Patent Application: US-201314099037-A

Abstract:
concentrations of endogenous steroids in ovarian follicular fluid are used to develop steroid profiles which provide means for the diagnosis and prognosis of endocrine - related conditions and for identifying and developing appropriate treatments for related conditions , including the identification and development of suitable protocols for in vitro fertilization , treatment and predictive strategies for successful ivf outcomes and selected uses of oocytes for ivf or embryonic stem cell procedures .

Description:
a4 androstenedione adf androgen - dominant follicles allopregn allopregnalone andr androgen auc area under curve cv coefficient of variation dhea dehydroepiandrostenedione 11dc 11 deoxycortisol e cortisone e1 estrone e2 estradiol e3 estriol edf estrogen - dominant follicles esi electrospray ionization estr estrogens f cortisol ff follicular fluid gc - ms gas chromatography mass spectrometry 17ohp 17 - hydroxyprogesterone 17ohpregn 17 - hydroxypregnenolone hplc high performance liquid chromatography hsd hydroxysteroid dehydrogenase ia immunoassay is internal standard ivf in - vitro fertilization lc - ms / ms liquid chromatography tandem mass spectrometry mrm multiple reaction monitoring m / z mass to charge ratio pregn pregnenolone prog progesterone pcos polycystic ovary syndrome ria radioimmunoassay rm regularly menstruating roc receiver operating characteristic sd standard deviation shbg sex hormone binding globulin te testosterone as used herein and in the appended claims , the singular forms “ a ”, “ an ”, and “ the ” include plural reference unless the context clearly dictates otherwise . for example , reference to “ a steroid ” includes a plurality of such steroids , and reference to the “ a steroid profile ” is a reference to one or more profiles , and so forth . as used herein , “ comprising ,” “ including ,” “ having ,” “ containing ,” “ characterized by ,” and grammatical equivalents thereof , are inclusive or open - ended terms that do not exclude additional , unrecited elements or method steps , but also include the more restrictive terms “ consisting of ” and “ consisting essentially of .” as used herein , “ successful pregnancy ” or “ viable pregnancy ” means the successful implantation of a fertilized ovum such that fetal development and birth are likely to result . as used herein , “ outcome ,” when used in association with “ in vitro fertilization ,” is inclusive of both viability of an oocyte and non - viability of an oocyte for in vitro fertilization . as used herein , “ successful outcome of in vitro fertilization ” means successful fertilization of an ovum that is suitable for implantation and intrauterine development . during the last decade , tandem mass spectrometry has become the method of choice for analyzing endogenous steroids . the methods used herein allow accurate quantitation of thirteen steroids from 40 μl of ff . analysis of these steroids using ia - based methods would require at least a few milliliters of ff , which is a sample size that is unrealistic for follicles during early follicular stage of the menstrual cycle or for follicles of women with pcos . in addition there are some pitfalls associated with use of immunoassays for analyzing ff samples . compared to serum , ff has significantly higher concentrations of some of the steroids , and the difference in concentrations may cause cross - reactivity that is not observed in the serum samples ( for which ia are typically validated ). another pitfall is related to the need of reducing the concentration of steroids into the range measurable by the ia by diluting the ff . the characteristics of the diluents could alter the binding of proteins thus affecting the observed concentrations in methods not including extraction steps prior to ia . the above problems are not relevant to the mass spectrometry - based methods . methods for the analysis of steroid patterns in ff samples from rm women twenty - one regularly menstruating ( rm ) women of caucasian decent were recruited for the study . the women attended the hospital for laparoscopic treatment of infertility presumably caused by pelvic adhesions . all women had regular cycles and normal ovaries on pelvic ultrasound examination , were in good general health and had not taken hormonal medication or oral contraceptives during the last three months before inclusion in the study . the study was approved by the ethics committees in donetsk state medical university ( ukraine ) and in uppsala university ( sweden ). in rm women , ff samples were obtained between days 4 and 7 of the follicular phase of a cycle during laparoscopic adhesiolysis . ff aspirated from ovarian follicles ( 5 - 8 mm diameter ) was pooled within each subject and centrifuged . size of the follicles was measured by transvaginal ultrasonography