1
University of Oslo, Norway The Faculty of Medicine Oslo, Autumn 2020
Diagnosing polycystic ovary syndrome (PCOS) and functional hypothalamic amenorrhea (FHA)
– a comparison
Emilie Eriksen Tansø and Marte Lenes Lindgren Class V17
Supervisor Péter Fedorcsák
UNIVERSITY OF OSLO, FACULTY OF MEDICINE
2
Abstract
The purpose of this thesis has been to review the pathophysiology and diagnostic criteria of polycystic ovary syndrome (PCOS) and functional hypothalamic amenorrhea (FHA), aiming to clarify what separates the two and if and how they overlap. We conducted a focused review of existing literature on PCOS and FHA in an effort to clarify the difference between the diagnoses.
Infertility affects one in seven couples, and 25 percent of infertility is caused by ovulation disorders. PCOS and FHA are common ovulation disorders that both can give absent or irregular menses and anovulatory infertility. The PCOS diagnosis is made based on the Rotterdam criteria and is defined as the presence of two or more of the following:
hyperandrogenism, ovulatory dysfunction, and polycystic ovarian morphologic features (PCOM), after ruling out other possible diagnoses. FHA describes amenorrhea with low to normal gonadotropins and hypoestrogenism, most often caused by low energy availability, excessive exercise or stress, or a combination thereof. The term “functional” implies that removal of stressors will restore ovarian function. It is a diagnosis of exclusion given after ruling out other reasons for amenorrhea and anovulation.
One of the main areas of confusion is the appearance of PCOM in FHA women, and it has been observed in as high as 30-50% of patients with FHA. Indeed, some of the women with FHA have multiple features of PCOS, including increased serum androgens when given gonadotropin stimulation, increased ovarian size and PCO-like ovarian morphology, as well as increased AMH levels. The overlap between PCOS phenotypes with FHA may present a confusing clinical picture and have raised questions as to whether FHA and PCOS can coexist or if PCOM in FHA just mimic PCOS.
Both PCOS and FHA are diagnoses of exclusion, but challenging these criteria is a new understanding that PCOS and FHA may coexist in some women and are not mutually exclusive. Concerns have also been raised about the Rotterdam criteria being too broad, leading to misdiagnosis of PCOS in women with FHA, if hypothalamic origin of amenorrhea is not recognized. It has been proposed that the threshold for antral follicle count (AFC) should be increased for better distinction of hypothalamic amenorrhea and PCOS. Several
3 researchers have emphasized that PCOM may commonly occur in FHA as well as in
asymptomatic patients and does not automatically mean that the patient has PCOS. Further research is needed on the topic as existing evidence is too scarce to draw firm conclusions.
The diagnosis will have impact on treatment strategies as the two disorders differ significantly in terms of treatment and pharmaceutical options. It is important to obtain comprehensive anamnesis, appropriate blood tests and a gynecological examination, including ultrasound. As both disorders are diagnoses of exclusion, this will also be helpful in excluding other
diagnoses.
4
Table of contents
1. Introduction ___________________________________________________________ 7 1.1. The ovaries and ovarian follicle pool ___________________________________ 8 1.2. Initial recruitment _________________________________________________ 10 1.3. Transition from primary stage to early antral stage _____________________ 16 1.4. Cyclic recruitment - Early antral to preovulatory transition ______________ 18 1.5. Menstrual cycle physiology and the hypothalamic-pituitary ovarian axis ___ 22 1.5.1. The uterus and menstruation ________________________________________ 24 2. Infertility and evaluation of ovulatory dysfunction ___________________________ 26 2.1. Evaluation of ovulatory dysfunction __________________________________ 27 2.2. Classification of amenorrhea ________________________________________ 28 3. Polycystic ovary syndrome (PCOS) ________________________________________ 30 3.1. Epidemiology _____________________________________________________ 30 3.2. Etiology __________________________________________________________ 32 3.3. Pathophysiology ___________________________________________________ 33 3.3.1. Theca cell dysfunction and PCOS ovarian steroid synthesis _______________ 33 3.3.2. Altered GnRH pulses and other extraovarian factors _____________________ 35 3.3.3. Granulosa cell dysfunction _________________________________________ 36 3.3.4. Hyperandrogenism and its effect on the ovaries _________________________ 37 3.4. Diagnostic evaluation ______________________________________________ 39 3.4.1. Hyperandrogenism _______________________________________________ 41 3.4.2. Ovulatory dysfunction _____________________________________________ 43 3.4.3. Polycystic ovarian morphologic features ______________________________ 44 3.5. Treatment ________________________________________________________ 45 3.5.1. Lifestyle modifications ____________________________________________ 45 3.5.2. Suppression of ovarian androgen secretion – combined oral contraceptives ___ 46 3.5.3. Metformin ______________________________________________________ 47 3.5.4. Antiandrogens - flutamide, finasteride, spironolactone ___________________ 48 3.5.5. Assessment and treatment of infertility ________________________________ 48 3.5.6. Aromatase inhibitors – Letrozole ____________________________________ 50 3.5.7. Clomiphene citrate _______________________________________________ 51 3.5.8. Gonadotropins ___________________________________________________ 51 3.5.9. Laparoscopic ovarian surgery _______________________________________ 51 3.5.10. In vitro fertilization _____________________________________________ 52 4. Functional hypothalamic amenorrhea (FHA) _______________________________ 53 4.1. Epidemiology _____________________________________________________ 53 4.2. Etiology __________________________________________________________ 54 4.3. Pathophysiology ___________________________________________________ 54 4.3.1. Functional reduction in GnRH drive __________________________________ 55 4.3.2. Neuroendocrine alterations _________________________________________ 56
5 4.3.3. Energy intake and expenditure mismatch ______________________________ 57 4.3.4. The effects of FHA on bone health ___________________________________ 58 4.3.5. The effects of prolonged hypoestrogenism on the cardiovascular system _____ 59 4.4. Diagnostic evaluation of FHA _______________________________________ 60 4.4.1. When should FHA be suspected? ____________________________________ 61 4.4.2. Anamnesis ______________________________________________________ 63 4.4.3. Clinical examination ______________________________________________ 64 4.4.4. Laboratory testing ________________________________________________ 65 4.4.5. Imaging ________________________________________________________ 66 4.5. Treatment ________________________________________________________ 66 4.5.1. Behavioral modification ___________________________________________ 67 4.5.2. Pharmacological treatment of FHA in women without current child-wish ____ 67 4.5.3. Pharmacological treatment of infertility _______________________________ 69 5. Discussion - The relationship between FHA and PCOS – Can they coexist? _______ 71 5.1. The Rotterdam criteria and PCOM __________________________________ 71 5.2. Can FHA and PCOS coexist? ________________________________________ 73 6. Concluding remarks ____________________________________________________ 77 7. References ____________________________________________________________ 79
List of Illustrations
Tables
Table 1: Intrinsic ovarian factors involved in preantral and antral follicle development. Obtained from Hannon and Curry, 2018 (13). ______________________________________________________________________ 17 Table 2 Classification of Amenorrhea according to WHO, 1970. Table remade after Castillón et al., 2018 (98).
