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valuation of urinary excretion of androgens conjugated to cysteine in humanregnancy by mass spectrometry
ndreu Fabregata, Josep Marcosa,b, Lorena Garrostasa, Jordi Seguraa,b, Oscar J. Pozoa,∗, Rosa Venturaa,b
Bioanalysis Research Group, IMIM, Hospital del Mar, Doctor Aiguader 88, 08003 Barcelona, SpainDepartment of Experimental and Health Sciences, Universitat Pompeu Fabra, Doctor Aiguader 88, 08003 Barcelona, Spain
r t i c l e i n f o
rticle history:eceived 31 August 2012eceived in revised form 10 January 2013ccepted 31 January 2013
eywords:regnancyndrogensass spectrometryopingrine
a b s t r a c t
Alterations in the maternal excretion of steroids during pregnancy are not restricted to the production ofprogesterone and estriol by the fetoplacental unit. Although there is a lack of longitudinal data on urinaryandrogen concentrations during pregnancy, some studies revealed that modifications in the excretionsof androgens might be significant. Recently, several testosterone metabolites excreted as cysteine conju-gates have been reported in human urine. We conducted a longitudinal study on androgens conjugatedwith cysteine and major androgens and estrogens excreted as glucuronides in three pregnant women bymass spectrometric techniques. The urinary concentrations obtained in samples weekly collected duringeach of the three trimesters and samples collected before pregnancy were compared. Results showeda significant increase in urinary estrogens and norandrosterone and a moderate decrease in the uri-nary concentrations for most of the androgens. The most significant exception to this behavior was therise observed for epitestosterone glucuronide when comparing basal levels with the first trimester. Cys-teinyl conjugates of testosterone metabolites showed a different behavior. Whereas 4,6-androstanedioneremained almost constant through the three trimesters, and �6-testosterone decreased as the majorityof androgens, the excretion profile of 1,4-androstanedione notably increased, reaching a maximum at thethird trimester. Alterations in the steroid profile are used in doping control analysis for the screening of
endogenous anabolic androgenic steroid misuse. In this study, the main parameters proposed for dopingcontrol have been determined for basal samples and samples collected in the first trimester and theyhave been compared. In spite of the limited number of cases, significant variations have been found inall pregnancies studied. These alterations have to be taken into consideration if anabolic steroids areincluded into the Athlete Biological Passport.
This article is part of a Special Issue entitled ‘Pregnancy and Steroids’.
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-diol; DHEA, dehydroepiandrosterone; DHT, 5�-dihydrotestosterone; MSTFA, N-o between testosterone and epitestosterone excreted as glucuronides.Institut de Recerca Hospital del Mar, Doctor Aiguader, 88, 08003 Barcelona, Spain.
Normal human pregnancy dramatically affects the maternalxcretion of steroids. These changes in urinary steroid composi-ion are mainly due to the fact that products of the feto-placentalnit have no other exit than the maternal serum and urine. Quan-itatively, the two major steroids produced in normal pregnancyre fetal 16�-hydroxy-DHEA sulfate, and placental progesterone.hose steroids are metabolized and excreted in maternal urine inhe forms of estriol, and pregnanediols, respectively. The biosyn-hetic routes for the pregnanediols and estriol are completelyifferent. Pregnanediols, are principally synthesized from mater-al cholesterol whereas estriol almost exclusively originates from
etal cholesterol via DHEA sulfate [1].In addition to those two main sources of changes, there are
ome other reasons that might vary the urinary steroidal pro-le. After the first five to six weeks of pregnancy, the estradiolnd estrone also increase due to the aromatization of androgenshat take place in the placenta [2]. Pregnancy also affects theypothalamic–pituitary–adrenal axis, resulting in progressive riseseveral hormones, which include corticotropin-releasing hormone,drenocorticotropic hormone, and cortisol [3–5]. Particularly, a 2-o 3-fold increase in urinary free cortisol levels during the secondnd third trimesters of pregnancy has been found [6].
However, there is a paucity of studies for the evaluation ofndrogens levels during pregnancy. It is known that nandrolones produced during the process of aromatization [7,8], which once
etabolized leads to the presence of low amounts of noran-rosterone in pregnancy urine specimens (urinary concentrationselow 10 ng/mL) [9,10]. The formation of 19-norandrosterone
s important in the antidoping field, since together with 19-oretiocholanolone it is the main phase I metabolite of severalanned anabolic steroids like nandrolone, norandrostenedione, andorandrostenediol [11–14].
