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 (IVF), treatment and predictive strategies for successful IVF outcomes and selected uses of oocytes for IVF or embryonic stem cell procedures.

Description:
TECHNICAL FIELD 
       [0001]    This invention relates to the field of biotechnology, and more particularly to the use of steroid profiles derived from analysis of ovarian follicular fluid as biomarkers for diagnosis of and/or prognosis for a subject&#39;s condition, and for predicting the viability of oocytes for selected biological procedures, especially in vitro fertilization. 
       BACKGROUND 
       [0002]    The references discussed herein are provided solely for the purpose of describing the field relating to the invention. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate a disclosure by virtue of prior invention. 
         [0003]    In women of fertile age, the ovarian follicles are the main source for the synthesis of estrogens; ovarian follicles also contribute to circulating androgens with the adrenal cortex serving as another source of circulating androgens. Follicular steroids are secreted by granulose and theca cells under the control of gonadotropins, and this hormonal microenvironment affects development of the follicles and oocyte viability (1). A higher concentration of estradiol (E2) in follicular fluid (FF) is associated with healthy mature follicles containing oocytes that are capable of meiosis, while higher concentrations of androgens are indicative of atretic changes (1, 2). With the introduction of in vitro fertilization (IVF) a number of studies have focused on analyzing FF from women receiving ovarian stimulation. The majority of these studies were undertaken to obtain prognostic parameters for the likelihood of a successful implantation (3). However, relatively few publications have focused on the steroid hormones present in FF of regularly menstruating (RM) women and the relationship of the steroids to follicular development (4). 
         [0004]    Polycystic ovary syndrome (PCOS) is one of the most common reproductive endocrine disorders, affecting about 5-8% of reproductive-age women, and is characterized by hyperandrogenism and anovulatory infertility (5). In PCOS patients, the chronic absence of ovulations results in accumulation in the ovaries of large number of atretic follicles, which produce the excess of androgens that leads to hyperandrogenism. In addition to reproductive abnormalities and hyperandrogenism, symptoms characteristic of PCOS may also include low FSH levels combined with high LH levels, obesity, hyperinsulinemia, type II diabetes, dyslipidemia, menstrual disorders, anovulation, hyperandrogenism, hirsutism, acne, a higher incidence of cardiovascular disease, and increased risk of endometrial and breast cancers. 
         [0005]    In PCOS, follicular development arrests at the stage of selection of the dominant follicle, at about 7-9 mm in diameter, which may be due in part to abnormal regulation of enzyme functions in the ovary. While the exact mechanism that blocks follicle development is not known, insulin imbalance, abnormalities in the enzymes involved in steroid hormone biosynthesis and genetic predisposition all appear to play a role. Local steroid production in the ovarian follicles is controlled by enzymes expressed in the ovaries that regulate conversion between the steroids (6, 7) ( FIG. 1 ). In PCOS, concentrations of androgens in the follicular fluid (FF) have been shown to be higher than in non-PCOS women (5). 
         [0006]    A number of studies have examined the relationship between concentrations of specific steroids in FF from women who have undergone ovarian stimulation protocols in preparation for IVF and association of steroid concentrations with IVF outcome. An increased cortisol/cortisone ratio (8, 9) and lower concentrations of cortisone in FF (8) has been associated with a positive outcome (i.e., successful pregnancy) of IVF in some studies, while others have failed to find any association between cortisone concentrations with IVF outcome (10). Higher concentrations of progesterone and progesterone/estradiol (E2) ratio in FF samples have been associated with positive outcome of IVF in one study (11), while lower progesterone concentrations were associated with positive outcome in another study (12). Higher E2/androstendione and E2/testosterone ratios have also been associated with positive outcome in IVF (13). Due to the variation in reported results from these studies, the association of concentrations of steroids in FF with IVF outcome has remained unclear. Previous studies have not attempted to examine the association between concentrations of multiple steroids and the outcome of IVF. 
         [0007]    Furthermore, the information on steroids present in FF and their concentrations in RM women is conflicting. In part this is related to the very limited sample volume of FF that may be obtained from follicles of RM women and the absence of sensitive and specific methods allowing simultaneous quantitative analysis of multiple steroids in such small samples. In previous studies (7-16), measurements of steroids in FF were performed using immunoassays (IA), which may have high cross-reactivity with structurally-related compounds (17), or using gas chromatography mass spectrometry (GC-MS) methods, which are more specific but require larger sample aliquots (18-19). Recent advancements in biological mass spectrometry helped overcome some of the problems associated with poor sensitivity and specificity of immunoassays and has enabled simultaneous accurate quantification of multiple analytes. 
         [0008]    Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) methods allow high sensitivity detection and accurate quantification of a large number of steroids using a small sample volume (20-25). Increased knowledge about the underlying mechanisms and processes involved in the regulation of the menstrual cycle and ovulation may help to understand anovulatory conditions, such as in PCOS, and help to tailor and fine-tune in vitro fertilization (IVF) regimens. In addition, knowledge of specific steroid profiles which are associated with PCOS and other endocrine disorders may be useful in providing a definitive diagnosis of a specific condition or guiding treatment. Identification of specific steroid profiles in FF associated with outcomes of successful or unsuccessful pregnancy following IVF treatments can also be used for predicting outcomes and selecting oocytes which have a greater probability of resulting in a successful pregnancy in IVF treatments; alternatively oocytes, which are identified as having a low probability of achieving viable pregnancy can be selected for use in generation of embryonic stem cells for related procedures, such as research or therapy. 
       SUMMARY OF THE INVENTION 
       [0009]    In accordance with the present invention, specific steroid profiles in FF are identified for diagnostic and prognostic use in identifying and treating conditions relating to ovarian function in women. The present invention determines the concentrations of endogenous steroids in FF and describes an association between the patterns of distribution of steroids in FF during the early follicular phase of the menstrual cycle and after ovarian stimulation for in vitro fertilization (IVF), thereby providing means for identifying potential strategies leading to successful outcomes of in vitro fertilization (IVF). The present invention also describes the steroid profiles in ovarian FF samples from women diagnosed with PCOS and in the early follicular phase of regularly menstruating women. The differences in concentrations of steroid hormones, the patterns of their distribution and differences in product/precursor ratios of steroids (illustrating relative enzyme activities), and the associations between concentrations of steroids in the FF and baseline characteristics are determined. 
         [0010]    The invention also relates to the use of a steroid profile as a diagnostic method for the identification of deficiencies or defects in one or more steroid synthesis pathway. For example, a low concentration of progesterone relative to the concentration of pregnenolone in FF samples may be indicative of a deficiency of 3βHSD. Thus, the steroid profiles of the invention provide diagnostic methods for identifying abnormal regulation in the steroid biosynthesis pathway. In addition, the identification of defects in the steroid biosynthesis pathway may also be used for selecting an appropriate IVF protocol, to predict outcome of IVF treatment, to select oocytes which are more likely to lead to a viable pregnancy and/or to modify an IVF protocol for improving chances of successful outcome. 
         [0011]    Diagnostic testing is more clinically useful when the results are related to an appropriate reference value. Comparing the pattern of distribution of steroids in the FF from PCOS and non-PCOS women provides a method for associating specific steroids or enzyme-regulating conversions that are important for normal ovarian regulation with abnormally regulated enzymes that characterize the follicular arrest in PCOS women. 
         [0012]    More particularly, accumulation in the ovaries of a large number of atretic follicles and an excess of androgens are characteristic, but not specific, markers of PCOS. Because of this, PCOS is considered a diagnosis of exclusion, meaning that the diagnosis is generated by the exclusion of other possible diseases causing similar symptoms. It is common practice to base diagnosis of PCOS on patient history, physical examination and semi-specific laboratory tests (e.g., LH-FSH ratio, free and total androgens). The testing is usually performed for the purpose of excluding other diseases which cause symptoms similar to PCOS. In contrast, the present invention identifies steroid profiles in the FF of women with PCOS and provides a comparison to the steroid concentrations observed in FF of RM women, thereby identifying specific biomarkers of PCOS ( FIGS. 3-4 ). Thus, the invention provides a more specific method for direct diagnosis of PCOS based on measurement of biomarkers in ovarian follicular fluid. 
         [0013]    The invention also provides steroid response profiles for ovarian stimulation during IVF treatment which allow a physician to choose the most suitable protocol, to select oocytes which are more likely to result in viable pregnancy, or to modify the protocol to obtain, diagnose, or prognose the successful outcome and avoid complications of the therapy or of the procedure as a whole. 
         [0014]    The invention provides values of steroid concentrations and ratios of concentrations of steroids in FF from women diagnosed with PCOS and from regularly menstruating women, thereby providing a diagnostic method for certain conditions and determination of appropriate treatment regimens. LC-MS/MS methods are highly sensitive and specific and allow simultaneous measurement of multiple steroids, and are, therefore, suitable methods for better understanding the underlying mechanism and/or processes involved in the regulation of the menstrual cycle, ovulation and anovulation. In addition, the invention provides a diagnostic and/or prognostic method that allows for identification of patients who are more likely to have a successful or unsuccessful outcome in IVF treatment, for selection of oocytes which are more likely to lead to viable pregnancy following IVF treatment, and the tailoring and fine-tuning of IVF-regimens to reach the goal of successful ovulation and pregnancy. 
         [0015]    The invention also provides a kit for determining a steroid profile comprising written instructions, at least one composition capable of use as an internal standard, and at least one reference standard. The kit may include a reference standard, wherein a steroid profile from a sample that differs from the reference is indicative of a disease condition or physiological state. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0016]      FIG. 1  depicts the pathway for biosynthesis of steroids, and the enzymes involved in the pathway; 
           [0017]      FIG. 2A  illustrates the distribution of median concentrations of steroids in FF of regularly menstruating women from androgen-dominant follicles, where androgen-dominant follicles are defined as having an E2/Te ratio &lt;4 (4); 
           [0018]      FIG. 2B  illustrates the distribution of median concentrations of steroids in FF of regularly menstruating women from estrogen-dominant follicles, where estrogen-dominant follicles are defined as having an E2/Te ratio &gt;4; 
           [0019]      FIG. 3A  illustrates the distribution of median concentrations of steroids in FF of healthy women; 
           [0020]      FIG. 3B  illustrates the distribution of median concentrations of steroids in FF of women diagnosed with PCOS; 
           [0021]      FIG. 4  shows ROC curves for six biomarkers of PCOS in FF samples; 
           [0022]      FIG. 5  illustrates comparative distributions of concentrations of 17-OH Progesterone (A), 17-OH Pregnenolone (B), Pregnenolone (C) and Total Pregnenolones (D) in subjects with a viable pregnancy and subjects with no viable pregnancy; 
           [0023]      FIG. 6  illustrates comparative distributions of concentrations of Estrone (A), Estradiol (B), Estriol (C) and Total Estrogens (D) in subjects with a viable pregnancy and subjects with no viable pregnancy; 
           [0024]      FIG. 7  illustrates comparative distributions of concentrations of DHEA (A), Androstenedione (B), hydroxyprogesterone (C) and Total Androgens (D) in subjects with a viable pregnancy and subjects with no viable pregnancy; 
           [0025]      FIG. 8  illustrates comparative distributions of concentrations of Cortisone (A), Cortisol (B), 11-Deoxycortisol (C) and Total Glucocoricoids (D) in subjects with a viable pregnancy and subjects with no viable pregnancy; 
           [0026]      FIG. 9  illustrates two distinct steroid profiles present within the group with no pregnancy or lost pregnancy outcomes. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0027]    A key to the abbreviations used herein is as follows:
       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       
 
         [0065]    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. 
         [0066]    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.” 
         [0067]    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. 
         [0068]    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. 
         [0069]    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. 
       Example I 
     Methods for the Analysis of Steroid Patterns in FF Samples from RM Women 
     Participants 
       [0070]    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). 
       Collection and Handling of Follicular Fluid Samples 
       [0071]    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. 
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                 TABLE 1 
               
             
             
               
                   
               
               
                 Anthropometric and reproductive characteristics of healthy 
               
               
                 women of fertile age (n = 21). 
               
             
          
           
               
                   
                 Variable 
                 Mean ± SD median [range] 
               
               
                   
                   
               
               
                   
                 Age (years) 
                   28 ± 3.2# 
               
               
                   
                 Height (cm) 
                 16.5 ± 6.2 
               
               
                   
                 Weight (kg) 
                 64.8 ± 10.4 
               
               
                   
                 BMI (kg/m2) 
                 23.9 ± 3.8 
               
               
                   
                 Parity 
                  2.1 ± 1.7 [1-8] 
               
               
                   
                 Average number of menstrual 
                 12/12 
               
               
                   
                 cycles during last 12 months 
               
               
                   
                 Menstrual cycle day at follicular 
                  6 [4-7] 
               
               
                   
                 fluid sampling 
               
               
                   
                 Menstrual cycle length (days) 
                 28 [21-32] 
               
               
                   
                 Hirsutism index ## 
                  3 [1-8] 
               
               
                   
                 Current smokers 
                  9/21 
               
               
                   
                   
               
               
                   
                 #range: 21-34 years; 
               
               
                   
                 ## Modified Ferriman and Gallwey scale 
               
             
          
         
       
     
       Reagents and Standards 
       [0072]    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. 
       LC-MS/MS Methods 
       [0073]    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, 
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                 TABLE 2 
               
             
             
               
                   
               
               
                 Outline of sample preparation and instrumental analysis for determination of concentrations of steroids in FF samples. 
               
