Study on Maternal, Fetal, and Amniotic Human Prolactin at Term VICTOR S. FANG AND MOON H. KIM1 Department of Medicine and Department of Obstetrics and Gynecology, the Pritzker School of Medicine, University of Chicago, Chicago, Illinois 60637 ABSTRACT. Prolactin present in maternal blood, fetal blood, and amniotic fluid samples obtained simultaneously at term was examined. The relative concentrations of prolactin in maternal and fetal serum samples were found to vary with each individual case. However, amniotic fluid samples contained a much higher level of prolactin than did both maternal and fetal serum samples in eveiy case. After gel filtration, three separate peaks of immunoreactive prolactin were observed, probably representing three different sizes of the hormone molecule. The main component was small prolactin, constituting at least 69% of the total prolactin immunoreactivity in blood samples and 90% in amniotic fluids. Prolactin was found to be very similar
I
T HAS been demonstrated that immunoreactive human prolactin (hPRL) present in blood samples is heterogeneous in terms of molecular size (1,2). The reported evidence indicates that the different prolactin components are secreted from the pituitary gland. However, the possibility of aggregation of "monomeric" prolactin or the physical conversion of "big" to "little" components has not been ruled out (1,2). During the course of gestation, serum hPRL levels in the mother rise steadily until the time of parturition (3). Levels of hPRL in fetal serum appear to rise in the same pattern (4). In addition, it has been found that amniotic fluid contains large amounts of prolactin (5,6), but the source of amniotic prolactin is not known. This study was carried out to examine hPRL in terms of the quantitative and qualitative differences of molecular heterogeneity in the maternal and fetal circulaReceived April 16, 1975. 1 Present address: Department of Obstetrics and Gynecology, the Ohio State University Hospitals, Columbus, Ohio 43210.
in cord venous and arterial sera, both quantitatively and in terms of heterogeneity. There were more noticeable differences in the percentages of medium and small prolactin present in serum samples than in amniotic fluid samples. Big prolactin was present in all samples; its percentage in maternal serum was slightly higher than its percentages in the other samples. These results suggest that: 1) The mother and fetus cany on independent secretion of prolactin during gestation. 2) The size heterogeneity of prolactin in amniotic fluid differs from maternal hPRL to a greater degree than it does from fetal hPRL. 3) The prolactin in amniotic fluid appears to be of fetal origin. (J Clin Endocrinol Metab 41: 1030, 1975)
tions and in amniotic fluid at the time of delivery. The results suggest that in all likelihood, amniotic hPRL is of fetal origin. Materials and Methods Serum and amniotic fluid samples. Five healthy women who progressed normally in the course of pregnancy were selected; the relevant obstetric information is provided in Table 1. Samples of clear amniotic fluid and maternal venous blood were collected simultaneously during the delivery. To avoid all possibility of contamination with serum hPRL, the amniotic fluid samples were collected when the amniotic membrane was bulging at the time of full dilation of the cervix in cases of vaginal delivery. A gauge 18 needle was introduced through the membrane to aspirate the amniotic fluid for each sample. Arterial and venous blood samples from the umbilical cord were drawn when the cord was clamped at both ends, after the baby was born. The blood samples were allowed to clot in a refrigerator, and then they were centrifuged on the same day at 4 C to separate the serum. The amniotic fluids were also kept refrigerated for the same length of time, and any settled debris was discarded. All samples were stored at — 50 C until used. In all cases, the storage time was less than 3 weeks.
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1031
MATERNAL, FETAL AND AMNIOTIC hPRL Column chromatography. The sample (1 ml of serum or fluid) was passed through a Sephadex G-100 column (1 x 180 cm) with phosphate buffer (0.01M) and saline (0.15M) solution (phosphate buffered saline), pH 7.4, in a cold room (4 C). Before the application of the sample, 2 ml of PBS containing 2.5% bovine serum albumin was added on the column, followed by 0.2 ml of PBS containing 0.1% blue dextran (average mol wt 2,000,000, purchased from Sigma Chemical Co., St. Louis, Mo.). After the addition of the sample, the effluent was collected in 1 ml (34 drops) fractions at the rate of 10 ml/h. A rather consistent void volume of 55 ± 2 ml was observed, as determined by the peak dye fraction. The eluates as well as the original samples which were diluted 20 x with PBS, were stored at — 20 C until assayed. In all cases, the period of frozen storage was less than 4 days.
