The concentration of inhibin B in follicular fluid: relation to oocyte maturation and embryo development

Chia-Lin Chang, Tzu-Hao Wang, Shang-Gwo Horng, Hsien-Ming Wu, Hsin-Shih Wang,1 and Yung-Kuei Soong

Department of Obstetrics and Gynaecology, Chang-Gung Memorial Hospital, Lin-Kou Medical Centre, Taipei, Taiwan, Republic of China


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: The objective of the present study was to investigate the correlation between inhibin B and estradiol levels in follicular fluid (FF) with the quality of subsequent embryo development from in-vitro fertilized oocytes aspirated from the same follicle. METHODS: A total of 156 infertile women undergoing controlled ovarian stimulation for IVF and embryo transfer was recruited to the present study. Prospectively, 233 FF samples and matched mature oocytes were studied. Concentrations of inhibin B and estradiol were determined by enzyme-linked immunosorbent assay (ELISA) and immunofluorometric assay (IFMA) respectively. RESULTS: Inhibin B levels in FF were significantly correlated with embryo scores on days 2 and 3 (48 and 72 h after oocyte retrieval). In contrast, both inhibin B and estradiol levels in FF were inversely related to age. Furthermore, FF inhibin B levels were inversely associated with serum FSH levels on day 3 of the menstrual cycle, which was believed to reflect the ovarian reserve. CONCLUSION: Inhibin B in FF may serve as an effective marker of follicular development and a useful predictor of quality of embryo. In addition, quality of oocyte is age-related and declines as age increases.

Key words: embryo score/estradiol/granulosa cell/inhibin B/oocyte maturation


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Inhibins are members of the transforming growth factor-ß (TGF-ß) superfamily (Massague, 1990Go; Kingsley, 1994Go). Structurally, inhibins are heterodimeric glycoprotein with a molecular weight of ~31–32 kDa. Bioactive inhibins consist of an {alpha}-subunit and one of two ß-subunits, ßA or ßB, linked by disulphide bonds (inhibin A = {alpha}ßA and inhibin B = {alpha}ßB) (Groome et al., 1996Go). Physiologically, inhibins are secreted by the granulosa cells of ovarian follicles and selectively inhibit the pituitary FSH secretion (Klein et al., 1996Go). In follicular phase, inhibin B is secreted from developing preantral and small antral follicles by the stimulation of FSH (Burger et al., 1998Go; Fraser et al., 1999Go), and further enhanced by the combination of FSH and insulin-like growth factor-I (IGF-I) (Welt and Schneyer, 2001Go). In contrast, inhibin A is not secreted from preantral follicles, but in small antral follicles under the stimulation of FSH (Welt and Schneyer, 2001Go). Collectively, inhibin B appears to play a more important paracrine role in developing follicles and a greater regulatory role with respect to FSH secretion than inhibin A (Magoffin and Jakimiuk, 1997Go).

In women with ovulatory cycles, the circulating inhibin B levels increase slowly and steadily in the early follicular phase, and reach a peak in the mid-follicular phase, then decrease progressively in the late follicular phase before ovulation. Shortly after the LH surge, there is a short-lived peak in the circulating inhibin B, which in turn declines to a low concentration for the remainder of the luteal phase (Groome et al., 1996Go). In contrast, the inhibin A levels are low in the early follicular phase, rise at the pre-ovulatory phase, and reach a peak in the mid-luteal phase (Groome et al., 1996Go; Wang et al., 2000Go).

Previous studies have shown that serum inhibin B is believed to be of predictive value in monitoring ovarian stimulation treatment for IVF (Hayes et al., 1998Go; Eldar-Geva et al., 2000Go). In addition, serum inhibin A may also act as a useful marker for monitoring the effects of gonadotrophin stimulation (Lockwood et al., 1996Go). Furthermore, concentrations of inhibin in FF, though not specified inhibin A or B, are greater in the fertilized group as compared with the unfertilized (Fowler et al., 1995Go), and are suggested to be used as an index of follicular maturation (Franchimont et al., 1990Go). However, the relationship between inhibin levels in follicular fluid (FF) and the quality of oocytes and embryos has not yet been explored.