________________________________________________________________________________________ 27 Table 3: Etiologies for amenorrhea, data obtained from (106, 107). __________________________________ 29 Table 4: Etiologies of amenorrhea, adapted from Gordon et al., 2017 (9). _____________________________ 60 Table 5: Laboratory patterns for common causes of anovulation, borrowed from Gordon et al., 2017 (9). ____ 65 Table 6: Results comparing FHA to FHA+PCOS adapted from Sum and Warren, 2009 (406). _____________ 74 Table 7: Comparison of PCOS and FHA. Based on information from previous chapters on PCOS and FHA. __ 77
Figures
Figure 1: Cross-section of an ovary, illustrating the different development stages of the follicle and its faith after ovulation. Redrawn after Jones, 1991 (11). _______________________________________________________ 8 Figure 2: The PI3K-signaling pathway in oocytes regulates initial recruitment and activation of primordial follicles. Binding of the growth factor ligand KIT to receptor tyrosine kinases recruits PI3K to the inner membrane of the oocyte. This stimulates the conversion of PIP2 to PIP3, resulting in increased PIP3 levels and Akt phosphorylation and thus activation. Activated Akt can further migrate to the cell nucleus, where it
suppresses FOXO3, which in turns promote primordial follicle growth. PTEN converts PIP3 back to PIP2, inhibiting the Akt-pathway. In the illustration green text color indicates stimulators of initial recruitment, while red text color indicates inhibitors of initial recruitment (13, 27). Phosphorylated Akt can also stimulate protein translation, cell growth and primordial follicle recruitment by activation mammalian target of rapamycin pathway (mTOR-pathway). Akt phosphorylates TSC1 and TSC2 (44, 45), which destabilizes the proteins and removes the inhibitory effect on the mTORC1 cascade. The result is protein translation and primordial follicle recruitment (13, 27). Redrawn after figures in Hannon and Curry, 2018 (13) and Hsueh et al., 2015 (27). ____ 13 Figure 3: Mechanical signals (i.e. fragmentation, incision, drilling, wedge resection) disrupts ovarian Hippo signaling and promotes follicle growth. The mechanical disruption leads to actin polymerization resulting in
6
nuclear translocation of Yes-associated protein (YAP), that interacts with TEAD and increases the expression of several downstream biochemical signals including CCN growth factors and apoptosis inhibitors. The result is follicle growth. Redrawn after Hsueh and Kawamura, 2020 (56). ____________________________________ 14 Figure 4: Selected primordial follicles develop to primary follicles under Akt and mTOR signaling, the initial recruitment. The primary follicles develop into secondary follicles, before acquiring an antrum reaching the early antral stage. Most early antral follicles undergo atresia, selected antral follicles supported by cyclic changes in FSH and LH reach preovulatory stage, releasing mature oocytes (cyclic recruitment). Redrawn after McGee and Hsueh, 2000 (10). ________________________________________________________________ 18 Figure 5: Theca and granulosa cell interactions. Redrawn after Hannon and Curry, 2018 (13). ____________ 20 Figure 6: The hypothalamus-pituitary-ovarian axis, redrawn after Padamanabhan et al., 2018 (82). _______ 23 Figure 7: Overview of the ovarian and menstrual cycle. Figure from Tortora and Derrickson, 2016 (92). ____ 25 Figure 8: Causes of infertility, data obtained from Hull et al., 1985 (97) and Kuohung and Hornstein, 2020 (96).
________________________________________________________________________________________ 26 Figure 9: The relationship between anovulation and amenorrhea. ___________________________________ 28 Figure 10: Normal ovarian steroid synthesis vs PCOS ovarian steroid synthesis. Adapted and redrawn after Rosenfield and Ehrmann, 2016 (116). __________________________________________________________ 34 Figure 11: The role of AMH in follculogenesis. Redrawn after Azziz et al., 2016 (109). ___________________ 36 Figure 12: The effect of androgens on folliculogenesis. The proposed hypothesis on androgens and its effect on primordial follicle recruitment includes androgen-induced expression of KIT ligand and androgen receptor- induced PI3K/Akt-pathway. As previously described androgen effect on preantral follicles include increased follicle growth by promoting hyperplasia of granulosa and theca-cell, as well as contributing to follicular survival by inhibiting pro-apoptotic proteins, thus preventing atresia. Androgens increase the expression FSH receptor in follicles, thus increases follicular sensitivity towards FSH, promoting follicle growth (167, 169-171).
Androgens also stimulate the expression of aromatase, a key steroidogenic enzyme (172). Synergistic effects exist between androgens and FSH. FSH alone promotes follicular growth, but additional androgens significantly increase follicular diameter (171). The synergistic effects of androgens on FSH actions may be explained by increased intracellular cAMP due to increased androgens without the exact mechanisms being known (167).
cAMP is a major intracellular mediator of FSHR-signaling, and increased cAMP levels will potentiate follicular cell proliferation and differentiation (173, 174). These effects together promote preantral follicle growth. In periovulatory follicles, androgens have been postulated to exert genomic actions influencing the ovulatory process (167). Redrawn after Prizant et al., 2014 (167). ___________________________________________ 38 Figure 13 PCOS phenotypes: A workshop by the National Institute of Health (NIH) in 2012 proposed the specification of PCOS phenotypes. The phenotypes are listed in decreasing order of diagnostic specificity.
Redrawn after Azziz et al., 2016 (188). _________________________________________________________ 40 Figure 14: The Ferriman-Gallwey scoring system. Original drawing by Rosenfield, 1986 (193). ___________ 42 Figure 15: Classification of PCOS by the Rotterdam Criteria gives rise to four phenotypes. FHA is marked as number five in red. The figure illustrates that women with FHA and PCOM meet the criteria for PCOS
phenotype 4/D. ____________________________________________________________________________ 72
«Jeg bekrefter at Emilie Eriksen Tansø og Marte Lenes Lindgren skrev oppgaven sammen med lik innsats fra begge.
Med hilsen, Peter Fedorcsak»
7
1. Introduction
Natural human reproduction starts when the spermatozoa travel through the cervix and uterine cavity to the fallopian tubes after being ejaculated into the vagina. If ovulation takes place, and the timing is right, the spermatozoa meet the ovum for fertilization. The fertilized egg, the embryo, then travels down the fallopian tubes to the uterine cavity where implantation
happens (1).
The process of reproduction is reliant upon the sperm being viable and transported up the reproductive tract and a successful ovulation. This is in turn influenced by endocrine factors, timing, frequency of sexual intercourse and health status (2). Deficiencies in one of the above factors can lead to infertility, defined as the inability of a couple to achieve pregnancy after 12 months of frequent unprotected intercourse (3).
Infertility affects one in seven couples, and 25 percent is caused by ovulation disorders (2, 4).
The World Health Organization (WHO) classifies ovulation disorders into several groups, and in this thesis we focus on group I and II disorders of ovulation, functional hypothalamic amenorrhea (FHA) and polycystic ovary syndrome (PCOS), respectively (2).
PCOS is the most common endocrine disorder in women (5) and accounts for the majority of anovulatory infertility (6, 7) and entails increased risk of adverse events under assisted reproduction treatment. The major clinical manifestations of PCOS include ovulatory dysfunction, hirsutism, polycystic ovaries and frequently infertility (6-8). FHA is another frequent cause of infertility. The term describes amenorrhea with low to normal
gonadotropins and hypoestrogenism, most often caused by low energy availability, excessive exercise or stress, or a combination thereof. The term “functional” implies that removal of stressors and causal behavioral factors will restore ovarian function. It is a diagnosis of exclusion given after ruling out other reasons for amenorrhea and anovulation (9).
