Reproductive Endocrinology and Hyperandrogenism

 The gonads serve as the endocrine part of the reproductive system, with ovaries in females and
testes in males.
 This results in primary and secondary sexual characteristics, the latter emerging during puberty.
- Development hinges on the activation of sex steroid production, which increases and changes
throughout puberty.
 These sex steroids encompass estrogens, androgens, and progestogens, all stemming from
cholesterol.
 These hormones share a structure featuring three six-carbon rings (perhydro phenanthrene) and
one five-carbon ring (cyclopentane).
 They are also categorized by the number of carbon atoms they contain
- C-18 for estrogens
- C-19 for androgens
- C-21 for corticosteroids and progestogens.

How is this system made?

 Sex hormone synthesis is controlled by the pulsatile release of hypothalamic gonadotropin
releasing hormone (GnRH).
- It stimulates the release of luteinizing hormone (LH) follicle stimulating hormone (FSH) into
the general circulation.
 TLH binds to target cells (Leydig or theca cells), boosting StAR protein expression.
 StAR facilitates cholesterol transfer to inner mitochondrial membrane, starting steroidogenesis -
the key step.
 Cholesterol becomes pregnenolone at this membrane.

FSH
Women

 FSH prompts follicle cell growth, spurs granulosa cells to convert androgens (from theca cells)
into estrogen.
 Growing follicles lead to increased estrogen by granulosa cells. LH and FSH jointly drive
granulosa and theca cells for follicle maturation, culminating in ovulation.
Men:

 FSH triggers Sertoli cells in seminiferous tubules, fostering spermatogonia development and the
spermatogenic pathway.
 Notably, FSH stimulates Sertoli cells to produce ABP (Androgen Binding Protein), which
concentrates androgens like testosterone in the tubule's lumen. This high androgen concentration
aids sperm maturation.
LH
 LH impacts women by activating androgen synthesis from theca cells, primarily during secondary
follicle maturation.
- It also stimulates progesterone production from the corpus luteum post-ovulation.
 In men, LH influences Leydig cells in the testes to produce testosterone.
- Both Sertoli and Granulosa cells can affect upper structures via steroids and inhibin, a
significant hormone regulating the gonadotropic axis pulsatility, especially in men.

HPG Axis and Reproductive Organ Function

 HPG axis controls testes and ovaries’ health.
 Testes have Leydig and seminiferous components.
- Leydig cells secrete testosterone.
- Seminiferous tubules contain spermatogenesis cells and Sertoli cells.
 Sertoli cells regulate germ cell differentiation, influenced by testosterone and FSH.
 Sertoli cells secrete androgen-binding globulin (ABP), maintaining androgen and estradiol levels
in sperm fluid.
 Sertoli cells produce inhibin, selectively inhibiting FSH.
 LH triggers Leydig cells to secrete testosterone.
 Feedback loop operates at pituitary and hypothalamus levels.
 Leydig cells secrete other hormones with paracrine effects.
 Spermatogenesis needs testosterone and FSH, mediated by Sertoli cell ABP secretion.

HYPOTHALAMUS-PITUITARY-GONADAL AXIS IN MEN - HPG axis

 This axis stimulates FSH and LH synthesis. It triggers their production via distinct mechanisms in
response to GnRH stimulus.
 It promotes synthesis and production of FSH and LH after receiving a stimulus that is given by
GnRH.
 The patterns of steroidogenic glands are dictated by cell-specific expression of specific
steroidogenic enzymes.
 The only enzyme not present at the level of adrenal gland are aromatase.
 There are two types of enzymes involved in steroidogenesis
- HSD: hydroxo-steroid-dehydrogenase
- Cytochrome b 450
 The synthesis of steroids always starts with the internalization of cholesterol within the inner
membrane of the mitochondria and the cleavage of the side chain of cholesterol.
 Two enzymes handle these steps:
- STAR (Thyroid Activation Receptor), with various family members depicted in the image.
- CYP11
 The end product of these steps is pregnenolone, a lipid that easily moves through the
mitochondria membrane to the endoplasmic reticulum. This molecule serves as the precursor to
all steroid products (androgens, progestins, estrogens, glucocorticoids, and mineralocorticoids).

