1. The measurement of alternative androgens in the investigation of PCOS Brian Keevil

2. Diagnosis and treatment of polycystic Ovary syndrome: An Endocrine Society Clinical Practice Guideline
Richard S. Legro, Silva A. Arslanian, David A. Ehrmann, KathleenM. Hoeger, M. Hassan Murad, Renato Pasquali, and Corrine K. Welt JCEM 2013;98:
‘Biochemical hyperandrogenism refers to an elevated
serum androgen level and typically includes
Testosterone
Free testosterone
Bioavailable testosterone’
The latest endocrine society guidelines make no reference to any other androgen for the diagnosis of PCOS than T.

3. Testosterone
SHBG T
60-80%
Free T
Total serum testosterone(T):
1-2%
Bioavailable
albumin T
20-40%
Changes in binding proteins greatly affect measured total T
genetic SHBG variants
estrogen treatment
critical illness and sepsis
T circulates in blood extensively bound to carrier proteins
Under normal, unstressed, conditions free serum T accounts for only 1% of total,
In males 80% T bound to SHBG, in females 60% bound to SHBG 3

4. Pre- and Post-menopausal Androgen Excess
Polycystic Ovarian Syndrome
Hyperthecosis
Obesity induced Hyperandrogenism
Ovarian or adrenal tumor
Congenital adrenal hyperplasia
Other: DRUGS, exogenous androgens,
Prolactinoma
Cushing’s syndrome
There are a variety of other disorders other than PCOS which can lead to raised androgens and these must be excluded, so we need more than just T in our diagnosis.

5. Diagnostic process Testosterone
Testosterone / SHBG =Free Androgen Index
Androstenedione + DHEAS
17OHP
The traditional diag=nostic pathway started with T but it is now recognised that FAI is more sensitive, A4 and DHEAS are useful for differentiating between ovarian and adrenal tumours cancers as a cause of the raised T .17OHP is useful for excluding CAH.

6. Immunoassay
So how do we measure these steroids, traditionally we have used IA. IA has suffered with poor specificity which used to be compensated for by solvent extraction before assay. This was abandoned in favour of direct assays to speed up the workflow in routine labs.

7. Main Immunoassay analysers
Main analysers support T, PRG E2 but are available only for high volume tests

8. Niche Immunoassay analysers
Immulite
Kryptor
The main analysers do not provide assays for A4, 17OHP or DHEAS and these have tro be provided for by niche analysers.
Ability to run one depends on reagent rental and workload to justify them placing the equipment
Liaison
iSYS

9. LC-MS/MS
                                                                                                                                                
We separate out compounds on a n LC column then ionise them and measure them in a mass spec. This is a very sensitive and specific measurement process but is comapratibvely slow comapred to IA.

10. Modern instruments are small and easy to use, all of our shift workers can use these instruments including at weekends.

11. Testosterone (female) UKNEQAS
Theer have been big improvements in some IA methods over the past few years but in my opinion LC-MS is still better. You can see in some of the QC returns that IA can still give variable performance
Kind permission of Finlay Mackenzie

12. Androstenedione UKNEQAS
The situation is worse for A4 where IA gives much higher results.
Kind permission of Finlay Mackenzie

13. Comparison of steroidogenic pathways among normoandrogenic and hyperandrogenic polycystic ovary syndrome patients and normal cycling women
262 women with hyperandrogenic PCOS
93 women with normoandrogenic PCOS
114 non PCOS women with normal cycles
NA and HA PCOS showed no difference in body weight
Both had higher insulin and LH than non PCOS
We know that PCOS can exist without an androgenic profile . This study from brazil looked at enzyme activity in 3 different groups, NA, HA and normal cycling women
De Medeiros J Obs Gynae Res 2014

14. Comparison of steroidogenic pathways among normoandrogenic and hyperandrogenic polycystic ovary syndrome patients and normal cycling women
HA PCOS patients
3βHSD activity higher (p<0.05)
17,20 lyase activity higher (p<0.0001)
Aromatase activity lower ( p<0.05)
Compared to NA PCOS patients and normal cycling women
Conversion of 17OHpreg to cortisol
was lower in the HA PCOS and
NA PCOS than normal cycling women
The reduced conversion of 17OH preg to cortisol favours the accumulation of androgen precursors. This provides good eveidence that A4 production is also increased in PCOS.
Häggström M, Richfield D (2014). "Diagram of the pathways of human steroidogenesis". Wikiversity Journal of Medicine 1 (1
De Medeiros J Obs Gynae Res 2014

15. Androgen profile through life in women with polycystic ovary syndrome: A Nordic multicenter collaboration study
651 women with PCOS
230 controls
Main outcome measures
Testosterone, Androstenedione, DHEAS
FAI, cFT
Women recruited at 6 health centres
Pinola etal JCEM 2015