The aim of this thesis is to review the pathophysiology and diagnostic criteria of PCOS and FHA, aiming to clarify what separates the two and if and how they overlap. Due to COVID- 19, we had to make some changes to our original plan which was to review how PCOS and FHA were diagnosed at Oslo University Hospital, and the possible consequences of an erroneous diagnosis when receiving fertility treatment. Hospital and university restrictions
8 impeded us to conduct the clinical study at this point in time, but hopefully this thesis, which is a focused review of existing literature on PCOS and FHA, will be a solid backbone for further work when time and the corona situation allow it.
We would like to say a special thank you to Péter Fedorcsák. He has been more than we could expect from a supervisor and has contributed with valuable advices, discussions and
encouragements throughout the process.
1.1. The ovaries and ovarian follicle pool
The major function of the ovaries, the female gonads, is generation and release of the oocyte possibly leading to fertilization and formation of an embryo. The ovaries also function as an endocrine organ producing hormones that allow for the development of female secondary sexual characteristics as well as supporting pregnancy and reproductive functions (10).
Figure 1: Cross-section of an ovary, illustrating the different development stages of the follicle and its faith after ovulation.
Redrawn after Jones, 1991 (11).
The ovaries are located on each side of the upper pelvic cavity. They consist of an inner medulla rich in vascular supply and an outer cortex housing the ovarian follicles. In the
cortex, numerous follicles at different stages of development are embedded in the stroma (12).
9 The basic functional units of the ovaries are the follicles. The follicles start to develop during fetal life when primordial follicles are formed. Primordial follicles are the most immature stage of a follicle´s development, and consists of primary oocytes, that is, female gametes, surrounded by a layer of single flat squamous pre-granulosa cells. Maturation of the oocytes, the oogenesis, is closely related to the folliculogenesis because factors produced by the oocytes are highly affecting the development of the surrounding granulosa and theca cells (12).
Primordial follicles are the only source of oocytes during a woman´s reproductive life, meaning that the total number of ovarian follicles is determined early in life and depletion of this pool leads to menopause and reproductive senescence. The newborn ovary contains one million follicles. At the time of puberty, an average of 200 000 follicles are remaining in the ovary, most of them maintained in a dormant state (10). The established follicle pool will diminish throughout life and the term “ovarian reserve” is related to the number of oocytes remaining in the ovaries (2).
Once the primordial follicle pool is established, the follicle is destined to one of four fates: 1) to remain dormant for a varying period of time as part of the ovarian reserve, 2) to directly undergo atresia, 3) to undergo initial recruitment and eventually reaching ovulation and 4) to undergo initial recruitment but then to later die through atresia (13). The fate of each follicle is controlled by endocrine and even more importantly diverse paracrine factors (14-16).
When initiated to grow, the activated primordial follicles develop into primary follicles, secondary and eventually antral follicles, gradually reducing the original follicle pool (10).
Most early antral follicles undergo atretic degeneration, but a few, under the cyclic
stimulation of gonadotropins that occur after puberty, will reach the preovulatory stage (17, 18). Out of the follicle pool, over 99% are lost via atresia, and under 1% ovulate, that is an average of 300 ovulations during the reproductive lifespan (13).
The final step of the ovarian ageing process is menopause. As the follicle pool diminishes with increasing age, it will dictate the onset of cycle irregularity and final cessation of menses. The remaining number of oocytes in the ovaries is closely related to a woman´s fertility and influences the chances of a successful pregnancy. Not only does the follicle count diminish with ageing, but the quality of the oocytes also falls, contributing to the progressive
10 decline in fertility (19). In addition to a declining ovarian reserve, there is also clear evidence of a lower rate of embryo implantation and increased chance of pregnancy loss with
increasing age (2).
1.2. Initial recruitment
The first stage of follicle development is called initial recruitment. During this stage the dormant primordial follicles are recruited into the primary follicle stage, entering the growing follicle pool in a continuous manner. The initial recruitment is a rate limiting step and only a few of the follicles will develop into a primary follicle, and as such the default fate for most of the primordial follicles is to remain dormant and immature (10). The recruitment of primordial follicles is highly regulated, ensuring a sustainable balance between growing and dormant follicle pools. The goal is to provide a mature and healthy oocyte for ovulation each month, while preventing exhaustion of the ovarian reserve (20).
During initial recruitment, the oocyte increases in size and acquires a zona pellucida, a thick membrane surrounding the oocyte that is required for fertilization (13). Reactivation of the oocyte genome causes the oocyte to secrete growth factors affecting the growth of the follicle.
The morphology of the granulosa cells changes from a squamous to cuboidal shape (12).
The initial recruitment is completed when the primordial follicle is transformed to a primary follicle containing a larger oocyte, zona pellucida, and a single cuboidal layer of granulosa cells. The process is estimated to take approximately 150 days in humans and is the longest part of the folliculogenesis (13).
Since the primordial follicles have not yet developed functional gonadotropin receptors (21- 24), initial recruitment is gonadotropin independent and relies on a balance between inhibiting and promoting intraovarian factors (13). Support for this includes an FSH receptor knock-out mouse model providing evidence that follicular development proceeded until arrest in the preantral stage (25). The primordial follicle recruitment is complex, involving several cell types and signaling molecules that converge on multiple signaling pathways. It is likely that the intricate coordination between these stimulatory and inhibitory substances determine the primordial follicles fate (13).
11 Under basal conditions, dormant primordial follicles are under constant inhibitory influences (13, 26). One of the signaling pathways involved in the control of the initial recruitment, is the phosphatidylinositol 3-Kinase (PI3K)/ Protein kinase B (Akt)-signaling pathway
(PI3K/Akt-pathway). Its critical role in the regulation of initial recruitment is well established (27), as it controls cell proliferation, survival, migration, metabolism and the initial
recruitment. Several proteins are involved in regulating the PI3K/Akt-signaling pathway.
Proteins promoting the signaling pathway includes mammalian target of rapamycin complex 1 (mTORC1), protein kinase B (Akt) and ribosomal protein s6 (rpS6), while inhibitory proteins includes forkhead box O3 (FOXO3), phosphatase and tensin homolog (PTEN), tuberous sclerosis 1 and 2 (TSC1 and TSC2) (13).
Studies have shown that a basal level of PI3K/Akt-signaling is needed for oocytes to survive.
If the balance between inhibitory and promoting factors is shifted towards factors inhibiting the PI3K/Akt-pathway, the oocytes will remain quiescence, while a shift towards PI3K promoting factors will induce follicle recruitment. As such, primordial follicles can be stimulated to undergo initial recruitment with the treatment of a PTEN inhibitor and PI3K stimulating phosphopeptide, a treatment regimen currently under investigation as a potential therapeutic option in vitro (13). Studies has shown that incubation of human ovarian tissue together with PTEN inhibitor or PI3K stimulating phosphopeptide generates a massive primordial follicle growth activation (28-32).
While intracellular PI3K signaling utilizes factors restricted to the oocyte, there are also extracellular factors influencing the PI3K/Akt-pathway. The mast/stem cell growth factor receptor KIT, a receptor tyrosine kinase, is thought to stimulate PI3K/Akt-signaling in the ovaries if activated by the binding of KIT ligand. The oocyte and the precursor theca cells express KIT, while the granulosa cells produce KIT ligand. As such, KIT-KIT ligand signaling involves multidirectional coordination (13). The binding of KIT ligand to KIT receptor has been shown to promote follicle development at the primary follicle stage.