Where does cholesterol originate?

  Cholesterol has various sources. Cells can synthesize their own cholesterol, or it can be absorbed
from the bloodstream.
 This absorption involves specific pathways: an endocytic pathway for LDL and a selective
pathway for HDL. Regardless of its origin, cholesterol is incorporated into the mitochondrial
membrane through a regulated signal cascade.
 This cascade can be triggered by gonadotropins (FSH and LH) or ACTH, originating from the
gonads or adrenal gland respectively.
 In the context of testicular steroidogenesis in Leydig cells, there are two pathways: a classic
(delta-5) pathway and an alternative one. It's important to highlight that some intermediary
hormones

Source of Sex Steroid in Men

 Sex steroids in men primarily originate from peripheral conversion of preandrogens.
 While some conversion occurs in the testis, the adrenal gland is the main site.
 Notably, DHEA (conjugated with an S group) is extensively converted into androgens within the
adrenal gland, serving as a reservoir for androgen synthesis in both genders.

The Ovary and Oocyte Maturation

 The ovary houses oocytes at various maturation stages from puberty onward.
 Initial stage: Primary oocytes encased by a few granulosa cells.
 Post-puberty, some of these begin maturing, triggering new granulosa cell growth.
 Upon becoming FSH-sensitive, the true maturation begins, culminating in ovulation.
 Granulosa cells multiply, forming layers and the outermost layer transforms into Theca cells.
Most units degenerate in the menstrual cycle; only one or two mature and ovulate. The follicle
secretes fluid, becoming a Graaf or antral follicle.
 Fluid pushes the oocyte to the ovary's edge until release - ovulation. The dominant follicle
successfully matures. Theca cells produce androgens, while granulosa cells yield estrogens and
progesterone.

Ovarian Follicle Development in Brief:

 Ovarian follicle = egg + granulosa cells.
 Follicles grow, some mature, others regress.
 Few mature follicles respond to FSH.
 Signal -> more granulosa cells & theca layer.
 One dominant follicle fully develops, others regress.
 FSH/LH and intra-ovarian factors control growth.
 LH stimulates theca, produces androgens.
 Androgens move to granulosa, converted to estrogens.
 Estrogens have systemic effects.
 Ovarian steroidogenesis: Shared process between cells.
FSH triggers initial steroid enzyme expression in granulosa cells, converting cholesterol to pregnenolone.
Aromatization enzymes for estrogen synthesis follow. Granulosa cells gain LH receptors, enhancing
aromatase due to LH sensitivity.
Theca cells, lacking full cholesterol activation, rely on diffused pregnenolone from granulosa cells. Theca
cells self-sufficiently produce androstenedione. Granulosa cells complete androgen conversion.
Androgens circulate, some becoming estrogens, influencing ovaries.
Luteal phase sees corpus luteum form from follicle remnants. Granulosa cells turn luteal, producing
progesterone with LH support.

Premenopausal and postmenopausal hormone levels

 In women of reproductive age, the bloodstream contains varying hormone levels based on their
reproductive stage.
 Androgens are produced by both the ovaries and adrenals.
 The primary androgen is DHEA-S, present in micrograms per ml, followed by DHEA in
nanograms per ml.
 Androstenedione circulates at about 1 ng/ml. Testosterone, dihydrotestosterone, and estrone are
present in even lower concentrations due to their position in the steroidogenesis pathway.
 Around 50% of reproductive-age women's total circulating testosterone comes from peripheral
conversion, mostly from DHEA or DHEA-S, while the remaining 50% is produced by the ovaries
and adrenals.
 The adrenal's role is noteworthy and relevant in women's health. Androstenedione is equally
produced by both sources.
 Dihydrotestosterone is minimally present in women and is mainly generated in peripheral cells.
In contrast, estrogen is primarily secreted by the ovaries, with only a small portion coming from
peripheral conversion.
 Menopause leads to significant changes, especially in estrogen levels. Estrogen depletion occurs
as the ovaries age.
 Androstenedione production remains relatively balanced between the two sources, but adrenal
testosterone production greatly decreases due to adrenal senescence.
 The adrenal's contribution to DHEA(-S) remains high as the ovaries cannot produce it.