correction factor of 1/5 was introduced, so that our results will be comparable to those reported by other laboratories. The assay sensitivity is 0.05 ng of the weight of the standard (73-4-27) or a concentration of 0.2 ng/ml as expressed in this report. The intra-assay variation is less than 5% and the inter-assay variation is less than 15% with samples containing hPRL ranging from 2 to 200 ng/ml. The assay is highly specific, since 10 fxg of human placental lactogen (Lederle 717340) and 100 IU of human chorionic gonadotropin did not cross-react, and 1 /xg of human growth hormone (NIH, A-21) cross-reacted with hPRL less than 0.1%.
Prolactin radioimmunoassay. Immunoreactive hPRL in each fraction and diluted sample was measured in a homologous radioimmunoassay system with a double-antibody technique for separation of the bound from the free hormone. A highly purified hPRL (72-11-23) and a highly specific rabbit anti-hPRL antiserum (65-5) were kindly donated by Dr. Henry Friesen. The procedure is essentially the same as described (7), with the following minor exceptions: a) Iodination of hPRL was earned out with a lactoperoxidase method (8) to specific activities of 60-120 /xCi//xg. The labeled hPRL prepared in this way could be stored at —50 C and used for at least 1 month without any deteriorating effect on immunochemical properties for radioimmunoassay. b) Due to the scarcity of the highly purified hPRL, a less pure hPRL preparation (Friesen 73-4-27) was used as reference standard. As the immunoreactive potency of this standard is 5 times less than the standard generally used, a
Results
The concentration of hPRL in serum and amniotic fluid samples collected from the five subjects at term are listed in Table 2. There were obvious differences in hPRL levels between maternal and fetal circulations. In three cases the maternal serum samples contained higher concentrations of hPRL than did the fetal samples, but in the other two cases, the opposite situation was observed. In all five cases, hPRL levels in each individual cord venous and arterial serum sample were very close. The concentrations of hPRL in amniotic fluids were 1.3-7.1 times higher than those in maternal sera and 1.7-7.8 times higher than those in cord sera. The Sephadex chromatography of maternal, fetal, and amniotic hPRL is shown in Fig. 1 with subject S.M. as the representative. In the chromatograms, three peaks of immunoreactivity, big, medium, and small hPRL, could be designated. The three peaks
TABLE 1. Obstetric data of the subjects studied Mother
Gravida
Gestation weeks
Labor
Delivery
Infant
W.L. S.M. I.M.B. C.L.M. G.R.
IV II III III I
40 41 41 38 41
Spontaneous Spontaneous Induced by Syntocinon2 None Spontaneous
Normal vaginal Normal vaginal Normal vaginal Cesarean section Cesarean section
Male, 3,200 g Male, 3,300 g Male, 3,025 g Female, 2,850 g Male, 3,960 g
Syntocinon is a synthetic oxytocin supplied by Sandoz.
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FANG AND KIM
1032
TABLE 2. Prolactin concentrations (ng/ml) in serum samples and amniotic fluids at term
Subject
Maternal venous serum
Cord venous serum
Cord arterial serum
W.L. S.M. I.M.B. C.L.M. C.R.
140 270 41 296 109
81 174 91 188 248
64 182
84 176 248
Amniotic fluid 568 304 292 520 504
appeared in the same effluent positions for all samples fractionated. The overall hPRL profiles were remarkably similar in both cord samples, but distinctly different in the maternal serum and amniotic fluid samples. The medium hPRL peak was proportionately larger in maternal serum than in the cord samples and was nearly absent in the amniotic fluid. The big hPRL peaks showed less variation among the different samples. Recovery of hPRL after gel filtration ranged from 65-90% of the total immunoreactive prolactin in the sample added. 125I-hPRL added on the column was eluted in a single peak coinciding with small hPRL. The prolactin heterogeneity of samples from all subjects expressed in percentage of big, medium, and small hPRL is summarized in Table 3. The main component in all samples was small hPRL, which constituted at least 69% of the total immunoreactive prolactin. In amniotic fluid, over 90% of the hormone was small hPRL; only a very small amount of medium hPRL could be detected. In the maternal sera, medium hPRL appeared as a second large peak. The percentage of big hPRL was rather constant in the various samples, and it was particularly so in cord sera and amniotic fluid. There was no consistent pattern of hPRL distribution among the four different samples examined from each individual.
