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MANUBAI NAGAMANI, M.D.* PAUL G. McDONOUGH, M.D. JAMES 0. ELLEGOOD, M.S.
VIRENDRA B. MAHESH, PH.D., D.PHIL.
Augusta, Georgza Concentrations of testosterone, dihydrotestosterone, androstenedione, progesterone, 17a-hydroxyprogesterone, and estradiol were measured by radioimmunoassay in the amniotic fluid ar.d maiemal peripherai biood obtained from normal pregnancies between 14 and 40 weeks oi gestation. There was a sex difference in t-he levels of aU the androgenic steroid$ in the amniotic fluid before 20 weeks wJth higher levels jn pregnancies with me!e fett-.•Jses. ~"rnniotic fil-.Jid 17a-hydroxyprogesterone levels were significantly elevated in a pregnancy with the fetus affected with congenital adrenal hyperplasia. The levels of all the steroids in the amniotic fluid were significantly elevated in the pregnancy with molar degener-ation of the placenta. There was a sex difference in the levels of dihydrotestosterone in the maternal peripheral blood before 20 weeks with higher levels in pregnancies with male fetuses. There was no correlation between the steroid levels in the maternal serum and amniotic fluid even though most of the samples of maternal serum were drawn at the sa.rns time as a.-nniocantasis. (AM. J. OasTEi. GYNECOL. 134:674, 1979.)
studies on the endocrine function of fetal gonads show that human fetal testis in contrast to ovary secretes significant amounts of testosterone at the period of sexual differentiation in utero.L 2 Jost, 3 by his experiments on fetal rabbits, had shown that the development of the male phenotype, except for regression of the miillerian system, depends on the secretion of testosterone by the fetal testis at this critical period. It is also clear from the work of Wilson and Lasnitzki 4 • 5 that testosterone itself is responsible for the differentiation of wolffian duct into epididymis, vas differens, and seminal vesicle, and dihydrotestosterone is probably the intraceiiuiar mediator in the differentiation of uroPREVIOUs
From the Departments of Endocrirwlogy and Obstetrics and Gynecology, Medical College of Georgia. Supported by General Research Support Grant No. 5507 RRo-5365-16 and Training Grant No.5 TO! HD OJ 12-09 from the National Institutes of Health. Received for publication August 3, 1978.
genital sinus and urogenital tubercle to prostate and external genitalia. Estradiol does not seem to play a significant role in the process of sexual differentiation and no significant amounts of this steroid had been detected in the fetal testis or ovaries at this stage of pregnancy.6 The fetus plays a considerable role in the formation of amniotic fluid in all stages of pregnancy. Before 20 weeks, amniotic fluid possibly bears a closer relationship to fetal plasma in its concentration of the steroids because of the free diffusion occurring through the fetal skin which had been shown by electron microscopy to be a meshiike structure with numerous microvilli. 7 After 20 weeks. fetal urine becomes an important source. 8 The present srudy was designed to measure various steroids in the amniotic fluid to see whether this approach could be utilized to study the hormonal events occurring in the fetus during different stages of pregnancy. Steroids were also measured in maternal peripheral blood for comparison.
Revised September 28, 1978. Accepted October 2, 1978. Reprint requests: Dr. V. B. Mahesh, Department of Endocrirwlogy, Medical College of Georgia, AugU!ita, Georgia 30901. *Present address: Department of Obstetrics and Gynecology, University of Texas Medical Branch, Galveston, Texas 77550.
674
Material and rnelhode One hundred one samples of amniotic fluid were obtained between 14 and 40 weeks of pregnancy when amniocentesis was done for cytogenetic studies in early pregnancy and for fetal maturity in late pregnancy. All the pregnancies were normal without any complica0002-9378179/140674+07$00.70/0
©
1979 The C. V. Mosby Co.
Volume 134 Number6
Maternal and amniotic steroids thrc.ughout pregnancy
675
Table I. Levels of testosterone, androstenedione, dihydrotestosterone, estradiol, progesterone, and 17a-hydroxyprogesterone in the amniotic fluid Steroids
14-20 wk.
26-40 wk.
0.188 ::!: 0.014* (0.086-0.477)t 0.022 ::!: 0.002* (0.006-0.064)
0.107::!: 0.015 (0.068-0.172) 0.056 ::!: 0.009* (0.021-0.132)
0.348 ::!: 0.032 (0.065-0.900) 0.156::!: 0.029* (0.011-0.810)
0.187::!: 0.027 (0.04-0.361) 0.331 ::!: 0.038* (0.137-0.492)
0.024 ::!: 0.003 (0.008-0.067) Undetectable
0.026 ::!: 0.006 (0.011-0.064) Undetectable
0.036 ± 0.009 (0.009-0.065) 0.048 ± 0.009 (0.011-0.141)
0.043 ± 0.009 (0.008-0.115) 0.067 ± 0.013 (0.018-0.147)
TestosterofU! (ng!ml):
M F
AndrosteP..edioP..e (ng/ml): M F Dihydrotesto\tero;u (ng/mlj: M F Estradiol (nglml): M F Progesteron~ (nglml):
M F 17-HydroxyprogesterofU! (nglml): M F
34.7 ± 4.1 (7-97) 30.1 ± 4.0 (6-148)
14.1 ± 3.1 (7-31) 13.3 ± 2.1 (4-20)
1.62 ± 0.15 (0.39-3.23) !.5! ± 0.! (0.44-2.66)
0.87 ± 0.15 (0.25-1.25) 0.69 ::!: 0.08 (0.31- !.47)
M = Pregnancies with male fetuses; F "" pregnancies with female fetuses. *Mean levels :':: S.E. tRange. *Significantly different from male fetuses.
