0021-972X/91/7204-0862$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1991 by The Endocrine Society ^_
Vol. 72, No. 4 Printed in U.S.A.
JIANPING QU, LEO VANKRIEKEN, CHRISTINE BRULET, AND KARL THOMAS Physiology of Human Reproduction Research Unit, Department of Obstetrics and Gynecobgy, University of Louvain, Brussels, Belgium
In the midterm of pregnancy, serum inhibin was elevated at average levels of 2.84 and 3.84 U/mL at 20 and 28 weeks, respectively. The peak level of inhibin (5.33 U/mL) was obtained at 38 weeks, which was an increase of 237% compared to that at 4 weeks. The average rate of increase in serum inhibin levels was 14.51% every 2-4 weeks (ranging from 8.1-20%). These findings suggest that circulating inhibin is useful marker during human pregnancy. (J Clin Endocrinol Metab 72: 862-866,1991)
ABSTRACT. In this study bioactive inhibin was measured in 112 serum samples from 103 pregnant women by a sensitive ovine pituitary cell culture system. Human inhibin activities were detected in a range between 0.02-5.28 U/mL at six dilutions by using serum from the 38-week pregnant women as a quality control. A remarkable increase in serum inhibin was observed from 4 to 38 weeks of pregnancy. The mean serum inhibin level was 1.58 U/mL at 4 weeks. Thereafter, inhibin levels increased progressively with the weeks of pregnancy (r = 0.988; P < 0.001).
I
women by a sensitive ovine pituitary cell culture system in order to observe the natural profile of circulating inhibin levels at various stages of pregnancy.
NHIBIN is a gonadal glycoproteic hormone, which regulates the production of FSH in the anterior pituitary gland (1, 2), Several studies (3,4) have demonstrated that inhibin is synthesized by the granulosa cells of the ovaries and is accumulated in the follicular fluid. Recently, it has been found that the peak level of circulating inhibin occurred in the midluteal phase of the menstrual cycle (5, 6), suggesting that inhibin may also be secreted by the human corpus luteum (7, 8), It is assumed that inhibin may play an important role in controlling FSH secretion and follicular growth. Human pregnancy is a complicated physiological event. Many hormones participate in the regulation of hormonogenesis in the chorionic system. The placenta synthesizes and secretes several peptide and steroid hormones that express bioactivities in the maternal-fetal compartment. Whether inhibin is also involved in the hormonal regulation during pregnancy has yet to be elucidated. Although some studies (9, 10) have revealed that circulating immunoactive inhibin was elevated at the beginning of pregnancy, little is known about the inhibin secretion pattern during the whole period of pregnancy. The main purpose of this study was to measure the bioactive inhibin levels in the sera of normal pregnant
Materials and Methods Subjects One hundred and twelve serum samples from 103 pregnant women were selected for this study. All cases were spontaneous pregnancies. The mean age was 29.50 yr (range, 18-43 yr). The diagnosis of pregnancy was indicated by the elevated serum hCG level 30 days after the last menstruation, followed by the confirmation of ultrasonography. All serum samples were divided into 10 groups according to age of pregnancy, covering the period of pregnancy from 4-38 weeks, with intervals of 2 or 4 weeks. There were 8-12 samples in each group. Inhibin in vitro bioassay The inhibin bioassay was based on the sheep pituitary cell culture system according to the method described by Tsonis et al. (11). The heads of sheep (1-1.5 yr old) were obtained from a local abattoir and transported to the laboratory within 1-2 h after slaughter. The pituitary glands were immediately removed and minced into small pieces using a scalpel blade. Tissue fragments were rinsed eight times in Dulbecco's phosphatebuffered saline (DPBS; Gibco, Grand Island, NY) supplemented with 0.1% BSA and 7.5 mM glucose (supplemented DPBS) and were then subjected to an enzymatic digestion with supplemented DPBS containing 0.5% trypsin for 30 min at 37 C with gentle stirring. Thereafter, the suspension of cells and clusters was further treated with 2 mM EDTA (Gibco) for 10 min in supplemented DPBS without calcium and magnesium.
July 3,1990. Address all correspondence and requests for reprints to: Jianping Qu, M.D., Physiology of Human Reproduction Research Unit, Department of Obstetrics and Gynecology, Faculty of Medicine, University of Louvain 5330, 53 Avenue Emmanuel Mounier, B-1200 Brussels, Belgium.
