Polycystic ovarian syndrome (PCOS)

Ovarian tumor

Nonclassic CAH
1.9. Epidemiology of amenorrhea
No evidence indicates that the prevalence of amenorrhea varies according to
national origin or ethnic group. However, local environmental factors related
to nutrition and the prevalence of chronic disease undoubtedly have an
effect. For instance, the age of the first menses varies by geographic
location, as demonstrated by a World Health Organization study comparing
11 countries, which reported a median age of menarche of 13-16 years
across centers )Lippincott Williams & Wilkins, 2005(.
Recent increases in the rates of childhood obesity around the world may also
contribute to earlier onset of menarche and increased prevalence of obesityrelated menstrual disorders, especially in areas where obesity is more
prevalent (Pandey and Bhattacharya, 2010). Exposure to environmental
toxins, namely hormonally active endocrine disruptors, may also result in
36
increased rates of menstrual and reproductive disorders in endemic areas
(Phillips and Foster, 2008).
1.10. Prognosis of amenorrhea
Loss of menstrual regularity has been associated with an increased risk of
wrist and hip fractures related to reduce bone density, even without the
development of amenorrhea. A later menarche and menstrual cycle intervals
longer than 32 days have both been associated with increased fracture rates
in later years. Young women with ovarian insufficiency that is unresponsive
to therapy require hormone replacement to maintain bone density. All of
which play an important role in building and maintaining bone mass. Late
menarche has been associated with a 3-fold increase in the risk of wrist
fracture (Adams and Nelson,2003).
In some cases, loss of menstrual regularity is an early sign of declining
fertility and impending premature ovarian failure. Also in some cases,
follicle depletion progresses to cause irreversible infertility. Approximately
10% of women evaluated for amenorrhea in a tertiary center are found to
have premature ovarian failure or primary ovarian insufficiency (Diaz,et al.,
2006).
1.11. Patient education
For patients with ovarian insufficiency that persists despite appropriate
evaluation and treatment, careful counseling is warranted to stress the need
for ongoing attention to the factors that help maintain bone density.
Hormone replacement therapy is important for these patients. Other factors
to consider are the need for adequate calcium and vitamin D intake (120037
1500 mg/d of elemental calcium and 1000 IU/d of vitamin D) and the need
for 20-30 minutes of weight-bearing exercise each day (Diaz,et al., 2006).
1.12. Amenorrhea clinical presentation
An adequate history includes childhood growth and development and other
areas, including height, weight charts and age at thelarche and menarche.
Ascertaining the age at menarche of the patient's mother and sisters is
advisable because the age at menarche in family members can occur within a
year of the age in others. The duration and flow of menses, cycle days, day
and date of last menstrual period, presence or absence of molimina (breast
soreness and mood change immediately before menses) are necessary pieces
of information (Alan, et al., 2002).
Any history of chronic illness, trauma, surgery, and medications is also
important. A sexual history should be obtained in a confidential manner.
Information regarding substance use, exercise, diet, home and school
situations, and psychosocial issues should be elicited. A comprehensive
review of symptoms should include vasomotor symptoms, hot flashes,
virilizing
changes,
galactorrhea,
headache,
fatigue,
palpitations,
nervousness, hearing loss, and visual changes (Alan, et al., 2002).
1.12.1. Primary amenorrhea
Absence of spontaneous menstruation before age 16 years is an indication
for a careful review of systems. The menstrual cycle should be viewed as a
vital sign. Inquiring about other aspects of growth and pubertal development
is important. An absence of any breast development or pubertal growth spurt
38
by age 13-14 years in girls is distinctly abnormal and requires investigation
(Deligeoroglou, et al., 2010).
Breast development, pubertal growth spurt and adrenarche are delayed or
absent in girls with hypothalamic pituitary failure. A distinguishing factor in
the case of isolated ovarian insufficiency or failure is that adrenarche occurs
normally, while estrogen-dependent breast development and the pubertal
growth spurt are absent or delayed (Sharma, et al., 2008).
Pregnancy could be the cause for primary amenorrhea. Determining whether
the patient is sexually active and whether she is using contraceptive methods
is important. In some cases, hormonal contraception itself may be the cause
of the amenorrhea (Darney, et al., 2008).
1.12.2. Secondary amenorrhea
Loss of menstrual regularity is an indication for a careful review of systems.
The menstrual cycle should be viewed as a vital sign. Loss of menstrual
regularity may be the first clear symptom heralding the onset of a major
illness or systemic disease. Viewing the menstrual cycle as a vital sign may
lead to earlier diagnosis of, and intervention for, several potentially lifethreatening disorders. The clinician need not wait for an arbitrarily defined
duration of amenorrhea to pass before taking corrective action
(Deligeoroglou, et al., 2010).