tions. Fifty-one samples were from pregnancies with male fetuses and 50 samples were from pregnancies with female fetuses. The sex of each fetus was determined at the time of deiivery or by karyotype of amniotic fluid cells if undelivered. Seventy-six samples were between 14 and 20 weeks of pregnancy and 25 samples were between 26 and 40 weeks of pregnancy. Additionally, one sample of fluid was obtained from a pregnancy ( 17 weeks) which ended in an abortion, and the abortus contained a fetus lvhose karyotype sho,ved triploidy (69, XXX) and extensive molar degeneration of the placenta. There were also four samples of amniotic fluid obtained from different stages of pregnancy in a patient who subsequently delivered a male fetus affected with congenital adrenal hyperplasia (21-hydroxylase deficiency). Sixty samples of maternal peripheral vein blood were obtained from different stages of normal pregnancies. The serum and amniotic fluid samples were stored at -20" C until assayed. Each sample was assayed for testosterone, androstenedione, dihydrotestosterone, estradiol, progesterone, and 17a-hydroxyprogesterone. Fetal age was obtained by subtracting two weeks from the time since the last menstrual pc:riod. If the last menstrual period was not known, the length of gestation was determined by ultrasonic measurement of fetal biparietal diameter. Student's t test and Duncan's muitirange analysis were used for statistical analysis. Hormone assays. All the steroids were measured by radioimmunoassay after fractionation of the steroids by Celite microcolumn chromatography as reported
earlier. 9 Three milliliters of amniotic fluid or 3 ml of serum was extracted twice with three volumes of ether after adding 3H tracers of progesterone, androstenedione, dehydroepiandrosterone, testosterone, and estradiol for recovery calculations. The extracts were dried under nitrogen and subjected to chromatography on microcelite column with ethylene and propylene glycol as stationary phase. The chromatographic separation was effective in removal of steroids that \vou!d interfere in the assay due to cross=reactivity. For example, progesterone and 17a-hydroxyprogesterone; androstenedione, dihydrotestosterone, and testosterone; and estrone and estradiol appeared in separate fractions and estriol was not eluted from the column. 9 The various fractions were collected and dried, and the residue was redissolved in 2 ml of assay buffer. Duplicate aliquots of different volumes of this were used in the assay and 0.1 ml was used for recovery calculations. Blank and control sera (pooled male serum to serve as internal control between assays) were run with each assay. The coefficient of variation between assays had been 5.4% for progesterone, 4.3% for androstenedione, 5.6% for testosterone, 5.8% for dihydrotestosterone, 8.5% for 17 a-hydroxyprogesterone, and 8.9% for estradiol. The precision, specificity, and sensitivity for the assay system had been reported previousiy. 9 3 H-progesterone, 3 H-testosterone, and 3 H-estradiol were added to amniotic fluid. After extTaction, Celite microcoiumn chromatography was carried out as previously described with the exception that 1 ml aliquots of the column eluate were collected.
676 Nagamani et al.
july 15, 1979 Am. J. Obstet. Gynecol.
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0.05). There was considerable overlap in levels in pregnancies with male fetuses and female fetuses (Fig. 2).
AeeuHs Amniotic fluid. The mean testosterone concentrations in 10 I samples of amniotic fluid are shown in Table I. At all stages of pregnancy, the levels of testosterone were significantly higher when the fetus was male and the difference was significant both in midpregnancy and in late pregnancy (p < 0.001). Highest levels were noted in the cases of a male fetus at 16 weeks. Between 14 and 20 weeks, the levels of testosterone in pregnancies with male fetuses varied from 0.086 to 0.477 ng/ml (mean 0.188 ± 0.014) and the levels with female fetuses varied from 0.006 to 0.064 ng/ml (mean 0.022 ± 0.002) with no overlap of vaJues (Fig. 1). Toward term, the difference in mean testosterone levels depending on the sex of the fetus persisted, but there was considerable overlap in values (Fig. 1).
Volume 134 Number 6
Maternal and amniotic steroids throughout pregnancy 677
Table II. Amniotic fluid steroids in a pregnancy with a male fetus having congenital adrenal hyperplasia of pregrumcy
T (ng/ml)
A (ng/ml)
DHT (nglml)
14 22 28 34
0.22 0.606 0.251 0.200
0.345 1.224 0.826
0.026
Week.~
0.040
Abbreviations: T testosterone, A = androstenedione, DHT OHP = 17a.-hydroxyprogesterone.
£2
p
(nglml)
(nglml)
0.106 0.067 0.046
15 15 20
I7a.-OHP (ng/ml)
8.23 20.68 2.93 3.67 progesterone, 17 a.-
dihydrotestosterone, E2
Table III. Amniotic fluid steroids in a pregnancy with hydatidiform degeneration of placenta (nglml)
.4 (ng/ml)
DHT (ng/ml)
(nglml)
p (nglml)
17a.-OHP (nglml)
0.129
0.482
0.050
0.466
160.0
6.24
0.022 ± 0.002
0.156 ± 0.029
Undetectable
0.048 ± 0.009
30.1 ± 0.4
1.51±0.1
T
Affected pregnancy Control
E,
See Table II fo:o abbreviations.
Progesterone levels in amniotic fluid are shown in Fig. 2 and Table I. The levels were significantly higher (p < 0.05) betwt!en 14 and 20 weeks than toward term. There was no difference in the levels depending on the sex of the fetus. The 17a-hydroxyprogesterone levels in the amniotic Huid also showed a similar trend with a higher level in early pregnancy (p < 0.01; Table I) with no sex difference in the levels. In the samples of fluid obtained from a patient who subsequently delivered a male fetus affected with congenital adrenal hyperplasia, the concentration of testosterone and androstenedione was higher than the control levels in one sample obtained at 22 weeks, but the levels in other samples were not significantly different from the control levels (Table II). The levels of progesterone, dihydrotestosterone, and estradiol in these samples were within the range of the control samples. However, 17a-hydroxyprogesterone levels were elevated above the control levels in all stages of pregnancy (Table II). The maximum elevation was at 22 weeks of pregnancy. In the sample of amniotic fluid obtained from a pregnancy in which there was extensive hydatidiform degeneration of the placenta, the level of progesterone, 17a-hydroxypmgesterone, testosterone, dihydrotestosterone, and estradiol were significantly higher than in control samples (Table III). Among the control pregnancies one fetus was found to be mongoloid (trisomy 21) by karyotype of amniotic fluid cells and this pregnancy was subsequently aborted. The steroid levels in the fluid obtained from this pregnancy were within the normal range. Maternal serum. All the steroids in maternal serum showed progres:1ive increase with increasing duration of gestation. The levels of estradiol varied from 0.6 to 22.6 ng/ml and che levels of progesterone varied from
Table IV. Levels of steroids in maternal serum Steroids
14-20 wk.
Testosterone (nglml): 0.744 ± 0.08* M F 0.935 ± 0.2 Androstenedione (ng/ml): M 1.87 ± 0.18 F 1.6 ± 0.35 Dihydrotestosterone (nglml): M 0.22 ± O.oJ F 0.14 ± 0.02t Estradiol (ng/ml): M 5.29 ± 0.5 F 4.6 ± 0.6 Progesterone (ng/ml): M 60.16 ± 9.3 F 51.00 ± 2.4 17-Hydroxyprogesterone (ng/ml): M 2.2 ± 0.2 F 2.5 ± 0.4
26-40 wk.
0.87 0.2 1.38 ± 0.3 3.05 ± 0.9 6.37 ± 1.8 0.42 ± 0.14 0.67 ± 0.17 8.26 ± 2 8.32 ± 2.2 76.73 ± 20 84.4 ± 24 5.2 ± 1.0 5.6 ± 1.0
M = Pregnancies with male fetus; F = pregnancies with female fetus. *Mean± S.E. tSignificantly different from pregnancies with male fetus.
