1 Department of Obstetrics and Gynecology, University of South Dakota School of Medicine, South Dakota, USA
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: body mass index/IVF outcome/leptin/predictive factors
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Several recent studies have investigated the impact of leptin in assisted reproduction cycles. For example, circulating leptin concentrations reportedly increase during gonadotrophin stimulation for IVF, apparently enhanced by the high oestradiol concentrations experienced during IVF cycles (Strowitski et al., 1998; Butzow et al., 1999
; Zhao et al., 2000
). It was shown in one study (Butzow et al., 1999
) that a large relative increase in leptin was associated with diminished ovarian response to gonadotrophin. Furthermore, leptin may influence outcomes from IVF. In a recent report (Mantzoros et al., 2000
), patients who became pregnant from IVF had lower mean follicular fluid concentrations of leptin than patients who did not become pregnant. Follicular fluid leptin concentrations have been negatively correlated with intrafollicular oxygen tension (Barroso et al., 1999
), which may negatively impact oocyte development and maturation. The objective of the present study was to determine whether IVF outcomes (e.g. fertilization, embryo development, implantation, and pregnancy) correlate with serum leptin concentration and/or BMI.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Assays
Serum leptin concentrations were measured by homologous radioimmunoassay using a human leptin radioimmunoassay kit (Linco Research Inc., St Charles, MO, USA) as previously described (Zhao et al., 2000). Duplicate 100 µl-volumes of serum were assayed for each sample. High and low end quality control specimens were placed at the beginning and the end of each assay. Inter- and intra-assay coefficients of variation were <8% at all concentrations. Serum oestradiol concentrations were measured using the DPC Immulite System® (Diagnostic Products Corp., Los Angeles, CA, USA) in a clinical laboratory according to standard protocol.
Data collection
Demographic and IVF cycle outcome data were recorded from patient charts and laboratory database. Data included female and male diagnoses, peak oestradiol concentrations, type of insemination (i.e. IVF or ICSI), number of mature (metaphase II) oocytes retrieved, fertilization and cleavage rates, embryo development on post- retrieval day 3 (mean number of blastomeres), number of superior quality embryos on day 3 post-retrieval (defined as eight blastomeres of equal size with good to excellent morphology), and blastocyst formation rate. Implantation rate was defined as the number of gestational sacs visible on ultrasound expressed per number of embryos transferred. In all cases, pregnancy rate is defined based on the presence of at least one gestational sac visible on ultrasound expressed per cycle (only cycles with a fresh embryo transfer were included in the study).
Statistical analyses
Unless otherwise indicated, all results are based on first attempt IVF cycles only. Thirty patients had repeat IVF cycles, and these were analysed separately. Statistical analyses were performed using SigmaStat 2.0 software (SPSS Inc., Chicago, IL, USA). Multiple logistic regression analyses were initially used to determine overall relationships among serum leptin concentration, BMI, and pregnancy rate. Based upon logistic regression analyses, patients were categorized by serum leptin:BMI ratio (see Results). Normally distributed parametric data were analysed by analysis of variance (ANOVA). Categorical data and parametric data that did not show normal distribution were analysed by 2 or KruskalWallis test as appropriate. When significance was found, ANOVA and KruskalWallis tests were followed by Duncan's or Dunn's post-hoc tests respectively. Significance was assumed at P
0.05 for all tests.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A negative relationship between follicular fluid concentrations of leptin and IVF pregnancy success was recently reported (Mantzoros et al., 2000). However, these authors found no association between pregnancy success and serum leptin concentrations, in contrast to the present data. This was somewhat surprising since serum and follicular fluid concentrations of leptin have been shown to be similar (Butzow et al., 1999
; Zhao et al2000
). Leptin was measured in serum at the end of gonadotrophin stimulation in the former study, whereas serum leptin was measured prior to stimulation in the present study. Serum leptin concentration has been reported to increase during gonadotrophin stimulation in association with rising oestradiol concentrations (Strowitski et al., 1998
; Zhao et al., 2000
). This may partly account for the difference in results between the two studies.