Amenorrhea can be due to pregnancy, anatomic defects of the outflow tract,
ovarian disorders, and pituitary or hypothalamic disorders. In some cases,
the cause is functional, meaning that the hypothalamic gonadotropinreleasing hormone (GnRH) pulse generator has shut down the reproductive
39
system in its role as an integrator of metabolic and psychogenic stress
(Doufas and Mastorakos, 2000).
Attributing the loss of menstrual regularity to a recent stressful life event is
tempting; however, this approach can delay the detection of significant
pathology that can have long-term health consequences. One study has
shown that one third of women in a control group report a significant
stressful life event in the preceding year (Loucks, et al., 2008).
Taking a careful patient history is paramount in deciphering potential
etiologies of secondary amenorrhea. Often, time constraints do not permit
practitioners to obtain a thorough history and review of symptoms on the
first visit. Scheduling a repeat visit to permit a more thorough evaluation
may be necessary. Another option is to use standardized history-taking
instruments to collect this information in preparation for a return visit. In
other cases, patients may be asked to keep a menstrual calendar and return in
3 months for reassessment. The importance of the ovary as an endocrine
organ that helps maintain bone density should be stressed to the patient to
help ensure proper follow-up (Gladwell and Malcolm, 2000).
1.12.3. Disorders of the outflow tract
A history of otherwise normal growth and pubertal development and cyclic
pelvic pain in association with primary amenorrhea suggests the possibility
of a congenital outflow tract abnormality such as imperforate hymen or
agenesis of the vagina, cervix, or uterus. These findings are also compatible
with the complete androgen resistance syndrome (Vaishali, et al., 2008).
40
Prior history of a surgical procedure involving the endometrial cavity,
especially if performed in the presence of infection, raises the possibility of
uterine synechiae (Asherman syndrome) (Hunt, et al., 2008).
1.12.4. Ovarian disorders
Symptoms of vaginal dryness, hot flashes, night sweats, or disordered sleep
may be a sign of ovarian insufficiency or premature ovarian failure. The
presence of these symptoms in young women demands further evaluation in
a timely manner. A prior history of chemotherapy or radiation therapy may
be associated with ovarian failure (Welt, 2008).
Autoimmune oophoritis may be associated with autoimmune adrenal
insufficiency, a potentially fatal condition that often manifests as vague and
nonspecific symptoms. Loss of menstrual regularity may be the first clear
symptom indicating a need for further evaluation to detect this condition
(Bakalov, et al., 2008).
1.12.5. Hypothalamic/pituitary disorders
Associated galactorrhea, headaches, or reduced peripheral vision could be a
sign of an anterior pituitary adenoma. These symptoms require immediate
further evaluation. However, secondary amenorrhea may be the only overt
symptom of a small prolactinoma (Dastur and Lalita, 1968).
An impaired sense of smell in association with primary amenorrhea and
failure of normal pubertal development may be related to isolated
gonadotropin deficiency, as is observed in persons with Kallmann syndrome
(Kallman and Schoenfeld, 1944).
41
Neurosarcoidosis can infiltrate the hypothalamus and/or pituitary and cause
hypogonadotropic hypogonadism, leading to disrupted menses. Sarcoidosis
can manifest insidiously, with development of mild fatigue, malaise,
anorexia, weight loss, and fever. Because 90% of patients with sarcoidosis
have pulmonary involvement at some stage of the disorder, cough and
dyspnea may be present. Hemachromatoses may manifest as weakness,
lassitude, weight loss and a change in skin color (Falsetti, et al., 2002).
A history of hemorrhage after childbirth with subsequent failure of regular
menses to return may be an indication of postpartum pituitary necrosis
(Sheehan syndrome). Failure of lactation is an even earlier sign. Detecting
this condition early is important because of the possible development of
associated central adrenal insufficiency, a potentially fatal condition
(Schrager and Sabo, 2001).
1.12.6. Functional hypothalamic impairment
Dieting with excessive restriction of energy intake, especially fat restriction
may lead to amenorrhea and associated bone loss. In extreme cases, the
process may advance to anorexia nervosa, a potentially fatal condition.
Associated symptoms are an intense fear of fatness and a body image that is
heavier than observed. Eating disorders can be restrictive in nature or can be
of a binge-eating/purging type (Miller, et al., 2006).
Orthorexia is characterized by obsession with eating healthy or organic
foods, often to the detriment of a patient’s health. This is currently classified
as an ―eating disorder not otherwise specified‖ in the DSM-IV-TR. Patients
with orthorexia may also restrict specific nutrients and calories from their
42
diet and develop amenorrhea and its long-term health consequences, namely,
low bone mineral density(Cartwright, 2004).
