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Relationship between salivary progesterone, 17-hydroxyprogesterone, and cortisol levels throughout the normal menstrual cycle of healthy postmenarcheal girls
FERTILITY AND STERILITY威 VOL. 76, NO. 3, SEPTEMBER 2001 Copyright ©2001 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Relationship between salivary progesterone, 17-hydroxyprogesterone, and cortisol levels throughout the normal menstrual cycle of healthy postmenarcheal girls Michael Gro¨schl, Dipl. Biol., Manfred Rauh, Ph.D., Patricia Schmid, and Helmuth-Gu¨nther Do¨rr, Prof. M.D. Klinik mit Poliklinik fu¨r Kinder und Jugendliche, Friedrich-Alexander-University, Erlangen-Nu¨rnberg, Germany
Objective: To determine the usefulness of salivary P and 17␣-hydroxyprogesterone (17-OHP) for the assessment of ovarian function. In addition, salivary cortisol (F) levels were measured to assess the role of the adrenal cortex throughout the menstrual cycle. Design: Prospective study. Setting: Outpatients in hospital for children and adolescents. Patient(s): Thirty young women with regular menstrual cycles. Intervention(s): Saliva collection in the early morning from day 1 of menstrual bleeding until next menses. Main Outcome Measure(s): Salivary P, 17-OHP, and F measured by RIAs. Result(s): During days 1–12 of the follicular phase, P and 17-OHP levels remained unchanged (P: 9 –29.3 pg/mL; 17-OHP: 8 –31 pg/mL). Thereafter, P increased exponentially from day 13 onward, reaching a plateau (mean ⫾ SEM, 70.1 ⫾ 9.0 pg/mL) between day 16 and 20, followed by a constant decrease until end of the cycle. The 17-OHP levels increased between day 14 and 17 (maximum: 45.8 ⫾ 4.5 pg/mL), decreasing rapidly thereafter. The F levels remained unchanged (follicular: 7.5 ⫾ 1.1 ng/mL; luteal 7.2 ⫾ 1.1 ng/mL). There was a significant correlation between P and 17-OHP (r2 ⫽ 0.43; P⬍.001). When calculating ratios of P/F and 17-OHP/F, linear regression yielded a much stronger correlation (r2 ⫽ 0.74; P⬍.001), although F did not show any correlation to P or 17-OHP. Conclusion(s): Changes in salivary 17-OHP levels throughout the menstrual cycle reflect ovarian but not adrenal function. (Fertil Steril威 2001;76:615–7. ©2001 by American Society for Reproductive Medicine.) Key Words: Saliva, progesterone, 17-hydoxyprogesterone, cortisol, menstrual cycle
Received December 14, 2000; revised and accepted March 15, 2001. Reprint requests: Michael Gro¨schl, Dipl. Biol., Klinik mit Poliklinik fu¨r Kinder und Jugendliche, Loschgestrasse 15, 91054 Erlangen, Germany (FAX: ⫹⫹49/9131/8533714; E-mail: michael.groeschl@ kinder.imed.uni-erlangen. de). 0015-0282/01/$20.00 PII S0015-0282(01)01960-4
The determination of salivary steroids is widely accepted as a reliable alternative to serum measurements. The main advantages are the noninvasiveness of saliva collection and a high correlation between salivary steroids and the nonprotein bound, physiologically active fraction of these hormones in serum (1, 2). The possibility of collecting frequent samples within short intervals even from outpatient volunteers enables studies with a high density of samples. During the menstrual cycle of healthy women, alterations of E2 and P levels are well documented, not only in serum but also in saliva (3–5). Thus, salivary P levels can be used to assess normal ovarian function. In clinical practice, the measurement of salivary 17␣hydroxyprogesterone (17-OHP) and cortisol
(F) is more common, as it has been shown that both steroids are excellent parameters to monitor the efficiency of therapy in patients with endocrine disorders, such as congenital adrenal hyperplasia due to 21-hydroxylase deficiency (6, 7). However, to the best of our knowledge, salivary 17-OHP levels have yet not been studied during the normal menstrual cycle. Therefore, we measured salivary P, 17-OHP, and F in healthy adolescents and young women to define the relationship between ovarian and adrenal steroids throughout the normal menstrual cycle.
