Leptin during assisted reproductive cycles: the effect of ovarian stimulation and of very early pregnancy

L. Unkila-Kallio1, S. Andersson1,2, H.A. Koistinen3, S.-L. Karonen4, O. Ylikorkala1 and A. Tiitinen1,5

1 Departments of Obstetrics and Gynaecology, 2 Pediatrics, 3 Medicine and 4 Clinical Chemistry, Helsinki University Central Hospital, Haartmaninkatu 2, FIN-00290 Helsinki, Finland


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Leptin may have a role in human reproduction. The impact of IVF and of very early pregnancy on serum leptin concentrations was studied in 66 infertile patients, of whom 19 became pregnant. Ovarian suppression was accompanied by a fall in leptin concentrations (21 ± 4%, mean ± SE; P < 0.01) from the mid-luteal phase, and ovarian stimulation by a rise (76 ± 8%; P < 0.0001) from suppression. The mid-luteal concentration of leptin after stimulation was 28 ± 7% higher than that during the preceding normal cycle (P < 0.001). Concentrations of leptin and oestradiol were related before treatment, at ovarian suppression and at 8 days after oocyte retrieval. In addition, the rises in leptin and oestradiol concentrations during stimulation were correlated, but only in those patients who became pregnant (r = 0.69; P = 0.001). Women with a successful pregnancy had higher concentrations of leptin (18.7 ± 4.8 µg/l) at 12 days after embryo transfer than those who had miscarriages (10.0 ± 1.9 µg/l; P < 0.001), or those failing to become pregnant (11.6 ± 1.2 µg/l; P < 0.0001). We concluded that leptin concentrations are influenced by ovarian function and that the relationship between leptin and oestrogen (but not a single leptin concentration), may be an important factor for the outcome of IVF.

Key words: GnRH agonist/gonadotrophin/IVF/oestradiol/progesterone


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Leptin, the product of the ob gene, primarily regulates energy homeostasis (Zhang et al., 1994Go). However, increasing evidence indicates that leptin may also act as a regulator in reproduction (Barash et al., 1996Go; Macut et al., 1998Go; Cunningham et al., 1999Go; Foster and Nagatani, 1999Go; Messinis and Milingos, 1999Go). This is also supported by data showing that congenital leptin deficiency in humans is associated with hypogonadotrophic hypogonadism (Strobel et al., 1998Go; Farooqi et al., 1999Go). A role for leptin in ovarian physiology is substantiated by the production of leptin (Antczak et al., 1997Go), and the presence of its receptors in human granulosa cells (Cioffi et al., 1997Go; Karlsson et al., 1997Go) and by the connections between leptin and oestrogen (Zachow and Magoffin, 1997Go; Agarwal et al., 1999Go), LH/human chorionic gonadotrophin (HCG) (Karlsson et al., 1997Go), and progesterone concentrations (Brannian et al., 1999Go) in human granulosa cells. In vivo, serum leptin concentrations rise during the follicular phase and reach their peak during the luteal phase of the spontaneous cycle (Hardie et al., 1997Go; Shimizu et al., 1997Go; Mannucci et al., 1998Go; Messinis et al., 1998Go; Riad-Gabriel et al., 1998Go). Cyclical changes in leptin have been attributed to the effect of oestrogen, a fact also supported by higher concentrations of leptin in women than in men (Saad et al., 1997Go). With regard to the role of leptin in ovarian disorders, e.g. polycystic ovarian disease, the data are inconsistent (Brzechffa et al., 1996Go; Caro, 1997Go; Rouru et al., 1997Go; El Orabi et al., 1999Go; Jacobs and Conway, 1999Go; Sir-Petermann et al., 1999Go). In contrast, previous data uniformly show that ovarian stimulation as a part of IVF programme is associated with elevated leptin concentrations (Butzow et al., 1997Go, 1999Go; Mannucci et al., 1998Go; Messinis et al., 1998Go; Strowitzki et al., 1998Go; Stock et al., 1999Go; Lindheim et al., 2000Go), whereas few data exist on leptin during the suppression phase of IVF (Lindheim et al., 2000Go). In addition, nothing is known of leptin during very early pregnancy, although placental cells are capable of producing leptin (Masuzaki et al., 1997Go; Senaris et al., 1997Go). Therefore, we studied the impact of an ovarian suppression–stimulation programme on leptin in infertile women who either did or did not become pregnant.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Study subjects
With the approval of the local Institutional Review Committee, 66 Caucasian women aged 23–41 years who were otherwise healthy but complained of primary (57%) or secondary (43%) infertility lasting for 2–16 years were enrolled in the study. The patients and their partners were thoroughly investigated (indices of ovulation, transvaginal ultrasonography, laparoscopy with assessment of tubal patency, semen analysis) for the cause of infertility. Of the patients, 29 had tubal occlusion, nine patients had moderate to severe endometriosis, and 28 patients were infertile due to some unexplained cause(s). A total of 18 patients (29%) were smokers. Body mass index (BMI, kg/m2) was calculated, and `underweight' was defined as BMI <=19.4 kg/m2, `normal weight' as BMI 19.5–26.4 kg/m2, and `overweight' as BMI >=26.5kg/m2 (Bianco et al., 1998; see Table IGo).