Menstrual Cycle
 The menstrual cycle involves four key compounds: two gonadotropins, estradiol, and
progesterone. It features intricate hormonal interactions among the hypothalamus, anterior
pituitary, and ovary. The cycle unfolds as follows:
1. FSH levels rise, initiating follicle growth in the early follicular phase.
2. This triggers a rise in estrogen levels in the bloodstream.
3. Positive feedback leads to increased LH levels, driven by estrogen production from the
follicle.
4. Estrogen peaks, followed by an LH peak. Ovulation typically occurs around 24 hours after
the estrogen peak, marking the Luteal phase.
5. The Luteal phase spans about 14 days with stable estrogen levels and gradual progesterone
increase post-ovulation. LH and FSH decline. High progesterone acts on the uterine wall. In
case of fertilization, LH is replaced by human chorionic gonadotropin.
6. In the follicular phase, estrogen stimulates uterine wall proliferation. In the subsequent phase,
it encourages glandular secretion, aiding embryo implantation if fertilization transpires.
 Slide 1: Ovarian Cycle Overview
The endometrial changes are tied to ovarian hormone
fluctuations, driven by hypothalamus and pituitary
signals from puberty. Monthly, follicles mature under
FSH, one ovulates, others regress. Follicle maturation
takes 14 days, named follicular/estrogenic phase due
to estrogen release. Estrogen thickens endometrium,
aligning with uterine proliferative phase.

Slide 2: Follicular Phase and Oocyte Modifications

- Granulosa cells surround oocyte forming granular membrane and refractive zona pellucida.
- Follicular cells secrete liquor follicle, forming antrum.
- Vesicular follicle holds oocyte, antrum, cumulus oophorous (at thickened granular
membrane).
- Theca cells layer around granulose membrane (theca interna/externa). Mature follicle
contains oocyte, antrum, granulosa, and theca cells. Follicle rupture signals phase end,
granulosa and theca transform into corpus luteum.
Slide 3: Ovulation and Secretory Phase

- LH surge (23h before ovulation) prompts oocyte release. In 28-day cycles, ovulation is day
14; longer cycles delay it.
- Post-ovulation, uterine secretory (lutein/progestin) phase starts. LH guides corpus luteum,
promoting uterine readiness for fertilized oocyte via progesterone, boosting endometrial
gland secretion.
- Adequate progesterone halts LH via direct and GnRH blocking. Waning LH triggers corpus
luteum regression.
Slide 4: Uterine Cycle

- Day 1: Menstrual cycle begins with bleeding.

- Uterine mucosa (ENDOMETRIUM) experiences 3 phases:
1. Menstrual phase: 4-5 days of bleeding.
2. Proliferative phase: Endometrium cell and gland growth, stimulated by estrogen. Lasts 10-11
days, aligns with ovarian estrogenic/follicular phase.
3. Secretory phase: Endometrial glands become active due to progesterone. Lasts 14 days.
- If embryo doesn't implant, corpus luteum regresses. Reduced progesterone levels lead to
endometrial degeneration, detachment, and new menstrual bleeding.

Infertility
 Unlike sterility, which is permanent, infertility can be reduced or reversed. As per WHO, it's a
pathology where conception doesn't happen after 1-2 years of regular unprotected sex.
  It's increasingly common, affecting around 50% of Italian couples, partly due to delayed
pregnancies.
 Both genders can experience infertility, with causes including unexplained factors. Some couples
can conceive with others but not together.
In Females

 Female infertility often results from aging, especially after 30, due to aging gametes.
 Quality of older oocytes decreases, alongside mechanical and inflammatory issues like
endometriosis.
 Females start with 7 million primordial cells before birth, but only around 40,000 reach puberty.
Only a fraction matures during cycles and respond to FSH.
In Males

 In males, puberty marks a distinct phase as they retain spermatogonia, perpetually undergoing
uneven divisions.
 Sperm maturation spans around 70 days. To sustain fertility, rapid cycles occur, maintaining
various spermatozoa developmental stages in seminiferous tubules concurrently. Initial mitosis
yields primary spermatocytes, which undergo meiosis for further differentiation.
 These germ cells are vulnerable to external factors impacting fertility during their crucial activity
over this extended period.
 Upon spermatid differentiation, cells are released into the tubule lumen, aided by Sertoli cells in
maturation. Along the tubules, they transform into active spermatozoa.
 Aging moderately affects male gamete vitality and function.

