Modulating effect of leptin on basal and follicle stimulating hormone stimulated steroidogenesis in cultured human lutein granulosa cells.

BACKGROUND: In vitro data have shown conflicting results in terms of the effect of leptin on granulosa cells steroidogenesis. AIM: The aim of the present study was to investigate the effect of low and high doses of leptin on basal and FSH-induced steroids secretion by human luteinized granulosa cells in culture. MATERIALS AND METHODS: Granulosa cells were obtained from normal women undergoing in vitro fertilization (IVF) treatment and were cultured in serum-free conditions for 72 h. A one-way analysis of variance design was set to study the effect of leptin on basal and FSH-induced steroidogenesis. RESULTS: Leptin affected basal estradiol and progesterone secretion in a dose-related manner. In particular, leptin at low concentrations stimulated the secretion of estradiol (1 and 10 ng/ml) and progesterone (10 ng/ml), while at a high concentration (100 ng/ml) it suppressed the secretion of both steroids. A dose-related effect of leptin on FSH-induced steroidogenesis was not evident, since only the suppressive effect of the high concentration of leptin (100 ng/ml) reached statistical significance for both steroids. CONCLUSIONS: These results demonstrate that leptin affects the secretion of steroids in luteinized granulosa cells in a dose-dependent manner. Although a physiological role for leptin is possible, it is suggested that this protein is a mediator of negative rather than positive influential interactions on ovarian function that may compromise fertility.

Endothelin-3 and PRL levels in the maternal and fetal circulation at delivery.

OBJECTIVE: The aim of the present cross-sectional study was to test the hypothesis that endothelin-3 (ET-3) is involved in PRL secretion via systemic hormonal interaction during labor. MATERIALS AND METHODS: Fifty healthy pregnant women with singleton pregnancies were included in the present study. At delivery, blood samples were drawn from umbilical vein and artery. At the same time, a blood sample was obtained from a peripheral vein of the mother. In all blood samples, plasma ET-3 and serum PRL concentrations were determined. The main outcome measures were the differences between maternal peripheral blood, umbilical artery and vein in terms of ET-3 and PRL levels, and the associations between ET-3 and PRL levels. RESULTS: ET-3 values (mean+/-SEM) in umbilical artery did not differ significantly from those in umbilical vein (4.94+/-0.27 vs 5.05+/-0.32 pg/ml) but were in both vessels significantly higher than in maternal vein (1.14+/-0.56 pg/ml, p<0.001). Serum PRL values showed similar patterns. There was a significant positive correlation of the ET-3 levels between umbilical artery and vein (r=0.906, p<0.001), but not between maternal peripheral venous blood and the umbilical vessels. Similar correlations were found for PRL values. However, no significant correlations were found between ET-3 and PRL levels in all vessels studied. CONCLUSIONS: The present study demonstrates for the first time that ET-3 levels are higher in fetal than in maternal circulation at term. The lack of correlation between ET-3 and PRL levels suggests that ET-3 does not play an important endocrine role in the control of maternal and fetal PRL secretion during labor.

Absence of leptin expression and secretion by human luteinized granulosa cells.

Whether leptin is secreted by the human ovary is not known. The available data on leptin gene (ob gene) expression by human granulosa cells are conflicting. The aim of the present study was first to re-examine the expression of leptin messenger RNA (mRNA) by human granulosa cells and second to investigate if these cells have the ability to secrete leptin in cultures. Human luteinized granulosa cells were obtained from normal women undergoing in vitro fertilisation treatment after ovarian stimulation and follicle aspiration. The expression of ob gene was studied by Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) both in primary granulosa cells treated immediately after oocyte recovery and in cells cultured up to 24 h under baseline and hormonally stimulated conditions (FSH: 100 ng/ml, LH: 100 ng/ml). ob mRNA transcripts were not detected in luteinized granulosa cells, while they were present in adipose tIssue cDNA. Actin gene expression was detected in all studied samples. Using a sensitive radioimmunoassay (lower limit of detection 0.05 ng/ml), leptin was undetectable in the culture media at all points during the 72 h cultures, while at the same time significant amounts of oestradiol and progesterone were produced particularly after the addition of androstendione (1 microM) to the incubation media. These results demonstrate for the first time that leptin is not secreted by human luteinized granulosa cells in cultures. From a physiological point of view, this may contribute to the development of the optimal follicular environment for oocyte maturation during the preovulatory period.

