1 Departments of Obstetrics and Gynaecology, 2 Pediatrics, 3 Medicine and 4 Clinical Chemistry, Helsinki University Central Hospital, Haartmaninkatu 2, FIN-00290 Helsinki, Finland
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Abstract |
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Key words: GnRH agonist/gonadotrophin/IVF/oestradiol/progesterone
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Introduction |
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Materials and methods |
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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, 12 days before HCG injection/35 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., 1996). 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 IV 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 IVI 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). 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.
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Results |
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Ovarian suppression was associated with a fall of 21 ± 4% in leptin (P < 0.01) (Figure 1). 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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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 1). 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 1). 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 2
). 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 2
) 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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Discussion |
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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., 2000) and also broadly similar (Matalliotakis et al., 2000
) 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., 2000) and on being increased during danazol and leuprolide depot treatment of patients with endometriosis (Matalliotakis et al., 2000
). 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., 1997
) 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., 1997
; Casabiell et al., 1998
) and that bilateral ovariectomy leads to reduced leptin concentrations in women (Messinis et al., 1999
). 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., 1998; Messinis et al., 1998
). 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., 1998
; Riad-Gabriel et al., 1998
; Teirmaa et al., 1998
; Butzow et al., 1999
; Lindheim et al., 2000
), 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., 1998
). 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., 1999
). Another explanation could be the direct stimulation of leptin synthesis by LH, as demonstrated by in-vitro experiments (Sivan et al., 1998
).
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., 1998), injected HCG could therefore have increased the release of leptin from the adipocytes and perhaps also from the granulosa cells (Antczak et al., 1997
; Cioffi et al., 1997
; Karlsson et al., 1997
). 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., 1999). However, leptin and progesterone have been found to be been related at the time of maximal stimulation prior to HCG injection (Butzow et al., 1999
). 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., 1997; Senaris et al., 1997
), and of significant rises in leptin during the late first, second and third trimester of pregnancy (Hardie et al., 1997
; Sivan et al., 1998
; Laivuori et al., 2000
), 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., 1989
). 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., 1999
). 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., 1999
), the trophoblasts (Masuzaki et al., 1997
; Senaris et al., 1997
), 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 3). 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., 2000
).
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.
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Acknowledgements |
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Notes |
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References |
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Submitted on August 21, 2000; accepted on December 11, 2000.