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Analytes > Reproductive > Flashcards

Reproductive Flashcards

1
Q

What is anti-Mullerian hormone?

A

Member of the TGF-beta family.
Homodimeric protein hormone consisting of 2 identical subunits

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2
Q

Physiological roles of AMH

A
  1. In male fetus - secreted by testicular Sertoli cells to inhibit growth and development of Mullerian structures
  2. 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
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3
Q

How is AMH measured in your lab

A

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.

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4
Q

Acceptable specimen types for AMH

A

Serum and Lithium heparin plasma, but NOT EDTA

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5
Q

A pt taking high dose biotin presents for AMH collection. What should the collector do?

A

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.

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6
Q

Indications for AMH testing

A

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

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7
Q

What should AMH NOT be used for and why?

A

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.

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8
Q

Advantages and limitations of AMH for PCOS

A

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

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9
Q

How can AMH be used in IVF protocols?

A

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

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10
Q

Forms of HCG

A
  1. Intact
  2. Free alpha subunit
  3. Free beta subunit
  4. Nicked - enzymatic cleavge of peptide bonds at position 44 and 45 of beta subunit and inactivates the hormone
  5. Nicked free beta
  6. Beta-core fragment - detectable only in urine, the predominant form in urine, the terminal degradation product of HCGbeta
  7. Hyperglycosylated - predominates in first 4 weeks of pregnancy
  8. Superglycosylated
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11
Q

What forms of HCG are found in urine

A

Predominantly beta core fragment but also unmodified hCG and hCGn

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12
Q

What is HCG?

A

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.

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13
Q

Where is HCG synthesized?

A

Syncitiotrophoblast and cytotrophoblast of the placenta. Minute amounts synthesised in pituitary in men and women.

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14
Q

How is HCG cleared

A

Liver and kidney

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15
Q

Describe the changes in HCG in pregnancy.

A

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.

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16
Q

How do HCG and LH differ?

A

HCG has an extra 20 amino acids. Same first 115 amino acids

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17
Q

How do POC HCG assays work?

A

Principle of immunochromatography
Detects HCG when concentration exceeds a certain threshold (usually 25 IU/L)

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18
Q

How are HCG assays classified by analytic specificity?

A

Assays that detect:
1. Only dimeric/intact HCG
2. HCG and HCG beta
3. HCG, HCG beta and HCG beta core fragment

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19
Q

Issues around standardising HCG assays

A
  1. 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.
  2. 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.
  3. Secondary standards provided by assay manufacturers vary greatly in terms of purity
  4. Hence HCG results from one assay cannot be directly compared to another.
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20
Q

Why does androstenedione require collection in a clot rather than serum separator tube?

A

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).

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21
Q

Testosterone method in your lab

A

Competitive chemiluminescent immunoassay. Acridinium ester label. Proprietary releasing agent to free bound testosterone from endogenous binding proteins.

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22
Q

Causes of a low testosterone in males

A

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

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23
Q

Major sources of testosterone in females

A

Ovaries, adrenal glands, peripheral conversion of precursors esp androstenedione to testo

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24
Q

Causes of a high testosterone in females

A

PCOS, CAH, Cushings, adrenal/ovarian tumours

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25
Q

Proportion of free testosterone in plasma?

A

<2.5%

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26
Q

Analytical interferences with testosterone in your lab

A

Icterus (conjugated hyperbilirubinaemia caused a negative interference, unconjugated hyperbilirubinaemia caused a positive interference)
Turbidity after freeze thaw cycle
Cross-reactivity with nandrolone decanoate, 11beta-hydroxytestosterone, 11ketotestosterone causing increased results
Possible cross-reactivity with other steroid analogues
Heterophile antibodies (either positive or negative interference)
Biotin at concentrations >30ng/mL can cause falsely increased results

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27
Q

Calculating FAI

A

FAI% = Testo (nmol/L) / SHBG (nmol/L) x 100

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28
Q

Testosterone reference method

A

ID-LC-MS/MS

29
Q

Clinical indications for DHEA-S requesting

A

Investigation of hyperandrogenism
Assessment of adrenarche and delayed puberty

30
Q

Source of DHEA-S

A

Adrenal glands, some may derive from male testes

31
Q

DHEA-S secretion through the lifespan?

A

Increases from 7th year of life then gradually declines after third decade

32
Q

Why is measurement of DHEA-S preferred over DHEA?

A

Much longer half life (24hrs) and slower turnover than DHEA, resulting in concentrations up to a thousandfold greater than DHEA. Circulates in an unbound for and does not exhibit significant diurnal variation.

33
Q

Causes of a high DHEA-S

A

PCOS
Adrenal hormone secreting tumour (>19umol/L)

34
Q

DHEA-S method in your lab

A

Competitive chemiluminscent immunoassay using streptavidin magnetic latex particles as the solid phase, biotin labeled anti-DHEAS antibody as the capture conjugate and an acridinium ester labeled DHEAS conjugate.

35
Q

Analytical considerations with DHEA-S method in your lab

A

Not validated in newborn population
Heterophilic antibody interference can cause falsely high or low results
Biotin can cause falsely increased results

36
Q

Oestradiol method in your lab

A

Competitive assay format with anti-estradiol antibody labeled with acridinium ester and an oestradiol-derivative capture solid phase.
Releasing agent used to release oestradiol from its binding proteins prior to addition of signal antibody.