862
Downloaded from https://academic.oup.com/jcem/article-abstract/72/4/862/2652773 by University of Otago user on 17 December 2018
Circulating Bioactive Inhibin Levels during Human Pregnancy
BIOACTIVE INHIBIN LEVELS DURING PREGNANCY
Results Sensitivity of inhibin bioassay of sheep pituitary cell culture The data of inhibin bioactivities in human and porcine inhibin reference preparations measured by sheep pituitary cell culture are shown in Fig. 1. The secretion of oFSH was inhibited in a linear fashion after the disaggregated pituitary cells were exposed to the various concentrations of inhibin preparations. The dose-response curves for the inhibition of oFSH secretion of the two inhibin standards were parallel. A greater response of the inhibition of oFSH secretion was induced by human inhibin. At the ID50 level, the doses of inhibin needed to suppress oFSH secretion in the cell culture were 0.36 U/ mL human inhibin and 3.89 U/mL porcine inhibin. Different inhibin bioactivities were measured in sheep, porcine, and human follicular fluids as well as in the sera from women at 38 weeks of pregnancy (Fig. 2). The highest inhibin bioactivity (ID50) was found in sheep follicular fluid, which was 4-fold higher than that in porcine follicular fluid (P < 0.001). In contrast, the inhibin activity (ID50) in human follicular fluid was 6fold lower than that in porcine follicular fluid (P < 0.001). The human inhibin concentration (ID50) in the follicular fluid was about 3.5-fold higher than that in pregnancy serum (P < 0.001). By using the sera from the 38-week pregnant women as a quality control, human inhibin activities were detected in a range between 0.025.28 U/mL at six serum dilutions ranging from 1:6 to 1:6000. The concentrations of residual steroid hormones in the pregnancy serum after the treatment with charcoal were shown in Table 1. The levels of estrone, E2, estriol, 100 5?
Human Porcine
80
C
o U 0)
60 40
X LL O
20
Statistics All data were expressed as the mean ± SEM. Unpaired Student's t test was applied to compare the values in different groups. The relationship of the variations was analyzed by a linear regression. The correlation coefficent of the regressions was tested by the method of Finney (13, 14), Results were considered statistically significant at a two-tailed value of P < 0.05.
-2
-1
1
Human and Porcine Inhibin Standard U/ml (Log)
FIG. 1. Effects of human and porcine inhibin standards on the release of FSH from sheep pituitay cells in culture. The oFSH dose-response curve is expressed as the percent inhibition from normal control values. Human: y = 36.468 - 30.833* (r = 0.978); porcine: y = 66.864 - 28.603* (r = 0.975).
Downloaded from https://academic.oup.com/jcem/article-abstract/72/4/862/2652773 by University of Otago user on 17 December 2018
The disaggregated pituitary cells in the supernatant were harvested and washed three times with supplemented DPBS. The cells were then resuspended at a final concentration of 3 X 106 cells/mL in the supplemented Dulbecco's Modified Eagle's Medium (DMEM; containing 10 mM NaHCO3 2 mM L-glutamin, 10% lamb serum, 2.5% fetal bovine serum, 20 mM HEPES, 50 IU/mL penicillin, and 50 /ig/mL streptomycin). Aliquots of cell suspension were distributed in the 24-well culture plates (100 A*L/well), which had been previously filled with 500 nL supplemented DMEM in each well. The pituitary cells were cultured for 48 h at 37 C under a humidified atmosphere of CO2. The viability of the disaggregated pituitary cells was counted by trypan blue exclusion. To measure the inhibin activity, test samples (100 ^L/well) were added to the pituitary cell culture after removal of the medium. Then, supplemented DMEM was added to the plates to a final volume of 600 /uL/well. To exclude the possible influence from steroid hormones, all test samples were pretreatedwith 1% (wt/vol) dextran-coated charcoal (Biomerieux, Marcy L'Etorte, France) to remove the steroids. For the serum and follicular fluid test samples, rabbit anti-17/3-estradiol (antiE2) and rabbit antiprogesterone (anti-P4) sera (UCB, Braine L'Alleud, Belgium) were added to the culture medium at an initial dilution of 1:100 to block the action of any residual steroid remaining in the samples. After the cells were cultured for another 48 h, the ovine FSH (oFSH) concentration in the cultured medium was measured by RIA. The oFSH RP-1 RIA kit were provided by the National Hormone and Pituitary Program (NIDDK, Bethesda, MD). Human sera (from 38-week pregnant women), human follicular fluid pool (from the in vitro fertilization program of our clinic), porcine follicular fluid pool (PFF 120-1675/22587; NIDDK), and ovine follicular fluid pool (OFF Cl 10/89; from this laboratory) were used as quality controls. Human inhibin (Medgenix, Fleurus, Belgium) and porcine inhibin (WHO 86/690, National Institute for Biological Standards, Hertfordshire, United Kingdom) were used as inhibin standards. All of the inhibin standards and quality controls were tested separately in triplicate at a six-dose range to generate the oFSH inhibition curve. The serum samples from the pregnant women were tested in triplicate at three dilutions. The oFSH dose-response curve was expressed as the percent inhibition of oFSH secretion compared to that in normal controls (1, 12). The inhibin activities in serum samples were expressed as units of human inhibin per mL, using human inhibin as the reference standard. In this bioassay system, the intraassay coefficient of variation (C.V.) was 6.8% for 30 assays (ED50), and the interassay coefficient of variation was 9.4% (EDM).
