Reproductive Flashcards
What is anti-Mullerian hormone?
Member of the TGF-beta family.
Homodimeric protein hormone consisting of 2 identical subunits
Physiological roles of AMH
- In male fetus - secreted by testicular Sertoli cells to inhibit growth and development of Mullerian structures
- In female post-puberty - released by preantral and small antral follicular granulosa cells to inhibit FSH-mediated recruitment and maturation of additional follicles, until selection of the dominant follicle occurs. May also titrate the oestrogen released by granulosa cells through desensitising the granulosa cells to FSH
How is AMH measured in your lab
One-step 2-site electrochemiluminescent immunoassay using biotin labelled capture antibody bound to streptavidin coated magnetic microparticles and ruthenium labelled detection antibodies. After incubation of sample with capture and detection antibodies, then an incubation with solid phase, microparticles are magnetically captured on to the surface of an electrode and the unbound sample washed away. A voltage is passed through the electrode which induces chemiluminescent emission which is measured by a photomultiplier.
Acceptable specimen types for AMH
Serum and Lithium heparin plasma, but NOT EDTA
A pt taking high dose biotin presents for AMH collection. What should the collector do?
Determine whether or not biotin has been taken in the past 8 hours, and if so, delay the test until the patient can stop biotin for at least 8 hours prior to the test.
Indications for AMH testing
Vary between manufacturers. Roche AMH assay has been validated for the following indications:
1. Prediction of ovarian hyperstimulation
2. Individual daily dose determination of follitropin delta of Ferring
3. Prediction of ovarian reserve (correlation with antral follicular count) - note POOR correlation, LOTS of scatter, interrater variability in AFC between sonographers and sites
Other indications:
1. Diagnosis of Disorders of Sex Development
2. Granulosa cell tumour marker - primary, recurrent, tumour size
Under investigation:
1. Surrogate biomarker of antral follicle count in PCOS
What should AMH NOT be used for and why?
As a marker of general fertility or biological clock/egg timer. Women with AMH in the lowest 20% have the same fecundability (ability to fall pregnant within 1 menstrual cycle) and fertlity (ability to fall pregnant in 12 menstrual cycles) as women with AMH levels in the middle 60% of the distribution.
Advantages and limitations of AMH for PCOS
Adv:
1. Correlation with antral follicle count
2. Increased in PCOS
3. Increase correlates with symptom severity
4. Consistent throughout menstrual cycle
Disadv:
1. Concentrations affected by weight, age, smoking, COCP use, ethnicity
2. Technical challenges - standardisation, speci handling
How can AMH be used in IVF protocols?
Normally, IVF is initiated by the stimulation of follicle development using GnRH agonists.
If AMH is high, the women is at a higher risk of ovarian hyperstimulation. Long-acting GnRH antagonists can be concurrently administered to titrate the action of the GnRH agonists.
If AMH is low, a “flare” protocol can be used, using several microdoses of a GnRH agonist to prevent premature LH surges that can work against a collection that is already likely to be suboptimal
Forms of HCG
- Intact
- Free alpha subunit
- Free beta subunit
- Nicked - enzymatic cleavge of peptide bonds at position 44 and 45 of beta subunit and inactivates the hormone
- Nicked free beta
- Beta-core fragment - detectable only in urine, the predominant form in urine, the terminal degradation product of HCGbeta
- Hyperglycosylated - predominates in first 4 weeks of pregnancy
- Superglycosylated
What forms of HCG are found in urine
Predominantly beta core fragment but also unmodified hCG and hCGn
What is HCG?
Human chorionic gonadotropin. A glycoprotein. Protein core consists of a heterodimer of alpha and beta subunits. When the hCG dimer is dissociated, activity is lost.
Where is HCG synthesized?
Syncitiotrophoblast and cytotrophoblast of the placenta. Minute amounts synthesised in pituitary in men and women.
How is HCG cleared
Liver and kidney
Describe the changes in HCG in pregnancy.
Intact HCG is produced, initially in a hyperglycosylated form for the first few weeks. Synthesis of betaHCG peaks at about 8-10 weeks gestation but production of alphaHCG continues to increase in proportion to the growing placenta.
How do HCG and LH differ?
HCG has an extra 20 amino acids. Same first 115 amino acids
How do POC HCG assays work?
Principle of immunochromatography
Detects HCG when concentration exceeds a certain threshold (usually 25 IU/L)
How are HCG assays classified by analytic specificity?
Assays that detect:
1. Only dimeric/intact HCG
2. HCG and HCG beta
3. HCG, HCG beta and HCG beta core fragment
Issues around standardising HCG assays
- Most assays are standardised to WHO Fourth international standard. This standard is not pure and contains substantial amounts of hCGn and hCGbeta. Some HCG assays over- or under-recognise these variants or may not detect them at all.
- Assays are not harmonised, in that they have different antibodies that recognise different epitopes on the different subunits and forms and therefore recognise HCG variants differently.
- Secondary standards provided by assay manufacturers vary greatly in terms of purity
- Hence HCG results from one assay cannot be directly compared to another.
Why does androstenedione require collection in a clot rather than serum separator tube?
Androstenedione can adsorb into the gel in a separator tube resulting in falsely low readings. If being run by LCMS, gel can cause interference with mass spec (noisy background).
Testosterone method in your lab
Competitive chemiluminescent immunoassay. Acridinium ester label. Proprietary releasing agent to free bound testosterone from endogenous binding proteins.
Causes of a low testosterone in males
Hypogonadism either hypogonadotropic or hypergonadotropic
Hypergonadotropic: testicular failure, mumps, Klinefelter’s
Hypogonadotropic: obesity, alcohol, opioids, diabetes mellitus, liver disease, critical illness, hyperprolactinaemia, hypopituitarism, Kallman’s
Major sources of testosterone in females
Ovaries, adrenal glands, peripheral conversion of precursors esp androstenedione to testo
Causes of a high testosterone in females
PCOS, CAH, Cushings, adrenal/ovarian tumours
