REPRODUCTIVE ENDOCRINOLOGYSpecialist Portfolio Seminar 23rd June 2014

Katie Jones

Sandwell and West Birmingham Hospitals NHS Trust

Overview

• Hypothalamic – pituitary – gonadal axis• Females• Males

• Overview of reproductive hormones• Pathophysiology & Investigation

• Female• Male

• Analytical considerations• Example cases• Questions

Hypothalamic-pituitary-gonadal axis

Hypothalamic-pituitary-gonadal axis

• Gonadotrophin axis most “fragile”

• Similar to other hormonal systems measurement of one hormone can be difficult to interpret

• Feedback loops

• Pulsatile secretion: GnRH, LH

Reproductive Hormones

• FSH• LH• Progesterone• Prolactin• Testosterone• Oestrogen (oestradiol)• SHBG

Sex steroids

http://www.sciencedirect.com/science/article/pii/S0197458009004072

Produced in ovary, testis and adrenal gland

Female reproductive axis

• Oestrogen usu –ve feedback inhibition of FSH and LH but positive feedback to produce LH surge & ovulation

Hypothalamus

Pituitary

Ovary

GnRH

LHFSH

ProgesteroneOestradiol

-

-

-

-

Menstrual cycle

• Timing of measurement!

• Avoid LH ‘surge’

• Detect progesterone peak

http://en.wikipedia.org/wiki/Ovulation

LH & FSH

FSH granulosa cell• Cell differentiation• ↑ Aromatase• ↑ LH receptors on granulosa cells• Actions amplified by oestradiol

LH theca cells• Androgen production

LH mature granulosa cells• Oestradiol & progesterone production

LH & FSH: 2 cell hypothesis

• Different enzyme expression• Aromatase only present in granulose cell

Theca cell

cholesterol

testosterone

androstenedione

Granulosa cell

testosterone

androstenedione

oestradioloestrone

aromatase

FSH

LH

cholesterol

progesterone

X

progesterone

Oestrogen & Progesterone

Oestradiol

• Maintains function of reproductive tract• Endometrial thickening

• Mucus secretion at ovulation

• Secondary sexual characteristics

• Systemic effects• Bone density

• Cardioprotective

Progesterone

• Prepares endometrium for implantation

• Essential for maintenance of early pregnancy

• Day 21 progesterone – indicates ovulation• SWBH reference range: follicular <1; luteal 1-16; post menopausal <1

• Window 2 days either side acceptable

• If menstruation is irregular, samples can be taken several times a week until

menstruation occurs

Prolactin

• Produced by lactotrophs of anterior pituitary• Negative control by dopamine• Positive feedback by oestrogens and TRH

• Direct effects• Milk production

• Measure in females with:• Oligo or amenorrhoea• Galactorrhoea• Subfertility

• Measure in males with• Hypogonadotrophic hypogonadism with unknown cause

Male reproductive axis

FSH & LH

LH Leydig cells• Androgen production

FSH seminiferous tubules (Sertoli cells)• Spermatogenesis

Testosterone & DHT

Testosterone• Male reproductive development (foetal & pubertal)

• Male secondary sex characteristics

• Spermatogenesis

• Bone mass (male & females)

• Also effects on erythropoiesis, lipids

Dihydrotestosterone• Peripheral conversion from testosterone by 5α-reductase (prostate,

skin)

• Testosterone has direct & indirect effects

• In some tissues testosterone is a prohormone for DHT

• Male reproductive development requires both

• DHT measurement indicated in investigating some DSD

Testosterone circadian rhythm

SHBG

• Sex Hormone Binding Globulin• Glycoprotein produced in the liver

• High affinity for testosterone & DHT• Testosterone:

• 44-65% bound in males• 66-78% in females

• Not important for DHEA/DHEA-S

• N.B. Albumin also binding protein:• DHEA & DHEA-S primarily bound to albumin• Testosterone & DHT: ~33-50% bound in males, ~20-30% in

females (‘bioavailable’)