863
QU ET AL.
864
JCE & M • 1991 Vol 72 • No 3
tween the antisera-treated and control groups (P > 0. 05).
100 -i 80
Changes in bioactive inhibin during pregnancy 60 40
X to
20
-
3
-
2
-
1
0
1
2
3
HPS, HFF, PFF, OFF ul/ml (Log)
FIG. 2. Effects of bioactive inhibin in sheep follicular fluid (OFF), porcine follicular fluid (PFF), human follicular fluid (HFF), and human pregnancy serum (at 38 weeks; HPS) on the release of oFSH from sheep pituitary cells in culture. OFF: y = 15.385 - 28.948* (r = 0.958); PFF: y = 32.680 - 29.276* (r = 0.969); HFF: y = 55.632 - 28.743* (r = 0.979); HPS: y = 70.011 - 26.739* (r = 0.982). TABLE 1. Concentrations of steroid hormones in pregnancy serum after charcoal treatment Treatment
Estrone (pmol/L)
(pmol/L)
E2
Estriol (pmol/L)
(nmol/L)
Charcoal Control
255.18 19822.48
197.83 58817.9
Vol. 72, No. 4 Printed in U.S.A.
JIANPING QU, LEO VANKRIEKEN, CHRISTINE BRULET, AND KARL THOMAS Physiology of Human Reproduction Research Unit, Department of Obstetrics and Gynecobgy, University of Louvain, Brussels, Belgium
In the midterm of pregnancy, serum inhibin was elevated at average levels of 2.84 and 3.84 U/mL at 20 and 28 weeks, respectively. The peak level of inhibin (5.33 U/mL) was obtained at 38 weeks, which was an increase of 237% compared to that at 4 weeks. The average rate of increase in serum inhibin levels was 14.51% every 2-4 weeks (ranging from 8.1-20%). These findings suggest that circulating inhibin is useful marker during human pregnancy. (J Clin Endocrinol Metab 72: 862-866,1991)
ABSTRACT. In this study bioactive inhibin was measured in 112 serum samples from 103 pregnant women by a sensitive ovine pituitary cell culture system. Human inhibin activities were detected in a range between 0.02-5.28 U/mL at six dilutions by using serum from the 38-week pregnant women as a quality control. A remarkable increase in serum inhibin was observed from 4 to 38 weeks of pregnancy. The mean serum inhibin level was 1.58 U/mL at 4 weeks. Thereafter, inhibin levels increased progressively with the weeks of pregnancy (r = 0.988; P < 0.001).
I
women by a sensitive ovine pituitary cell culture system in order to observe the natural profile of circulating inhibin levels at various stages of pregnancy.