Binding of KIT ligand on precursor theca cells has been shown to induce transition into theca cells, while binding of KIT ligand on KIT receptor on the primordial follicle oocyte initiate oocyte growth and development (13).
In addition to KIT-ligand, other stimulating factors has shown to increase initial recruitment
12 such as basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), keratinocyte growth factor (KGF), bone morphogenetic proteins 4 and 7 (BMP4 and BMP7), and platelet- derived growth factor (PDGF). Studies has shown that blocking of the stimulating factors bFGF and LIF causes a reduction in the activation of primordial follicles (13).
Another regulator known for its importance in primordial follicle activation as well as
regulation of cell growth, proliferation and differentiation is mTOR signaling (33). mTOR is a serine/threonine kinase, that forms two different kinds of complexes: the rapamycin sensitive mTOR complex 1 (mTORC1) and the rapamycin insensitive mTOR complex 2 (mTORC2) (34-36). mTOR plays a vital role in several processes needed for cell division and growth.
Under influences such as nutritional factors, stress, oxygen, energy and other signals mTOR will promote anabolic processes such as protein synthesis, lipid and nucleotide biogenesis and limiting autophagy (37-39).
In the oocyte, mTOR signaling is an important mechanism of primordial follicle activation and conversely suppression of mTORC1 activity has been shown to be a prerequisite for maintaining primordial follicles in dormancy. TSC1 and TSC2 are negative regulators of mTORC1 and studies using mutant mice indicated that oocyte-specific deletions of TSC1 and TSC2 promotes growth of all primordial follicles and following exhaustion of the entire follicle pool (33, 40). As previously mentioned, oocyte-specific deletion of the PTEN gene, increases Akt activity and activation of primordial follicles (41). It has been found that simultaneously deletions of TSC1 and PTEN leads to synergistic effects in terms of oocyte growth and follicle activations, indicating that increased Akt signaling and mTORC1 activation have synergistic effects (40).
While FOXO3 suppression is the main effect of increased Akt signaling, mTORC1 is also downstream of Akt and part of the PI3K/Akt/mTOR-pathway. As illustrated in figure 2, Akt can through phosphorylation destabilize TSC1/2 and remove the inhibitory effect on
mTORC1 (42, 43). This promotes protein translation and overactivation of primordial follicles (33, 40).
13
Figure 2: The PI3K-signaling pathway in oocytes regulates initial recruitment and activation of primordial follicles. Binding of the growth factor ligand KIT to receptor tyrosine kinases recruits PI3K to the inner membrane of the oocyte. This stimulates the conversion of PIP2 to PIP3, resulting in increased PIP3 levels and Akt phosphorylation and thus activation.
Activated Akt can further migrate to the cell nucleus, where it suppresses FOXO3, which in turns promote primordial follicle growth. PTEN converts PIP3 back to PIP2, inhibiting the Akt-pathway. In the illustration green text color indicates
stimulators of initial recruitment, while red text color indicates inhibitors of initial recruitment (13, 27). Phosphorylated Akt can also stimulate protein translation, cell growth and primordial follicle recruitment by activation mammalian target of rapamycin pathway (mTOR-pathway). Akt phosphorylates TSC1 and TSC2 (44, 45), which destabilizes the proteins and removes the inhibitory effect on the mTORC1 cascade. The result is protein translation and primordial follicle recruitment (13, 27). Redrawn after figures in Hannon and Curry, 2018 (13) and Hsueh et al., 2015 (27).
Recent studies have suggested that Hippo signaling also plays a role in follicle activation under non-physiological conditions (28). The Hippo signaling pathway is a major suppressor of tissue overgrowth and essential in maintaining optimal organ size (46-49).
The Hippo signaling pathway involves several negative growth regulators in a kinase cascade that inactivates key Hippo signaling effectors, YAP (yes-associated protein) and TAZ
(transcriptional coactivator with PDZ-binding motif) (28, 50). The kinase cascade is
mechanoresponsive, and the inactivation of YAP and TAZ maintain the basal state and inhibit organ growth (28, 51). The Hippo signaling pathway is thought to sense internal mechanical stress, and mechanical signaling maintain the quiescent state of primordial follicles and inhibit follicle activation (52, 53). In order for transcription of growth factors to occur, nuclear localization of YAP is necessary (54). Disruption of Hippo signaling leads to the
14 translocation of non-phosphorylated YAP into the nucleus where it interacts with TEAD proteins (Transcription factors containing the TEA/ATTS DNA binding domain). This interaction increases the expression of several downstream factors, and among them CCN proteins that stimulate cell growth, proliferation and survival (55).
Figure 3: Mechanical signals (i.e. fragmentation, incision, drilling, wedge resection) disrupts ovarian Hippo signaling and promotes follicle growth. The mechanical disruption leads to actin polymerization resulting in nuclear translocation of Yes- associated protein (YAP), that interacts with TEAD and increases the expression of several downstream biochemical signals including CCN growth factors and apoptosis inhibitors. The result is follicle growth. Redrawn after Hsueh and Kawamura, 2020 (56).
The ovaries express key Hippo signaling genes in follicles at different stages. Follicle growth has been demonstrated using a murine model, following ovarian fragmentation and allo- transplantation disrupting the Hippo pathway (28). Findings by Kawamura et al. (28) have demonstrated that mechanical signals induced by ovarian fragmentation could increase
biochemical signals leading to early follicle growth. Recent studies on human ovarian cortical fragments have confirmed that activators of PI3K/Akt-signaling acts in synergy with
disruption of the Hippo signaling pathway (57). The exact mechanisms of the link between mechanical signaling and the PI3K/Akt-pathway has not been fully elucidated (54), but the result is accelerated primordial follicle recruitment (57).
15 Transcriptional and histological analyses have revealed that surrounding granulosa cells with its secretion of extracellular matrix proteins can compress the primordial follicles leading to a state of high mechanical pressure (58). Similar to the fragmentation approach described by Kawamura et al. (28), the compressed primordial follicles became activated upon loosening the structure through enzymes degrading the extracellular matrix, while follicle dormancy was restored by adding exogenous pressure (58). Mechanical tension due to rigid sclerotic
capsules in some PCOS ovaries can inhibit follicle development. Resection of the ovarian wedge/capsule or diathermy/laser drilling in PCOS patients results in follicle growth (28, 59).
Studies by Kawamura et al. (28), suggests that damage on PCOS ovaries by cutting or drilling could enhance actin polymerization disrupting Hippo signaling, in turn promoting follicle growth. They suggest that local administration of actin polymerization drugs and CCN growth factors could be a possible treatment regime for PCOS patients.
Anti-Mullerian hormone (AMH) is thought to be one of the suppressors keeping primordial follicles dormant, inhibiting initial recruitment. AMH is a paracrine factor secreted by growing follicles. The mechanisms of the initial recruitment suppression by AMH is largely unknown as controversy exist to whether primordial follicles express receptors for AMH (13).
As growing follicles secrete AMH, follicular AMH levels peaks at the preantral and early antral follicular stages. When entering preovulatory follicle development, the AMH level declines. In vitro treatment with AMH in humans and rodent ovarian samples resulted in increased number of primordial follicles and decreased numbers of growing follicles (60, 61).