JCE & M Vol 41
1975 No 6
gest that the secretions of hPRL in the mother and fetus before parturition are independent of each other. The noticeable differences in medium and small hPRL distributions of maternal and cord blood samples (Table 3) further support the notion that prolactin secreted into maternal and fetal circulations is different. Furthermore, the quantitative and qualitative resemblance of cord arterial and venous hPRL also indicates that the fetus has maintained its own prolactin secretion before delivery. Siler-Khodr et al. (9) recently reported that the adenohypophyses from normal fetuses obtained at 5-40 weeks fetal life were able
S3-
FIG. 1. Chromatograms of hPRL from mother (S.M.) venous, cord venous, and arterial serum samples and from amniotic fluid collected at the time of delivery. The peak blue dextran fraction is indicated by j . The percentages of big, medium, and small hPRL are given.
Discussion The differences in the relative concentrations of hPRL between maternal and fetal circulations at term (Table 2) strongly sug-
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MATERNAL, FETAL AND AMNIOTIC hPRL to synthesize and release immunoreactive prolactin when cultured in vitro. Other studies on the ontogenesis of prolactin secretion indicated that fetal hyperprolactinemia is due to the underdevelopment of the hypothalamic regulatory mechanism before birth (4). The occurrence of maternal hyperprolactinemia has been attributed to the progressive increase in blood levels of estrogens, because in rhesus monkeys, plasma prolactin levels remained relatively low throughout pregnancy, due to the low production of estrogens during this period (10). Since the human feto-placental unit functions cooperatively and actively in the biosynthesis of steroids, including estrogens (11), the human fetal pituitary gland may react to the stimulation of sulfoconjugated or free estrogenic steroids by secreting consistently large amounts of prolactin. However, the source of prolactin in amniotic fluid remains puzzling. In spite of relatively low levels of prolactin in maternal and fetal blood in rhesus monkeys, prolactin levels in amniotic fluid are high (10). In our studies, the low percentage of big hPRL in amniotic fluid samples resembled more closely cord arterial sera than maternal serum samples (Table 3). This suggests that fetal pituitary prolactin has been excreted into, and concentrated in, amniotic fluid. The low percentage of medium hPRL might be explained by its possible conversion into big and small hPRL, since such changes seemed to occur in vitro by storage (2). However, other evidence suggests that polypeptide hormones of different molecular sizes are synthesized in the gland and released directly into the bloodstream. These included proinsulin and insulin (12), proparathormone and parathormone (13), proglucagon and glucagon (14), big and heptadecapeptide gastrin (15), big and small forms of growth hormone (16), and big and small forms of prolactin (1). Therefore, unless the prolactin present in amniotic fluid was secreted from sources other than the pituitary gland, its origin is more likely to
1033
TABLE 3. Prolactin heterogeneity of serum samples and amniotic fluids at term (%) Samples Maternal venous serum W.L. S.M. I.M.B. C.L.M. G.R. Mean ± SD Cord venous serum W.L. S.M. I.M.B. C.L.M. G.R. Mean ± SD Cord arterial serum W.L. S.M. I.M.B. C.L.M. G.R. Mean ± SD Amniotic fluid W.L. S.M. I.M.B. C.L.M. G.R. Mean ± SD
Big
Medium
10.6
17.9 26.8 16.0 16.7 5.2 16.5 ± 7.7
3.7
12.3 1.6 1.4
5.9 ± 5.2
7.1 2.7 5.0
9.4 9.2 7.8
0.3
5.7
0.5
3.1 ± 2.9
8.0 2.2 5.8 1.7 1.5
3.8 ± 2.9 6.1 5.1 1.9
8.0
8.0 ± 1.5
15.2 8.8 6.7
0.5 10.5 8.3 ± 5.4
2.0
4.5 0.4 0.6 0 0.5
3.4 ± 2.1
1.2 ± 1.9*1
1.8
Small
71.5 69.5 71.7 81.7 93.4 77.6 ± 10.1
83.5 88.1 87.2 94.0 91.5 88.9 ± 4.0
76.8 89.0 87.5 97.8 88.0 87.9 ± 7.5 89.4 94.4 97.5 98.2 97.5 95.4 ± 3.7*
* P < 0.005 by Student's t test compared with maternal venous serum samples. t P < 0.05 by Student's t test compared with both cord serum samples.
