0021-972X/90/7105-1294$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1990 by The Endocrine Society

Vol. 71, No. 5 Printed in U.S.A.

Inhibin Suppresses Human Chorionic Gonadotropin Secretion in Term, but not First Trimester, Placenta MICHAEL S. MERSOL-BARG, KURT F. MILLER, CAROL M. CHOI, ARTHUR C. LEE, AND MOON H. KIM Department of Obstetrics and Gynecology, Ohio State University, Columbus, Ohio 43210

ABSTRACT. Inhibin is produced by the placenta, with serum concentrations rising throughout pregnancy. In contrast, hCG serum concentrations peak in the first trimester and are 80-90% lower at term. This study was designed to determine the effect of inhibin on hCG secretion both early and late in gestation. Villus tissue from 3 term and 3 first trimester (8-10 week) placentas was maintained in an in vitro explant culture model for 5 days. Tissue from each placenta was incubated with control medium in 24 replicate wells for the first 72 h. During the final 48 h, 12 wells received control medium, and 12 wells received medium containing 1% rabbit antiserum raised against the asubunit (residues 1-32) of the human inhibin peptide. The antiserum demonstrated biological activity by increasing serum FSH concentrations in an immature female rat bioassay. The relative increase in hCG secretion at the conclusion of days 4

and 5 in control and antiserum-treated groups for each first trimester and term placenta were compared to pretreatment hCG concentrations on day 3. The relative increases in hCG secretion of first trimester control groups on day 4 (mean ± SD, 34 ± 11%) and day 5 (63 ± 23%) were compared to those in antiserum-treated groups on day 4 (39 ± 13%) and day 5 (54 ± 5%) and showed no significant difference between groups on either day. The same comparison in term cultures showed the relative increases in hCG secretion of control groups on day 4 (31 ± 10%) and day 5 (64 ± 50%) to be significantly lower than those in antiserum-treated groups on day 4 (100 ± 41%) and day 5 (150 ± 108%; P < 0.001). These findings suggest that inhibin suppresses hCG secretion in term, but not first trimester, placentas. (J Clin Endocrinol Metab 7 1 : 1294-1398, 1990)

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ONSIDERING the totipotential nature of placental tissue, the discovery of a growing number of proteins secreted by the trophoblast should be of no surprise. The qualitative and quantitative secretory patterns of hCG have been well described (1). More recently, inhibin was discovered to be produced by the placenta (2-4), specifically the cytotrophoblast (5). Concentrations of inhibin in serum rise throughout pregnancy (6). This is in contrast to the pattern of hCG, which peaks between the eighth and tenth weeks of gestation, then rapidly falls to levels 10-20% of the peak concentration (1). The diverging secretory patterns of these two hormones may merely be coincidental or may reflect the presence of a regulatory interaction. Inhibin selectively suppresses FSH release from the pituitary (7). Inhibin could participate in the regulatory mechanism of another peptide hormone, specifically hCG. Placental GnRH and inhibin have been proposed as local regulators of hCG secretion, with placental GnRH being stimulatory and inhibin causing suppression. This hypothesis was based on studies using a term trophoblast monolayer primary culture (6, 8).

Placentas of different gestational ages, at which hCG secretion is maximal and minimal, were obtained and cultured using an in vitro explant model. We raised bioactive rabbit antiserum against the human amino a(1-32) portion of the inhibin peptide. Polyclonal antisera raised against the amino-terminal portion of bovine, porcine, and human inhibin a-chains have been shown to block inhibin activity in vitro and in vivo (9-11). The antiserum was used to neutralize inhibin activity in the in vitro placental explant culture model; the end point was its effect on hCG secretion. This study was undertaken to compare the responses of first trimester and term placentas to bioactive antisera. Examining possible alterations in hCG secretion induced by the inhibin antisera at these two gestational ages may increase our understanding of inhibin's role in the endocrinology of pregnancy.

Materials and Methods Preparation of antiserum

Received May 4, 1990. Address requests for reprints to: Michael S. Mersol-Barg, M.D., K9, Henry Ford Hospital, 2799 W. Grand Boulevard, Detroit, MI 48202.

