1 Division of Endocrinology, Department of Internal Medicine, Beth Israel Deaconess Medical Center, Boston, MA and 2 Obstetrics and Gynecology-Epidemiology Center, Brigham and Women's Hospital, Boston, MA, USA
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: assisted reproduction/fertility/follicular fluid/leptin/polycystic ovarian syndrome
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although it was originally thought that leptin affects reproduction by regulating the hypothalamicpituitarygonadal axis, recent in-vitro observations indicate that leptin may also have direct intra-ovarian actions. In addition, although a previous study has shown that follicular fluid leptin may be associated with implantation potential (Cioffi et al., 1997), no previous study has examined the role of leptin in predicting successful pregnancy. These in-vitro observations have yet to be confirmed in vivo, however. Using specimens obtained as part of a collaborative study of predictors of success after in-vitro fertilization (IVF) or gamete intra-fallopian transfer (GIFT), serum and follicular fluid leptin concentrations were compared in three groups. Concentrations of leptin were examined in women who succeeded in becoming pregnant within three cycles of IVF or GIFT, in women who failed to become pregnant within three cycles, and in women with PCOS. Because there is some evidence that smoking may both decrease leptin (Mantzoros, 1999
) and affect IVF success (Van Voorhis et al., 1996
), the study of the role of leptin in reproduction was restricted to women who were non-smokers.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Biological specimens obtained from each patient included serum samples that were collected as part of monitoring the ovarian stimulation regimen, and follicular fluid that was collected at the time of oocyte retrieval. Since leptin is secreted in a pulsatile fashion and has a diurnal rhythm (Licinio et al., 1997, 1998
) a morning blood sample was withdrawn wherever possible in this study to minimize diurnal variation. Leptin concentrations are not influenced significantly by a preceding meal (Considine et al., 1996
), and thus samples in this study were not necessarily fasting samples. Only the last serum obtained immediately before administration of human chorionic gonadotrophin (pre-HCG specimen) was saved. This specimen generally correlated with the highest oestradiol concentration during the cycle as measured at the clinic laboratories. Follicular fluid was also collected at the time of oocyte retrieval. To avoid contamination with the flushing medium, only fluid from the first aspiration of the dominant follicle was saved. This meant that it was not possible to assay leptin concentrations in individual follicles and correlate it with oocyte quality. Follicular fluid was inspected visually, and no visible contamination was detected.
From the 1244 couples enrolled in the study and observed for a total of 2895 cycles, four groups were selected which were defined as follows. The `assisted reproduction techniques success' group included women who succeeded in becoming pregnant within three cycles, as previously described (Hall et al., 1999), had a follicular fluid collected in their first cycle, and excluded women who had PCOS or were smokers. From 360 potentially eligible women, a random sample of 60 was chosen. Of these women, 53 actually had a sufficient amount of follicular fluid left available for analysis. The `assisted reproduction techniques failure' group included women who failed to become pregnant after three cycles, had a follicular fluid collected in their first cycle, and excluded women who had PCOS or were heavy smokers. Three months was chosen as the time interval because most women would quit after failing three times. From 294 potentially eligible women, a random sample of 60 was selected; among these women, 50 had sufficient follicular fluid for analysis. The `PCOS' group included all 22 women who had follicular fluid available and who were diagnosed as having a primary or secondary infertility diagnosis of PCOS. Because there is some evidence that smoking may both decrease leptin (Mantzoros, 1999
) and affect IVF success (Van Voorhis et al., 1996
), a fourth group was added to the study to evaluate the potential effect of smoking on follicular fluid leptin concentrations. The `smokers' group included women who were current or former smokers and who had accumulated more than 10 pack-years of smoking, but excluded women who had PCOS. From 157 potential women in this group, a random sample of 60 was selected, of whom 48 actually had sufficient follicular fluid for analysis. The `PCOS' and `smokers' groups included both women who had succeeded and who had failed after assisted reproduction techniques. Sufficient residual pre-HCG serum was available in 35 of the women in the successful group, 34 in the failure group, 16 in the PCOS group, and 38 in the smokers group.