It is also known that some disorders can modify the excretion ofndrogens in the maternal urine. That is the case in aromatase defi-iencies, where an excess in androgen production is observed [15],nd the case of a pregnancy carrying a fetus affected by P450 oxi-oreductase deficiency, where an abnormal elevated synthesis of�-androgens through an alternative pathway has been described16,17].
It is well documented that the main route of metabolismor testosterone, androstenedione and DHEA leads to the forma-ion of androsterone, etiocholanolone, and androstanediol, whichre eliminated through the urine as glucuronides and sulfates18]. However, the metabolism of androgens is not restrictedo those major components. Recently, it was demonstrated that
inor amounts of 4,6-androstadien-3,17-dione, 4,6-androstadien-7�-ol-3-one and 1,4-androstadien-3,17-dione conjugated withysteine are also urinary testosterone metabolites [19–21].
Since these cysteine conjugates have been proposed as markersor the detection of testosterone abuse, it is important to evalu-
Please cite this article in press as: A. Fabregat, et al., Evaluation of urinary emass spectrometry, J. Steroid Biochem. Mol. Biol. (2013), http://dx.doi.org/
te whether their concentrations vary during the first trimester ofregnancy.
On the other hand, the addition of extra double bonds tondrostendione had been reported as an efficient way to decrease
aromatase activity. Particularly, it has been reported that while theaddition of a C1–C2 double bond to androstendione suffices to gen-erate a suicide substrate inhibitor for aromatase, the addition of aC6–C7 double bond produces a competitive aromatase inhibitorbut does not cause a mechanism-based inactivation of the enzyme[22,23]. These results suggest that 4,6-androstadien-3,17-dione isproduced in the human body via phase I metabolism. Therefore, thisbiotransformation can be potentially considered as an endogenousmechanism for the regulation of the aromatase activity.
The goal of the study was to evaluate the variations in theexcretion of polyunsaturated testosterone related compound dur-ing pregnancy. The variation in the excretion of these androgensis compared with the excretion of the major androgen metabolitesand estrogens.
2. Materials and methods
2.1. Chemicals and reagents
1,4-Androstadien-3,17-dione, 19-norandrosterone, etiochola-nolone, etiocholanolone-d5 (internal standard), testosterone-d3(internal standard) and epitestosterone were obtained from NMI(Pymble, Australia). 4,6-Androstadien-3,17-dione, 17-hydroxy-4,6-androstadien-3-one (�6-testosterone), androsterone andestradiol were purchased from Steraloids (Newport, USA).Methandienone (internal standard), 5�-androstane-3�,17�-diol (5�-diol), 5�-androstane-3�,17�-diol (5�-diol), estriol,5�-dihydrotestosterone (DHT), testosterone, dehydroepiandros-terone (DHEA), androstenedione and estrone were acquired fromSigma–Aldrich (St. Louis, MO, USA). Androsterone-�-glucuronide-2,2,4,4-d4 (internal standard) was obtained from Orphachem(Saint-Beuzire, France). The �-glucuronidase preparation (fromEscherichia coli type K12) was purchased from Roche Diagnostics(Mannheim, Germany).
Analytical grade potassium carbonate, hydrochloric acid, di-sodium hydrogen phosphate, sodium hydrogen phosphate andtert-butyl-methyl ether, ammonium iodide, sodium hydroxide,acetonitrile and methanol (LC gradient grade), formic acid, ammo-nium formate (LC/MS grade) and cyclohexane were obtainedfrom Merck (Darmstadt, Germany). N-Methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) was from Karl Bucher ChemischeFabrik GmbH (Waldstetten, Germany) and 2-mercaptoethanol wasfrom Sigma–Aldrich (St. Louis, MO, USA).
Milli Q water was obtained using a Milli-Q purification sys-tem (Millipore Ibérica, Barcelona, Spain). The Sep-Pak® Vac RC(500 mg) C18 cartridges were purchased from Waters (Milford,Massachusetts, USA).
2.2. Instrumentation
The specific gravity of all samples was measured using an UG-�urine specific gravity refractometer (Atago, Japan).