Clinically, serum AMH levels are used to assess ovarian function, women´s fertility and reproductive lifespan. During a woman´s lifetime, the serum AMH level peaks at puberty with the highest number of maturing follicles, with a subsequent decline with age until the AMH level is undetectable at menopause. For in vitro fertilization outcomes, serum AMH levels are used to predict the number of antral follicles that possibly can be stimulated by gonadotropins. It is also a positive correlation between the present number of primordial follicles in the ovary and the serum AMH levels (13).
16
1.3. Transition from primary stage to early antral stage
During the final stages of initial recruitment the stromal cells known as the precursor theca cells migrate to the follicle and condense around the follicle (10). The formation of this theca layer is dependent upon growth differentiation factor-9 (GDF-9), which is produced by the oocyte (12). The theca layers will form the theca interna and externa. The single layer of granulosa cells in the primary follicle undergo mitosis, leaving the follicle with two to six layers of cuboid shaped granulosa cells, promoted by R-Spondin2 (28), GDF-9 and BMP15 secreted by the oocyte (62-64). The granulosa cells also undergo profound changes and progressively acquire differentiated characteristics, making them capable of producing steroids establishing the preantral follicle. This process takes roughly 120 days in humans (13).
The follicles continue to grow and once reaching the preantral stage, a basement membrane and two layer of theca cells form. The theca interna is the inner layer adjacent to the basement membrane separating theca and granulosa cells. This layer consists of steroid producing cells.
Theca externa, the outer layer consists of non-steroidogenic fibroblast-like cells. The theca cells become increasingly vascularized, providing the follicle with direct access to nutrients and hormones (13).
As the granulosa cells continue to proliferate to form multiple layers and vascularization of the theca layer increases, fluid filled spaces in the granulosa cell layer starts to develop as a result of secretory products by the granulosa cells and substances brought in through the bloodstream. The follicular fluid is similar to blood serum and contains steroid and protein hormones, enzymes, electrolytes and anticoagulants. Once the follicle is filled with fluid creating an antral cavity the follicle has reached the antral stage (13). Antrum formation depends on gonadotropins, stimulating the production and secretion of polysaccharides and proteins by the granulosa cells (65). The antrum physically separates the granulosa cells into a cumulus and a mural population. The cumulus cells surround the oocyte while the mural cells are close to the follicle wall. The oocytes remain arrested in prophase of meiosis as they continue to grow (10).
17 The transition from primary to early antral stage are similar to initial recruitment, largely driven by intraovarian factors and signaling pathways (13). Intrinsic ovarian factors involved in preantral follicle development is presented in table 1.
Factor Source Action
Activins Granulosa cells Stimulate granulosa cell proliferation
AMH Granulosa cells Inhibitory factor on small follicle development BMP15 Oocyte Stimulate granulosa cell proliferation
BMP4/7 Theca cells Modulate FSH signaling to increase estradiol levels, prevent premature luteinization
Connexins 37 and 43 Granulosa cells and granulosa-oocyte junction
Communication between granulosa cells to granulosa cells.
Knockout have inhibited development beyond primary stage
Cyclin D2 Granulosa cells FSH stimulated factor that controls granulosa cell proliferation GDF9 Oocyte Theca cell recruitment. Knockouts have inhibited development
beyond primary stage Hedgehog signaling
members
Granulosa cells and theca cells
Proper theca cell function
IGF1/GFR Granulosa cells Enhance granulosa cell responsiveness to FSH
KGF Theca cells Modulating communication between theca cells and granulosa cells
KIT/KIT-ligand Granulosa cells and oocyte Continued follicle development, oocyte growth, and theca cell organization
NTF5/BDNF/NTRK2 Granulosa cells and oocyte Knockouts have impaired development beyond primary stage WT1 Granulosa cells Inhibitory factor on small follicle development
Table 1: Intrinsic ovarian factors involved in preantral and antral follicle development. Obtained from Hannon and Curry, 2018 (13).
The follicular growth from primordial to secondary follicles is gonadotropin-independent (12), supported by the evidence that early follicles are formed in the complete absence of gonadotropins (13). The gonadotropin-independent stage is largely controlled by intraovarian factors. The ovarian follicle development can as such be divided in two phases, the
gonadotropin-independent stage and the gonadotropin dependent stage (27). As illustrated in the Figure 4, the gonadotropin-independent stage starts with the maturation of primordial follicles and ends before the follicle develops an antrum (66).
18
Figure 4: Selected primordial follicles develop to primary follicles under Akt and mTOR signaling, the initial recruitment.
The primary follicles develop into secondary follicles, before acquiring an antrum reaching the early antral stage. Most early antral follicles undergo atresia, selected antral follicles supported by cyclic changes in FSH and LH reach preovulatory stage, releasing mature oocytes (cyclic recruitment). Redrawn after McGee and Hsueh, 2000 (10).
Some studies suggests that treatment with FSH and LH mediates preantral follicle growth (27, 67, 68) and that theca and granulosa cells contain LH and FSH-receptors at early stages of development. Thus, the middle transition from preantral to antral can be thought of as gonadotropin responsive (13).
1.4. Cyclic recruitment - Early antral to preovulatory transition
Before puberty, the fate of the growing follicles is atretic demise. Cyclic recruitment, in contrast to initial recruitment, starts after onset of puberty and requires increased levels of FSH during each reproductive cycle (10). This phase of the folliculogenesis is the most rapid, lasting for about 14-20 days and leads to the development of a dominant follicle (13). Since the secretion of FSH is cyclic, this period is referred to as cyclic recruitment. During cyclic recruitment a small cohort of antral follicles are rescued from atresia and survive to further develop into preovulatory follicles (10). The preovulatory follicles are the only follicles capable of ovulation and they are also the major producers of estradiol in women, which is required for further follicle development (13).
19 After completing the initial recruitment and transitioning from primary stage to early antral stage, the ovary contains a cohort of early antral follicles that are gonadotropin responsive and able to be stimulated by FSH for final maturation to the preovulatory stage. After puberty, stimulation by gonadotropins is the major survival factor, and without FSH stimulation, the early antral follicles undergo atresia, and thus further development of the early antral follicle is now gonadotropin dependent (10). Several studies have analyzed the growth-promoting (27, 69) and anti-atretic actions (27, 70) of FSH on antral follicles. The rising level of FSH during the late luteal/early follicular stage of the menstrual cycle is the major stimulator for preovulatory follicle development (27). The PI3K-Akt pathway described under initial recruitment is also though to play an important role in the stimulation of granulosa cell
differentiation of antral follicle (71) and oocyte maturation by FSH (72). Activin promotes the FSH-responsiveness of granulosa cells (73), while AMH lowers FSH-sensitivity (74).
During cyclic recruitment the early antral follicles acquire a single antral cavity and as theca vascularization increases, so does the size of the antral cavity. The granulosa cells, under FSH stimulation, will proliferate, leading to a massive increase in the size of the maturing antral follicle (13). The granulosa cells differentiate into different subpopulations, largely driven by a concentration gradient of oocyte-derived growth differentiating factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15). The outer layer of the granulosa cells, the mural
granulosa cells, are the most steroidogenically active cells, producing estradiol. The cumulus granulosa cells surrounding the oocyte actively participate in the maturation of the oocyte, promoting its growth and development. The mural granulosa cells are the primary site of sex steroid hormone synthesis as the follicle matures. The cumulus granulosa cells remain intimately associated with the oocyte, the first layer surrounding the oocyte is the corona radiata. Both the corona cells and cumulus cells communicate with the oocyte trough extensions (65).
