Higher cortisol:cortisone ratios in the preovulatory follicle of completely unstimulated IVF cycles indicate oocytes with increased pregnancy potential

S.D. Keay1, C.R. Harlow2, P.J. Wood3, J.M. Jenkins4 and D.J. Cahill4,5

1 The Sir Quinton Hazell Molecular Medicine Research Centre, Department of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, 2 Department of Reproductive and Developmental Sciences, University of Edinburgh Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9ET, 3 Regional Endocrine Unit, Department of Chemical Pathology, Duthie Building, Southampton General Hospital, Tremona Road, Southampton, SO16 6YD and 4 Division of Obstetrics and Gynaecology, University of Bristol, St Michaels Hospital, Southwell Street, Bristol BS2 8EG, UK


    Abstract
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
BACKGROUND: Conception following gonadotrophin-stimulated IVF and embryo transfer has been associated with a higher intrafollicular cortisol:cortisone ratio and decreased metabolism of cortisol to cortisone. The role of glucocorticoids in human oocyte maturation is not fully understood, but active glucocorticoid (cortisol) may be important. This study relates intrafollicular cortisol and cortisone concentrations to oocyte fertilization and embryo implantation in unstimulated cycles. METHODS: Patients aged <40 years with favourable sperm underwent unstimulated IVF–embryo transfer. Study 1 related intrafollicular cortisol levels to oocyte and IVF outcome: (i) fertilized, pregnant (n = 9); (ii) fertilized, not pregnant (n = 21); and (iii) unfertilized (n = 12). Study 2 was a case–control study of 27 patients (same outcome groups of equal size) which measured intrafollicular cortisol, cortisone and the cortisol:cortisone ratio. RESULTS: Conception cycles demonstrated higher cortisol concentrations compared with the fertilized group (study 1) [median (95% confidence interval): 299 (249–330) versus 227 nmol/l (185–261); P < 0.05] and higher cortisol:cortisone ratios when compared with the unfertilized group (study 2) [7.38 (5.23–9.19) versus 3.56 (1.75–7.46) respectively; P = 0.02]. Of the women with cortisol:cortisone ratios greater than the outcome independent mean of 5.90, 58% conceived compared with only 13% with ratios <5.90 (P < 0.02). CONCLUSION: Higher cortisol:cortisone ratios in conception cycles suggest that active glucocorticoid may be important for final oocyte maturation and embryo implantation in unstimulated cycles.

Key words: 11ß-HSD/cortisol/cortisone/natural cycle IVF


    Introduction
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Ovarian cortisol concentrations and the enzyme 11ß-hydroxysteroid dehydrogenase (11ß-HSD) which catalyses the interconversion of cortisol (active glucocorticoid) and cortisone (inactive steroid) have been studied extensively in relation to oocyte quality and IVF outcome. Interest was raised by the observation that detectable conversion of cortisol to cortisone following prolonged in-vitro culture of granulosa-lutein cells was associated with very low IVF pregnancy rates (Michael et al., 1993aGo,1995Go). A similar experiment using freshly isolated granulosa-lutein cells found no correlation between cortisol metabolism and the clinical outcome of IVF (Thomas et al., 1998Go). Cortisol to cortisone interconversion is determined by the expression of the two known isoforms of 11ß-HSD. Type 1 is NADPH-dependent and acts predominantly as a reductase, generating cortisol from cortisone, and type 2 is a NAD+-dependent dehydrogenase converting cortisol to cortisone (Tannin et al., 1991Go; Albiston et al., 1994Go).

Transcripts of type 1 mRNA have been detected in human luteinized granulosa cells (Michael et al., 1997Go; Smith et al., 1997Go; Tetsuka et al., 1997Go), cumulus cells and oocytes (Smith et al., 2000Go), suggesting that the cumulus–oocyte complex is capable of locally controlling the availability of active glucocorticoid. The enzyme 11ß-HSD is developmentally and differentially regulated in human granulosa cells. Type 2 11ß-HSD is expressed in the follicular phase (favouring cortisol to cortisone conversion), but following the onset of the LH surge a change in isoform expression occurs with type 1 11ß-HSD being expressed (Tetsuka et al., 1997Go) which favours cortisol generation.