In the work reported here, FF and matched oocytes were collected simultaneously to study further the correlation among the inhibin B and estradiol levels in FF as well as the embryo development. In addition, the association between oocyte quality and age was also investigated.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Subjects and protocols of ovulation induction
A total of 156 infertile women undergoing controlled ovarian stimulation for IVF was recruited in the present study. Indications for IVF included tubal absence or occlusion, ovulation disorders, endometriosis and unexplained infertility, whereas polycystic ovarian syndrome (PCOS) and male factors were excluded. Age of patients ranged from 25–41 years (mean age, 35.4). Body mass index [weight (kg) divided by height squared (m)] was 23.7 ± 3.8 (range, 19.1–28.7) kg/m2. All patients gave written informed consent to participate in the study, and the experimental design was approved by the local ethics committee.

The protocol for controlled ovarian stimulation included the pituitary down-regulation with subcutaneous injection of GnRH agonist (1 mg/day, Lupron®; Abbott Laboratories, North Chicago, IL, USA) started on the 21st day of the previous menstrual cycle. Subcutaneous administration of recombinant human (r-hFSH) (150–450 IU/day, Gonal-F®; Laboratories Serono SA, Aubonne, Switzerland) was initiated on the third day of the menstruation, based on the women's age and previous response of ovulation induction. HCG (10 000 IU, Pregnyl®; NV Organon, Oss, The Netherlands) was applied i.m. 34–36 h before oocyte retrieval, and ovulation induction was monitored by transvaginal sonography.

During oocyte retrieval, FF without contamination of blood and matched oocyte from each single follicle were collected individually. Follicle aspirates which were not clear and contaminated with blood were excluded. Oocytes were evaluated, immediately after retrieval, for nuclear maturity and graded as metaphase II, metaphase I, or prophase I. FF was stored at -70°C until subsequent assays for inhibin B and estradiol.

Laboratory evaluation of embryos
Oocytes were cultured in Petri dishes in IVF-20® (Vitrolife, Göteborg, Sweden) at 37°C in a humidified 5% CO2/95% air environment. The semen was processed using 80% Percoll (Sigma Chemicals, St Louis, MO, USA) discontinuous gradient centrifugation at 800 g for 15 min. Insemination with 10 000– 15 000 progressively motile sperm for each oocyte was performed 4–6 h after oocyte retrieval. Following IVF, the resulting embryos were cultured in IVF-20® at 37°C under 5% CO2 in air.

Quality of embryo was assessed by the embryo score described previously (Steer et al., 1992Go). Briefly, a quality score of each embryo on days 2 and 3 was produced by the morphological grade of the embryo multiplied by the number of blastomeres. Morphologically, embryos were graded as follows: grade 4, equal sized symmetrical blastomeres; grade 3, uneven blastomeres with <10% extracellular fragmentation; grade 2, uneven blastomeres with 10–50% extracellular fragmentation; grade 1, >50% blastomeric fragmentation with unequal sized blastomeres.

Enzyme-linked immunosorbent assay (ELISA) for inhibin B
Concentrations of inhibin B in FF were determined by a commercial ELISA kit (Serotec, Oxford, UK). The assay employed the quantitative sandwich enzyme immunoassay technique. Samples of FF were diluted 1:500 in inhibin-free fetal calf serum prior to measurement. The minimum detection limit of assay was 15.6 pg/ml. The intra-assay coefficients of variation were 7.7% at 75.2 pg/ml, and 6.4% at 185 pg/ml (n = 8). The interassay coefficients of variation were 8.6% at 68.3 pg/ml and 7.3% at 196 pg/ml (n = 6).

Immunofluorometric assays (IFMA) for estradiol and FSH
Estradiol levels in FF were measured by immunofluorometric assays (Pharmacia, Turku, Finland). The detection limit of the estradiol assay was 13.6 pg/ml. The intra-assay coefficients of variation were 7.2% at 152 pg/ml and 5.3% at 446 pg/ml (n = 16). The interassay coefficients of variation were 9.1% at 163 pg/ml and 7.5% at 465 pg/ml (n = 12).

