molecular pathways for development of PCOS

1. Polycystic Ovary Syndrome
Ekbal Mohamed Abo-Hashem (MD )
Professor of Clinical Pathology
Mansoura University-Egypt

3. Introduction
Polycystic ovary syndrome (PCOS) is a complex and heterogeneous
disorder, affecting approximately 7-10 per cent of women in reproductive
age. It is characterized by :
• Chronic anovulation,
• Hyperandrogenemia,
• Altered LH: FSH ratio (>2/3:1) and
• Polycystic ovaries.
The syndrome is a major cause of anovulatory infertility .
The aetiopathogenesis of this syndrome is likely to be mutifactorial
consisting of genetic and environmental components.

8. Insulin resistance (IR) is now known to be intrinsic to this disorder, present in
approximately 50-70 per cent of these women independent of obesity, and
contributing in a major way to its pathogenesis.
Women with PCOS are frequently obese which contributes an extrinsic
component of IR.
• It is known that IR progresses towards the development of compensatory
hyperinsulinemia, which drives hyperandrogenemia in these women.

9. • Excess androgen levels lead to menstrual disturbances, development of
ovarian cysts, hirsutism and other related disorders. IR also increases the risk
for development of glucose intolerance, type 2 diabetes mellitus (T2DM),
hypertension, dyslipidaemia and cardiovascular abnormalities in these women
.

11. • In PCOS the “central paradox” is that the ovary remains sensitive to insulin
action to produce androgens , (steroidogenic activity ) , in spite of systemic
insulin resistant state; where classical target organs of insulin as well as ovary
remain resistant to its metabolic activity (selective insulin resistance ).
• The number and affinity of insulin receptors are optimal in different insulin
target tissues and ovary, and also no structural and mutational abnormalities
could be detected in the PCOS women.
• Thus a post-receptor binding defect in the insulin signalling pathway appears to
play an important role in the etiology of selective IR.

12. Signalling pathways of insulin action

13. Abbreviations:
PIP2-phosphatidylinositol 4,5-bisphosphate, PIP3- phosphatidylinositol 3,4,5-triphosphate, mTOR- mammalian
target of rapamycin, Raptor-regulatory associated protein of mTOR, Glut4- glucose transporter type 4, GSK3-
glycogen synthase kinase 3, PDK1/2-phosphoinositide dependent kinase1/2, p70S6K- ribosomal protein S6
kinase, MEK- mitogen-activated protein kinase kinase, ERK1/2- extracellular signal regulated kinase ½, SHP-2-
SH2 domain containing protein tyrosine phosphatase.

14. 1. Binding of insulin to its receptor results in autophosphorylation and tyrosine
kinase activation of the receptor which further phosphorylates other
downstream mediators [insulin receptor substrate (IRS) and Src homology
domain containing transforming protein 2 (Shc)]. These mediators then
differentially activate various downstream signalling proteins.
2. Phosphatidylinositol 3-Kinase (PI3K) plays a major role in glucose transport,
glycogenesis and protein synthesis.
3. On the other hand, Grb2/SOS (growth factor receptor-bound receptor 2/ Son of
sevenless) complex activates mitogen-activated protein kinase pathway
(MAPK) playing a crucial role in mitogenic response.
4. Another pathway via inositol glycan generation has been suggested which may
play a vital role in steroidogenesis.

15. Several hypotheses have been proposed to explain the paradox between insulin
augmentation of androgen synthesis in the ovary despite the systemic insulin
resistant state .
The first one suggests that the steroidogenic action of insulin might be mediated via
IGF-1 receptor or hybrid insulin/IGF-1 receptor.

16. The second hypothesis proposes that the signaling cascade for metabolic action of
insulin in the ovary becomes divergent from the one, which mediates steroidogenic
action after binding of insulin to INSR.
Insulin may activate steroidogenesis via several pathways, either via cross talk with
LH-induced cAMP accumulation, which in turn might activate PI3K activity or MAPK
pathway or via alternate pathways of insulin signaling.

17. The third hypothesis is the serine phoshorylation theory, based on the observation
that serine phosphorylation of the main regulatory enzyme of androgen biosynthesis
i.e., P450c17 appears to modulate its 17, 20 lyase activity and subsequent androgen
production. In a subgroup of PCOS women, IR appears to be related to excess
serine phosphorylation of the β subunit of INSR.