The oocyte is arrested in meiosis, sustained by maintained levels of cAMP within the oocyte.
The cumulus granulosa cells express a receptor with guanylyl cyclase activity, NPR2. Mural granulosa cells produce and release c-type natriuretic peptide (CNP) a ligand that can activate NPR2 on the cumulus cells. Activation of NPR2 increases intracellular levels of cGMP.
Through gap junctions between the cumulus cells and the oocyte, the cGMP can diffuse from its cumulus origin into the oocyte to suppress the activity of phospodiesterases (PDE3),
20 resulting in maintained intra-oocyte cAMP levels and thus inhibition of meiotic resumption of oocytes (27, 65).
When the ovulation is triggered by the gonadotropin surge, the surge decreases CNP levels and thus it will stimulate the activation of phosphodiesterases within the oocyte. This will in turn reduce the levels of cAMP and allows the oocyte to resumption of meiosis (65).
Steroidogenesis requires both theca and granulosa cells, and both gonadotropins from the anterior pituitary gland. Theca cells produce androgens under control of LH signaling, but do not have the ability to synthesize estrogens such as estradiol. Granulosa cells, which produce estradiol under control of FSH signaling, do not have the ability to synthesize androgens needed for production of estradiol, but can convert androgens that diffuse from the theca cells across the basement membrane to estrogen, primarily estradiol. FSH increases the levels of aromatase, the enzyme needed to produce estrogens from androgens, in granulosa cells, thus stimulating estradiol production. Estradiol can then act within the follicle or can be released in circulation. As increasing FSH levels mature antral follicles, estradiol levels also increase during this period. Insulin-like growth factors (IGF) and activin has also been found to increase the level of estradiol (13).
Figure 5: Theca and granulosa cell interactions. Redrawn after Hannon and Curry, 2018 (13).
21 The hypothalamus and pituitary gland, receives negative feedback from estrogen, causing a decrease in FSH levels. Inhibin B, a granulosa cell-produced peptide hormone also inhibits FSH secretion by the anterior pituitary gland. As this is occurring, a dominant preovulatory follicle is growing and diverges from the other antral follicles in the cohort (13).
During cyclic recruitment, selection of follicles is a continuous process (27). The result of this selection is the emergence of a preovulatory follicle with a functional oocyte for fertilization (10). During the perimenstrual period, increasing levels of circulating FSH and thus FSH stimulation, rescues a cohort of about 10 antral follicles from apoptosis. Among these, one of the leading follicles grows faster than the rest and produces higher levels of estrogens and inhibins (10, 75). Normally one Graafian follicle is formed each month for ovulation (14).
The dominant follicle is likely to be more sensitive to gonadotropins, and more efficient in its utilization of gonadotropins (76). The exact reasons behind why one follicle emerges as dominant is unclear, but its though that increasing granulosa cell numbers and therefore enhanced FSH and LH receptor expression may play a role (77-79). The rapidly growing dominant follicle also produces higher levels of autocrine and paracrine growth factors that stimulate vascularization and FSH responsiveness (10, 80). In sum these mechanisms create a dwindling FSH environment and a local positive selection mechanism (13). Furthermore, the dominant follicle´s increased responsiveness to FSH stimulates the expression of both FSH and LH receptors on granulosa cells as the follicle develop towards ovulation (10, 81). LH receptors on granulosa cells are required for ovulation, thus providing the dominant follicle with a fail-safe mechanism to ensure the eventual ovulation. The dominant follicle further enhances its dominance, by producing estrogens and inhibins that suppress pituitary FSH released during the mid-follicular phase. As a result, the remaining growing antral follicles are deprived of adequate FSH stimulation and will suffer the faith of atresia (10).
The dominant follicle continues to grow and will reach a threshold level in its production of estradiol leading to positive feedback to the hypothalamic-pituitary axis, initiating the LH- surge leading to ovulation. The rapid increase in LH, known as the preovulatory LH surge, induces several events in the follicles, including resumption of the meiosis in the oocyte, expansion of the cumulus granulosa cells, breakdown of the follicular wall and the
differentiation of follicular cells to luteal cells. These events lead to ovulation of the mature oocyte for fertilization, and the remaining theca and granulosa cells creates the corpus luteum.
22 The periovulatory period, from the LH surge to the oocyte release, lasts for about 36 hours in humans (13).
1.5. Menstrual cycle physiology and the hypothalamic-pituitary ovarian axis
The cyclical reproductive function starts after puberty and is dependent on a carefully
coordinated hypothalamic-pituitary-ovarian axis (HPO-axis), also called the reproductive axis (82). The hypothalamic-pituitary ovarian axis responds to hormonal and neural signals as well as external factors (83).
The normal menstrual cycle, defined as the interval between two menses, normally last between 26 and 32 days (84). Depending on the organ under examination it can be divided into an ovarian cycle and a uterine cycle.For the ovarian cycle the first phase is called the follicular phase, while the corresponding phase for the uterus cycle is called the proliferative phase. The second phase is for the ovaries called the luteal phase, while for the uterus cycle it is called the secretory phase. The luteal phase lasts for around 14 days (1).
The menstrual cycle is under hormonal control by gonadotropin-releasing hormone (GnRH), released by the hypothalamus in a pulsatile matter that in turn stimulate the gonadotropes in the pituitary to release follicle-stimulating hormone (FSH) and luteinizing hormone (LH).
These hormones travel by the blood to the ovaries and stimulate folliculogenesis and steroid and peptidergic hormone secretion (82). The abovementioned hormones together orchestrate the different events of the reproductive cycle ensuring appropriate timing and successful reproduction (85).
23
Figure 6: The hypothalamus-pituitary-ovarian axis, redrawn after Padamanabhan et al., 2018 (82).
GnRH is released in a pulsatile matter, and it has been shown that GnRH pulses occur every 90 minutes at the beginning of the follicular phase, increasing to every 60 minutes at the end of the follicular phase. During luteal phase pulses are found to occur every 240
minutes. The pulsatility of GnRH release from the hypothalamus is directly correlated with the release of LH (84).
As previously mentioned FSH stimulates follicular growth and maturation during the follicular phase, starting from first day of menstruation. One or two days before the onset of menstruation, slight but significant increases in FSH secretion can be identified, as a result of declining ovarian hormone production, especially inhibin A and estradiol (82). During the latter part of the follicular phase, progressively increasing estradiol levels inhibit FSH release prior to the midcycle gonadotropin surge. As previously described, this is advantageous for the dominant follicle that is more sensitive to gonadotropin due to a higher density of FSH receptors than the other developing follicles. Thus they will be deprived of adequate FSH stimulation and will go to the process of atresia (82).
24 At the end of the follicular phase, estradiol exerts a positive feedback, which stimulates the midcycle gonadotropin surge, initiating ovulation. Even though a surge in both LH and FSH occurs, the surge in LH has the most significant role in the ovulation process (84).
LH exerts its role in three ways; 1) it stops the proliferation of granulosa cells, 2) it induces differentiation of granulosa cells enabling them to secrete progesterone and become the corpus luteum, and 3) lastly it resumes the oocyte meiosis (86).