37
Q

Interferences with the oestradiol method in your lab

A

Biotin (but levels required to cause interference are probably beyond that seen with high dose biotin supplementation)
Fulvestrant (ER antagonist used to treat breast cancer) can cause falsely increased results
Cross-reactivity with oestradiol valerate and possibly other steroid analogues
Heterophile antibodies could cause falsely increased or decreased results

38
Q

Reference method for oestradiol

A

Isotope-dilution GCMS

39
Q

What is FSH?

A

Glycoprotein hormon with 2 subunits. Alpha subunit similar to LH, HCG and TSH. Beta subunit confers biochemical specificity

40
Q

FSH method in your lab

A

2-site chemiluminescent immunoassay with paramagnetic particles and acridinium ester label. Anitbodies specific to beta-subunit of FSH

41
Q

Limitations of FSH method in your lab

A

Heterophile antibodies
Robust to high dose hook effect up to FSH concentration of 1000 IU/L

42
Q

LH method in your lab

A

2-site chemiluminescent immunoassay with paramagnetic particles and acridinium ester label. Anitbodies specific to beta-subunit of intact LH molecule

43
Q

Causes of an increased LH

A

Administration of GnRH, clomiphene citrate, human menopausal gonadotropin
Midcycle
PCOS
Menopause/POI
Heterophile antibodies

44
Q

Causes of a decreased LH

A

Exogenous sex hormones (including COCP)
Heterophile antibodies
Robust to high dose hook effect to LH of 18 000 IU/L

45
Q

Physiological functions of LH in females

A

Theca cells of ovary: Stimulate production of androgens that FSH converts to oestradiol during follicular phase
Graafian follicle: Acts synergistically with FSH to cause ovulation during midcycle peak
Corpus luteum: Stimulates formation of corpus luteum after ovulationa nd progesterone secretion during luteal phase.

46
Q

Physiological functions of LH in males

A

Leydig cells: stimulates testo production

47
Q

Physiological functions of FSH in females

A

Stimulate follicle development and production of oestrogens during follicular phase
Act synergistically with LH to cause ovulation

48
Q

Physiological functions of FSH in males

A

Sertoli cells: stimulate spermatogenesis

49
Q

Physiological role of progesterone

A

Prepare endometrium for blastocyst implantation and maintenance of pregnancy

50
Q

Source of progesterone

A

In F, major sources are corpus luteum and placenta. Minor sources are adrenal cortex in men and women, and testes in men.

51
Q

How do progesterone levels change during pregnancy?

A

Increase throughout until 3rd trimester

52
Q

Progesterone method in your lab

A

Competitive chemiluminescent immunoassay. Anti-progesterone antibody labelled with acridinium ester. Progesterone derivative covalently coupled to paramagnetic particles in the solid phase.

53
Q

Limitations of the progesterone method in your lab

A

DHEA supplements may cause falsely increased results (do LCMS)
Possible that other steroid analogues could cause cross-reactivity
Heterophile antibodies

54
Q

Why use unconjugated estriol instead of total estriol in prenatal screening?

A

More specific for identifying a fetus with Down syndrome

55
Q

How much estriol circulates in plasma unconjugated?

A

10%

56
Q

How does unconjugated estriol travel in blood (low solubility in water)?

A

Bound strongly to SHBG

57
Q

Where is estriol conjugated? To what? Why?

A

In the maternal liver. Glucuronate and sulfate conjugates. To permit renal clearance

58
Q

Biosynthesis of uE3 requires functioning…

A

Fetal adrenal
Fetal liver
Placenta

59
Q

Methods for uE3 analysis

A

Ultrasensitive RIA
Automated immunoassays

60
Q

Where is inhibin A produced and what is its physiological function

A

Placenta
Granulosa cells of the ovary
Role is to suppress FSH secretion

61
Q

Methods for Inhibin A detection

A

Total inhibin (inhibin A, B and precursors) - ELISA, RIA
Dimeric inhibin A - immunoassays using monoclonal antibodies which are more specific than total inhibin assays and provide better screening performance

62
Q

Why is PAPP-A not useful in second trimester?

A

In Down syndrome, PAPP-A will be low in first trimester but normal by second trimester.

63
Q

What is PAPP-A?

A

A zinc-containing metalloproteinase glycoprotin produced particularlly by placental tissues throughout gestation

64
Q

Physiological role of PAPP-A

A

Regulat actin of insulin-like growth factor II by cleaving its binding protein (IGFBP-4) and increasing bioavailable forms

65
Q

PAPP-A method in your lab

A
66
Q

Pre-analytical factors affecting PAPP-A measurement.

A

Serum specis in glass tubes preferred. Increase in PAPP-A with plastic versus glass tubes. Significant decrease in heparin (masks antgenic sites) and EDTA (chelates Zn, changing protein conformation) tubes. Need refrigeration.

67
Q

Clinical utility of fetal fibronectin testing

A

Concentration in cervical and vaginal secretions a test to aid in predict in preterm delivery. NPV high - if negative, <1% chance of preterm delivery. However, the PPV is low. As the pretest probability of spontaneous PTB is already low, having a test with high NPV is not useful. A more useful test would have a high PPV to better separate women who will deliver prematurely from the larger number of women with similar signs and symptoms who will not.

68
Q
A

Analytes (15 decks)

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