NHIBIN is a gonadal glycoproteic hormone, which regulates the production of FSH in the anterior pituitary gland (1, 2), Several studies (3,4) have demonstrated that inhibin is synthesized by the granulosa cells of the ovaries and is accumulated in the follicular fluid. Recently, it has been found that the peak level of circulating inhibin occurred in the midluteal phase of the menstrual cycle (5, 6), suggesting that inhibin may also be secreted by the human corpus luteum (7, 8), It is assumed that inhibin may play an important role in controlling FSH secretion and follicular growth. Human pregnancy is a complicated physiological event. Many hormones participate in the regulation of hormonogenesis in the chorionic system. The placenta synthesizes and secretes several peptide and steroid hormones that express bioactivities in the maternal-fetal compartment. Whether inhibin is also involved in the hormonal regulation during pregnancy has yet to be elucidated. Although some studies (9, 10) have revealed that circulating immunoactive inhibin was elevated at the beginning of pregnancy, little is known about the inhibin secretion pattern during the whole period of pregnancy. The main purpose of this study was to measure the bioactive inhibin levels in the sera of normal pregnant
Materials and Methods Subjects One hundred and twelve serum samples from 103 pregnant women were selected for this study. All cases were spontaneous pregnancies. The mean age was 29.50 yr (range, 18-43 yr). The diagnosis of pregnancy was indicated by the elevated serum hCG level 30 days after the last menstruation, followed by the confirmation of ultrasonography. All serum samples were divided into 10 groups according to age of pregnancy, covering the period of pregnancy from 4-38 weeks, with intervals of 2 or 4 weeks. There were 8-12 samples in each group. Inhibin in vitro bioassay The inhibin bioassay was based on the sheep pituitary cell culture system according to the method described by Tsonis et al. (11). The heads of sheep (1-1.5 yr old) were obtained from a local abattoir and transported to the laboratory within 1-2 h after slaughter. The pituitary glands were immediately removed and minced into small pieces using a scalpel blade. Tissue fragments were rinsed eight times in Dulbecco's phosphatebuffered saline (DPBS; Gibco, Grand Island, NY) supplemented with 0.1% BSA and 7.5 mM glucose (supplemented DPBS) and were then subjected to an enzymatic digestion with supplemented DPBS containing 0.5% trypsin for 30 min at 37 C with gentle stirring. Thereafter, the suspension of cells and clusters was further treated with 2 mM EDTA (Gibco) for 10 min in supplemented DPBS without calcium and magnesium.
July 3,1990. Address all correspondence and requests for reprints to: Jianping Qu, M.D., Physiology of Human Reproduction Research Unit, Department of Obstetrics and Gynecology, Faculty of Medicine, University of Louvain 5330, 53 Avenue Emmanuel Mounier, B-1200 Brussels, Belgium.
862
Downloaded from https://academic.oup.com/jcem/article-abstract/72/4/862/2652773 by University of Otago user on 17 December 2018
Circulating Bioactive Inhibin Levels during Human Pregnancy
BIOACTIVE INHIBIN LEVELS DURING PREGNANCY
Results Sensitivity of inhibin bioassay of sheep pituitary cell culture The data of inhibin bioactivities in human and porcine inhibin reference preparations measured by sheep pituitary cell culture are shown in Fig. 1. The secretion of oFSH was inhibited in a linear fashion after the disaggregated pituitary cells were exposed to the various concentrations of inhibin preparations. The dose-response curves for the inhibition of oFSH secretion of the two inhibin standards were parallel. A greater response of the inhibition of oFSH secretion was induced by human inhibin. At the ID50 level, the doses of inhibin needed to suppress oFSH secretion in the cell culture were 0.36 U/ mL human inhibin and 3.89 U/mL porcine inhibin. Different inhibin bioactivities were measured in sheep, porcine, and human follicular fluids as well as in the sera from women at 38 weeks of pregnancy (Fig. 2). The highest inhibin bioactivity (ID50) was found in sheep follicular fluid, which was 4-fold higher than that in porcine follicular fluid (P < 0.001). In contrast, the inhibin activity (ID50) in human follicular fluid was 6fold lower than that in porcine follicular fluid (P < 0.001). The human inhibin concentration (ID50) in the follicular fluid was about 3.5-fold higher than that in pregnancy serum (P < 0.001). By using the sera from the 38-week pregnant women as a quality control, human inhibin activities were detected in a range between 0.025.28 U/mL at six serum dilutions ranging from 1:6 to 1:6000. The concentrations of residual steroid hormones in the pregnancy serum after the treatment with charcoal were shown in Table 1. The levels of estrone, E2, estriol, 100 5?
Human Porcine
80
C
o U 0)
60 40
X LL O
20
Statistics All data were expressed as the mean ± SEM. Unpaired Student's t test was applied to compare the values in different groups. The relationship of the variations was analyzed by a linear regression. The correlation coefficent of the regressions was tested by the method of Finney (13, 14), Results were considered statistically significant at a two-tailed value of P < 0.05.