MATERIALS AND METHODS Saliva was obtained from 30 healthy female adolescents and young women (age, 15–22 615
years) with regular menstrual cycles. First day of menstruation was defined as day 1 of the cycle. Collection took part daily (6:30 – 8:00 AM) at home, using the Salivette device (Sarstedt, Nu¨rnbrecht, Germany). Samples were collected until the onset of the next menses. Each volunteer had normal menstrual cycles for at least 2 years. Menarche occurred between 12.2 and 14.1 years. None of the volunteers took contraceptives. Duration of the cycles was 29 ⫾ 2 days (range, 26 –31 days). Body mass indices were in normal ranges (19.5–24.7 kg/m2). Sleep duration was between 7 and 10 hours. Samples were frozen immediately and stored at ⫺25°C until measurement. All volunteers gave informed consent to participate in this study, which was approved by the local ethics committee.
FIGURE 1 Course of morning salivary progesterone during menstrual cycles of healthy young women. Data are presented as mean ⫾ SD.
We used commercially available radioimmunoassays (Diagnostic Systems Laboratories, Sinsheim, Germany) adapted for the determination of salivary steroids (8, 9). Sensitivity of the P assay was 12 pg/mL; the intra-assay coefficient of variation (CV) was 5.2%. For 17-OHP, sensitivity was 15 pg/mL; CV was 6.9%. Sensitivity of the RIA for F was 0.3 ng/mL; CV was 5.8%. Statistical comparison between follicular and luteal steroid values was performed using an unpaired t-test. Alterations during the course of the cycle were calculated by analysis of variance with post-hoc Bonferroni’s multiple comparison test. Correlation between matched steroid values was calculated by linear regression. All data are presented either as mean ⫾ SD or total range.
Gro¨schl. Salivary steroids during the menstrual cycle. Fertil Steril 2001.
RESULTS Differences between follicular and luteal P levels were highly significant (P⬍.001). The P levels were consistently low during the major part of the follicular phase (days 1–12 of the menstrual cycles, 25.2 ⫾ 2.2 pg/mL, 18.0 –74.0 pg/ mL) and increased beginning with day 13 up to 585 pg/mL. A high plateau was reached between days 16 and 20 (70.1 ⫾ 9.0 pg/mL), then values decreased constantly (P⬍.001) to follicular levels (Fig. 1). A similar, but less distinctive, biphasic course could be observed for salivary 17-OHP. Significantly lower values (P⬍.001) were measured during the follicular phase (20.5 ⫾ 1.8 pg/mL, 15– 65 pg/mL) in comparison to the luteal phase (45.8 ⫾ 4.5 pg/mL, 25–240 pg/mL). An increase of 17-OHP between day 16 and 18 was followed by a constant decrease, finally reaching follicular levels at day 28 (Fig. 2). In contrast, salivary F levels did not significantly change throughout the menstrual cycle (P⫽.60). During the follicular phase, values remained in the same range as during the luteal phase (7.46 ⫾ 1.1 ng/mL vs. 7.23 ⫾ 1.1 ng/mL). To study relationships between the measured steroids, linear correlations were calculated. Matched values of P and 17-OHP yielded a correlation of r2 ⫽ 0.43 with a regression of y ⫽ 0.4x ⫹ 13.9. To exclude adrenal influence, P and 616
Gro¨schl et al.
Salivary steroids during the menstrual cycle
17-OHP values were divided through the matched F values. Linear regression between the ratios P/F and 17-OHP/F yielded a much stronger correlation of r2 ⫽ 0.78 (y ⫽ 0.8x ⫹ 0.28). There was no significant relationship between salivary cortisol and the two other steroids from matched measurements.
