Free and protein-bound steroids in amniotic fluid of midpregnancy CHUNG H. WU, M.D. :vt!CHAEL T. MENNUTI, M.D. (~fORGE
MIKHAIL, M.D.
Philarle/fJhia, Pe11nsylvania Ninety-seven amniotic fluid (AF) specimens, 67 with male fetuses and 30 with female fetuses, were obtained by amniocentesis at 14 to 18 weeks of gestation for analysis of free and protein-bound estrone (E,), estradiol (E2 ), androstenedione (A), testosterone (T), and progesterone (P). Ten maternal plasma (MP) samples of comparable gestational age were analyzed for the same free and protein-bound steroids. Significantly lower total E,, E2 , A, and T, but not P. levels were found in AF when compared to MP. The percentage of free steroids was higher in AF than in MP, while the percentage of specific protein-bound steroids was lower in the AF than in MP. Free (unbound) steroid levels of E, and P were higher in AF than in MP, whereas levels of E2 were lower in AF than in MP. The free A and T levels in MP were lower than in AF with male fetuses, yet higher than in AF with female fetuses. Possible biologic activity of steroids in this fluid may depend on the dynamic balance between the free, the specific protein-bound, and the nonspecific protein-bound steroids. (AM. J. 0BSTET. GYNECOL. 133:666, 1979.)
PRo o u c TI oN of steroid hormones increases greatly during pregnancy when compared to production in rhe nonpregnant state. The placenta plays the major role in steroid secretion during the second and third trimesters of human pregnancy. Steroid hormone concentrations in the maternaJI-; and fetal 6-~ plasma have been examined, yet relatively little information is available regarding steroids in amniotic fluid. Plasma proteins, including albumin, 10 testosteroneestradiol-binding globulin (TeBG), 11 and corticosteroid-binding globulin (CBG), 12 have been identified in amniotic fluid. The unconjugated plasma steroid hormones are known to be bound largely to proteins, either specific binding protein such as TeBG 13 and CBG, 14 or nonspecific binding protein such as albumin. The biologic activity of steroid has been attributed to the free'' or the free plus albumin-bound steroid. 16 During pregnancy the specific steroid binding proteins, both TeBG 17 and GBG, 1'1 increase in the maternal plasma. and the free and the albumin-bound fraction
THE
From the Department of' Obstetrics and Gynecology. Univtrsity of Prmruylvania School of Medicinr. Spun10red by the Sooetyfor Gynecologic lnvest1gatiun. Reprint request.;: Chung H. Wu, M.D., Department of' Obstetrics and Gynecology, Hospital of the University of' Penmylvania, 3400 Spruce St., Phikulelphia, Pennsylvania 19104.
666
of steroid decrease. Thus the biologically active steroid concentrations lag behind the progressive increase of total plasma steroid concentration as pregnancy advances. In this study we measured the concentration of unconjugated steroid hormones, including estrone (E 1), estradiol (E 2), androstenedione (A), testosterone (T), and progesterone (P), as well as their free and protein-bound fractions in the amniotic fluid of midpregnancy.
Materials and methods Amniotic fluid specimens (n 97) were obtained during mid pregnancy (14 to 18 weeks of gestation) by amniocentesis for genetic studies. Following low-speed centrifugation, the cellular sediments were subjected to cell culture for karyotype analysis, while the supernatant fluids were stored at 20° C for steroid assav. The total concentrations of steroid hormones, including E~o £ 2, A, T, and P, were measured by their respective radiommunoa~says (RIAs). 18 - 20 These RIAs for plasma steroids were evaluated for application to amniotic fluid steroids. Partial purification of P extract was carried out by Sephadex LH-20 column chromatography in benzene: methanol (95: 5) system prior to RIA. The sensitivity of RI As was I 0 pg/tube forE 1 and E2 , 20 pg/tube for A and T, and 50 pg/tube fur P. The coefficient:; of variation of intra- and interassay res pee0002-9378/79/060666+07$00.70/0
©
1979 The C. V. Mosby Co.
Volume 133 Number 6
Free and protein-bound steroids in amniotic fluid of midpregnancy
r I
1-
700
MIKHAIL, M.D.