The synthetic peptide replicate of human inhibin, a-(l-32), was purchased from Bachem, Inc. (Torrence, CA). The aminoterminal sequence is Ser-Thr-Pro-Leu-Met-Ser-Trp-Pro-TrpSer-Pro-Ser-Ala-Leu-Arg-Leu-Leu-Gln-Arg-Pro-Pro-Glu-GluPro-Ala-Ala-His-Ala-Asn-Cys-His-Arg. The synthetic peptide

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INHIBIN SUPPRESSES PLACENTAL hCG was conjugated to BSA (Sigma Chemical Co., St. Louis, MO) using a bifunctional reagent, 6-maleimidocaproyl-iV-hydroxysuccinimide ester (MCS), according to the procedures of Lee et al. (12). The carrier protein BSA was first reacted with the active ester end of the bifunctional reagent MCS. The aminoterminal peptide of human a-inhibin sequence 1-32 was then coupled to the MCS-modified BSA through its thiol group of the side-chain of residue 30 (Cysteine). The conjugate thus formed was used in the immunization of rabbits without purification. These conjugates contained five peptides coupled per 105 daltons BSA. Five sexually mature 3-kg female New Zealand White rabbits were used for active immunization. The initial dose of conjugate (1.0 mg/rabbit- immunization) was dissolved in 0.5 mL physiological saline and emulsified with an equal amount of complete Freund's adjuvant (Difco Laboratories, Detroit, MI). Subsequent inoculations contained incomplete Freund's adjuvant (Difco Laboratories). Each animal received a 1.0-mL im injection in its hindquarter. Inoculations were administered three times at 25-day intervals. Blood samples (20-40 mL) were collected weekly by intracardiac puncture, commencing 21 days after initial inoculation. Sera were stored at -20 C. Serum MB2 8-15-89 was used in these experiments. Immunoneutralization Twenty-eight-day-old female Sprague-Dawley rats were divided into four groups. Antisera against synthetic peptide human a-(l-32) (group 1) or nonimmune rabbit serum (NRS) as a control (group 2) were injected ip in a dose of 5 mL/kg BW under light halothane anesthesia. Bilateral oophorectomy (group 3) or sham procedures (group 4) were performed under halothane anesthesia. Blood samples were obtained 6 h later by intracardiac puncture under halothane anesthesia. Serum from each animal was collected and stored at —20 C. FSH concentrations were measured by RIA. Animals were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Tissue preparation Placental tissue was obtained under sterile conditions from 6 women, 3 undergoing elective cesarean section at term and 3 undergoing dilation and evacuation for sociomedical reasons at 8-10 weeks gestation. Gestational age was determined by last menstrual period and measurement of fetal crown-rump length by real time transabdominal ultrasound on the day of the procedure. Villus tissue was collected from multiple cotyledons of term placenta and dissected free from membranes, decidua, vessels, fibrin, and calcium deposits. First trimester villi were separated from vessels and membranes. Tissue was transported in ice-cold Dulbecco's Modified Eagle's Medium (Gibco, Grand Island, NY) containing 5 mg glucose/mL, 100 U/mL penicillin, and 100 ng/mL streptomycin (DMEM). Sterile conditions were maintained throughout the entire course of the experiment. Tissue was sharply minced into pieces approximately 1 mm3 in size. The pooled minced tissue was rinsed three times in DMEM. Five pieces of first trimester tissue or 10 pieces of term tissue were placed into 40 x 36 grid mesh stainless steel wire