Major psychiatric disorders such as depression, obsessive-compulsive, or
schizophrenia may cause amenorrhea. Symptoms associated with these
conditions may be detected upon review of systems. In these cases,
secondary amenorrhea may be due to the psychiatric disorder itself, as these
are chronic disease states, or amenorrhea may be related to necessary
medications, such as antipsychotic or antiepileptic drugs (Karno, et al.,
1988)
Autoimmune adrenal insufficiency is a rare disorder and a potentially fatal
condition, often manifesting as vague and gradually evolving nonspecific
symptoms such as fatigue, anorexia, and weight loss. Occasionally, an acute
crisis can become life threatening, owing to the sudden interruption of a
normal or hyperfunctioning adrenal or pituitary gland or a sudden
interruption of adrenal replacement therapy. Clinical suspicion mandates
appropriate diagnostic screening and early intervention with sodium chloride
– containing fluids and hydrocortisone replacement. Long-term management
of patients with adrenal insufficiency requires an experienced specialist as
management can be challenging. All clinicians should have some basic
knowledge of when to suspect and begin the diagnostic workup of suspected
acute adrenal failure (Arlt and Allolio, 2003).
Amenorrhea may herald the onset of other autoimmune endocrine disorders
such as hyperthyroidism, hypothyroidism, or autoimmune lymphocytic
hypophysitis. The same is true for other endocrine disorders such as Cushing
syndrome or pheochromocytoma. A careful review of symptoms may help
43
uncover these disorders. Strenuous exercise related to a wide variety of
athletic activities can be associated with the development of amenorrhea.
Elicit a history regarding the type of exercise activity and its duration per
week. Both extreme thinness or rapid weight loss and morbid obesity or
rapid weight gain may result in amenorrhea by altering pulsatile GnRH
release (Eldar-Geva,et al.,2010).
History of excessive food intake may be due to Prader-Wili Syndrome. Or
leptin deficiency, both of which cause both extreme obesity and amenorrhea
(Blüher,et al.,2010).
Women with hypothalamic amenorrhea have lower serum leptin
concentrations, which may contribute to their low gonadotropin secretion.
Leptin administration resulted in improvement of the reproductive axis in
one study of women with functional hypothalamic amenorrhea (Welt,et al.,
2004).
Kisspeptin, a neural signal that acts directly on GnRH neurons to stimulate
neuronal firing, and which may act downstream from leptin as an integrator
of metabolic cues to the GnRH pulse generator, is also down regulated in
cases of hypothalamic amenorrhea. Interestingly, exogenous administration
of kisspeptin to women with hypothalamic amenorrhea acutely stimulates
gonadotropin secretion, an effect similar to what is seen with leptin
administration )Jayasena, et al., 2009(.
1.12.7. Chronic diseases
Malnutrition and cirrhosis associated with alcoholism may cause loss of
menstrual regularity. Human Immuno-deficiency Virus (HIV) disease or
44
other types of immune-deficiency states may induce systemic infection,
lipodystrophy, or other chronic health complications, leading to loss of
menstrual regularity. Occult malignancy with progressive weight loss and a
catabolic state may lead to loss of menstrual regularity. A careful review of
systems may help uncover such a disorder. Sickle cell disease (Erb, et
al.,2008( and thalassemia are associated with amenorrhea )Karabulut, et
al.,2010).
Type 1 and type 2 diabetes may both be associated with disordered menses
(Livshits, et al., 2009).
Epilepsy itself, as well as antiepileptic medications, are associated with
reproductive dysfunction in women. The etiology of menstrual cycle
abnormalities in epileptic females may vary and includes polycystic ovarian
syndrome (PCOS), hypothalamic amenorrhea, and hyperprolactinemia
(Bauer, et al., 2008).
Chronic kidney disease requiring hemodialysis is associated with loss of
menstrual cyclicity and vitamin D deficiency, putting patients at high risk of
bone mineral density loss(Weisinger, et al., 2000).
1.13. Hormonal studies
Hormonal studies may include assays of prolactin, FSH, LH, estradiol,
thyroid hormones, or androgens.
1.13.1. Prolactin
Prolactin levels in excess of 200 ng/mL are not observed except in the case
of prolactin-secreting pituitary adenoma (prolactinoma). In general, the
45
serum prolactin level correlates with the size of the tumor (Berne and levy,
1993).
Psychotropic drugs, hypothyroidism, stress, and meals can also raise
prolactin levels. Repeatedly elevated prolactin levels require further
evaluation if the cause is not readily apparent (Lee, et al., 2012).
1.13.2. FSH, LH and estradiol
An FSH level of approximately 40 mIU/mL is indicative of ovarian
insufficiency. However, this is assay-dependent and some patients have a
lower menopausal level of FSH. If a repeat value in 1 month confirms this
finding and amenorrhea still persists, then the diagnosis of premature
ovarian failure/primary ovarian insufficiency is confirmed. LH levels are
elevated in cases of 17, 20 lyase deficiencies, 17-hydroxylase deficiency,
and premature ovarian failure (Davis, 1996).