A significant rise in intrafollicular cortisol occurs immediately prior to ovulation in women suggesting that the steroid may exert a physiological role in oocyte maturation and ovulation (Harlow et al., 1997Go). In other species, cortisol is required for final maturation (Greeley et al., 1986Go) but the findings of studies relating follicular fluid (FF) cortisol levels to oocyte maturity in women undergoing gonadotrophin stimulation are conflicting (Fateh et al., 1989Go; Andersen and Hornnes 1994Go).

Taken together, the localization and functional studies reported above imply that type 1 11ß-HSD expression in granulosa cells and its ability to convert cortisone to cortisol is important in follicular and oocyte development. Furthermore, the observation in gonadotrophin-stimulated IVF of higher FF cortisol:cortisone ratios in pregnancy cycles compared with non-pregnant (Michael et al., 1999Go) supports the concept of cortisol having a physiological role in oocyte maturation and embryo implantation.

The aim of this study was to measure the relative concentrations of cortisol and cortisone in individual preovulatory follicles in completely unstimulated cycles and relate these levels to oocyte fertilization and embryo implantation as indices of developmental competence.


    Material and methods
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Management of IVF patients
Infertile patients attending the Reproductive Medicine Clinic were recruited to a study of pituitary–ovarian endocrinology, follicular growth and IVF in completely unstimulated cycles, approved by the local research ethics committee. All women had been fully investigated, including laparoscopy. Women <40 years old with unexplained infertility (>3 years duration), tubal infertility or minor endometriosis [minimal or mild by revised American Fertility Society scoring; (American Fertility Society, 1985Go)] were included in the study. The men had favourable sperm function and all other infertility investigations were normal. This study has been previously described fully (Cahill et al., 1995Go,1997Go).

In brief, IVF was undertaken in an unstimulated cycle. Follicular diameter was monitored by daily transvaginal ultrasonography from day 9 of the cycle until oocyte retrieval, measuring the dominant follicle in three perpendicular planes to calculate the mean diameter. Serum estradiol (E2) and LH concentrations were measured daily until the dominant follicle reached 14 mm in diameter. Thereafter, the sampling frequency for LH increased to every 4 h (08:00–24:00 h, but not 04:00 h) and follicle size and serum E2 were monitored daily until oocyte retrieval was undertaken. Oocyte recovery was undertaken 34 h after the onset of the spontaneous LH surge, calculated as a rise of 180% above the baseline. At oocyte retrieval, blood free FF was collected and frozen at –70°C until assayed. Laboratory methods for sperm preparation, oocyte and embryo culture, and embryo transfer were performed as previously described (Wardle et al., 1985Go).

We selected FF from three patient groups in the above study. The groups were: (i) unfertilized; (ii) fertilized where the oocyte fertilized and a cleaving embryo was transferred but did not implant; and (iii) pregnant, where the oocyte fertilized and the cleaving embryo implanted and fetal heart pulsation was identified on transvaginal ultrasound.

Study 1
A comparison was made between nine patients who conceived, 21 patients whose oocyte fertilized but did not implant and 12 patients in whom the oocyte did not fertilize with respect to their intrafollicular cortisol concentrations. Having identified differences between the groups, we proceeded to a more detailed study of intrafollicular glucocorticoid concentrations.

Study 2
The nine patients who conceived were then matched with the next nine unfertilized and nine fertilized patients by date of sample collection. These 27 patients had follicular fluid cortisol and cortisone levels assayed and the intrafollicular cortisol:cortisone ratio calculated.

Assays
Cortisol assay
Cortisol assays were performed using a direct radioimmunoassay with sheep anti-cortisol anti-serum HPS-631-IG and a cortisol-3-(o-carboxymethyl) oxime-histamine-[125I] tracer (Moore et al., 1985Go). Cross-reactivity with cortisone was 1.2%, with progesterone <0.1% and with 17{alpha}-hydroxyprogesterone 2.4%.