Age and Oocytes

 Oocyte age significantly impacts fertility.
 Incidence increases with age: from 1 in 900 at 30, when pregnancies peak, to 1 in 25 at 45.
 Miscarriage rates rise, notably at 40 (34% vs. 18% at ≤30). Diminished oocytes and sperm quality
compound the issue.
 Uterine morphology, inflammation, and fallopian tube blockages also affect fertility.
 Natural selection heightens miscarriage risk due to malformations: 18% (30-39y), 34% (≈40y),
10% (<30y).
 Fewer and lower-quality oocytes contribute. Around 38, oocyte numbers decline, often yielding
abnormal embryos.
 Assisted reproduction success hinges on age; older women yield fewer, chromosomally altered
oocytes.

Age and Uterus

 Placental issues can lead to miscarriage, age-related complications arise.
 Fibroids limit embryo implantation. Lesions pre-fertilization can obstruct uterine arteries.
 Uterine mispositioning affects fetus. Inflammation, like endometriosis, hampers uterine activity.

Excess Androgens in Females: Causes and Symptoms

Cause: Female hyperandrogenism, characterized by elevated levels of androgens (including testosterone),
can be attributed to various factors. The primary contributor, accounting for 70% of cases, is Polycystic
Ovary Syndrome (PCOS), a common endocrine disorder affecting up to 10% of women in reproductive
age. Additional causes include adrenal hyperplasia, Cushing's disease, specific cancers, and certain
medications.
Symptoms: The manifestations of hyperandrogenism resemble those of PCOS and encompass acne,
alopecia (hair loss), and hirsutism (excessive body hair). This condition presents two distinct phenotypes:
1. Biochemical Phenotype: This involves identifying elevated levels of testosterone and other
androgens in the bloodstream.
2. Clinical Phenotype: Clinical presentation involves hirsutism or alopecia. Remarkably, only half
of hyperandrogenic women exhibit visible clinical symptoms.

Causes of Female Hyperandrogenism

The diagnosis of PCOS is established by excluding all other potential causes, highlighting its complex
nature. Several factors can contribute to female hyperandrogenism:

- Congenital Adrenal Hyperplasia: This arises from deficiencies in enzymes related to

glucocorticoid synthesis in the adrenal glands.
- Cushing's Syndrome: This condition involves the adrenal glands, leading to excess production of
glucocorticoids, and sometimes androgens.
- Tumors: Certain tumors can trigger increased androgen production.
- Ovarian Hypethecosis: Hyperproliferation of Theca cells within the ovaries can contribute to
androgen excess.
- Idiopathic Hirsutism: In cases where the source is unknown, the term "idiopathic" is used to
describe excessive hair growth.

PCOS
 PCOS, or Polycystic Ovary Syndrome, is a common condition affecting around 10% of
individuals, varying by ethnicity.
 Prevalence is lowest in the Far East and highest in the Mediterranean Area within Europe.
 Discovered in 1935 by gynecologists linking amenorrhea to polycystic ovaries, PCOS is linked to
a 50-60% higher risk of type 2 diabetes in affected women.
 Diagnosing PCOS involves excluding other causes, as its origin is uncertain. Criteria include
menstrual irregularities, PCO morphology, and androgen excess, though focus initially centered
on ovaries rather than hormone levels.
 The Rotterdam criteria (2003) remain the most widely used diagnostic tool, requiring the
presence of any two of these three criteria:
1. Oligo-anovulation
2. Biochemical or clinical hyperandrogenism
3. Polycystic ovaries

Ovarian Dysfunction in PCOS: Menstrual Irregularities and Ovulation Issues

Menstrual Cycle Irregularities:

- Mild-moderate oligomenorrhea (>35-day cycle)

- Severe oligomenorrhea (>60-day cycle)
 - Amenorrhea (no menstruation for ≥6 months)

Ovulation Problems:

- Chronic or cyclic anovulation (elevated LH)

- Polymenorrhea/oligomenorrhea (frequent bleeding, no ovulation)
- Early pubarche
- Oligomenorrhea is present in 100% of those with menstrual irregularities.