Oestradiol plus progesterone treatment increases serum leptin concentrations in normal women.

BACKGROUND: Previous studies have alluded to a role for both oestradiol and progesterone in the secretion of leptin from fat cells in the human, although direct evidence has yet to be obtained. The study aim was to assess serum leptin concentrations in normally cycling women receiving exogenous oestradiol and progesterone. METHODS: Normally cycling women were investigated in an untreated spontaneous cycle (control, n = 10), a cycle treated with oestradiol (oestradiol cycle, n = 10) and a cycle treated with oestradiol plus progesterone (oestradiol+progesterone cycle, n = 6). Oestradiol was given to the women through skin patches on cycle days 2, 3 and 4, and progesterone intravaginally on cycle days 3, 4 and 5. Serum concentrations of leptin, oestradiol, progesterone, FSH and LH were measured in daily blood samples. RESULTS: During the treatment, serum oestradiol and progesterone concentrations increased significantly. In the oestradiol cycles, leptin concentrations were not affected by treatment and did not differ from those in controls. In the oestradiol+progesterone cycles, leptin concentrations (mean +/- SEM) increased in all women from cycle day 3 (8.6 +/- 1.1 ng/ml) to days 5 (12.2 +/- 1.8 ng/ml, P < 0.01) and 6 (11.9 +/- 2.0, P < 0.05), and were at these points significantly higher than in the control cycles (P < 0.05). The mean percentage increase from day 3 to the peak concentration on days 5 or 6 was 62.6 +/- 6.8%. Leptin concentrations returned to the pretreatment value on day 7, together with the concentrations of oestradiol and progesterone. In the oestradiol+progesterone cycles, leptin concentrations correlated significantly with oestradiol and progesterone concentrations, but not with FSH and LH concentrations. CONCLUSIONS: These results show, for the first time, that leptin secretion can be stimulated in women by the administration of oestradiol plus progesterone. This may explain the increased concentrations of leptin during the luteal phase of the normal menstrual cycle.

Treatment of normal women with oestradiol plus progesterone prevents the decrease of leptin concentrations induced by ovariectomy.

To study the role of oestradiol and progesterone in the secretion of leptin, 21 normally ovulating women were recruited from those scheduled for ovariectomy plus hysterectomy performed in mid-follicular phase of the cycle. Seven of the women were used as controls and received no hormonal treatment post-operatively. Another seven women received oestradiol (oestradiol group) and the remaining seven women received oestradiol plus progesterone (oestradiol plus progesterone group). Serum leptin values showed a temporal but significant increase 24 h after the operation and were significantly correlated with the cortisol and progesterone values, which increased temporarily at 12 h. At that time a marked decline in oestradiol concentrations was seen. After the temporal increase, leptin values in the controls and the oestradiol group decreased significantly up to day 4 (P: < 0.05), while in the oestradiol plus progesterone group they increased (P: < 0.01) and were significantly higher than in the other two groups (P: < 0.05). Body mass index (BMI) was the most important variable accounting for the changes in leptin values post-operatively, but in the oestradiol plus progesterone group progesterone correlated significantly with leptin independently of BMI. These results suggest that progesterone and cortisol can stimulate leptin secretion in women regardless of oestradiol concentrations.

Leptin concentrations in normal women following bilateral ovariectomy.

