Increased follicular fluid total and free cortisol levels during the luteinizing hormone surge

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1 FRTILITY AND STRILITY Copyright" 1997 American Society for Reprodctive Medicine Pblished by lsevier Science Inc. Vol. 68, No.1, Jly 1997 Printed on acid-free paper in U S. A. Increased folliclar flid total and free cortisol levels dring the lteinizing hormone srge Christopher R. Harlow, Ph.D.*t Jlian M. Jenkins, D.M.*:j: Robert M. L. Winston, F.R.C.O.G. University ofbristol, Bristol, and University oflondon, Institte of Obstetrics and Gynaecology, London, United Kingdom Objective: To determine the changes in folliclar flid (FF) total and free cortisol dring the LH srge in natrally ovlating women. Patient(s): Twenty-six women having diagnostic laparoscopy dring the folliclar phase of normal menstral cycles were selected. Intervention(s): Blood samples were collected 1 day before, the day of, and 1 day after srgery and the reslts of serm z and LH were sed to divide the cycles retrospectively into pre- and post-lh srge grops. Folliclar flid was collected dring laparoscopy. Main Otcome Measre(s): Serm P, total and free cortisol, and FF volme, z, P, total cortisol, and free cortisol were measred on the day of srgery. Res1t(s): Median serm and FF P levels were significantly higher in the post-lh srge grop compared with the pre-lh srge grop (.54 verss 1.54 ng/ml [1.7 verss 4.85 nmol! L] and 5.3 verss 28. j.lg/ml [15.8 verss 88. j.lmol!l], respectively). Folliclar flid volme also increased significantly after the srge (2.5 verss 4.5 ml.). Median serm total and free and percent free cortisol were higher after the srge, althogh not significantly. In contrast, FF total, free, and percent free levels increased dramatically between pre- and post-lh srge samples (4.41 verss 43.6 ng/ml [16. verss 158 nmol!l],.138 verss 6.68 ng/ml [.5 verss 24.2 nmol/l], and 3.3% verss 15.%, respectively; P <,5). Conclsion(s): An increase in total and free cortisol occrs in the follicle dring the LH srge. Cortisol and its reglation by l1,b-hydroxysteroid dehydrogenase therefore may exert a physiologic role in oocyte matration or ovlation. (Fertil Sterilv 1997;68: by American Society for Reprodctive Medicine.) Key Words: Cortisol, estradiol, folliclar flid, ll,b-hydroxysteroid dehydrogenase, LH srge, progesterone It long has been recognized that glcocorticoid secretion, as a physiologic response to stress, cold have adverse effects on a woman's natral fertility. The mechanisms proposed inclde actions on hypothalamic pititary fnction leading to redced go- Received October 21,1996; revisedand acceptedmarch 7,1997. * University Department of Hospital Medicine, Division of Obstetrics and Gynaecology, St Michael's Hospital. t Spported by a Medical Research Concil (London, United Kingdom) Training Fellowship. t Reprint reqests: Jlian M. Jenkins, D.M., University Department of Hospital Medicine, Division of Obstetrics and Gynaecology, St. Michael's Hospital, Sothwell Street, Bristol BS2 8G, United Kingdom (Fax: ; JULIAN.JNKINS@BRISTOL.AC.UK). University of London, Institte of Obstetrics and Gynaecology, Hammersmith Hospital. 48 nadotropin secretion, as well as direct effects of cortisolon the ovary reslting in redced estrogen biosynthesis, redced pregnenolone synthesis by inhibition of cholesterol side-chain cleavage, and disrption of ovlation by inhibition of plasminogen activation (1-4). However, evidence now is accmlating that sggests that cortisol may have beneficial effects on oocyte matration. In fish, cortisol stimlates oocyte matration (5). Folliclar flid (FF) levels of cortisol at the time of oocyte recovery for IVF are two to three times higher in follicles yielding matre compared with immatre oocytes (6). Rat granlosa cell P prodction is enhanced by glcocorticoids, probably by stimlation of 3,B-hydroxysteroid dehydrogenase (3,8HSD) and inhibition of 2aHSD activity (7). Cortisol metabolism, in particlar the reglation /97/$17. PH S15-282(97)73-3