Serum estradiol levels undergo wide fluctuations during the normal
menstrual cycle. During the early follicular phase of the menstrual cycle,
levels may be lower than 50 pg/ mL. During the preovulatory estradiol
surge, levels in the range of 400 pg/ mL are not uncommon. In healthy
menopausal women, estradiol levels are routinely lower than 20pg/m
(Nachtigall, et al., 2012).
46
1.14. Thyroid hormone
1.14.1. Thyroid gland
The thyroid gland is one of larger endocrine gland, weighting 2-3 grams in
neonates and 18-60 grams in adults, and increased in pregnancy, with
abutterfly-shaped organ it composes of two cone-like lobes or wing s, lobus
dexter (right lobe)and lobus sinister (left lobe), connected via the isthmus
(Nawaz, 2012) . The thyroid isthmus is variable in presence and size, can
change shape and size, and can encompass a cranially extending pyramid
lobe (lobus pyramidalisor processus pyramidalis), remnant of the
thyroglossal duct. Thyroid gland is situated on anterior side of the neck,
lying against and around the larynx and trachea, reaching posteriorophly the
esophagus and carotid sheath. It starts cranially at the oblique line on the
thyroid cartilage (just below the laryngeal prominence, or Adam′s apple),
and extends inferiorly to approximately the fifth or sixth tracheal ring. It is
difficult to demarcate the gland′s upper and lower border with vertebral
levels because it moves position in relation to these during swallowing
(Aggrawal, 2011). It covered by a thin fibrous sheath, the capsula glandulae
thyroidea, composed of an internal and external layer. The external layer is
interiorly continuous with the lamina pretrachealis fasciae cervicalis and
posteriorolaterally continuous with the carotid sheath. The gland is covered
interiorly with infrahyoid muscles and laterally with the sternocleidomastoid
muscle also known as sternomastoid muscle. On the posterior side, the gland
is fixed to the cricoid and tracheal cartilage and cricopharyngeus muscle by
thickening of the fascia to form the posterior suspensory ligament of
Berry(Yalcin, et al ., 2006). The thyroid glands firm attachment to the
underlying trachea is the reason behind its movement with swallowing. The
47
thyroid is supplied with arterial blood from the superior thyroid artery, a
branch of the external carotid artery, and the inferior thyroid artery, a branch
of thyrocervical trunk, and sometimes by the thyroid ima artery, branching
directly from the subclavian artery. The blood is drained via superior thyroid
veins, draining in the internal jugular vein, and via inferior thyroid veins,
draining via the plexus thyroideus impar in the left branchiocephalic vein
(Stephen and Saffron, 2001).
1.14.2. Thyroid physiology
The primary function of the thyroid is production of the hormones
triiodothyronin (T3), thyroxin (T4), and calcitonin. Up to 80% of the T4 is
converted to T3 by peripheral organs such as the liver, kidney and spleen. T3
is several times more powerful than T4, which is largely aprohormone
(Stephen and Saffron, 2001).
1.14.3. Type of thyroid hormones
Disorders of the thyroid gland may result in menstrual irregularities;
however, for it to present as primary amenorrhea is uncommon, measure
thyrotropin and free thyroxin (T4), if symptoms of hypothyroidism or
hyperthyroidism are present.
The thyroid gland has the unique capacity of concentrating iodine. It takes
up from plasma by active Transport mechanism, and is controlled by the
thyroid stimulating hormone (TSH) secreted by the anterior pituitary
signaled by thyroid releasing hormone (TRH).
The iodine is rapidly
converted to inorganic iodine and at once taken up by the tyrosine residue of
specific thyroid protein, thymoglobulin. The iodinated tyrosine in it is
rapidly transformed into active hormones, tiiodothyronine (T3) and
48
tetraiodothyronine (T4) through the intermediate steps of monoiodotyrosine
(MIT) and diiodotyrosine (DIT) and their coupling to form thyronine residue
(Jack, 2002).
(і) Thyroid stimulating hormone (TSH): is thyrotropin family hormone such
as Luteinizing hormone (LH) and follicle stimulating hormone (FSH), Each
of them is glycoprotein consisting of two subunits (alpha and beta subunit),
The β subunit carries the TSH-specific immunological and biological
information, whereas the α-chain carries species-specific information and
has an identical amino acid sequence to the α-chains of LH, FSH and hCG
(Pierce and Parsons, 1981).
TSH has a stimulating action in all stages of thyroid hormone formation and
secretion; it also has a proliferative effect. The determination of TSH severs
as the initial test in thyroid diagnostics. Even very slight changes in the
concentrations of the free thyroid hormone bring about much greater
opposite changes in the TSH level. Accordingly, TSH is very sensitive and
specific parameter for assessing thyroid function and is particularly suitable
for early detection or exclusion of disorders in the central regulation between
the hypothalamus, pituitary and thyroid (Shvaraj, et al ., 2006).