             
          
           
               
                   
                 Sample 
                 Mass transitions, m/z (Collision energy, V) 
               
             
          
           
               
                 Analyte 
                 IS 
                 preparation 
                 LC column 
                 LC conditions 
                 Quantitative 
                 Qualitative 
               
               
                   
               
               
                 Pregnenolone 
                 d 4 -Pr 
                 20 μL of follicular 
                 Synergy Fusion RP, 
                 Mobile phase: 70% 
                 332 to 86 (40 V) 
                 332 to 300 (30 V) 
               
               
                 (Pr) 
                   
                 fluid (FF) extracted 
                 50 × 2 mm, 5 μm 
                 methanol, 30% 
                   
                   
               
               
                 17-OH-pregnen- 
                 d 3 -17OHPr 
                 by SPE, derivatized 
                 (Phenomenex). 
                 formic acid, 5 
                 348 to 330 (5 V) 
                 348 to 312 (20 V) 
               
               
                 olone (17OHPr) 
                   
                 with hydroxylamine, 
                   
                 mM, flow rate 
                   
                   
               
               
                 17-OH-proges- 
                 d 8 -17OHP 
                 derivative extracted 
                   
                 250 μL/min 
                 361 to 124 (45 V) 
                 361 to 112 (45 V) 
               
               
                 terone (17OHP) 
                   
                 with MTBE 
                   
                   
                   
                   
               
               
                 11-deoxy- 
                 d 2 -11DC 
                   
                   
                   
                 377 to 124 (42 V) 
                 377 to 112 (42 V) 
               
               
                 cortisol (11DC) 
                   
                   
                   
                   
                   
                   
               
               
                 Testosterone 
                 d 3 -Te 
                   
                   
                   
                 304 to 124 (40 V) 
                 304 to 112 (40 V) 
               
               
                 (Te) 
                   
                   
                   
                   
                   
                   
               
               
                 DHT 
                 d 3 -Te 
                   
                   
                   
                 304 to 253 (32 V) 
                 304 to 213 (32 V) 
               
               
                 DHEA 
                 d 3 -Te 
                   
                   
                   
                 306 to 255 (40 V) 
                 306 to 215 (40 V) 
               
               
                 Androstanedioe 
                 d 3 -Te 
                   
                   
                   
                 304 to 286 (30 V) 
                 304 to 271 (30 V) 
               
               
                 Androstenedioe 
                 d 3 -Te 
                   
                   
                   
                 317 to 124 (40 V) 
                 317 to 124 (45 V) 
               
               
                 Progesterone 
                 d 8 -17OHP 
                   
                   
                   
                 345 to 124 (40 V) 
                 345 to 112 (40 V) 
               
               
                 Allopregnan- 
                 d 4 -Allo 
                   
                   
                   
                 334 to 86 (48 V) 
                 334 to 316 (25 V) 
               
               
                 olone (Allo) 
                   
                   
                   
                   
                   
                   
               
               
                 Hydroxy- 
                 d 8 -170HP 
                   
                   
                   
                 304 to 124 (40 V) 
                 304 to 112 (40 V) 
               
               
                 progesterone 
                   
                   
                   
                   
                 and 346 to 124 (40 V) 
                 and 346 to 112 (40 V) 
               
               
                 Cortisol (F) 
                 d 4 -F, 
                 10 μL of FF 
                 Luna Phenyl-hexyl 
                 Mobile phase: 50% 
                 363 to 121 (35 V) 
                 363 to 97 (45 V) 
               
               
                 Cortisone (E) 
                 d 3 -E 
                 extracted with 
                 50 × 2 mm, 5 μm 
                 methanol; 50% 
                 361 to 163 (35 V) 
                 361 to 163 (25 V) 
               
               
                   
                   
                 MTBE, evaporated, 
                 particles 
                 water, 5 mM; flow 
                   
                   
               
               
                   
                   
                 reconstituted 
                 (Phenomenex). 
                 rate 300 μL/min 
                   
                   
               
               
                 Estrone (E1) 
                 d 4 -E1 
                 10 μL of FF, 
                 2D LC: 1 st  dimension 
                 Gradient 90% water 
                 504 to 156 (75 V) 
                 504 to 171 (45 V) 
               
               
                 17α-estradiol 
                 d 3 -17βE2 
                 extracted with 
                 separation C1, 
                 to 50% water (in 
                 506 to 156 (75 V) 
                 506 to 171 (45 V) 
               
               
                   
                   
                 MTBE derivatized 
                 2 nd  dimension 
                 methanol) 
                   
                   
               
               
                 17β-estradiol (E2) 
                 d 3 -17βE2 
                 with dansyl chloride 
                 Germini C6 100 × 
                 Gradient 50% water 
                 506 to 156 (75 V) 
                 506 to 171 (45 V) 
               
               
                 Estriol (E3) 
                 d 3 -E3 
                   
                 2 mm, 3 μm (both 
                 to 85% water (in 
                 522 to 156 (75 V) 
                 544 to 171 (45 V) 
               
               
                   
                   
                   
                 Phenomenex). 
                 acetonitrile), flow 
                   
                   
               
               
                   
                   
                   
                   
                 rate 600 μL/min 
               
               
                   
               
             
          
         
       
     
         [0074]    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. 
         [0075]    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. 
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                 TABLE 3 
               
             
             
               
                   
               
               
                 Median values of concentration of steroids in FF samples 
               
               
                 collected at oocyte retrieval from women undergoing ovarian stimulation 
               
               
                 determined with LC-MS/MS and IA methods, comparing values from 
               
               
                 the present study and values reported in published studies. 
               
             
          
           
               
                   
                   
                 De Sutter 
                   
                 Bergh 
                   
               
               
                   
                 Present 
                 et al 
                 Andersen 
                 et al 
                 Smitz et al 
               
               
                   
                 study 
                 1991 (14) 
                 1993 (13) 
                 1995 145) 
                 2007 (16) 
               
               
                   
                   
               
             
          
           
               
                 Foll.diam, mm 
                 &gt;15 
                 Na 
                 &gt;12 
                 &gt;12 
                 na 
               
               
                 Method 
                 LC- 
                 LC- 
                 IA 
                 IA 
                 IA 
               
               
                   
                 MS/MS 
                 Spectr. 
               
               
                 17OHP 
                 520 
                 460 
               
               
                 DHEA 
                 2.7 
                   
                   
                 4.8 
               
               
                 A4 
                 6.8 
                 19.3 
                 14.1 
                 18.6 
                 14.6 
               
               
                 Te 
                 0.3 
                   
                 2.9 
                 5.5 
                 4.4 
               
               
                 E1 
                 24 
                   
                   
                 29 
               
               
                 E2 
                 240 
                 390 
                 594 
                 373 
                 431 
               
               
                 Cortisol 
                 53 
                   
                   
                   
                 188 
               
               
                 Cortisone 
                 12 
                   
                   
                   
                 18 
               
               
                   
               
               
                 Conc. are in ng/mL.; 
               
               
                 na = data not available; 
               
               
                 LC-Spectr = liquid chromatography-spectrophotometry 
               
             
          
         
       
     
         [0076]    The distribution pattern of steroid concentrations in androgen-dominant follicles (n=13) and estrogen dominant follicles (n=8) was also analyzed, as illustrated in  FIG. 2A  and  FIG. 213 . 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 ( FIG. 2 ). 
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                 TABLE 4 
               
             
             
               
                   
               
               
                 Variables showing significant differences between FF samples 
               
               
                 from androgen dominant (ADF) and estrogen dominant (EDF) follicles 
               
               
                 from RM women. 
               
             
          
           
               
                   
                 ADF 
                 EDF 
               
               
                   
                   
               
             
          
           
               
                   
                 N 
                 13 
                 8 
               
             
          
           
               
                   
                 A4 
                 590 
                 (330-890) 
                 300 
                 (180-410)** 
               
               
                   
                 Te 
                 25 
                 (15-54) 
                 7.5 
                 (6.0-21)** 
               
               
                   
                 E2 
                 14 
                 (2.0-43) 
                 190 
                 (33-490)*** 
               
               
                   
                 E1 
                 22 
                 (3.3-97.1) 
                 83 
                 (15.5-139.9)* 
               
               
                   
                 E2/E1-ratio 
                 0.42 
                 (0.15-2.44) 
                 2.16 
                 (0.81-6.64)** 
               
               
                   
                   
               
               
                   
                 Conc. in ng/mL; Median (5th-95th percentile) 
               
               
                   
                 Significance of differences between the groups denoted by: 
               
               
                   
                 *p &lt; 0.05, 
               
               
                   
                 **p &lt; 0.01, and 
               
               
                   
                 ***p &lt; 0.001, respectively. 
               
             
          
         
       
     
         [0077]    The concentrations of various steroids from FF samples taken from RM women were determined and are shown in Table 5, below. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Concentrations of steroids in FF samples of RM women 
               
               
                 measured by LC-MS/MS. Median (5th-95th percentile). 
               
             
          
           
               
                   
                 RM women 
               
               
                   
                   
               
             
          
           
               
                   
                 Number 
                 21 
               
             
          
           
               
                   
                 Pregnenolone (Pregn) 
                 52 
                 (16-89) 
               
               
                   
                 17OH pregnenolone (17OH Pregn) 
                 32 
                 (4.4-60) 
               
               
                   
                 17OH progesterone (17OHP) 
                 180 
                 (65-310) 
               
               
                   
                 11 deoxycortisol (11DC) 
                 4.1 
                 (1.8-6.6) 
               
               
                   
                 Cortisol (F) 
                 17 
                 (3.9-38) 
               
               
                   
                 Cortisone (E) 
                 32 
                 (19-47) 
               
               
                   
                 Dehydroepiandrosterone (DHEA) 
                 86 
                 (34-190) 
               
               
                   
                 Adrostenedione (A4) 
                 420 
                 (200-830) 
               
               
                   
                 Testosterone (Te) 
                 18 
                 (6.2-43) 
               
               
                   
                 Androstanedione (A) 
                 2.0 
                 (0.6-6.2) 
               
               
                   
                 Androgens total 
                 534,013 
                 (252-997) 
               
               
                   
                 Estrone (E1) 
                 34 
                 (3.3-140) 
               
               
                   
                 Estradiol (E2) 
                 31 
                 (2.6-302) #   
               
               
                   
                 Estriol (E3) 
                 0.47 
                 (0.1-2.3) #   
               
               
                   
                 Estrogens total 
                 66 
                 (11-388) #   
               
               
                   
                 F/E ratio 
                 0.55 
                 (0.14-1.19) 
               
               
                   
                 E2/E1 ratio 
                 0.66 
                 (0.15-3.51) #   
               
               
                   
                 E2/Te ratio 
                 1.5 
                 (0.12-42) #   
               
               
                   
                   
               
               
                   
                   # one result was excluded as outlier (using Mahalanobis test). 
               
               
                   
                 Concentrations in ng/mL. 
               