-2
-1
1
Human and Porcine Inhibin Standard U/ml (Log)
FIG. 1. Effects of human and porcine inhibin standards on the release of FSH from sheep pituitay cells in culture. The oFSH dose-response curve is expressed as the percent inhibition from normal control values. Human: y = 36.468 - 30.833* (r = 0.978); porcine: y = 66.864 - 28.603* (r = 0.975).
Downloaded from https://academic.oup.com/jcem/article-abstract/72/4/862/2652773 by University of Otago user on 17 December 2018
The disaggregated pituitary cells in the supernatant were harvested and washed three times with supplemented DPBS. The cells were then resuspended at a final concentration of 3 X 106 cells/mL in the supplemented Dulbecco's Modified Eagle's Medium (DMEM; containing 10 mM NaHCO3 2 mM L-glutamin, 10% lamb serum, 2.5% fetal bovine serum, 20 mM HEPES, 50 IU/mL penicillin, and 50 /ig/mL streptomycin). Aliquots of cell suspension were distributed in the 24-well culture plates (100 A*L/well), which had been previously filled with 500 nL supplemented DMEM in each well. The pituitary cells were cultured for 48 h at 37 C under a humidified atmosphere of CO2. The viability of the disaggregated pituitary cells was counted by trypan blue exclusion. To measure the inhibin activity, test samples (100 ^L/well) were added to the pituitary cell culture after removal of the medium. Then, supplemented DMEM was added to the plates to a final volume of 600 /uL/well. To exclude the possible influence from steroid hormones, all test samples were pretreatedwith 1% (wt/vol) dextran-coated charcoal (Biomerieux, Marcy L'Etorte, France) to remove the steroids. For the serum and follicular fluid test samples, rabbit anti-17/3-estradiol (antiE2) and rabbit antiprogesterone (anti-P4) sera (UCB, Braine L'Alleud, Belgium) were added to the culture medium at an initial dilution of 1:100 to block the action of any residual steroid remaining in the samples. After the cells were cultured for another 48 h, the ovine FSH (oFSH) concentration in the cultured medium was measured by RIA. The oFSH RP-1 RIA kit were provided by the National Hormone and Pituitary Program (NIDDK, Bethesda, MD). Human sera (from 38-week pregnant women), human follicular fluid pool (from the in vitro fertilization program of our clinic), porcine follicular fluid pool (PFF 120-1675/22587; NIDDK), and ovine follicular fluid pool (OFF Cl 10/89; from this laboratory) were used as quality controls. Human inhibin (Medgenix, Fleurus, Belgium) and porcine inhibin (WHO 86/690, National Institute for Biological Standards, Hertfordshire, United Kingdom) were used as inhibin standards. All of the inhibin standards and quality controls were tested separately in triplicate at a six-dose range to generate the oFSH inhibition curve. The serum samples from the pregnant women were tested in triplicate at three dilutions. The oFSH dose-response curve was expressed as the percent inhibition of oFSH secretion compared to that in normal controls (1, 12). The inhibin activities in serum samples were expressed as units of human inhibin per mL, using human inhibin as the reference standard. In this bioassay system, the intraassay coefficient of variation (C.V.) was 6.8% for 30 assays (ED50), and the interassay coefficient of variation was 9.4% (EDM).
863
QU ET AL.
864
JCE & M • 1991 Vol 72 • No 3
tween the antisera-treated and control groups (P > 0. 05).
100 -i 80
Changes in bioactive inhibin during pregnancy 60 40
X to
20
-
3
-
2
-
1
0
1
2
3
HPS, HFF, PFF, OFF ul/ml (Log)
FIG. 2. Effects of bioactive inhibin in sheep follicular fluid (OFF), porcine follicular fluid (PFF), human follicular fluid (HFF), and human pregnancy serum (at 38 weeks; HPS) on the release of oFSH from sheep pituitary cells in culture. OFF: y = 15.385 - 28.948* (r = 0.958); PFF: y = 32.680 - 29.276* (r = 0.969); HFF: y = 55.632 - 28.743* (r = 0.979); HPS: y = 70.011 - 26.739* (r = 0.982). TABLE 1. Concentrations of steroid hormones in pregnancy serum after charcoal treatment Treatment
Estrone (pmol/L)
(pmol/L)
E2
Estriol (pmol/L)
(nmol/L)
Charcoal Control
255.18 19822.48
197.83 58817.9