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LC–MS/MS analyses were performed in a triple quadrupole(Quattro Premier XE) mass spectrometer equipped with anorthogonal Z-spray-electrospray ionization source (ESI) (WatersAssociates, Milford, MA, USA) interfaced to an UPLC system, Acquity
Waters Associates) for the chromatographic separation. Drying gass well as nebulising gas was nitrogen. The desolvation gas flowas set to approximately 1200 L/h and the cone gas flow to 50 L/h.
cone voltage of 25 V, and a capillary voltage of 3.0 kV were usedn positive ionization mode. The nitrogen desolvation temperature
as set to 450 ◦C and the source temperature to 120 ◦C.The LC separation was performed using an Eclipse Plus C18 col-
mn (50 mm × 2.1 mm i.d., 1.8 �m) (Agilent, Palo Alto, CA, USA), at aow rate of 300 �L/min. Water and methanol both with formic acid0.01%) and ammonium formate (1 mM) were selected as mobilehase solvents. A gradient program was used; the percentage ofrganic solvent was linearly changed as follows: at 0 min, 45%; at.50 min, 45%; at 8.5 min, 90%; at 9 min, 90%; at 9.5 min, 45%; at1 min, 45%.
Analytes were determined by a selected reaction monitor-ng (SRM) method including two transitions for each compoundTable 1).
GC–MS/MS was performed on an Agilent 7890A gas chromato-raph equipped with a 7693 autosampler, a split/splitless capillarynlet and an Agilent 7000A Series Triple Quadrupole GC/MS, usinghe Selected Reaction Monitoring acquisition mode.
The GC was equipped with a capillary column (HP-Ultra 1,6 mm × 0.2 mm i.d., with a 0.11 �m film thickness) from J&W (Agi-
ent Technologies, USA). The oven temperature was programmed asollows: the initial temperature was 80 ◦C, maintained for 0,2 min,hen increased at 70 ◦C/min to 183 ◦C, then at 5 ◦C/min to 220 ◦C,hen at 50–310 ◦C, and maintained at the final temperature for
min. The transfer line was kept at 280 ◦C. Helium was used as car-ier gas at a constant flow rate of 0.8 mL/min. One microliter of thenal extract was injected in split mode (split ratio 1/30). Nitrogenas used as collision gas at a flow rate of 1.5 mL/min, and helium as
quenching gas at a flow rate of 2.25 mL/min. The electron impactource was kept at 220 ◦C and the quadrupoles at 180 ◦C.
Analytes were determined by a SRM method including two tran-itions for each compound (Table 1).
.3. Sample preparation
The method used for LC–MS/MS determination was based on theethod described and validated elsewhere [24]. Briefly, after addi-
ion of 50 �L of ISTD (methandienone at 1 �g/mL), 2.5 mL of urineas basified by addition of 300 �L of KOH (6 M). The mixture waseated at 60 ◦C for 15 min, followed by a liquid–liquid extractionith 6 mL of tert-butylmethylether. The sample was centrifuged
nd the organic layer separated and evaporated. The residue wasissolved into 150 �L of a mixture of water:acetonitrile (1:1, v/v).inally, 10 �L were directly injected into the LC–MS/MS system.
For GC–MS, the procedure for preparing the samples is basedpon the currently used screening methods in routine doping con-rol of the so-called total fraction, which includes the determinationf glucuronides plus unconjugated excreted steroids [25]. Briefly,.5 mL of urine were added with an internal standard solutionontaining androsterone-d4-glucuronide, etiocholanolone-d5, andestosterone-d3. The reconstituted extracts were hydrolysed with0 �L of �-glucuronidase from E. coli at pH 7 for 1 h at 55 ◦C. Afterooling to room temperature, pH was increased to approximately.5 by the addition of 250 �L of a 25% K2CO3 solution. Subsequentlyamples were extracted with 6 mL of tert-butylmethylether andentrifuged. Finally the organic layer was evaporated, and deriva-ized with 50 �L of MSTFA/NH4I/2-mercaptoethanol (1000:2:6,/w/v) by heating at 60 ◦C for 20 min.
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.4. Subjects and urine samples
Urine samples from three pregnant women were collectedrom the 5th week of pregnancy until delivery. Additionally, for
PRESSy & Molecular Biology xxx (2013) xxx– xxx 3
volunteers 1 and 2, daily spot samples were collected before thepregnancy started during one menstrual cycle. Aliquots of 50 mLurine were frozen at −20◦C until analysis.