19. Increased androgen production by ovarian cells is the classical endocrine phenotype
of PCOS. The genomic and molecular studies demonstrated multiple alterations in
the steroidogenic machinery of theca cells from PCOS women viz., overexpression
of various proteins including luteinizing hormone(LH) receptor,INSR, lipoprotein
receptor (HDL and LDL ),steroidogenic acute regulatory protein (StAR ),P450 side
chain cleavage (P450 scc), 3 beta hydroxysteroid dehydrogenase(3 beta-HSD),and
cytochromeP450 C17(CYP-17). All these contribute to excess production of
progesterone(P4), 17 alpha hydroxyprogesterone and testosterone as compared to
normal theca cells .
Studies with ovarian tissues

20. Identification of INSR throughout the ovary and the ability of insulin to stimulate
biosynthesis of androgens, estrogen and P4 in ovarian cell culture suggest that
the ovary is another target organ of insulin action . These findings indicate that
insulin may play a role in normal follicular development and hence ovarian
dysfunction states are manifested in a variety of insulin resistant states .

21. Insulin rather than sex steroids appears to be the major regulator of SHBG
production .These effects of altering insulin levels were seen only in women with
PCOS (and not in normal women ) . This observation suggests that polycystic
ovarian changes (e.g., theca cell hyperplasia) and/or disordered gonadotropin
secretion (e.g., increased LH levels) are a prerequisite for these reproductive
actions of insulin . Insulin has a substantially greater effect on T production in
theca cells isolated from women with PCOS

22. • Lowering insulin levels with the insulin- sensitizing drugs (ISDs), metformin and
the thiazolidinediones (TZDs) can reduce circulating androgen levels, increase
SHBG levels, and restore ovulatory menstrual cycles in women with PCOS .
• Abnormalities in apparent 17,20-lyase activity have improved in parallel with
reduced circulating insulin levels, consistent with insulin-mediated stimulation of
this enzyme .
• Estrogen levels have also been reported to decrease during ISD therapy in
PCOS , suggesting that insulin has direct stimulatory effects on multiple
steroidogenic pathways. Some of the effects of ISDs to lower circulating
androgen levels are most likely secondary to direct effects of these agents on
steroidogenesis.

23. Resistin:

24. Adipose tissue factors in pathogenesis of PCOS :
Adipose tissue functions as a highly specialized endocrine and paracrine organ
producing an array of adipokines like adiponectin, leptin, resistin, TNF-α etc.
which affect insulin sensitivity. These adipokines may have a relation with IR and
obesity in PCOS .

26. Genetic factors in PCOS
Evidence from twin and family based studies have demonstrated an increased
prevalence of PCOS and its phenotypic features in the relatives of women with
PCOS, suggesting genetic factors underlying the syndrome.
PCOS is more likely a complex genetic diseases with at least several
susceptibility genes .

27. Most of the genetic studies have focused on candidate gene approach, selecting
genes from multiple logical signaling pathways, implicated in the pathogenesis of
PCOS, such as genes involved in steroid hormone biosynthesis and metabolism,
insulin signaling, gonadotropin action and its regulation, and pro-inflammatory
genes .

28. PCOS candidate genes :
On the basis of their known or expected role in the pathogenesis of PCOS,
numerous PCOS candidate genes have been proposed and studied. Attention has
been focused on defects in:
(i) ovarian androgen biosynthesis and action;
(ii) insulin secretion and action;
(iii) hypothalamic-pituitary axis;
(iv) adipose tissue biochemical disturbances.

29. • Altered expression of several genes involved in insulin function like PDK4
(pyruvate dehydrogenase kinase 4), PIGH (phosphatidylinositolglycan class H)
and UDP-GalNAcPP (UDP-GalNAc pyrophosphorylase) and some genes
involved in insulin mitogenic pathway, mainly those in MAPK pathway, such as
RPS6KA2 .
• PDK4 has recently been shown to play a role in the pathogenesis of insulin
resistance in T2DM . Similar studies with oocyte revealed the presence of
putative binding sites for androgen receptor (AR), PPAR-γ, PPAR-γ/Retinoic X
Receptor (RXR) in the promoter regions of many genes differentially expressed
in PCOS.
Genetic variants involved in insulin resistance in PCOS

30. • PPAR-γ is a ligand-activated transcription factor involved in glucose and lipid
metabolism is known to modulate insulin signalling pathway. Over expression of
PPAR-γ gene in PCOS ovary and the presence of PPAR-γ binding sites in
differentially expressed genes suggest its role in pathogenesis of the syndrome

31. Genes involved in ovarian androgen production and action :
Increased transcriptional activity of genes encoding steroidogenic enzymes could
lead to upregulation of androgen biosynthesis. The most widely studied PCOS .
Candidate genes from this group are CYP17 gene (cytochrome P450 17-
hydroxylase/17, 20-desmolase gene), CYP21 gene (cytochrome P450 21-
hydroxylase gene) and CYP11A gene (cytochrome P450 side-chain cleavage
enzyme gene) (15q23-q24).