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Table I. Means (± SE) baseline and treatment-related characteristics and outcome of study patients undergoing an IVF treatment
 
The IVF programme was started with pituitary down-regulation; a gonadotrophin-releasing hormone (GnRH) agonist (nafarelin, Synarela®; Syntex, Södertälje, Sweden, 400 µg twice a day) was administered as a nasal spray starting in the mid-luteal phase (cycle day 22.3 ± 0.3). Two weeks later, after onset of withdrawal bleeding, ovarian suppression was confirmed by the following criteria: (i) all ovarian follicles were <10 mm in diameter; (ii) the thickness of the endometrial double layer was <5 mm; and/or (iii) the serum concentration of oestradiol was <100 pmol/l. Four patients had to continue the down-regulation for an additional week, one patient for 3 weeks, and one patient for 4 weeks, before the criteria for suppression were met. Ovarian stimulation was initiated by giving 150–225 IU daily of either human menopausal gonadotrophins (HMG) (Humegon®; Organon, Oss, The Netherlands, n = 15; or Pergonal®; Laboratories Serono S.A., Aubonne, Switzerland, n = 19, containing equal amounts of FSH and LH) or of purified urinary FSH (containing only FSH) (Fertinorm HP®; Laboratories Serono S.A., n = 14; or Follegon®; Organon, n = 18). Concomitantly with the start of the gonadotrophin regimen, the GnRH dosage was halved and continued until the HCG injection (10 000 IU, Pregnyl®; Organon). Ovarian stimulation was monitored by assessment of serum oestradiol concentration, and the dosage of gonadotrophin was adjusted accordingly. The injection of HCG took place if at least three follicles were >16 mm in diameter. Oocyte retrieval was carried out through transvaginal aspiration 36 h after HCG injection. Ova were fertilized in vitro, and a maximum of two embryos were transferred 2 (n = 48) or 3 days (n = 11) after oocyte retrieval. Fertilization failed to produce any embryos in three patients, and embryo transfer was not carried out in an additional four women because of the signs of ovarian hyperstimulation syndrome (OHSS; serum oestradiol >=1500 pmol/l and the presence of more than 20 follicles). Thus, embryo transfer was carried out in 59 patients. After embryo transfer, daily micronized progesterone (600 mg, Lugesteron®; Leiras, Turku, Finland) was given intravaginally. Clinical pregnancy (= no bleeding, with consistently rising HCG concentrations) was achieved in 18 women (BMI range 17.9–37.6 kg/m2) of whom 13 carried to term. A marked (>50 IU/l), but transient rise, in HCG 12 days after embryo transfer followed by a profuse bleeding soon thereafter was defined as a subclinical abortion.