8.6 to 288.8 ng/ml. Testosterone levels ranged from 0.2 to 3.2 ng/ml with a mean level of 1.01 ± 0.1 ng/ml. Androstenedione levels varied from 1.7 to 21.2 ng/ml with a mean level of 3.29 ± 0.51 ng/ml. The mean levels in pregnancies with male and female fetuses are given in Table IV. We did not find any difference in the levels of these steroids depending on the sex of the fetus. The levels of dihydrotestosterone ranged from 0.04 to 2.02 ng/ml with the mean value of 0.35 ± 0.05 ng/ml. The mean level of dihydrotestosterone in the maternal serum in the pregnancies with male fetuses before 20 weeks was 0.22 ± 0.01 ng/ml which was significantly higher than the mean level of 0.14 ± 0.02 ng noted in pregnancies with female fetuses before 20
678 Nagamani et al.
Am.
weeks of gestation (p < 0.02). There was, however, considerable overlap of values. The concentration of 17a-hydroxyprogesterone in maternal serum before 34 weeks ranged from 0. 78 to 4.07 ng/ml with a mean nu/mL Tht>rP 2 --o·----IPvPI of ----·- -----. --------- incrt>ase ------ was a twofold --- 0 - -n-± -- 2 --·-after 34 weeks with a mean level of 5.4 ± 0.6 ng/ml. The 17a-hydroxyprogesterone level in maternal serum in the pregnancy with the fetus affected with congenital adrenal hyperplasia was significantly higher than the control levels after 34 weeks of pregnancy. The 17a-hydroxyprogesterone levels during pregnancy have been reported by us previously. 25 --~-
~
Comment This study compares six steroids in the amniotic fluid of pregnancies with male and female fetuses along with a comparison with steroids circulating in maternal blood. Higher testosterone concentrations in pregnancies with male fetus as compared to female fetus were found with no overiap of vaiues between 14 and 20 weeks of pregnancy. This sex difference persisted in iate pregnancy but with considerable overlap of values. Reyes and associates 10 found significantly higher testosterone levels in the fetal serum when the fetus was male than when it was female. Abramovich and Row" found a similar difference in the testosterone concentrations of serum of male and female fetuses between 12 and 18 lAJeeks, but at term t..ltere was no significant difference. Our results on amniotic fluid testosterone levels seem to follow the same pattern as that reported for fetal serum. Similar results have been reported recently by Warne and colleagues. 12 Judd and coworkers13 also found significantly higher levels of testosterone in the amniotic fluid from pregnancies with male fetuses at midterm. The source of amniotic fluid androstenedione is not very clear. The presence of sex difference in the levels suggests that at least part of this steroid comes from the fetal gonads. In pregnancies with male fetuses we found significantly higher levels of androstenedione between 14 to 20 weeks at which time the fetal testis has been shown to be the site of active steroidogenesis. Robinson and associates 14 have reported similar observations on the sex difference in the levels of androstenedione in midpregnancy. The levels reported by them were three to four times higher than our values. The antiserum for androstenedione used by them was a different one, and no explanation for this difference is available. Close to term, we found significantly higher levels in pregnancies with female fetuses. We are not aware of any report on amniotic androstenedione levels in late pregnancies. Riverola and co-workers 15 did not find any sex difference in the androstenedione levels in the umbilical vein sera of term infants. Payne
J.
Juiy i!J, i97'1 Obstet. Gyneco!.
and Jaffe, ' 6 by in vitro incubation studies ol fet~1l ovaries with 3 H-pregnenolone sulfate, found androstenedione as one of the major C 19 metabolite hmned by the ovaries, although they huled to detect any estradiol. Fetal adrenals also had been shown to be capable of producing androstenedioneH The third androgen we measured in the amniotic fluid was dihydrotestosterone. Dihydrotestosterone has been shown to be the steroid involved in the differentiation of urogenital sinus to prostate and external genitals. The rate of conversion of testosterone to dihydrotestosterone has been shown to be most rapid in the tissues of urogenital sinus and urogenital tubercle. 1 Significant amounts of dihydrotestosterone were present in the amniotic fluid of pregnancies with male fetuses and it was below the limit of detectability of the assay in the pregnancies with female fetuses. Dawood and Saxena 17 were not able to lind any dihydrotestosterone amniotic fluid. Robinson and colieagues 14 found significantly higher amniotic fluid estradiol concentration in pregnancies with female fetuses at midterm. In addition, the levels of estradiol reported by them were higher than ours. We did not find any significant sex difference in the levels of this steroid and the reason for the discrepancy is not clear at this time. The source of estradiol in the amniotic fluid is still speculative. Payne and Jaffe, 16 by in vitro incubation studies with radioactive precursors, failed to detect any estradiol in the fetal ovaries. Reyes and associates 6 found little or no estradiol in the fetal testis, ovaries, or adrenals. Fetal granulosa cells lacked ultrastructural features associated with steroidogenic activity, 18 and theca interna, a principal source of steroid hormones in the mature ovary, does not differentiate until the follicle development is well established. It seems likely that placenta is probably the principal source of the estradiol in the amniotic fluid. Progesterone levels in amniotic fluid were higher between 14 and 20 weeks of pregnancy than toward term, with no sex difference in the levels. Our results are comparable to those of Warne and associates 12 with the exception that the decrease in progesterone at term observed by them in pregnancies with male fetuses was not observed by us. Hageman and Kittinger 19 reported that the progesterone concentrations in the umbilical vein were always higher than in the umbilical artery. Billair and co-workers 20 i~ected 3H-pregnenolone sulfate during midgestation and reported 50% extraction of progesterone by the fetus. Placenta via the fetus is probably the principal source of progesterone in the amniotic fluid. Hageman and Kittinger 19 found that the progesterone concentrations in the umbilical vein at term did not show any sex difference but the umbilical arteriovenous difference at term was higher for fe-
Volume 134 Number 6
male fetuses than for maie fetuses. 1 nese authors speculated tha1t the female fetuses seem to metabolize progesterone to a greater extent than male fetuses. Attempts had been made to diagnose congenital adrenal hyperplasia in utero by measuring different steroids like 17-ketosteroids and pregnanetriol in the amniotic fluid but the results have not been uniform. 2 1. 22 Frasier and associates 23 have reported that testosterone concentrations in the amniotic fluid from a pregnancy in which the fetus was affected with congenital adrenal hyperplasia were within the range found in control pregnancies. This is in agreement with our results of normal testosterone levels in the affected pregnancy with the exception of the sample at 22 weeks of pregnancy. Measurement of 17a-hydroxyprogesterone levels in the amniotic fluid had been shown to be more promising in the prenatal diagnosis of this disorder. 