- Email: [email protected]
Receptors for and myometrial responses to oxytocin and vasopressin in preterm and term human pregnancy: Effects of the oxytocin antagonist atosiban
Bossmar et al.
oxytocin antagonist atosiban on pretenn uterine activity in the human. AM] OBSTET GYNECOL 1994;170:474-8. 22. Sheldrick EL, Flick-Smith HC. Effect of ovarian honnones on oxytocin receptor concentrations in explants of uterus from ovariectomized ewes. ] Reprod FertiI1993;97:241-5. 23. Soloff MS, Fernstrom MA, Periyasami S, Soloff S, Baldwin S, Wieder M. Regulation of oxytocin receptor concentration in rat uterine explants by estrogen and progesterone. Can] Biochem Cell Bioi 1983;61 :625-30.
December 1994 Am J Obstet Gynecol
24. Sheldrick EL, Flint AP. Endocrine control of uterine oxytocin receptors in the ewe. ] Endocrinol 1985; 106:249-58. 25. Randolph GW, Fuchs A. Pulsatile administration enhances the effect and reduces the dose of oxytocin required for induction of labor. Am] Perinatol 1989;6: 159-66. 26. Cummiskey KC, Dawood MY. Induction of labor with pulsatile oxytocin. AM] OBSTET GYNECOL 1990;163:186874.
Receptors for and myometrial responses to oxytocin and vasopressin in preterm and term human pregnancy: Effects of the oxytocin antagonist atosiban Thomas Bossmar, MD," Mats Akerlu~d, MD, PhD," Guido Fantoni, MD; Jazec Szamatowicz, MD,b Per Melin, PhD,d and Mario Maggi, MD, PhDc
Lund and Malmo, Sweden, Florence, Italy, and Bialystok, Poland OBJECTIVE: Our purpose was to study myometrial oxytocin and type V1 vasopressin receptors, the in vitro contractile effects of these hormones, and the influence of an oxytocin antagonist. STUDY DESIGN: Women delivered by cesarean section preterm (n = 51) and at term (n = 71), with and without labor contractions, gave myometrium for the estimation of oxytocin and V1 vasopressin receptors. The in vitro myometrial effects of the peptides and the influence on these of the competitive oxytocin receptor blocking agent 1-deamino-2-D-Tyr(OEt)-4-Thr-8-0rn-oxytocin were also tested. RESULTS: The median concentration of oxytocin receptors was 116 fmol/mg protein (range 15 to 372 fmol/mg protein) in patients delivered preterm not in labor, 134 fmol/mg protein (27 to 1421 fmol/mg protein) in the beginning of labor, and 46 fmol/mg protein (9 to 140 fmol/mg protein) in advanced labor. At term the corresponding concentrations were 172 (25 to 629),223 (24 to 414), and 70 (21 to 92) fmol/mg protein. The concentration of V1 vasopressin receptors also decreased in advanced labor. In advanced labor after oxytocin infusion a reduction in the concentration of the receptor for this hormone was observed, which appeared to be related to the duration and dose of treatment. Oxytocin receptors did not vary between women with different indications for cesarean section. The oxytocin effects in vitro and the degree of inhibition by the antagonist of oxytocin responses correlated with the concentration of oxytocin receptors but not with that of V1 vasopressin receptors. No correlation was seen between the response to vasopressin and concentrations of oxytocin or V1 vasopressin receptors. CONCLUSIONS: The effect of oxytocin on the myometrium in pregnancy is mediated by an oxytocin receptor, whereas vasopressin acts on both oxytocin and vasopressin receptors. The initiation of labor both preterm and at term may be primarily related to increased release of oxytocin, which is locally produced in the uterus and not detectable in the plasma, but oxytocin and vasopressin receptors may playa role in the regulation of labOr. The analog 1-deamino-2-D-Tyr(OEt)-4-Thr-8-0rn-oxytocin, which blocks both the oxytocin and the V1 vasopressin receptor, should inhibit labor both preterm and at term, the former confirming results of recent clinical studies in Sweden and the United States. (AM J OBSTEr GVNECOL 1994;171:1634-42.)
Key words: Oxytocin and VI vasopressin receptors, myometrium, preterm pregnancy, term pregnancy
From the Departments of Obstetrics and Gynecology, University Hospitals of Lund" and Bialystok: the Endocrinology Unit, University of Florence,' and Ferring Pharmaceuticals." Supported by Swedish Medical Research Council grant No. B93-17X06571-11, Polish Research Council grant No. KBN-447299203, the University of Lund, Sweden, Ferring Pharmaceuticals, and R. W. Johnson Pharmaceutical Research Institute.