The present results demonstrate a stronger relationship between leptin and pregnancy success when leptin concentrations are expressed relative to BMI, i.e. the leptin:BMI ratio. The coefficient of determination (r2 = 0.546) for the linear regression analysis of leptin versus BMI indicates that only about half of the variability in leptin concentrations in this patient population was related to BMI. Therefore the stronger correlation between pregnancy success and leptin:BMI ratio may suggest that other determinants of leptin variability influence pregnancy outcome independently or interactively with leptin. When circumstances exist that elevate leptin concentrations more than would be predicted by body mass alone, fertility may be more greatly impacted. Conversely, when leptin concentrations are less than predicted by body mass, fertility may still be good even in obese patients. This is supported by the data in that several patients with BMI 30, i.e. obese, became pregnant if their serum leptin concentration was less than predicted by the regression equation. However, very few patients became pregnant when their baseline leptin was
30 ng/ml, even if their BMI was relatively low.
Many factors have been shown to influence leptin secretion in animal and human models, including insulin (Kolaczynski et al., 1996; Carmina et al., 1999
), and glucose metabolism (Chin-Chance et al., 2000
; Wellhoener et al., 2000
). Recent studies in human subjects (Chin-Chance et al., 2000
; Wellhoener et al., 2000
) have provided compelling evidence that energy balance, i.e. caloric intake, is a major determinant of leptin secretion, and affects circulating leptin concentrations more than serum insulin or glucose concentrations (Wellhoener et al., 2000
). It's possible that women may be able to improve their fertility through dietary and lifestyle modifications that lower circulating leptin concentrations.
High concentrations of oestrogen experienced during gonadotrophin stimulation cycles are associated with elevated leptin concentrations (Strowitzki et al., 1998; Zhao et al., 2000). In a preliminary study of 35 IVF cycles included in the final study, serum leptin was measured on the day of HCG administration. Pregnancy success appeared to be more closely associated with baseline leptin concentrations than with stimulated concentrations, although the two were very strongly correlated. Butzow et al. (1999) reported that a large relative increase in leptin concentrations during gonadotrophin stimulation was associated with diminished ovarian response (Butzow et al., 1999
). Pregnancy rates in our preliminary study were very similar in patients whose leptin increased by 20% or more (26/35; pregnancy rate = 30.8%) versus those whose leptin remained relatively unchanged (9/35; pregnancy rate = 33.3%; unpublished data).
It remains to be elucidated how elevated leptin concentrations negatively impact IVF outcomes. Since there was no effect of leptin on the number of mature oocytes retrieved, number of fertilized zygotes or fertilization rate, diminished outcomes in patients with high leptin:BMI ratios would appear to be a function of post-fertilization events (e.g. embryo development, ability of embryos to implant, or uterine receptivity), although these may be pre-determined if oocyte development or maturation are impaired. The fact that patients with a high leptin:BMI ratio had fewer superior quality embryos on day 3 post-retrieval, despite starting out with the same number of fertilized zygotes, suggests an effect on oocyte developmental competence and/or embryo development.
Several pieces of evidence suggest that leptin may affect folliculogenesis and thus oocyte development. Leptin can modulate follicular steroidogenesis (Karlsson et al., 1997; Spicer and Francisco, 1997
, 1998
; Zachow and Magoffin, 1997
; Brannian et al., 1999
; Kitawaki et al., 1999
) and a strong inverse correlation was demonstrated between follicular fluid leptin concentrations and intrafollicular oxygen concentration (Barroso et al., 1999
). Moreover, leptin may have direct receptor-mediated actions on developing oocytes. Although leptin receptor expression at the molecular concentration has apparently not yet been confirmed in human oocytes, mouse metaphase II (MII) and germinal vesicle (GV) stage oocytes express both mRNA and protein of the long isoform (OB-Rl) of the leptin receptor (Matsuoka et al., 1999
). Physiological concentrations of leptin resulted in tyrosine phosphorylation (i.e. activation) of STAT3 in mouse MII oocytes (Matsuoka et al., 1999
). Moreover, leptin and STAT3 exhibit polarized co-localization in human oocytes and early cleaving embryos (Cioffi et al., 1997
; Antczak and Van Blerkom, 1997
), and leptin induced activation of mitogen-activated protein kinase (MAPK) in a mouse embryonic cell line (Takahashi et al.1997
). Taken together, these results support the view that leptin is a physiological regulator of oocyte developmental competence acting indirectly via the follicle and directly via actions on the oocyte. It is therefore quite possible that elevated leptin concentrations might have a negative impact on oocyte developmental competence in IVF cycles.