Cortisone assay
Cortisone was extracted into chloroform and measured in the extracts using rabbit anti-cortisone antiserum N-137 and a 21 acetyl-cortisone-3-(o-carboxymethyl) oxime-histamine-[125I] tracer. Cross-reactivities of cortisol, progesterone and 17{alpha}-hydroxyprogesterone were each <0.1% (Wood et al., 1996Go).

Statistical analysis
As the cortisol and cortisone concentrations were not normally distributed the Mann–Whitney U-test was used for group comparison. Results are presented as medians and 95% confidence intervals (CI) (Minitab v.10 Minitab, Pennsylvania University, USA). P < 0.05 was considered statistically significant.


    Results
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Study 1
A significantly higher FF cortisol concentration was measured in follicles yielding an oocyte that fertilized and implanted compared with follicles whose oocyte fertilized but did not implant [median (95% CI): 299 (249–330) versus 227 nmol/l (185–261) respectively; P < 0.05] (Figure 1Go). When the data were pooled irrespective of the outcome of the oocyte for all 42 patients, eight of the nine conception cycles were associated with intrafollicular cortisol levels above the outcome independent mean of 246.7 nmol/l [8/21 (38.1%) versus 1/21 (4.8%) respectively; {chi}2 = 6.93, P = 0.008)].



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Figure 1. Total follicular fluid cortisol concentrations in unstimulated cycles related to oocyte fertilization and implantation. Bar represents median value. *P < 0.05

 
Study 2
The nine patients who conceived were matched with the next nine unfertilized and nine fertilized patients and the ratio of cortisol:cortisone calculated for each patient and related to the outcome measures (Figure 2Go). For these groups, the median age (range) was 29 (29–33), 29 (28–39) and 30 years (29–34) respectively.



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Figure 2. Preovulatory follicular fluid glucocorticoid concentrations related to result of oocyte retrieved from unstimulated cycle.(a) Cortisol, (b) cortisone, (c) ratio cortisol:cortisone. Bar represents median value. *P < 0.05; **P = 0.02.

 
A similar trend existed for differences in the cortisol concentrations between the pregnant and fertilized groups [median (95% CI): 319 (272–367) versus 263 nmol/l (152–320) respectively] but did not reach statistical significance (P = 0.07) reflecting the smaller numbers studied. Cortisone concentrations were significantly higher in the unfertilized group compared with the pregnant group [median (95% CI): 74 (51–158) versus 38 nmol/l (35–62) respectively; P < 0.05] with the fertilized group being intermediate at 51 nmol/l (26–80). No pregnancy occurred in the eight cycles with intrafollicular cortisone concentrations above the outcome independent mean of 73.1 nmol/l compared with 9/19 with cortisone levels below the mean cortisone value ({chi}2 = 5.67, P < 0.02).

The cortisol:cortisone ratio was significantly higher in the pregnant compared with the unfertilized group [7.38 (5.23–9.19) versus 3.56 (1.8–7.5); P = 0.02]. Overall conception occurred in seven of the 12 cycles (58.3%) with an intrafollicular cortisol:cortisone concentration above the outcome independent mean of 5.90 compared with 2/15 (13.3%) ({chi}2 = 6.02, P < 0.02)


    Discussion
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
This preliminary study in unstimulated cycles related the relative concentrations of cortisol and cortisone to the clinical endpoints of oocyte fertilization and implantation. Samples from completely unstimulated cycles are difficult to obtain in practice and hence our study was limited by relatively small numbers. However, natural cycle IVF provides a unique model whereby the intrafollicular glucocorticoid concentrations can be directly related to the outcome of the individual oocyte. We used a simple study design within a larger research programme and acknowledge that restricting the sample selection to nine in each outcome group reduced the power of the study.