24 • 25 Previous reports on steroidogenic function of trophoblastic tissue in molar pregnancy are limited. Toeh and co-workers 26 found very high concentrations of progesterone in the mole vesicular fluid. Dawood and co-workers 27 • 211 have reported elevated levels of progesterone and e~;tradioi in maternai peripherai biood. Presently we do not have any data available on the levels of androgenic steroids in molar tissue. We found elevated levels of progesterone, estradiol, testosterone 17a-hydroxyprogesterone, and dihydrotestosterone in the amniotic fluid from the pregnancy which ended in an abortion and showed extensive hydatiform degeneration of the placenta (Table III). The mean level of testosterone in the maternal serum is comparable to the levels reported by other investigations during pregnancy. 29 This increase could be explained by the increased steroid-binding globulin levels during pregnancy and increased binding. The production rate of testosterone during pregnancy had been shown to be not significantly different from the rate seen in nonpregnant state while the metabolic clearance rate is significantly reduced because of increased binding. 30 The level of androstenedione was also higher in the pregnant serum compared to the levels noted in cycling females. 9 Gandy31 found similar increased levels of androstenedione during pregnancy, while Rivarola and associates 15 did not find any significant increase. Dihydrotestosterone was also found in higher concentrations during pregnancy than in the nonpregnant state. 9 The pr~::~duction rate of dihydrotestosterone had been reported to be similar in pregnant and cycling women. 31 It is interesting to find increased levels of dihydrotestosterone in the maternal peripheral blood in pregnancies with male fetus than in pregnancies with female fetuses. This raises the possibility that the
Maternal and amniotic steroids throughout pregnancy 679
fetus might be contributing to some of this steroid in the maternal serum. It is hard to explain why this difference did not persist throughout the late stages of pregnancy. Another steroid in the maternal serum to which the fetus seems to contribute significantly is 17a-hydroxyprogesterone in later stages of pregtlancy. T}-l...is is shown by the fact that increased levels were found in our patient with congenital adrenal hyperplasia and marked fall in levels were noted by Tulchinsky and Simmer32 in anencephaly and intrauterine death of the fetus. Our results on the levels of progesterone in the maternal serum are comparable to the levels reported by other investigators. 33 • 34 It is fairly well established that placenta is the main site of progesterone production in mid- and late pregnancy. 35 • 36 Estradiol is mainly produced by the placenta by way of aromatization of androgenic steroids. Tulchinsky and Korenman 37 found that maternal dehydroepiandrosterone sulfate contributed to 60% of estradiol that is formed during pregnancy. Lindberg and associates 38 measured the estradiol and estriol levels in ten healthy pregnant women and the ieveis reported by these authors are close to our results. In conclusion, the maternal compartn1ent does not seem to contribute significantly to the steroids in amniotic fluid since we did not find any correlation between the levels in the maternal serum and amniotic fluid. Several steroids in the amniotic fluid were found in higher concentration in early pregnancy (taking into account the sex of the fetus) while the levels in the maternal serum showed increasing levels with increasing duration of gestation. Since the concentration of steroids found in the amniotic fluid does not correlate with the levels in maternal serum, it leads to the speculation that the fetus is probably the major source of various steroids in the amniotic fluid. There are two possible explanations for the observations of higher concentration of steroids in the amniotic fluid in midpregnancy than in late pregnancy. First, the increased concentrations may indicate increased production of these steroids by the fetus at the critical period of sexual differentiation in midpregnancy. Second, it may be due to the difference in the sources of the amniotic fluid in different stages of pregnancy. In early pregnancy, the steroids possibly enter the amniotic fluid by way of passive diffusion through the fetal skin at which time the steroid levels in the amniotic fluid may closely reflect the levels in the fetal serum. After 20 weeks, the fetal skin gradually becomes thick and cornified and fetal urine becomes an important source. Studies are now underway to correlate the steroid levels in umbilical cord blood and amniotic fluid.
680 Nagamani et al. Am.
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and metabolism during male sexual differentiation in the human embryo,]. Clin. Endocrinol. Metab. 58:113, 1974. 2. Payne, A. H., and Jaffe, R. B.: Androgen formation from pregnenolone sulfate by fetal, neonatal, prepubertal and adult human testis, J. Clin. Endocrinol. Metab. 40:102, 1975. 3. Jost, A.: Problems of fetal endocrinology: The gonadal and hypophyseal hormones, Recent Prog. Horm. Res. 8:379, 1953. 4. Wilson, I. D., and Lasnitzki. 1.: Dihvdrotestosterone formation in fetal tissues of th~ rabbit 'and rat, Endocrinology 89:659, 1971. 5. Wilson,]. D.: Testosterone uptake by the urogenital tract of rabbit embryo, Endocrinology 92: 1192, 1973. 6. Reyes, F. 1., Winter, J. S. D., and Faiman, C.: Studies on human sexual development. I. Fetal gonadal and adrenal sex steroids,]. Clin. Endocrinol. Metab. 57:74, 1973. 7. Lind, T., Parkin, F. M., and Cheyne, G. A.: Biochemical and cytoiogicai changes in iiquor amnii with advancing gestation, Br. J. Obstet. Gynaecol. 76:673, 1969. 8 . .Jeffcoate, T. N. A., and Scott, _f. S.: Polyhydramnios and -oligohydramnios, Can. Med. Assoc. J. 80:77, 1959. 9. Parker, R. C., Ellegood, J. 0., and Mahesh, V. B.: Methods of multiple steroid radioimmunoassay, J. Steroid Biochem. 6:1, 1975. 10. Reyes, F. I., Boroditsky, R. S., Winter, J. S. D., and Faiman, C.: Studies on human sexual development. II. Fetal and maternal serum gonadotropin and sex steroid concentrations,]. Clin. Endocrinol. Metab. 58:612, 1974. i L. Abramovich, D. R., and Row, D.: Fetal plasma testosterone levels in mid pregnancy and at term. Relationship to fetal sex, J. Endocrinol.56:621, 1973. 12. Warne, G. 1., Faiman, C. Reyes, F. 1., and Winter,]. S.D.: Studies on human sexual development. V. Concentrations of testosterone, 17a-hydroxyprogesterone and · progesterone in human amniotic fluid throughout gestation, J. Clin. Endocrinol. Metab. 44:934, 1977. 13. Judd, H. L., Robinson, J. D., Young, P. E., and Jones, 0. W.: Amniotic fluid testosterone levels in mid pregnancy, Obstet. Gynecol. 48:690, 1976. 1.41 J~.
15.
16. 17. 18. 19. 20.
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25.
26.
27.
28. 29. 30.
31. 32.
1:.1.1
L.,JUUe:S, V. Vl'.,
and Yen, S.C.: Amniotic fluid androgens and estrogens in mid gestation, J. Clin. Endocrinol. Metab. 45:755, 1977. Rivarola, M.A., Forest, M.D., and Migeon, C.J.: Testosterone, androstenedione and dehydroepiandrosterone in plasma during pregnancy and delivery: Concentration and protein binding, J. Clin. Endocrinol. Metab. 82:34, 1968. Payne, A. H., and Jaffe, R. B.: Androgen formation from pregnenolone sulfate by the human fetal ovary, J. Clin. Endocrine!. Metab. !9:300, 1974. Dawood, M. V., and Saxena, B. B.: Testosterone and dihydrotestosterone in maternal and cord blood and in atnniotic fluid, AM. j. 0BSTET. GYNECOL. 129:37, 1977. Gondos, B., Bhiraleus, P., and Hobel, C. J.: Ultrastructural observations on germ cells in human fetal ovaries, AM. j. 0BSTET. GYNECOL. 110:644, 1971. Hagmen, F. C., and Kittinger, G. W.: The influence of fetal sex on the levels of plasma progesterone in the human fetus, J. Clin. Endocrinol. Metab. 56:389, 1973. Billair, R. B., Jassani, M., Saarikoski, S., and Little, B.: Pregnenolone sulfate metabolism in vivo and uterine ex-
33.