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Received for publication December 7,1993; accepted May 13,1994. Reprint requests: Mats Akerlund, MD, PhD, Department of Obstetrics and Gynecology, University Hospital, S-221 85 Lund, Sweden. Copyright © 1994 by Mosby-Year Book, Inc. 0002-9378194 $3.00 + 0 6/1/57532
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The human myometrium in pregnancy possesses oxytocin and type VI vasopressin receptors on which these peptides may act, I but data are conflicting regarding whether there is a significant increase in the plasma level of oxytocin or vasopressin before the onset of labor. 2. 3 However, oxytocin originates not only in the hypothalamus-posterior pituitary, but also in the decidua, amnion, chorion, and placenta:' 5 and hormone released from the latter sources may not necessarily be reflected in an increased plasma level. An increased myometrial oxytocin receptor concentration, resulting in a more pronounced effect of the oxytocin being available, has also been suggested as mechanism for labor induction, I. 6. 7 but it is not completely clear how receptors will change during the process of labor, vary between different obstetric complications, and be influenced by oxytocin treatment. In women in preterm labor the amount of data supporting the theory of an elevated receptor concentration as a common cause of contractions was very limited and estimations were performed for only a few of the different causes of this syndrome. I. 6. 7 Furthermore, the regulation of these receptors during different stages of preterm labor is virtually unknown. The therapeutic effect in this condition of the competitive oxytocin receptor blocking agent I-deamino-2-D-Tyr(OEt)-4Thr-8-0rn-oxytocin S - 1O is in agreement with a role of these receptors in the initiation of preterm labor contractions. The compound should be effective in the presence of both unchanged and elevated concentrations of oxytocin at the receptor. In this investigation we studied myometrial oxytocin and VI vasopressin receptors extensively in pregnant women undergoing cesarean section for a wide variety of indications, both preterm and at term and both before and during labor. These results were correlated to clinical data and findings in studies of contractile effects of the posterior pituitary hormones and the inhibition by I-deamino-2-D-Tyr(OEt)-4-Thr-8-0rnoxytocin of oxytocin responses of isolated myometrium from the same women. Material and methods
Subjects. Myometrial tissue samples were obtained from 122 white women who were undergoing cesarean section. They were all well informed of the purpose and procedure of the investigation and gave consent for participation. The study was approved by the local ethics committee. In 51 women the cesarean section was performed before the end of the thirty-sixth week of pregnancy. Twenty-seven were not in labor, with a cervical dilatation of 0 to 1.5 cm and zero to one contractions per 10 minutes, 18 in the beginning of labor with a cervical dilatation of 1.5 to 3 cm and one to two contractions per 10 minutes, and six were in advanced labor with a
Bossmar et al.
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more pronounced cervical change or higher frequency of contractions. The number of women delivered at term in the corresponding groups were 45, 9, and 17. At operation a specimen of myometrial tissue with a length of approximately 3 cm, a width of 0.7 x 0.5 cm, and a weight of about 2 gm was obtained from the upper rim of the transverse isthmic incision, this being located at the same part of the uterus irrespective of the presence of labor contractions. The material was immediately divided in two parts, one of which was placed in oxygenated Krebs-Ringer solution at 0° C and the other deep-frozen at - 80° C. The former part was used for myometrial contractility studies within 12 hours of operation. Membrane preparations. Details of the membrane preparation techniques were given previously. I. Jl In short, the previously frozen uterine specimens were first put in one type of buffer (10 mmoVL Tris-hydrochloric acid, pH 7.4, containing 1.5 mmoVL ethylenediaminetetraacetic acid, 0.5 mmoVL dithiothreitol, 1 mmoVL benzamidine, 0.01% bacitracin, and 0.002% soybean trypsin inhibitor) at 4° C and homogenized. The homogenate was centrifuged at 1000g for 10 minutes and the resulting supernatant centrifuged at 160,000g for 30 minutes at 4° C. The resulting pellet was dispersed in a second type of buffer (buffer II; 50 mmoVL Tris maleate, pH 7.6, containing 10 mmoVL magnesium sulfate, 1 mmoVL benzamidine, 