DISCUSSION The concentrations of adrenal and ovarian steroids during the menstrual cycle are related to changes in the activities of steroidogenic p-450 enzymes (10). Folliculogenesis begins in the late luteal phase of the preceding cycle and continues during the transition between luteal and follicular phases. The ongoing demise of the corpus luteum is associated with a rapid decline in levels of E2, P, and inhibin. With the onset of menstruation, the values of these hormones reach minimum levels. Whereas E2 levels increase again during the follicular phase, P levels still remain low. The dominance of E2 leads to growth and maturation of the follicle. The increasing pulse frequency of LH secretion, followed by an outstanding LH peak on day 13 of the regular cycle, causes ovulation. The luteinization of the theca granulosa cells after ovulation leads to an increasing ability to synthesize progesterone. The described pattern of serum P levels is also reflected in Vol. 76, No. 3, September 2001
FIGURE 2 Course of morning salivary 17-hydroxyprogesterone during menstrual cycles of healthy young women. Data are presented as mean ⫾ SD.
matched P and 17-OHP levels, but no correlation to corresponding F levels. This correlation was much stronger, when ratios of P/F and 17-OHP/F were calculated to exclude the adrenal contribution to the 17-OHP values. Our results suggest the conclusion that changes of salivary 17-OHP levels during the menstrual cycle predominantly reflect ovarian and not adrenal function. Therefore, salivary 17-OHP could be used as an additional endocrine parameter to assess ovarian function. References
Gro¨schl. Salivary steroids during the menstrual cycle. Fertil Steril 2001.
saliva samples. Our data on salivary P correspond well with published data (3, 4, 11, 12). Age-dependent variations (13) were not expected, as the young women participating in our study were all of similar age. At present, to assess endocrine function of the ovaries, other steroids than P and E2 have not been used. In contrast to previously published studies, we also measured salivary 17-OHP and F levels during the cycle. These steroid hormones were routinely measured in many endocrine laboratories as they are important parameters for monitoring patients with endocrine disorders such as congenital adrenal hyperplasia due to 21-hydroxylase deficiency (6, 7, 14, 15) or Cushing’s syndrome (16, 17). Our data show a biphasic course of salivary 17-OHP during the menstrual cycle similar to P. In contrast, salivary F levels did not differ between follicular and luteal phases. Thus, we found a significant linear correlation between
FERTILITY & STERILITY威
1. Kirschbaum C, Hellhammer DH. Salivary cortisol in psychobiological research: an overview. Neuropsychobiology 1989;22:150 – 69. 2. Vining RF, McGinley RA, Maksvytis JJ, Ho KY. Salivary cortisol: a better measure of adrenal cortical function than serum cortisol. Ann Clin Biochem 1983;20:329 –35. 3. Bourque J, Sulon J, Demey PE, Sodoyez JC, Gaspard U. A simple, direct radioimmunoassay for salivary progesterone determination during the menstrual cycle. Clin Chem 1986;32:948 –51. 4. Connor ML, Sanford LM, Howland BE. Saliva progesterone throughout the menstrual cycle and late pregnancy. Can J Physiol Pharmacol 1982;60:410 –3. 5. Lipson SF, Ellison PT. Reference values for luteal progesterone measured by salivary radioimmunoassay. Fertil Steril 1994;61:448 – 54. 6. Hughes IA, Read GF. Control in congenital adrenal hyperplasia monitored by frequent saliva 17OH-progesterone measurements. Horm Res 1984;19:77– 85. 