Volunteer 1 was a 34 years-old Caucasian woman with a BMI of25.2 kg/m2. She was taking Cariban (10 mg doxylamine succinateand 10 mg pyridoxine hydrochloride/8 h), Natalben (1 capsule/day)and Clamoxyl (750 mg/day) during the 36 weeks of pregnancy. Asa consequence of being diagnosed with gestational diabetes, shefollowed a low carbohydrates diet during the last fifteen weeks ofgestation. She delivered at 40 weeks of gestation a healthy babygirl.
Volunteer 2 was a 35 years-old Caucasian woman with a BMI of22.6 kg/m2. She was taking pyridostigmine (120 mg/day) during thewhole pregnancy. She did not follow nay-specific diet and deliveredat 40 weeks of gestation a healthy baby girl.
Volunteer 3 was a 35 years-old Caucasian woman with a BMI of25.9 kg/m2. She did not take any medication during the pregnancy,and did not follow any specific diet. She delivered at 39 weeks ofgestation a healthy baby boy.
In all three cases, fetus growth throughout the course of thepregnancy was within the normal ranges according to ecographicand biochemical data.
Ethical approval for the study had been granted by the EthicalCommittee of our Institute (Comité Ètic d’Investigació Clínica CEIC-Parc de Salut Mar, Barcelona, Spain). The three subjects participatingin the study gave their written informed consent prior to inclusion.
2.5. Statistical analysis
Statistical analyses were performed using SPSS version 18 (Jul30, 2009). Results are presented as the mean ± standard deviation.
For the statistical analysis, a T-student and its non-parametric analogous Mann–Whitney U test were used.The normality of the different markers evaluated in thisstudy for the doping control field (T/E, 5�-diol/5�-diol,1,4-androstadien-3,17-dione/4,6-androstadien-3,17-dione,1,4-androstadien-3,17-dione/�6-testosterone and 4,6-androstadien-3,17-dione/�6-testosterone) was set using theKolmogorov–Smirnov test. Statistical significance was taken asP < 0.05.
In order to reduce variability due to urine dilution, the urinaryconcentrations of the analytes were normalized by means of theurinary density. The concentrations per sample were corrected tospecific gravity of 1.020 applying
Eight androgens (androsterone, etiocholanolone, 5�-diol, 5�-diol, DHT, androstendione, DHEA and testosterone) and epitestos-terone were quantified after glucuronide hydrolysis by theGC–MS/MS method. Samples were separated in four groups (basal,1st, 2nd, and 3rd trimester of pregnancy) in order to make theresults handier. The average concentration and the standard devi-ation for each androgen are summarized in Table 2.
Basal concentrations for androgens in the two volunteers werebetween the normal ranges of urinary concentrations established
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for females [26]. The main exception was volunteer 2, which exhib-ited extremely low urinary concentrations for testosterone andDHT. The detection of these analytes in this volunteer was accom-plished by increasing the amount of urine to 5 mL.
T: retention time, CE: collision energy, ISTD: internal standard.
During pregnancy, different trends were observed for thendrogens studied (Fig. 1). Thus, constant concentrations duringregnancy were observed for several androgens like testosteroner androstendione where no statistical differences were foundmong the samples collected in the different trimesters. Otherndrogens like etiocholanolone, androsterone, 5�-diol, 5�-diol andHEA exhibited moderate decreases (up to 2–3 times) during in
Please cite this article in press as: A. Fabregat, et al., Evaluation of urinary emass spectrometry, J. Steroid Biochem. Mol. Biol. (2013), http://dx.doi.org/
he third trimester of pregnancy compared to basal levels. Despitef being produced in the human fetal adrenal cortex [27], andn the placenta [28], it has been reported that DHEA sulphaten maternal serum fell by 50% at mid gestation [29]. Our results
able 2verage urinary concentration and standard deviation of androgens.
regarding the excretion of DHEA glucuronide are in agreement withthat.
It is also significant the slight difference between the behaviourof 5� (5�-diol and androsterone) and 5� (5�-diol and etio-cholanolone) metabolites. Thus, the decrease observed for the5�-metabolites was slightly higher than the decrease observed for5�-metabolites. These preliminary results imply a variation during
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pregnancy of the normally very stable 5�/5� ratios.The most distinct behaviour was observed for epitestosterone.