Serum samples
Blood samples were collected in the morning, after an overnight fast. Sample I was collected in the mid-luteal phase before the start of pituitary down-regulation (cycle day 22.3 ± 0.3, n = 66) (= `basal concentration'); sample II, collected in the presence of ovarian suppression (n = 66); sample III, collected in the presence of ovarian stimulation, 1–2 days before HCG injection/3–5 days before oocyte retrieval (n = 62); sample IV, collected at the time of oocyte retrieval (n = 66); sample V, collected 8 days after oocyte retrieval (n = 64); sample VI, collected 14 days after oocyte retrieval (n = 60). Sera separated by centrifugation (2000 rpm for 15 min) were kept frozen (–20°C) until analysis.

Assays
All serum samples were assayed for leptin with radioimmunoassay (Linco Research, St Charles, MO, USA) (Ma et al., 1996Go). The detection limit of this assay was 0.5 µg/l, and intra- and inter-assay coefficients of variation were 4.7 and 2.6% respectively at low concentrations (2.8 µg/l), and 3.8 and 2.2% respectively at medium concentrations (15.6 µg/l). Serum samples I–V were also analysed for oestradiol using radioimmunoassay (Spectria® direct estradiol [125I], and serum samples IV and V for progesterone, Spectria® progesterone [125I]; Orion diagnostica, Espoo, Finland). In addition, serum samples I and II were measured for LH and serum samples I–VI for HCG by time-resolved immunofluorometric assays (IFMA; Delfia®; Wallac, Turku, Finland).

Statistical analyses
Leptin concentrations were normally distributed (Kolmogorov Smirnov Normality Test), whereas the distributions of other hormones were skewed; therefore these data were subject to log10-transformation. However, all data are given as arithmetic mean (± SE). {chi}2 test, and analysis of (co)variance (AN(C)OVA), using BMI as a co-variate for leptin data) and analysis of variance (ANOVA) for repeated measurements (BMI groups as a factor for leptin data) were used when appropriate. The differences found in ANOVA were tested with Fisher's protected least squares difference (PLSD) post-hoc test. Simple regression (relations between relative changes in leptin and other hormones) and multiple regression (to assess the effect of BMI on the relationship between the concentrations of leptin and steroid hormones) were used to analyse the effects between continuous variables. P < 0.05 was considered to be statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In the whole series, basal leptin concentrations were correlated with BMI (r = 0.85, P < 0.0001). This concentration of leptin in patients who were underweight was lower (6.4 ± 0.8 µg/l, range 3.1–11.9 µg/l) than that in those of normal weight (12.0 ± 0.7 µg/l, range 5.0–25.3 µg/l, P < 0.01), or in those who were overweight (25.7 ± 2.8 µg/l, range 15.5–39.3 µg/l, P < 0.0001). Moreover, the difference in leptin between normal and overweight groups was significant (P < 0.0001). Basal leptin concentrations in unexplained infertility (12.1 ± 1.5 µg/l), in endometriosis (13.1 ± 3.0 µg/l), and in tubal occlusion (14.0 ± 1.3 µg/l) were similar. Neither age (<=35 years or >35 years), primary or secondary character of infertility, nor smoking (yes/no) were significant factors for basal leptin concentrations. In addition to BMI, oestradiol was an independent factor for basal leptin by multiple regression analysis (r = 0.16, P = 0.01).

Ovarian suppression was associated with a fall of 21 ± 4% in leptin (P < 0.01) (Figure 1Go). This leptin fall did not correlate with falls in LH (46 ± 5 %) or oestradiol (91 ± 1%). Relatively, the fall in leptin seemed higher in patients with normal weight (21 ± 4%) and with overweight (26 ± 7%) than in the underweight patients (12 ± 10%), but no statistical difference emerged between these changes. In the presence of the lowest oestradiol concentrations, oestradiol was positively correlated with leptin (r = 0.20, P = 0.01).