34. 35. 36.
J.
july 15, 1979 Obstet. Gynecol.
traction at mid gestation. J _ Clin. Endocrino!. Metab. 59:27, 1974. Nichols, J.: Antenatal diagnosis of adrenocortical hyperplasia, Lancet 1:1151, 1969. Merkatz, I. R., New, M. 1., Peterson, R. E., and Searman, M. P.: Prenatal diagnosis of adrenogenital syndrome by amniocentesis, J. Pediatr. 75:977, 1969. Frasier, S. D., Weiss, B. A., and Oston, R. H.: Amniotic fluid testosterone: Implications for prenatal diagnosis of congenital adrenal hyperplasia, J. Pediatr. 84:738, 1974. Frasier, S.D., Thorneycroft, I. H., Weiss, B. A., and Horton, R.: Elevated amniotic fluid concentration of 17ahydroxyprogesterone in congenital adrenal hyperplasia, ]. Pediatr. 86:310, 1975. Nagamani, M., McDonough, P. G., Ellegood, J. 0., and Mahesh, V. B.: Maternal and amniotic fluid 17a-hydroxyprogesterone levels during pregnancy: Diagnosis of congenital adrenal hyperplasia in utero, AM. J. 0BSTET. GYNECOL. 150:791, 1978. Teoh, E. S., Das, N. D., Dawood, M. Y., and Ratnam, S. S.: The source of circuiating progesterone and 17ahydroxyprogesterone in hydatidiform mole, Acta Endocrinol. 71:773, 1972. Dawood, M. Y., Ratnam, S. S., and Teoh, E. S.: Serum estradiol-17/3 and serum human chol'ionic gonadotropin in patients with hydatidiform moles, AM. J. OBSTET. GYNECOL. ll9:904, 1974. Dawood, M. Y.: Progesterone concentrations in the sera of patients with intact and aborted hydatidiform moles, AM.j. 0BSTET. GYNECOL. ll9:911, 1974. Demish, K., Grant,]. K., and Black, H.: Plasma testosterone in women in iate pregnancy and after deiivery,]. Endocrinol. 42:477, 1968. Sally, J. M., Forest, M. G., Morera, A. M., and Bertraud, J .: Metabolic clearance rate and blood productions rate of testosterone and dihydrotestosterone in normal subjects during pregnancy and in hyperthyroidism, J. Clin. Invest. 51:1226, 1972. Gandy, H. M.: Androgens, in Fuchs F., and Klopper, A., editors: Endocrinology of Pregnancy, New York, 1977, Harper & Row, Publishers, p. 145. Tulchinsky, D., and Simmer, H.: Sources of plasma 17ahydroxyprogesterone in human pregnancy, j. Clin. Endocrinol. Metab. 35:799, 1972. Johansson, E. D. B., and Jonasson, L. E.: Progesterone ieveis in amniotic fiuid and piasma from women. I. Leveis during normal pregnancies, Acta Obstet. Gynecol. Scand. 50:339, 1971. Wiest, W. G.: Estimation of progesterone in biological tissues and fluids from pregnant women by double isotope derivative assay, Steroids 10:279, 1967. Lurie, A. 0., Reid, D. E., and Vi1lee, C. A.: The role of the fetus and placenta in maintainance of plasma progesterone, i\M. ]. OssTET. GYNECOL. 96:67-o, !966. Ryan, K. J., Meigs, R., and Petro, Z.: The formation of progesterone by human placenta, AM. J. OssTET. GY-·----
ftL'!.I':.'.,.,C
1fiCC
NJ!.LUL. :ru;U/U 1 1!:1UU.
37. Tulchinsky, D., and Korenman, S. G.: The plasma estradiol as an index of fetoplacental function, J. Ctin. Invest. 50:1490, 1971. 38. Lindberg, B. S., Johansson, E. D. B., and Nilsson, B. A.: Plasma levels of nonconjugated estrone, estradiol, 17{3 estradiol during uncomplicated pregnancy, Acta Obstet. Gynecol. Scand. (Suppl.) 52: I, 1974.
• '...l •L L .._ StefOluS tnrougnOUt
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MANUBAI NAGAMANI, M.D.* PAUL G. McDONOUGH, M.D. JAMES 0. ELLEGOOD, M.S.
VIRENDRA B. MAHESH, PH.D., D.PHIL.
Augusta, Georgza Concentrations of testosterone, dihydrotestosterone, androstenedione, progesterone, 17a-hydroxyprogesterone, and estradiol were measured by radioimmunoassay in the amniotic fluid ar.d maiemal peripherai biood obtained from normal pregnancies between 14 and 40 weeks oi gestation. There was a sex difference in t-he levels of aU the androgenic steroid$ in the amniotic fluid before 20 weeks wJth higher levels jn pregnancies with me!e fett-.•Jses. ~"rnniotic fil-.Jid 17a-hydroxyprogesterone levels were significantly elevated in a pregnancy with the fetus affected with congenital adrenal hyperplasia. The levels of all the steroids in the amniotic fluid were significantly elevated in the pregnancy with molar degener-ation of the placenta. There was a sex difference in the levels of dihydrotestosterone in the maternal peripheral blood before 20 weeks with higher levels in pregnancies with male fetuses. There was no correlation between the steroid levels in the maternal serum and amniotic fluid even though most of the samples of maternal serum were drawn at the sa.rns time as a.-nniocantasis. (AM. J. OasTEi. GYNECOL. 134:674, 1979.)
studies on the endocrine function of fetal gonads show that human fetal testis in contrast to ovary secretes significant amounts of testosterone at the period of sexual differentiation in utero.L 2 Jost, 3 by his experiments on fetal rabbits, had shown that the development of the male phenotype, except for regression of the miillerian system, depends on the secretion of testosterone by the fetal testis at this critical period. It is also clear from the work of Wilson and Lasnitzki 4 • 5 that testosterone itself is responsible for the differentiation of wolffian duct into epididymis, vas differens, and seminal vesicle, and dihydrotestosterone is probably the intraceiiuiar mediator in the differentiation of uroPREVIOUs
From the Departments of Endocrirwlogy and Obstetrics and Gynecology, Medical College of Georgia. Supported by General Research Support Grant No. 5507 RRo-5365-16 and Training Grant No.5 TO! HD OJ 12-09 from the National Institutes of Health. Received for publication August 3, 1978.
genital sinus and urogenital tubercle to prostate and external genitalia. Estradiol does not seem to play a significant role in the process of sexual differentiation and no significant amounts of this steroid had been detected in the fetal testis or ovaries at this stage of pregnancy.6 The fetus plays a considerable role in the formation of amniotic fluid in all stages of pregnancy. Before 20 weeks, amniotic fluid possibly bears a closer relationship to fetal plasma in its concentration of the steroids because of the free diffusion occurring through the fetal skin which had been shown by electron microscopy to be a meshiike structure with numerous microvilli. 7 After 20 weeks. fetal urine becomes an important source. 8 The present srudy was designed to measure various steroids in the amniotic fluid to see whether this approach could be utilized to study the hormonal events occurring in the fetus during different stages of pregnancy. Steroids were also measured in maternal peripheral blood for comparison.