Contrarily to the androgens, urinary concentrations of epitestos-terone increased during pregnancy up to three times in the first
ig. 1. Trend observed in the urinary concentrations (a) androsterone, (b) etiocholand (i) AED in volunteer 1.
rimester (Table 2, Fig. 1). After this first increase, urinary epitestos-erone remained almost constant during the rest of pregnancy. Theole of epitestosterone in human body remains still unknown [30],nd this different behaviour support the fact that its role is notelated to the androgens’ one.
.2. Estrogens and norandrosterone
Urinary concentrations of estrogens (estradiol, estrone andstriol) excreted free or conjugated with glucuronide werebtained (Table 3). Although estrogens are only partially excreteds glucuronides, this detection provides useful information abouthe trend on the excretion of these compounds. Thus, estradiolnd estrone have a similar behaviour increasing approximately 100old between the basal levels and the levels obtained in the thirdrimester. These results are in agreement with those previouslyeported in the literature [31]. Since both estradiol and estrone areroduced in the placenta and can be interconverted by the enzyme7�-hydroxysteroid dehydrogenases [32], a similar behaviour it isxpected. This is confirmed by a satisfactory correlation betweenhe concentrations found for both compounds (Fig. 2).
Contrarily to estrone and estradiol, urinary estriol is origi-
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ated from 16�-hydroxy-DHEA sulphate, which is hydrolyzedy placental steroid sulphatase, oxidized by 3�-hydroxysteroidehydrogenase/isomerase, and finally aromatized, largely glu-uronidated and excreted in maternal urine [33]. Therefore,
no linear correlation between estrone/estradiol and estriol isexpected. This absence of correlation is confirmed by the resultspresented in this study (Fig. 2). The increase observed for urinaryestriol concentrations was larger than the other estrogens. Thus,concentrations up to 1000 times higher than the basal levels wereobserved in samples collected during third trimester (Table 3).
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6 A. Fabregat et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2013) xxx– xxx
F ol, (b)( ione v
btttSaonphte
ig. 2. Correlation observed between urinary concentration of (a) estrone vs estradie) 1,4-androstadien-3,17-dione vs epitestosterone and (f) 1,4-androstadien-3,17-d
The presence of norandrosterone in urine during pregnancy haseen reported in several studies [7–10]. For this reason, norandros-erone was also analysed in these samples (Table 3). Accordingo previous publications, urinary concentrations of norandros-erone increased during pregnancy reaching values up to 4 ng/mL.imilar results were obtained in our study. Norandrosterone waslready detected during the first trimester and maximum valuesf around 5 ng/mL were obtained in the last weeks of the preg-ancy. It has been postulated that norandrosterone can be a side
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roduct of the synthesis of estrogens after aromatization [9]. Thisypothesis is supported by our results by the fact that a satisfac-ory correlation was observed between urinary concentrations ofstradiol and norandrosterone (Fig. 2). This correlation shows that
estrone vs estriol, (c) norandrosterone vs estradiol, (d) �6-testosterone vs 5�-diol,s estrone.
norandrosterone and estrogens urinary concentrations increased atthe same pace, suggesting that the generation of both compoundsis guided by the same pathway.
3.3. Cysteinyl conjugates of testosterone metabolites
The occurrence in urine of several testosterone metabolitesexcreted as cysteine conjugates has been recently reported [20,34].
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Although they have been reported to be good markers for andro-gens misuse [19,21], their function and origin is still unclear [33].The behaviour of their urinary concentrations during pregnancycan provide additional information about them.
The detection of three of these metabolites (1,4-androstandien-,17-dione, 4,6-androstadien-3,17-dione and �6-testosterone) inrine samples were performed by an indirect method based onhe release of the cysteinyl group after alkaline treatment [24].rinary concentrations for the three metabolites obtained in the
amples collected before pregnancy (Table 4) were in the normalange observed in population studies [19]. Even volunteer 2 whoxhibited extremely low urinary concentrations of testosteronead values in the normal range for cysteinyl conjugates metabo-
ites.Each metabolite exhibited different trend during pregnancy.
hus, urinary concentrations of 4,6-androstadiendione remainedlmost constant during pregnancy (Table 4) and no significantifferences were observed between the three trimesters. Theehaviour of this metabolite was found to be similar to thatbserved for testosterone. Thus, whereas in volunteers 2 and
both 4,6-androstadiendione and testosterone remained con-tant, a slight increase in the urinary concentrations of both,6-androstadiendione and testosterone was observed in the firstrimester of volunteer 1.