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boats (Johnson Wire Works, Montreal, Quebec, Canada). One prepared boat was placed into each culture well (Cell Wells, six polystyrene 35-mm wells/plate, Corning Glassware, Corning, NY) with 3 mL medium. Twelve replicate control wells and 12 replicate treatment wells were prepared for each placenta. Control medium contained DMEM with 2% NRS. Treatment medium contained DMEM, 1% NRS, and 1% inhibin a(1-32) rabbit antiserum developed in our laboratory. All media were sterilized by ultrafiltration using 0.22-Mm Micropore filters (Millipore, Bedford, MA). Plates were incubated for 5 days in an atmosphere of 95% air-5% CO2 at 37 C, with daily medium changes. Control wells received DMEM with 2% NRS on all 5 days. Treatment wells received DMEM with 2% NRS for the first 72 h, then DMEM with 1% NRS and 1% antiserum for the final 48 h. Spent medium from each plate was removed with a sterile pipet and centrifuged at 3300 X g for 15 min, and the supernatant was stored at —20 C. At the conclusion of 5 days, tissue from each well was blotted dry, heated to 60 C for 1 h, and allowed to cool to room temperature for 3 h. Dry weights were then recorded. Concentrations of hCG in medium were measured by RIA. We have observed that hCG secretion declines during the first 2 days in control cultures. First trimester and term explant hCG secretion is maintained at a relatively constant rate from days 3 through 5 in our system. It is for this reason that day 3 was selected to determine baseline hCG production and was used as the reference for relative changes in hCG secretion on days 4 and 5. RIAs hCG. A double antibody RIA was used to measure hCG in the medium from the placental explant cultures. Rabbit antiserum to the whole hCG peptide was generously provided by Dr. Vernon Stevens, Ohio State University (Columbus, OH). The second antibody, a sheep antirabbit 7-globulin, was obtained commercially (Pel-Freeze, Inc., Rogers, AR). An immunochemical grade hCG (CR-125; biological potency, 11,900 IU/mg relative to the Second International Reference Preparation) was used as the standard and tracer and was kindly provided by Dr. Salvatore Raiti, National Hormone and Pituitary Program (Baltimore, MD). The cross-reaction with human LH was 20%, that with hCGa was less than 1%, that with hCG/3 was less than 2%, and that with human FSH was less than 5%. Between- and within-assay coefficients of variation were 5.9% and 4.9%, respectively. The lower limit of sensitivity was 4.8 mlU/mL. First trimester medium was diluted 1:100, and term medium was undiluted. Duplicate assays of 100-jiL aliquots were used in the RIA. The hCG values were expressed as milliinternational units per mL medium and adjusted for dry tissue weight (milligrams). Results were expressed in terms of the relative increase in hCG secretion (percent increase in hCG) on days 4 and 5 (during treatment) compared to that on day 3 (pretreatment). FSH. Serum FSH levels were measured using the rat FSH RIA kit provided by Dr. Salvatore Raiti, National Hormone and Pituitary Program, NIDDK. NIDDK rFSH RP-2 was used as the standard. The lower limit of sensitivity was 7.5 mlU/mL.

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Statistical analysis Concentrations of FSH in the immunoneutralization experiment were analyzed by analysis of variance. Differences among group means were identified by use of Duncan's multiple range test. Placenta explant culture data for each placenta were analyzed by analysis of variance, with treatment effects tested by well within treatment. Subsequently, analysis of variance was performed using data from all of the placentas. Means were compared with Duncan's multiple range test. All statistical analyses were performed using release 6.03 of SAS for the personal computer (13).

Results

2

3 Days

4

5

FIG. 2. Daily hCG secretion by three first trimester placentas (n = 36) and three term placenta (n = 36) in control cultures over the entire 5 days. Values were adjusted for dry tissue weight and represent the mean ± SD. Note the different scales.

Immunoneutralization The effect of immunoneutralization of endogenous

inhibin on serum FSH levels in the immature female rat by antiserum against inhibin a-(1-32) is shown in Fig. 1. The antiserum induced a dramatic increase in serum FSH compared to NRS and sham groups (P < 0.001). FSH levels in the antiserum-treated group were the same as those in the oophorectomized group. hCG secretion

A composite profile of hCG secretion for term and first trimester placentas is shown in Fig. 2. Values for each day include data from all three term and three first trimester placentas (n = 36 each). First trimester hCG secretion was approximately 50-fold greater than term secretion for the entire 5 days. A wide variation was observed in baseline levels of hCG secretion between placentas of the same gestational age. Replicate culture wells (n = 24) of any given placenta were more consistent in hCG secretion than were wells from different placentas. Therefore, the response of each placenta to inhibin Rat FSH Bioassay ou