2 by l1,6hsd, also may be important in the normal development of the ovlatory follicle. Ths, Michael and co-workers (8) demonstrated that l1,6hsd was ndetectable in cltred hman granlosa cells of all 22 women in the stdy who became pregnant, whereas detectable levels of 11,6HSD were fond in 25 of 32 cases in which there was no pregnancy. These reslts were confirmed in a sbseqent stdy involving 172 treatment cycles (9). The messenger RNA (mrna) encoding type 1 l1,6hsd has been identified in ovarian tisses (1), and this isoform has been demonstrated to act predominantly as an 11-ketoredctase (converts cortisone to cortisol), with a low affinity for cortisol (K m, 7.5 /Lg/mL [27 /LMD (11). Ths, the presence of ll,6hsd activity wold be expected to be accompanied by high levels of intrafolliclar cortisol. However, a second renal isoform, 11,6HSD2, has been identified, with a high affinity for cortisol (Km> 11 ng/ml [4 nmd (12), and others have fond evidence of an ovarian isoform of 11,6HSD with a similarly high affinity for cortisol (Michael A, Piercy G, Stedman B, dwards CRW, Seckl JR, Cooke AB, abstract). It is believed that free cortisol, rather than the protein-bond fraction, is biologically active in vivo (13). The level offree cortisol in circlation is generally < 1% of the total and may fall as low as 1% dring estrogen administration or pregnancy (14). Free cortisol in the preovlatory follicle was calclated from the levels oftotal cortisol and sex steroids, cortisol-binding globlin (CBG), sex-steroid binding globlin and albmin, and the binding affinities for varios steroids to these and was fond to be 1 fold higher than in serm (22% verss 2.1%) (15). However, these findings have not been confirmed by direct measrement offree cortisol in FF, and there are no reports of intrafolliclar cortisol before the LH srge. The aims of the present stdy therefore were to examine the changes in circlating and FF cortisol dring the LH srge in women ndergoing diagnostic laparoscopy dring the preovlatory phase of nstimlated cycles. Total and free cortisol, P, and 2 were measred, together with serm LH and folliclar volme. Sbjects MATRIALS AND MTHODS Twenty-six women having diagnostic laparoscopy for fertility investigation dring the late folliclar phase of the cycle were recrited into the stdy. All women had reglar menstral history with median cycle length ranging between 27 and 33 days and a maximm variation in cycle length for each individ- Vol. 68, No.1, Jly 1997 al of 5 days over the previos 6 months. They had apparently normal ovarian fnction, as assessed by midlteal phase P levels in a previos cycle that were>1 ng/ml (conversion factor to SI nit, 3.18). All women had given their informed consent, and the project had the approval of the Hammersmith and Qeen Charlotte's Special Health Athority Medical thics Committee. Blood Samples Folliclar matration was determined retrospectively by the measrement of serm 2 and LH in blood samples collected the day before (12: noon to 6: P.M.), the day of (7: A.M. to 9: A.M.), and the day after (8: A.M. to 12: noon) srgery. Women were divided into pre- and post-lh srge grops on the basis of their 2 levels and when their LH srge began. The LH srge was defined as a 1.8 fold increase in baseline levels or a vale of 2 miui ml (conversion factor to SI nit, 1.). Women in the pre-lh srge grop had 2 levels >22 pg/ml (6 pmolll) on the day of srgery and an LH srge that was detected on the day after srgery. The post LH srge grop had variable 2 levels and an LH srge that began on the day of srgery. Blood samples from the day of srgery were stored at - 2 C for sbseqent measrement of P and cortisol. Folliclar Aspiration Folliclar aspiration was performed dring the diagnostic laparoscopy sing a doble-channel IVF aspiration