be attributed to the fetal pituitary gland than to the mother's. In humans, the concentration of amniotic fluid prolactin is highest at the beginning of the third trimester (32-weeks gestation) and decreases afterwards (3,17), while blood concentrations of prolactin in both the mother and fetus continue to rise. The drastic increase in the volume of amniotic fluid after the second trimester (18) might be a contributory factor for the dilution of amniotic fluid prolactin. In spite of the volume change, human chorionic gonadotropin (hCG) maintained a stable concentration in amniotic fluid up to term (17). This difference between hCG and hPRL
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JCE & M • 1975 Vol 41 • No 6
FANG AND KIM
levels in amniotic fluid during gestation indicates that hPRL, unlike hCG, is not a placental product of chorionic origin. The question of the role of the epithelial cells of the amniotic membrane in the possible secretion of prolactin should be investigated. The significant disparity in distributions of big, medium, and small hPRL between maternal serum and amniotic fluid (Table 3) suggests different origins of the hormone. It also suggests that amniotic fluid hPRL is probably not a product of the maternal pituitary gland. Our conclusion that maternal prolactin is not transferred to the amniotic fluid is consistent with the finding by Friesen et at. (10) that no isotope appeared in the amniotic fluid or in the fetus after 125I-PRL was injected to a pregnant monkey. The most plausible source of amniotic fluid hPRL seems to be from the fetal pituitary gland by excretion through, but poor tubular reabsorption by, the immature kidneys. The hormone in the amniotic sac is probably concentrated by the dynamic turnover of the fluid involving fetal swallowing. This conclusion is supported by evidence that polyhydramnios with reduced rate of fetal swallowing of the fluid in utero was often found with low prolactin concentrations in amniotic fluid (10), probably due to a lack of the concentrating process. Acknowledgments The authors are grateful to Dr. Henry G. Friesen, the University of Manitoba, Winnipeg, Canada, for his generous gift of human prolactin and antiserum to hPRL. We also appreciate the excellent technical assistance of Miss Bong-Ja Kim, and the secretarial help of Miss Constance Balint.
References 1. Rogol, A. D., and S. W. Rosen,./ Clin Endocrinol Metab 38: 714, 1974. 2. Suh, H. K., and A. G. Frantz, / Clin Endocrinol Metab 39: 928, 1974. 3. Tyson, J. E., P. Hwang, H. Guyda, and H. G. Friesen, Am J Obstet Gynecol 113: 14, 1972. 4. Aubert, M. L., M. M. Grumbach, and S. L. Kaplan, Endocrinology 92: A-49, 1973 (Abstract). 5. Friesen, H., G. Tolis, R. Shiu, and P. Hwang, In Pasteels, J. L., and C. Robyn (eds.), Human Prolactin, International Congress Series No. 308, Excerpta Medica, Amsterdam, 1973, p. 11. 6. Badawi, M., F. R. Perez-Lopez, and C. Robyn, Ada Endocrinol [Suppl] (Kbh) 177: 237, 1973 (Abstract). 7. Hwang, P., H. Guyda, and H. Friesen, Proc Natl Acad Sci USA 68: 1902, 1971. 8. Rogol, A. D., and S. W. Rosen, J Clin Endocrinol Metab 39: 379, 1974. 9. Siler-Khodr, T. M., L. L. Morgenstern, and F. C. Greenwood, J Clin Endocrinol Metab 39: 891, 1974. 10. Friesen, H., P. Hwang, H. Guyda, G. Tolis, J. Tyson, and R. Myers, In Boyns, A. R., and K. Griffiths (eds.), Prolactin and Carcinogenesis, Alpha Omega Alpha Publishing, Cardiff, Wales, 1972, p. 64. 11. Diczfalusy, E., Fed Proc 23: 791, 1964. 12. Steiner, D. F., J. L. Clark, D. Nolan, A. H. Rubenstein, E. Margoliash, B. Alten, and P. E. Oyer, Recent Prog Hormone Res 25: 207, 1969. 13. Wong, E. T., and A. W. Lindall, Proc Soc Exp Biol Med 148: 387, 1975. 14. Noe, B. D., and G. E. Bauer, Endocrinology 89: 642, 1971. 15. Berson, S. A., and R. S. Yalow, Gastroenterology 60: 215, 1971. 16. Stachura, M. E., and L. A. Frohman, Science 187: 447, 1975. 17. Perez-Lopez, F. R., E. Canas, M. L'Hermite, E. Lopez, M. C. Roncero, and C. Robyn, Intern Res Commun System 2: 1101, 1974. 18. Abramovich, D. R., In Shearman, R. P. (ed.), Human Reproductive Physiology, Blackwell Scientific Publications, Oxford, 1972, p. 258.