Serum levels of FSH on day 3 of the menstrual cycle were determined by immunofluorometric assays (Pharmacia, Turku, Finland). The detection limit of the estradiol assay was 0.5 mIU/ml. The intra-assay coefficients of variation were 6.3% at 2.4 mIU/ml and 7.1% at 6.6 mIU/ml (n = 8). The interassay coefficients of variation were 8.5% at 2.5 mIU/ml and 9.7% at 7.5 mIU/ml (n = 6).

Statistical analysis
In order to explore the relationship among inhibin B and estradiol in FF, embryo scores on days 2 and 3, as well as age, analysis using linear regression was employed. In all cases, a P value < 0.05 was considered statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
A total number of 306 FF samples without contamination of blood and matched oocytes with mature nuclei at metaphase II was collected. After IVF, 233 fertilized oocytes with two pronuclei were obtained (fertilization rate 76%). Thereafter, 54 fertilized oocytes failed to develop into embryos before day 2 (48 h after oocyte retrieval) and another 20 embryos were characterized by arrested growth between days 2 and 3 (48–72 h after oocyte retrieval), resulting in 179 and 159 embryos studied on days 2 and 3 respectively.

Inhibin B levels in FF were significantly related to embryo scores on days 2 and 3 (n = 179, P < 0.000 001 and n = 159, P < 0.000 1 respectively) (Figure 1Go). Notably, inhibin B levels in FF from which 54 oocytes failed to develop into embryos were relatively low [49.1 ng/ml (median), ranged from 10.9–103.8 ng/ml], although they were overlapped with those from which developed into embryos. In FF, inhibin B showed a positive correlation with estradiol (P < 0.02) (Figure 2Go). Nevertheless, no association was found between estradiol levels in FF and embryo scores on days 2 and 3 (Figure 3Go).



View larger version (18K):
[in this window]
[in a new window]
 
Figure 1. The relationship between (a) inhibin B levels in follicular fluid (FF) and embryo score on day 2 (48 h after oocyte retrieval) (n = 179), and (b) inhibin B levels in FF and embryo score on day 3 (72 h after oocyte retrieval) (n = 159). d.f. represents degree of freedom.

 


View larger version (20K):
[in this window]
[in a new window]
 
Figure 2. The correlation between inhibin B and estradiol levels in follicular fluid (FF). d.f. was 231, analysed by lineal regression.

 


View larger version (20K):
[in this window]
[in a new window]
 
Figure 3. The relationship between (a) estradiol levels in follicular fluid (FF) and embryo score on day 2 (48 h after oocyte retrieval) (n = 179), and (b) estradiol levels in FF and embryo score on day 3 (72 h after oocyte retrieval) (n = 159). NS = not significant.

 
In FF, both inhibin B and estradiol levels showed an inverse correlation with age (P < 0.02 and P < 0.05 respectively) (Figure 4a,bGo). In addition, there was an inverse association between inhibin B levels in FF and serum concentrations of FSH on the third day (day 3) of the menstrual cycle (P < 0.0001) (Figure 5aGo). On the contrary, no relation was found between estradiol levels in FF and serum FSH levels on day 3 of the menstrual cycle (Figure 5bGo).



View larger version (22K):
[in this window]
[in a new window]
 
Figure 4. The association between (a) inhibin B levels in follicular fluid (FF) and age, and (b) estradiol levels in FF and age. d.f. was 231, analysed by lineal regression.

 


View larger version (23K):
[in this window]
[in a new window]
 