device, and accessible follicles > 12 mm in diameter were aspirated into 1-mL plastic tbes (Falcon, Becton-Dickinson, Cowley, Oxford, United Kingdom). Folliclar flid with or withot minor blood contamination and granlosa cells was separated by centrifgation (15 X g, 5 mintes), and the clear FF was stored at -2 C ntil assayed. Serm LH, 2, and P Measrement Lteinizing hormone was determined sing the LH ter RIA kit (Sereno Diagnostics Ltd., Woking, Srrey, United Kingdom), which had an interassay coefficient of variation (CV) of 1.6%. stradiol was measred by a direct RIA kit (R-155; Steranti Research Ltd., St. Albans, Herts, United Kingdom), which had an interassay CV of 9.1%. Progesterone was determined sing the DPC assay (roidpc Ltd., Glyn Rhonwy, Llanberis, Caemarfon, Gwynedd, United Kingdom). The interassay CV was 9.4%. stradiol and P Measrement in FF Folliclar flid was dilted p to 1:1, with assay bffer (. 1 M phosphate-bffered saline) Harlow et al. Folliclar flid total and free cortisol 49

3 before assay sing previosly described RIA methods (16). Cortisol Measrement Total (free and protein-bond) cortisol in serm and FF was measred withot extraction. Cortisol assays were performed on five samples in each grop sing the DPC Coat-a-Cont assay (roidpc Ltd.), Interassay CV was 6.3%. Relevant cross-reactivity of the antiserm with other steroids was cortisone,.6%; P,.15%; and 17a-hydroxyprogesterone (17-HP), <.5%. Validation of the assay for measrement of cortisol in FF was achieved by making serial diltions of an FF pool containing high endogenos cortisol concentrations with cortisol-free serm. The pool showed excellent linearity on diltion (r =.96) that was parallel to the standard crve over the working range of the assay. Measrement of Free Cortisol Free cortisol was separated from protein-bond cortisol sing the Centrifree microseparation device (Amicon Ltd., Stonehose, Glocestershire, United Kingdom) as described previosly (17), before assay as above. In brief, prewarmed serm or FF samples (1 ml) were loaded into the top compartment of the device and centrifged in a 35 fixed-angle rotor at 37 C for 3 mintes at 2, X g. The size exclsion filter allows free cortisol to pass into a filtrate collection cp. The volme of the original sample recovered in the filtrate was 83.8% ± 1.5% (mean ± SM, n = 8), which was acconted for when calclating the amont of free cortisol in each sample. Recovery of cortisol was determined by the addition of approximately 5, cpm 125I-cortisol and 2.88 ng (1 nmol) nlabeled cortisol to 1 ml serm. The mean percent recovery of radioactivity from for separation devices was 83.% ± 4.6% (SM), the remaining radioactivity being detected in the filters. A previos stdy (17) showed excellent correlation of the method with eqilibrim dialysis (r =.94), althogh the slope of the regression (1.43) indicated an overestimate in comparison with eqilibrim dialysis. Statistical Analysis Hormonal levels and FF volme were not normally distribted. Reslts therefore were presented as median and 95% confidence intervals (CIs). Comparisons between medians were made by the Mann Whitney U test, which was chosen becase of the small nmber of observations in some cases and the absence of a normal distribtion that was not correctable by log transformation. RSULTS Sex steroid and LH levels and FF volme reslts are presented in Figre 1. There was no difference in the median serm or FF 2levels before and after the LH srge, althogh there was a greater variability in the levels after the srge. In contrast, serm and FF P were significantly higher after the onset A soot 3.5 r'l ::: 3 r I '6 I I :J 3 > -= 2.5 ' '6.; 2 1 i21 GO ~ 1.5 ~ 11 I '3 1.\1 'is,.5 I B 2.5 r *** I Figre 1 Serm and FF levels of 2 (conversion factor to SI nit, 2.724) (A) and P (conversion factor to SI nit, 3.145) (B) and serm LH and FF volme (e) in women on the day of diagnostic laparoscopy, either before (open bars) or after (solid bars) the onset of the LH srge. Bars represent the median vales, with the vertical lines being the 95% CIs and the nmbers at the base of each bar being the nmber of observations. **p <.1, ***p <.1; compared with pre-lh srge vale (Mann-Whitney U test). OJ ~ 3 :J 2 > 1 -= GO s c.! 2 ls 1.5 GO.! > > a ' ii 11 :2 " '3 ~.5 1 j ol 15 1 ': 1 J1 ', C 5 ** 5 ~ 4 fl J :J ] 3.23 :I: > J ' '5 ~ 2 ~ 2f IJl '3 := I '] ~ 1 t r *** 11 ** 11 5 Harlow et al, Folliclar flid total and free cortisol Fertility and Sterility"