16. Androgen profile through life in women with polycystic ovary syndrome: A Nordic multicenter collaboration study
Androgen levels in women with PCOS decreased with age towards menopause. Difference between PCOS and normals narrowed in individual variation increased as they approached menopause. Testo, FAI nad cFT were significantly higher in women with PCOS aged yrs adjusted for BMI.
Pinola et al JCEM 2015

17. Androgen profile through life in women with polycystic ovary syndrome: A Nordic multicenter collaboration study
The best predictive factors for having PCOS were cFT(>0.4 ng/dl), FAI(>2), and A4 (>277ng/dL).
Women with PCOS also had high androgens even after menopause. The best predictors at all ages were A4 , FAI and cFT. The use of cFT is better than FAI for diagnosing women lower than 40 yrs
Pinola et al JCEM 2015

18. Adrenal Androgen Excess and Body Mass Index in Polycystic Ovary Syndrome
No significant difference with T between obese and non obese patients. They saw higher androgens in PCOS compared to controls but significantly higher A4, DHEA and DHEAS in non obese PCOS individuals
Moran JCEM 2015

19. The Impact of Simultaneous Measurement of Testosterone and Androstenedione in Women with Suspected Androgen Excess
We had just moved over to LC-MS in 2005 because we were not happy with IA. We set up simultaneous analysis of T and a4 and collated the first years results
1436 female samples received in the laboratory for assessments of androgen status, 560 had at least one raised androgen
Duxbury et al Clin Chem 2007

20. Hyperandrogenaemia predicts metabolic phenotype in PCOS; the utility of serum androstenedione O‘Reilly MW, Taylor AE, Crabtree NJ, HughesBA, Capper F, Crowley RK, Stewart PM, Tomlinson JW,* and Arlt W
86 PCOS patients with confirmed diagnosis
56 patients had high T and high A4
30 patients had normal T
20 patients had high A4
10 patients had normal A4
This interseting paper actually defines a role for A4 in PCOS. By predicting a metabolic phenotype. Discuss the phenotype.
J Clin Endocrinol Metab Mar; 99(3): 1027–1036.

21. NA/HT n=0
HA/HT n=56
NA/NT n=10
HA/NT n=20
O’Reilly MW JCEM 2014

22. A B C D O’Reilly MW et al. JCEM 2014
NA/NT had normal steroid excretion but insulin resistance
HA/NT had elevated steroid excretion and also insulin resistance
A4 identifies a subset of individuals at risk of the metabolic syndrome
O’Reilly MW et al. JCEM 2014

23. Testosterone vs Androstenedione n=4329 female samples
So whats teh scale of teh problem, we audited our results and found that amongst 4000 samples sent to our lab approx 65 had this HA/NT phenotype.

24. Testosterone to Dihydrotestosterone ratio as a New Biomarker for an Adverse Metabolic Phenotype in the Polycystic Ovary Syndrome
275 premenopausal PCOS patients - median age 26 years
35 controls - median age 35 years
Munzker JCEM 2015

25. Testosterone to Dihydrotestosterone ratio as a New Biomarker for an Adverse Metabolic Phenotype in the Polycystic Ovary Syndrome
These data provide evidence for a strong link between tt/DHT ratio and an adverse metabolic phenotype in patients with PCOS. The correlation was only found in PCOS patients suggesting the TT/DHT ratio to be anew biomarker for an adverse metabolic phenotype.
In PCOS patients alone the TT/DHT ratio was significantly higher in
Those with metabolic syndrome, obesity, IGT and insulin resistance
Munzker JCEM 2015

26. Testosterone to Dihydrotestosterone ratio as a New Biomarker for an adverse Metabolic phenotype in the Polycystic Ovary Syndrome
PCOS patients showed significantly higher values of
Testosterone p<0.001
Free testosterone p<0.001
Free DHT p<0.001
Compared to healthy controls
The TT/DHT ratio was significantly higher in PCOS patients.
Munzker JCEM 2015

27. Multiplexed steroids A4 2pmol/L ( 0.57 ng/mL)
Testo 2 pmol/L ( 0.57 ng/mL)
17OHP 4 pmol/L ( 1.3 ng/mL)
So here we have the trace from an LCMs instrument showing the seaparation of 5 potentailly usefiul steroids. They can all be measured simultaneously for no extra cost unlike IA where you would hav eto run 5 separate assays. we now run this as a routine clinical method in our lab.
DHEA nmol/L (1.8 ng/mL)
DHT nmol/L ( 0.1 ng/mL)

28. Diurnal variation of serum androgens in females

29. Androgen Profile in Young Women
n=159 adolescent girls
normal weight
eumenohrreic
55 indeterminate
53 follicular
51 luteal
Upper reference limits
Luteal phase
T= ng/mL (2.0 nmol/L)
FAI = 6.75
Folicular phase
T= ng/mL (1.56 nmol/L)
FAI = 4.71
Higher T and 17OHP were seen in the luteal phase needing different reference ranges.
Fanelli JCEM 2013