Figure 5. The relationship between (a) inhibin B levels in follicular fluid (FF) and serum FSH levels on the third day (day 3) of the menstrual cycle, and (b) estradiol levels in FF and serum FSH levels on the day 3 of the menstrual cycle. d.f. was 231, analysed by lineal regression.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The present results, for the first time, demonstrated that inhibin B levels in FF were significantly correlated with embryo scores on days 2 and 3 (P < 0.000001 and P < 0.0001 respectively) (Figure 1Go), indicating the close relationship between inhibin B and embryo quality. Physiologically, the combination of circulating inhibin B and estradiol signals to the hypothalamus/pituitary to suppress pituitary FSH secretion, leading to an important advantage of the dominant follicle over the cohort follicles. In regularly cycling women, inhibin B levels in FF elevate rapidly and progressively as follicle diameter increases to 13 mm and decrease in follicles 15–17 mm in diameter (Magoffin and Jakimiuk, 1997Go) followed by a short-lived peak at the time of ovulation (Groome et al., 1996Go). It is also evident that serum concentrations of inhibin B are significantly higher on the day of follicle aspiration in patients who have a greater number of oocytes retrieved (Enskog et al., 2000Go). In addition, changes in FF inhibin B levels correlate closely with the pattern of circulating inhibin B (Groome et al., 1996Go). Thus, it would be attractive to speculate that, clinically, FF inhibin B may reflect the ovarian response and further predict the quality of embryo in women undergoing stimulation of ovulation for IVF programmes.

A previous study failed to show the relationship between inhibin A and B levels in FF and the quality of oocytes and found little effect on oocyte quality of fertilization (Lau et al., 1999Go). In the present study, although FF inhibin B did not correlate with oocyte quality, it did so with the quality of embryos which were fertilized (Figure 1Go). This might suggest that some other variable is dominating the actual fertilizability of the oocytes, but that oocytes made in a follicle with well-functioning granulosa cells, once fertilized, are better fitted to start embryonic development.

Reproductive capacity in women is believed to be age-related and declines dramatically beyond the fourth decade of life. In this respect, oocyte quality is most likely the primary determinant of reproductive potential (Klein and Sauer, 2001Go). Results from the present study showed that concentrations of both inhibin B and estradiol in FF were inversely associated with age (P < 0.02 and P < 0.05 respectively) (Figure 4a,bGo). In addition, inhibin B levels in FF appeared to be an indicator of oocyte quality, since they were significantly correlated with embryo scores on days 2 and 3 (P < 0.000001 and P < 0.0001 respectively) (Figure 1Go). Collectively, these findings demonstrate a notion that oocyte quality declines as the age of women progresses.

Serum FSH levels on day 3 of the menstrual cycle, but not inhibin B, are believed to be an indicator of ovarian reserve (Corson et al., 1999Go). It is also evident that age and day 3 FSH, rather than inhibin B, are the stronger predictors for successful pregnancy (Creus et al., 2000Go). In addition, follicular phase inhibin B secretion is decreased in older ovulatory women (aged 40–45 years) who demonstrate a monotrophic FSH rise, implying a diminished follicular pool in older women (Klein et al., 1996Go). Moreover, decreased serum inhibin B throughout the menstrual cycle is suggested to be an early marker of the decline in follicle number across reproductive ageing (Welt et al., 1999Go). In the present study, an inverse correlation was observed between day 3 serum FSH and inhibin B levels in FF (P < 0.0001) (Figure 5aGo), further suggesting that the age-related decline in oocyte quality can be reflected by inhibin B levels in FF at the time of oocyte aspiration.

Ontogenically, inhibin is a product of granulosa cells and serves as a protein marker of granulosa-cell function (McLachlan et al., 1986Go). This is further confirmed by a previous study demonstrating that inhibins A and B levels in FF increase as follicles grow and develop (Magoffin and Jakimiuk, 1997Go). In contrast, production of estradiol in the human ovary is through a co-operation of theca and granulosa cells (Erickson, 1996Go). In clinical practice, serum or plasma estradiol is an established variable of follicular development. A close correspondence has been reported between circulating inhibin and estradiol concentrations in women undergoing ovulation induction in the IVF programme (McLachlan et al., 1986Go), suggesting that both circulating inhibin and estradiol can be used as a monitoring indicator of ovarian response during ovulation induction.

Although serum estradiol has been used to monitor the follicular growth during controlled ovarian stimulation, estradiol levels in FF may not play a role in predicting the quality of oocytes and embryos. This is supported by the results from the present study that estradiol levels in FF showed no relation to embryo scores on both days 2 and 3 (Figure 3Go). In Figure 2Go, there was a positive association between FF inhibin B levels and estradiol concentrations (P < 0.02), suggesting that both inhibin B and estradiol in FF reflect the follicular function. However, as described previously, inhibin is a product of granulosa cells and estradiol is predominantly a theca-cell product (McLachlan et al., 1986Go) (Erickson, 1996Go). Thus, unlike inhibin B, FF estradiol may not be a strong indicator for predicting the embryo quality.