4 A I I 2 ~ 1 ~ II> :J 16.s 8 II> '.s ' () ~ 12 6 () iii iii :2 'tl :2 8 4 ~ :> 16 '3 VI 4 ;: 2 I",. I- r ~ 14 * :J II> 4.s 12 II>.s ' ' :e 1., ~3 () 8 ~ 'tl.:: 2 ;a 6 :> 16 '3 4 VI 1 := ' 2 C G 16 G ~ ~ ~ ~ 1.5 ] 12 ' () ~ () [-- 'tl U 1 ~ 8 ;;; 16 '3 VI o -=.5 ', 4 I I Figre 2 Serm and FF levels oftotal cortisol (Al, free cortisol (B) (conversion factor to SI nits, 3.625), and percent free cortisol (C) in a sbgrop offive of the women in Figre 1 on the day of diagnostic laparoscopy, either before or after the onset of the LH srge. Bars represent the median vales, with the vertical lines being the range of vales. "P <.5 compared with pre-lh srge vale (Mann-Whitney U test). of the LH srge. As expected, serm LH levels were significantly higher after the LH srge onset. Folliclar flid volme increased by 65% in the post-lh srge grop. Serm and FF total cortisol, free cortisol, and percent free cortisol are shown in Figre 2. All three levels were higher after the srge in serm, bt the differences were not significant. By contrast, there were significantly higher levels after the srge in FF, Vol. 68, No.1, Jly 1997 I * * the median being 1-fold, 5-fold, and 5-fold greater, respectively, than before the srge. DISCUSSION To or knowledge, this is the first report of direct measrements of free cortisol in hman FF, and it reveals dramatic increases in free and total cortisol after the onset ofthe LH srge. The median level of serm total cortisol before and after the LH srge was very similar to that reported by Fateh et al (6) in gonadotropin-treated women at the time of oocyte recovery for NF. However, the level of total cortisol in FF before the srge was 1-fold lower than that observed after the srge and 4-fold lower than fond dring the folliclar phase by Fateh et al (6). After the srge, we fond levels that were somewhatlower than those observed by Fateh et al (6) in gonadotropin-treated women and more in keeping with the levels reported by Dehennin et al (18) and Yding Andersen and Hornnes (15). One of the problems of measring cortisol in FF is the possibility of interference of other steroids, notably P and 17-HP, which are present in levels p to 1,-fold higher than those of cortisol in a preovlatory follicle. ven a minor cross-reactivity of the cortisol antiserm with progestins cold alter significantly the apparent cortisol concentration, as pointed ot by Dehennin (19). The antibody sed in the present stdy cross-reacts with P (.15%) and 17-HP «.5%), bt there was no correlation between cortisol and P levels in FF, sggesting that cross-reaction was not affecting the cortisol level Free cortisol has not been measred previosly in FF by a direct method. We fond very low levels (median,.138 ng/ml [.5 nmolll]) before the LH srge. However, the levels we observed after the LH srge were some 5-foldhigher. They are similar to those calclated previosly, based on the levels of total cortisol, sex steroids, and binding proteins in preovlatory follicles of gonadotropin-treated women at the time of oocyte recovery for IVF (15). The large increase in total cortisol after the srge may reslt from changes in cortisol metabolism. Michael et al (8) proposed that low l1,bhsd activity and high intrafolliclar cortisol might be necessary for complete matration of the oocyte, and or observations ofincreasingcortisol levels spportthisidea. Frther spport for a change in cortisol metabolism may come from stdies of cortisol:cortisone ratios. Crrently, we are ndertaking a stdy to examine the levels of intrafolliclar cortisone. The high percentage of free cortisol in FF (median, 15.%) compared with serm (median, 2.1%), which represents the biologically active form (13), is probably a reslt of the rapid post-lh srge rise in P and Harlow et al. Folliclar flid total and free cortisol 51