7. Young MC, Robinson JA, Read GF, Riad FD, Hughes IA. 17OHprogesterone rhythms in congenital adrenal hyperplasia. Arch Dis Child 1988;63:617–23. 8. Gro¨schl M, Biskupek SJ, Rauh M, Do¨rr HG. Measurement of cortisol in saliva using a commercial radioimmunoassay developed for serum. J Lab Med 2000;6/7:314 – 8. 9. Gro¨schl M, Rauh M, Biskupek SJ, Do¨rr HG. Practicability of commercial methods for the measurement of 17-hydroxyprogesterone and progesterone in human saliva. J Lab Med 2001;1/2:36 – 42. 10. Sano Y, Suzuki K, Arai K, Okinaga S, Tamaoki B. Changes in enzyme activities related to steroidogenesis in human ovaries during the menstrual cycle. J Clin Endocrinol Metab 1981;52:994 –9. 11. Choe JK, Khan DF, Dawood MY. Progesterone and estradiol in the saliva and plasma during the menstrual cycle. Am J Obstet Gynecol 1983;147:557– 62. 12. Finn MM, Gosling JP, Tallon DF, Madden AT, Meehan FP, Fottrell PF. Normal salivary progesterone levels throughout the ovarian cycle as determined by a direct enzyme immunoassay. Fertil Steril 1988;50: 882–7. 13. Lipson SF, Ellison PT. Normative study of age variation in salivary progesterone profiles. J Biosoc Sci 1992;24:233– 44. 14. Arisaka O, Shimura N, Nakayama Y, Arisaka M, Yabuta K. Salivary 17-hydroxyprogesterone concentration in monitoring of the treatment of congenital adrenal hyperplasia. Dtsch Med Wochenschr 1988;113: 1913–5. 15. Do¨rr HG, Sippell WG. Adrenogenital syndrome with 21-hydroxylase deficiency. Monatsschr Kinderheilkd 1993;141:609 –21. 16. Castro M, Elias PC, Quidute AR, Halah FP, Moreira AC. Out-patient screening for Cushing’s syndrome: the sensitivity of the combination of circadian rhythm and overnight dexamethasone suppression salivary cortisol tests. J Clin Endocrinol Metab 1999;84:878 – 82. 17. Raff H, Raff JL, Findling JW. Late-night salivary cortisol as a screening test for Cushing’s syndrome. J Clin Endocrinol Metab 1998;83:2681– 6.
617
Relationship between salivary progesterone, 17-hydroxyprogesterone, and cortisol levels throughout the normal menstrual cycle of healthy postmenarcheal girls Michael Gro¨schl, Dipl. Biol., Manfred Rauh, Ph.D., Patricia Schmid, and Helmuth-Gu¨nther Do¨rr, Prof. M.D. Klinik mit Poliklinik fu¨r Kinder und Jugendliche, Friedrich-Alexander-University, Erlangen-Nu¨rnberg, Germany
Objective: To determine the usefulness of salivary P and 17␣-hydroxyprogesterone (17-OHP) for the assessment of ovarian function. In addition, salivary cortisol (F) levels were measured to assess the role of the adrenal cortex throughout the menstrual cycle. Design: Prospective study. Setting: Outpatients in hospital for children and adolescents. Patient(s): Thirty young women with regular menstrual cycles. Intervention(s): Saliva collection in the early morning from day 1 of menstrual bleeding until next menses. Main Outcome Measure(s): Salivary P, 17-OHP, and F measured by RIAs. Result(s): During days 1–12 of the follicular phase, P and 17-OHP levels remained unchanged (P: 9 –29.3 pg/mL; 17-OHP: 8 –31 pg/mL). Thereafter, P increased exponentially from day 13 onward, reaching a plateau (mean ⫾ SEM, 70.1 ⫾ 9.0 pg/mL) between day 16 and 20, followed by a constant decrease until end of the cycle. The 17-OHP levels increased between day 14 and 17 (maximum: 45.8 ⫾ 4.5 pg/mL), decreasing rapidly thereafter. The F levels remained unchanged (follicular: 7.5 ⫾ 1.1 ng/mL; luteal 7.2 ⫾ 1.1 ng/mL). There was a significant correlation between P and 17-OHP (r2 ⫽ 0.43; P⬍.001). When calculating ratios of P/F and 17-OHP/F, linear regression yielded a much stronger correlation (r2 ⫽ 0.74; P⬍.001), although F did not show any correlation to P or 17-OHP. Conclusion(s): Changes in salivary 17-OHP levels throughout the menstrual cycle reflect ovarian but not adrenal function. (Fertil Steril威 2001;76:615–7. ©2001 by American Society for Reproductive Medicine.) Key Words: Saliva, progesterone, 17-hydoxyprogesterone, cortisol, menstrual cycle