Causes of a high SHBG

• High oestrogen (female sex, pregnancy, HRT, OCP)

• Hyperthyroidism

• Liver disease

• Anorexia (?insulin/IGF-1)

• Anticonvulsant drugs phenytoin and phenobarbitone (hepatic enzyme induction)

Causes of a low SHBG

• High androgen (male sex, androgen use)

• Hypothyroidism

• Insulin resistance

• Obesity

• Diabetes

• Cushing’s disease

Effect of Age

• high in childhood, decrease in puberty (male>female),

• increase in elderly male, decrease in post-menopausal female

Infertility

• Defined as failure to conceive after 12 months of regular sexual intercourse without contraception

• Estimated that infertility affects 1 in 7 heterosexual couples (UK)

• Causes:• unexplained infertility (25%)• ovulatory disorders (25%)• tubal damage (20%)• uterine or peritoneal disorders (10%)• factors in the male causing infertility (30%)

• ~40% of cases disorders are found in both the man & woman

Infertility - female

• Role for biochemical investigations:• Confirming ovulation (regular & irregular cycles):

progesterone• Investigating irregular menstrual cycles: FSH & LH• Ovulatory disorders: prolactin• Predicting response to IVF stimulation protocol: AMH & FSH

• Women with regular monthly menstrual cycles are likely to be ovulating

• In women with prolonged irregular menstrual cycles measurement timing of progesterone may need to be adjusted e.g. day 28 of a 35-day cycle & repeated weekly thereafter until the next menstrual cycle starts

Infertility - male

• Semen analysis (WHO guidelines)

• Endocrine:• Testosterone• LH• FSH

Hyperprolactinaemia

Common causes:• Dopamine antagonists (dopamine negative feedback)• Stress• Pregnancy

Macroprolactin• IgG complex• Low bioactivity• Should be screened to avoid unnecessary investigations

Oligo- or amenorrhoea

• Oligomenorrhoea = menstrual cycle length > 6 weeks but <6 months

• Amenorrhoea = absence of menstruation or cycle length >6 months

• Ovulation is infrequent

Ovulation disorders

• Hypothalamic pituitary failure • hypothalamic amenorrhoea or hypogonadotrophic hypogonadism• E.g. low BMI, excessive exercise

• Hypothalamic-pituitary-ovarian dysfunction• predominately polycystic ovary syndrome• Hyperprolactinaemia• Hypothyroidism (TRH prolactin)

• Ovarian failure• Primary – cause often unknown• Secondary e.g. post radiotherapy, surgery• “Premature” if <40y (definition can vary)

PCOS

• Two of the following:

• Oligo- or an-ovulation

• Clinical &/or biochemical evidence of hyperandrogenism

• Polycystic ovaries (ultrasound scan)

• And other causes excluded (e.g. late onset CAH, adrenal / ovarian tumours, Cushing’s syndrome)

• therefore biochemical tests usually performed

• Testosterone & androstenedione often raised, DHEA-S may also be raised

• LH may be raised, FSH normal

• Similar presentation possible with late onset CAH: • measure 17α-hydroxyprogesterone concentrations (SST)

PCOS

• Management dependent upon the desired outcome e.g. promote fertility, reduce hirsutism etc

• Weight loss• OCP• Metformin

Female: excess androgens

• Hirsutism• Excessive androgen-dependent hair growth

• Note difference with hypertrichosis• Normal/mildly raised androgens

• Virilisation• usually marked increase in androgens• manifestations include temporal hair recession, clitoromegaly,

increased muscle mass, breast atrophy, deepening of voice, oligo/amenorrhoea

• Grossly elevated testosterone (>5 nmol/L) with sudden onset hirsutism/virilisation more worrying

Pregnancy

• Oestradiol: increases throughout• Progesterone: increases throughout• Prolactin: increases throughout• LH & FSH: suppressed

• N.B. Total testosterone also increases (up to 10X pre-pregnancy values) during pregnancy• SHBG increases during 1st trimester• Free testosterone should remain constant