             
          
         
       
     
       Example II 
     Steroid Profiles in FF from Women with and without PCOS 
     Participants 
       [0078]    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. 
         [0079]    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. 
         [0080]    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. 
         [0081]    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. 
         [0082]    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. 
         [0083]    Clinical data and hormone concentrations for individual study participants are given in Table 6, below, 
         [0000]                                                                                                                                      TABLE 6                                           Sum ocf                                                               concentra-       Total                               Sample       17 OH       Total   Total   tions (A4 +       Pregnen-   Total           Total               ID   Group   Pregn   E1   ANDR   steroids   DHEA + 17OHP)   A4   olones   GC   DHEA   E3   ESTR   11DC   Cortiso               37   Normal   4.38   34.1   1104.2   1142.7   1160.2   992   20.78   41.56   34.2   0.82   49.1   5.06   3.9       38   Normal   48.4   55.1   997.3   1100.8   1165   829   106.5   61.17   132   0.494   77.4   5.17   28.1       39   Normal   82.1   143   454.8   679.9   629   251   170.7   43.45   197   1.01   216.8   3.25   17.5       45   Normal   4.41   3.24   627.6   635.3   647.4   562   13.61   35.01   26.8   1.16   5.6   3.37   3.84       46   Normal   13.3   134   483.2   630.5   696.7   398   53.9   60.35   61.7   3.67   439.7   5.05   13.3       53   Normal   36.7   31.1   650.9   718.7   858.4   549   85.7   60.15   81.4   0.721   85.8   4.85   21.6       54   Normal   15.3   3.26   354.9   373.5   402   289   67.2   87.3   48.5   0.451   15.1   1.6   57       71   Normal   54.5   66.6   758.3   879.4   975   599   121.8   80.91   128   0.402   102.5   5.51   32.7       73   Normal   49.6   11.8   511.2   572.6   677   384   114.6   56.08   118   0.378   51.1   3.08   21.5       89   Normal   59.9   18.1   734.3   812.3   884   531   129.1   56.19   185   0.0809   20.7   3.59   20.5       91   Normal   43.5   21   486.0   550.5   618   353   109.8   89.58   117   0.34   54.8   2.58   38.3       92   Normal   11.5   22.5   267.7   301.7   371   220   43.5   36.24   39   0.148   53.1   1.84   16.9       107   Normal   18.3   11.2   806.2   835.7   878.9   700   41.3   56.6   62.9   0.367   18.7   4.2   16.8       108   Normal   14.8   70.6   252.1   337.5   557.3   198   114.3   36.07   46.3   10.9   669.5   6.57   10.5       117   Normal   31.9   80.3   534.0   646.2   732.8   424   87.7   53.67   85.8   2.11   197.4   3.87   10       119   Normal   28.8   6.25   526.3   561.4   595.5   420   54.3   61.96   88.7   0.091   10.5   2.96   32       120   Normal   35.8   65.3   862.5   963.6   1013   716   95.3   46.61   101   0.243   92.6   6.01   12       121   Normal   38.5   143   774.7   956.2   1061   639   119.2   53.86   117   0.694   171.4   6.56   11.5       122   Normal   45.5   22.3   786.6   854.4   895   594   96.9   64.9   163   0.0997   25.7   4.1   13.8       123   Normal   17.9   84.8   220.8   323.5   387.3   164   55.7   36.76   48.3   2.26   385.1   2.51   5.95       124   Normal   26.8   124   433.8   584.6   638.9   343   72.5   59.09   83.9   1.54   381.5   4.79   13       6   PCOS   14.3   2.1   239.7   415.8   274.3   193   27.4   91.29   36.2   1.67   12.3   1.79   59.8       8   PCOS   47.7   9.51   990.9   1386.8   1130   807   95.8   104.31   147   0.482   19.8   6.21   57.8       9   PCOS   64.6   5.54   1170.5   1558.7   1303   898   119.4   61.45   234   0.492   36.4   5.35   16.1       20   PCOS   71.8   26.2   1177.3   1620.8   1315   913   139.7   90.27   220   0.374   31.5   5.97   40.2       29   PCOS   79.9   7.66   1145.2   1728.3   1393   928   147.2   124.3   177   0.32   23.6   8.1   66.7       32   PCOS   35.5   5.41   1300.6   1589.9   1404   1100   64.9   59.81   153   0.274   13.5   6.01   15.9       35   PCOS   43.1   3.13   801.8   1058.7   890   631   66   70.32   142   0.168   3.6   5.32   17.8       51   PCOS   48.1   10.8   606.4   967.9   747.5   499   107.4   69.64   86.5   0.696   22.5   4.14   32.2       52   PCOS   71.8   4.31   862.9   1339.3   1090   722   161.9   49.17   120   0.428   17.3   3.87   20.5       56   PCOS   82.4   11.1   1526.5   2043.6   1731   1240   160.2   69.07   234   0.54   30.8   7.37   29       57   PCOS   53.1   24.7   863.8   1325.6   1048   657   112.8   61.23   176   0.119   72.8   4.63   13.3       63   PCOS   63   23.6   851.4   1243.9   990   646   118.6   70.85   171   0.0909   30.0   4.35   23.3       65   PCOS   96.5   17.1   1579.2   2122.4   1807   1270   175   66.72   263   0.416   27.5   9.42   12.6       72   PCOS   66.8   11.2   851.0   1424.1   1108   688   144.9   88.43   140   0.465   59.8   5.43   32       79   PCOS   66.8   8.24   1025.1   1434.6   1237   828   109.6   54.7   176   0.133   12.3   5   17.3       80   PCOS   68.5   9.2   841.2   1259.2   1041   679   121.4   57.07   139   0.113   16.5   5.77   14.9       82   PCOS   72.8   35.6   1285.2   1921.2   1523   1040   132.1   50.54   213   2.75   183.4   7.48   9.16       84   PCOS   103   12.2   1578.0   2382.9   2016   1310   193.8   104.5   225   0.184   25.6   15.5   31.5       85   PCOS   124   25.4   1348.3   2505.7   2006   1100   255   153.4   210   0.387   53.0   10.6   89.6       87   PCOS   61.2   6.83   883.1   1243.1   1022   698   114.2   65.35   154   0.145   10.5   4.05   19.1       90   PCOS   90.1   7.08   998.8   1411.6   1181   769   148   49.72   206   0.0886   9.1   4.92   16.1       96   PCOS   48.9   36.4   992.3   1526.2   1167   810   102.2   49.89   130   0.318   154.7   6.19   5.7       97   PCOS   40.7   0   920.5   1272.3   1068   769   84.8   94.06   126   0   0.0   5.66   32.1       101   PCOS   37.5   10.4   529.5   821.6   631.1   414   68.7   80.48   96.1   0.066   22.0   3.68   49.1       109   PCOS   35.6   22.5   681.0   1010.6   786   515   75.2   96.88   141   0.191   27.5   4.28   35.2       110   PCOS   75.5   16.7   1254.9   1817.4   1558   1090   125.5   67.4   134   0.162   35.6   10.1   16.6       112   PCOS   59.1   21.1   508.1   859.8   619   317   123.3   82.11   181   0.119   25.2   2.11   40.1                                            Total   Ratio   Ratio Total   Ratio   Ratio           Ratio       Sample       Andro-           17   Pro-   11DC/   ESTR/Total   DHEA/   Cortisol/   Ratio   Ratio   17OH Pregn/       ID   Cortisone   stanediol   E2   Pregn   OH P   gestines   17OHP   ANDR   17OHP   Cortisone   E2/E1   E2/Te   Pregn               37   32.6   7   14   16.4   134   134   0.038   0.04   7.81   0.12   0.42   0.201   0.27       38   27.9   3   22   58.1   204   204   0.025   0.08   2.73   1.01   0.40   0.661   0.83       39   22.7   1   73   88.6   181   181   0.018   0.48   2.40   0.77   0.51   11.761   0.93       45   27.8   5   1   9.2   58.6   58.6   0.058   0.01   6.08   0.14   0.36   0.034   0.48       46   42   5   302   40.6   237   237   0.021   0.91   4.64   0.32   2.25   16.413   0.33       53   33.7   2   54   49   228   228   0.021   0.13   2.22   0.64   1.74   2.842   0.75       54   28.7   2   11   51.9   64.5   64.5   0.025   0.04   3.17   1.99   3.50   0.722   0.29       71   42.7   6   36   67.3   248   248   0.022   0.14   2.35   0.77   0.53   1.414   0.81       73   31.5   1   39   65   175   175   0.018   0.10   2.38   0.68   3.30   4.518   0.76       89   32.1   2   3   69.2   168   168   0.021   0.03   3.09   0.64   0.14   0.159   0.87       91   48.7   2   34   66.3   148   148   0.017   0.11   2.69   0.79   1.60   2.376   0.66       92   17.5   1   31   32   112   112   0.016   0.20   3.39   0.97   1.36   3.756   0.36       107   35.6   3   7   23   116   116   0.036   0.02   3.44   0.47   0.63   0.176   0.80       108   19   1   588   99.5   313   313   0.021   2.66   3.13   0.55   8.33   85.465   0.15       117   39.8   2   115   55.8   223   223   0.017   0.37   2.69   0.25   1.43   5.157   0.57       119   27   2   4   25.5   86.8   86.8   0.034   0.02   3.08   1.19   0.66   0.266   1.13       120   28.6   3   27   59.5   196   196   0.031   0.11   2.82   0.42   0.42   0.635   0.60       121   35.8   1   28   80.7   305   305   0.022   0.22   3.04   0.32   0.19   1.539   0.48       122   47   3   3   51.4   138   138   0.030   0.03   3.58   0.29   0.15   0.124   0.89       123   28.3   3   298   37.8   175   175   0.014   1.74   2.70   0.21   3.51   49.750   0.47       124   41.3   1   256   45.7   212   212   0.023   0.88   3.13   0.31   2.06   41.626   0.59       6   29.7   1   9   13.1   45.1   45.1   0.040   0.05   2.53   2.01   4.08   0.946   1.09       8   40.3   7   10   48.1   176   176   0.035   0.02   3.08   1.43   1.03   0.323   0.99       9   40   5   30   54.8   171   171   0.031   0.03   3.62   0.40   5.49   0.913   1.18       20   44.1   5   5   67.9   182   182   0.033   0.03   3.06   0.91   0.19   0.126   1.06       29   49.5   6   16   67.3   288   288   0.028   0.02   2.22   1.35   2.04   0.455   1.19       32   37.9   9   8   29.4   151   151   0.040   0.01   4.31   0.42   1.45   0.202   1.21       35   47.2   3   0   22.9   117   117   0.045   0.00   3.29   0.38   0.08   0.010   1.88       51   33.3   4   11   59.3   162   162   0.026   0.04   1.80   0.97   1.02   0.636   0.81       52   24.8   5   13   90.1   248   248   0.016   0.02   1.67   0.83   2.92   0.792   0.80       56   32.7   10   19   77.8   257   257   0.029   0.02   2.84   0.89   1.73   0.456   1.06       57   43.3   4   48   59.7   215   215   0.022   0.08   3.31   0.31   1.94   1.798   0.89       63   43.2   3   6   55.6   173   173   0.025   0.04   2.71   0.54   0.27   0.200   1.13       65   44.7   4   10   78.5   274   274   0.034   0.02   2.73   0.28   0.58   0.239   1.23       72   51   2   48   78.1   280   280   0.019   0.07   2.10   0.63   4.29   2.346   0.86       79   32.4   1   4   42.8   233   233   0.021   0.01   2.63   0.53   0.47   0.198   1.56       80   36.4   2   7   52.9   223   223   0.026   0.02   2.03   0.41   0.78   0.339   1.29       82   33.9   2   145   59.3   270   270   0.028   0.14   2.93   0.27   4.07   4.866   1.23       84   57.5   5   13   90.8   481   481   0.032   0.02   2.18   0.55   1.08   0.346   1.13       85   53.2   11   27   131   696   696   0.015   0.04   1.69   1.68   1.07   0.978   0.95       87   42.2   4   4   53   170   170   0.024   0.01   2.52   0.45   0.52   0.128   1.15       90   28.7   2   2   57.9   206   206   0.024   0.01   2.29   0.56   0.27   0.088   1.56       96   38   5   118   53.3   227   227   0.027   0.16   2.66   0.15   3.24   2.500   0.92       97   56.3   0   0   44.1   173   173   0.033   0.00   3.10   0.57       0.000   0.92       101   27.7   3   12   31.2   121   121   0.030   0.04   2.56   1.77   1.11   0.697   1.20       109   57.4   2   5   39.6   130   130   0.033   0.04   3.96   0.61   0.21   0.213   0.90       110   40.7   5   19   50   334   334   0.030   0.03   1.77   0.41   1.12   0.719   1.51       112   39.9   1   4   64.2   121   121   0.017   0.05   3.06   1.01   0.19   0.468   0.92                    
Women with PCOS had higher BMI values, serum testosterone, Te/SHBG-ratio and a hirsutism index compared to RM women, as shown in Table 7, below.
 
         [0000]                                  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            
Comparison of Median Values in PCOS Vs. RM Women
 
         [0084]      FIG. 3  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), 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 Median concentration of steroids in FF (ng/mL) of PCOS 
               
               
                 women and RM women. 
               
             
          
           
               
                   
                 PCOS 
                 RM 
                 PCOS vs. 
                   
               
               
                   
                 N = 27 
                 N = 21 
                 RM 
                 P value* 
               
               
                   
                   
               
             
          
           
               
                 Estrone 
                 11.0 
                 34.1 
                 − 
                 0.0016 
               
               
                 Estradiol 
                 10.5 
                 30.5 
                 − 
                 0.032 
               
               
                 Estriol 
                 0.3 
                 0.5 
                 − 
                 0.028 
               
               
                 Dehydroepiandrosterone 
                 154.0 
                 85.8 
                 + 
                 &lt;0.0001 
               
               
                 17 hydroxypregnenolone 
                 64.6 
                 31.9 
                 + 
                 &lt;0.0001 
               
               
                 Androstenedione 
                 769.0 
                 424.0 
                 + 
                 0.0003 
               
               
                 Testosterone 
                 26.7 
                 18.0 
                 + 
                 0.024 
               
               
                 Androstanedione 
                 3.6 
                 2.0 
                 + 
                 0.024 
               
               
                 17 hydroxyprogesterone 
                 206.0 
                 175.0 
                 + 
                 0.17 
               
               
                 Pregnenolone 
                 55.6 
                 51.9 
                 + 
                 0.49 
               
               
                 Total androgens 
                 991.0 
                 534.0 
                 + 
                 &lt;0.0001† 
               
               
                 Total estrogens 
                 25.4 
                 77.4 
                 − 
                 &lt;0.006‡ 
               
               
                 Ratio total estrogens/ 
                 0.028 
                 0.11 
                 − 
                 0.0004§ 
               
               
                 total androgens 
               
               
                 11 deoxycortisol 
                 5.4 
                 4.1 
                 + 
                 0.007 
               
               
                 Cortisol 
                 23.3 
                 16.8 
                 + 
                 0.030 
               
               
                 Cortisone 
                 40.3 
                 32.1 
                 + 
                 0.004 
               
               
                   
               
               
                 *Non-parametric test (Wilcoxon test); 
               
               
                 †adj for BMI, p &lt; 0.0001: 
               
               
                 ‡Adj for BMI: p &lt; 0.005; 
               
               
                 §Adj for BMI: p &lt; 0.01. 
               