Urinary concentrations of �6-testosterone gradually decreaseduring pregnancy, and concentrations in the third trimester wereround three times lower than the basal ones (Table 4). Theseesults are similar to those obtained for other androgens andetabolites like androsterone, etiocholanolone or both androstan-
iols (Table 2). In fact a good correlation between �6-testosteronend these compounds was obtained (Fig. 2), suggesting that theiotransformation leading to the formation of �6-testosterone isomehow related to the process of generating the tetrahydro andro-en metabolites.
Contrary to 4,6-androstadiendione, �6-testosterone and mostf the androgens, it was observed that urinary concentrationsf 1,4-androstadiendione increased during pregnancy. Concentra-ions in the last trimester were around five times higher thanhe basal levels (Table 4). Among the compounds analysed inhis study, only epitestosterone and estrogens exhibited similarehaviour. Therefore, the presence of 1,4-androstadiendione inrine could be related either with epitestosterone or with estro-ens. In order to have more information about this relationship,he correlation between 1,4-androstadiendione and both epitestos-erone and estrogens were performed (Fig. 2). No correlationas found between 1,4-androstadiendione and epitestosteronehereas some correlation was obtained between urinary concen-
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rations of 1,4-androstadiendione and estrogens in two of the threeolunteers. This fact suggests that, similarly to norandrosterone,,4-androstadiendione is formed as a side product in the conversionf testosterone to estrogens.
3.4. Diagnostic ratios for doping control analysis
Doping control analysis is one of the fields in which steroid pro-file plays a crucial role. Thus, detection of abnormal variation inthe steroid profile is one of the most powerful screening tools forthe detection of the misuse of endogenous androgenic anabolicsteroids. Several ratios between endogenous steroids have beenreported as useful for this purpose [26].
The first approach for the detection of the misuse of endogenousandrogenic anabolic steroids was the use of population thresholdvalues. In this approach, a sample is suspicious if it exceeds the pop-ulation threshold. However, using this methodology the detectionof the misuse of endogenous androgenic anabolic steroids by sub-jects with low basal values is difficult. A more promising approachis the use of individual threshold values in which thresholds areindividually established for each athlete by the compilation of alldata reported for him/her. These values are characteristic of eachathlete and are part of the Athlete Biological Passport [35]. Usingthis approach, a sample becomes suspicious when it exceeds theestablished individual threshold values.
In this context, it is important to know how the steroid profilevaries in common scenarios like early pregnancy. For this reason,the steroid profile for samples collected during the first trimesterof pregnancy was evaluated and compared to that obtained forsamples collected before pregnancy.
3.4.1. T/EThe main characteristic ratio used in the screening of
testosterone misuse is the ratio between testosterone andepitestosterone excreted as glucuronides (T/E). Thus, an increase ofT/E is observed after testosterone administration. However, varia-tions of T/E have been reported in common scenarios like alcoholconsumption [36]. These scenarios have to be taken into accountbefore reporting an altered T/E.
In the three pregnancies studied, significant differences in theT/E values were observed when comparing the results obtainedbefore and during the first trimester of pregnancy (Table 5). Thus,whereas testosterone remained almost constant for the completepregnancy, a significant raise was observed for epitestosterone dur-ing the first week. Due to this fact, a substantial decrease on T/E wasobserved even in the fifth week of pregnancy. This decrease of theT/E goes in the opposite direction than the administration of testos-
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terone (increase of the T/E) and therefore it is not likely to producefalse positive results in doping control analysis. Nonetheless, theyhave to be taken into account as a source of variation of the ath-lete steroid profile included in the Athlete Biological Passport. A
Fig. 3. Values of (a) T/E and (b)1,4-androstandien-3,17-dio
ossibility to assist interpretation would consist of measuringuman chorionic gonadotropin in all doping control samples.
.4.2. 5˛-Androstandiol/5ˇ-androstandiolThe ratio 5�-diol/5�-diol is one of the ratios between andro-
ens, which remain more constant in longitudinal studies [37].his ratio is affected by the administration of some endogenousndrogenic anabolic steroids like DHT [38]. Regarding pregnancy,lthough the concentration of both 5�-diol and 5�-diol decreasedlready in the first trimester (Table 2), the ratio 5�-diol/5�-diolemained constant almost during the complete pregnancy and noignificant differences are observed between basal values and thosebtained in the first trimester of pregnancy (Table 5). Therefore,arly pregnancy would not affect in the evaluation of 5�-diol/5�-iol values for doping control analysis.