Ovarian Morphology:

- In PCOS, ovaries have multiple large follicles on the periphery, differing from normal ovaries
with follicles within the parenchyma.

PCO Morphology (Rotterdam’s criteria):

 Clinical indicators include hirsutism; androgenic alopecia and acne are no longer considered
specific markers for defining hyperandrogenism.
 Hirsutism: Excessive terminal hair in androgen-dependent areas; a chronic issue with
psychological and metabolic impacts. Associated with a high risk of insulin resistance.
 Etiology: Hirsutism arises from elevated androgens in the bloodstream, increased peripheral
sensitivity, or heightened androgen precursor activation. Determining the primary cause can be
challenging.
 Differentiating Terminal and Vellus Hair: Terminal hair causes hirsutism and follows a male
pattern in areas like face, chest, abdomen, and back. Vellus hair is finer and usually found in these
areas in women.

Alopecia
 Progressive non-scarring miniaturization of hair follicles in defined areas of the head (scalp), in
genetically predisposed males and females.
 HAIR GROWTH CYCLE: Alopecia scale depends on the hair growth cycle. Here's a brief
explanation of its four phases:
1. Anagen: Hair growth and color development.
2. Catagen: Growth pause.
3. Telogen: Shedding, followed by new hair growth.
4. Kenogen: Interval between shedding and new growth. In androgenic alopecia, hormonal
imbalance can lengthen this phase.

Pathogenesis
 Pathogenesis involves evaluation through scales. Notable features are:
1. Follicular miniaturization
2. Prolonged Kenogen phase (empty follicles)

Biochemical hyperandrogenism
Identifying elevated androgens. Which androgens? How?

 For women, assessing androgen excess starts with total testosterone measurement.
  Most testosterone binds to sex-hormone-binding globulin (SHBG), usually low in overweight
hyperandrogenic patients. This can mask excess androgens.
 To overcome this, until direct free testosterone assays emerge, SHBG and calculations are used to
estimate free testosterone.
 Testosterone is a steroid, so a lipid, and does not circulate in the free form, but it is mainly bone
to SHBG (sex-hormone-binding-globulin)

Beyond Testosterone Measurement: Role of Laboratory

 In assessing elevated androgens in BS, excluding alternate causes is pivotal.
 High T levels warrant tumor investigation; DHEAS measurement, secreted solely by adrenals,
determines it. Positive DHEAS indicates tumor possibility, else further tests and imaging.
 Moderate testosterone levels, typical in PCOs, necessitate excluding adrenal steroidogenesis
enzymatic deficiency. In pediatric cases, notably newborns, 21-hydroxylase deficit is key.
 Hyperandrogenism and metabolic syndromes intertwine through insulin. Insulin influences Theca
cell and Granulosa cell response, impacting androgen secretion and FSH sensitivity.
 Excess androgens from Theca cells lead to hirsutism. Lower Granulosa cell sensitivity reduces
androgen-to-estrogen conversion, disrupting circulation. Ovulation falters, affecting Granulosa
cells.
 This affects axis pulsatility, as androgens hinder estrogen feedback to the hypothalamus.
 Result: excess androgens, but normal/increased estrogens, based on Granulosa cell FSH
sensitivity. LH and FSH imbalances follow: elevated LH, normal/reduced FSH.
 Insulin and LH foster Theca cell androgen synthesis, meant for Granulosa cell estrogen
conversion. If FSH-insensitive Granulosa cells obstruct, overall axis suffers, thwarting ovulation.

Congenital Adrenal Hyperplasia

 It is a genetic condition causing excessive androgens. It stems from a defect in 21-Hydroxylase,
hindering cortisol synthesis.
 Cortisol is vital for survival, along with aldosterone derived from progesterone.
 Affected patients lack mineralocorticoids, leading to increased androgen production, mainly
testosterone.
 The hypothalamus-pituitary unit reacts by enlarging the adrenal glands, futilely trying to make
glucocorticoids.
 High androgens, notably 17OH-progesterone, result, leading to 21deoxycortisol formation.
Detecting this compound is challenging but essential for accurate diagnosis, distinguishing it
from PCOS and aiding prenatal screenings.
 Research aims to quantify it using mass spectrometry.
 Newborn screening occasionally catches the related 17 OH PS elevation, but this is rare. Milder
cases allow some cortisol and androsterone production, eventually causing hyperandrogenism in
women due to surplus androgen precursors.

