Predictive value of serum and follicular fluid leptin concentrations during assisted reproductive cycles in normal women and in women with the polycystic ovarian syndrome

Christos S.Mantzoros1,3, Daniel W.Cramer2, Rebecca F.Liberman2 and Robert L.Barbieri2

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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Leptin is an adipocyte-derived hormone which plays a central role in the regulation of body weight and energy homeostasis and in signalling to the brain that adequate energy stores are available for reproduction. Although leptin may affect reproduction by regulating the hypothalamic–pituitary–gonadal axis, recent in-vitro observations indicate that leptin may also have direct intra-ovarian actions. Leptin concentrations were measured in women who succeeded in becoming pregnant within three cycles of in-vitro fertilization (IVF) or gamete intra-fallopian transfer (n = 53), in women who failed to become pregnant within three cycles (n = 50), and in women with polycystic ovarian syndrome (PCOS) (n = 22). It was found that lower follicular fluid leptin concentrations were a marker of assisted reproduction treatment success in normal women. Women with PCOS had higher leptin concentrations than women without such a diagnosis, but this was due to their higher body mass index (BMI). After adjustment for age and BMI, women with PCOS who became pregnant tended to have lower mean follicular fluid leptin concentrations than women with PCOS who did not succeed at becoming pregnant. Further studies exploiting the strengths of the IVF model are needed to assess whether the prognostic role for follicular fluid leptin in human reproduction is independent of other factors, and to elucidate the underlying mechanisms.

Key words: assisted reproduction/fertility/follicular fluid/leptin/polycystic ovarian syndrome


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Leptin, the protein product of the obesity (ob) gene, is mainly expressed in adipocytes, and plays a central role in the regulation of body weight and energy homeostasis by signalling the amount of energy stores. In addition, several observations in animals and humans suggest that leptin plays a key role in signalling that adequate energy stores are available for reproduction. Congenital deficiency of leptin occurs in ob/ob mice who are obese and infertile, and leptin treatment of these mice increases luteinizing hormone (LH) concentrations and reproductive organ weights (Barash et al., 1996Go). Obesity and infertility also occur in db/db mice with defective leptin receptors (Houseknecht and Portocarrero, 1998Go). While only a distinct minority of human obesity is linked to leptin deficiency or leptin receptor mutations, individuals with leptin gene mutations are obese and remain prepubertal with hypogonadotrophic hypogonadism (Clement et al., 1998Go; Mantzoros, 1999Go). In general, however, obesity is a leptin-resistant state in humans such that higher leptin concentrations are found in more obese women, an observation which appears to account for the high leptin concentrations found in women with the polycystic ovarian syndrome (PCOS) (Brzechffa et al., 1996Go; Chapman et al., 1997Go; Laughlin et al., 1997Go; Mantzoros et al., 1997aGo; Rouru et al., 1997Go). At the other extreme, the adverse effects of food restriction and low weight on reproductive health might also be mediated through leptin. In normal prepubertal rodents, leptin administration is able partially to reverse the effect of food restriction in delaying pubertal maturation (Cheung et al., 1997Go). In anorexic women who are amenorrhoeic, leptin concentrations are low (Kopp et al., 1997Go), increase with feeding, and track well with LH and resumption of menstruation, when leptin concentrations exceed a threshold of about 2 ng/ml (Hebebrand et al., 1997Go; Audi et al., 1998Go; Ballauff et al., 1999Go).