             
          
         
       
     
       Multiple Logistic Regression Analysis and ROC Analysis 
       [0085]    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. 
         [0086]      FIG. 4  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 Ratios of Concentrations of Steroid Products/Precursors in the Pathway 
       [0087]    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). 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                 Ratio of concentrations of steroids product/precursors of the 
               
               
                 pathway values for the groups used as markers of enzyme activities 
               
               
                 in PCOS and non-PCOS women. 
               
             
          
           
               
                   
                 Steroid product/precursor 
                   
                 Control 
               
               
                   
                 concentration 
                 PCOSN = 
                 women 
               
               
                 Enzyme 
                 ratios 
                 27 
                 N = 21 
               
               
                   
               
             
          
           
               
                 3βHSD 
                 17OHProg/17OHPregn 
                 3.45 b   
                 6.21 
               
               
                 CYP21 
                 11DC/17OHProg 
                 0.028 0.06   
                 0.022 
               
               
                 CYP11 
                 F/11DC 
                 4.72 
                 4.0 
               
               
                 11βHSD type 1 and 2 
                 E/F 
                 1.78 
                 1.81 
               
               
                 CYP17 
                 DHEA/17OHPregn 
                 2.66 a   
                 3.08 
               
               
                 CYP17 
                 A4/17OHProg 
                 3.73 a   
                 2.41 
               
               
                 CYP17 
                 17OHPregn/Pregn 
                 1.13 c   
                 0.60 
               
               
                 3βHSD 
                 A4/DHEA 
                 4.89 
                 4.92 
               
               
                 17HSD3 
                 Te/A4 
                 0.035 
                 0.040 
               
               
                 CYP19 
                 E1/A4 
                 0.014 c   
                 0.067 
               
               
                 CYP19 
                 E2/Te 
                 0.455 a   
                 1.54 
               
               
                 17βHSD1 type 1 and 2 
                 E2/E1 
                 1.08 
                 0.66 
               
               
                   
               
               
                 *Non-parametric test (Wilcoxon two-group test); 
               
               
                   a p &lt; 0.05, 
               
               
                   b p &lt; 0.01, 
               
               
                   c p &lt; 0.001 
               
             
          
         
       
     
         [0088]    When six product/precursor ratios, illustrating enzyme activities in the pathway of steroid biosynthesis ( FIG. 1 ) 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. 
         [0089]    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. 
         [0090]    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. 
       Example III 
     Analysis of Steroid Profiles in Ovarian FF Following Ovarian Stimulation in Women Undergoing IVF Treatment 
     Participants 
       [0091]    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. 
       Follicle Fluid Collection and Analysis 
       [0092]    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. 
         [0093]    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. 
         [0000]    
       
         
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 10 
               
               
                   
               
               
                   
                   
                 Previous 
                 Starting 
                   
                   
                   
                 Number 
                 Urine HCG 
                   
               
               
                 Sample 
                 Age at start 
                 IVF 
                 Dose of 
                 Total dose 
                 # days with 
                 hCG given 
                 oocytes 
                 (positive or 
                   
               
               
                 # 
                 of stimulation 
                 attempts 
                 FSH (IU) 
                 FSH (IU) 
                 FSH given 
                 at day 
                 retrieved 
                 negative) 
                 Ultrasound result 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 6642 
                 33 
                 4 
                 200 
                 1900 
                 10 
                 10 
                 11 
                 + 
                 Viable fetus 
               
               
                 6653 
                 35 
                 3 
                 300 
                 2700 
                 9 
                 9 
                 10 
                 − 
                 No pregnancy 
               
               
                 6654 
                 26 
                 1 
                 125 
                 875 
                 10 
                 10 
                 11 
                 + 
                 Positive pregnancy 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 test, no viable fetus 
               
               
                 6655 
                 38 
                 1 
                 250 
                 3050 
                 13 
                 13 
                 11 
                 − 
                 No pregnancy 
               
               
                 6658 
                 30 
                 2 
                 150 
                 1350 
                 9 
                 9 
                 3 
                 + 
                 Viable fetus 
               
               
                 6659 
                 30 
                 1 
                 100 
                 700 
                 7 
                 7 
                 3 
                 − 
                 No pregnancy 
               
               
                 6660 
                 25 
                 1 
                 300 
                 3600 
                 12 
                 12 
                 12 
                 + 
                 Viable fetus 
               
               
                 6661 
                 30 
                 2 
                 250 
                 3500 
                 14 
                 14 
                 10 
                 − 
                 No pregnancy 
               
               
                 6662 
                 35 
                 1 
                 225 
                 3150 
                 14 
                 14 
                 19 
                 − 
                 No pregnancy 
               
               
                 6663 
                 35 
                 2 
                 425 
                 7225 
                 17 
                 17 
                 6 
                 − 
                 No pregnancy 
               
               
                 6664 
                 38 
                 3 
                 225 
                 1575 
                 7 
                 7 
                 5 
                 − 
                 No pregnancy 
               
               
                 6643 
                 39 
                 4 
                 450 
                 5400 
                 12 
                 12 
                 3 
                 − 
                 No pregnancy 
               
               
                 6665 
                 39 
                 5 
                 375 
                 3750 
                 10 
                 10 
                 5 
                 − 
                 No pregnancy 
               
               
                 6667 
                 30 
                 2 
                 150 
                 1650 
                 11 
                 11 
                 7 
                 + 
                 Viable fetus 
               
               
                 6670 
                 38 
                 1 
                 100 
                 1350 
                 12 
                 12 
                 2 
                 − 
                 No pregnancy 
               
               
                 6671 
                 38 
                 2 
                 250 
                 2300 
                 11 
                 11 
                 8 
                 − 
                 No pregnancy 
               
               
                 6672 
                 38 
                 2 
                 150 
                 1650 
                 11 
                 11 
                 11 
                 + 
                 Positive pregnancy 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 test, no viable fetus 
               
               
                 6673 
                 36 
                 3 
                 300 
                 3900 
                 13 
                 13 
                 12 
                 + 
                 Viable fetus 
               
               
                 6674 
                 27 
                 2 
                 150 
                 2200 
                 13 
                 12 
                 7 
                 + 
                 Viable fetus 
               
               
                 6675 
                 37 
                 2 
                 150 
                 1575 
                 12 
                 12 
                 19 
                 − 
                 No pregnancy 
               
               
                 6676 
                 30 
                 1 
                 100 
                 725 
                 10 
                 10 
                 20 
                 − 
                 No pregnancy 
               
               
                 6677 
                 33 
                 1 
                 200 
                 2800 
                 14 
                 14 
                 4 
                 − 
                 No pregnancy 
               
               
                 6644 
                 31 
                 2 
                 150 
                 2100 
                 14 
                 14 
                 9 
                 − 
                 No pregnancy 
               
               
                 6678 
                 38 
                 3 
                 300 
                 3900 
                 13 
                 13 
                 10 
                 + 
                 Viable fetus 
               
               
                 6686 
                 34 
                 3 
                 300 
                 3600 
                 12 
                 12 
                 8 
                 − 
                 No pregnancy 
               
               
                 6688 
                 35 
                 1 
                 150 
                 1800 
                 12 
                 12 
                 11 
                 + 
                 Positive pregnancy 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 test, no viable fetus 
               
               
                 6689 
                 39 
                 5 
                 300 
                 3600 
                 12 
                 12 
                 10 
                 − 
                 No pregnancy 
               
               
                 6690 
                 31 
                 2 
                 150 
                 1800 
                 13 
                 12 
                 11 
                 + 
                 Positive pregnancy 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 test, no viable fetus 
               
               
                 6691 
                 33 
                 3 
                 105 
                 1260 
                 12 
                 11 
                 10 
                 + 
                 Viable fetus 
               
               
                 6692 
                 35 
                 1 
                 300 
                 2700 
                 9 
                 9 
                 2 
                 − 
                 No pregnancy 
               
               
                 6693 
                 39 
                 1 
                 200 
                 1250 
                 7 
                 7 
                 7 
                 − 
                 No pregnancy 
               
               
                 6694 
                 37 
                 3 
                 187.5 
                 1875 
                 10 
                 10 
                 21 
                 + 
                 Viable fetus 
               
               
                 6695 
                 38 
                 2 
                 300 
                 3300 
                 11 
                 11 
                 7 
                 + 
                 Viable fetus 
               
               
                 6645 
                 35 
                 4 
                 450 
                 4500 
                 11 
                 11 
                 9 
                 − 
                 No pregnancy 
               
               
                 6698 
                 31 
                 1 
                 100 
                 1000 
                 10 
                 10 
                 12 
                 − 
                 No pregnancy 
               
               
                 6699 
                 31 
                 1 
                 150 
                 1650 
                 11 
                 11 
                 6 
                 − 
                 No pregnancy 
               
               
                 6700 
                 33 
                 1 
                 200 
                 2800 
                 11 
                 11 
                 11 
                 + 
                 Viable fetus 
               
               
                 6701 
                 25 
                 4 
                 450 
                 4050 
                 9 
                 9 
                 13 
                 + 
                 Viable fetus 
               
               
                 6702 
                 30 
                 2 
                 300 
                 3600 
                 12 
                 12 
                 6 
                 − 
                 No pregnancy 
               
               
                 6703 
                 36 
                 3 
                 450 
                 5400 
                 12 
                 12 
                 6 
                 − 
                 No pregnancy 
               
               
                 6704 
                 39 
                   
                 225 
                 1800 
                 8 
                 8 
                 12 
                 − 
                 No pregnancy 
               
               
                 6705 
                 39 
                   
                 225 
                 4275 
                 12 
                 12 
                 5 
                 + 
                 Viable fetus 
               
               
                 6646 
                 28 
                 1 
                 250 
                 2650 
                 12 
                 12 
                 11 
                 − 
                 No pregnancy 
               
               
                 6647 
                 39 
                 3 
                 300 
                 3600 
                 12 
                 12 
                 3 
                 − 
                 No pregnancy 
               
               
                 6648 
                 24 
                 1 
                 150 
                 1800 
                 12 
                 12 
                 13 
                 + 
                 Viable fetus 
               
               
                 6649 
                 35 
                 1 
                 125 
                 1250 
                 14 
                 14 
                 6 
                 − 
                 No pregnancy 
               
               
                 6650 
                 31 
                 3 
                 300 
                 3300 
                 11 
                 11 
                 6 
                 − 
                 No pregnancy 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 11 
               
               
                   
               
               
                 Steroid measurements (ng/mL), ratios, and outcomes for subjects undergoing IVF treatment. 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 Hydroxy- 
                   
                   
                   
                   
                   
                   
               
               
                 Sample 
                   
                 17-OH 
                 17-OH 
                   
                   
                   
                   
                   
                   
                 proges- 
                   
                   
                   
                   
                   
                   
               
               
                 ID 
                 Pregn 
                 Pregn 
                 Preg 
                 11-DC 
                 F 
                 E 
                 DHEA 
                 A4 
                 Te 
                 terone 
                 A4 
                 E1 
                 E2 
                 E3 
                 Preg 
                 Allopregn 
               
               
                   
               
               
                 6642 
                 504 
                 2.79 
                 714 
                 2.59 
                 64 
                 12.40 
                 1.06 
                 2.20 
                 0.02 
                 10.85 
                 0.00 
                 15.60 
                 114 
                 2.38 
                 13200 
                 5.17 
               
               
                 6643 
                 591 
                 15.20 
                 3350 
                 14.25 
                 42 
                 23.20 
                 2.77 
                 12.10 
                 0.45 
                 38.93 
                 0.00 
                 46.10 
                 154 
                 7.48 
                 11100 
                 1.64 
               
               
                 6644 
                 174 
                 1.39 
                 607 
                 3.13 
                 64 
                 12.25 
                 0.35 
                 1.75 
                 0.05 
                 7.43 
                 0.00 
                 19.20 
                 111 
                 2.80 
                 6720 
                 3.44 
               
               
                 6645 
                 1030 
                 4.36 
                 1305 
                 5.90 
                 64 
                 14.55 
                 0.87 
                 3.15 
                 0.03 
                 15.32 
                 0.67 
                 27.60 
                 145 
                 5.33 
                 14300 
                 4.98 
               
               
                 6646 
                 331 
                 3.30 
                 1125 
                 4.73 
                 40 
                 13.65 
                 1.39 
                 15.25 
                 0.41 
                 12.78 
                 0.72 
                 14.50 
                 113 
                 3.18 
                 14500 
                 5.66 
               