.4.3. Ratios between cysteinyl metabolitesSeveral ratios between cysteinyl metabolites have been
eported as useful for the detection of the misuse of sev-ral endogenous anabolic androgenic steroids in differentdministration forms [19,21]. Thus, an increase of 1,4-ndrostandiendione is observed after testosterone administrationnd 1,4-androstandiendione/4,6-androstadiendione and 1,4-ndrostandiendione/�6-testosterone have been used for theetection of the misuse of oral testosterone, dermal testosteronend DHT [19,21]. Additionally, increases of �6-testosteronend 4,6-androstendiendione have been reported after DHEAdministration and �6-testosterone/1,4-androstandiendionend 4,6-androstendiendione/1,4-androstandiendione have beenuggested as ratios for the detection of DHEA misuse. The ratio,6-androstandiendione/�6-testosterone was found to be verytable in population studies and was not substantially affected byteroid administration [19]. Up to our knowledge, no studies inrder to evaluate factors affecting the profile of these ratios have
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een performed.Most of these ratios have been found to be affected by early
regnancy. The substantial rise of 1,4-androstandiendione andhe decrease of �6-testosterone significantly varied the basal
b)
6-testosterone before pregnancy and in the first trimester.
results for ratios between cysteinyl metabolites. As an exam-ple, values for the ratio 1,4-androstandiendione/�6-testosteroneduring the first trimester were up to three times larger thanthe obtained in basal samples (Table 5, Fig. 3). Other ratios like1,4-androstandiendione/4,6-androstandiendione were also signif-icantly affected in the first trimester of pregnancy. Even thevery stable ratio 4,6-androstandiendione/�6-testosterone wassubstantially affected during pregnancy. This effect was notobserved with other 17-hydroxyl/17-oxo couples like testos-terone/androstendione or estradiol/estrone which were found tobe stable during the whole pregnancy.
These preliminary results advise that early pregnancy has to betaken into account if using ratios between cysteinyl conjugates forthe detection of endogenous steroids misuse.
4. Conclusions
A longitudinal study of the steroid profile (androgens, mainestrogens excreted as glucuronides and cysteinyl conjugates oftestosterone) during pregnancy has been performed. As expected,our study demonstrated a rise in urinary estrogens and norandros-terone during pregnancy, together with a moderate decrease inmost of the androgens. A relevant increase of urinary epitestos-terone concentrations were found even in the first week ofpregnancy. This preliminary result is especially relevant for anti-doping control purposes since epitestosterone is usually used ascommon reference marker for endogenous steroid misuse.
Every studied testosterone metabolite excreted as cysteinylconjugate showed a different behavior. Thus, constant urinaryconcentrations of 4,6-androstadiendione were observed duringpregnancy. This behaviour was similar to the observed for testos-terone. In the case of urinary concentrations of �6-testosterone,they gradually decreased during pregnancy, following a sim-ilar behaviour with other major testosterone metabolites like
xcretion of androgens conjugated to cysteine in human pregnancy by10.1016/j.jsbmb.2013.01.014
androsterone, etiocholanolone or both androstandiols. Finally,a substantial increase of 1,4-androstandiendione was observedduring pregnancy. This behaviour is similar to the obtainedwith the estrogens and norandrosterone. A possible explanation
or this behaviour is that, similarly to norandrosterone, 1,4-ndrostadiendione is formed as a side product in the conversionf testosterone to estrogens although more research is needed inrder to corroborate this hypothesis.
Thus, urinary steroid profile is modified during pregnancy andome of these modifications are already noticeable during the firsteek of pregnancy. Therefore, they should be considered whensing the athlete’s biological passport for detecting the misuse ofnabolic steroids in the anti-doping control field.
Due to the expected high interindividual variability, these pre-iminary conclusions need to be confirmed by following up a largerumber of pregnancies.
cknowledgements
This work was supported by grant from Instituto de Salud Car-os III FEDER, (CP10/00576). Financial support of WADA (11A22OP)nd grant by the Generalitat de Catalunya (2009GR00492 to theesearch team) are also acknowledged. Collaboration of M. Merino,. Haro, M. Serra, S. Saborit and S. González in providing samples
s acknowledged.
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