Revised September 28, 1978. Accepted October 2, 1978. Reprint requests: Dr. V. B. Mahesh, Department of Endocrirwlogy, Medical College of Georgia, AugU!ita, Georgia 30901. *Present address: Department of Obstetrics and Gynecology, University of Texas Medical Branch, Galveston, Texas 77550.
674
Material and rnelhode One hundred one samples of amniotic fluid were obtained between 14 and 40 weeks of pregnancy when amniocentesis was done for cytogenetic studies in early pregnancy and for fetal maturity in late pregnancy. All the pregnancies were normal without any complica0002-9378179/140674+07$00.70/0
©
1979 The C. V. Mosby Co.
Volume 134 Number6
Maternal and amniotic steroids thrc.ughout pregnancy
675
Table I. Levels of testosterone, androstenedione, dihydrotestosterone, estradiol, progesterone, and 17a-hydroxyprogesterone in the amniotic fluid Steroids
14-20 wk.
26-40 wk.
0.188 ::!: 0.014* (0.086-0.477)t 0.022 ::!: 0.002* (0.006-0.064)
0.107::!: 0.015 (0.068-0.172) 0.056 ::!: 0.009* (0.021-0.132)
0.348 ::!: 0.032 (0.065-0.900) 0.156::!: 0.029* (0.011-0.810)
0.187::!: 0.027 (0.04-0.361) 0.331 ::!: 0.038* (0.137-0.492)
0.024 ::!: 0.003 (0.008-0.067) Undetectable
0.026 ::!: 0.006 (0.011-0.064) Undetectable
0.036 ± 0.009 (0.009-0.065) 0.048 ± 0.009 (0.011-0.141)
0.043 ± 0.009 (0.008-0.115) 0.067 ± 0.013 (0.018-0.147)
TestosterofU! (ng!ml):
M F
AndrosteP..edioP..e (ng/ml): M F Dihydrotesto\tero;u (ng/mlj: M F Estradiol (nglml): M F Progesteron~ (nglml):
M F 17-HydroxyprogesterofU! (nglml): M F
34.7 ± 4.1 (7-97) 30.1 ± 4.0 (6-148)
14.1 ± 3.1 (7-31) 13.3 ± 2.1 (4-20)
1.62 ± 0.15 (0.39-3.23) !.5! ± 0.! (0.44-2.66)
0.87 ± 0.15 (0.25-1.25) 0.69 ::!: 0.08 (0.31- !.47)
M = Pregnancies with male fetuses; F "" pregnancies with female fetuses. *Mean levels :':: S.E. tRange. *Significantly different from male fetuses.
tions. Fifty-one samples were from pregnancies with male fetuses and 50 samples were from pregnancies with female fetuses. The sex of each fetus was determined at the time of deiivery or by karyotype of amniotic fluid cells if undelivered. Seventy-six samples were between 14 and 20 weeks of pregnancy and 25 samples were between 26 and 40 weeks of pregnancy. Additionally, one sample of fluid was obtained from a pregnancy ( 17 weeks) which ended in an abortion, and the abortus contained a fetus lvhose karyotype sho,ved triploidy (69, XXX) and extensive molar degeneration of the placenta. There were also four samples of amniotic fluid obtained from different stages of pregnancy in a patient who subsequently delivered a male fetus affected with congenital adrenal hyperplasia (21-hydroxylase deficiency). Sixty samples of maternal peripheral vein blood were obtained from different stages of normal pregnancies. The serum and amniotic fluid samples were stored at -20" C until assayed. Each sample was assayed for testosterone, androstenedione, dihydrotestosterone, estradiol, progesterone, and 17a-hydroxyprogesterone. Fetal age was obtained by subtracting two weeks from the time since the last menstrual pc:riod. If the last menstrual period was not known, the length of gestation was determined by ultrasonic measurement of fetal biparietal diameter. Student's t test and Duncan's muitirange analysis were used for statistical analysis. Hormone assays. All the steroids were measured by radioimmunoassay after fractionation of the steroids by Celite microcolumn chromatography as reported
earlier. 9 Three milliliters of amniotic fluid or 3 ml of serum was extracted twice with three volumes of ether after adding 3H tracers of progesterone, androstenedione, dehydroepiandrosterone, testosterone, and estradiol for recovery calculations. The extracts were dried under nitrogen and subjected to chromatography on microcelite column with ethylene and propylene glycol as stationary phase. The chromatographic separation was effective in removal of steroids that \vou!d interfere in the assay due to cross=reactivity. For example, progesterone and 17a-hydroxyprogesterone; androstenedione, dihydrotestosterone, and testosterone; and estrone and estradiol appeared in separate fractions and estriol was not eluted from the column. 9 The various fractions were collected and dried, and the residue was redissolved in 2 ml of assay buffer. Duplicate aliquots of different volumes of this were used in the assay and 0.1 ml was used for recovery calculations. Blank and control sera (pooled male serum to serve as internal control between assays) were run with each assay. The coefficient of variation between assays had been 5.4% for progesterone, 4.3% for androstenedione, 5.6% for testosterone, 5.8% for dihydrotestosterone, 8.5% for 17 a-hydroxyprogesterone, and 8.9% for estradiol. The precision, specificity, and sensitivity for the assay system had been reported previousiy. 9 3 H-progesterone, 3 H-testosterone, and 3 H-estradiol were added to amniotic fluid. After extTaction, Celite microcoiumn chromatography was carried out as previously described with the exception that 1 ml aliquots of the column eluate were collected.
676 Nagamani et al.
july 15, 1979 Am. J. Obstet. Gynecol.
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0.05). There was considerable overlap in levels in pregnancies with male fetuses and female fetuses (Fig. 2).
AeeuHs Amniotic fluid. The mean testosterone concentrations in 10 I samples of amniotic fluid are shown in Table I. At all stages of pregnancy, the levels of testosterone were significantly higher when the fetus was male and the difference was significant both in midpregnancy and in late pregnancy (p < 0.001). Highest levels were noted in the cases of a male fetus at 16 weeks. Between 14 and 20 weeks, the levels of testosterone in pregnancies with male fetuses varied from 0.086 to 0.477 ng/ml (mean 0.188 ± 0.014) and the levels with female fetuses varied from 0.006 to 0.064 ng/ml (mean 0.022 ± 0.002) with no overlap of vaJues (Fig. 1). Toward term, the difference in mean testosterone levels depending on the sex of the fetus persisted, but there was considerable overlap in values (Fig. 1).
Volume 134 Number 6
Maternal and amniotic steroids throughout pregnancy 677
Table II. Amniotic fluid steroids in a pregnancy with a male fetus having congenital adrenal hyperplasia of pregrumcy
T (ng/ml)
A (ng/ml)
DHT (nglml)
14 22 28 34
0.22 0.606 0.251 0.200
0.345 1.224 0.826
0.026
Week.~
0.040
Abbreviations: T testosterone, A = androstenedione, DHT OHP = 17a.-hydroxyprogesterone.
