Intro to PCOS

Arguably the most prevalent medical condition in women Patients with PCOS have systemic metabolic manifestations with multiple symptomatology: endocrine, gynaecological, diabetic, dermatological, eating disorder, psychiatry Insulin resistance (IR) likely to have a life-long impact on patient. Annual economic cost of diagnosis and treatment of PCOS in USA recently calculated to be $4.36 billion and 40% of this was IR/T2D-related

The ovary contains increased numbers (>12) of small antral follicles (2-9mm) visible on u/s There is a disorder of follicle growth at all stages Possibly increased proportion of primordial follicles & increased number of activated (primary) follicles Arrested antral follicle growth before they mature Lower rates of atresia » antral follicles persist (visible on u/s) In some cases there is a failure of dominant follicle selection and therefore anovulation The main problem with PCO is that it has Disorder of follicle growth occurs across all stages.

First described in 1935 by Stein and Leventhal - syndrome described as obesity, hirsutism and anovulation in the presence of bilaterally enlarged sclerocystic ovaries. Discovered that many women have the change in the ovaries and do not necessarily have the symptoms Spectrum of presentation has led to lack of consensus regarding the definition Diagnosis of exclusion i.e. disorders that mimic PCOS: congenital adrenal hyperplasia (most common is deficiency of 21-hydroxylase → ↑17-hydroxyprogesterone & androgens) Hyperprolactinemia, thyroid disease, Cushing’s syndrome Ovarian hyperthecosis (very rare) - nests of luteinized theca cells Ovarian hyperthecosis – excess androgen production due to nests of luteinized theca cells scattered throughout ovarian stroma. Ovary slightly enlarged but devoid of antral follilces – hence distinct from PCOS. Serum testo levels>women with PCOS but

There are 3 criteria: Polycystic ovaries, ovulatory dysfunction, hyper androgenism. Need 2 out of these 3 for a PCOS diagnosis Polycystic ovaries:(either 12 or more follicles measuring 2-9mm diameter and/or increased ovarian volume >10ml in either ovary & no DF >10mm). Technique and equipment dependent. T/V imaging not always appropriate Hyper androgensim: Needs clinical/biochemical evidence: assays not standardized across labs; normative data not clearly defined; clinical hyperandrogenism difficult to quantify; ethnicity. Clinical hyperandrogenism includes hirsutism, acne, or male pattern alopecia or biochemical signs of hyperandrogenism i.e. elevated levels of total or free testosterone. Ovulatory dysfunction: Oligomenorrhea and anovulation Frequent bleeding <21d or infrequent bleeding >35d. To confirm ovulation serum progesterone level at mid-luteal phase (d21-22) of cycle (values ≥7ng/ml needed for regular luteal function)

By Ultrasound: Normal no more than 5 follicles in an ovary with a small amount of stroma in a woman with regular cycles PCO In at least one ovary ≥ 12 follicles of 2-9mm diameter arranged peripherally around an enlarged core of dense stroma - ovarian volume >10mls, without a dominant follicle PCOS PCO on scan plus one or more symptoms The terms cyst and follicles used interchangeably but not correct – what you seen in PCO are NOT cysts but follicles – has lead to confusion. Scanning is not a science but an art ie. very operator dependent. It is easy to miss follicles and it also depends on where you are in the cycle. Also age – adolescents can have multi-follicular morphology until puberty is complete. Ideal would be to scan sequentially.

Most women with PCO probably have regular or almost regular cycles Most women with PCOS and cycle problems have oligomenorrhoea There are a number of candidates for follicle arrest androgens, intra-follicular inhibitors eg AMH defect in apoptosis dysregulated gonadotrophin secretion (both FSH and LH) Main difference between ov and anov is also the level of insulin resistance Adult rhesus macaques fed a western style diet (high fat/sugar) & exposed to chronically elevated T from pre-puberty to menopause altered small AF numbers, morphology and transcriptome (Bishop CV et al, 2015, Fertil.Steril.)

Familial aggregation Sisters more likely to be affected first-degree relatives have higher rates of metabolic abnormalities (including insulin resistance, decreased beta-cell function etc) Male relatives of women with PCOS increased prevalence of metabolic syndrome & obesity compared to general US male population Monozygotic twins twice as likely to both have PCOS than dizygotic. common finding of raised androgen led to belief that PCOS is caused by an inherited disorder -most likely in the steroid biosynthetic pathway Many candidate genes were investigated: all ‘obvious’ ones ruled out Complex polygenic disease – involves subtle interaction with environmental factors (intra- & extra-uterine) Definitely runs in families Because of hyperandrogenimia thought that gene in biosynthetic pathway for androgens was responsible. GWAS study in China: 1st Genome-wide association study (GWAS) identified causative genes in Han Chinese women (2011) 744 women with PCOS &amp; 895 controls 3 loci linked and candidate genes within these loci are: LHCGR FSHR THADA….linked to T2D DENND1A ...linked with obesity Confirming the biological relevance of PCOS-associated variants by molecular analysis (e.g. expression analysis, targeted genetic disruption in cell culture or organism) is critical to confirming findings from GWAS – rarely done.

Theca cells were transfected with DENND1A isoform and treated with/without forskolin (to stimulate cAMP) Measured production of various androgens and progestegins DENND1A overexpression recapitulated hyperandrogenic theca cell function

Consistent feature of PCOS is disordered gonadotrophin secretion leading to downstream ovarian consequences Elevated/upper-normal mean LH Low/low-normal FSH Rapid GnRH frequency → favouring rapid LH pulse secretion Elevated LH pulse frequency and increased LH:FSH ratio impairs downstream ovarian folliculogenesis and alters steroid hormone production. In PCOS the “FSH threshold” that is required for follicle maturation is not reached (due to this dysregulated gonadotrophins and also increased AMH, which alters FSH sensitivity) causing follicles to arrest in antral stages. What may be more important in PCOS is the LH:FSH ratio.

