REPRODUCTIVE ENDOCRINOLOGY
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1 Overview REPRODUCTIVE ENDOCRINOLOGY Specialist Portfolio Seminar 23 rd June 2014 Katie Jones Sandwell and West Birmingham Hospitals NHS Trust 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 1
2 Reproductive Hormones Sex steroids FSH LH Progesterone Prolactin Testosterone Oestrogen (oestradiol) SHBG Produced in ovary, testis and adrenal gland Female reproductive axis Menstrual cycle - Hypothalamus - - GnRH Pituitary - Timing of measurement! FSH LH Avoid LH surge Ovary Detect progesterone peak Oestradiol Progesterone Oestrogen usu ve feedback inhibition of FSH and LH but positive feedback to produce LH surge & ovulation 2
3 LH & FSH FSH granulosa cell Cell differentiation Aromatase LH receptors on granulosa cells Actions amplified by oestradiol LH & FSH: 2 cell hypothesis Different enzyme expression Aromatase only present in granulose cell FSH LH cholesterol LH theca cells Androgen production LH mature granulosa cells Oestradiol & progesterone production cholesterol progesterone X androstenedione Granulosa cell testosterone aromatase oestrone oestradiol progesterone androstenedione testosterone Theca cell Oestrogen & Progesterone Prolactin 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 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 3
4 Male reproductive axis FSH & LH LH Leydig cells Androgen production FSH seminiferous tubules (Sertoli cells) Spermatogenesis Testosterone & DHT Testosterone circadian rhythm 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 4
5 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 Infertility - female 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 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 5
6 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 Ovulation disorders Oligomenorrhoea = menstrual cycle length > 6 weeks but <6 months Amenorrhoea = absence of menstruation or cycle length >6 months Ovulation is infrequent 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) 6
7 PCOS 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 Management dependent upon the desired outcome e.g. promote fertility, reduce hirsutism etc Weight loss OCP Metformin 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) Female: excess androgens Pregnancy Hirsutism Excessive androgen-dependent hair growth Note difference with hypertrichosis Normal/mildly raised androgens Oestradiol: increases throughout Progesterone: increases throughout Prolactin: increases throughout LH & FSH: suppressed Virilisation usually marked increase in androgens manifestations include temporal hair recession, clitoromegaly, increased muscle mass, breast atrophy, deepening of voice, oligo/amenorrhoea N.B. Total testosterone also increases (up to 10X prepregnancy values) during pregnancy SHBG increases during 1 st trimester Free testosterone should remain constant Grossly elevated testosterone (>5 nmol/l) with sudden onset hirsutism/virilisation more worrying 7
8 Male hypogonadism 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 Primary (i.e. testicular dysfunction) Genetic Klinefelter s syndrome (most commonly 46,XXY) Cryptorchidism 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 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 8
9 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) Analytical considerations Steroids: Total or free hormone (usually total) Detection limits (esp oestradiol pmol/l levels early follicular phase) Prolactin macro-prolactin cross-reactivity variable effects of PEG on assay Testosterone Oestradiol Progesterone SHBG 9
10 Assays: testosterone 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) 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 ( Case 1 Male age 63y Clinical details:?subfertility O/E: no abnormality Testosterone 39.5 nmol/l ( ) Case 1 Further investigation: AFP & hcg: within reference range USS testes: NAD CT abdomen: 1cm adrenal lesion MRI adrenal: inconclusive Referred to Endocrinology On repeat: Testosterone 28.0 nmol/l ( ) SHBG nmol/l ( ) Endocrinology: High SHBG calculated free testosterone Why is SHBG high? 10
11 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 Case 1 conclusion Case 2: Raised testosterone? Testosterone 30.5 nmol/l ( ) SHBG nmol/l ( ) Albumin 38 g/l (35-50) Calculated free testosterone 0.26 nmol/l ( ) Cause for raised SHBG? TFTs ok Suspected Hep B carrier Male, age 22y Testosterone 5 nmol/l ( ) Add on gonadotrophins: LH 1 IU/L FSH 2 IU/L DB phones GP Possible anabolic steroid use Inappropriately low Learning point Importance of binding proteins / active hormone concentrations 11
12 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/L Follicular 3-8: Luteal 1-5: Ovulatory 3-17: Post menopausal LH <1 IU/L Follicular 2-12: luteal 1-14: ovulatory 9-89: post menopausal 5-62 Oestradiol 9823 Foll : luteal : ov peak: : 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 LH 10 IU/L Follicular 2-12: luteal 1-14: ovulatory 9-89: post menopausal 5-62 Case 5 Male, age 52y Testosterone 5.5 nmol/l ( ) Repeat at 9am Testosterone 6.0 nmol/l Clinical details: ED Add FSH/LH Both low Likely PCOS 12
13 Case 5 Add: Prolactin TSH / ft4 Cortisol 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) Suggest endocrine referral 13
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