performed during laparoscopic adhesiolysis . the samples were transferred in microcentrifuge tubes and stored at − 70 ° c . until analysis . clinical and anthropometrics characteristics of participating women are listed in table 1 , below . testosterone ( te ), estrone ( e1 ), 17βe2 , 17αe2 , estriol ( e3 ), pregnenolone ( pregn ), 17 hydroxypregnenolone ( 17 - ohpregn ), 17 hydroxyprogesterone ( 17ohp ), 11 deoxycortisol ( 11dc ), cortisol ( f ), cortisone ( e ), progesterone ( prog ), allopregnalone ( allopregn ), hydroxylamine , formic acid , trifluoroacetic acid , dansyl chloride and sodium carbonate were purchased from sigma chemical company ( st . louis , mo .). androstenedione ( a4 ), dehydroepi - androsterone ( dhea ), dihydrotestosterone ( dht ) and androstanedione ( a ) were purchased from steraloids inc . ( newport , r . i .). the internal standards ( is ) were deuterium ) labeled analogs of the steroids d 3 - te , d 3 - pregn , d 2 - 11dc , d 8 - 17ohp , d 3 - 17ohpregn , d 3 - e ( cambridge isotope laboratories , andover , mass . ); and d 4 - e1 , d 3 - e2 , d 3 - e3 and d 4 allopregn ( cdn isotopes , toronto , on ). methanol , acetonitrile , and methyl - tert - butyl ether ( mtbe ) were all hplc grade from vwr ( west chester , pa .). all other chemicals were of the highest purity commercially available . concentrations of all steroids in ff were determined using . lc - ms / ms based methods ( 20 - 25 ). estrogens were analyzed as dansyl derivatives ( 23 , 24 ); ketosteroids were analyzed as oxime derivatives ( 21 - 22 ), cortisol and cortisone were analyzed as non - derivatized ( 20 ). the hplc system consisted of series 1200 hplc pumps ( agilent , santa clare , calif . ); a 10 - port switching valve , a vacuum degasser and an autosampler htc pal ( leap technologies , nc ) equipped with a fast wash station . an api 4000 ( applied biosystems / mds sciex ) tandem mass spectrometer was used in the positive ion mode with a turboionspray ™ ion source . sample preparation , chromatographic separation conditions , and mass transitions used in the methods have been previously described ( 20 - 25 ) and are summarized in table 2 , below , the quadrupoles q1 and q3 were tuned to unit resolution and the mass spectrometer conditions were optimized for maximum signal intensity of each steroid . two mass transitions were monitored for each steroid and the steroid &# 39 ; s is . concentrations of each steroid were determined using the primary mass transitions ; specificity of the analysis for each steroid in every sample was evaluated by comparing concentrations determined using the primary and secondary mass transitions of each steroid and the steroid &# 39 ; s is ( 26 ). quantitative data analysis was performed using analyst ™ 1 . 4 . 2 software ( applied biosystems / mds sciex ). the assays showed within - run variation of less than 10 % and between - run variation of less than 12 %. calibration curves were generated with every set of samples using six calibration standards ; three quality control samples were included with every set of samples . concentrations of steroids in ff fluid of women after ovarian stimulation , obtained using lc - ms / ms methods , were compared to values observed in three studies ( 13 - 16 ) using ia methods and one study using liquid chromatography followed by spectrophotometric detection ( 14 ). the comparison of steroid concentrations is shown in table 3 , below . values obtained by lc - ms / ms methods were usually lower , and in some cases were considerably lower than those obtained by the other techniques , especially for testosterone ( e . g ., up to 18 - fold difference ). these differences are likely due to cross - reactivity of ia methods intended for performing measurements in specific matrices ( i . e ., serum ) rather than in ff , and suggest the necessity of using highly specific methods for performing measurements of steroids in ff samples . the distribution pattern of steroid concentrations in androgen - dominant follicles ( n = 13 ) and estrogen dominant follicles ( n = 8 ) was also analyzed , as illustrated in fig2 a and fig2 . androgen - dominant follicles ( adf ) were defined as having an e2 / te ratio & lt ; 4 , and estrogen - dominant follicles ( edf ) were defined as follicles with the e2 / te ratio & gt ; 4 ( 26 ). steroids for which significant differences were demonstrated between adf and edf are given in table 4 , below . compared to adf , edf had significantly higher concentration of e2 , significantly higher e2 / e1 - ratio and significantly lower concentrations of a4 and te , ( table 4 ). in adf , a4 was the dominating steroid ( 56 . 4 %), followed by 17 - ohp and dhea . in edf , a4 was also the dominating steroid ( 30 . 