0.01% bacitracin, and 0.002% soybean trypsin inhibitor) and then spun again at 160,000g for 30 minutes. The final pellet was then resuspended in buffer II and divided into 0.5 ml aliquots, frozen in solid carbon dioxide, and stored at - 80° C until assayed. Concentration of protein was determined with Bio-Rad protein reagent (Bio-Rad Laboratories, Munchen, Germany). Binding assays. As previously described, I, II aliquots of membranes (0.3 mg/ml) were incubated with ligands in buffer II in the presence of 0.1% bovine serum albumin at 22° C in tubes containing tritiated d(CH 2 )s Tyr-Me-arginine vasopressin (50.7 Cilmmol, New England Nuclear; Boston) for 60 minutes. This arginine vasopressin analog binds specifically to the VI vasopressin receptor. Aliquots were also incubated in tubes containing tritiated oxytocin (36 Cilmmol, New England Nuclear) for 180 minutes. Tritiated d(CH 2 )sTyr-Me-arginine vasopressin and tritiated oxytocin were present at 0.7 nmoVL in tubes containing increasing concentrations (0.1 to lOOOO nmoVL) of the corresponding unlabeled peptides and at 0.03 to 0.7 nmoVL in tubes without unlabeled ligands (final volume 0.25 ml). All measurements were performed in triplicate. Mter incubation membranes were filtered through Whatman GF/B filters (Clifton, N.J.) that had been presoaked in ice-cold 50 mmoVL Tris with a pH of 7.4 and in 0.1 % bovine serum albumin with the Brandel M-48R 48-well cell harvester (Gaithersburg, Md.). Fil-
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December 1994 Am J Ob,tet Gynecol
OTreceptor (fmol/mg protein)
.1421
600
400
200
I
--+--
-.
i
O~-----r--------r--------r--------r-------~----
before
beginning noOT
beginning OT
advanced noOT
advanced OT
State of labor
Fig. 1. Concentrations of the oxytocin (OT) receptor (median is indicated) of women delivered by cesarean section preterm while not in labor with cervical dilatation of 0 to 1.5 cm and zero to one contraction per 10 minutes, in beginning of labor with cervical dilatation of 1.5 to 3 cm and one to two contractions per 10 minutes, and in advanced labor with more pronounced cervical change or higher frequency of contractions. Subjects in latter two groups who had or had not received oxytocin (OT) infusion for inducing or stimulating labor contractions are accounted for separately. In women who had not received oxytocin, concentrations in beginning of labor and in advanced labor differed significantly.
ters were washed twice with 3 ml of ice-cold 50 mmoVL Tris with a pH of 7.4 and placed in liquid scintillation vials. Radioactivity retained by filters was measured in a liquid scintillation counter after overnight incubation in 10 ml of scintillation fluid. Oxytocin receptors were studied with homologous competition curves for oxytocin in the presence of 2 nmoVL d(CH2)5Tyr-Me-arginine vasopressin to mask the VI vasopressin site. Similarly, the density of VI arginine vasopressin receptors was investigated with homologous displacement curves for d(CH2)5Tyr-Mearginine vasopressin in the presence of 5 nmol!L oxytocin to block the oxytocin receptors. 1. 11 In 42 uterine specimens receptor density was evaluated with two- or three-point Scatchard plots, freezing the dissociation constant values at 0.5 nmol!L for oxytocin curves and at 0.16 nmoVL for d(CH2)5Tyr-Me-arginine vasopressin curves. Myometrial contractility studies. Myometrial segments with a standardized size of 2 x 2 x 10 mm, all with the same muscle fiber direction, were prepared for final dissection under a microscope at tenfold magnification. "Six strips from each patient were prepared. They were mounted in organ baths containing 10 ml
of Krebs-Ringer solution at pH 7.4 at a temperature of 37° C and aerated with carbogen. After an adaptation period of at least 30 minutes isometric contractions at a resting tension of 2 mN were recorded with a Grass force transducer (model IT 03). The effect of 30 nmol!L oxytocin and arginine vasopressin and the inhibitory effect of the same concentration of I-deamino- 2 -D-Tyr( 0 Et )-4-Thr-8-0rn -oxytocin on the responses of oxytocin were tested on the preparations. 12 The oxytocin and vasopressin responses were measured as the area under the curve during a 10minute period after administration. The antagonist was added to the tissue bath 1 minute before the oxytocin administration and the effect expressed as percent inhibition of the oxytocin response. Repeated washing was used between each drug administration. Statistical methods. Results obtained in different groups of women were compared by Mann-Whitney testing. Spearman's rank test was used for analysis of correlations.