Received December 14, 2000; revised and accepted March 15, 2001. Reprint requests: Michael Gro¨schl, Dipl. Biol., Klinik mit Poliklinik fu¨r Kinder und Jugendliche, Loschgestrasse 15, 91054 Erlangen, Germany (FAX: ⫹⫹49/9131/8533714; E-mail: michael.groeschl@ kinder.imed.uni-erlangen. de). 0015-0282/01/$20.00 PII S0015-0282(01)01960-4
The determination of salivary steroids is widely accepted as a reliable alternative to serum measurements. The main advantages are the noninvasiveness of saliva collection and a high correlation between salivary steroids and the nonprotein bound, physiologically active fraction of these hormones in serum (1, 2). The possibility of collecting frequent samples within short intervals even from outpatient volunteers enables studies with a high density of samples. During the menstrual cycle of healthy women, alterations of E2 and P levels are well documented, not only in serum but also in saliva (3–5). Thus, salivary P levels can be used to assess normal ovarian function. In clinical practice, the measurement of salivary 17␣hydroxyprogesterone (17-OHP) and cortisol
(F) is more common, as it has been shown that both steroids are excellent parameters to monitor the efficiency of therapy in patients with endocrine disorders, such as congenital adrenal hyperplasia due to 21-hydroxylase deficiency (6, 7). However, to the best of our knowledge, salivary 17-OHP levels have yet not been studied during the normal menstrual cycle. Therefore, we measured salivary P, 17-OHP, and F in healthy adolescents and young women to define the relationship between ovarian and adrenal steroids throughout the normal menstrual cycle.
MATERIALS AND METHODS Saliva was obtained from 30 healthy female adolescents and young women (age, 15–22 615
years) with regular menstrual cycles. First day of menstruation was defined as day 1 of the cycle. Collection took part daily (6:30 – 8:00 AM) at home, using the Salivette device (Sarstedt, Nu¨rnbrecht, Germany). Samples were collected until the onset of the next menses. Each volunteer had normal menstrual cycles for at least 2 years. Menarche occurred between 12.2 and 14.1 years. None of the volunteers took contraceptives. Duration of the cycles was 29 ⫾ 2 days (range, 26 –31 days). Body mass indices were in normal ranges (19.5–24.7 kg/m2). Sleep duration was between 7 and 10 hours. Samples were frozen immediately and stored at ⫺25°C until measurement. All volunteers gave informed consent to participate in this study, which was approved by the local ethics committee.
FIGURE 1 Course of morning salivary progesterone during menstrual cycles of healthy young women. Data are presented as mean ⫾ SD.
We used commercially available radioimmunoassays (Diagnostic Systems Laboratories, Sinsheim, Germany) adapted for the determination of salivary steroids (8, 9). Sensitivity of the P assay was 12 pg/mL; the intra-assay coefficient of variation (CV) was 5.2%. For 17-OHP, sensitivity was 15 pg/mL; CV was 6.9%. Sensitivity of the RIA for F was 0.3 ng/mL; CV was 5.8%. Statistical comparison between follicular and luteal steroid values was performed using an unpaired t-test. Alterations during the course of the cycle were calculated by analysis of variance with post-hoc Bonferroni’s multiple comparison test. Correlation between matched steroid values was calculated by linear regression. All data are presented either as mean ⫾ SD or total range.