8 %), followed by 17 - ohp and e2 ( fig2 ). study subjects were recruited and investigated at the donetsk regional center of mother and child care , donetsk , ukraine . ff from 27 women with pcos and 21 regularly cycling women without pcos were included in this study . the diagnosis of pcos was based on amenorrhea or oligomenorrhea (& lt ; 10 cycles per year ), a characteristic ovarian image on ultrasound examination (≧ 10 small follicles per plane , in association with a marked ovarian stroma ) ( 27 ). hirsutism , was assessed by a modified version of the protocol used by ferriman and gallwey ( 28 ) and women with a score of ≧ 8 were considered clinically hirsute . bmi was calculated as weight ( kg ) divided by height ( m ) squared . all the ultrasound examinations were performed transabdominally or transvaginally ( 3 . 5 and 5 mhz sector probe , respectively ; kranzbühler gmbh , germany ). the pcos patients were treated for infertility by ovarian wedge resection and ff was collected during that surgery . control subjects were women with infertility presumably caused by pelvic adhesions . these women had regular menstrual cycles and normal ovaries on pelvic ultrasound examination . all subjects were in good general health and had not taken hormonal medication or oral contraceptives during the preceding three months prior to inclusion in the study . ultrasound images from women diagnosed with pcos and controls were blindly evaluated by two independent swedish gynecological ultrasound experts . sampling was performed between days 3 and 7 in the follicular phase in rm women ( controls ) and at any day in oligo -/ amenorrheic patients . ff from women diagnosed with pcos and ff from follicles having a diameter of 5 - 8 mm in control women were pooled within each subject and centrifuged . follicle size was measured by transvaginal ultrasonography performed during laparoscopic surgery ( wedge resection for pcos women ) or adhesiolysis ( controls ). the samples were kept frozen at below − 20 ° c . until used for analysis . the reagents and standards for ff analysis were the same as described in example 1 , above . likewise , the lc - ms / ms methods were the same as described above in example 1 . baseline comparisons between the study groups ( pcos and rm women ) were assessed using non - parametric wilcoxon two - group tests for continuous variables and chi - square test . associations between variables were accessed using the spearman rank correlation test . multiple logistic regression analysis was used to explore the putative independent effects of measured hormones and product / precursor ratios ( enzyme activities ) with regard to presence of pcos . receiver operating characteristic ( roc ) curves , were plotted for evaluation of steroids biomarkers of pcos in ff samples . for every statistically significant result cited , the p value was less than 0 . 05 , unless otherwise specified . statistical analyses were performed using the jmp software ( sas institute inc ., nc , usa ). values of steroid concentrations and the ratios of steroid concentration are expressed as median and range , unless otherwise stated . clinical data and hormone concentrations for individual study participants are given in table 6 , below , table 7 anthropometric and reproductive characteristics of pcos women and rm women of fertile age . pcos control ( n = 27 ) ( n = 21 ) mean ± sd mean ± sd variable median [ range ] median [ range ] age ( years ) 25 ± 3 . 5 #, b 28 ± 3 . 2 ## height ( cm ) 164 ± 6 . 4 165 ± 6 . 2 weight ( kg ) 73 . 5 ± 14 . 9 64 . 8 ± 10 . 4 bmi ( kg / m 2 ) 27 . 2 ± 5 . 2 b 23 . 9 ± 3 . 8 parity ( n ) 1 . 4 ± 0 . 9 2 . 1 ± 1 . 7 average number of menstrual cycles 6 / 12 [ 0 - 9 ] 12 / 12 during last 12 months menstrual cycle day of follicular fluid na 6 [ 4 - 7 ] sampling menstrual cycle length ( days ) na 28 [ 21 - 32 ] serum shbg ( nmol / l ) 42 . 8 ± 31 67 . 0 ± 27 hirsutism index ### 9 [ 6 - 24 ] c 3 [ 1 - 8 ] serum testosterone ( nmol / l ) 2 . 69 ± 1 . 2 b 1 . 6 ± 0 . 7 serum t / shbg 0 . 11 ± 0 . 2 c 0 . 03 ± 0 . 02 current smokers ( n ) 9 / 27 9 / 21 # range : 21 - 34 years ; ## range : 19 - 32 years ; ### modified ferriman and gallwey scale ; a p & lt ; 0 . 05 , b p & lt ; 0 . 01 , c p & lt ; 0 . 001 fig3 shows pie diagrams of distribution of median concentrations of measured steroids in ff of rm women ( a ) and ff of women diagnosed with pcos ( b ). in ff from women diagnosed with pcos , as compared to ff from rm women , concentrations of total androgens were significantly higher ( p & lt ; 0 . 0001 ), whereas concentrations of total estrogens ( p & lt ; 0 . 01 ) and the ratio of total - estr / total - andr ( p & lt ; 0 . 001 ) were significantly lower . all of these tests remained statistically significant after adjustment for differences in bmi , as set forth in table 8 , below . in addition , in ff of women diagnosed with pcos , concentrations of 11 deoxycortisol , dhea , 17 hydroxypregnenolone , androstenedione , testosterone , androstandione , cortisol and cortisone were significantly higher and concentrations of e1 , e2 and e3 were significantly lower compared to samples from rm women ( table 8 ). in pcos women , bmi was negatively associated with ff concentrations of total estrogens ( r =− 0 . 