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Figure 1. Mean (± SE) serum concentrations of (A) leptin, in respect to (B) body mass index (BMI), and those of (C and D) oestradiol in 66 women undergoing an IVF programme. I = mid-luteal phase, before the start of gonadotrophin-releasing hormone (GnRH) agonist; II = ovarian suppression; III = ovarian stimulation, 3–5 days before oocyte retrieval (OPU) or 8 and 14 days after retrieval. The arrow indicates time of HCG injection. Analysis of variance for repeated measurements (BMI grouping as covariate); Fisher's PLSD: *P < 0.05, **P < 0.001, ***P < 0.0001; the effect of BMI grouping: #P < 0.05, ##P < 0.001, ###P < 0.0001. Split analysis of BMI groups (B and D): Fisher's PLSD significant between marked points (a), between consecutive points (b).

 
Ovarian stimulation was followed by significant increases in leptin concentrations (Figure 1Go; P < 0.0001). This rise was on average 45 ± 6% at sampling time III and reached the maximum of 76 ± 8% at the time of oocyte retrieval. A rise of 20% or more in leptin during stimulation occurred in 56% of the underweight patients, 83% of the normal weight and in every overweight patient (P < 0.05). No linear relationship existed between daily serum leptin and oestradiol or progesterone concentrations during ovarian stimulation, or between leptin and number of ovarian follicles. The rise in leptin during stimulation with HMG (containing LH) (90 ± 11%) did not significantly differ from that with purified FSH (62 ± 10%).

In the presence of high progesterone values (69.7 ± 7.8 nmol/l), 8 days after oocyte retrieval, leptin concentrations were similar to those seen at oocyte retrieval (Figure 1Go). The concentration of leptin was not correlated with that of progesterone but was positively correlated with oestradiol (r = 0.17, P < 0.05). The mid-luteal concentration of leptin after ovarian stimulation (16.5 ± 1.5 µg/l) was 28 ± 7% higher than that at the same phase during the preceding cycle (13.1 ± 0.9 µg/l; P< 0.001).

By 12 days after embryo transfer, when endogenous HCG becomes detectable if pregnancy occurs, the concentration of leptin fell (P < 0.01, Figure 1Go). Relatively, the decrease in leptin concentrations from oocyte retrieval to 14 days after oocyte retrieval (i.e. 12 days after embryo transfer) tended to be larger in women failing to become pregnant (25 ± 4%) and in those with miscarriages (27 ± 7%) than in those women with successful pregnancies (8 ± 6%) (Figure 2Go). A successful outcome of pregnancy was associated with a higher concentration of leptin in the first days of gestation (18.7 ± 4.8 µg/l; Figure 2Go) than in those experiencing early miscarriages (10.7 ± 1.7 µg/l, P < 0.001) or no implantation (11.6 ± 1.2 µg/l, P < 0.0001). In the three women with twin pregnancies, leptin concentrations (20.4 ± 1.9 µg/l) were comparable with those seen with singleton pregnancies (18.2 ± 6.3 µg/l). The concentration of HCG was not an independent factor for leptin (r = 0.12).



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Figure 2. Leptin (mean ± SE) according to outcome of IVF. For timing of sampling, see Figure 1Go. Analysis of variance for repeated measurements was not significant. *Leptin 14 days after oocyte retrieval (OPU). Analysis of covariance (body mass index as a covariate, P < 0.001), Fisher's protected least squares difference (PLSD) post-hoc test P < 0.01 between successful pregnancy and miscarriage; P < 0.0001 between successful pregnancy and no pregnancy.