£2
p
(nglml)
(nglml)
0.106 0.067 0.046
15 15 20
I7a.-OHP (ng/ml)
8.23 20.68 2.93 3.67 progesterone, 17 a.-
dihydrotestosterone, E2
Table III. Amniotic fluid steroids in a pregnancy with hydatidiform degeneration of placenta (nglml)
.4 (ng/ml)
DHT (ng/ml)
(nglml)
p (nglml)
17a.-OHP (nglml)
0.129
0.482
0.050
0.466
160.0
6.24
0.022 ± 0.002
0.156 ± 0.029
Undetectable
0.048 ± 0.009
30.1 ± 0.4
1.51±0.1
T
Affected pregnancy Control
E,
See Table II fo:o abbreviations.
Progesterone levels in amniotic fluid are shown in Fig. 2 and Table I. The levels were significantly higher (p < 0.05) betwt!en 14 and 20 weeks than toward term. There was no difference in the levels depending on the sex of the fetus. The 17a-hydroxyprogesterone levels in the amniotic Huid also showed a similar trend with a higher level in early pregnancy (p < 0.01; Table I) with no sex difference in the levels. In the samples of fluid obtained from a patient who subsequently delivered a male fetus affected with congenital adrenal hyperplasia, the concentration of testosterone and androstenedione was higher than the control levels in one sample obtained at 22 weeks, but the levels in other samples were not significantly different from the control levels (Table II). The levels of progesterone, dihydrotestosterone, and estradiol in these samples were within the range of the control samples. However, 17a-hydroxyprogesterone levels were elevated above the control levels in all stages of pregnancy (Table II). The maximum elevation was at 22 weeks of pregnancy. In the sample of amniotic fluid obtained from a pregnancy in which there was extensive hydatidiform degeneration of the placenta, the level of progesterone, 17a-hydroxypmgesterone, testosterone, dihydrotestosterone, and estradiol were significantly higher than in control samples (Table III). Among the control pregnancies one fetus was found to be mongoloid (trisomy 21) by karyotype of amniotic fluid cells and this pregnancy was subsequently aborted. The steroid levels in the fluid obtained from this pregnancy were within the normal range. Maternal serum. All the steroids in maternal serum showed progres:1ive increase with increasing duration of gestation. The levels of estradiol varied from 0.6 to 22.6 ng/ml and che levels of progesterone varied from
Table IV. Levels of steroids in maternal serum Steroids
14-20 wk.
Testosterone (nglml): 0.744 ± 0.08* M F 0.935 ± 0.2 Androstenedione (ng/ml): M 1.87 ± 0.18 F 1.6 ± 0.35 Dihydrotestosterone (nglml): M 0.22 ± O.oJ F 0.14 ± 0.02t Estradiol (ng/ml): M 5.29 ± 0.5 F 4.6 ± 0.6 Progesterone (ng/ml): M 60.16 ± 9.3 F 51.00 ± 2.4 17-Hydroxyprogesterone (ng/ml): M 2.2 ± 0.2 F 2.5 ± 0.4
26-40 wk.
0.87 0.2 1.38 ± 0.3 3.05 ± 0.9 6.37 ± 1.8 0.42 ± 0.14 0.67 ± 0.17 8.26 ± 2 8.32 ± 2.2 76.73 ± 20 84.4 ± 24 5.2 ± 1.0 5.6 ± 1.0
M = Pregnancies with male fetus; F = pregnancies with female fetus. *Mean± S.E. tSignificantly different from pregnancies with male fetus.
8.6 to 288.8 ng/ml. Testosterone levels ranged from 0.2 to 3.2 ng/ml with a mean level of 1.01 ± 0.1 ng/ml. Androstenedione levels varied from 1.7 to 21.2 ng/ml with a mean level of 3.29 ± 0.51 ng/ml. The mean levels in pregnancies with male and female fetuses are given in Table IV. We did not find any difference in the levels of these steroids depending on the sex of the fetus. The levels of dihydrotestosterone ranged from 0.04 to 2.02 ng/ml with the mean value of 0.35 ± 0.05 ng/ml. The mean level of dihydrotestosterone in the maternal serum in the pregnancies with male fetuses before 20 weeks was 0.22 ± 0.01 ng/ml which was significantly higher than the mean level of 0.14 ± 0.02 ng noted in pregnancies with female fetuses before 20
678 Nagamani et al.
Am.
weeks of gestation (p < 0.02). There was, however, considerable overlap of values. The concentration of 17a-hydroxyprogesterone in maternal serum before 34 weeks ranged from 0. 78 to 4.07 ng/ml with a mean nu/mL Tht>rP 2 --o·----IPvPI of ----·- -----. --------- incrt>ase ------ was a twofold --- 0 - -n-± -- 2 --·-after 34 weeks with a mean level of 5.4 ± 0.6 ng/ml. The 17a-hydroxyprogesterone level in maternal serum in the pregnancy with the fetus affected with congenital adrenal hyperplasia was significantly higher than the control levels after 34 weeks of pregnancy. The 17a-hydroxyprogesterone levels during pregnancy have been reported by us previously. 25 --~-
~
Comment This study compares six steroids in the amniotic fluid of pregnancies with male and female fetuses along with a comparison with steroids circulating in maternal blood. Higher testosterone concentrations in pregnancies with male fetus as compared to female fetus were found with no overiap of vaiues between 14 and 20 weeks of pregnancy. This sex difference persisted in iate pregnancy but with considerable overlap of values. Reyes and associates 10 found significantly higher testosterone levels in the fetal serum when the fetus was male than when it was female. Abramovich and Row" found a similar difference in the testosterone concentrations of serum of male and female fetuses between 12 and 18 lAJeeks, but at term t..ltere was no significant difference. Our results on amniotic fluid testosterone levels seem to follow the same pattern as that reported for fetal serum. Similar results have been reported recently by Warne and colleagues. 12 Judd and coworkers13 also found significantly higher levels of testosterone in the amniotic fluid from pregnancies with male fetuses at midterm. The source of amniotic fluid androstenedione is not very clear. The presence of sex difference in the levels suggests that at least part of this steroid comes from the fetal gonads. In pregnancies with male fetuses we found significantly higher levels of androstenedione between 14 to 20 weeks at which time the fetal testis has been shown to be the site of active steroidogenesis. Robinson and associates 14 have reported similar observations on the sex difference in the levels of androstenedione in midpregnancy. The levels reported by them were three to four times higher than our values. The antiserum for androstenedione used by them was a different one, and no explanation for this difference is available. Close to term, we found significantly higher levels in pregnancies with female fetuses. We are not aware of any report on amniotic androstenedione levels in late pregnancies. Riverola and co-workers 15 did not find any sex difference in the androstenedione levels in the umbilical vein sera of term infants. Payne
J.