53 ; p = 0 . 006 ), 17ohprog ; (− 0 . 40 ; 0 . 04 ), and e2 (− 0 . 57 ; 0 . 003 ) and marginally associated with e2 / e1 ratio (− 0 . 38 ; 0 . 056 ). hirsutism index was positively associated with ft concentrations of te ( 0 . 51 ; 0 . 006 ). in regularly menstruating women , bmi was negatively associated with concentration of pregn (− 0 . 51 ; 0 . 018 ), among the three estrogens tested , e1 was strongly associated with the presence pcos . when tested alone , e1 yielded auc = 0 . 77 ; p = 0 . 009 . the association was even stronger than for the total concentration of estrogens . among the pregnenolones tested , 17ohpregn had the strongest , significant and independent association with pcos ( p = 0 . 0491 ), followed by pregn ( p = 0 . 061 ), 17ohpregn ( auc = 0 . 84 ; p = 0 . 0007 ) and total andr ( auc = 0 . 84 ; p = 0 . 0010 ). when evaluated in the same model , e1 and 17ohpregn yielded an auc of 0 . 95 , and both steroids had significant independent effects , although it was stronger for 17ohpregn ; p = 0 . 031 and p = 0 . 0026 , respectively . total andr and total estr , when included in the same model , yielded an auc = 0 . 87 ; both being independent predictors but a stronger relationship was observed for total andr , p = 0 . 0044 and p = 0 . 044 , respectively . fig4 shows examples of roc curves for potential steroid biomarkers of pcos identified herein ( only markers with auc & gt ; 0 . 75 are shown ). the greatest sensitivity and specificity out of the identified potential biomarkers was the ratio of 17ohpregn / pregn , followed by concentrations of dhea , 17ohpregn , androstanedione , the ratio of total estrogens / total androgens and the concentration of estrone . the predictive ability of the biomarkers for determination of pcos improves when they are used in combination . thus , the invention includes use of individual biomarkers , ratios of concentrations of the steroid biomarkers , and all combinations of the steroid biomarkers . comparison of the product / precursor ratios , as markers of the enzyme activities in the ovarian follicles , as shown in table 9 , below , showed that women with pcos had a higher activity of cyp17 - linked enzymes , favoring higher concentrations of 17ohpregn and a4 . in addition , ratios of e1 / a4 and e2 / te were five times and 3 times lower , respectively , in pcos women , indicating a reduced ovarian activity of cyp19 - linked enzymes ( aromatase ). when six product / precursor ratios , illustrating enzyme activities in the pathway of steroid biosynthesis ( fig1 ) were evaluated simultaneously , the auc reached 0 . 99 . however , the only significant and independent ratio was 17ohpregn / preg , p = 0 . 021 . when evaluated alone , 17ohpregn / pregn yielded auc = 0 . 95 , p = 0 . 0027 . the optimal cut - off value for the 17ohpregn / pregn ratio was found to be 0 . 89 and yielded a sensitivity of 89 % and a specificity of 90 %. when e1 / a4 ( cyp19 ) and 17ohpregn / pregn ( cyp 17 ) were included in the same model , the auc = 0 . 96 . however , only the 17ohpregn / pregn ratio had an independent effect ( p = 0 . 019 ), suggesting the strong impact of increased cyp17 activity in ff of the pcos patients . in roc analysis , the highest values of auc were found for 17ohpregn / pregn , a4 / 17ohprog , total andr , dhea , a4 and the ratio of total andr / total estr , all pointing to higher activity of cyp17 and a lower activity of cyp19 in women diagnosed with pcos as compared to women without pcos . the distribution of concentrations ( table 8 ), product / precursor ratios ( table 9 ) and the roc analysis suggest higher activity of the enzyme cyp17 and a lower activity of the enzyme cyp19 ( aromatase ) in women diagnosed with pcos . the results of the present study favor the hypothesis of a reduced activity / inhibition of aromatase enzyme in the ovaries of pcos women compared with rm women . the present data also indicates a strong influence of increased cyp17 activity leading to increasing concentrations of ff androgens . analysis of steroid profiles in ovarian ff following ovarian stimulation in women undergoing ivf treatment follicular fluid was sampled from patients attending ivf treatment at uppsala university hospital ( uppsala , sweden ). reasons for infertility in these patients included male factor infertility , tubal factor infertility , non - ovarian endometriosis and unexplained infertility . most currently , the treatment protocol consists of pituitary down - regulation by gnrh analog ( suprecur : sanofi - avensis ) employing the “ long ” protocol initiated at the mid - luteal phase ( 1200 micrograms / day , intranasal administration ). recombinant fsh ( puregon : schering - plough ) was injected daily ( 100 - 450 iu / day ) starting on cycle day 3 ( subcutaneous injection ). dose adjustment was performed , when necessary , from cycle day 7 . human chorionic gonadotropin ( hcg ) ( pregnyl : schering - plough ), 10 , 000 iu , was administered when one or more follicles reached a diameter of & gt ; 17 mm , additional details and modifications being included in table 10 . transvaginal oocyte retrieval was performed under ultrasound guidance 36 - 38 hours after hcg administration . follicles larger than 15 mm in diameter were aspirated . ff samples were kept frozen at − 20 ° c . until analysis . the reagents and standards for follicular fluid analysis were the same as described previously in example i . likewise , the lc - ms / ms methods for this aspect of the invention were the same as previously described in example i . thirteen subjects had a positive outcome ( viable fetus by ultrasound and delivered babies ) following ivf treatment , while the remaining 33 subjects had a negative outcome . negative outcomes included failure to become pregnant ( 29 subjects ) and spontaneous abortion following a positive pregnancy test ( 4 subjects ). stimulation protocols and ivf methodology did not correlate with outcome ( data not shown ). table 10 , below , shows information on the participants and the treatments . table 11 shows concentrations of steroids in ff samples of women undergoing ivf treatment , and ratios of concentrations of the steroids and ivf outcome . fig5 - 8 show graphical representations of observed values for steroid concentrations associated with both positive and negative ivf outcomes . median values for concentrations of steroids and ratios were grouped for the subjects based on the outcomes ( viable pregnancy vs . no viable pregnancy ), along with the central 90th percentile of these values , as shown in table 12 , below . the percent difference between the 5th percentile and 95th percentile values associated with each group were also determined . this analysis reveals differences in the distribution of the values for specific analytes between the groups . in comparison to the group with viable pregnancies , negative outcomes were associated with an altered distribution of steroid concentration . steroids for which 95th percentile values were markedly elevated by approximately 50 % or more in the group with no viable pregnancy , compared with those with viable pregnancy , were 17 - oh progesterone , 17 - oh pregnenolone , pregnenolone and total pregnenolones ( pregnenolone and 17 - oh pregnenolone ), indicating that higher concentrations of these steroids in ff may serve as markers predictive of a decreased probability of viable pregnancy . analytes for which 5th percentile values were decreased by 20 % or more in the group with no viable pregnancy , compared with those with viable pregnancy , were e1 , e2 , e3 , dhea , a4 , cortisol , cortisone , total estrogens ( estrone , estradiol and estrone ), and total glucocorticoids ( cortisol , cortisone ). the 95th percentile values for a4 and total androgens ( a4 , dhea , and te ) were also markedly decreased in this group . thus , lower concentrations of one or more of these steroids in ff may also be an indicator of a decreased likelihood of viable pregnancy . for some analytes , particularly hydroxyprogesterone ( a chromatographic peak which chilled at relative retention times of 0 . 89 relative to progesterone and 1 . 15 relative to 17 - hydroxyprogesterone and possessing the same characteristic mass transitions as progesterone and 17 - hydroxyprogesterone ), 11dc , estrone , pregnenolone , androstenedione , total andr , as well as the ratio 17oh - pregnenolone ; pregnenolone and the ratio estradiol / estrone , it appears that both elevated and lowered values are associated with a decreased likelihood of viable pregnancy . to determine the frequency of the steroid levels occurring outside of the distribution of the values observed in the group with no viable pregnancies compared to the viable pregnancy group , data were evaluated as follows : the minimum and maximum observed values for concentration of each steroid or ratios of concentrations of steroids in the group with viable pregnancies were determined , and the number of samples from the group with no viable pregnancy which fell outside of this range , were calculated , as shown in table 13 , below . values from the group with no viable pregnancy which were above the maximum values seen in the group with viable pregnancy were designated “ out of range high ”, and those which were below the minimum values were designated “ out of range low .” a chi - square test was performed to determine statistical significance of the findings . the results of this