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I
T HAS been demonstrated that immunoreactive human prolactin (hPRL) present in blood samples is heterogeneous in terms of molecular size (1,2). The reported evidence indicates that the different prolactin components are secreted from the pituitary gland. However, the possibility of aggregation of "monomeric" prolactin or the physical conversion of "big" to "little" components has not been ruled out (1,2). During the course of gestation, serum hPRL levels in the mother rise steadily until the time of parturition (3). Levels of hPRL in fetal serum appear to rise in the same pattern (4). In addition, it has been found that amniotic fluid contains large amounts of prolactin (5,6), but the source of amniotic prolactin is not known. This study was carried out to examine hPRL in terms of the quantitative and qualitative differences of molecular heterogeneity in the maternal and fetal circulaReceived April 16, 1975. 1 Present address: Department of Obstetrics and Gynecology, the Ohio State University Hospitals, Columbus, Ohio 43210.
in cord venous and arterial sera, both quantitatively and in terms of heterogeneity. There were more noticeable differences in the percentages of medium and small prolactin present in serum samples than in amniotic fluid samples. Big prolactin was present in all samples; its percentage in maternal serum was slightly higher than its percentages in the other samples. These results suggest that: 1) The mother and fetus cany on independent secretion of prolactin during gestation. 2) The size heterogeneity of prolactin in amniotic fluid differs from maternal hPRL to a greater degree than it does from fetal hPRL. 3) The prolactin in amniotic fluid appears to be of fetal origin. (J Clin Endocrinol Metab 41: 1030, 1975)
tions and in amniotic fluid at the time of delivery. The results suggest that in all likelihood, amniotic hPRL is of fetal origin. Materials and Methods Serum and amniotic fluid samples. Five healthy women who progressed normally in the course of pregnancy were selected; the relevant obstetric information is provided in Table 1. Samples of clear amniotic fluid and maternal venous blood were collected simultaneously during the delivery. To avoid all possibility of contamination with serum hPRL, the amniotic fluid samples were collected when the amniotic membrane was bulging at the time of full dilation of the cervix in cases of vaginal delivery. A gauge 18 needle was introduced through the membrane to aspirate the amniotic fluid for each sample. Arterial and venous blood samples from the umbilical cord were drawn when the cord was clamped at both ends, after the baby was born. The blood samples were allowed to clot in a refrigerator, and then they were centrifuged on the same day at 4 C to separate the serum. The amniotic fluids were also kept refrigerated for the same length of time, and any settled debris was discarded. All samples were stored at — 50 C until used. In all cases, the storage time was less than 3 weeks.
1030
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1031
MATERNAL, FETAL AND AMNIOTIC hPRL Column chromatography. The sample (1 ml of serum or fluid) was passed through a Sephadex G-100 column (1 x 180 cm) with phosphate buffer (0.01M) and saline (0.15M) solution (phosphate buffered saline), pH 7.4, in a cold room (4 C). Before the application of the sample, 2 ml of PBS containing 2.5% bovine serum albumin was added on the column, followed by 0.2 ml of PBS containing 0.1% blue dextran (average mol wt 2,000,000, purchased from Sigma Chemical Co., St. Louis, Mo.). After the addition of the sample, the effluent was collected in 1 ml (34 drops) fractions at the rate of 10 ml/h. A rather consistent void volume of 55 ± 2 ml was observed, as determined by the peak dye fraction. The eluates as well as the original samples which were diluted 20 x with PBS, were stored at — 20 C until assayed. In all cases, the period of frozen storage was less than 4 days.
correction factor of 1/5 was introduced, so that our results will be comparable to those reported by other laboratories. The assay sensitivity is 0.05 ng of the weight of the standard (73-4-27) or a concentration of 0.2 ng/ml as expressed in this report. The intra-assay variation is less than 5% and the inter-assay variation is less than 15% with samples containing hPRL ranging from 2 to 200 ng/ml. The assay is highly specific, since 10 fxg of human placental lactogen (Lederle 717340) and 100 IU of human chorionic gonadotropin did not cross-react, and 1 /xg of human growth hormone (NIH, A-21) cross-reacted with hPRL less than 0.1%.