To study the relationships between gonadal steroids and leptin, 20 women with normal cycles were investigated during the postoperative period following a laparotomy. Fourteen women underwent bilateral ovariectomy plus total hysterectomy either in the mid- to late follicular phase (n = 7, group 1) or in the early to midluteal phase (n = 7, group 2). The remaining six of the 20 women underwent cholocystectomy in the early to midfollicular phase of the cycle and were used as controls (group 3). In all three groups, serum leptin values decreased rapidly up to post-operative day 4. Then, leptin values increased significantly only in group 3 (P < 0.05). Leptin values before and after the operation showed significant positive correlations with body mass index (BMI), oestradiol and progesterone. However, with multiple regression analysis, BMI was the only parameter significantly correlated with leptin in group 3 (days 0 and 4-7), whereas in groups 1 and 2 progesterone and BMI showed independent significant correlations with leptin (days 0 and 8, r = 0.601 and r = 0.602 respectively). These results demonstrate for the first time a significant reduction in leptin concentrations in normal women following bilateral ovariectomy. Although BMI seems to be the predominant factor, it is also suggested that oestradiol and progesterone may participate in the control of leptin production during the human menstrual cycle.

Leptin concentrations in the follicular phase of spontaneous cycles and cycles superovulated with follicle stimulating hormone.

It has been reported that oestradiol may play a role in the production of leptin from adipocytes. To investigate this relationship further, nine normally ovulating women were studied during two menstrual cycles, i.e. an untreated spontaneous cycle and a cycle treated with follicle stimulating hormone (FSH) from cycle day 2 until the day of human chorionic gonadotrophin (HCG) injection. Serum leptin values on cycle day 2 did not differ significantly between the spontaneous and the FSH cycles. In the spontaneous cycles, leptin values declined gradually and significantly up to day 7 and then increased progressively up to the day of luteinizing hormone (LH) surge onset, at which point they achieved the highest values. In the FSH cycles, serum leptin values increased gradually and significantly up to day 6, remaining stable thereafter, and were in the midfollicular phase significantly higher than in the spontaneous cycles. Significant positive correlations were found between mean values of leptin and mean values of oestradiol during the second half of the follicular phase in the spontaneous cycles and during the first half in the FSH cycles. A significant negative correlation was found between these two parameters in the spontaneous cycles during the first half of the follicular phase. Serum leptin levels were significantly higher in the midluteal than in the follicular phase in both cycles. These results demonstrate for the first time significant changes in leptin values during the follicular phase of the human menstrual cycle and a significant increase during superovulation induction with FSH. It is suggested that oestradiol may be involved in the regulation of leptin production in women.

Changes in gonadotrophin response to gonadotrophin releasing hormone in normal women following bilateral ovariectomy.

OBJECTIVE: Pituitary responsiveness to GnRH varies throughout the normal menstrual cycle. We have investigated whether there are differences in the ovarian mechanisms which regulate gonadotrophin secretion between the follicular and the luteal phase of the cycle. DESIGN: Normally ovulating women were studied during the first week following hysterectomy plus bilateral ovariectomy performed either in the mid- to late follicular phase (follicle size 16 mm) or in the early to midluteal phase (5 days post LH peak). The response of LH to a single dose of 10 micrograms GnRH was investigated 2 hours before the operation and every 12 hours after the operation until postoperative day 4 and every 24 hours until day 8. PATIENTS: Fourteen normally cycling premenopausal women with normal FSH (< 10 IU/l). Seven women were ovariectomized in the follicular and 7 in the luteal phase. MEASUREMENTS: Pituitary response to GnRH was calculated as the net increase in FSH (delta FSH) and LH (delta LH) at 30 minutes above the basal value. RESULTS: Basal levels of FSH and LH before the operation were significantly lower in the luteal than the follicular phase (P < 0.05), while those of oestradiol (E2) were similar. Also, similar were delta LH and delta FSH values. Serum progesterone and immunoreactive inhibin (Ir-inhibin) concentrations before the operation were higher in the luteal than the follicular phase (P < 0.05). Following the operation, serum E2, progesterone and Ir-inhibin values declined dramatically, while basal FSH and LH as well as delta FSH values showed a gradual and significant increase. The percentage increase in FSH and LH values (mean +/- SEM) on day 8 after the operation was similar in the follicular (453 +/- 99% and 118 +/- 35% respectively) and the luteal phase (480 +/- 71% and 192 +/- 45% respectively). In contrast to delta FSH, delta LH values after a temporal increase 12 hours from the operation, remained stable in the follicular phase and declined significantly in the luteal phase up to day 4. CONCLUSIONS: Basal gonadotrophin secretion during the normal menstrual cycle is predominantly under a negative ovarian effect. It is suggested that in contrast to FSH, the secretion of LH in response to GnRH is controlled by different ovarian mechanisms during the two phases of the menstrual cycle.