 
Patients who became pregnant with the treatment showed a significant correlation (P < 0.001) between ovarian stimulation-induced rise in leptin and maximal oestradiol value (Figure 3Go). Similarly, these patients with upcoming clinical pregnancies showed positive correlations between the rise in leptin and follicle count (r = 0.62, P = 0.006), oocyte count (r = 0.74, P < 0.001), and a marginal correlation with progesterone at oocyte retrieval (17.4 ± 1.3 nmol/l; r = 0.46, P = 0.05). All leptin responses to IVF were independent of the cause of infertility.



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Figure 3. Relationship between rises in leptin and peak oestradiol concentrations during ovarian stimulation in women becoming clinically pregnant and those failing to.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Marked changes were found in serum leptin concentrations during an ovarian suppression–stimulation programme. In addition, a significant correlation was observed between rises in leptin and oestrogen but only in patients achieving a clinical pregnancy. Moreover, women with very early pregnancies with successful outcomes exhibited higher leptin concentrations after embryo transfer than those failing to achieve a pregnancy or those who later miscarried.

Leptin concentrations before IVF treatment were highly dependent on BMI, whereas infertility characteristics were not factors in this regard. However, patients with untreated endometriosis have shown both higher (Matarese et al., 2000Go) and also broadly similar (Matalliotakis et al., 2000Go) serum leptin concentrations, compared with healthy controls. Furthermore, leptin responses to ovarian suppression and stimulation were similar in all infertility groups and in all weight groups.

Nafarelin was used to induce ovarian suppression, starting in the mid-luteal phase, and we noticed a significant fall in leptin in our patients. This finding is in conflict with recent data on leptin concentrations being unaffected during leuprolide acetate-induced ovarian suppression (Lindheim et al., 2000Go) and on being increased during danazol and leuprolide depot treatment of patients with endometriosis (Matalliotakis et al., 2000Go). The cause of these discrepancies may be related to the different GnRH agonists used, to different degrees of hypo-oestrogenism, or to too small number of patients studied. In normal cycles leptin concentrations also fall significantly from the luteal phase to the early follicular phase (Hardie et al., 1997Go) and, thus, the fall in leptin from luteal phase concentrations to ovarian suppression in our IVF programme may not solely be dependent on the GnRH analogue. However, in-vitro studies demonstrate that hypo-oestrogenism reduces the synthesis of leptin in adipocytes (Shimizu et al., 1997Go; Casabiell et al., 1998Go) and that bilateral ovariectomy leads to reduced leptin concentrations in women (Messinis et al., 1999Go). Thus, the bulk of evidence supports the conclusion that hypo-oestrogenism is associated with a fall in circulating leptin.

Ovarian stimulation by HMG/FSH was accompanied by a significant rise in leptin, as in some previous studies (Mannucci et al., 1998Go; Messinis et al., 1998Go). Since the peak leptin concentration occurred after the oestrogen surge, it is suggested that the oestrogen surge rather than FSH itself was probably the main cause of this leptin rise. However, as in most previous studies on leptin and oestrogen (Mannucci et al., 1998Go; Riad-Gabriel et al., 1998Go; Teirmaa et al., 1998Go; Butzow et al., 1999Go; Lindheim et al., 2000Go), it was not possible to demonstrate a linear relationship between daily oestradiol and daily leptin concentrations during ovarian stimulation, although such a relationship has been described during normal cycles (Messinis et al., 1998Go). The stimulation of leptin by oestrogen may be a complex, time-dependent process and, therefore, a daily relationship between leptin and oestrogen does not necessarily become detectable. The mass of adipose tissue contributes to oestrogenicity through the extraglandular aromatization of oestrogen precursors to oestrogens and, therefore, obesity may also increase leptin concentrations through endogenous hyper-oestrogenism. The slightly higher rise in leptin following administration of post-menopausal FSH with LH activity, rather than purified FSH may be a result of the stimulation of the granulosa cells by LH leading to increased oestrogen synthesis (Filicori et al., 1999Go). Another explanation could be the direct stimulation of leptin synthesis by LH, as demonstrated by in-vitro experiments (Sivan et al., 1998Go).