Prolactin radioimmunoassay. Immunoreactive hPRL in each fraction and diluted sample was measured in a homologous radioimmunoassay system with a double-antibody technique for separation of the bound from the free hormone. A highly purified hPRL (72-11-23) and a highly specific rabbit anti-hPRL antiserum (65-5) were kindly donated by Dr. Henry Friesen. The procedure is essentially the same as described (7), with the following minor exceptions: a) Iodination of hPRL was earned out with a lactoperoxidase method (8) to specific activities of 60-120 /xCi//xg. The labeled hPRL prepared in this way could be stored at —50 C and used for at least 1 month without any deteriorating effect on immunochemical properties for radioimmunoassay. b) Due to the scarcity of the highly purified hPRL, a less pure hPRL preparation (Friesen 73-4-27) was used as reference standard. As the immunoreactive potency of this standard is 5 times less than the standard generally used, a
Results
The concentration of hPRL in serum and amniotic fluid samples collected from the five subjects at term are listed in Table 2. There were obvious differences in hPRL levels between maternal and fetal circulations. In three cases the maternal serum samples contained higher concentrations of hPRL than did the fetal samples, but in the other two cases, the opposite situation was observed. In all five cases, hPRL levels in each individual cord venous and arterial serum sample were very close. The concentrations of hPRL in amniotic fluids were 1.3-7.1 times higher than those in maternal sera and 1.7-7.8 times higher than those in cord sera. The Sephadex chromatography of maternal, fetal, and amniotic hPRL is shown in Fig. 1 with subject S.M. as the representative. In the chromatograms, three peaks of immunoreactivity, big, medium, and small hPRL, could be designated. The three peaks
TABLE 1. Obstetric data of the subjects studied Mother
Gravida
Gestation weeks
Labor
Delivery
Infant
W.L. S.M. I.M.B. C.L.M. G.R.
IV II III III I
40 41 41 38 41
Spontaneous Spontaneous Induced by Syntocinon2 None Spontaneous
Normal vaginal Normal vaginal Normal vaginal Cesarean section Cesarean section
Male, 3,200 g Male, 3,300 g Male, 3,025 g Female, 2,850 g Male, 3,960 g
Syntocinon is a synthetic oxytocin supplied by Sandoz.
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FANG AND KIM
1032
TABLE 2. Prolactin concentrations (ng/ml) in serum samples and amniotic fluids at term
Subject
Maternal venous serum
Cord venous serum
Cord arterial serum
W.L. S.M. I.M.B. C.L.M. C.R.
140 270 41 296 109
81 174 91 188 248
64 182
84 176 248
Amniotic fluid 568 304 292 520 504
appeared in the same effluent positions for all samples fractionated. The overall hPRL profiles were remarkably similar in both cord samples, but distinctly different in the maternal serum and amniotic fluid samples. The medium hPRL peak was proportionately larger in maternal serum than in the cord samples and was nearly absent in the amniotic fluid. The big hPRL peaks showed less variation among the different samples. Recovery of hPRL after gel filtration ranged from 65-90% of the total immunoreactive prolactin in the sample added. 125I-hPRL added on the column was eluted in a single peak coinciding with small hPRL. The prolactin heterogeneity of samples from all subjects expressed in percentage of big, medium, and small hPRL is summarized in Table 3. The main component in all samples was small hPRL, which constituted at least 69% of the total immunoreactive prolactin. In amniotic fluid, over 90% of the hormone was small hPRL; only a very small amount of medium hPRL could be detected. In the maternal sera, medium hPRL appeared as a second large peak. The percentage of big hPRL was rather constant in the various samples, and it was particularly so in cord sera and amniotic fluid. There was no consistent pattern of hPRL distribution among the four different samples examined from each individual.