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•1 6 0 -

i

T

I 40LL

E 2 20-

1

i

n. SH

OVX

NRS

AS

FlG. 1. Serum FSH levels from immature rat bioassay comparing the oophorectomized group (OVX; n = 7), the sham control group (SH; n = 5), the NRS control group (n = 6), and the group treated with antiserum against inhibin a-(l-32) (AS; n = 5). Values represent the mean ± SD.

antisera is presented in Fig. 3. Values on days 4 and 5 are expressed as percent increase (mean ± SD) from day 3. These data show no significant difference between control and treatment groups in any of the first trimester placentas for either day 4 or 5. Each term placenta had a significant increase in hCG secretion in treatment groups compared to controls on days and 5 (P < 0.001). Similar observations are found in combined data from the three first trimester and three term placentas shown in Table 1. The mean percent increase in hCG secretion in term placentas treated with inhibin antiserum was significantly greater than that in control cultures on both days 4 and 5 (P < 0.001). No significant difference was seen between treatment and control cultures in first trimester placentas on either day 4 or 5.

Discussion The physiological factors involved in the regulation of hCG production in normal placental trophoblast during human gestation are not clearly understood. Maternal serum concentrations of many placental proteins, such as human placental lactogen, (14) pregnancy-specific 0glycoprotein (14), and hCGa (15), rise exponentially during the first trimester, but increase at a slower rate until term. The placenta is the major source of inhibin during pregnancy, and secretion of inhibin rises in a similar fashion (5). This pattern parallels the growth rate of placental mass, suggesting that the increase in trophoblastic mass accounts for the increased production of peptide hormones. (16). In contrast, maternal serum hCG levels exhibit a different pattern from the other peptide hormones. Maternal serum levels of hCG rise exponentially during the first trimester, peaking 8-10 weeks after the last menses, declining to a nadir at approximately 18 weeks gestation, then remaining relatively constant until delivery (1, 14). Inhibin has been suggested as a suppressive regulator of hCG synthesis and secretion. Petraglia and colleagues (5) added medium containing antiserum

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INHIBIN SUPPRESSES PLACENTAL hCG

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First Trimester Placenta 1

Placenta 2

Placenta 3

200

FIG. 3. Percent increase in hCG secretion on days 4 and 5 relative to that on pretreatment day 3 by individual placentas. Control cultures are compared to cultures treated with antisera to inhibin a-(l-32). Each value represents the mean ± SD of 12 replicate culture determinations. Note the change of scale in term placenta 3.

Days

Days

Control TABLE 1. Comparison of the percent increase in hCG secretion by treatment, gestational age, and day Control First trimester Term

Antiserum-treated

Day 4

Day 5

Day 4

Day 5

34 ± 11 31 ± 10

63 ± 2 3 64 ± 5 0

39 ± 13 100 ± 41

54 ± 5 150 ± 108

Percent increase from day 3 (mean ± SD). Each mean represents 3 observations. Each observation represents the mean of 12 replicate determinations. raised against porcine inhibin « - ( l - 2 5 ) peptide to h u m a n

primary term placental monolayer cultures and observed an increase in hCG secretion. Indirect immunofluorescence staining with the same antiserum revealed the cytotrophoblast to be immunoreactive, suggesting these cells to be the source of inhibin. We chose to use an in vitro system, using a tissue explant model that would preserve the histological architecture and autocrine milieu of the placental tissue as it exists in vivo, yet permit the introduction of substances in a controlled experimental environment. A dispersed trophoblast cell culture system was not chosen due to normal transformation of cytotrophoblasts to syncytiotrophoblasts within 3 days (17). Assuming that inhibin is secreted from cytotrophoblasts and hCG is secreted from syncytiotrophoblasts, separation of these two cell types may disrupt normal autocrine interaction, should any exist. There may also be a greater loss of cytotrophoblasts as they form syncytiotrophoblasts in culture, thereby diminishing the source of inhibin available in the culture system. In our preliminary experiments cultures of first trimester and term explants were maintained for 6 days. Tissues were examined before and after the 6-day culture under transmission electron microscopy. Comparing tis-