               
                 6647 
                 582 
                 2.30 
                 626 
                 1.99 
                 32 
                 9.71 
                 0.39 
                 1.89 
                 0.09 
                 6.99 
                 0.55 
                 14.60 
                 112 
                 2.00 
                 14200 
                 5.28 
               
               
                 6648 
                 582 
                 2.97 
                 955 
                 3.47 
                 34 
                 10.59 
                 0.70 
                 2.45 
                 0.03 
                 8.63 
                 0.00 
                 12.70 
                 106 
                 2.59 
                 22000 
                 4.21 
               
               
                 6649 
                 319 
                 3.47 
                 1755 
                 12.35 
                 63 
                 19.45 
                 1.87 
                 17.10 
                 0.64 
                 27.37 
                 0.00 
                 131.00 
                 149 
                 4.65 
                 13200 
                 3.02 
               
               
                 6650 
                 206 
                 2.18 
                 1210 
                 9.01 
                 47 
                 11.60 
                 1.14 
                 34.20 
                 2.08 
                 21.40 
                 0.00 
                 46.10 
                 148 
                 5.81 
                 6120 
                 2.52 
               
               
                 6653 
                 382 
                 1.95 
                 460 
                 2.07 
                 40 
                 11.90 
                 1.18 
                 1.52 
                 0.06 
                 6.39 
                 0.00 
                 59.90 
                 90 
                 2.80 
                 11700 
                 5.75 
               
               
                 6654 
                 498 
                 3.83 
                 617 
                 4.01 
                 57 
                 19.00 
                 2.78 
                 37.55 
                 0.58 
                 11.47 
                 0.84 
                 18.80 
                 125 
                 3.49 
                 5830 
                 4.77 
               
               
                 6655 
                 1825 
                 77.15 
                 2445 
                 8.51 
                 45 
                 15.35 
                 14.90 
                 6.85 
                 0.14 
                 32.33 
                 0.00 
                 42.60 
                 162 
                 5.81 
                 11300 
                 0.69 
               
               
                 6658 
                 367 
                 3.46 
                 885 
                 3.08 
                 43 
                 12.55 
                 0.67 
                 3.61 
                 0.08 
                 10.14 
                 0.00 
                 17.70 
                 110 
                 3.31 
                 13500 
                 6.79 
               
               
                 6659 
                 665 
                 3.67 
                 908 
                 4.23 
                 70 
                 11.25 
                 0.74 
                 2.57 
                 0.06 
                 12.88 
                 0.00 
                 21.50 
                 146 
                 4.49 
                 10500 
                 2.41 
               
               
                 6660 
                 487 
                 4.67 
                 1595 
                 10.70 
                 64 
                 20.60 
                 1.36 
                 13.80 
                 0.44 
                 21.80 
                 0.00 
                 45.40 
                 154 
                 7.00 
                 16300 
                 3.42 
               
               
                 6661 
                 183 
                 4.18 
                 911 
                 4.06 
                 85 
                 14.20 
                 1.85 
                 45.60 
                 3.15 
                 15.93 
                 0.65 
                 30.50 
                 121 
                 1.61 
                 3650 
                 1.55 
               
               
                 6662 
                 759 
                 11.05 
                 1535 
                 4.26 
                 22 
                 7.89 
                 1.29 
                 3.54 
                 0.03 
                 18.90 
                 0.00 
                 20.50 
                 126 
                 3.52 
                 12400 
                 1.90 
               
               
                 6663 
                 373 
                 2.54 
                 1185 
                 7.20 
                 53 
                 13.20 
                 
                           
                 
                 3.36 
                 0.07 
                 15.53 
                 0.00 
                 44.00 
                 147 
                 7.04 
                 10600 
                 5.63 
               
               
                 6664 
                 482 
                 5.71 
                 1615 
                 8.48 
                 35 
                 12.10 
                 
                           
                 
                 2.82 
                 0.04 
                 17.72 
                 0.00 
                 40.80 
                 146 
                 7.04 
                 12100 
                 2.44 
               
               
                 6665 
                 27 
                 1.00 
                 726 
                 6.63 
                 72 
                 7.34 
                 0.28 
                 7.38 
                 0.37 
                 10.41 
                 0.00 
                 11.30 
                 107 
                 1.85 
                 6940 
                 2.61 
               
               
                 6667 
                 454 
                 5.99 
                 991 
                 2.76 
                 44 
                 15.50 
                 1.53 
                 3.17 
                 0.07 
                 12.93 
                 0.00 
                 13.80 
                 109 
                 2.88 
                 13400 
                 3.04 
               
               
                 6670 
                 753 
                 16.75 
                 2910 
                 14.70 
                 38 
                 10.95 
                 1.71 
                 10.30 
                 0.36 
                 37.37 
                 0.00 
                 30.40 
                 158 
                 6.23 
                 14000 
                 3.15 
               
               
                 6671 
                 130 
                 2.07 
                 748 
                 3.46 
                 22 
                 7.48 
                 0.74 
                 4.25 
                 0.07 
                 11.03 
                 0.00 
                 22.70 
                 115 
                 3.44 
                 9130 
                 3.61 
               
               
                 6672 
                 809 
                 5.64 
                 1115 
                 6.94 
                 42 
                 10.35 
                 0.59 
                 1.98 
                 0.05 
                 14.78 
                 0.00 
                 34.70 
                 146 
                 7.49 
                 12200 
                 4.39 
               
               
                 6673 
                 441 
                 3.28 
                 1005 
                 4.30 
                 38 
                 10.70 
                 0.63 
                 2.36 
                 0.03 
                 10.54 
                 0.00 
                 16.50 
                 111 
                 2.91 
                 8600 
                 4.02 
               
               
                 6674 
                 169 
                 2.77 
                 1012 
                 6.95 
                 70 
                 13.20 
                 1.86 
                 22.55 
                 0.85 
                 16.06 
                 0.00 
                 36.70 
                 149 
                 5.78 
                 4870 
                 1.87 
               
               
                 6675 
                 305 
                 1.85 
                 1210 
                 11.04 
                 49 
                 9.13 
                 0.70 
                 29.45 
                 2.08 
                 20.42 
                 0.00 
                 35.10 
                 161 
                 6.06 
                 5970 
                 3.75 
               
               
                 6676 
                 351 
                 3.04 
                 1080 
                 7.38 
                 32 
                 10.75 
                 0.55 
                 18.60 
                 0.62 
                 13.59 
                 0.00 
                 47.20 
                 141 
                 5.32 
                 14100 
                 3.10 
               
               
                 6677 
                 171 
                 4.67 
                 903 
                 8.99 
                 47 
                 11.45 
                 1.64 
                 147.00 
                 8.23 
                 12.94 
                 2.77 
                 19.30 
                 109 
                 0.48 
                 3730 
                 1.33 
               
               
                 6678 
                 179 
                 3.28 
                 852 
                 5.10 
                 51 
                 28.05 
                 3.03 
                 117.50 
                 4.41 
                 19.20 
                 0.00 
                 32.40 
                 123 
                 2.28 
                 3360 
                 1.02 
               
               
                 6686 
                 572 
                 2.50 
                 629 
                 1.89 
                 42 
                 7.78 
                 0.48 
                 1.53 
                 0.01 
                 6.86 
                 0.00 
                 6.37 
                 67 
                 1.73 
                 11800 
                 4.03 
               
               
                 6688 
                 717 
                 9.74 
                 1755 
                 3.36 
                 38 
                 26.65 
                 1.34 
                 4.04 
                 0.15 
                 13.39 
                 0.00 
                 17.70 
                 104 
                 2.10 
                 12700 
                 2.02 
               
               
                 
                           
                 
                 337 
                 3.67 
                 1005 
                 5.25 
                 45 
                 13.15 
                 0.48 
                 2.30 
                 0.03 
                 16.19 
                 
                           
                 
                 29.10 
                 148 
                 5.32 
                 10000 
                 1.63 
               
               
                 6690 
                 306 
                 3.31 
                 807 
                 2.14 
                 33 
                 12.45 
                 0.65 
                 7.41 
                 0.31 
                 6.16 
                 0.00 
                 20.00 
                 78 
                 1.22 
                 18000 
                 8.02 
               
               
                 6691 
                 333 
                 2.19 
                 648 
                 4.08 
                 69 
                 11.10 
                 0.58 
                 3.00 
                 0.07 
                 10.49 
                 0.00 
                 22.10 
                 138 
                 3.63 
                 8810 
                 3.89 
               
               
                 6692 
                 510 
                 3.24 
                 583 
                 1.68 
                 68 
                 15.45 
                 0.54 
                 0.83 
                 0.02 
                 5.94 
                 0.00 
                 7.45 
                 80 
                 1.30 
                 8670 
                 4.16 
               
               
                 6693 
                 177 
                 1.67 
                 1130 
                 7.83 
                 44 
                 13.65 
                 0.62 
                 7.94 
                 0.23 
                 15.64 
                 0.00 
                 39.80 
                 156 
                 5.50 
                 7540 
                 2.23 
               
               
                 6694 HO 
                 331 
                 1.97 
                 669 
                 3.60 
                 50 
                 11.25 
                 0.57 
                 3.26 
                 0.05 
                 9.40 
                 0.00 
                 22.60 
                 135 
                 3.67 
                 8350 
                 4.09 
               
               
                 6695 HO 
                 202 
                 1.43 
                 941 
                 6.47 
                 58 
                 19.55 
                 0.84 
                 46.20 
                 2.09 
                 18.59 
                 0.72 
                 31.40 
                 143 
                 5.46 
                 5620 
                 1.92 
               
               
                 6696 HO 
                 406 
                 4.17 
                 646 
                 2.06 
                 29 
                 10.40 
                 0.71 
                 1.48 
                 0.03 
                 6.74 
                 0.43 
                 5.92 
                 81 
                 1.76 
                 10500 
                 6.17 
               
               
                 6699 HO 
                 351 
                 4.72 
                 1415 
                 9.54 
                 57 
                 14.10 
                 0.61 
                 7.83 
                 0.07 
                 17.13 
                 0.00 
                 26.90 
                 144 
                 4.81 
                 8730 
                 2.51 
               
               
                 6700 HO 
                 604 
                 3.07 
                 1610 
                 6.54 
                 75 
                 15.10 
                 0.63 
                 3.28 
                 0.04 
                 20.77 
                 0.00 
                 23.20 
                 144 
                 4.33 
                 7770 
                 4.47 
               
               
                 6701 HO 
                 333 
                 3.06 
                 1099 
                 6.90 
                 47 
                 12.45 
                 1.66 
                 9.63 
                 0.23 
                 18.83 
                 0.00 
                 40.60 
                 152 
                 6.71 
                 7910 
                 1.17 
               
               
                 6702 HO 
                 453 
                 2.53 
                 1080 
                 5.40 
                 39 
                 15.35 
                 0.68 
                 1.63 
                 0.02 
                 11.15 
                 0.00 
                 15.30 
                 121 
                 2.63 
                 12900 
                 1.83 
               
               
                 6703 HO 
                 
                           
                 
                 10.80 
                 1845 
                 8.66 
                 38 
                 11.10 
                 1.28 
                 4.67 
                 0.11 
                 26.18 
                 0.00 
                 40.50 
                 154 
                 5.67 
                 10200 
                 2.85 
               
               
                 6704 HO 
                 355 
                 2.31 
                 620 
                 2.44 
                 52 
                 10.60 
                 0.81 
                 1.97 
                 0.03 
                 7.86 
                 0.32 
                 11.00 
                 96 
                 1.85 
                 6340 
                 4.07 
               
               
                   
               
             
          
           
               
                   
                   
                   
                 Total 
                   
                   
                   
                   
                   
                 Ratio 
                 Ratio 
                   
                   
                   
               
               
                 Sample 
                 Total 
                 Total 
                 ANDR (with 
                 Total 
                 Total 
                 Total 
                 Ratio 
                 Ratio 
                 17OH-Pregn/ 
                 17OH Preg/ 
                 Ratio 
                 Ratio 
                 Ratio 
               
               
                 ID 
                 ESTR 
                 ANDR 
                 isomer) 
                 OC 
                 
                           
                 
                 
                           
                 
                 Preg/E3 
                 E/F 
                 Pregn 
                 Pregn 
                 E2/E1 
                 E3/E2 
                 E3/E1 
               
               
                   
               
               
                 6642 
                 132 
                 3.29 
                 14 
                 75.90 
                 13914 
                 
                           
                 
                 5546 
                 0.20 
                 0.006 
                 1.42 
                 7.31 
                 0.02 
                 0.15 
               
               
                 6643 
                 20           
                 15.32 
                 54 
                 64.80 
                 14450 
                 606 
                 1484 
                 0.56 
                 0.026 
                 5.67 
                 3.34 
                 0.05 
                 0.16 
               
               
                 6644 
                 133 
                 2.14 
                 10 
                 75.85 
                 7527 
                 175 
                 2400 
                 0.19 
                 0.008 
                 4.64 
                 5.78 
                 0.03 
                 0.15 
               