Significantly higher intrafollicular cortisol:cortisone ratios have been reported in gonadotrophin-stimulated IVF conception cycles, but as multiple embryos were transferred in each patient it was not possible to determine which follicle or oocyte gave rise to the implanting embryo (Michael et al., 1999Go). High intrafollicular cortisol:cortisone ratios may reflect either low rates of cortisol oxidation, as previously correlated with conception by IVF–embryo transfer (Michael et al., 1993aGo), or high rates of cortisone reduction to cortisol. The cortisol:cortisone ratios measured in this study are similar to those reported in gonadotrophin-stimulated cycles (Michael et al., 1995Go). Furthermore, the intrafollicular cortisol:cortisone ratios are considerably lower than the typical plasma ratio of 10:1. This suggests that the ovarian isoforms of 11ß-HSD favour low levels of oxidation of cortisol to cortisone rather than the reduction of cortisone to cortisol. Higher cortisol levels were detected in follicles yielding mature compared with immature oocytes (Fateh et al., 1989Go), but in a further study no correlation existed between intrafollicular cortisol concentrations and oocyte maturity, oocyte fertilization or embryo cleavage (Andersen and Hornnes, 1994Go). Glucocorticoids in follicular fluid are derived from serum, there being no 21 hydroxylase present within the human ovary (Hillier, 1994Go). We are unable to comment on circulating levels as these were not measured in our study, but it is likely that the balance of intrafollicular cortisol and cortisone is determined by 11ß-HSD isoform expression.

Andersen et al. described the problems of measuring cortisol in FF with potential cross-reactivity from progestins in FF, particularly 17{alpha}-hydroxyprogesterone (Andersen et al., 1999Go). With 2.4% cross-reactivity in our assay, up to 12 nmol/l may be accounted for by 17{alpha}-hydroxyprogesterone, but this does not alter our findings of observed differences between groups.

Embryo implantation is an indirect indicator of oocyte quality as the study was restricted to young patients with favourable sperm function. Our finding of higher intrafollicular cortisol levels and successful implantation suggests that exposure to a higher cortisol concentration may be important for oocyte maturation. Cortisone concentrations were significantly higher in follicles where the oocyte failed to fertilize. Our observation is consistent with high levels of cortisol oxidation demonstrated in immature follicles where the measured 11ß-HSD activity increases cortisone levels (Yong et al., 2000Go).

Oocytes with reduced fertilization in vitro are a feature of minor endometriosis (Cahill and Hull, 2000Go) which is suggestive of a functional defect in the preovulatory follicle or oocyte. This condition is associated with significantly lower total intrafollicular cortisol levels, indicating a possible subtle disturbance of cortisol metabolism (Smith et al., 2002Go). We demonstrated an improvement in clinical outcome in gonadotrophin-stimulated IVF with dexamethasone co-treatment (Keay et al., 2001Go). Dexamethasone's mechanism of action is unclear and it is not known whether this observation represents a direct ovarian or a systemic effect of the synthetic glucocorticoid down-regulating endogenous glucocorticoids.

Progesterone is a potent inhibitor of 11ß-HSD activity and may influence 11ß-HSD isoform expression (Michael et al., 1993bGo) and therefore the relative concentrations of cortisol and cortisone within the follicle. We have not related our results to progesterone concentrations, but in earlier work we have demonstrated a significant rise in free cortisol concentration following the onset of the LH surge (Harlow et al., 1997Go). Whether this represents an alteration in cortisol metabolism or displacement of cortisol from cortisol-binding globulin by progesterone is not understood.

In summary, our data that suggest higher intrafollicular cortisol:cortisone ratios are associated with improved embryo implantation in completely unstimulated IVF cycles and that glucocorticoids may influence follicular development and oocyte maturation. Furthermore, the observation of significantly higher intrafollicular cortisone concentrations in unfertilized oocytes suggests relative immaturity of the oocytes and is consistent with reports of the predominance of type 2 11ß-HSD activity in immature follicles. The present study is preliminary and a further large study is necessary to verify our findings.


    Notes
 
5 To whom correspondence should be addressed. E-mail: d.j.cahill{at}bristol.ac.uk Back


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Submitted on January 11, 2002; resubmitted on April 4, 2002; accepted on May 8, 2002.