JCE & M Vol 41
1975 No 6
gest that the secretions of hPRL in the mother and fetus before parturition are independent of each other. The noticeable differences in medium and small hPRL distributions of maternal and cord blood samples (Table 3) further support the notion that prolactin secreted into maternal and fetal circulations is different. Furthermore, the quantitative and qualitative resemblance of cord arterial and venous hPRL also indicates that the fetus has maintained its own prolactin secretion before delivery. Siler-Khodr et al. (9) recently reported that the adenohypophyses from normal fetuses obtained at 5-40 weeks fetal life were able
S3-
FIG. 1. Chromatograms of hPRL from mother (S.M.) venous, cord venous, and arterial serum samples and from amniotic fluid collected at the time of delivery. The peak blue dextran fraction is indicated by j . The percentages of big, medium, and small hPRL are given.
Discussion The differences in the relative concentrations of hPRL between maternal and fetal circulations at term (Table 2) strongly sug-
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MATERNAL, FETAL AND AMNIOTIC hPRL to synthesize and release immunoreactive prolactin when cultured in vitro. Other studies on the ontogenesis of prolactin secretion indicated that fetal hyperprolactinemia is due to the underdevelopment of the hypothalamic regulatory mechanism before birth (4). The occurrence of maternal hyperprolactinemia has been attributed to the progressive increase in blood levels of estrogens, because in rhesus monkeys, plasma prolactin levels remained relatively low throughout pregnancy, due to the low production of estrogens during this period (10). Since the human feto-placental unit functions cooperatively and actively in the biosynthesis of steroids, including estrogens (11), the human fetal pituitary gland may react to the stimulation of sulfoconjugated or free estrogenic steroids by secreting consistently large amounts of prolactin. However, the source of prolactin in amniotic fluid remains puzzling. In spite of relatively low levels of prolactin in maternal and fetal blood in rhesus monkeys, prolactin levels in amniotic fluid are high (10). In our studies, the low percentage of big hPRL in amniotic fluid samples resembled more closely cord arterial sera than maternal serum samples (Table 3). This suggests that fetal pituitary prolactin has been excreted into, and concentrated in, amniotic fluid. The low percentage of medium hPRL might be explained by its possible conversion into big and small hPRL, since such changes seemed to occur in vitro by storage (2). However, other evidence suggests that polypeptide hormones of different molecular sizes are synthesized in the gland and released directly into the bloodstream. These included proinsulin and insulin (12), proparathormone and parathormone (13), proglucagon and glucagon (14), big and heptadecapeptide gastrin (15), big and small forms of growth hormone (16), and big and small forms of prolactin (1). Therefore, unless the prolactin present in amniotic fluid was secreted from sources other than the pituitary gland, its origin is more likely to
1033
TABLE 3. Prolactin heterogeneity of serum samples and amniotic fluids at term (%) Samples Maternal venous serum W.L. S.M. I.M.B. C.L.M. G.R. Mean ± SD Cord venous serum W.L. S.M. I.M.B. C.L.M. G.R. Mean ± SD Cord arterial serum W.L. S.M. I.M.B. C.L.M. G.R. Mean ± SD Amniotic fluid W.L. S.M. I.M.B. C.L.M. G.R. Mean ± SD
Big
Medium
10.6
17.9 26.8 16.0 16.7 5.2 16.5 ± 7.7
3.7
12.3 1.6 1.4
5.9 ± 5.2
7.1 2.7 5.0
9.4 9.2 7.8
0.3
5.7
0.5
3.1 ± 2.9
8.0 2.2 5.8 1.7 1.5
3.8 ± 2.9 6.1 5.1 1.9
8.0
8.0 ± 1.5
15.2 8.8 6.7
0.5 10.5 8.3 ± 5.4
2.0
4.5 0.4 0.6 0 0.5
3.4 ± 2.1
1.2 ± 1.9*1
1.8
Small
71.5 69.5 71.7 81.7 93.4 77.6 ± 10.1
83.5 88.1 87.2 94.0 91.5 88.9 ± 4.0
76.8 89.0 87.5 97.8 88.0 87.9 ± 7.5 89.4 94.4 97.5 98.2 97.5 95.4 ± 3.7*
* P < 0.005 by Student's t test compared with maternal venous serum samples. t P < 0.05 by Student's t test compared with both cord serum samples.