               
                 6645 
                 178 
                 4.72 
                 20 
                 78.40 
                 15605 
                 1034 
                 2683 
                 0.23 
                 0.004 
                 1.27 
                 5.25 
                 0.04 
                 0.19 
               
               
                 6646 
                 131 
                 17.76 
                 31 
                 53.35 
                 15625 
                 334 
                 4560 
                 0.34 
                 0.010 
                 3.40 
                 7.79 
                 0.03 
                 0.22 
               
               
                 6647 
                 129 
                 2.92 
                 10 
                 42.11 
                 14825 
                 584 
                 7100 
                 0.30 
                 0.004 
                 1.08 
                 
                           
                 
                 0.02 
                 0.14 
               
               
                 6648 
                 121 
                 3.18 
                 12 
                 44.09 
                 22965 
                 584 
                 8494 
                 0.32 
                 0.005 
                 1.66 
                 8.35 
                 0.02 
                 0.20 
               
               
                 6649 
                 285 
                 19.61 
                 47 
                 102.80 
                 14955 
                 322 
                 
                           
                 
                 0.23 
                 0.011 
                 5.50 
                 
                           
                 
                 0.03 
                 0.04 
               
               
                 6650 
                 200 
                 37.41 
                 59 
                 58.50 
                 7330 
                 208 
                 1053 
                 0.25 
                 0.011 
                 5.87 
                 3.21 
                 0.04 
                 0.13 
               
               
                 6653 
                 153 
                 2.76 
                 9 
                 52.25 
                 12160 
                 354 
                 4179 
                 0.29 
                 0.005 
                 1.20 
                 1.51 
                 0.03 
                 0.05 
               
               
                 6654 
                 147 
                 41.75 
                 53 
                 76.45 
                 6447 
                 
                           
                 
                 1670 
                 0.33 
                 0.006 
                 1.24 
                 
                           
                 
                 0.03 
                 0.19 
               
               
                 6655 
                 210 
                 21.89 
                 54 
                 60.15 
                 13745 
                 1902 
                 1945 
                 0.34 
                 0.042 
                 1.34 
                 
                           
                 
                 0.04 
                 0.14 
               
               
                 6658 
                 131 
                 4.33 
                 14 
                 55.95 
                 14595 
                 370 
                 4109 
                 0.29 
                 0.009 
                 2.71 
                 4.21 
                 0.03 
                 0.19 
               
               
                 6659 
                 172 
                 3.36 
                 16 
                 81.10 
                 11408 
                 668 
                 2339 
                 0.16 
                 0.006 
                 1.37 
                 4.79 
                 0.03 
                 0.21 
               
               
                 6660 
                 208 
                 15.60 
                 37 
                 85.00 
                 17895 
                 492 
                 2329 
                 0.32 
                 0.010 
                 3.28 
                 3.59 
                 0.05 
                 0.15 
               
               
                 6661 
                 153 
                 51.25 
                 67 
                 98.90 
                 4561 
                 187 
                 2267 
                 0.17 
                 0.023 
                 4.98 
                 3.97 
                 0.01 
                 0.05 
               
               
                 6662 
                 150 
                 4.86 
                 24 
                 29.29 
                 13935 
                 770 
                 3523 
                 0.37 
                 0.015 
                 2.02 
                 6.15 
                 0.03 
                 0.17 
               
               
                 6663 
                 198 
                 4.31 
                 20 
                 56.05 
                 11785 
                 
                           
                 
                 1506 
                 0.25 
                 0.007 
                 3.18 
                 3.34 
                 0.05 
                 0.16 
               
               
                 6664 
                 194 
                 3.66 
                 21 
                 46.75 
                 13715 
                 488 
                 1719 
                 0.35 
                 0.012 
                 3.35 
                 
                           
                 
                 0.05 
                 0.17 
               
               
                 6665 
                 120 
                 8.02 
                 18 
                 75.94 
                 7668 
                 26 
                 3751 
                 0.10 
                 0.037 
                 26.82 
                 9.47 
                 0.02 
                 0.16 
               
               
                 6667 
                 126 
                 4.76 
                 18 
                 59.55 
                 14391 
                 460 
                 4653 
                 0.35 
                 0.013 
                 2.18 
                 7.90 
                 0.03 
                 0.21 
               
               
                 6670 
                 195 
                 12.36 
                 50 
                 48.75 
                 16910 
                 769 
                 2247 
                 0.29 
                 0.022 
                 3.87 
                 5.20 
                 0.04 
                 0.20 
               
               
                 6671 
                 141 
                 5.06 
                 16 
                 29.53 
                 9878 
                 132 
                 2654 
                 0.34 
                 0.016 
                 5.77 
                 5.07 
                 0.03 
                 0.15 
               
               
                 6672 
                 188 
                 2.83 
                 17 
                 52.70 
                 13315 
                 614 
                 
                           
                 
                 0.24 
                 
                           
                 
                 
                           
                 
                 4.21 
                 0.05 
                 0.22 
               
               
                 6673 
                 130 
                 3.01 
                 14 
                 48.35 
                 9605 
                 444 
                 2955 
                 0.28 
                 0.007 
                 2.28 
                 6.73 
                 0.03 
                 0.16 
               
               
                 6674 
                 191 
                 25.25 
                 41 
                 83.55 
                 5882 
                 172 
                 
                           
                 
                 0.19 
                 
                           
                 
                 5.99 
                 4.06 
                 0.04 
                 0.16 
               
               
                 6675 
                 202 
                 32.23 
                 53 
                 57.98 
                 7180 
                 307 
                 
                           
                 
                 
                           
                 
                 
                           
                 
                 3.97 
                 4.59 
                 0.04 
                 0.17 
               
               
                 6676 
                 194 
                 19.77 
                 33 
                 42.55 
                 15190 
                 354 
                 
                           
                 
                 0.34 
                 
                           
                 
                 3.11 
                 2.99 
                 0.04 
                 0.11 
               
               
                 6677 
                 129 
                 157.64 
                 171 
                 56.60 
                 4633 
                 176 
                 7659 
                 0.24 
                 0.027 
                 5.28 
                 5.65 
                 
                           
                 
                 0.03 
               
               
                 6678 
                 158 
                 124.94 
                 144 
                 68.65 
                 4212 
                 182 
                 1474 
                 0.46 
                 0.018 
                 4.77 
                 3.60 
                 0.02 
                 0.07 
               
               
                 6686 
                 95 
                 2.02 
                 9 
                 50.13 
                 12429 
                 574 
                 6821 
                 
                           
                 
                 0.004 
                 1.10 
                 13.59 
                 0.02 
                 0.27 
               
               
                 6688 
                 124 
                 5.52 
                 19 
                 64.65 
                 14455 
                 726 
                 6048 
                 0.70 
                 0.014 
                 2.45 
                 
                           
                 
                 
                           
                 
                 0.12 
               
               
                 
                           
                 
                 182 
                 3.42 
                 20 
                 58.60 
                 11005 
                 341 
                 1880 
                 0.29 
                 0.011 
                 2.98 
                 
                           
                 
                 0.04 
                 0.18 
               
               
                 6690 
                 99 
                            .37 
                 15 
                 45.70 
                 16807 
                 309 
                 13115 
                 0.37 
                 0.011 
                 2.64 
                 
                           
                 
                 0.02 
                 0.06 
               
               
                 6691 
                 164 
                 3.65 
                 14 
                 69.85 
                 9458 
                 
                           
                 
                 
                           
                 
                 0.19 
                 0.007 
                 1.95 
                 
                           
                 
                 
                           
                 
                 0.17 
               
               
                 6692 
                 88 
                 1.39 
                 7 
                 63.60 
                 9253 
                 513 
                 
                           
                 
                 0.23 
                 0.008 
                 1.14 
                 10.68 
                 0.02 
                 0.17 
               
               
                 6693 
                 201 
                            .80 
                 24 
                 57.70 
                 8670 
                 179 
                 1371 
                 0.31 
                 0.009 
                 
                           
                 
                 3.92 
                 0.04 
                 0.14 
               
               
                 6694 HO 
                 161 
                 3.86 
                 13 
                 68.90 
                 9019 
                 333 
                 2275 
                 0.20 
                 0.006 
                 2.02 
                 5.97 
                 
                           
                 
                 0.16 
               
               
                 6695 HO 
                 180 
                 49.84 
                 68 
                 77.80 
                 6561 
                 203 
                 1029 
                 0.34 
                 0.007 
                 4.65 
                 4.55 
                 0.04 
                 0.17 
               
               
                 6696 HO 
                 
                           
                 
                 2.65 
                 9 
                 39.60 
                 11348 
                 412 
                 
                           
                 
                 0.36 
                 
                           
                 
                 2.08 
                 
                           
                 
                 0.02 
                 0.30 
               
               
                 6699 HO 
                 178 
                            .51 
                 26 
                 71.30 
                 10145 
                 355 
                 1815 
                 0.25 
                 0.013 
                 4.04 
                 
                           
                 
                 0.03 
                 0.17 
               
               
                 6700 HO 
                 172 
                 3.94 
                 25 
                 89.60 
                 8380 
                 607 
                 1794 
                 0.20 
                 0.005 
                 2.67 
                 
                           
                 
                 0.03 
                 0.19 
               
               
                 6701 HO 
                 200 
                 11.51 
                 30 
                 59.55 
                 9009 
                 336 
                 1179 
                 0.26 
                 
                           
                 
                 3.30 
                 3.73 
                 0.04 
                 0.16 
               
               
                 6702 HO 
                 139 
                 2.31 
                 13 
                 54.80 
                 13960 
                 456 
                 4905 
                 0.39 
                 
                           
                 
                 2.34 
                 7.91 
                 0.02 
                 0.17 
               
               
                 6703 HO 
                 203 
                 6.00 
                 32 
                 48.85 
                 12045 
                 
                           
                 
                 1176 
                 0.29 
                 0.012 
                 1.97 
                 3.80 
                 
                           
                 
                 0.21 
               
               
                 6704 HO 
                 109 
                 3.12 
                 11 
                 62.40 
                 6960 
                 357 
                 3427 
                 0.20 
                 0.007 
                 1.75 
                 
                           
                 
                 0.02 
                 0.17 
               
               
                   
               
             
          
           
               
                   
                   
                 Ratio Total 
                 Ratio 
                   
                 Ratio Total 
                 Ratio Total 
                 Ratio Total 
                 Ratio 
                   
                   
               
               
                   
                 Sample 
                 ANDR/Total 
                 Preg/17OH 
                 Ratio 
                 progestines/ 
                 progestines/ 
                 progestines/ 
                 Pregn/ 
                 Ratio 
                   
               
               
                   
                 ID 
                 ESTR 
                 
                           
                 
                            /E2 
                 total ESTR 
                 total OC 
                 total ANDR 
                 Allopregn 
                 A4/11OC 
                 
                           
                 
               
               
                   
               
               
                   
                 6642 
                 0.02 
                 18.49 
                 115.79 
                 105.43 
                 183 
                 4226 
                 97.49 
                 
                           
                 
                 Viable fetus 
               
               
                   
                 6643 
                 0.07 
                 3.31 
                 
                           
                 
                 69.61 
                 223 
                 943 
                 380.37 
                 
                           
                 
                 No pregnancy 
               
               
                   
                 6644 
                 0.02 
                 
                           
                 
                 
                           
                 
                 56.59 
                 
                           
                 
                 3510 
                 50.58 
                 0.56 
                 No pregnancy 
               
               
                   
                 6645 
                 0.03 
                 10.96 
                 96.62 
                 87.70 
                 199 
                 3306 
                 206.83 
                 0.53 
                 No pregnancy 
               
               
                   
                 6646 
                 0.14 
                 12.69 
                 
                           
                 
                 119.57 
                 
                           
                 
                 
                           
                 
                 58.48 
                 3.23 
                 No pregnancy 
               
               
                   
                 6647 
                 0.02 
                 22.68 
                 126.79 
                 115.29 
                 352 
                 5076 
                 110.13 
                 0.95 
                 No pregnancy 
               
               
                   
                 6648 
                 0.03 
                 22.80 
                 
                           
                 
                 189.34 
                 521 
                 
                           
                 
                 
                           
                 
                 0.71 
                 Viable fetus 
               
               
                   
                 6649 
                 0.07 
                 7.52 
                 88.59 
                 52.54 
                 145 
                 763 
                 
                           
                 
                 1.35 
                 No pregnancy 
               
               
                   
                 6650 
                 0.19 
                 5.06 
                 41.35 
                 36.67 
                 125 
                 196 
                 81.75 
                 
                           
                 
                 No pregnancy 
               
               
                   
                 6653 
                 0.02 
                 
                           
                 
                 129.42 
                 79.43 
                 233 
                 4414 
                 66.43 
                 0.73 
                 No pregnancy 
               
               
                   
                 6654 
                 0.28 
                 9.46 
                 46.64 
                 43.77 
                 84 
                 154 
                 104.40 
                 
                           
                 
                 Lost pregnancy 
               
               
                   
                 6655 
                 0.10 
                 4.62 
                 69.75 
                 65.32 
                 229 
                 628 
                 
                           
                 
                 
                           
                 
                 No pregnancy 
               
               
                   