Impaired uterine receptivity is also possible. Human endometrium expresses leptin receptor (Alfer et al., 2000; Kitawaki et al.2000
). Although the presence of leptin was not obligatory for implantation in mice (Mounzih et al., 1998
), the effect of high leptin on endometrial receptivity and implantation is unknown. In the present study, uterine receptivity was not directly evaluated. Endometrial thickness was not related to serum leptin concentration or leptin:BMI ratio, and the miscarriage rate was similar in all groups. Moreover, cleavage and implantation rates in frozen embryo transfer cycles were lower in women with high leptin:BMI in their fresh cycle, suggesting that either embryo quality was impaired or that endometrial receptivity was poor in both fresh and frozen embryo transfer cycles. The data would seem to favour an embryonic origin in explaining the reduced implantation associated with elevated leptin, but the effect of leptin on uterine receptivity and placentation requires investigation.
Data from repeat IVF cycles suggest that women with a low leptin:BMI ratio have a high probability of pregnancy success during second IVF attempts if their first cycle was unsuccessful. In contrast, women with a high leptin:BMI ratio had very poor success in repeat cycles. Thus the leptin:BMI ratio may be very useful in counselling patients considering repeat IVF attempts. Remarkably in the eight women who had repeat cycles in which their leptin:BMI ratio differed from their first cycle, pregnancy success appeared to be better in the cycles with the lower leptin:BMI ratio. Confirmation of this in a larger cohort of patients would provide hope that women may be able to improve their chances of pregnancy by lowering their leptin concentrations, possibly through dietary and lifestyle modifications, before repeated attempts at IVF.
An important consideration in interpreting the results of this study is that only single, non-fasting measurements of serum leptin were made during each IVF cycle. Leptin variability derives primarily from body fat mass and exhibits a wide range of physiological concentrations from 12 ng/ml up to well over 50 ng/ml. Within the context of the intersubject variation, leptin concentrations within an individual vary over time, dependent upon circadian and ultradian patterns of secretion and fed state. Although serum samples were drawn at the same time of day (08001000 h), patients were not instructed to fast. In general, the normal within-individual diurnal variations are of a much smaller magnitude than the inter-individual variation determined by significant differences in body adiposity. The relative fluctuation between minimum and maximum daily concentrations appears to be somewhere between 21% (Schoeller et al., 1997) and about 50% (Boden et al., 1997
; van Aggel-Leijssen et al., 1999
). Considine et al. (1996) reported no significant change in serum leptin before or after regular meals (Considine et al., 1996
), although in a larger study leptin increased on average about 25% within 6 h following a high calorie meal (Dallongeville et al., 1998
). Leptin secretion also follows ultradian pulsatile patterns in men and women (Licinio et al., 1998
). Leptin pulse amplitude in women, although greater than in men, is generally <50% (Licinio et al., 1998
; Sir-Peterman et al., 1999
). Mean baseline non-fasting serum leptin concentrations in low versus high leptin:BMI groups in the present study population differed by nearly five-fold. Variations in leptin concentrations due to secretory pulsatility or pre- versus post-prandial conditions cannot account for the large differences between patient groups. Moreover, all serum specimens were drawn between 0800 and 1000 h, which is the time of day when circulating leptin concentrations would be expected to be at their nadir (Schoeller et al., 1997
; van Aggel-Leijssen et al., 1999
). Therefore it seems highly unlikely that diurnal variation in leptin concentrations had a significant impact on the correlation between leptin:BMI and implantation and pregnancy outcomes observed in the present study. Nevertheless confirmation of these results in a controlled prospective study comparing IVF outcomes with fasting leptin concentrations is warranted.