Leptin concentrations were highest at the time of oocyte retrieval when patients had become exposed to the effect of administered HMG/FSH and HCG. Because HCG can stimulate the release of leptin by the adipocytes in vitro (Sivan et al., 1998Go), injected HCG could therefore have increased the release of leptin from the adipocytes and perhaps also from the granulosa cells (Antczak et al., 1997Go; Cioffi et al., 1997Go; Karlsson et al., 1997Go). Arguing against this speculation is the fall in leptin concentrations and the lack of correlation between the concentrations of leptin and HCG at oocyte retrieval and during the first days of pregnancy.

These data revealed no correlation between leptin and progesterone concentrations during ovarian stimulation at oocyte retrieval and are in accordance with a recent study (Stock et al., 1999Go). However, leptin and progesterone have been found to be been related at the time of maximal stimulation prior to HCG injection (Butzow et al., 1999Go). In the current study, the missing relationship between leptin and progesterone during the luteal phase of an IVF cycle may relate to the exogenous progesterone administered, although the dosage was similar for each patient. Thus in-vivo data regarding the relationship between leptin and progesterone remain conflicting.

In view of the trophoblastic production of leptin (Masuzaki et al., 1997Go; Senaris et al., 1997Go), and of significant rises in leptin during the late first, second and third trimester of pregnancy (Hardie et al., 1997Go; Sivan et al., 1998Go; Laivuori et al., 2000Go), we expected to see rising concentrations of leptin in women during very early pregnancy. It was, therefore, a surprise that the leptin concentrations decreased during the first days of gestation, although less so than in non-pregnant women. Unfortunately, we did not measure oestrogen concentrations in this phase; however, previous studies have shown that they decrease during very early gestation (Speroff et al., 1989Go). Thus, the early pregnancy-associated fall in leptin could be a consequence of a fall in oestrogen. Regardless of the reason, this fall in leptin may be biologically meaningful, because leptin diminishes the stimulatory effect of HCG on progesterone production in vitro (Brannian et al., 1999Go). Thus, the falling concentrations of leptin in very early pregnancy may help the corpus luteum to produce maximum amounts of progesterone. The higher concentration of leptin in pregnant (compared with non-pregnant) women may be a reflection of stronger oestrogenic stimulation on leptin production from the adipocytes, luteinized granulosa cells (Kitawaki et al., 1999Go), the trophoblasts (Masuzaki et al., 1997Go; Senaris et al., 1997Go), or from all of them.

The data presented here suggest that the increase in leptin secretion during ovarian stimulation is related to the peak oestrogen concentration for a successful outcome of treatment (Figure 3Go). This is a novel finding and supports the view that leptin has a function in reproduction, at least in assisted reproduction. In addition, the data imply that a single serum concentration of leptin before or during stimulation does not predict the outcome of the treatment. However, a low concentration of leptin in follicular fluid at oocyte retrieval has been associated with success in assisted reproduction cycles (Mantzoros et al., 2000Go).

In conclusion, leptin concentrations decreased significantly from the normal mid-luteal phase to ovarian suppression and rose significantly following ovarian stimulation, regardless of BMI. These alterations in leptin may be caused by the parallel changes in oestrogenic milieu. The relationship between the rise in oestradiol (the number of follicles/oocytes developed) and leptin during ovarian stimulation may be an important factor for a successful IVF outcome. In addition, a successful outcome of pregnancy was associated with high concentrations of leptin at 12 days after embryo transfer.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We are grateful to Veikko A.Koivisto for his valuable co-work and comments in preparing this manuscript. This work was supported by grants from the Helsinki University Central Hospital Research Funds and from the Finnish Foundation of Obstetrical and Gynaecological Research.


    Notes
 
5 To whom correspondence should be addressed. E-mail: aila.tiitinen{at}hus.fi Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on August 21, 2000; accepted on December 11, 2000.