30. Androgen Profile in Young Women
51 luteal – 18 anovultory
31 ovulatory
Anovulatory cycles are frequent in the first few years after menarche . Based on progesterone (<2ng/ml and >3 ng/mL ) 2 distinct ovulatory populations were seen. Differences in androgen profiles seen in anovulatory cycles such that T and A4 were higher and 17OHP was lower. The increase in T is accompanied by a decrease in E2 suggesting decreased aromatase activity and by general reduced actrivity of corpus lutem. Both LH and FSH tended to be higher unlike PCOS where FSH is lower.
Fanelli JCEM 2013

31. Conclusions
Testosterone is not the only useful biochemical marker for PCOS.
Androstenedione and DHT may have important roles to play
LC-MS/MS is a powerful tool for measuring multiple useful steroids

32. *Thought to reflect circulating free and tissue levels of steroid
Steroids in saliva
Saliva only contains free unbound steroids
Moves easily into saliva via passive diffusion
Neutral steroids unaffected by increased flow rates
Free-T
Free-T
*Thought to reflect circulating free and tissue levels of steroid
Testosterone is freely filtered by salivary ducts and is unaffected by flow rates, saliva contains only the free fraction and salivary testo is thought to be a better bio marker of tissue levels of T.
Blood capillary
Salivary duct
Cortisol in saliva approximates very well to the free plasma concentration.
Lipid insoluble steroids in saliva e.g DHEAS make up <1% of the free plasma concentration

33. Saliva collection unstimulated passive drool Recommended protocol:
Mouth rinsed
No food intake 30 mins prior to collection
Volunteers had not brushed teeth within one hour before collection
Saliva allowed to pool in the mouth for 5 min
Aim to collect minimal volume of saliva of 1 mL
Saliva was drooled down plastic straw into collection vessel
Freeze/thaw before analysis to break down mucins
We collect saliva by passive drool because many of the commercial absorbent swab collection devices have problems with adsorption and give poor recoveries. Passive drool is very simple and cheap and after freeze thawing gives reasonably clean samples.

34. Sample stability At 4o C At ambient temperature After freeze / thawing
0.1
0.2
0.3
0.4 1 2 3 4 5
Days at Room Temp
Testosterone (nmol/L)
After freeze / thawing
We looked at the stability of saliva samples and found that testo measured by LC-MS/MS was not only stable for up to at least 5 days at 4 c but was also stable at room temperature. This should allow samples to be safely sent to the lab in the post. Testo is also quite stable for up to 5 freeze thaw cycles.

35. Diurnal variation in females
Saliva
P=0.01
Sal-T pmol/L
And as in the male samples sal T was seen to exhibit a marked diurnal variation with early morning samples (blue bars) being significantly higher than evening samples (green bars). This re-inforces the need to standardise sample collection times preferably around 0900 when you will the greatest sensitivity . am
Mean = 25.7 pmol/L (SD=15 pmol/L) pm
Mean = 15.1 pmol/L (SD=7.5 pmol/L)

36. LC-MS/MS Reference intervals (Age range 16-74)
Female n=96
Male n=96
We established reference ranges in saliva samples and found good demarcation between the male and female reference ranges. The median female range is pmol/L and the median male range is pmol/l approx 10 fold higher.
Mean= 16.0
Median=13.7
SD=9.2
95% confidence interval 5-46 pmol/L
Mean = 215
Median= 205
SD= 79.8
95% confidence interval pmol/L

37. National survey into sexual attitudes and lifestyle (NATSAL) Salivary testosterone
Women n=1276
Men n= 1074

38. Healthy population-women sal-T (pmol/L)
Mean sd median (IQR)
( )
( )
( )
( )
( )
( )
(n=1276)

39. Conclusions
T can be reliably and accurately measured in saliva by LC-MS/MS in both male and female samples
Significant diurnal variations in salivary T levels are detectable in both males and females
Instruments with high sensitivity are needed to measure in the female range.
We need to explore the application of these techniques in different patient groups to establish their clinical utility.
T in saliva is stable –good for storage and transport
In conclusion the LC-MS has validated well and gives accurate and precise results in both male and female samples. The LC-MS ref ranges are much lower than RIA wich is not surprising because it mirrors the results found in serum samples, although the female testo results are even more pronounced. Instruments with high anlaytical sensitivity are needed to measure the much lower concentrations found in the female samples. This study has proven the concept of using salivary testo measured by LC-MS in normal individuals. We still need to explore the application of these techniques to various clinical groups and we have some studies already underway.

40. Acknowledgemnts
Laura Owen
Jo Adaway
Fred Wu
Natsal team