Results Receptor estimations. Oxytocin bound with a dissociation constant of 0.60 ± 0.39 nmol!L (~ean ± SD,
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OTreceptor (fmollmg protein)
600
400
y
200
..:!.....
..·
·
-....· y
.
.
-,I
O~-----T--------~-------'--------~--------~--beginning beginning advanced advanced before OT no OT noOT OT
State of labor
Fig. 2. Concentrations of oxytocin (OT) receptor (median is indicated) of women delivered by cesarean section at term while not in labor with cervical dilatation of 0 to 1.5 cm and zero to one contraction per 10 minutes, in beginning of labor with cervical dilatation of 1.5 to 3 cm and one to two contractions per 10 minutes, and in advanced labor with more pronounced cervical change or higher frequency of contractions. Subjects in latter two groups who had or had not received oxytocin infusion for inducing or stimulating labor contractions are accounted for separately. In women who had not received oxytocin, concentrations in beginning of labor and in advanced labor differed significantly.
n = 81) to the oxytocin site, whereas d(CH2)2Tyr-Mearginine vasopressin showed a dissociation constant of 0.34 ± 0.18 nmoVL (n = 46) for the VI vasopressin site. No differences were seen between the various groups of patients. Concentrations of the oxytocin receptor in different stages of labor in women delivered preterm are shown in Fig. 1 and those in term-delivered women in Fig. 2, and the statistical significances of observed differences are shown in Table I. The interindividual variation was large, particularly in women in the beginning of labor both preterm and at term. In patients delivered preterm who had not received oxytocin treatment the median concentration of the oxytocin receptor of those not in labor was 116 fmoVmg protein (range 15 to 372 fmoVmg protein), in the beginning of labor 134 fmoVmg protein (27 to 1421 fmoVmg protein), and in advanced labor it had decreased significantly to 46 fmoVmg protein (9 to 140 fmoVmg protein). In women delivered at term and not having received oxytocin treatment the receptor concentration in the group not in labor was 172 fmoVmg protein (25 to 629 fmoVmg protein) and in the beginning of labor 223 fmoVmg
protein (24 to 414 fmoVmg protein). In the advanced labor it had again decreased to a median of 70 fmoVmg protein (range 21 to 92 fmoVmg protein), a decrease that was also observed for preterm and term patients taken together (Table I). There were no statistically significant differences in oxytocin receptor concentration between women in preterm and term labor in corresponding stages of labor (Figs. 1 and 2). In none of the groups with indications for cesarean section was the concentration of oxytocin receptors significantly different from that in other women at the same stage of labor (Tables II and III). The concentrations of the oxytocin receptor did not correlate significantly with the pregnancy weeks (Ts = 0.11) or the age of the patients (Ts = 0.08). Concentrations of the VI vasopressin receptor in women who had not received oxytocin treatment did not vary significantly between those in pre term and in term labor (Table IV), between different stages of labor (Fig. 3), or between women with different indications for cesarean section (not shown). Concentrations of oxytocin and VI vasopressin receptors in women who had been treated by oxytocin
1638 Bossmar at al.
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Am J Obstet Gynecol
Table I. Significance of differences
Preterm Term All
Beginning vs advanced
Before vs advanced
Before vs beginning
p= P=
p = 0.043
0.065 0.026 P = 0.002
NS NS NS
P= P=
0.086 0.001
NS, Not significant.
Table II. Myometrial oxytocin receptor concentrations (femtomoles per milligram of protein, median values with number of subjects in parentheses) in women with various indications for cesarean section before term and in different stages of labor State of labor Advanced
Beginning Indication for cesarean section
Before
Preeclampsia Placenta previa Abruptio placentae Disproportion-breech presentation Diabetes mellitus Fetal distress Poor obstetric history Fetal growth retardation Multipregnancy-polyhydramnios Premature rupture of membranes Previous cesarean section Other
127 (7) 163 (1) 108 50 15 132 167 124 213 116
No oxytocin
(1) (3) (1) (3) (2) (3) (1) (5)
1
Oxytocin
No oxytocin
I
Oxytocin
88 (2) 45 (1) 92 (3) 163 (5) 65 (2) 93 (1)
74 (2) 288 (3)
28 (1) 76 (1) 46 (1)
94 (1)
478 (1)
No significant difference was seen between any of the groups.