5 17-HP, steroids that wold displace cortisol from its binding proteins (15). Given that CBG levels in FF are similar to serm levels (2), it is not srprising that cortisol bond to CBG is mch lower in FF compared with serm (48% verss 94%), whereas the amont bond to albmin is mch higher (3% verss 3.6%) (15). Given the low binding affinity of cortisol for albmin (2-fold less than that of'p) (15), it is reasonable to assme that >5% of the total cortisol in FF may be effectively available as a biologically active molecle. The role of cortisol in the preovlatory follicle is nclear. Ben-Rafael et al. (21) showed enhancement of hman granlosa cell 2 and P prodction by cortisol, bt only at spraphysiologic levels. By contrast, Michael et al. (3) demonstrated significant inhibition oflh-stimlated pregnenolone synthesis by cortisol at 28.8 ng/ml (l nmol/l), An effect of cortisolon steroidogenesis in the follicle therefore is possible, bt the availability of cortisol may be reglated by the activity of l1,bhsd (3). Becase the predominant direction of action of l1,bhsd is dependent on the relative abndance of the isoforms present, frther clarification ofthe natre of the ovarian l1,bhsd isoforms is necessary. Crrently, we are investigating the occrrence ofmrna for type 1 and 2 ll,bhsd in hman granlosa cells. Cortisol also may reglate processes involved in celllar remodeling occrring at ovlation. For example, glcocorticoids inhibit plasminogen activator in rat granlosa cells (4), and cortisol inhibits macrophage collagenase secretion (22). Or observation of changing cortisol dring folliclar matration sggests a physiologic role for this steroid in the follicle and also a mechanism by which the elevation of cortisol that occrs in response to stress may affect ovarian fnction directly. Althogh there is no evidence that stress directly increases intrafolliclar cortisol, physiologic (serm) levels of cortisol were shown to inhibit granlosa cell LHstimlated pregnenolone synthesis in vitro (3). We previosly showed higher serm cortisol and state anxiety in women having IVF treatment (23), and others have shown increased cortisol in response to emotional stressors in infertile women (24, 25). In conclsion, we have shown that intrafolliclar total and free cortisol levels increase dring the LH srge and that free cortisol reaches levels that may exert a physiologic role in oocyte matration or ovlation. The mechanisms reglating the availability of cortisol in the follicle are not flly nderstood bt may inclde alterations in the activity of the varios isoforms of l1,bhsd and release of cortisol from its binding proteins by the increasing levels of progestins as ovlation approaches. Acknowledgment. We thank Hardev Jeer, B.Sc., University of London, Institte of Obstetrics and Gynaecology, for his technical assistance with RIAs. RFRNCS 1. Cnningham GR, Caperton M Jr, Goldzeiher JW. Antiovlatory activity of synthetic corticoids, J Clin ndocrinol Metab 1975;4: Hseh AJW, rickson GF. Glcocorticoid inhibition of FSHindced estrogen prodction in cltred granlosa cells. Steroids 1978; 32: Michael A, Pester LA, Crtis P, Shaw RW, dwards CRW, Cooke BA. Direct inhibition of ovarian steroidogenesis by cortisol and the modlatory role of 1113-hydroxysteroid dehydrogenase. Clin ndocrinoi1993;38: Harlow CR, Coombs RJ, Hodges JK, Jenkins N. Modlation of plasminogen activation by glcocorticoid hormones in the rat granlosa cell. J ndocrinol 1987; 114: Greeley MS