analysis suggest that elevated concentrations of 17 - oh progesterone , pregnenolone , 17 - oh pregnenolone , and total pregnenolones in ff are significantly less likely to be associated with a viable pregnancy , as illustrated in table 14 , below . lower concentrations of e2 , e3 , a4 , hydroxyprogesterone , 17 - ohprog , 11 - dc , e and total androgens and total estrogens in ff are also significantly less likely to be associated with a viable pregnancy , as suggested in table 15 , below . in addition , elevated ratios of 17 - oh pregnenolone / pregnenolone and a lowered ratio of 17 - oh progesterone / pregnenolone also appear to be indicative of a decreased likelihood of viable pregnancy . the invention thus provides analytical means for determining the viability of oocytes for ivf based on analyzing follicular fluid samples and determining steroid profiles therefrom . the invention also provides means for determining which oocytes are unlikely to produce favorable ivf outcomes , thereby enabling the determination of the usefulness of such oocytes for stem cell protocols . several distinct profiles of steroid distribution in ff were observed within the group of samples from women who did not become pregnant , as shown in fig9 . one group is characterized by an elevated concentration of pregn and its immediate metabolites , 17ohpreg and 17ohp ( profile 1 ). this profile appears to indicate an enhanced rate of steroidogenesis coupled with a deficiency in the activity of the enzymes required for biosynthesis of sex steroids . subjects who exhibited higher concentrations of pregnenolone and its metabolites in ff were also likely to have elevated concentration of 11 - dc . this profile is characterized by lower activity of enzymes cyp11 , cyp17 , 17βhsd , cyp19 , and 3βhsd , as shown in table 15 , below . another distinct steroid profile observed in ff of women who did not become pregnant was associated with reduced concentrations of the progestines , sex steroids 11 - dc and e ( profile 2 ). this profile is characterized by lower activity of the enzymes cyp17 , 3βhsd , cyp21 , increased activity of the enzymes cyp11 and cyp19 . ratios of concentrations of the steroids which indicate these changes in enzyme activities are shown in table 16 . the invention provides novel descriptions of steroid concentrations in ff from women diagnosed with pcos and from regularly menstruating women , thereby providing means for determining the underlying causes in more detail . simultaneous measurement of multiple steroids provides a better understanding of the underlying mechanisms and processes involved in the regulation of the menstrual cycle , ovulation and anovulation . in addition , the invention provides diagnostic and / or prognostic methods that allow for the tailoring and fine - tuning of ivf regimens to reach the goal of successful ovulation and pregnancy . the invention provides a panel of laboratory tests that provide a diagnostic test for pcos and related conditions or diseases relating to ovarian function , such as hyperandrogenism , reproductive abnormalities , infertility , menstrual disorders , anovulation , and can be useful for identification of the underlying deficiencies in ovarian function which are the cause of these and similar conditions . the invention also provides a diagnostic and / or prognostic test that may be used to refine stimulation regimens during fertility treatment , such as ivf , for selecting oocytes having a higher probability of achieving viable pregnancy , as well as for selecting oocytes which have low probability of achieving viable pregnancy , and , therefore , can be used for other purposes , such as production of embryonic stem cells for research or therapy . the invention further provides a method of analyzing the output or affect of potential drug candidates on ovarian function . while this invention has been described in certain embodiments , the present invention can be further modified within the spirit and scope of this disclosure . this application is therefore intended to cover any variations , uses , or adaptations of the invention using its general principles . further , this application is intended to cover such departures from the present disclosure as come within known or customary practice in the art to which this invention pertains and which fall within the limits of the appended claims . all references , including publications , patents , and patent applications , cited herein , and contained in the following list , are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein . 1 . speroff l , et al , ( 1999 ), chapter 6 : regulation of the menstrual cycle . in : mitchell c , editor . clinical gynecologic endocrinology and infertility . 6th ed . philadelphia , pa . : lippincott williams & amp ; wilkins ; 202 - 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