M Antiserum treated

sues of the same gestational age, there were no histological differences between pre- and postculture explants. There was no evidence of necrosis, intracellular vacuolization, or breakdown in the integrity of intracellular organelles. The distributions of mononuclear cells (cytotrophoblasts) and multinuclear cells (syncytiotrophoblasts) were similar. There was a great prevalence of nontrophoblastic cells (fibroblasts) and cellular deposits (fibrin and calcium) in term tissue not found in first trimester tissue. A recent study examined mononuclear trophoblasts from first trimester and term placentas in cell culture. Kato and Braunstein (18) reported that although the rates of differentiation from cytotrophoblasts to syncytiotrophoblasts were the same at both gestational ages, hCG secretion in first trimester cultures was approximately 10-fold greater than that for term cultures. These findings suggest that despite similar morphological differentiation of first trimester and term trophoblast cells, quantitative differences in hCG secretion are due to intrinsic differences in function that are dependant upon the age of the placenta. In other preliminary studies (data not shown), preterm and term cultures were exposed to 50 ^mol/L 8-bromocAMP daily for 6 days. hCG secretion increased exponentially after the second day of culture and reached levels approximating those reported previously using a similar culture model (19). These levels of hCG secretion were well above those measured in the present study. This information assured that an increase in hCG secretion observed would not be blunted due to the tissue approaching maximal hCG output. A problem reported by others using placental explant cultures is the high degree of variation in hCG secretion among replicate culture wells (20, 21). We reduced vari-

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ation by using 24 replicate wells for each placenta. Significant variation was observed in hCG secretion among placentas of the same gestational age; therefore, the experimental design of this study focused on the response of individual placentas to inhibin antiserum and compared placentas in terms of relative increase in hCG secretion from baseline levels (day 3). The biological neutralizing activity of this antisera was established in vivo, thereby providing validation for its use in vitro. Observations from the explant cultures show a significant increase in hCG secretion of term placentas in the antiserum-treated group compared to that in controls. No significant effect was observed in the first trimester placental cultures. These findings lend support to the hypothesis that inhibin has a suppressive influence on hCG secretion. First trimester explants may not produce inhibin in amounts great enough to suppress hCG secretion, thereby accounting for the lack of response to inhibin antiserum. Although hCG secretion increases in term placental explants in the presence of inhibin antiserum, the levels are not nearly as great as basal levels in first trimester explants. This may be due in part to the lower number of trophoblastic cells per mg tissue in term placenta. The delivery and diffusion of antibodies to critical cells may be inefficient in this system, resulting in incomplete immunoneutralization and continued suppression of hCG by inhibin. Perhaps a dispersed cell culture system would circumvent this problem. A recent study shows high hCG levels along with high inhibin serum levels in patients with hydatidiform mole (22). While high hCG levels in the presence of high inhibin levels may be the result of a unique neoplastic process, this finding suggests that other suppressing and/ or stimulating factors are involved and act independently or in concert with inhibin to regulate hCG secretion. The data observed in this study suggest that inhibin suppresses hCG secretion at term. Further investigation into the biochemical mechanism of this action is needed. Acknowledgments The authors wish to express gratitude to Drs. Chad Friedman and Vernon Stevens for their invaluable advice and encouragement. We thank Dr. Mervyn Samuel and his staff for their assistance in obtaining placental tissue for this study. We also wish to thank Ms. Nancy Carney, Ms. Patricia Hennessey, and Ms. Kathleen Fischer for their excellent technical assistance in raising the antiserum used in this study.

References 1. Braunstein GD, Rasor J, Adler D, Danzer N, Wade ME. Serum human chorionic gonadotropin levels throughout normal pregnancy. Am J Obstet Gynecol. 1976;126:678-81.