The pre-HCG serum oestradiol concentrations and oocyte count were obtained from the treatment abstract. Leptin concentrations were analysed in the pre-HCG serum specimen using a competitive radioimmunoassay as described previously (Mantzoros et al., 1997b). Because it was not clear which cycle should be chosen for the `assisted reproduction techniques failure' group, and in order to minimize the effect of multiple IVF cycles on leptin concentrations, the first cycle specimens were chosen for each group. However, a separate analysis was performed on the subgroup of women who had `successful assisted reproduction techniques' and who conceived in their first cycle. Thus, leptin was measured in the serum specimen immediately preceding the ovulatory stimulus and in follicular fluid from the lead follicle, as performed in studies of similar design in the past (Hall et al., 1999
). `Smokers' and women with PCOS were excluded from both the `assisted reproduction techniques success' and the `assisted reproduction techniques failure' groups to allow these latter two groups to be combined as a comparison group for the `smokers' and `PCOS' groups. Differences in leptin concentrations and other quantitative variables among women in the various groups were analysed by means of t-tests and generalized linear models. All analyses were performed using the SAS system (SAS Institute, Cary, NC, USA).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Correlations between age, BMI, oestradiol, number of oocytes retrieved, serum and follicular fluid leptin concentrations and number of ampoules of gonadotrophins used are shown in Table II. Several of the parameters of interest were interrelated by univariate analysis. Age was inversely correlated with the pre-HCG serum oestradiol, number of oocytes recovered and serum leptin (borderline statistical significance) and was positively correlated with the amount of gonadotrophins required. BMI was strongly and positively correlated with follicular fluid and serum leptin. Serum oestradiol was strongly correlated with oocytes recovered, and both oestradiol and oocytes were negatively correlated with the amount of gonadotrophins administered. No significant correlations were observed between serum or follicular fluid leptin and serum oestradiol, number of oocytes retrieved, or amount of gonadotrophins administered. However, the follicular fluid and the serum leptin concentrations were highly correlated (r = 0.83). Although a recent study has shown a decrease in serum leptin concentrations in normal premenopausal women following bilateral ovariectomy (Messinis et al., 1999
), this correlation may indicate that the same factors influencing serum leptin concentrations, such as oestrogens and insulin concentrations (Geisthovel et al., 1998
; Messinis et al., 1999
), may also influence follicular fluid leptin concentrations.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
During assisted reproduction, the pituitary is bypassed. In the present study of women undergoing assisted reproduction, the focus was on whether follicular fluid and/or serum leptin concentrations predict successful pregnancy outcome. It was found that women who succeeded in becoming pregnant within three cycles of IVF or GIFT had significantly lower leptin concentrations in follicular fluid compared with women who failed to become pregnant within three cycles, even after adjustment for age and BMI. During a normal cycle, leptin is normally highest at the time of the LH surge (Messinis et al., 1998; Teirmaa et al., 1998
), and in this study leptin was measured in follicular fluid obtained at the time of oocyte retrieval, after HCG had been administered (equivalent to the LH surge). Since virtually all women undergoing IVF in this study received a standard 10 000 unit dose of HCG, it seems unlikely that differences in leptin concentrations were due to an effect of chorionic gonadotrophins. Moreover, the fact that leptin concentrations were lower in women with subsequently successful assisted reproduction suggests that factors operating independent of gonadotrophin administration are of major importance.
Thus, it is necessary to address the effect of leptin at the ovarian level to explain why follicular fluid leptin was inversely correlated with assisted reproduction techniques success. Leptin receptors are expressed in human ovaries, and leptin has been shown to inhibit LH as well as insulin-like growth factor (IGF)-I-stimulated oestradiol production in granulosa/thecal cells (Karlsson et al., 1997). A similar inhibitory effect on insulin-induced oestradiol and progesterone production was observed in bovine ovaries (Spicer and Francisco, 1998
). Other data in rodents suggest that leptin does not interfere with FSH-induced oestradiol production, but does block the additive effect of IGF-I on FSH-induced oestradiol production (Zachow and Magoffin, 1997
). Since leptin does not alter the effect of IGF-I on progesterone production, it has been suggested that leptin interferes primarily with aromatase expression or activity in humans (Agarwal et al., 1999
). Taken together, these data indicate that high leptin concentrations in the ovary may suppress oestradiol production and interfere with the development of dominant follicles and oocyte maturation. Although no correlation was found between either serum or follicular fluid leptin concentration and peak serum oestradiol during the assisted reproduction techniques cycle, it may be that the follicular fluid oestradiol concentrationswhich were not measured in this studyare much more important than serum oestradiol concentrations, as suggested by the above studies, though this remains to be shown by future studies.