(іі) The thyroid hormone thyroxin (T4): is physiologically part of the
regulating secretion of the thyroid gland and has an effect on general
metabolism. The major fraction of the total thyroxin is bound to transport
proteins (prealbumin, and albumin). The free thyroxin (fT4) is the
physiologically active thyroxin component.
The determination of free thyroxin is an important element in clinical routine
diagnostic. Free thyroxin (fT4) is measured together with TSH when thyroid
49
function disorders are suspected. The determination of fT4 is also suitable
for monitoring thyrosuppressive therapy (Walter and Boron, 2003).
(ііі) Triiodothyronin (T3): is one of the thyroid hormones present in serum
which regulates metabolism. Determination of this hormone concentration is
important
for
the
diagnostic
differentiation
of
hyperthyroidism,
hypothyroidism. The major fraction of total triiodothyronin (fT3) is bound to
the transport proteins (prealbumin, and albumin). Free triiodothyronin (fT 3)
is the physiologically active form of the thyroid hormone, triiodothyronin
(T3). The determination of free triiodothyronin (fT3) has advantage of being
independent of changes in the concentrations and binding properties of the
binding proteins (Walter and Boron, 2003).
50
1.15. Rationale and objectives
1.15.1. Rationale
Some authors suggest the absence of menses for 6 months constitutes
amenorrhea, but the basis for this recommendation is unclear.
For a postmenarchal girl or a reproductive-aged woman to experience a
menstrual cycle interval of more than 90 days is statistically unusual.
Practically speaking, this should be an indication for an evaluation to seek
the cause (Barbara, 2002).
Increase plasma FSH, LH, and prolactin concentration are recognized by
risk factors for primary and secondary amenorrhea. Hyperprolactinemia is
one of the most striking modifications of biological parameters occurring in
infertile women (Barbara, et al., 2002).
To the best of our knowledge there has been previous study established to
assess gonadotrophic hormones, progesterone and prolactin levels among
infertile women (Braide, et al., 2002). Therefore; the present study was
conducted mainly to assess hormonal profile among amenorrheic Sudanese
woman less than 40 years.
51
1.15.2. Objectives
General objectives
To assess hormonal profile among amenorrheic Sudanese women under 40
years
Specific objectives
1. To identify the causes of amenorrhea among Sudanese women under
40years.
2. To determine the incidence of thyroid disturbance in Sudanese women
with amenorrhea.
3. To measure serum levels of FSH, LH, TSH and prolactin among
amenorrheic Sudanese women under 40years.
4. To determine the relationship between past immunization and fertility
hormone in Sudanese women with amenorrhea.
52
CHAPTER TWO
Materials and methods
2. Materials and methods
2.1. Study duration
The study was conducted between May 2012 and June 2014.
2.2. Study design and sample size
Cross-sectional survey of 200 amenorrheic Sudanese women under 40 years
and106 healthy individuals with no history of hormonal disturbance or any
other diseases were used as control subjects.
2.3. Study setting
The study was conducted in National Ribat University Hospital, Omdurman
Maternity Hospital, Reproductive Health Care Center and Soba University
Hospital.
2.4. Study population
The study included those who had pervious history of hormonal disturbance
and normal healthy participants as control group.
2.5. Inclusion criteria
Test group: Sudanese women under40 years of age with amenorrhea.
Control group: age matched women.
2.6. Exclusion criteria
Women who had been receiving anticoagulant therapy, or with cardiac,
renal diseases, thyroid disturbance or any other chronic disease.
53
2.7. Sampling technique
Blood specimens were collected under sterile conditions into sterile plain
containers and the serum was separated into another plain container.
Samples were stored at -70 Ċ till the time of analysis.
2.8. Tools of data collection
Structural interviewing questionnaire was designed to collect and maintain
all valuable information cases and controls.
2.9. Ethical consideration
Ethical approval for the study was obtained from ethical committee of
Federal Ministry of Health and National Ribat University.
2.10. Organization
The lab work of this study was done in National Ribat University
Hospital, which is a well-organized and equipped hospital.
2.11. Excepted out comes
By the end of the study we should identify the effects of hormonal
disturbance in infertile Sudanese women that would require treatment
and more care.
2.12. Specimen collection and preparation
Grossly heamolyzed, icteric or lipaemic specimens were avoided. Blood
was collected by venipuncture, allowed to clot, and separated to serum by
stored at -70 Ċ till the time of analysis. centrifugation at room
54
temperature. Specimens were capped and stored at -70 Ċ till the time of
analysis.
2.13. Statistical analysis
Data was analyzes using Statistical Package for Social Science (SPSS)
version 20. P. value <0.05 was considered as significant.