Male hypogonadism

The diagnosis of hypogonadism is based upon• Appropriate symptoms• Measurement of testosterone in the morning on >1 occasion

• Ideally 9am, in practice 7-11am

• Prepubertal testicular failure• Lack of sexual maturation• Increased arm span (delayed epiphyseal closure)

• Post-pubertal symptoms less obvious• Decreased libido, impotence, infertility• Increased oestradiol/testosterone ratio can lead to

gynaecomastia

Male hypogonadism

Primary (i.e. testicular dysfunction)• Genetic

• Klinefelter’s syndrome (most commonly 46,XXY)

• Cryptorchidism

Secondary (i.e. pituitary or hypothalamic dysfunction)• Congenital

• Kallman’s syndrome

• Acquired

• N.B. Can see both testicular and pituitary dysfunction e.g. haemochromatosis

Investigation

• LH & FSH• differentiate primary or secondary (hyper- & hypo-gonadotropic

respectively)

• Prolactin • If testosterone <5.2 nmol/L• or when secondary hypogonadism suspected

Treatment:• Testosterone replacement

Male ageing

• Male ‘andropause’• Reduced testosterone with ageing• Debate over treating borderline low testosterone in ageing

male• Improved muscle mass, BMD• Possible effects on mood, QOL, insulin resistance & diabetes• Possible risks of prostate Ca, CVD• Awaiting long term outcome studies

Dynamic function testing

• hCG stimulation test • Distinguish primary testicular failure from gonadotrophin

deficiency• Confirm presence of testicular tissue (cryptorchidism)• Combined pituitary and testicular dysfunction e.g.

haemochromatosis

• GnRH stimulation test• Same principle• Rarely used

Interpretation of reproductive hormones

Consider:• Age• Sex (gender reassignment)• Pubertal status• Pregnancy• Medications

• OCP, anabolic steroids• Testosterone replacement (high variation possible with injections)• Prostate cancer Tx (suppressed testosterone)

• Free hormone levels (SHBG)• Timing

• E.g. Within menstrual cycle • E.g. Testosterone circadian rhythm

• Peak 4-8am, nadir 4-8pm• Request 9am testosterone

• Pituitary hormones LH, FSH – are these appropriate?

Analytical considerations

Peptides: 2 site immunoassays• FSH• LH

(shared alpha subunit, also hCG and TSH)

• Prolactin• macro-prolactin cross-reactivity variable• effects of PEG on assay

• SHBG

Analytical considerations

Steroids:• Total or free hormone (usually total)• Detection limits (esp oestradiol – pmol/L levels early

follicular phase)

• Testosterone• Oestradiol• Progesterone

Assays: testosterone

Total testosterone• Reference method: GC-MS

• Immunoassay

• Require displacement of steroid from binding protein

• Good agreement with GCMS in male range but poor agreement at low end

• For prepubertal & female testosterone direct IA should not be used

• Extraction prior to IA may give improved results

• Usually some (3-5%) cross-reactivity with DHT - ?importance

• DHT concentrations usually <20% of testosterone

• E.g. Architect Testo II

• V high cross-reactivity nandrolone (19 nortestosterone, synthetic steroid)

• LC-MS/MS (greater specificity)

Assays: testosterone

Free testosterone:• Equilibrium dialysis/ultrafiltration• Bioavailable testosterone (non-SHBG T) – precipitate SHBG-T

with ammonium sulphate• Immunoassay (gives lower values than ED)

• Estimated:• FAI (female): T/SHBG * 100

(Assumes binding capacity of SHBG greatly exceeds testosterone concentration therefore unsuitable for males)

Note ref range dep on T & SHBG method.• Calculated free testosterone (male): Vermuelen eqn

Uses T, SHBG, albumin (http://www.issam.ch/freetesto.htm)

Case 1

• Male age 63y• Clinical details: ?subfertility• O/E: no abnormality

• Testosterone 39.5 nmol/L (8.0-30.0)

On repeat:• Testosterone 28.0 nmol/L (8.0-30.0)• SHBG 103.4 nmol/L (11.0-78.8)

Case 1

• Further investigation:• AFP & hCG: within reference range• USS testes: NAD• CT abdomen: 1cm adrenal lesion• MRI adrenal: inconclusive• Referred to Endocrinology

• Endocrinology:• High SHBG calculated free testosterone• Why is SHBG high?