This study confirmed that women with PCOS have higher leptin concentrations than women without such a diagnosis, and that this association appears to be due to the positive correlation between leptin and BMI. It was found that women with PCOS who became pregnant had lower mean follicular fluid leptin concentrations than those with PCOS who did not succeed at becoming pregnant (P = 0.16) after adjustment for age and BMI. The relatively smaller number of patients in the PCOS success and failure groups could explain the fact that although the difference of mean leptin concentrations in these two groups was similar in magnitude to that observed in normal women, this difference did not achieve statistical significance in the PCOS groups. Thus, the prognostic significance of follicular fluid leptin may also pertain in women with PCOS although future larger studies with a larger number of subjects would be necessary to prove this hypothesis at the conventional P = 0.05 level of statistical significance.
Another, secondary focus of this study was the possible association between smoking and follicular fluid leptin (Mantzoros et al., 1998). In this study it was found that leptin concentrations in current and former heavy smokers undergoing assisted reproduction techniques were not different compared with non-smokers. However, the inclusion of past heavy smokers with the current smokers may have diluted any effect of current smoking on leptin and gonadotrophin administration and thus may have altered any association between smoking and leptin. This remains to be tested by future studies.
This study has several limitations, including the focus on reproductive success in non-smokers only, which may affect the generalizability of these findings. None of these limitations however could have affected the validity of the data. As women with fertility problems sufficient to require IVF were studied, our results may not be generalizeable to spontaneous ovulatory cycles in normal women. Thus, the role of leptin in normal reproduction needs to be studied by similar studies in the future. With regard to the prognostic significance of serum or follicular fluid leptin on IVF success, specimens were used from the first IVF cycle, even though conception may have occurred in a subsequent cycle for those women who eventually succeeded. However, when analysis was restricted to the cycle from which follicular fluid had been obtained, the results were almost identical, revealing a role for leptin in predicting successful outcome. In addition, no difference between first cycle and second and third cycle pregnancies was detected. Thus, the fact that only first cycle follicular fluid leptin was measured reinforces the potential value of leptin as a long-term predictor of success during an IVF treatment course. The distinct possibility exists that the negative effect of leptin may be even more prevalent in natural cycles, but this remains to be shown. Although this study extends previous in-vitro data and indicates for the first time a role for follicular fluid leptin as a predictor of pregnancy success in humans, it does not provide information regarding the potential mechanisms through which leptin may operate. Although, it would be reasonable to speculate that the same mechanisms shown in the in-vitro studies may also operate in humans, direct measurements of other follicular fluid hormonal factors, such as androgens, oestrogens and growth factors, by future studies is warranted. Another potential mechanism could be a direct leptin action on the developing embryo (Van Blerkom et al., 1995; Antczak and Van Blerkom, 1999
). Finally, leptin concentrations measured in this study were drawn irrespective of leptin's pulsatile secretion and to a certain extent irrespective of the timing of the meals. Potential misclassification due to these factors would be non-differential misclassification that would tend to depress the effect estimates towards the null, thus making it more difficult to achieve statistical significance.
In summary, it was shown that low follicular fluid leptin concentrations were a marker of success after IVF. Future studies exploiting the strengths of the IVF model will be useful in further delineating the important role for follicular fluid leptin in normal and assisted human reproduction. The interactions of follicular fluid leptin concentrations with sex steroids, including but not limited to oestradiol and the growth hormoneIGF-I system, as well as the possible direct and independent actions of leptin in the ovaries, require further study in order to elucidate the mechanisms underlying those observations made in this study.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antczak, M. and Van Blerkom, J. (1999) Temporal and spatial aspects of fragmentation in early human embryos: possible effects on developmental competence and association with the differential elimination of regulatory proteins from polarized domains. Hum. Reprod., 14, 429447.