Impaired negative regulation of GnRH pulse generator High Testosterone impairs negative feedback by Progesterone in presence of oestradiol Proof: block AR with flutamide → progesterone then able to↓ LH & FSH Also see that in late puberty girls without HA respond to progesterone with ↓LH pulse frequency o/n, which did not occur in HA girls at same pubertal stage The higher LH will drive thecal cell hyperplasia and the hyper-androgenemia, but HA is also intrinsic and can be independent of LH.

Increased androgen Most consistent biochemical abnormality in women with PCOS is hypersecretion of androgens ideal to measure free (T) i.e. SHBG and total testosterone to work out free T anov> ov>normal increased androgen production by the ovary, even in ovPCO Increased LH leads to increased androgen production May lead to hirsutism and acne consistently reported as most distressing symptoms in young women

Testosterone converted to DHT at hair follicle DHT more potent androgen 5a-reductase may be higher in PCOS Not just absolute levels of testosterone per se but the sensitivity to AR – see this with acne Testosterone converted to DHT at hair follicle by 5alpha-reductase For treatment, sometimes give 5alpha-inhibitors Excess androgens coming from either the ovary and/or cells of adrenal cortex. Which cells in ovary – theca? Suppress ovary with GnRH analogues and measure adrenal androgens. Then suppress adrenals with dex. and measure. In normal women the adrenal glands and ovaries secrete androgens in response to ACTH and LH respectively. Approximately half the androgen production stems from direct secretion and half from peripheral conversion by enzymes in skin, liver and adipose tissue. In women with PCOS the ovary is main source of androgen, though adrenals do contribute in about 30-50% of women

Theca grown in vitro and steroids measured. Be aware that it’s a log scale. Androstenedione is the main androgen from ovary and not testo. Comparing the androgen output/1000 theca cells from normal nad PCOS. Remember it’s not just androgens that are higher but also progesterone Stable phenotype of PCOS theca CYP17 promoter is more active CYP17 mRNA degrades more slowly

Both 17α-hydroxylase and C17-20-lyase reside on a single protein and are encoded by a single gene, namely, CYP17 Due to ↑in number of arrested follicles will see a slight ↑E2 but not DF levels in spite of ↑T – why? Levels of AR may not be increased in GC, hence cannot bind excess T No massive increase in aromatase levels

Hughesden 1982 counted follicle numbers in sections from 17 PCOs and 17 normal ovaries found 2x all of the growing stages in PCO First was pathologist Hughesden – who spent his life slicing ovaries and counting follicles – know now there were errors in his counting methods, but still borne out by other studies that an increase even in preantral follicles. This finding supports the idea that primary follicles are stockpiled in PCOS. Hence the question: does PCOS cause more primordial follicles to start growing or does it prevent atresia of preantral follicles already stimulated to grow? If recruitment were accelerated, then PCOS ovaries would be expected to contain fewer primordial follicles. This was not found. Nor were atretic preantral follicles identified in the control and PCOS ovaries. Taken together, these results support the hypothesis that primary follicles in PCOS ovaries are growing more slowly than normal. Webber et al (2003)..Lancet counted follicles in biopsies six times more primary follicles no significant increase in primordials Maciel GA et al, (2004)…JCEM Counted follicles in sections ‘stockpiling’ of primary follicles

Androgens seem likely candidate for increasing follicle numbers early in folliculogenesis Androgens involved in stimulating primordial follicle initiation and increasing number of small antral follicles LH hypersecretion amplifies androgen production by theca AR expression found in GC at all follicle stages Increased numbers at the primary stage persist to antral stages? Low/normal FSH:LH ratio reduce normal maturation Lower rate of atresia Intra-ovarian factors involving follicular recruitment & growth also contribute AMH & others TGF-β superfamily members AMH production high from granulosa cells of PCO

Excess foetal T (exposure of female animals to elevated androgens in utero) induces PCOS-like traits that manifest in offspring during adulthood: Sheep models had increased LH pulsatility and impaired E2/P feedback Offspring of T-exposed monkey mothers after puberty → LH hypersecretion, ovulatory dysfunction, hyper-androgenism and IR 50% have enlarged ovaries and increased follicles counts Adolescent girls with HA have similar pattern of rapid LH pulse secretion before menarche Obesity in pubertal girls also alters LH pulses Pregnant women with PCOS? Maternal T is raised but is fetus exposed? High levels of SHBG & aromatase activity in placenta, prevent maternal T crossing over Maybe excess secretion coming from foetus itself?

Exposure of developing hypothalamus to excess androgen before final programming of steroid feedback and other regulatory mechanisms alters GnRH pulsatility and feedback

Excess AMH in utero may affect development of female foetus This AMH arises from mother and not the foetus In women with normal fertility AMH levels would drop during pregnancy In pregnant women with PCOS AMH levels are elevated Treated pregnant mice with AMH → altered neuroendocrine phenotype of female offspring and induced PCOS-like phenotype

Polycystic ovaries are genetically acquired and may have a foetal origin There is a defect in the way follicles grow There is a basic defect in steroid metabolism The gene/s causing this are becoming known Endocrine disturbances result in miscarriage, anovulation, infertility and hyper-androgenism