Gro¨schl. Salivary steroids during the menstrual cycle. Fertil Steril 2001.
RESULTS Differences between follicular and luteal P levels were highly significant (P⬍.001). The P levels were consistently low during the major part of the follicular phase (days 1–12 of the menstrual cycles, 25.2 ⫾ 2.2 pg/mL, 18.0 –74.0 pg/ mL) and increased beginning with day 13 up to 585 pg/mL. A high plateau was reached between days 16 and 20 (70.1 ⫾ 9.0 pg/mL), then values decreased constantly (P⬍.001) to follicular levels (Fig. 1). A similar, but less distinctive, biphasic course could be observed for salivary 17-OHP. Significantly lower values (P⬍.001) were measured during the follicular phase (20.5 ⫾ 1.8 pg/mL, 15– 65 pg/mL) in comparison to the luteal phase (45.8 ⫾ 4.5 pg/mL, 25–240 pg/mL). An increase of 17-OHP between day 16 and 18 was followed by a constant decrease, finally reaching follicular levels at day 28 (Fig. 2). In contrast, salivary F levels did not significantly change throughout the menstrual cycle (P⫽.60). During the follicular phase, values remained in the same range as during the luteal phase (7.46 ⫾ 1.1 ng/mL vs. 7.23 ⫾ 1.1 ng/mL). To study relationships between the measured steroids, linear correlations were calculated. Matched values of P and 17-OHP yielded a correlation of r2 ⫽ 0.43 with a regression of y ⫽ 0.4x ⫹ 13.9. To exclude adrenal influence, P and 616
Gro¨schl et al.
Salivary steroids during the menstrual cycle
17-OHP values were divided through the matched F values. Linear regression between the ratios P/F and 17-OHP/F yielded a much stronger correlation of r2 ⫽ 0.78 (y ⫽ 0.8x ⫹ 0.28). There was no significant relationship between salivary cortisol and the two other steroids from matched measurements.
DISCUSSION The concentrations of adrenal and ovarian steroids during the menstrual cycle are related to changes in the activities of steroidogenic p-450 enzymes (10). Folliculogenesis begins in the late luteal phase of the preceding cycle and continues during the transition between luteal and follicular phases. The ongoing demise of the corpus luteum is associated with a rapid decline in levels of E2, P, and inhibin. With the onset of menstruation, the values of these hormones reach minimum levels. Whereas E2 levels increase again during the follicular phase, P levels still remain low. The dominance of E2 leads to growth and maturation of the follicle. The increasing pulse frequency of LH secretion, followed by an outstanding LH peak on day 13 of the regular cycle, causes ovulation. The luteinization of the theca granulosa cells after ovulation leads to an increasing ability to synthesize progesterone. The described pattern of serum P levels is also reflected in Vol. 76, No. 3, September 2001
FIGURE 2 Course of morning salivary 17-hydroxyprogesterone during menstrual cycles of healthy young women. Data are presented as mean ⫾ SD.
matched P and 17-OHP levels, but no correlation to corresponding F levels. This correlation was much stronger, when ratios of P/F and 17-OHP/F were calculated to exclude the adrenal contribution to the 17-OHP values. Our results suggest the conclusion that changes of salivary 17-OHP levels during the menstrual cycle predominantly reflect ovarian and not adrenal function. Therefore, salivary 17-OHP could be used as an additional endocrine parameter to assess ovarian function. References
Gro¨schl. Salivary steroids during the menstrual cycle. Fertil Steril 2001.