Audi, L., Mantzoros, C.S., Vidal-Puig, A. et al. (1998) Leptin in relation to the resumption of menses in women with anorexia nervosa. Mol. Psychiatry, 3, 544547.[ISI][Medline]
Ballauff, A., Ziegler, A., Emons, G. et al. (1999) Serum leptin and gonadotropin levels in patients with anorexia nervosa during weight gain. Mol. Psychiatry, 4, 7175.[ISI][Medline]
Barash, I.A., Cheung, C.C., Weigle, D.S. et al. (1996) Leptin is a metabolic signal to the reproductive system. Endocrinology, 137, 31443147.[Abstract]
Brzechffa, P.R., Jakimiuk, A.J., Agarwal, S.K. et al. (1996) Serum immunoreactive leptin concentrations in women with polycystic ovary syndrome. J. Clin. Endocrinol. Metab., 81, 41664169.[Abstract]
Chapman, I.M., Wittert, G.A. and Norman, R.J. (1997) Circulating leptin concentrations in polycystic ovary syndrome: relation to anthropometric and metabolic parameters. Clin. Endocrinol. (Oxf.), 46, 175181.[ISI][Medline]
Cheung, C.C., Thornton, J.B., Kuijper, J.L. et al. (1997) Leptin is a metabolic gate for the onset of puberty in the female rat. Endocrinology, 138, 855858.
Cioffi, J.A., Van Blerkom, J., Antczak, M. et al. (1997) The expression of leptin and its receptors in pre-ovulatory human follicles. Mol. Hum. Reprod., 3, 467472.[Abstract]
Clement, K., Vaisse, C., Lahlou, N. et al. (1998) A mutation in the human leptin receptor gene causes obesity and pituitary dysfunction. Nature, 292, 398401.
Considine, R.V., Sinha, M.K., Heiman, M.L. et al. (1996) Serum immunoreactive-leptin concentrations in normal-weight and obese humans. N. Engl. J. Med., 334, 292295.
Cramer, D.W., Powers, D.R., Oskowitz, S.P. et al. (1999) Gonadotropin releasing hormone agonist use in assisted reproduction cycles: the influence of long and short regimens on pregnancy rates. Fertil. Steril., 72, 8389.[ISI][Medline]
Escobar-Morreale, H.F., Serrnao-Gotarredona, J. and Varela, C. (1997) Circulating leptin concentrations in women with hirsutism. Fertil. Steril., 68, 898906.[ISI][Medline]
Finn, P.D., Cunningham, M.J., Pau, K.Y. et al. (1998) The stimulatory effect of leptin on the neuroendocrine reproductive axis of the monkey. Endocrinology, 139, 46524662.
Geisthovel, F., Meysing, A. and Brabant, G. (1998) C-peptide and insulin, but not C19-steroids, support the predictive value of body mass index on leptin in serum of premenopausal women. Hum. Reprod., 13, 547553.[Abstract]
Hall, J.E., Welt, C.K. and Cramer, D.W. (1999) Inhibin A and Inhibin B reflect ovarian function in assisted reproduction but are less useful at predicting outcome. Hum. Reprod., 14, 409415.
Hebebrand, J., Blum, W.F., Barth, N. et al. (1997) Leptin levels in patients with anorexia nervosa are reduced in the acute stage and elevated upon short-term weight restoration. Mol. Psychiatry, 2, 330334.[ISI][Medline]
Houseknecht, K.L. and Portocarrero, C.P. (1998) Leptin and its receptors: regulators of whole-body energy homeostasis. Domest. Anim. Endocrinol., 15, 457475.[ISI][Medline]
Karlsson, C., Lindell, K., Svensson, E. et al. (1997) Expression of functional leptin receptors in the human ovary. J. Clin. Endocrinol. Metab., 82, 41444148.