Although it was originally thought that leptin affects reproduction by regulating the hypothalamic–pituitary–gonadal 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., 1997Go), 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, 1999Go) and affect IVF success (Van Voorhis et al., 1996Go), the study of the role of leptin in reproduction was restricted to women who were non-smokers.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Begun in 1994, the Boston Collaborative IVF study was a prospective study of couples undergoing the assisted reproduction techniques of IVF or GIFT at three infertility clinics in greater Boston (Boston IVF, the Brigham and Women's IVF Center, and the Reproductive Science Center of Boston) (Cramer et al., 1999Go). In a protocol approved by the Brigham and Women's Hospital Human Research Committee, information was obtained on epidemiological variables and details on each couple's treatment and outcome. Women were enrolled with their first IVF or GIFT cycle at one of these sites and followed until they became pregnant, or had completed up to six failed cycles. Approximately 65% of couples approached agreed to participate in the study. Medical and lifestyle information, including demographics, smoking history, anthropometry and duration of infertility, was collected using self-administered questionnaires. Body mass index (BMI) was calculated from weight (in kg) divided by height (in m2). Details of treatment were abstracted from the medical record, including: use of gonadotrophin hormone releasing agonists; dosage, type and duration of gonadotrophins used (most patients were on a long protocol); and details of cycle monitoring including oestradiol concentrations and follicle counts. The number of oocytes retrieved and fertilized was recorded, as was the number of embryos transferred. Although clinical pregnancy was defined as discharge from the IVF clinic with a viable pregnancy on ultrasound, all but one of the pregnancies in this series resulted in a liveborn.

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., 1997Go, 1998Go) 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., 1996Go), 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., 1999Go), 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, 1999Go) and affect IVF success (Van Voorhis et al., 1996Go), 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., 1997bGo). 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., 1999Go). `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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Values (means ± SE) for quantitative variables in the first IVF or GIFT cycle are shown in Table IGo. Age did not differ significantly among the four groups, although the women with PCOS tended to be younger. Not unexpectedly, BMI was highest among women with PCOS (P < 0.001 compared with all others). The oestradiol concentration, as measured in the pre-HCG serum, and number of oocytes retrieved were also highest in the PCOS group, although only the latter was statistically significant (P < 0.005 compared with all others). Neither the serum oestradiol concentration nor the number of oocytes retrieved differed significantly among the remaining three groups. Unadjusted follicular fluid leptin concentrations were lowest among women in the IVF success group (P = 0.01) and highest among women with PCOS (P = 0.01). Pre-HCG sera in the first cycle were less frequently available yielding a smaller number of subjects; however, in the groups with serum available, leptin concentrations followed a pattern similar to that for the follicular fluid.


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Table I. Means (± SE) baseline and treatment-related characteristics and leptin concentrations in categories of women undergoing assisted reproduction techniques (ART)
 
Excluding the high concentrations noted for women with PCOS, neither serum nor follicular fluid leptin concentrations varied significantly by primary infertility diagnosis; nor did the distribution of primary infertility diagnosis differ among the success, failure, or smoker groups (data not shown). In the three groups without PCOS, male factor and tubal factor were the most common primary infertility diagnoses. Finally, the amount of gonadotrophins used was similar in all groups except the women with PCOS, who required a lower amount of gonadotrophins, about six ampoules less. The difference was of borderline significance after adjustment for age and BMI.

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 IIGo. 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., 1999Go), this correlation may indicate that the same factors influencing serum leptin concentrations, such as oestrogens and insulin concentrations (Geisthovel et al., 1998Go; Messinis et al., 1999Go), may also influence follicular fluid leptin concentrations.


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Table II. Pearson correlationsa between age, BMI, serum oestradiol concentrations, number of oocytes retrieved, and serum and follicular fluid leptin concentrations
 
Table IIIGo shows t-tests for comparisons that were of a priority interest for serum and follicular fluid leptin concentrations between various groups. P-values based on non-paired t-tests are shown in the first column, with P-values adjusted for age and BMI in the second column. Age and BMI were considered the most important adjustment variables, given the importance of these variables in predicting both leptin concentrations and IVF success. Importantly, the assisted reproduction techniques success group had lower follicular fluid and serum leptin concentrations compared with the assisted reproduction techniques failure group; however, only the follicular fluid concentrations differed significantly and remained significant after adjusting for BMI and age (P = 0.017; Table IIIGo). In the IVF success subgroup of 26 women who succeeded in becoming pregnant in the first cycle, the follicular fluid leptin concentrations were also significantly lower compared with those in the failure group (11.9 versus 17.0 ng/ml, P = 0.011). This difference remained statistically significant after adjustment for age and BMI.