infusion before cesarean section and who were in the beginning of or in advanced labor are shown in Figs. 1 through 3 and in Tables II through IV. There were no statistically significant differences in oxytocin receptor concentrations between oxytocin-treated women preterm or at term or within these groups between those who had or had not received oxytocin treatment but who were in corresponding stages of labor (Figs. 1 and 2). For oxytocin-treated, pre term- and term-delivered patients taken together there was a significant difference between the oxytocin receptor concentrations in the beginning of labor (median) 119 fmoVmg protein (46 to 643 fmoVmg protein) and in advanced labor of 43 fmoVmg protein (17 to 105 fmoVmg protein)
total dose, duration of infusion, and interval between stopped infusion and operation are shown in Table V. The difference between the groups of shorter and longer durations of oxytocin infusion and groups of lower and higher total doses of oxytocin was not statistically significant. The cervical status did not differ between these groups. The total dose and duration of oxytocin treatment did not influence the concentration of VI vasopressin receptors, and no influence of the interval between stopped infusion and operation on either receptor was seen. The correlation between the concentration of oxytocin receptors and the durati{)n of oxytocin infusion had a coefficient, T., of -0.54
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Table III. Myometrial oxytocin receptor concentrations (femtomoles per milligram protein, median values with number of subjects in parentheses) in women with various indications for cesarean section at term and in different stages of labor State of labor Beginning Indication for cesarean section
Before
Preeclampsia Placenta previa Abruptio placentae Disproportion-breech presentation Diabetes mellitus Fetal distress Poor obstetric history Fetal growth retardation Primary uterine inertia Labor dystocia Multipregnancy-polyhydramnios Previous cesarean section Other
629 (1) 108 (3)
No oxytocin
143 (10) 99 (2)
I
Advanced Oxytocin
126 (3) 414 (I)
189 (7) 240 (2)
119 (3)
No oxytocin
I
92 (1) 57 (1)
39 (3)
52 (2)
34 (5)
93 (1) 167 (2) 187 (13) 160 (5)
Oxytocin
61 (I) 39 (2) 81 (I)
223 (I)
No significant difference was seen between any of the groups.
Table IV. Concentration of vasopressin VI receptors (femtomoles per milligram protein; median values, range, and number of observations are indicated) of women delivered by cesarean section pre term or at term while not in labor with cervical dilatation of 0 to 1.5 cm and zero to one contraction per 10 minutes, in beginning oflabor with cervical dilatation of 1.5 to 3 cmand one to two contractions per 10 minutes, and in advanced labor with more pronounced cervical change or higher frequency of contractions* State of labor
Pre term delivery
Term delivery
Before Beginning, no oxytocin Beginning, oxytocin Advanced, no oxytocin Advanced, oxytocin
116 (57-252, n = 16) 116 (41-207, n = 6)
105 (33-291, n = 24) 108 (34-135, n = 4) 148 (103-193, n = 2) 65 (n = I) 72 (5-111, n = 9)
115 (94-139, n = 3) 91 (n = I)
No significant differences were seen between preterm~ and term-delivered patients. *Subjects in·the latter group who had or had not received oxytocin infusion for inducing or stimulating labor contractions are accounted for separately.
correlation was seen between the responses to vasopressin and concentrations of oxytocin receptor (rs = 0.03) or the VI vasopressin receptor (rs = 0.07). The inhibitory effect of l-deamino-2-D-Tyr(OEt)-4Thr-8-0rn-oxytocin on oxytocin responses correlated with concentrations of the oxytocin receptor (rs = 0.39, P = 0.007) but not with those of the VI vasopressin receptor (rs = 0.40). Comment The lack of difference in oxytocin or VI vasopressin receptor affinity among different states of labor in this investigation is in agreement with a previous report l and supports the concept that an up regulation in such affinity is not involved in mechanisms oflabor induction. In the current study the concentration of oxytocin receptors correlated with the in vitro effects of oxytocin on uterine strips from the same women. This confirms the validity of the techniques used.
The fact that the in vitro effects of oxytocin correlated with concentrations of the oxytocin but not the VI vasopressin receptor confirms the previous finding that oxytocin stimulates uterine contractile activity in pregnant women by a specific effect on the oxytocin receptor.1 The lack of correlation between the in vitro effects of vasopressin and concentrations of the oxytocin or VI vasopressin receptor is also in agreement with previous data indicating that the action of this peptide is not confined to only one receptor but involves both the oxytocin and VI vasopressin sites. I The concentrations of the oxytocin receptor in different stages of term labor were generally in agreement with those found previously, I but in spite of a numeric difference between women before and in the beginning of labor and a material that was in all about 10 times larger than that of the previous studies we could not confirm statistically the hypothesis that an increase in oxytocin receptors is involved in the initiation of labor contrac-
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V1 VPreceptor (fmol/mg protein) 300
.' 200
...