be attributed to the fetal pituitary gland than to the mother's. In humans, the concentration of amniotic fluid prolactin is highest at the beginning of the third trimester (32-weeks gestation) and decreases afterwards (3,17), while blood concentrations of prolactin in both the mother and fetus continue to rise. The drastic increase in the volume of amniotic fluid after the second trimester (18) might be a contributory factor for the dilution of amniotic fluid prolactin. In spite of the volume change, human chorionic gonadotropin (hCG) maintained a stable concentration in amniotic fluid up to term (17). This difference between hCG and hPRL
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1034
JCE & M • 1975 Vol 41 • No 6
FANG AND KIM
levels in amniotic fluid during gestation indicates that hPRL, unlike hCG, is not a placental product of chorionic origin. The question of the role of the epithelial cells of the amniotic membrane in the possible secretion of prolactin should be investigated. The significant disparity in distributions of big, medium, and small hPRL between maternal serum and amniotic fluid (Table 3) suggests different origins of the hormone. It also suggests that amniotic fluid hPRL is probably not a product of the maternal pituitary gland. Our conclusion that maternal prolactin is not transferred to the amniotic fluid is consistent with the finding by Friesen et at. (10) that no isotope appeared in the amniotic fluid or in the fetus after 125I-PRL was injected to a pregnant monkey. The most plausible source of amniotic fluid hPRL seems to be from the fetal pituitary gland by excretion through, but poor tubular reabsorption by, the immature kidneys. The hormone in the amniotic sac is probably concentrated by the dynamic turnover of the fluid involving fetal swallowing. This conclusion is supported by evidence that polyhydramnios with reduced rate of fetal swallowing of the fluid in utero was often found with low prolactin concentrations in amniotic fluid (10), probably due to a lack of the concentrating process. Acknowledgments The authors are grateful to Dr. Henry G. Friesen, the University of Manitoba, Winnipeg, Canada, for his generous gift of human prolactin and antiserum to hPRL. We also appreciate the excellent technical assistance of Miss Bong-Ja Kim, and the secretarial help of Miss Constance Balint.
References 1. Rogol, A. D., and S. W. Rosen,./ Clin Endocrinol Metab 38: 714, 1974. 2. Suh, H. K., and A. G. Frantz, / Clin Endocrinol Metab 39: 928, 1974. 3. Tyson, J. E., P. Hwang, H. Guyda, and H. G. Friesen, Am J Obstet Gynecol 113: 14, 1972. 4. Aubert, M. L., M. M. Grumbach, and S. L. Kaplan, Endocrinology 92: A-49, 1973 (Abstract). 5. Friesen, H., G. Tolis, R. Shiu, and P. Hwang, In Pasteels, J. L., and C. Robyn (eds.), Human Prolactin, International Congress Series No. 308, Excerpta Medica, Amsterdam, 1973, p. 11. 6. Badawi, M., F. R. Perez-Lopez, and C. Robyn, Ada Endocrinol [Suppl] (Kbh) 177: 237, 1973 (Abstract). 7. Hwang, P., H. Guyda, and H. Friesen, Proc Natl Acad Sci USA 68: 1902, 1971. 8. Rogol, A. D., and S. W. Rosen, J Clin Endocrinol Metab 39: 379, 1974. 9. Siler-Khodr, T. M., L. L. Morgenstern, and F. C. Greenwood, J Clin Endocrinol Metab 39: 891, 1974. 10. Friesen, H., P. Hwang, H. Guyda, G. Tolis, J. Tyson, and R. Myers, In Boyns, A. R., and K. Griffiths (eds.), Prolactin and Carcinogenesis, Alpha Omega Alpha Publishing, Cardiff, Wales, 1972, p. 64. 11. Diczfalusy, E., Fed Proc 23: 791, 1964. 12. Steiner, D. F., J. L. Clark, D. Nolan, A. H. Rubenstein, E. Margoliash, B. Alten, and P. E. Oyer, Recent Prog Hormone Res 25: 207, 1969. 13. Wong, E. T., and A. W. Lindall, Proc Soc Exp Biol Med 148: 387, 1975. 14. Noe, B. D., and G. E. Bauer, Endocrinology 89: 642, 1971. 15. Berson, S. A., and R. S. Yalow, Gastroenterology 60: 215, 1971. 16. Stachura, M. E., and L. A. Frohman, Science 187: 447, 1975. 17. Perez-Lopez, F. R., E. Canas, M. L'Hermite, E. Lopez, M. C. Roncero, and C. Robyn, Intern Res Commun System 2: 1101, 1974. 18. Abramovich, D. R., In Shearman, R. P. (ed.), Human Reproductive Physiology, Blackwell Scientific Publications, Oxford, 1972, p. 258.
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