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2. Mayo KE, Cerelli GM, Spiess J, et al. Inhibin a-subunit cDNA's from porcine ovary and human placenta. Proc Natl Acad Sci USA. 1986;83:5849-53. 3. McLachlan RI, Healy DL, Robertson DM, Burger HG, de Kretser DM. The human placenta: a novel source of Inhibin. Biochem Biophys Res Commun. 1986;140:485-90. 4. Healy DL, McLachlan RI, Robertson DM, de Kretser DM, Burger HG. Inhibin: circulating levels in women during ovulation induction and detection in human placenta by specific radioimmunoassay. Ann NY Acad Sci. 1988;541:162-78. 5. Petraglia F, Sawchenko P, Lim ATN, Rivier J, Vale W. Localization, secretion, and action of inhibin in human placenta. Science. 1987;237:187-9. 6. McLachlan RI, Healy DL, Lutjen PJ, Findlay JK, de Kretser DM, Burger HG. The maternal ovary is not the source of circulating inhibin levels during human pregnancy. Clin Endocrinol. 1987;27:663-8. 7. Miyamoto K, Hasegawa Y, Fukuda M, et al. Isolation of porcine follicular fluid inhibin of 32K daltons. Biochem Biophys Res Commun. 1985;129:396-403. 8. Petraglia F, Vaughan J, Vale W. Inhibin and activin modulate the release of gonadotropic-releasing hormone, human chorionic gonadotropin, and progesterone from cultured human placental cells. Proc Natl Acad Sci USA. 1989;86:5114-7. 9. Findlay JK, Tsonis CG, Doughton B, et al. Immunization against the amino-terminal peptide (aN) of the alpha43 subunit of inhibin impairs fertility in sheep. Endocrinology. 1989;124:3122-4. 10. Rivier C, Vale W. Inhibin: measurement and role in the immature female rat. Endocrinology. 1987;120:1688-90. 11. Saito S, Roche PC, McKormick DJ, Ryan RJ. Synthetic peptide segments of inhibin-a and /5-subunits: preparation and characterization of polyclonal antibodies. Endocrinology. 1989;125:898-905. 12. Lee AC, Powell J, Tregear G, Niall H, Stevens V. A method for preparing -hCG COON peptide-carrier conjugates of predictable composition. J Mol Immunol. 1980;17:749-56. 13. SAS Institute. SAS User's Guide, release 6.03 edition. Cary: SAS Institute; 1988. 14. Braunstein GD, Rason JL, Engvall E, Wade ME. Interrelationships of human chorionic gonadotropin, human placental lactogen, and pregnancy-specific-/3i-glycoprotein throughout normal human gestation. Am J Obstet Gynecol. 1980;138:1205-13. 15. Benveniste R, Scommegna A. Human chorionic gonadotropin asubunit in pregnancy. Am J Obstet Gynecol. 1981;141:952-61. 16. Chard T. Placental synthesis. Clin Obstet Gynecol. 1986;13:44767. 17. Kliman H, Nestler JE, Sermasi E, Sanger JM, Strauss III JF. Purification, characterization and in vitro differentiation of trophoblasts from human term placentae. Endocrinology. 1986; 118:1567-82. 18. Kato Y, Braunstein GD. Purified first and third trimester placental trophoblasts differ in in vitro hormone secretion. J Clin Endocrinol Metab. 1990;70:1187-92. 19. Haning Jr RV, Breault PH, Mahendra VD, Hackett RJ, Pouncey CL. Effect of fetal sex, stage of gestation, dibutyryl cyclic adenosine monophosphate and gonadotropin releasing hormone on secretion of human chorionic gonadotropin by placental explants in vitro. Am J Obstet Gynecol. 1988;159:1332-7. 20. Huot RI, Foidart J-M, Stromberg K. Effects of culture conditions on the synthesis of human chorionic gonadotropin by placental organ cultures. In Vitro. 1979;15:497-502. 21. Haning Jr RV, Choi L, Kiggens AJ, Kuzma DL, Summerville JW. Effects of dibutyryl adenosine 3',5'-monophosphate, luteinizing hormone-releasing hormone, and aromatase inhibitor on simultaneous outputs of progesterone, 17/3-estradiol, and human chorionic gonadotropin by term placental explants. J Clin Endocrinol Metab. 1982;55:213-8. 22. Yohkaichiya T, Fukaya T, Hushiai H, Yajima A, de Kretser DM. Inhibin: a new circulating marker of hydatidiform mole? Br Med J. 1989;298:1684-6.

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Inhibin suppresses human chorionic gonadotropin secretion in term, but not first trimester, placenta.

Inhibin is produced by the placenta, with serum concentrations rising throughout pregnancy. In contrast, hCG serum concentrations peak in the first tr...
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