saliva samples. Our data on salivary P correspond well with published data (3, 4, 11, 12). Age-dependent variations (13) were not expected, as the young women participating in our study were all of similar age. At present, to assess endocrine function of the ovaries, other steroids than P and E2 have not been used. In contrast to previously published studies, we also measured salivary 17-OHP and F levels during the cycle. These steroid hormones were routinely measured in many endocrine laboratories as they are important parameters for monitoring patients with endocrine disorders such as congenital adrenal hyperplasia due to 21-hydroxylase deficiency (6, 7, 14, 15) or Cushing’s syndrome (16, 17). Our data show a biphasic course of salivary 17-OHP during the menstrual cycle similar to P. In contrast, salivary F levels did not differ between follicular and luteal phases. Thus, we found a significant linear correlation between
FERTILITY & STERILITY威
1. Kirschbaum C, Hellhammer DH. Salivary cortisol in psychobiological research: an overview. Neuropsychobiology 1989;22:150 – 69. 2. Vining RF, McGinley RA, Maksvytis JJ, Ho KY. Salivary cortisol: a better measure of adrenal cortical function than serum cortisol. Ann Clin Biochem 1983;20:329 –35. 3. Bourque J, Sulon J, Demey PE, Sodoyez JC, Gaspard U. A simple, direct radioimmunoassay for salivary progesterone determination during the menstrual cycle. Clin Chem 1986;32:948 –51. 4. Connor ML, Sanford LM, Howland BE. Saliva progesterone throughout the menstrual cycle and late pregnancy. Can J Physiol Pharmacol 1982;60:410 –3. 5. Lipson SF, Ellison PT. Reference values for luteal progesterone measured by salivary radioimmunoassay. Fertil Steril 1994;61:448 – 54. 6. Hughes IA, Read GF. Control in congenital adrenal hyperplasia monitored by frequent saliva 17OH-progesterone measurements. Horm Res 1984;19:77– 85. 7. Young MC, Robinson JA, Read GF, Riad FD, Hughes IA. 17OHprogesterone rhythms in congenital adrenal hyperplasia. Arch Dis Child 1988;63:617–23. 8. Gro¨schl M, Biskupek SJ, Rauh M, Do¨rr HG. Measurement of cortisol in saliva using a commercial radioimmunoassay developed for serum. J Lab Med 2000;6/7:314 – 8. 9. Gro¨schl M, Rauh M, Biskupek SJ, Do¨rr HG. Practicability of commercial methods for the measurement of 17-hydroxyprogesterone and progesterone in human saliva. J Lab Med 2001;1/2:36 – 42. 10. Sano Y, Suzuki K, Arai K, Okinaga S, Tamaoki B. Changes in enzyme activities related to steroidogenesis in human ovaries during the menstrual cycle. J Clin Endocrinol Metab 1981;52:994 –9. 11. Choe JK, Khan DF, Dawood MY. Progesterone and estradiol in the saliva and plasma during the menstrual cycle. Am J Obstet Gynecol 1983;147:557– 62. 12. Finn MM, Gosling JP, Tallon DF, Madden AT, Meehan FP, Fottrell PF. Normal salivary progesterone levels throughout the ovarian cycle as determined by a direct enzyme immunoassay. Fertil Steril 1988;50: 882–7. 13. Lipson SF, Ellison PT. Normative study of age variation in salivary progesterone profiles. J Biosoc Sci 1992;24:233– 44. 14. Arisaka O, Shimura N, Nakayama Y, Arisaka M, Yabuta K. Salivary 17-hydroxyprogesterone concentration in monitoring of the treatment of congenital adrenal hyperplasia. Dtsch Med Wochenschr 1988;113: 1913–5. 15. Do¨rr HG, Sippell WG. Adrenogenital syndrome with 21-hydroxylase deficiency. Monatsschr Kinderheilkd 1993;141:609 –21. 16. Castro M, Elias PC, Quidute AR, Halah FP, Moreira AC. Out-patient screening for Cushing’s syndrome: the sensitivity of the combination of circadian rhythm and overnight dexamethasone suppression salivary cortisol tests. J Clin Endocrinol Metab 1999;84:878 – 82. 17. Raff H, Raff JL, Findling JW. Late-night salivary cortisol as a screening test for Cushing’s syndrome. J Clin Endocrinol Metab 1998;83:2681– 6.
617