Changes in pituitary response to gonadotropin-releasing hormone following bilateral ovariectomy in women treated with follicle-stimulating hormone.

Superovulation- induction in women attenuates the pituitary response to gonadotropin-releasing hormone (GnRH). The aim of this study was to assess the duration of the suppressing activity of the ovaries on the pituitary. Eighteen normally ovulating women received treatment with follicle-stimulating hormone (FSH, 225 IU/day) on cycle days 2, 3 and 4. On cycle day 4, six women underwent hysterectomy plus bilateral ovariectomy (group A), another six women underwent hysterectomy without ovariectomy (group B) and the remaining six women underwent no operation (group C). The women of group C were also investigated during a preceding untreated spontaneous cycle (group D). The response of luteinizing hormone (LH) to an intravenous injection of 10 micrograms GnRH was investigated on cycle days 2, 3, 4 (2 and 12 h after clamping of the infundibulopelvic and/or round ligaments), 5, 6 and 7 in all four groups. The response of LH to GnRH at 30 min (delta LH) was significantly attenuated as early as 12 h from the onset of FSH treatment (groups A, B and C), while estradiol and inhibin concentrations started to increase later (group C). In group C (no operation), the attenuation of delta LH values continued throughout the study period, while in groups A and B the initial attenuation was followed by a marked increase in delta LH values within 2 h from the operation. The increase in group A was twice the value in group B. Following this, delta LH values in group B were attenuated again within the next 24 h, while in group A they remained for the rest of the postoperative period significantly higher than in group B. In conclusion, it was found that the factor that mediates the suppressing effect of superovulated ovaries on the pituitary has a short-lasting (< 2 h) attenuating activity in the circulation.

Effect of follicle stimulating hormone or human chorionic gonadotrophin treatment on the production of gonadotrophin surge attenuating factor (GnSAF) during the luteal phase of the human menstrual cycle.

OBJECTIVE: Although there is much in-vivo evidence for the existence of a gonadotrophin surge attenuating factor (GnSAF), its source and identity remain unknown. We have studied the control of GnSAF production by FSH and hCG during the luteal phase of the cycle. DESIGN: Normally cycling women were investigated in three cycle. Starting on day 5 after the midcycle LH peak, the women received i.m. injections of placebo (1st cycle control), hCG at a dose of 750 IU per day (2nd cycle) and FSH at a dose of 225 IU per day (3rd cycle) for five consecutive days. The response of LH to a single i.v. dose of 10 microg GnRH (GnSAF bioactivity) was investigated several times during the experimental period. PATIENTS: Six normally ovulating women with long-standing unexplained infertility were studied. The women were used as their own controls during the cycle treated with placebo. MEASUREMENTS: Pituitary response to GnRH was calculated as the net increase in LH at 30 minutes (deltaLH) above the basal value. RESULTS: Serum concentrations of FSH and hCG increased significantly during the second and 3rd cycles respectively. Compared with the control cycles, deltaLH was significantly attenuated as early as 12 hours from the onset of FSH injections. In contrast, basal concentrations of oestradiol (E2) and immunoreactive inhibin started to increase 48 hours after the first injection of FSH, while progesterone values remained similar to those in the controls. During treatment with hCG, no attenuation was seen in deltaLH values, while those of E2, progesterone and inhibin showed a significant increase. CONCLUSIONS: These results demonstrate that during the luteal phase of the human menstrual cycle, FSH, but not LH, stimulates the production of gonadotrophin surge attenuating factor. It is suggested that the source of gonadotrophin surge attenuating factor at that stage of the cycle is a cohort of small follicles rather than the corpus luteum.