Jr, Calder DR, Taylor MH, Hols H, Wallace RA. Oocyte matration in mmmichog (Fndls heteroclits): effects of steroids on germinal vesicle breakdown of intact follicles. Gen Comp ndocrinoi1986;62: Fateh M, Ben-Raphael Z, Benadiva CA, Mastroianni L Jr, Flickinger GL. Cortisol levels in hman folliclar flid. Fertil Steril1989;51: Adashi Y, Jones PBC, Hseh AJW. Synergistic effect of glcocorticoids on the stimlation of progesterone by folliclestimlating hormone in cltred rat granlosa cells. ndocrinology 1981; 19: Michael A, Gregory L, Walker SM, Antoniw JW, Shaw RW, dwards CRW, et al. Ovarian 1113-hydroxysteroid dehydrogenase: potential predictor of conception by in vitro fertilization and embryo transfer. Lancet 1993;342: Michael A, Gregory L, Piercy C, Walker SM, Shaw RW, Cooke BA. Ovarian 1113-hydroxysteroid dehydrogenase activity is inversely related to the otcome of in vitro fertilizationembryo transfer treatment cycles. Fertil SteriI1995;64: Tannin GM, Agarwal AK, Monder C, New MI, White PC. The hman gene for 1113-hydroxysteroid dehydrogenase. Strctre, tisse distribtion and chromosomal localization. J BioI Chern 1991;266: Monder C, Lakshami V. vidence for kinetically distinct forms of corticosteroid 1113-hydroxysteroid dehydrogenase in rat liver microsomes. J Steroid Biochern 1989; 32: Mercer WR, Krozowski ZS. Localization of an 11jJ-hydroxysteroid dehydrogenase activity to the distal nephron. vidence for the existence of two species in the rat kidney. ndocrinology 1992; 13: Slanwhite WR Jr, Lockie GN. Inactivity in vivo oftranscortin-bond cortisol. Science 1962; 135: Sandberg AA, Slanwhite WR Jr. Transcortin: corticosteroidbinding protein of plasma. II. Levels in varios conditions and the effects of estrogens. J Clin Invest 1959;38: Yding Andersen C, Hornnes P. Intrafolliclar concentrations of free cortisol close to folliclar rptre. Hm Reprod 1994;9: Hillier SG, van den Boogaard AMJ, Reichert L Jr, van Hall V. Intraovarian sex steroid hormone interactions and the reglation of folliclar matration: aromatization of androgens by hman granlosa cells in vitro. J Clin ndocrinol Metab 198;5: MacMahon W, Thompson J, Bowers W, Sgotas D. A simplified ltrafiltration method for determination of serm free cortisol. Clin Chim Acta 1983; 131: Dehennin L, Nahol K, Scholler R. Steroid 21-hydroxylation 52 Harlow et al. Folliclar flid total and free cortisol Fertility and Sterility"

6 by hm an preovlatory follicles from stimlated cycles: a mass spect rometrical stdy of deoxycorticosterone, 21-hydroxypregnenolone and ll-deoxycortisol in folliclar flid. J Steroid Biochem 1987;26: Dehennin L. Cortisol determination in folliclar flid. Fertil Steril 1989;54: Yding Andersen C. Levels of steroid-binding proteins and steroids in hman preovlatory follicle flid and serm as predictors of sccess in in vitro fertiliz ation-embryo transfer treatm ent. J Clin ndocrinol Metab 199;71: Ben-Rafael Z, Benadiva CA, Garcia CJ, Flickinger GL. Cortisol stimlation of est radiol and progestero ne secretion by hman granlosa cells is independent of follicle-stimlating hormone effects. Fertil SteriJ 1988;49: Werb Z. Biochemical actions of glcocorticoids on macrophages in cltre. Specific inhibition of elastase, collagenase and plasminogen acti vator secret ion and effects on other metabolic fnctions. J xp Med 1978; 147: Harl ow CR, Fahy UM, Talbot WM, Wardle PG, Hll MGR. Stress and stress-relate d horm ones dring in-vitro fertilization treatment. Hm Reprod 1996;11: Dernyttena ere K, Nijs P, vers-kiebooms G, Koninckx PH. The effect of a specific emotional stressor on prolactin, cort isol and testosterone concentrations in women varies with th eir tra it anxiety. Fertil Steril 1989;52: Modell, Goldstein D, Reyes FI. ndocrine and behavioral responses to psychological stress in hyperandrogenic women. Fert il Steril 199;53: Vol. 68, No.1, Jly 1997 Harlow et al, Folliclar flid total and free cortisol 53