'1' 100
.I. I
.,' I
o~----~------~--------~------~------~---advanced beginning beginning advanced before OT noOT noOT OT
State of labor
Fig. 3. Concentrations of VI vasopressin (VP) receptor (median is indicated) of women delivered by cesarean section preterm and at term, taken together, while not in labor with cervix dilatation of 0 to 1.5 cm and zero to one contraction per 10 minutes, in beginning of labor with cervical dilatation of 1.5 to 3 cm and one to two contractions per 10 minutes, and in advanced labor with more pronounced cervical change or higher frequency of contractions. Subjects in latter two groups who had or had not received oxytocin (OT) infusion for inducing or stimulating labor contractions are accounted for separately. Concentration in advanced labor in oxytocin-treated patients differed significantly from that of women before or in beginning of labor who had not received oxytocin treatment.
tions.l. 6, 7 A significant increase between concentrations before and in the beginning of labor did not even occur when values from pre term- and term-delivered patients were pooled in such a comparison. One explanation for this lack of difference is the wide interindividual variation in our results, something that may have been related to the fact that the material of the current study was obtained from patients with the full spectrum of indications for cesarean section, whereas the women giving myometrium for the previous studies had only a limited number of diagnoses. 1. 6. 7 In the preterm condition there was also a numeric but insigificant increase in oxytocin receptor concentrations when women not in labor were compared with subjects in the beginning of labor. Previous assumptions that an elevated concentration of this receptor is an important factor in the cause of preterm labor was supported by results from a small patient material, limited to only a few causes and not confirmed statistically, 1,6,7 whereas again the myometrial samples for the current study were obtained from about 10 times more patients who represented the full spectrum of indications for preterm cesarean section. In our material very high concentrations of the oxytocin receptor were seen
in some women in the beginning of labor, suggesting a role of those receptors in the initiation of labor in some cases. However, in view of the widespread interindividual variation in concentrations it is inconceivable that an up-regulation of receptors is a common mechanism for induction of the uterine activity in preterm labor, a condition seen in conjunction with some very different obstetric diseases. A more general factor in the initiation of labor than an increased level of the myometrial oxytocin receptors may instead be a raised release of oxytocin of uterine-fetoplacental origin not detectable in the plasma." 5 The lower oxytocin receptor concentration in advanced labor than in the beginning of labor found in the current study is in agreement with previous reports in the human. 1 , 6, 7 This finding suggests that oxytocin is a less important uterine stimulator in advanced labor than in early stages of the delivery process. The decrease in receptor concentration may be related to a down-regulating effect of oxytocin on its own receptor. An increase in circulating oxytocin during labor, if existing, is certainly limited,2, 3 but a more substantial release of oxytocin from uterine and fetoplacental sources 4 , 5 may be the cause of lowered oxytocin recep-
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1641
Table V. Concentration of oxytocin and vasopressin VI receptors (femtomoles per milligram protein; median values, range, and number of observations are indicated) of women delivered by cesarean section while in advanced labor with cervix dilated > 3 cm or with more than two contractions per IO minutes who had received oxytocin infusion for inducing or stimulating labor contractions; degree of cervical dilatation in different groups is also indicated Dose, duration, and interval between stopped infosion and operation
Total oxytocin dose s5 nmol >5 nmol Duration of oxytocin infusion s3 hr >3 hr Interval between stopped infusion and operation s30 min >30 min
Cervical dilatation
Oxytocin
Vasopressin Vl
6 (5-lO, n 9 (4-lO, n
= 6) = 6)
81 (17-105,n = 7) 34 (21-57, n = 6)
36 (5-9, n = 5) 80 (56-111, n = 5)
6 (5-10, n 9 (4-10, n
= 6) = 6)
81 (17-105, n = 7) 34 (21-57, n = 6)
36 (5-91, n = 5) 80 (56-111, n = 5)
7 (4-lO, n 8 (5-lO, n
= 8) = 4)
57 (21-94, n = 9) 32 (17-lO5, n = 4)
56 (5-111, n 80 (72-95, n
= 7) = 3)