2.14. Reagents and materials used in ELISA:
1. Microtiterwells, 12x8 strips, 96 wells:
2. Wells coated with anti-Prolactin, LH, FSH, TSH, T3 and T4.
3. Standards (6 vials).
4. Enzyme Conjugate, 1vial ready to use.
5. Substrate Solution, 1 vial, ready to use.
6. Stop Solution, 1 vial, ready to use.
7. A microplate calibrated reader.
8. Calibrated variable precision micropipettes.
9. Absorbent paper.
10.Timer.
11.Semi logarithmic graph paper or software for data reduction.
55
2.15. Assay procedure
2.15.1. General remarks
- All reagents and specimens were allowed to come to room temperature
before use. All reagents were mixed without foaming.
- Once the test had been started, all steps were completed without
interruption.
-New disposal plastic pipette tips for each standard control or sample had
been used in order to avoid cross contamination.
- Before starting the assay, capswere removed, all needed wells secured
in holder, etc. That would ensure equal elapsed time for each pipetting
step without interruption.
- As a general rule the enzymatic reaction was linearly proportional to
time and temperature.
-Pipetting of all standards, samples, and controls was completed with 6
minutes.
2.15.2. Test procedure:
-The desired number of microtiter wells was secured in the holder.
-25 µl of each standard, control and sample was dispensed with new
disposable tips into appropriate wells.
-100μl enzyme conjugate wasdispensed into each well.
-Wells were incubated for 30 minutes at room temperature.
56
-The contents of the wells were briefly shacked out, rinsed 5times with
distilled water (300µl per well) andstriked sharply on absorbent paper to
remove residual droplets.
-100µl of substrate solution was added to each well.
- Wells were incubated for 10 minutes at room temperature.
-The enzymatic reaction was stopped by adding 50µl of Stop Solution to
each well.
-The absorbance (OD) of each well was determined at 450±10 nm with
microtiter plate reader. The wells were read within 10 minutes after adding
the stop solution.
2.15.3. Calculations:
-The average absorbance values for each set of standards, controls
and patients were calculated.
-Using semi-logarithmic graph paper, a standard curve was
constructed by plotting the mean absorbance obtained from each
standard against its concentration with absorbance value on the
vertical(y) axis and concentration on the horizontal (x) axis.
-Using the mean absorbance value for each sample the corresponding
concentration from the standard curve was determined.
-The concentration of the samples was read directly from that
standard curve. Samples with concentrations higher than that of the
highest standard were further diluted or reported as >200ng/ml. For
57
the calculation of the concentrations this dilution factor had took into
account.
2.15.4 Quality control
The precision and accuracy of all method in this study were checked each
time a patch was analyzed by including commercially prepared control done
by using normal and pathological human sera for hormonal profile result ±
SD of the target value of the control sera were accepted.
2.15.5. Data analysis:
The data were analyzed using statistical package social studies (IBM SPSSversion 20). The mean, standard deviation were obtained T test was used to
compare between means; linear regression analysis was used to assess
correlation (R) between FSH. LH and prolactin with both age and duration
of amenorrhea considered to be statistically significant at (P ≤ 0.05).
58
CHAPTER THREE
Results
3. Results
This cross- sectional study was conducted in National Ribat University
Hospital, Omdurman Maternity Hospital, Reproductive Health Care Center
and Soba University Hospital. Data were analyzed using IBM SPSS version
20.
The study revealed that the mean age in the amenorrheic women was (33.5 ±
6.0 years), versus (29.2±5.9 years) in the control group, as shown below in
Table (3.1).
The prevalence of primary amenorrhea among the study group was8
(4%).The prevalence of secondaryamenorrhea among the study group was
192 (96%) as shown in the Table (3.2). The mean age at menarche in the
secondary amenorrheic women was (13.8±3.2 years), classified as;menarche
at15 years in 83 women (41.5 %), menarche at 14 years in 47 women (23.5
%), menarche at 13 years in 27 women (13.5 %), menarche at 12 years in10
women (5 %), menarche at 16 years in 16women (8%), menarche at 17 years
in 4 women (2%), menarche at 9 years in 3women (1.5 %), menarche at18
years in 1 woman (0.5 %) and menarche at 25 years in 1 woman (0.5 %),
Table (3.3); while the mean menarche age in the control group was (14.2±1
year); Table (3.4) & Table (3.5).
Among the amenorrheic women 74 (37%) were non vaccinated and
126(63%) were vaccinated, of them 80 (40%) were vaccinated against
meningitis,39 (19.5%), against tetanus , 2 (1%) against hepatitis, 3 (1.5%)
against tetanus & meningitis 1 (0.5%) against tetanus & hepatitis and 1
(0.5%) against meningitis & hepatitis, Table (3.6) ; but the vaccination in the
control group was revealed as 40(37.7%) vaccinated against meningitis, 43
59
(40.6%) vaccinated against tetanus, 3(2.8%) vaccinated against hepatitis, 14
(13.2%) vaccinated against tetanus &meningitis, 6 (5.7%) vaccinated
against tetanus & hepatitis and 0 (0.0%) vaccinated against meningitis and
hepatitis; Table (3.7)& Table (3.8).