                 6658 
                 0.03 
                 13.68 
                 123.64 
                 111.40 
                 261 
                 3372 
                 53.96 
                 1.17 
                 Viable fetus 
               
               
                   
                 6659 
                 0.02 
                 
                           
                 
                 71.92 
                 
                           
                 
                 141 
                 
                           
                 
                 275.73 
                 0.61 
                 No pregnancy 
               
               
                   
                 6660 
                 0.06 
                 10.22 
                 105.84 
                 
                           
                 
                 211 
                 1147 
                 142.40 
                 1.29 
                 Viable fetus 
               
               
                   
                 6661 
                 0.33 
                 4.01 
                 30.17 
                 29.79 
                 46 
                 89 
                 118.06 
                 11.25 
                 No pregnancy 
               
               
                   
                 6662 
                 0.03 
                 
                           
                 
                 96.41 
                 92.89 
                 474 
                 2870 
                 399.47 
                 0.83 
                 No pregnancy 
               
               
                   
                 6663 
                 0.02 
                 8.95 
                 72.11 
                 59.51 
                 175 
                 2733 
                 
                           
                 
                 0.47 
                 No pregnancy 
               
               
                   
                 6664 
                 
                           
                 
                 7.49 
                 
                           
                 
                 70.75 
                 
                           
                 
                 3744 
                 197.54 
                 0.33 
                 No pregnancy 
               
               
                   
                 6665 
                 0.07 
                 9.57 
                 64.86 
                 
                           
                 
                 97 
                 
                           
                 
                 10.36 
                 1.11 
                 No pregnancy 
               
               
                   
                 6667 
                 0.04 
                 13.53 
                 122.94 
                 114.50 
                 
                           
                 
                 
                           
                 
                 149.34 
                 1.15 
                 Viable fetus 
               
               
                   
                 6670 
                 0.06 
                 4.81 
                 
                           
                 
                 
                           
                 
                 347 
                 1368 
                 238.89 
                 
                           
                 
                 No pregnancy 
               
               
                   
                 6671 
                 0.04 
                 12.21 
                 79.39 
                 69.96 
                 334 
                 1953 
                 35.87 
                 1.23 
                 No pregnancy 
               
               
                   
                 6672 
                 0.01 
                 10.94 
                 
                           
                 
                 70.75 
                 253 
                 5059 
                 135.61 
                 0.29 
                 Lost pregnancy 
               
               
                   
                 6673 
                 0.02 
                 8.56 
                 77.48 
                 73.65 
                 199 
                 
                           
                 
                 109.58 
                 0.55 
                 Viable fetus 
               
               
                   
                 6674 
                 0.13 
                 
                           
                 
                 32.68 
                 30.72 
                 70 
                 233 
                 
                           
                 
                 3.24 
                 Viable fetus 
               
               
                   
                 6675 
                 0.16 
                 4.93 
                 37.06 
                 35.52 
                 124 
                 223 
                 81.33 
                 2.87 
                 No pregnancy 
               
               
                   
                 6676 
                 0.10 
                 12.94 
                 100.00 
                 78.49 
                 357 
                 768 
                 113.23 
                 2.52 
                 No pregnancy 
               
               
                   
                 6677 
                 1.22 
                 4.13 
                 34.22 
                 35.97 
                 79 
                 29 
                 128.57 
                 18.35 
                 No pregnancy 
               
               
                   
                 6678 
                 0.79 
                 3.95 
                 27.32 
                 26.71 
                 48 
                 34 
                 
                           
                 
                 23.04 
                 Viable fetus 
               
               
                   
                 6686 
                 0.02 
                 15.77 
                 136.25 
                 131.24 
                 248 
                 
                           
                 
                 141.94 
                 0.61 
                 No pregnancy 
               
               
                   
                 6688 
                 0.04 
                 7.24 
                 122.12 
                 116.76 
                 224 
                 2619 
                 354.70 
                 1.20 
                 Lost pregnancy 
               
               
                   
                 
                           
                 
                 
                           
                 
                 
                           
                 
                 67.57 
                 60.33 
                 1854 
                 3221 
                 206.75 
                 0.44 
                 No pregnancy 
               
               
                   
                 6690 
                 
                           
                 
                 
                           
                 
                 205.13 
                 
                           
                 
                 
                           
                 
                 2009 
                 38.09 
                 3.46 
                 Lost pregnancy 
               
               
                   
                 6691 
                 0.02 
                 
                           
                 
                 63.64 
                 57.70 
                 135 
                 2591 
                 85.48 
                 0.74 
                 Viable fetus 
               
               
                   
                 6692 
                 0.02 
                 14.87 
                 106.92 
                 104.73 
                 111 
                 
                           
                 
                 122.60 
                 0.50 
                 No pregnancy 
               
               
                   
                 6693 
                 0.04 
                 
                           
                 
                 48.33 
                 43.07 
                 150 
                 
                           
                 
                 
                           
                 
                 1.01 
                 No pregnancy 
               
               
                   
                 6694 HO 
                 0.02 
                 12.49 
                 61.85 
                 55.92 
                 131 
                 2324 
                 80.93 
                 0.56 
                 Viable fetus 
               
               
                   
                 6695 HO 
                 0.28 
                 5.97 
                 39.30 
                 36.48 
                 
                           
                 
                 132 
                 105.21 
                 7.14 
                 Viable fetus 
               
               
                   
                 6696 HO 
                 0.03 
                 12.39 
                 130.43 
                 128.69 
                 267 
                 
                           
                 
                 
                           
                 
                 0.72 
                 No pregnancy 
               
               
                   
                 6699 HO 
                 0.05 
                 6.17 
                 60.63 
                 57.09 
                 142 
                 
                           
                 
                 134.29 
                 0.62 
                 No pregnancy 
               
               
                   
                 6700 HO 
                 0.02 
                 4.63 
                 53.96 
                 54.68 
                 105 
                 2380 
                 135.12 
                 0.50 
                 Viable fetus 
               
               
                   
                 6701 HO 
                 0.06 
                 7.20 
                 52.04 
                 45.15 
                 
                           
                 
                 782 
                 264.62 
                 1.38 
                 Viable fetus 
               
               
                   
                 6702 HO 
                 0.02 
                 12.17 
                 106.81 
                 100.48 
                 
                           
                 
                 
                           
                 
                 247.54 
                 0.30 
                 No pregnancy 
               
               
                   
                 6703 HO 
                 0.03 
                 5.53 
                 66.23 
                 59.29 
                 247 
                 1988 
                 
                           
                 
                 0.53 
                 No pregnancy 
               
               
                   
                 6704 HO 
                 0.03 
                 10.23 
                 66.25 
                 64.12 
                 112 
                 2228 
                 87.10 
                 0.81 
                 No pregnancy 
               
               
                   
               
               
                             indicates data missing or illegible when filed 
               
             
          
         
       
     
       Median Values and Percentiles 
       [0094]      FIGS. 5-8  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. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 12 
               
             
             
               
                   
               
               
                 Median 5 th  and 95 th  percentile of concentrations (ratios of concentrations) of steroids 
               
               
                 measured in FF in groups with viable pregnancy and no viable pregnancy. 
               
             
          
           
               
                   
                 Viable pregnancy 
                 No viable pregnancy 
                 % Difference 
               
             
          
           
               
                 Analyte (concentrations in ng/mL) 
                 Median 
                 5 th   
                 95th 
                 Median 
                 5th 
                 95th 
                 5th 
                 95th 
               
               
                   
               
             
          
           
               
                 17OHProgesterone 
                 990.5 
                 660.3 
                 1601 
                 1090 
                 603.1 
                 2631 
                  −9% 
                 64% 
               
               
                 Hydroxyprogesterone 
                 12.9 
                 9.1 
                 21.1 
                 13.6 
                 6.3 
                 34.3 
                 −31% 
                 63% 
               
               
                 11deoxycortisol 
                 4.30 
                 2.69 
                 8.46 
                 5.40 
                 1.95 
                 13.11 
                 −28% 
                 55% 
               
               
                 Total estrogens 
                 161 
                 124 
                 202 
                 153 
                 92 
                 209 
                 −26% 
                  3% 
               
               
                 Pregnenolone 
                 367 
                 175 
                 591 
                 382 
                 154 
                 975 
                 −12% 
                 65% 
               
               
                 Estrone 
                 22.60 
                 13.36 
                 42.64 
                 27.60 
                 7.02 
                 52.28 
                 −47% 
                 23% 
               
               
                 Estradiol 
                 135 
                 108 
                 153 
                 126 
                 80 
                 159 
                 −26% 
                  4% 
               
               
                 Estriol 
                 3.67 
                 2.34 
                 6.83 
                 3.52 
                 1.27 
                 7.48 
                 −46% 
                 10% 
               
               
                 Androstenedione 
                 3.28 
                 2.29 
                 74.72 
                 4.25 
                 1.50 
                 40.77 
                 −35% 
                 −45%  
               
               
                 17OHPregnenolone 
                 3.06 
                 1.75 
                 5.20 
                 3.57 
                 1.77 
                 15.90 
                  1% 
                 206%  
               
               
                 Total androgens 
                 4.3 
                 3.1 
                 79.9 
                 5.5 
                 2.1 
                 45.5 
                 −33% 
                 −43%  
               
               
                 DHEA 
                 0.84 
                 0.58 
                 2.32 
                 0.81 
                 0.37 
                 2.77 
                 −35% 
                 19% 
               
               
                 Cortisol 
                 58.3 
                 36.0 
                 72.0 
                 44.1 
                 26.3 
                 76.3 
                 −27% 
                  6% 
               
               
                 Cortisone 
                 12.55 
                 10.66 
                 23.58 
                 12.10 
                 7.66 
                 20.95 
                 −28% 
                 −11%  
               
               
                 Total glucocorticoids 
                 69.9 
                 46.6 
                 89.0 
                 58.5 
                 35.6 
                 89.7 
                 −24% 
                  1% 
               
               
                 Progesterone 
                 8600 
                 4266 
                 18580 
                 10600 
                 4990 
                 14380 
                  17% 
                 −23%  
               
               
                 Total pregnenolones 
                 370 
                 178 
                 594 
                 384 
                 158 
                 983 
                 −11% 
                 66% 
               
               
                 Ratio 17OH-Pregnenolone/ 
                 0.007 
                 0.005 
                 0.017 
                 0.010 
                 0.004 
                 0.031 
                 −15% 
                 82% 
               
               
                 Pregnenolone 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Ratio 17OH Progesterone/ 
                 2.67 
                 1.56 
                 5.26 
                 2.98 
                 1.13 
                 6.08 
                 −28% 
                 16% 
               
               
                 Pregnenolone 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Ratio estradiol/estrone 
                 6.21 
                 3.59 
                 8.08 
                 5.09 
                 2.40 
                 11.85 
                 −33% 
                 47% 
               
               
                 Ratio estriol/estradiol 
                 0.028 
                 0.020 
                 0.045 
                 0.031 
                 0.015 
                 0.050 
                 −26% 
                 11% 
               
               
                 Ratio estriol/estrone 
                 0.173 
                 0.120 
                 0.206 
                 0.166 
                 0.042 
                 0.240 
                 −65% 
                 17% 
               
               
                 Ratio Pregnenolone/ 
                 109.6 
                 70.1 
                 218.8 
                 118.1 
                 37.2 
                 376.0 
                 −47% 
                 72% 
               
               
                 Allopregnenolone 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Ratio A4/11deoxycortisol 
                 1.15 
                 0.53 
                 13.50 
                 0.82 
                 0.32 
                 10.12 
                 −40% 
                 −25%  
               
               
                   
               
             
          
         
       
     
         [0095]    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. 
         [0096]    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. 
         [0097]    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. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 13 
               
             
             
               
                   
               
               
                 Maximum of minimum values of concentrations of steroids 
               
               
                 (ng/mL) or ratios of concentrations of steroids observed in group of patients with 
               
               
                 viable pregnancy and number of samples with values of the markers above and 
               
               
                 below the distribution observed in the group of patients with no viable pregnancy. 
               