32. The transmission disequilibrium test (TDT), a type of family-based association
testing, has been successfully used with a candidate gene approach to map PCOS
susceptibility genes . The TDT examines association in the presence of linkage by
assessing transmission of alleles from parents to affected offspring
Using the TDT approach, an allele of a dinucleotide repeat D19S884 on
chromosome 19p13.2 was linked and associated with the PCOS reproductive
phenotype

33. Mechanisms for the Association of Insulin Resistance and PCOS
Insulin as a reproductive hormone
Direct ovarian actions of insulin on steroidogenesis are evident ,and
the insulin signaling pathway is important for the control of ovulation.
Ovarian insulin action on steroidogenesis is preserved, despite resistance to
insulin’s metabolic actions in PCOS .In granulosa-lutein cells isolated from
ovaries of women with classic PCOS, insulin action on glucose metabolism is
significantly decreased, whereas insulin action on steroidogenesis is unchanged
compared with granulosa-lutein cells from control women .

34. • Insulin action on theca cell androgen production is evident only at
supraphysiological insulin concentrations. Theca cells from women with PCOS
are more responsive to the androgen stimulating actions of insulin than those
from control women .
• Under physiological circumstances, insulin most likely acts as a co-gonadotropin
to increase LH induced androgen synthesis in theca cells as well as to enhance
FSH-induced estrogen production and LH-induced luteinization in granulosa
cells .
• Insulin can also enhance GnRH-mediated LH and FSH release from cultured rat
pituitary cells

35. • Suppressing insulin levels with diazoxide resulted in a decrease in circulating T
levels in women with PCOS, independent of alterations in LH release .
• SHBG levels also increased with suppression of insulin levels by diazoxide ,
consistent with an important role for insulin as a negative regulator of SHBG
production .

36. • Lowering insulin levels with the insulin- sensitizing drugs (ISDs), metformin and
the thiazolidinediones (TZDs), can also reduce circulating androgen levels,
increase SHBG levels, and restore ovulatory menstrual cycles in women with
PCOS .
• Insulin rather than sex steroids appears to be the major regulator of SHBG
production

37. • Abnormalities in apparent 17,20-lyase activity have improved in parallel with
reduced circulating insulin levels, consistent with insulin-mediated stimulation of
this enzyme .
• Estrogen levels have also been reported to decrease during insulin-sensitizing
drug (ISD) therapy in PCOS , suggesting that insulin has direct stimulatory
effects on multiple steroidogenic pathways.
• Some of the effects of ISDs to lower circulating androgen levels are most likely
secondary to direct effects of these agents on steroidogenesis.

38. There is a dose response effect of TZDs on ovulatory function in PCOS , whereas
such an effect has not been investigated for metformin. These observations suggest
that insulin resistance contributes to anovulation in PCOS. Some of the human
studies suggest that hyperinsulinemia/insulin resistance-mediated reductions in
pituitary sensitivity to GnRH contribute to anovulation in PCOS.
Lowering insulin levels with ISDs has resulted in decreases in DHEAS levels in
PCOS women . This insulin effect appears to be a direct action of insulin to increase
adrenal sensitivity

39. In summary, investigation of the association between insulin resistance and
PCOS has revealed that insulin is a reproductive as well as a metabolic hormone.
It functions as a co-gonadotropin through its cognate receptor to modulate
ovarian steroidogenesis. This action is preserved despite resistance to the
metabolic actions of insulin in the periphery as well as in the ovary, an example of
selective insulin resistance.

40. Hormonal changes in PCOS

41. Hormonal changes during the normal menstrual cycle

46. Diagnostic criteria for PCOS :
• NICHD : Both hyperandrogenism and chronic anovulation
• Rotterdam :Two of the following: hyperandrogenism, chronic anovulation, and
PCOS .
• Androgen Excess AE-Society : Hyperandrogenism plus ovarian dysfunction
indicated by oligoanovulation and/or PCO
• All criteria require exclusion of other disorders: hyperprolactinemia, nonclassic
congenital adrenal 21-hydroxylase deficiency, thyroid dysfunction, androgen
secreting neoplasms, and Cushing’s syndrome.