Causes of a high SHBG

• High oestrogen (female sex, pregnancy, HRT, OCP) • Hyperthyroidism• Liver disease• Anorexia (?insulin/IGF-1)• Anticonvulsant drugs phenytoin and phenobarbitone (hepatic

enzyme induction)

Free Testosterone

Calculated alternatives:• Bioavailable testosterone

• Free testosterone and Albumin-bound testosterone• Bio T = fT + k*A*fT (A = albumin)

• Free androgen index (female)

• Calculated free testosterone• Vermuelen eqn (most accessible, most widely quoted, verified against

gold standard fT measurement)• Online calculator (& explanation) at http://www.issam.ch/freetesto.htm

Case 1 conclusion

Raised testosterone?

• Testosterone 30.5 nmol/L (8.0-30.0)

• SHBG 123.2 nmol/L (11.0-78.8)

• Albumin 38 g/L (35-50)

• Calculated free testosterone 0.26 nmol/L (0.23-0.64)

Cause for raised SHBG?

• TFTs ok

• Suspected Hep B carrier

Learning point

• Importance of binding proteins / active hormone concentrations

Case 2:

• Male, age 22y• Testosterone 5 nmol/L (8.0 – 30.0)

• Add on gonadotrophins:• LH 1 IU/L• FSH 2 IU/L

• DB phones GP• Possible anabolic steroid use

Inappropriately low

Anabolic steroids

• Synthetic derivatives of testosterone: aim to maximise anabolic effects & minimise androgenic effects• Testosterone 1:1• Stanozolol 30:1 anabolic:androgenic

• High serum androgens cause suppression of gonadotrophin release• Serum testosterone, LH, FSH & SHBG - all low

• Males: hypogonadotophic hypogonadism• Intra-testicular levels low (testicular levels usually >50x serum levels)

impaired spermatogenesis, testicular atrophy• Reversible effects, but 3-12 months for full axis restoration

• Females: virilisation• Some changes may be permanent (facial hair, voice deepening, scalp hair

loss)

• Can use hCG to ‘kickstart’ axis (N.B. hCG may be abused simultaneously)

Case 3

• Female age 28y• Secondary amenorrhoea

• FSH <1 IU/LFollicular 3-8: Luteal 1-5: Ovulatory 3-17: Post menopausal 27-133• LH <1 IU/LFollicular 2-12: luteal 1-14: ovulatory 9-89: post menopausal 5-62

• Oestradiol 9823

Foll 77-921: luteal 77-1145: ov peak: 140-2382: post menopaus <103

• Pregnancy test: positive

Case 4

• Female age 23y• Oligomenorrhoea, hirsute

• Testosterone 3.3 nmol/L (<2.9)

• FSH 5 IU/L Follicular 3-8: Luteal 1-5: Ovulatory 3-17: Post menopausal 27-133

• LH 10 IU/L

Follicular 2-12: luteal 1-14: ovulatory 9-89: post menopausal 5-62

• Likely PCOS

Case 5

• Male, age 52y• Testosterone 5.5 nmol/L (9.9-27.8)

• Repeat at 9am• Testosterone 6.0 nmol/L• Clinical details: ED

• Add FSH/LH• Both low

Case 5

Add:• Prolactin• TSH / fT4• Cortisol

• Suggest endocrine referral

References / Further Reading

• NICE CG156: Fertility (2013)

• Guidelines on the management of sexual problems in men: the role of androgens (British Society for Sexual Medicine 2010)