Philarle/fJhia, Pe11nsylvania Ninety-seven amniotic fluid (AF) specimens, 67 with male fetuses and 30 with female fetuses, were obtained by amniocentesis at 14 to 18 weeks of gestation for analysis of free and protein-bound estrone (E,), estradiol (E2 ), androstenedione (A), testosterone (T), and progesterone (P). Ten maternal plasma (MP) samples of comparable gestational age were analyzed for the same free and protein-bound steroids. Significantly lower total E,, E2 , A, and T, but not P. levels were found in AF when compared to MP. The percentage of free steroids was higher in AF than in MP, while the percentage of specific protein-bound steroids was lower in the AF than in MP. Free (unbound) steroid levels of E, and P were higher in AF than in MP, whereas levels of E2 were lower in AF than in MP. The free A and T levels in MP were lower than in AF with male fetuses, yet higher than in AF with female fetuses. Possible biologic activity of steroids in this fluid may depend on the dynamic balance between the free, the specific protein-bound, and the nonspecific protein-bound steroids. (AM. J. 0BSTET. GYNECOL. 133:666, 1979.)
PRo o u c TI oN of steroid hormones increases greatly during pregnancy when compared to production in rhe nonpregnant state. The placenta plays the major role in steroid secretion during the second and third trimesters of human pregnancy. Steroid hormone concentrations in the maternaJI-; and fetal 6-~ plasma have been examined, yet relatively little information is available regarding steroids in amniotic fluid. Plasma proteins, including albumin, 10 testosteroneestradiol-binding globulin (TeBG), 11 and corticosteroid-binding globulin (CBG), 12 have been identified in amniotic fluid. The unconjugated plasma steroid hormones are known to be bound largely to proteins, either specific binding protein such as TeBG 13 and CBG, 14 or nonspecific binding protein such as albumin. The biologic activity of steroid has been attributed to the free'' or the free plus albumin-bound steroid. 16 During pregnancy the specific steroid binding proteins, both TeBG 17 and GBG, 1'1 increase in the maternal plasma. and the free and the albumin-bound fraction
THE
From the Department of' Obstetrics and Gynecology. Univtrsity of Prmruylvania School of Medicinr. Spun10red by the Sooetyfor Gynecologic lnvest1gatiun. Reprint request.;: Chung H. Wu, M.D., Department of' Obstetrics and Gynecology, Hospital of the University of' Penmylvania, 3400 Spruce St., Phikulelphia, Pennsylvania 19104.
666
of steroid decrease. Thus the biologically active steroid concentrations lag behind the progressive increase of total plasma steroid concentration as pregnancy advances. In this study we measured the concentration of unconjugated steroid hormones, including estrone (E 1), estradiol (E 2), androstenedione (A), testosterone (T), and progesterone (P), as well as their free and protein-bound fractions in the amniotic fluid of midpregnancy.
Materials and methods Amniotic fluid specimens (n 97) were obtained during mid pregnancy (14 to 18 weeks of gestation) by amniocentesis for genetic studies. Following low-speed centrifugation, the cellular sediments were subjected to cell culture for karyotype analysis, while the supernatant fluids were stored at 20° C for steroid assav. The total concentrations of steroid hormones, including E~o £ 2, A, T, and P, were measured by their respective radiommunoa~says (RIAs). 18 - 20 These RIAs for plasma steroids were evaluated for application to amniotic fluid steroids. Partial purification of P extract was carried out by Sephadex LH-20 column chromatography in benzene: methanol (95: 5) system prior to RIA. The sensitivity of RI As was I 0 pg/tube forE 1 and E2 , 20 pg/tube for A and T, and 50 pg/tube fur P. The coefficient:; of variation of intra- and interassay res pee0002-9378/79/060666+07$00.70/0
©
1979 The C. V. Mosby Co.
Volume 133 Number 6
Free and protein-bound steroids in amniotic fluid of midpregnancy
r I
1-
700