             
          
           
               
                   
                 No viable pregnancy 
               
               
                   
                 N = 33 
               
             
          
           
               
                   
                   
                 No. of samples 
                 No. of samples 
               
               
                   
                 Viable Pregnancy 
                 in group above 
                 in group below 
               
               
                   
                 N = 13 
                 the distribution 
                 the distribution 
               
             
          
           
               
                   
                 Maximum 
                 Minimum 
                 seen in viable 
                 seen in viable 
               
               
                 Analyte in ng/mL 
                 observed value 
                 observed value 
                 pregnancy 
                 pregnancy 
               
               
                   
               
             
          
           
               
                 17OHProgesterone 
                 1610 
                 648 
                 8 
                 0 
               
               
                 11DC 
                 10.70 
                 2.59 
                 4 
                 6 
               
               
                 Pregnenolone 
                 604 
                 169 
                 8 
                 1 
               
               
                 17OHPregnenolone 
                 5.99 
                 1.43 
                 6 
                 0 
               
               
                 E1 
                 45.40 
                 12.70 
                 4 
                 5 
               
               
                 E2 
                 154 
                 106 
                 0 
                 6 
               
               
                 E3 
                 7.00 
                 2.28 
                 0 
                 8 
               
               
                 A4 
                 117 
                 2.2 
                 0 
                 8 
               
               
                 Hydroxyprogesterone 
                 8.63 
                 21.60 
                 5 
                 8 
               
               
                 Cortisone 
                 28.05 
                 10.6 
                 0 
                 6 
               
               
                 Cortisol 
                 74.5 
                 33.5 
                 2 
                 2 
               
               
                 DHEA 
                 3.03 
                 0.57 
                 1 
                 5 
               
               
                 Total estrogens 
                 206.4 
                 121.3 
                 3 
                 6 
               
               
                 Total androgens 
                 125 
                 3.01 
                 1 
                 7 
               
               
                 Total glucocorticoids 
                 89.6 
                 44.1 
                 2 
                 4 
               
               
                 Total pregnenolones 
                 607.1 
                 171.8 
                 8 
                 2 
               
               
                 (pregnenolone + 17OH- 
               
               
                 pregnenolone) 
               
               
                 Ratio 17OH-Pregnenolone/Pregnenolone 
                 0.02 
                 0.01 
                 6 
                 8 
               
               
                 Ratio 17OH Progesterone/Pregnenolone 
                 5.99 
                 1.42 
                 2 
                 8 
               
               
                 Ratio E2/E1 
                 8.35 
                 3.39 
                 5 
                 4 
               
               
                 Ratio E3/E2 
                 0.05 
                 0.02 
                 1 
                 2 
               
               
                 Ratio E3/E1 
                 0.21 
                 0.07 
                 4 
                 5 
               
               
                 Ratio 
                 284.62 
                 53.98 
                 5 
                 4 
               
               
                 Pregnenolone/Allopregnenolone 
               
               
                 Ratio A4/11deoxycortisol 
                 23.04 
                 .05 
                 0 
                 5 
               
               
                   
               
             
          
         
       
     
         [0098]    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. 
         [0099]    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. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 14 
               
             
             
               
                   
               
               
                 Percent of samples in group with no viable pregnancy, which 
               
               
                 have concentration or ratio of concentrations of steroids above the 
               
               
                 distribution in group with viable pregnancy and p-values for 
               
               
                 significance of the observed differences between the groups 
               
               
                 (Chi-Square test). 
               
             
          
           
               
                   
                 % of samples out 
                   
               
               
                   
                 of range high in 
               
               
                   
                 group with no 
               
               
                 Analyte 
                 viable pregnancy 
                 p-value 
               
               
                   
               
               
                 17OHProgesterone 
                 24% 
                 0.0003 
               
               
                 Pregnenolone 
                 24% 
                 0.0002 
               
               
                 Total pregnenolones 
                 24% 
                 0.0002 
               
               
                 17OHPregnenolone 
                 18% 
                 0.0006 
               
               
                 Hydroxyprogesterone 
                 18% 
                 0.0014 
               
               
                 11DC 
                 12% 
                 0.0032 
               
               
                 Estrone 
                 12% 
                 0.0032 
               
               
                 Estradiol 
                  9% 
                 0.0087 
               
               
                 Ratio 17OH-Pregnenolone/Pregnenolone 
                 18% 
                 0.0006 
               
               
                 Ratio E2/E1 
                 15% 
                 0.0013 
               
               
                 Ratio Pregnenolone/Allopregn 
                 15% 
                 0.0014 
               
               
                 Ratio E3/E1 
                 12% 
                 0.0031 
               
               
                   
               
             
          
         
       
     
         [0100]    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. 
         [0101]    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. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 15 
               
             
             
               
                   
               
               
                 Percent of samples in the group of patients with no viable 
               
               
                 pregnancy with concentration or ratio of concentrations of steroids below 
               
               
                 the distribution in the group with viable pregnancy and p-values for 
               
               
                 significance of the observed differences between the groups 
               
               
                 (Chi-square test). 
               
             
          
           
               
                   
                 % of samples out of 
                   
               
               
                   
                 range low high in 
               
               
                   
                 group with no viable 
               
               
                 Analyte 
                 pregnancy 
                 p-value 
               
               
                   
               
               
                 Estriol 
                 30% 
                 0.0001 
               
               
                 Androstenedione 
                 24% 
                 0.0002 
               
               
                 Hydroxyprogesterone 
                 24% 
                 0.0002 
               
               
                 Total androgens 
                 21% 
                 0.0003 
               
               
                 Estradiol 
                 18% 
                 0.0006 
               
               
                 17OHProgesterone 
                 18% 
                 0.0006 
               
               
                 11DC 
                 18% 
                 0.0006 
               
               
                 Total estrogens 
                 18% 
                 0.0006 
               
               
                 Cortisone 
                 18% 
                 0.0006 
               
               
                 DHEA 
                 15% 
                 0.0013 
               
               
                 Estrone 
                 15% 
                 0.0014 
               
               
                 Total glucocorticoids 
                 12% 
                 0.0032 
               
               
                 Ratio 17OH Progesterone/Pregnenolone 
                 24% 
                 0.0002 
               
               
                 Ratio E3/E1 
                 15% 
                 0.0013 
               
               
                 Ratio A4/11deoxycortisol 
                 15% 
                 0.0014 
               
               
                 Ratio E2/E1 
                 12% 
                 0.0032 
               
               
                 Ratio Pregnenolone/Allopregn 
                 12% 
                 0.0032 
               
               
                   
               
             
          
         
       
     
         [0102]    Association of Steroid Profiles with IVF Outcome 
         [0103]    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  FIG. 9 . 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. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 16 
               
             
             
               
                   
               
               
                 Median values of the ratios of concentrations of steroid 
               
               
                 product/precursors of the pathway used as markers of enzyme activities in 
               
               
                 women with viable pregnancy and no pregnancy (profile types 1 and 2). 
               
             
          
           
               
                   
                 Steroid product/precursor 
                   
                   
                   
               
               
                 Enzyme 
                 concentration ratios 
                 Viable pregnancy 
                 Profile 1 
                 Profile 2 
               
               
                   
               
             
          
           
               
                 3βHSD 
                 17OHProg/17OHPregn 
                 2.67 
                 2.45 
                 1.75 
               
               
                 CYP21 
                 11DC/17OHProg 
                 0.0057 
                 0.0043 
                 0.0032 a   
               
               
                 CYP11 
                 Cortisol/11DC 
                 11.40 
                 5.02 b   
                 19.54 b   
               
               
                 11βHSD type 1 and 2 
                 Cortisone/Cortisol 
                 0.284 
                 0.343 a   
                 0.227 
               
               
                 CYP17 
                 DHEA/17OHPregn 
                 0.29 
                 0.14 b   
                 0.19 a   
               
               
                 CYP17 
                 A4/17OHProg 
                 0.0046 
                 0.0028 
                 0.0030 
               
               
                 CYP17 
                 17OHPregn/Pregn 
                 0.007 
                 0.0146 b   
                 0.007 
               
               
                 3βHSD 
                 A4/DHEA 
                 5.21 
                 3.64 
                 3.16 
               
               
                 17HSD3 
                 Te/A4 
                 3.50 
                 3.63 
                 4.22 
               
               
                 CYP19 
                 E1/A4 
                 5.18 
                 5.79 
                 5.60 
               
               
                 CYP19 
                 E2/Te 
                 9.27 
                 5.44 
                 12.67 b   
               
               
                 17βHSD1 type 1 and 2 
                 E2/E1 
                 6.21 
                 3.80 a   
                 8.70 
               
               
                   
               
               
                 *Non-parametric test (Wilcoxon two-group test); 
               
               
                   a p &lt; 0.05, 
               
               
                   b p &lt; 0.01 
               
             
          
         
       
     
         [0104]    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. 
         [0105]    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. 
         [0106]    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. 
         [0107]    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; 202-246.   2. Brailly S, et al. (1981), Androgens and progestins in the human ovarian follicle: differences in the evolution of preovulatory, healthy nonovulatory, and atretic follicles  J. Clin. Endocrinol. Metab  53:128-134.   3. Itskovitz J, et al. (1991), Relationship of follicular fluid prorenin to oocyte maturation, steroid levels, and outcome of in vitro fertilization  J. Clin. Endocrinol. Metab  72:165-171,   4. Van Dessel H J H M, et al. (1996), Normal human follicle development: and evaluation of correlations with oestradiol, androstenedione and progesterone levels in individual follicles.  Clin Endocrinol  (Oxf) 44:191-198.   5. Franks S, (1989), Polycystic ovary syndrome: a changing perspective.  Clin. Endocrinol  ( Oxf ), 31(1):87-120.   6. Hillier S G, et al. (1980), Intraovarian sex steroid hormone interactions and the regulation of follicular maturation: aromatization of androgens by human granulosa cells in vitro.  J Clin Endocrinol Metab,  50(4):640-647.   7. Hillier S G, et al. (1981), Control of preovulatory follicular estrogen biosynthesis in the human ovary.  J Clin Endocrinol Metab,  52(5):847-856.   8. Lewicka S, et al. (2003), Cortisol and cortisone in human follicular fluid and serum and the outcome of IVF treatment.  Hum Reprod.  18:1613-7.   9. Michael, A E, et al. (1999) Relationship between ovarian cortisol:cortisone ratios and the clinical outcome of in vitro fertilization and embryo transfer (IVF-ET). Clin Endocrinol 51:535-40.   10. Andersen C Y et al. (1999), Assessment of the follicular cortisol:cortisone ratio. Hum Reprod. 14:1562-8.   11. Basuray R, et al. (1988) High progesterone/estradiol ratio in follicular fluid at oocyte aspiration for in vitro fertilization as a predictor of possible pregnancy. Fertil Steril. 49:1007-11.   12. Franchimont P, et al. (1989) Correlation between follicular fluid content and the result of in vitro fertilization and embryo transfer. I. Sex steroids. Fertil Steril. 52:1006-11   13. Andersen C Y, et al. (1993) Characteristics of human follicular fluid associated with successful conception after in vitro fertilization. J Clin Endocrinol Metab. 77:1227-34.   14. De Sutter P, et al. (1991). Correlation between follicular fluid steroid analysis and maturity and cytogenetic analysis of human oocytes that remained unfertilized after in vitro fertilization.  Fertil Steril  55:958-963.   15. Bergh C, Carlström K, Selleskog U, Hillensjö, T (1996), Effect of growth hormone on follicular fluid androgen levels in patients treated with gonadotropins before in vitro fertilization.  Eur J Endocrinol  134:190-196.   16. Smitz J, et al. p (2007), Endocrine profile in serum and follicular fluid differs after ovarian stimulation with HP-hMG or recombinant FSH in IVF patients.  Hum Reprod  22:676-687.   17. Taieb J, et al. (2002), Limitations of steroid determination by direct immunoassay,  Clin Chem  48:583-585.   18. Dehennin L. (1990), Estrogens, androgens, and progestins in follicular fluid from preovulatory follicles: identification and quantification by gas chromatography/mass spectrometry associated with stable isotope dilution.  Steroids.  55:181.4.   19. Dehennin L, et al, (1987), Androgen and 19-norsteroid profiles in human preovulatory follicles from stimulated cycles: an isotope dilution-mass spectrometric study.  J Steroid Biochem  26:399-405.   20. Kushnir M M, et al. (2004), Cortisol and cortisone analysis in serum and plasma by atmospheric pressure photoionization tandem mass spectrometry.  Clin Biochem,  37(5):357-362.   21. Kushnir M M, et al., (2006), Development and performance evaluation of a tandem mass spectrometry assay for 4 adrenal steroids.  Clin Chem,  52(8):1559-1567.   22. Kushnir M M, et al. (2006), Performance characteristics of a novel tandem mass spectrometry assay for serum testosterone.  Clin Chem,  52(1):120-128.   23. Kushnir M M, et al. (2008), A tandem mass spectrometry assay for estrone and estradiol in serum of postmenopausal women, men and children.  American J Clin Path,  129:530-539.   24. M M Kushnir, et al. (2007). High sensitivity tandem mass spectrometry test for serum estrogens.  Clin Chem  53:A183.   25. Kushnir M M, et al. (2008), Steroid Profiles in Ovarian Follicular Fluid from Regularly Menstruating Women and from Women after Ovarian Stimulation,  Clin. Chem  55:519-526.   26. Kushnir M M, et al. (2005), Assessing analytical specificity in quantitative analysis using tandem mass spectrometry.  Clin Biochem,  38(4):319-327.   27. Klein N A, et al. Soules M R (1996), Ovarian Follicular Development and the Follicular Fluid Hormones and Growth Factors in Normal Women of Advanced Reproductive Age.  J Clin Endocrinol Metab  81:1946-1951.   28. Adams J, et al. (1986), Prevalence of polycystic ovaries in women with anovulation and idiopathic hirsutism.  Br Med J  ( Clin Res Ed ) 293(6543):355-359.   29. Ferriman D, and Gallwey G, (1961), Clinical assessment of body hair growth in women.  J Clin Endoc Metab  21:1440-1447.