The study also showed that mean of the prolactin in the amenorrheic women
was (121.5± 265 ng/ml) versus (15.0±6.0 ng/ml) in the control group; with
P. value (0.000). The mean of the FSH in the amenorrheic women was (50.3
±34.0 IU/ml), versus (8.7 ± 31.3 IU/ml) in the control group; with P.value
(0.000). The mean of LHin the amenorrheic women was (34.89±22.7
IU/ml), versus (6.27± 2.55 IU/ml) in the control womenwith P.value (0.000).
The mean of TSH in the amenorrheic women was (1.7±2.0 IU/ml) versus
(1.2±0.7 IU/ml) in the control group with P.value (0.004), Table (3.9).
60
Table (3.1): Descriptive study of mean age of amenorrheic Sudanese
women and their control.
Age (years)
N
Mean (years)
STD
Amenorrheic women
200
33.5
6.0
Controls
106
29.2
5.9
61
Table (3.2): descriptive study of type of amenorrhea among
amenorrheic Sudanese women.
Type of amenorrhea
Frequency
Percent (%)
Primary amenorrhea
8
4
Secondary amenorrhea
192
96
Total
200
100 %
62
Table (3-3): descriptive study of experience of cycle (menarche) among
amenorrheic Sudanese women under study.
Year of menarche
Frequency
Percent (%)
9
3
1.5
10
1
0.5
12
10
5.0
13
27
13.5
14
47
23.5
15
83
41.5
16
16
8.0
17
4
2.0
18
1
0.5
25
1
0.5
Total
192+8=200
96+4=100
63
Table (3.4): Descriptive study of experience of cycle (menarche) among
control group.
Year of menarche
Frequency
Percent (%)
10
2
1.9
12
2
1.9
13
16
15.2
14
38
35.8
15
38
35.8
16
10
9.4
Total
106
100
64
Table (3.5): Comparison of mean of menarche in the test group and
their control.
Menarche (years)
N
Mean (years)
STD
Amenorrheic women
200
13.82
3.2
Controls
106
14.2
1.09
65
Table (3.6): descriptive study of vaccination among amenorrheic
Sudanese women.
subject
Frequency
Percent
Vaccinated women
126
63%
Non vaccinated women
74
37%
Total
200
100%
66
Table (3.7): Descriptive study of the type of vaccination among
amenorrheic Sudanese women.
Type
Frequency
Percent
Meningitis
80
40%
Tetanus
39
19.5%
Hepatitis
2
1%
Tetanus /Meningitis
3
1.5%
Tetanus /Hepatitis
1
0.5%
Meningitis /Hepatitis
1
0.5%
200
100%
Total
67
Table (3.8): Descriptive study of the type of vaccination among control
group.
Type
Frequency
Percent
Meningitis
40
37.7%
Tetanus
43
40.6%
Hepatitis
3
2.8%
Tetanus /meningitis
14
13.2%
Tetanus /hepatitis
6
5.7%
Meningitis /hepatitis
0
0%
106
100%
Total
68
Table (3.9): Comparison study of hormonal profile among
amenorrheic women and their controls.
Parameters
Prolactin
FSH
LH
TSH
N
Mean
Std. Deviation
P.value
patients
200
121.518
265.1284
0.000
controls
106
15.093
6.0739
patients
controls
patients
controls
patients
200
106
200
106
200
50.339
8.719
34.891
6.271
1.749
34.0496
31.2937
22.7162
2.5500
2.0265
Controls
106
1.273
.7695
69
0.000
0.000
0.004
CHAPTER FOUR
Discussion, Conclusion, recommendations
4.1. Discussion
Failure to ovulate normally is responsible for approximately 40% of
infertility in women as reported by Speroff et al (1994). In this study 96% of
infertile Sudanese women with early amenorrhea have secondary
amenorrhea that raises many questions about the etiology of this condition,
which probably associated with hormonal disturbance.This study shows
significant increase of the gonadotrophins -FSH &LH-in the infertile
amenorrheic Sudanese women, this finding is similar to that reported by
Adegokeet al(2007).The increases of LH and FSH in primary infertile and
secondary infertile subjects also recorded by Adegokeet al (2007).The
disturbance
of
these
gonadotropins
affects
the
function
of
the
luteinizinghormone in female, which causes release of the ovum from the
ovarian follicle whichmatures under the influence of follicle stimulating
hormone and induces the formation of the corpus luteum from the ruptured
follicles. The corpus luteum then secretes progesterone and estradiol under
the influence of pulsatile LH as reported by Tietz(2001). Thevariability in
LH/FSH ratio is another important cause of ovarian dysfunction and
infertility as written byCusson et al(2005). FSH levels are higher in
resistance ovarian syndrome, which occurs very rarely in younger
menstruating infertile women. It is only distinguished from primary ovarian
follicle by intermittent resolution of the ovaries and normalization of FSH
levels as reported by Balen and Michelmore(2002).Hypersecretion of LH is
associated with menstrual disturbances and infertility; that results in reduces
conception rates and increases rate of miscarriages in both natural and
assisted conception as reported by Balen et al (1993).