In conclusion, inhibin B in FF may reflect the ovarian response and serve as an effective marker of follicular development and quality of oocyte/embryo during controlled ovulation stimulation. We also demonstrate, deduced from the present results, that quality of oocyte is age-related and declines as age increases.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors are indebted to Drs Chi-Long Lee, Chun-Kai Chen, Chia-Wei Wang and Hong-Yuan Huang for providing invaluable clinical samples. We also acknowledge the contribution of Mei-Li Wang, Chieh-Yu Lin and Yi-Ju Tsai for their excellent technical assistance. This study has been supported by the Chang Gung Memorial Hospital to T.H.W. (CMRP-890) and the National Science Council, Taiwan, to T.H.W. (NSC89-2314-B-182A-103, NSC90-2314-B-182A-020, NSC90-2314-B-182A-150) and to H.S.W. (NSC89-2314-B-182-041, NSC89-2314-B-182–142, NSC89-2314-B-182–077, NSC90-2314-B-182–040, NSC90-2314-B-182–107).


    Notes
 
1 To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, Medical School of Chang-Gung University, Chang-Gung Memorial Hospital, Lin-Kou Medical Centre, Taipei, Taiwan, Republic of China. E-mail: hswang86{at}ms17.hinet.net Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Burger, H.G., Groome, N.P. and Robertson, D.M. (1998) Both inhibin A and B respond to exogenous follicle-stimulating hormone in the follicular phase of the human menstrual cycle. J. Clin. Endocrinol. Metab., 83, 4167–4169.[Abstract/Free Full Text]

Corson, S.L., Gutmann, J., Batzer, F.R., Wallace, H., Klein, N. and Soules, M.R. (1999) Inhibin-B as a test of ovarian reserve for infertile women. Hum. Reprod., 14, 2818–2821.[Abstract/Free Full Text]

Creus, M., Penarrubia, J., Fabregues, F., Vidal, E., Carmona, F., Casamitjana, R., Vanrell, J.A. and Balasch, J. (2000) Day 3 serum inhibin B and FSH and age as predictors of assisted reproduction treatment outcome. Hum. Reprod., 15, 2341–2346.[Abstract/Free Full Text]

Eldar-Geva, T., Robertson, D.M., Cahir, N., Groome, N., Gabbe, M.P., Maclachlan, V. and Healy, D.L. (2000) Relationship between serum inhibin A and B and ovarian follicle development after a daily fixed dose administration of recombinant follicle-stimulating hormone. J. Clin. Endocrinol. Metab., 85, 607–613.[Abstract/Free Full Text]

Enskog, A., Nilsson, L. and Brannstrom, M. (2000) Peripheral blood concentrations of inhibin B are elevated during gonadotrophin stimulation in patients who later develop ovarian OHSS and inhibin A concentrations are elevated after OHSS onset. Hum. Reprod., 15, 532–538.[Abstract/Free Full Text]

Erickson, G.F. (1996). Physiologic basis of ovulation induction. Semin. Reprod. Endocrinol., 14, 287–297.[ISI][Medline]

Fowler, P.A., Fahy, U., Culler, M.D., Knight, P.G., Wardle, P.G., McLaughlin, E.A., Cunningham, P., Fraser, M., Hull, M.G. and Templeton, A. (1995) Gonadotrophin surge-attenuating factor bioactivity is present in follicular fluid from naturally cycling women. Hum. Reprod., 10, 68–74.[Abstract]

Franchimont, P., Hazee-Hagelstein, M.T., Charlet-Renard, C., Jaspar, J.M., Hazout, A., Salat-Baroux, J., Schatz, B. and Demerle, F. (1990) Correlation between follicular fluid content and the results of in vitro fertilization and embryo transfer. II. Inhibin and aromatase inhibitor activity. J. Clin. Endocrinol. Metab., 71, 748–754.[Abstract]