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Table III. Pairwise comparisons of follicular fluid and serum leptin concentrations in selected subgroups with and without adjustment for BMI and age
 
Table IIIGo also indicates that serum and follicular fluid leptin were significantly higher in the PCOS group compared with all `controls' (successes and failures combined) (P = 0.01 for follicular fluid leptin and P = 0.03 for serum leptin). However, neither of these differences persisted after adjustment for BMI and age as previously described (Brzechffa et al., 1996Go; Chapman et al., 1997Go; Laughlin et al., 1997Go; Mantzoros et al., 1997aGo; Rouru et al., 1997Go) indicating that, similarly to the serum leptin concentrations, the higher follicular fluid leptin concentrations were due to the higher BMI of these women. Serum and follicular fluid leptin concentrations were then compared in those women who succeeded and those who failed to become pregnant among women with PCOS. Similarly to normal women, there was a tendency for follicular fluid leptin, adjusted for age and BMI, to be lower for women with PCOS, who were successful in getting pregnant after assisted reproduction techniques (P = 0.16). Finally, follicular fluid and serum leptin concentrations were slightly higher in smokers, but did not differ significantly compared with controls in either adjusted or unadjusted analyses. Leptin concentrations were slightly (but not significantly) lower in current compared with past smokers (data not shown).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cloning of the leptin gene and its receptor has advanced our understanding of the communication between adipose tissue and the hypothalamic–pituitary–ovarian axis, but many details remain to be clarified. Leptin receptors have been identified in the primate hypothalamus and anterior pituitary (Finn et al., 1998Go), and leptin induces luteinizing hormone releasing hormone (LHRH) release from the hypothalamus, and LH and follicle stimulating hormone (FSH) from the anterior pituitary (Yu et al., 1997Go; Finn et al., 1998Go). Leptin concentrations are increased during a natural menstrual cycle, correlate with LH at several time points (Teirmaa et al., 1998Go), and are highest at the time of the LH surge (Messinis et al., 1998Go). In addition, studies employing frequent blood sampling indicate that leptin pulsatility correlates with LH pulsatility in normal humans (Licinio et al., 1998Go). These observations suggest a role for leptin in regulation of the hypothalamic–pituitary–gonadal axis (Mantzoros, 1999Go). However, leptin is itself influenced by gonadotrophins and/or the resulting changes of oestradiol concentrations. During treatment with a gonadotrophin releasing hormone agonist, for example, leptin is increased during the initial stimulatory phase and then decreased during the suppressive phase (Escobar-Morreale et al., 1997Go), and leptin concentrations are increased in women using exogenous oestrogens (Mantzoros, 1999Go). Thus, there appears to be a feedback loop linking the hypothalamic–pituitary–gonadal axis and the leptin system and, in addition, recent accumulating evidence also suggests that leptin may exert direct actions at the level of the ovaries (Mantzoros, 1999Go).

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., 1998Go; Teirmaa et al., 1998Go), 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., 1997Go). A similar inhibitory effect on insulin-induced oestradiol and progesterone production was observed in bovine ovaries (Spicer and Francisco, 1998Go). 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, 1997Go). 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., 1999Go). 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 concentrations—which were not measured in this study—are 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., 1998Go). 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., 1995Go; Antczak and Van Blerkom, 1999Go). 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 hormone–IGF-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
 
We would like to thank Drs Douglas R.Powers, Mark Hornstein and Patricia McShane for data collection and helpful discussions.


    Notes
 
3 To whom correspondence should be addressed at: Division of Endocrinology, Beth Israel Deaconess Medical Center, RN 325, 99 Brookline Ave, Boston, MA, USA Back


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 Discussion
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Submitted on August 11, 1999; accepted on December 1, 1999.