Kopp, W., Blum, W.F., von Prittwita, S. et al. (1997) Low leptin levels predict amenorrhea in underweight and eating disordered females. Mol. Psychiatry, 2, 335340.[ISI][Medline]
Laughlin, G.A., Morales, A.J. and Yes, S.S. (1997) Serum leptin levels in women with polycystic ovary syndrome: the role of insulin resistance/hyperinsulinemia. J. Clin. Endocrinol. Metab., 82, 16921696.
Licinio, J., Mantzoros, C., Negrao, A.B. et al. (1997) Human leptin levels are pulsatile and inversely related to pituitary-adrenal function. Nature Med., 3, 575579.[ISI][Medline]
Licinio, J., Negrao, A.B., Mantzoros, C. et al. (1998) Synchronicity of frequently sampled, 24-h concentrations of circulating leptin, luteinizing hormone, and estradiol in healthy women. Proc. Natl Acad. Sci. USA, 95, 25412546.
Mantzoros, C.S. (1999) The role of leptin in human obesity and disease: a review of current evidence. Ann. Intern. Med., 130, 871881.
Mantzoros, C.S., Dunaif, A. and Flier, J.S. (1997a) Leptin concentrations in the polycystic ovary syndrome. J. Clin. Endocrinol. Metab., 82, 16871691.
Mantzoros, C.S., Flier, J.S., Lesem, M.D. et al. (1997b) Cerebrospinal fluid leptin in anorexia nervosa: correlation with nutritional status and potential role in resistance to weight gain. J. Clin. Endocrinol. Metab., 82, 18451851.
Mantzoros, C.S., Liolios, A.D., Tritos, N.A. et al. (1998) Circulating insulin concentrations, smoking, and alcohol intake are important independent predictors of leptin in young healthy men. Obes. Res., 6, 179186.[Abstract]
Messinis, I.E., Milingos, S., Zikopoulos, K. et al. (1998) Leptin concentrations in the follicular phase of spontaneous cycles and cycles superovulated with follicle stimulating hormones. Hum. Reprod., 13, 11521156.[Abstract]
Messinis, I.E., Milingos, S.D., Alexandris, E. et al. (1999) Leptin concentrations in normal women following bilateral ovariectomy. Hum. Reprod., 14, 913918.
Rouru, J., Anttila, L., Koskinen, P. et al. (1997) Serum leptin concentrations in women with polycystic ovary syndrome. J. Clin. Endocrinol. Metab., 82, 16971700.
Spicer, L.J. and Francisco, C.C. (1998) Adipose obese gene product, leptin, inhibits bovine ovarian thecal cell steroidogenesis. Biol. Reprod., 58, 207212.[Abstract]
Teirmaa, T., Luukkaa, V., Rouru, J. et al. (1998) Correlation between circulating leptin and luteinizing hormone during the menstrual cycle in normal-weight women. Eur. J. Endocrinol., 139, 190194.[ISI][Medline]
Van Blerkom, J., Davis, P., Merriam, J. et al. (1995) Nuclear and cytoplasmic dynamics of sperm penetration, pronuclear formation and microtubule organization during fertilization and early preimplantation development in the human. Hum. Reprod. Update, 1, 429461.[Abstract]
Van Voorhis, B.J., Dawson, J.D., Stovall, D.W. et al. (1996) The effects of smoking on ovarian function and fertility during assisted reproduction cycles. Obstet. Gynecol., 88, 785791.
Yu, W.H., Walczewska, A., Karanth, S. et al. (1997) Nitric oxide mediates leptin-induced luteinizing hormone-releasing hormone (LHRH) and LHRH and leptin-induced LH release from the pituitary gland. Endocrinology, 138, 50555058.
Zachow, R.J. and Magoffin, D.A. (1997) Direct intraovarian effects of leptin: impairment of the synergistic action of insulin-like growth factor I on follicle stimulating hormone dependent estradiol 17 beta production by rat ovarian granulosa cells. Endocrinology, 69, 847850.
Submitted on August 11, 1999; accepted on December 1, 1999.