The onset of LH surge occurs 35-44h before the ovulation, while the peak of the LH surge precedes ovulation by 10-12h (87). A decrease of estradiol is observed after the ovulation, while pulsatile LH secretions promote production of estradiol and progesterone during the luteal phase. If pregnancy occurs, the placenta secretes hCG which stimulates progesterone secretion from the corpus luteum. Progesterone has an essential role in preparing uterus for the implantation of the embryo, inducing secretory changes in the uterine cavity (84).
1.5.1. The uterus and menstruation
The uterus has several critical roles in reproduction functioning as the site of fetal growth and development. The uterine cervix functions as a barrier, inhibiting passage of bacteria and other infectious agents into the uterus, but it also, under the appropriate hormonal conditions it allows for the passage of sperm from the vagina into the uterus. The uterine environment is important for capacitation of the sperm, and the peristaltic contractions in the uterus help to move the sperm toward the oocyte (88). The secretions of the uterine glands nourish and maintain the embryo until implantation occurs about 9 days (6-10 days) after fertilization (89, 90).
The uterine wall changes throughout menstrual cycle that can be divided into three phases; the proliferative phase, the secretory phase and the menstrual phase. The proliferative phase corresponds with the follicular phase of the ovarian cycle. In this phase the estrogen-
dependent proliferation of glandular structures and vasculature the endometrium occurs. The endometrium lining increases 10-fold in thickness and mitotic activity is prominent (84).
After ovulation the secretory phase occurs, and the thickness of the endometrium is close to constant. Hallmarks of the secretory phase are tortuosity of the vessels and secretory activity
25 within the glands. The result of the secretory activity is edema, that can be seen within the stroma of the endometrium at the time of implantation of the blastocyst. At the same time decidualization occurs. Derivates from the stromal cells of the endometrium is called decidua and is rich in glycogen and lipid content. The decidual cells have the ability to produce autocrine and paracrine regulatory peptides and is involved in the implantation and
placentation. The implantation occurs 8 days after ovulation, equal to 21-23 of the cycle (84).
If pregnancy does not occur, the corpus luteum degenerate into corpus albicans, and stops the secretion of estrogen and progesterone. Following, the endometrial cells undergo apoptosis and the spiral arteries contract resulting in hemorrhage into the stroma and release of inflammatory mediators. The result of the decrease in estradiol and progesterone is
menstruation, bleedings due to shedding of the inner layer of the uterus, the endometrium, normally lasting for three to six days (91).
Figure 7: Overview of the ovarian and menstrual cycle. Figure from Tortora and Derrickson, 2016 (92).
26
2. Infertility and evaluation of ovulatory dysfunction
Infertility is clinically defined as the inability of a couple to conceive after one year or more of frequent unprotected sex (3). It is estimated that infertility affects around 13-15% of heterosexual couples (93), 5.5%, 9.4% and 19.7% respectively at ages 25-29 years, 30-34 years and 35-39 years (2, 94). In developed countries infertility can be explained by female factors in 30% of cases, male factors in 20%, combined female and male in 40% and in 10%
no obvious etiologic factor can be found (95).
While some causes of infertility are easy to identify such as azoospermia, bilateral tubal obstruction or longstanding amenorrhea, the situation is more complex for most couples with for example not absent but reduced numbers of sperm, partially obstructed tubes, intermittent ovulation and so on. Also adding to the complexity is the uncertain predictive validity of the tests used, and therefore an uncertain causal relationship between an abnormal infertility test and the actual cause of infertility (96). One population-based study by Hull et al. (97) reported the following results and must, due to the abovementioned difficulties be seen as a rough proxy of the frequency of the causes of infertility:
Figure 8: Causes of infertility, data obtained from Hull et al., 1985 (97) and Kuohung and Hornstein, 2020 (96).
27
2.1. Evaluation of ovulatory dysfunction
Ovulatory dysfunction accounts for about 21 percent of couples presenting with infertility (97) and is defined as abnormal, irregular or absent ovulation, most frequently presented clinically by loss of regular menstrual cycles (98). The World Health Organization (WHO) categorized amenorrhea due to ovulatory dysfunction into seven categories (98), as given in the table below.
CLASSIFICATION OF AMENORRHEA ACCORDING TO THE WHO, 1970
CATEGORY Origin of the problem Diseases
GROUP I Hypothalamus-pituitary axis failure Anorexia nervosa, Kallman syndrome GROUP II Hypothalamus-pituitary dysfunction Polycystic ovary syndrome (PCOS)
GROUP III Ovarian failure hypothyroidism
GROUP IV Genital tract malformations or alteration Early ovarian failure
Rokitansky-Küster-Hauser syndrome, Asherman syndrome
GROUP V Prolactinomas Prolactin pituitary tumor
GROUP VI Functional hyperprolactinemia Maternal lactation GROUP VII Hypothalamus-pituitary tumors, non-
prolactin secreting
Pituitary adenomas
Table 2 Classification of Amenorrhea according to WHO, 1970. Table remade after Castillón et al., 2018 (98).
Women with ovulatory dysfunction may have either anovulation or oligo-ovulation referring to the absence of ovulatory cycles or shift between ovulatory cycles and anovulation
respectively (99). Amenorrhea and oligo-amenorrhea are the most frequent clinical signs, with amenorrhea describing the total absence of menses and oligo-amenorrhea as infrequent menstrual periods (98).
In women who are not receiving hormonal contraceptives, a regular menstrual cycle serves as an indication that a successful ovulation finds place each month (98), as most cases of
amenorrhea, especially secondary amenorrhea, are anovulatory. Despite regular menses being an indication of a successful ovulation, anovulation and amenorrhea are not mutually inclusive states. Amenorrhea can occur despite normal ovulation if the uterus is incapable of responding to the cyclic hormonal stimulation (100). This also applies for genital anatomic
28 abnormalities causing outflow obstruction despite normal hormonal stimulation (101). Even though it is rare, eumenorrheic anovulation can occur (102). Shedding of the endometrium requires tissue proliferation promotes by sufficient amounts of circulating estrogen.
Ovulation itself is not a required precondition for estrogen production (100).
The cause of anovulation and amenorrhea will have consequences for treatment. In the figure below we attempt to illustrate that even though amenorrhea often is a sign of anovulation (the overlapping zone), there are outliers where anovulation and amenorrhea can exist on its own, not being mutual inclusive states.
Figure 9: The relationship between anovulation and amenorrhea.
2.2. Classification of amenorrhea
Amenorrhea is defined as the absence of menses (103). Spontaneous and regular menses are dependent on an intact and coherent hypothalamic-pituitary-ovarian axis. The endometrium also needs to be able to respond to steroid hormone stimulation and the female genital outflow tract needs to be intact (1). The menstrual cycle can be influences by different environmental stressors but missing a single menstruation often do not represent any significant pathology.
On the other hand, prolonged and persistent absence of menses may be pathologic (104).
Pregnancy, the most common cause of amenorrhea must be ruled out in all cases of
amenorrhea. Other physiological causes of amenorrhea may be lactation and menopause. If amenorrhea occurs apart from these causes, an etiological assessment must be carried out (105).
29 Amenorrhea is often classified as primary or secondary and describe whether amenorrhea occurred before or after menarche respectively (103). The first menses, called menarche, occurs at mean age of 12,5 years (105).