There are other limitations to this study. For example, not all patients received the same type of gonadotrophin and dosages varied, although the three leptin:BMI groups were proportionate for each type and duration of stimulation. Moreover, some patients had blastocyst transfers (day 5 or 6) whereas most had day 3 embryo transfers. Trends in pregnancy rates among leptin:BMI patient groups were not altered by gonadotrophin type or day of embryo transfer, although the limited data suggests the possibility of a greater impact of elevated leptin on implantation and pregnancy outcomes in blastocyst transfers.
In summary, baseline non-fasting serum leptin:BMI ratio appears to be highly predictive of IVF outcomes. The data suggest that elevated leptin has a negative impact on oocyte and/or embryo quality leading to impaired implantation and reduced probability of pregnancy success. Confirmation of this relationship may assist clinicians in counselling patients and improving the success of assisted reproduction.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antczak, M. and Van Blerkom, J. (1997) Oocyte influences on early development: the regulatory proteins leptin and STAT3 are polarized in mouse and human oocytes and differently distributed within the cells of the pre-implantation stage embryo. Mol. Hum. Reprod., 3, 10671086.[Abstract]
Barroso, G., Barrionuevo, M., Rao, P. et al. (1999) Vascular endothelial growth factor, nitric oxide, and leptin follicular fluid concentrations correlate negatively with embryo quality in IVF patients. Fertil. Steril., 72, 10241026.[ISI][Medline]
Boden, G., Chen, X., Kolaczynski, J.W. and Polansky, M. (1997) Effects of prolonged hyperinsulinemia on serum leptin in normal human subjects. J. Clin. Invest., 100, 11071113.
Brannian, J.D., Zhao, Y. and McElroy, M. (1999) Leptin inhibits gonadotrophin-stimulated granulosa cell progesterone production by antagonizing insulin action. Hum. Reprod., 14, 14451448.
Butzow, T.L., Moilanen, J.M., Lehtovirta, M. et al. (1999) Serum and follicular fluid leptin during in vitro fertilization: relationship among leptin increase, body fat mass, and reduced ovarian response. J. Clin. Endocr. Metab., 84, 31353139.
Carmina, E., Ferin, M., Gonzalez, F. et al. (1999) Evidence that insulin and androgens may participate in the regulation of serum leptin concentrations in women. Fertil. Steril., 72, 926931.[ISI][Medline]
Chin-Chance, C., Polonsky, K.S. and Schoeller, D.A. (2000) Twenty-four-hour leptin concentrations respond to cumulative short-term energy imbalance and predict subsequent intake. J. Clin. Endocr. Metab., 85, 26852691.
Cioffi, J.A., Van Blerkom, J., Antczak, M. et al. (1997) The expression of leptin and its receptor in pre-ovulatory human follicles. Mol. Hum. Reprod., 3, 467472.[Abstract]
Clarke, I.J., and Henry, B.A. (1999) Leptin and reproduction. Rev. Reprod., 4, 4855.
Considine, R.V., Sinha, M.K., Heiman, M.L. et al. (1996) Serum immunoreactive-lepti concentrations in normal-weight and obese humans. N. Engl. J. Med., 334, 324325.
Dallongeville, J., Hecquet, B., Lebel, P. et al. (1998) Short term response of circulating leptin to feeding and fasting in man: influence of circadian cycle. Int. J. Obes. Relat. Metab. Disord., 22, 728733.[Medline]
Jacobs, H.S. and Conway, G.S. (1999) Leptin, polycystic ovaries and polycystic ovary syndrome. Hum. Reprod. Update, 5, 166171.