Juiy i!J, i97'1 Obstet. Gyneco!.
and Jaffe, ' 6 by in vitro incubation studies ol fet~1l ovaries with 3 H-pregnenolone sulfate, found androstenedione as one of the major C 19 metabolite hmned by the ovaries, although they huled to detect any estradiol. Fetal adrenals also had been shown to be capable of producing androstenedioneH The third androgen we measured in the amniotic fluid was dihydrotestosterone. Dihydrotestosterone has been shown to be the steroid involved in the differentiation of urogenital sinus to prostate and external genitals. The rate of conversion of testosterone to dihydrotestosterone has been shown to be most rapid in the tissues of urogenital sinus and urogenital tubercle. 1 Significant amounts of dihydrotestosterone were present in the amniotic fluid of pregnancies with male fetuses and it was below the limit of detectability of the assay in the pregnancies with female fetuses. Dawood and Saxena 17 were not able to lind any dihydrotestosterone amniotic fluid. Robinson and colieagues 14 found significantly higher amniotic fluid estradiol concentration in pregnancies with female fetuses at midterm. In addition, the levels of estradiol reported by them were higher than ours. We did not find any significant sex difference in the levels of this steroid and the reason for the discrepancy is not clear at this time. The source of estradiol in the amniotic fluid is still speculative. Payne and Jaffe, 16 by in vitro incubation studies with radioactive precursors, failed to detect any estradiol in the fetal ovaries. Reyes and associates 6 found little or no estradiol in the fetal testis, ovaries, or adrenals. Fetal granulosa cells lacked ultrastructural features associated with steroidogenic activity, 18 and theca interna, a principal source of steroid hormones in the mature ovary, does not differentiate until the follicle development is well established. It seems likely that placenta is probably the principal source of the estradiol in the amniotic fluid. Progesterone levels in amniotic fluid were higher between 14 and 20 weeks of pregnancy than toward term, with no sex difference in the levels. Our results are comparable to those of Warne and associates 12 with the exception that the decrease in progesterone at term observed by them in pregnancies with male fetuses was not observed by us. Hageman and Kittinger 19 reported that the progesterone concentrations in the umbilical vein were always higher than in the umbilical artery. Billair and co-workers 20 i~ected 3H-pregnenolone sulfate during midgestation and reported 50% extraction of progesterone by the fetus. Placenta via the fetus is probably the principal source of progesterone in the amniotic fluid. Hageman and Kittinger 19 found that the progesterone concentrations in the umbilical vein at term did not show any sex difference but the umbilical arteriovenous difference at term was higher for fe-
Volume 134 Number 6
male fetuses than for maie fetuses. 1 nese authors speculated tha1t the female fetuses seem to metabolize progesterone to a greater extent than male fetuses. Attempts had been made to diagnose congenital adrenal hyperplasia in utero by measuring different steroids like 17-ketosteroids and pregnanetriol in the amniotic fluid but the results have not been uniform. 2 1. 22 Frasier and associates 23 have reported that testosterone concentrations in the amniotic fluid from a pregnancy in which the fetus was affected with congenital adrenal hyperplasia were within the range found in control pregnancies. This is in agreement with our results of normal testosterone levels in the affected pregnancy with the exception of the sample at 22 weeks of pregnancy. Measurement of 17a-hydroxyprogesterone levels in the amniotic fluid had been shown to be more promising in the prenatal diagnosis of this disorder. 24 • 25 Previous reports on steroidogenic function of trophoblastic tissue in molar pregnancy are limited. Toeh and co-workers 26 found very high concentrations of progesterone in the mole vesicular fluid. Dawood and co-workers 27 • 211 have reported elevated levels of progesterone and e~;tradioi in maternai peripherai biood. Presently we do not have any data available on the levels of androgenic steroids in molar tissue. We found elevated levels of progesterone, estradiol, testosterone 17a-hydroxyprogesterone, and dihydrotestosterone in the amniotic fluid from the pregnancy which ended in an abortion and showed extensive hydatiform degeneration of the placenta (Table III). The mean level of testosterone in the maternal serum is comparable to the levels reported by other investigations during pregnancy. 29 This increase could be explained by the increased steroid-binding globulin levels during pregnancy and increased binding. The production rate of testosterone during pregnancy had been shown to be not significantly different from the rate seen in nonpregnant state while the metabolic clearance rate is significantly reduced because of increased binding. 30 The level of androstenedione was also higher in the pregnant serum compared to the levels noted in cycling females. 9 Gandy31 found similar increased levels of androstenedione during pregnancy, while Rivarola and associates 15 did not find any significant increase. Dihydrotestosterone was also found in higher concentrations during pregnancy than in the nonpregnant state. 9 The pr~::~duction rate of dihydrotestosterone had been reported to be similar in pregnant and cycling women. 31 It is interesting to find increased levels of dihydrotestosterone in the maternal peripheral blood in pregnancies with male fetus than in pregnancies with female fetuses. This raises the possibility that the
Maternal and amniotic steroids throughout pregnancy 679
fetus might be contributing to some of this steroid in the maternal serum. It is hard to explain why this difference did not persist throughout the late stages of pregnancy. Another steroid in the maternal serum to which the fetus seems to contribute significantly is 17a-hydroxyprogesterone in later stages of pregtlancy. T}-l...is is shown by the fact that increased levels were found in our patient with congenital adrenal hyperplasia and marked fall in levels were noted by Tulchinsky and Simmer32 in anencephaly and intrauterine death of the fetus. Our results on the levels of progesterone in the maternal serum are comparable to the levels reported by other investigators. 33 • 34 It is fairly well established that placenta is the main site of progesterone production in mid- and late pregnancy. 35 • 36 Estradiol is mainly produced by the placenta by way of aromatization of androgenic steroids. Tulchinsky and Korenman 37 found that maternal dehydroepiandrosterone sulfate contributed to 60% of estradiol that is formed during pregnancy. Lindberg and associates 38 measured the estradiol and estriol levels in ten healthy pregnant women and the ieveis reported by these authors are close to our results. In conclusion, the maternal compartn1ent does not seem to contribute significantly to the steroids in amniotic fluid since we did not find any correlation between the levels in the maternal serum and amniotic fluid. Several steroids in the amniotic fluid were found in higher concentration in early pregnancy (taking into account the sex of the fetus) while the levels in the maternal serum showed increasing levels with increasing duration of gestation. Since the concentration of steroids found in the amniotic fluid does not correlate with the levels in maternal serum, it leads to the speculation that the fetus is probably the major source of various steroids in the amniotic fluid. There are two possible explanations for the observations of higher concentration of steroids in the amniotic fluid in midpregnancy than in late pregnancy. First, the increased concentrations may indicate increased production of these steroids by the fetus at the critical period of sexual differentiation in midpregnancy. Second, it may be due to the difference in the sources of the amniotic fluid in different stages of pregnancy. In early pregnancy, the steroids possibly enter the amniotic fluid by way of passive diffusion through the fetal skin at which time the steroid levels in the amniotic fluid may closely reflect the levels in the fetal serum. After 20 weeks, the fetal skin gradually becomes thick and cornified and fetal urine becomes an important source. Studies are now underway to correlate the steroid levels in umbilical cord blood and amniotic fluid.
680 Nagamani et al. Am.
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