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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 References References 1. Acosta A and Wringht G L (1983). Luteinizing hormone (LH) enzyme immunoassay. 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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