Difference in oxytocin receptor concentration between patients with smaller or higher total doses of oxytocin and shorter or longer durations of oxytocin infusion was not statistically significant (p = 0.086 in both cases), nor were any other significant differences observed among various groups. tor concentration as delivery progresses. A further confirmation of the existence of such a mechanism of down-regulation is the finding of a tendency for reduced oxytocin receptor concentration in women who had received oxytocin infusion for ~ 3 hours or of a total dose > 5 nmol in comparison with those having had shorter treatment or lower total dose. A timerelated magnitude of down-regulation of oxytocin receptors is in agreement with some animal data. I' The vasopressin receptor concentration did not differ between women before and in the beginning of labor, but the vasopressin VI receptor may still playa role for the induction of uterine activity in pregnant women, the fetus secreting substantial amounts of vasopressin during stressful situations such as labor. 14 A down-regulating effect of vasopressin, presumably of fetoplacental origin, on the VI vasopressin site may also have caused the lowered concentration of this receptor in advanced labor after oxytocin treatment. The group in advanced labor without oxytocin treatment was too small to enable any statistical comparison, but the mean value in this group was in accord with that in the women having had such treatment, which supports this theory. The analog I-deamino-2-D-Tyr(OEt)-4-Thr-8-0rnoxytocin inhibited the effects of oxytocin in vitro to a degree that perfectly correlated with the concentration of oxytocin receptors. This again confirms that oxytocin stimulates uterine contractile activity in pregnant women by a specific effect on the oxytocin receptor. I The fact that inhibition of the in vitro effects of oxytocin was seen on myometrium from women in preterm labor with a variety of different causes supports the observation of a consistent therapeutic effect of the analog in this condition. s,1O However, although not demonstrated in the current study, this analog also blocks the VI vasopressin receptor l2 • 15 and in primary dysmenorrhea, a
condition in which an increased circulating level of va sopr~ssin plays a causal role,16 I-deamino-2-D-Tyr(OEt)-4Thr-8-0rn-oxytocin inhibits contractions, increases uterine blood flow, and alleviates the pain. 17 If vasopressin effects on the uterus contribute to the contractile activity in preterm labor, any stimulating action of this hormone on the uterus should consequently also be blocked by the analog. This again is in agreement with the inhibition by I-deamino-2-D-Tyr(OEt)-4-Thr-8Orn-oxytocin in pre term labor.solO REFERENCES 1. Maggi M, Del Carlo P, Fantoni G, et al. Human myome-
2.
3. 4. 5. 6.
7.
trium during pregnancy contains and responds to VI VP receptors as well as oxytocin receptors. J Clin Endocrinol Metab 1990;70: 1142-54. Fuchs AR, Romero R, Keefe 0, et al. Oxytocin secretion and human parturition: pulse frequency and duration increase during spontaneous labor in women. AM J OBSTET GYNECOL 1991;165:1515-23. Thornton SS, Davison JM, Baylis PH. Plasma oxytocin during the first and second stages of spontaneous human labour. Acta Endocrinol 1992;126:425-9. Lefebvre. DL, Giaid A, Bennett H, et al. Oxytocin gene expression in rat uterus. Science 1992;256:1553-5. Chibbar R, Miller FD, Mitchell BF. Synthesis of oxytocin in amnion, chorion and decidua may influence the timing of human parturition. J Clin Invest 1993;91:185-92. Fuchs AR, Fuchs F, Husslein P, Soloff MS. Oxytocin receptors and human parturition: a dual role for oxytocin in the initiation of labour. Science 1982;215:1396-8. Fuchs AR, Fuchs F, Husslein P, Soloff MS. Oxytocin receptors in pregnant human uterus. AM J OBSTET GYNECOL 1984; 150:734-41.
8. Akerlund M, Stromberg P, Hauksson A, et al. Inhibition of uterine contractions of premature labour with an oxytocin analogue. Results from a pilot study. Br J Obstet Gynaecol 1987;94: lO40-4.
.
9. Andersen LF, Lyndrup J, Akerlund M, Melin P. OT receptor blockade: a new principle in the treatment of the preterm labour? Am J Perinatol 1989;6: 196-9. 10. Goodwin TM, Paul R, Silver H, et al. The effect of the
Castro, Hobel, and Gornbein
oxytocin antagonist atosiban on preterm uterine activity in the human. AM] OBSTET GYNECOL 1994;170:474-8. 11. Maggi M, Magini A, Fiscella A, et al. Sex steroid modulation of neurohypophysial hormone receptors in human nonpregnant myometrium. ] Clin Endocrinol Metab 1992;74:385-92. 12. Melin P, Trojnar ], Johansson B, et al. Synthetic antagonists of the myometrial response to oxytocin and vasopressin. ] Endocrinol 1986; 111: 125-31. 13. Engstrom T, Atke A, Vilhardt H. Oxytocin receptors and contractile response of the myometrium after long term infusion of prostaglandin F2"" indomethacin, oxytocin and an oxytocin antagonist in rats. Regul Pept 1988;20:65-72.