Karlsson, C., Lindell, K., Svensson, E. et al. (1997) Expression of functional leptin receptors in the human ovary. J. Clin. Endocr. Metab., 82, 41444148.
Kitawaki, J., Koshiba, H., Ishihara, H. et al. (2000) Expression of leptin receptor in human endometrium and fluctuation during the menstrual cycle. J. Clin. Endocr. Metab., 85, 19461950.
Kitawaki, J., Kusuki, I. Koshiba, H. et al. (1999) Leptin directly stimulates aromatase activity in human luteinized granulosa cells. Mol. Hum. Reprod., 5, 708713.
Kolaczynski, J.W., Nyce, M.R., Considine, R.V. et al. (1996) Acute and chronic effects of insulin on leptin production in humans: studies in vivo and in vitro. Diabetes, 5, 699701.
Licinio, J., Negrao, A.B., Mantzoros, C. et al. (1998) Sex differences in circulating human leptin pulse amplitude: clinical implications. J. Clin. Endocr. Metab., 83, 41404147.
Mantzoros, C.S., Cramer, D.W., Liberman, R.F. et al. (2000) Predictive value of serum and follicular fluid leptin concentrations during assisted reproductive cycles in normal women and women with the polycystic ovarian syndrome. Hum. Reprod., 15, 539544.
Matsuoka, T., Tahara, M., Yokoi, T et al. (1999) Tyrosine phosphorylation of STAT3 by leptin through leptin receptor in mouse metaphase 2 stage oocyte. Biochem. Biophys. Res. Comm., 256, 480484.[ISI][Medline]
Mounzih, K., Qiu, J. Ewart-Toland, A. et al. (1998) Leptin is not necessary for gestation and parturition but regulates maternal nutrition via a leptin resistance state. Endocrinology, 139, 52595262.
Schoeller, D.A., Cella, L.K., Sinha, M.K. and Caro, J.F. (1997) Entrainment of the diurnal rhythm of plasma leptin to meal timing. J. Clin. Invest., 100, 18821887.
Sir-Peterman, T., Maliqueo, M., Palomino, A. et al. (1999) Episodic leptin release is independent of luteinizing hormone secretion. Hum. Reprod., 14, 26952699.
Spicer, L.J. and Francisco, C.C. (1997) The adipose gene product, leptin: evidence of a direct inhibitory role in ovarian function. Endocrinology, 138, 33743379.
Spicer, L.J. and Francisco, C.C. (1998) Adipose gene product, leptin, inhibits bovine ovarian thecal cell steroidogenesis. Biol. Reprod., 58, 207212.[Abstract]
Strowitski, T., Kellerer, M., Capp, E. et al. (1998) Increase in serum leptin concentration in women undergoing controlled ovarian hyperstimulation for assisted reproduction. Gynecol. Endocrinol., 12, 167169.[ISI][Medline]
Takahashi, Y., Okimura, Y., Mizuno, I. et al. (1997) Leptin induces mitogen-activated protein kinase-dependent proliferation of C3H10T1/2 cells. J. Biol. Chem., 272, 1289712900.
van Aggel-leijssen, D.P., van Baak, M.A., Tenebaum, R. et al. (1999) Regulation of average 24 h human plasma leptin concentration; the influence of exercise and physiological changes in energy balance. Int. J. Obes. Relat. Metab. Disord., 23, 151158.[Medline]
Wellhoener, P., Fruehwald-Schultes, B., Kern, W. et al. (2000) Glucose metabolism rather than insulin is a main determinant of leptin secretion in humans. J. Clin. Endocr. Metab., 85, 12671271.
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 oestradiol-17ß production by rat ovarian granulosa cells. Endocrinology, 138, 847850.
Zhao, Y., Kreger, D.O., Brannian, J.D. (2000) Serum leptin concentrations in women during gonadotrophin stimulation cycles. J. Reprod. Med., 45, 121125.[ISI][Medline]
Submitted on February 28, 2001; accepted on June 5, 2001.