70
The results of this study also reveal significantly high TSH in infertile
women compare with their controls. This is known as hypothyroidism, the
presence of high levels of TSH lead to low levels of thyroid hormone and
interfere the ovulation, which impairs fertility. Hypothyroidism can affect
fertility due to anovulatory cycle, luteal phase defect, hyperprolactinemia
and sex hormone imbalance as reported by Poppeet al (2008).
The rate of depletion of the ovarian follicle store hastens at around the age of
37 years as reported by Yu and Yap (2003), but in this study the mean age of
the study group is only (33.5 ± 6.0 years); which raises many questions
again. Hyperprolactinemia as a common endocrine disorder with an
incidence of 9–17%; in this study there is a significant increase in mean of
serum prolactin in the infertile amenorrheic group compared to their control.
In humans, hyperprolactinemia is associated with a marked reduction in the
frequency and amplitude of LH pulses as reported by Bohnet et al (1976)
and Matsuzaki et al (1994), indirectly suggesting that both the brain and
pituitary might be targets for prolactin. The increase observed in prolactin
may be the cause of high TSH concentration in the infertile subjects as
written by Bayrak et al (2005).
Hormone imbalances may be due to exposure to some toxic chemicals that
can interfere with normal implantation of the fertilized egg and failure to
maintain pregnancy, or even chronic anovulation or amenorrhea,as reported
by Hul (1984) and Cramer and Wise (2000).
71
4.2. Conclusion
The age at menarche in secondary amenorrheic Sudanese women
significantly decreases, 40% of vaccinated amenorrheic women immunize
against meningitis. Serum FSH, LH, prolactin and TSH significantly
increase in the amenorrheic Sudanese women.
In Sudanese women with infertility associated with amenorrhea; fertility
hormones beside TSH should be measured, but the real cause of the early
cessation of the cycle in the reproductive age should be widely searched.
72
4.3. Recommendation
This study recommends that:
1. Fertility hormones including luteinizing hormone (LH), follicle
stimulating hormone (FSH), prolactin and thyroid stimulating hormone
should be used as diagnostic tools in women under fertility diagnosis.
2. Young women with ovarian insufficiency that is unresponsive to therapy
require hormone replacement to maintain bone density.
3. Further studies are required including more sample size, for assessing the
causes of early amenorrhea among infertile Sudanese women.
4. More research is recommended to specify the role of vaccination on
fertility and early amenorrheic in Sudanese women.
73
CHAPTER FIVE
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APPENDEIXES
Questionnaire
Name:………………………………………………………………………..
Age ………………………………….
Address……………………………………………………………………….
Occupation…………………………………………………………………….
Tribe……………………………………………………………………………
Phone No…………………………………………………………………………...
Marital status: - single
Married
divorced
widow
Number of pregnancies………
Children……..
Abortion…..
Thyroid disturbance: - Yes
Experience of cycle
yes………
Cycle:
irregular
regular
No
when……….
No…………….
frequency …………….
Duration of cycle…… days
Vaccination after cycle experience: - No
Yes
Period of amenorrhea…………years…….
months…….
Expected cause of amenorrhea: surgery
contraceptive
others……………………………
74
type of vaccine
Agreement consent
I ……………………………………………………………………………..
After the explanation of the study nature and I had enough time for
assurance and all my questions were sufficiency answered, I knew that I
have the right to stop this study at any time without losing the right of
medical care or any other rights.
I voluntary accept to participate in this study
Signature………………………………………
Date……………………………………………
75
‫بسن هللا الشحوي الشح٘ن‬
‫إقرار موافقه‬
‫اًب ‪..........................................................................‬‬
‫بؼذ اى حن ششح الذساسَ ّأػط٘ج فشطَ كبفَ٘ لالسخفسبس ّلذ حوج االجببَ ػلٔ كل اسئلخٔ بظْسٍ‬
‫ّاػحَ فِوج اى لٔ الحك فٔ الخْلف ػي ُزٍ الذساسَ فٔ آ ّلج هي غ٘ش اى افمذ الحك فٔ‬
‫الشػبَٗ الطبَ٘ اّ آ حمْق اخشٓ‪.‬‬
‫أّافك ؽْػ٘ب ببلوشبسكَ فٔ ُزٍ الذساسَ‬
‫الخْل٘غ‪.............................................................:‬‬
‫الخبسٗخ‪.............................................................:‬‬
‫‪76‬‬