Pros and Cons of Total Testosterone Measurement & Suitable Methods

SHBG measurement is essential to determine free testosterone. The optimal method for total testosterone
assessment includes:
 - Radioimmunoassay (RIA) with sample extraction and chromatography (obsolete).
- Electrochemiluminescence immunoassays, commonly used and versatile.
- Gas chromatography – mass spectrometry (GC-MS), complex yet effective.
- Liquid chromatography – tandem mass spectrometry (LC-MS/MS), highly specific and used in
our lab
Interpreting results requires various considerations, notably the patient's age, which applies to both
testosterone and DHT.

Circulating Steroid Hormone Levels in Women

 This overview focuses on the presence of major steroid hormones in the blood, either in their free
state or bound to specific proteins like SHBG for androgens and estrogens, or CBG for cortisol.
 While DHT and Testosterone are primarily bound to SHBG, albumin also plays a significant role.
 The "Free Hormone Theory" posits that only unbound T is active and can bind to androgen
receptors in target tissues. However, this theory has been questioned, with an alternative
suggesting both free T and weakly bound T contribute to androgen effects.
 "Bioavailable T" encompasses free T and weakly bound T, supported by the belief that albumin-
bound T can dissociate in capillaries, particularly in tissues with longer blood transit times.
 Measuring free testosterone levels poses challenges due to their extremely low concentrations in
blood.
 Techniques like ultrafiltration can work, but routine methods often yield unreliable results. SHBG
has functions in modulating free and bioavailable sex steroid hormones. DHT's binding affinity to
SHBG is notably higher than that of testosterone and estradiol.
Measuring Free Testosterone:

 The primary biomarker for female hyperandrogenism is free testosterone, but its measurement is
complex.
 Direct methods such as direct equilibrium dialysis and ultrafiltration are accurate but resource-
intensive. Routine methods like direct RIA are simpler but less reliable.
Measuring Bioavailable Testosterone:

 Measuring T in saliva is a potential method due to the diffusion of non-SHBG-bound T across

capillaries and salivary ducts.
 High-sensitivity assays are required due to saliva levels representing a small fraction of
circulating levels. However, this approach is not yet routine.

Quantifying Free Testosterone:

 Several formulas exist, with the Vermeulen formula being the most accurate but complex.
 Clinicians often use the Free Androgen Index (FAI) [total testosterone] x 100 / [SHBG] due to its
simplicity. Yet, FAI tends to overestimate cFT when SHBG levels are low, as seen in conditions
like PCOS.

Insulin's Role in PCOS:

  While insulin isn't a primary diagnostic factor, it does contribute to metabolic issues in a notable
portion of women with PCOS
 Insulin operates akin to LH, stimulating androgen secretion by Theca cells. This fuels a
detrimental loop of hyperandrogenism and hyperinsulinemia.
 In insulin resistance, multiple tissues, except the ovary, are affected, amplifying androgen
production.
 Insulin and chorionic gonadotropin work in tandem, potentiating insulin's anabolic influence.
Conversely, androgen excess worsens insulin resistance, affecting energy metabolism, promoting
inflammation, and impacting adipose tissue.
 The cycle perpetuates: many diagnosed women have both insulin resistance and
hyperandrogenism, making their chronological sequence unclear. Treatment strategies address
both PCOS and metabolic aspects.
Obesity's Impact

 Obesity compounds insulin resistance and PCOS. Visceral fat accumulation, common in PCOS,
fosters metabolic syndromes like type 2 diabetes.
 While not a diagnostic criterion, obesity significantly affects PCOS outcomes. Obese PCOS
individuals are more prone to various diseases.
 Mechanisms involve insulin issues, obesity-related inflammation, and androgen excess, alongside
genetic factors and phenotype variations.