Fraser, H.M., Groome, N.P. and McNeilly, A.S. (1999) Follicle-stimulating hormone-inhibin B interactions during the follicular phase of the primate menstrual cycle revealed by gonadotropin-releasing hormone antagonist and antiestrogen treatment. J. Clin. Endocrinol. Metab., 84, 1365–1369.[Abstract/Free Full Text]

Groome, N.P., Illingworth, P.J., O'Brien, M., Pai, R., Rodger, F.E., Mather, J.P. and McNeilly, A.S. (1996) Measurement of dimeric inhibin B throughout the human menstrual cycle. J. Clin. Endocrinol. Metab., 81, 1401–1405.[Abstract]

Hayes, F.J., Hall, J.E., Boepple, P.A. and Crowley, W.F. Jr (1998) Clinical review 96: differential control of gonadotropin secretion in the human: endocrine role of inhibin. J. Clin. Endocrinol. Metab., 83, 1835–1841.[Free Full Text]

Kingsley, D.M. (1994) The TGF-beta superfamily: new members, new receptors, and new genetic tests of function in different organisms. Genes Dev., 8, 133–146.[ISI][Medline]

Klein, J. and Sauer, M.V. (2001) Assessing fertility in women of advanced reproductive age. Am. J. Obstet. Gynecol., 185, 758–770.[ISI][Medline]

Klein, N.A., Illingworth, P.J., Groome, N.P., McNeilly, A.S., Battaglia, D.E. and Soules, M.R. (1996) Decreased inhibin B secretion is associated with the monotropic FSH rise in older, ovulatory women: a study of serum and follicular fluid levels of dimeric inhibin A and B in spontaneous menstrual cycles. J. Clin. Endocrinol. Metab., 81, 2742–2745.[Abstract]

Lau, C.P., Ledger, W.L., Groome, N.P., Barlow, D.H. and Muttukrishna, S. (1999) Dimeric inhibins and activin A in human follicular fluid and oocyte–cumulus culture medium. Hum. Reprod., 14, 2525–2530.[Abstract/Free Full Text]

Lockwood, G.M., Muttukrishna, S., Groome, N.P., Knight, P.G. and Ledger, W.L (1996) Circulating inhibins and activin A during GnRH-analogue down-regulation and ovarian hyperstimulation with recombinant FSH for in-vitro fertilization-embryo transfer. Clin. Endocrinol. (Oxf.), 45, 741–748.[ISI][Medline]

Magoffin, D.A. and Jakimiuk, A.J. (1997) Inhibin A, inhibin B and activin A in the follicular fluid of regularly cycling women. Hum. Reprod., 12, 1714–1719.[Abstract]

Massague, J. (1990) The transforming growth factor-beta family. Annu. Rev. Cell Biol., 6, 597–641.[ISI]

McLachlan, R.I., Robertson, D.M., Healy, D.L., de Kretser, D.M. and Burger, H.G. (1986) Plasma inhibin levels during gonadotropin-induced ovarian hyperstimulation for IVF: a new index of follicular function? Lancet, i, 1233–1234.

Steer, C.V., Mills, C.L., Tan, S.L., Campbell, S. and Edwards, R.G. (1992) The cumulative embryo score: a predictive embryo scoring technique to select the optimal number of embryos to transfer in an in-vitro fertilization and embryo transfer programme. Hum. Reprod., 7, 117–119.[Abstract]

Wang, H.S., Wang, T.H. and Soong, Y.K. (2000) Cyclic changes in serum levels of insulin-like growth factor binding protein-1 in women treated with clomiphene citrate and tamoxifen. Gynecol. Endocrinol., 14, 236–244.[ISI][Medline]

Welt, C.K., McNicholl, D.J., Taylor, A.E. and Hall, J.E. (1999) Female reproductive aging is marked by decreased secretion of dimeric inhibin. J. Clin. Endocrinol. Metab., 84, 105–111.[Abstract/Free Full Text]

Welt, C.K. and Schneyer, A.L. (2001) Differential regulation of inhibin B and inhibin A by follicle-stimulating hormone and local growth factors in human granulosa cells from small antral follicles. J. Clin. Endocrinol. Metab., 86, 330–336.[Abstract/Free Full Text]

Submitted on January 9, 2002; accepted on March 11, 2002.