Primary amenorrhea is when menarche has not occurred by age 15, but normal growth and sexual characteristics are present (106). If normal growth and secondary sexual characteristics are absent, the assessment should be done before age 14 (105).
Secondary amenorrhea is clinically defined as the absence of menses after 3 months of amenorrhea, in women who already had menses. PCOS and FHA are the most common causes of secondary amenorrhea. It should be mentioned that causes of secondary amenorrhea also can present as primary amenorrhea (107). The table below present the etiologies of primary and secondary amenorrhea.
Primary amenorrhea Secondary amenorrhea
Gonadal dysgenesis, incl Turner syndrome 〜 43%
Müllerian agenesis 〜 15%
Physiologic delay of puberty 〜 14%
Polycystic ovary syndrome (PCOS) 〜 7%
Isolated gonadotropin – releasing hormone (GnRH) deficiency 〜 5%
Transverse vaginal septum 〜 3%
Weight loss/anorexia nervosa 〜 2%
Hypopituitarism 〜 2%
Polycystic ovary syndrome (PCOS) 〜 30%
Functional hypothalamic amenorrhea 〜 35%
Primary ovarian insufficiency (POI) 〜 10%
Hyperprolactinemia 〜 13%
“Empty sella” 〜 1.5%
Sheehan syndrome 〜 1.5%
Cushing´s syndrome 〜 1%
Intrauterine adhesions 〜 7%
Other 〜 1%
Table 3: Etiologies for amenorrhea, data obtained from (106, 107).
30
3. Polycystic ovary syndrome (PCOS)
Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in reproductive- aged women. It accounts for the majority cases of anovulatory infertility and hirsutism (108).
PCOS is heterogenous in its presentation and is defined as a syndrome, where the major clinical and biochemical manifestations include ovulatory dysfunction, polycystic ovaries, and hyperandrogenism (8). Due to its heterogeneity in clinical presentation, PCOS can be divided into several phenotypes (109). Metabolic abnormalities, especially insulin resistance with compensatory hyperinsulinemia, are often present in of PCOS patients (110). The etiology of PCOS is largely unknow, but there are increasing grounds for PCOS being a complex multigenic disorder with epigenetic and environmental influences including but not limited to diet and lifestyle factors (111).
3.1. Epidemiology
PCOS affects approximately 6-10% of women of reproductive age, but prevalence is dependent on the diagnostic criteria used. In a 2016 meta-analysis of 24 population studies (112), the rate of PCOS when using Rotterdam criteria was 10 percent, making PCOS one of the most common endocrine disorders in women.
Women with PCOS are at increased risk of infertility and reproductive abnormalities (113). In a community-based study, infertility was noted by 72% of women reporting PCOS compared to 16% among non-PCOS women (110).
PCOS has long-term health implications with increased risk of infertility, but also metabolic syndrome, diabetes mellitus type 2, endometrial hyperplasia and cancer, cardiovascular abnormality, obstructive sleep apnea, depression and anxiety (6, 114-116).
Of women with PCOS two thirds have metabolic dysfunction, and therefore an increased risk of type 2 diabetes mellitus and possibly cardiovascular disease (113). Women with PCOS often have significantly reduced insulin sensitivity that occurs independently or additive to obesity. Among women with PCOS, insulin resistance affects 50-70%, but 95% of those who are obese (109). The incidence of metabolic syndrome, gestational diabetes mellitus, impaired glucose tolerance and type 2 diabetes mellitus are increased in women with PCOS when
31 compared to BMI-matched and age-matched non-PCOS (117). Out of PCOS women with impaired glucose intolerance, 2% progress to diabetes mellitus type 2 per year, while 16%
progress from being normoglycemic to having impaired glucose tolerance (118).
PCOS is associated with cardiovascular abnormalities and risk factors for development of cardiovascular disease later in life (5). Increased carotid intima-media thickness (119, 120) and coronary artery calcification are associated with PCOS (121, 122). However, definite proof that the abovementioned risk factors bring about cardiovascular disease in women with PCOS is lacking (123). Also, many patients with PCOS, especially the group with normal body weight, show no cardiovascular risk factors (124, 125).
The relationship between PCOS and obesity is complex, and strong evidence of associations between the two is lacking. A systematic review meta-analysis concluded that obesity was more prevalent in PCOS women compared to non-PCOS women (126). However, most of the conducted studies recruited patients from hospitals and specialist clinics. Studies on
unselected patient populations have suggested a more equal BMI distribution between non- PCOS and PCOS patients (127, 128). This indicates that the obesity among PCOS patients may be driven by self-referral (109, 129). In the United States, studies on unselected patient populations found no significant association between PCOS prevalence and BMI (130), while another similar study in Turkey showed increasing rates of PCOS with increasing BMI (131).
While different countries differ in mean population BMI, there are no relationship found between PCOS prevalence in different countries. This indicates that obesity does not drive the development of PCOS (132). Also, no associations have been found between PCOS and gene variants in genes involved in obesity (133, 134).
Increased risk of endometrial abnormalities including carcinoma is another complication of PCOS. The hyperinsulinemia due to insulin resistance and increased levels of estrogens causes proliferation of the endometrium (109). The risk of endometrial cancer among women with PCOS is estimated to be 2.7 times higher than for non-PCOS patients, with an estimated life time risk as high as 9% (7, 135). Women with PCOS may also be at increased risk of ovarian and breast cancer but is not well-documented (109).
Women with PCOS suffer from depression and anxiety in a greater extent and more severely than non-PCOS women (136-138), regardless of phenotype and BMI (138, 139). A meta-
32 analysis found that PCOS patients have more severe emotional stress, than non-PCOS
patients. Even though hirsutism, obesity and infertility are linked to the increased emotional distress, they cannot fully and consistently account for the high prevalence of depression and anxiety (137).
Patients with PCOS and their infants will be at increased risk of perinatal complications such as gestational diabetes, preterm labor and pre-eclampsia. Infants from PCOS patients have a higher risk of being born large for gestational age, aspirate meconium during birth, and a low Apgar score (<7) at 5 minutes (140).
3.2. Etiology
PCOS is a complex multifactorial disorder thought to arise from the interaction between genetic and environmental factors (111). The first clinical symptoms arise when mature gonadotropin levels are reached during puberty. The development of PCOS can be viewed as a “two-hit” process. The “first-hit” whereby PCOS arises from a congenital programmed predisposition, that becomes manifest in the presence of a “second-hit” postnatal provocative factor (116). The congenital factors encompass either genetic or acquired factors (e.g.
maternal exposure to drugs affecting the fetus), while the postnatal provoking factor most often is hyperinsulinemia due to insulin resistance, either congenital or acquired due to obesity. The above mentioned complex interactions in general mimic a dominant autosomal inheritance pattern with variable penetrance (141). In twin studies, hereditability of PCOS has been estimated to exceed 70 percent (142). 30-50% of women with PCOS have a mother or sister with the PCOS (143).
Large studies have identified close to 100 susceptibility genes related to PCOS (144). With PCOS being a complex polygenic disease, high-throughput genome-wide association studies (GWAS) provide a better and more comprehensive, discovery-driven approach to explore the genetics of PCOS (109). One of the major candidates identified by GWAS studies to date is the DENND1A.V2, an intronic locus linked to PCOS in most populations (145, 146).
DENND1A.V2 protein is overexpressed in PCOS theca cells and encodes for a protein that stimulates steroidogenesis (147-149).