December 1994 Am J Obstet Gynecol
14. Chard T, Hudson CN, Edwards CRW, Boyd NHR. Release of oxytocin and vasopressin by the human foetus during labour. Nature 1971;234:352-4. 15. Hauksson A, Akerlund M, Melin P. Uterine blood flow and myometrial activity at menstruation, and the action of vasopressin and a synthetic antagonist. Br] Obstet Gynaecol 1988;95:898-904. 16. Akerlund M, Stromberg P, Forsling ML. Primary dysmenorrhoea and vasopressin. Br ] Obstet Gynaecol 1979;86: 484-7. 17. Akerlund M. Can primary dysmenorrhoea be alleviated by a vasopressin antagonist? Acta Obstet Gynaecol Scand 1987;66:459-61.
Plasma levels of atrial natriuretic peptide in normal and hypertensive pregnancies: A meta-analysis Lony C. Castro, MD,' Calvin J. Hobel, MD,' and Jeffrey Gornbein, DrPhb
Los Angeles, California OBJECTIVE: Our goals were (1) to use meta-analysis to determine whether pregnancy and the puerperium are accompanied by alterations in plasma atrial natriuretic peptide levels when compared with the nonpregnant state and (2) to evaluate the additional effects of hypertensive disease during pregnancy on plasma atrial natriuretic peptide levels. STUDY DESIGN: Articles measuring atrial natriuretic peptide levels during pregnancy were reviewed. Data from articles meeting inclusion criteria were abstracted, and a meta-analysis was performed with the use of the maximum likelihood methods of Jennrich and Schluchter (Biometrics 1986;42:805-20). RESULTS: The mean atrial natriuretic peptide level in nonpregnant control subjects was 28.7 pg/ml (95% confidence interval 22.5 to 36.7). The mean plasma atrial natriuretic peptide level rose 41% to 40.5 pg/ml (95% confidence interval 31.7 to 51.8) in the third trimester (p < 0.0001). It was 71.1 pg/ml (95% confidence interval 51.2 to 98.7) or 148% greater than the mean nonpregnant level during the first week post partum (p < 0.0001). Compared with levels in pregnant control subjects, plasma atrial natriuretic peptide levels increased 52% to 52.1 pg/ml (95% confidence interval 32.9 to 82.5) in women with gestational hypertension (p < 0.005) and 130% to 78.8 pg/ml (95% confidence interval 52.3 to 118.8) in women with preeclampsia (p < 0.0001). Chronic hypertension did not significantly alter atrial natriuretic peptide levels. CONCLUSIONS: The 41 % increase in atrial natriuretic peptide levels in the third trimester suggests that atrial stretch receptors sense the expanded blood volume as normal to moderately increased. The rise in atrial natriuretic peptide during the first week post partum is consistent with known hemodynamic changes and suggests that atrial natriuretic peptide may be involved in the postpartum diuresis. The marked increase in plasma atrial natriuretic peptide levels observed in preeclampsia is not likely to result from elevated arterial pressures alone but may reflect underlying factors unique to this disease process. (AM J OBSTET GVNECOL 1994;171 :1642-51.)
Key words: Atrial natriuretic peptide, pregnancy, preeclampsia, hypertension, blood volume From the Division of Maternal-Fetal-Medicine, Department of Obstetrics and Gynecology, Cedars-Sinai Medical Center, a and the Department· of Biomathematics, b The University of California, Los Angeles, School of Medicine. Supported IJy United Cerebral Palsy Association grant No. R-40790 and IJy the Cigarette and Tobacco Surtax Fund of the State of California through the Tobacco Related Disease Research Program, grant No. 1KT96. Presented in part at the Eighth World Congress of Hypertension ill Pregnancy, Buenos Aires, Argentina, November 9, 1992.
1642
Received for publication January 12, 1994; revised July 26, 1994; accepted August 9, 1994. Reprint requests: Lony C. Castro, MD, Department of Obstetrics and Gynecology, Room 1738, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Los Angeles, CA 90048. Copyright © 1994 by MoslJy-Year Book, Inc. 0002-9378/94 $3.00 + 0 6/1/59680
oxytocin antagonist atosiban on pretenn uterine activity in the human. AM] OBSTET GYNECOL 1994;170:474-8. 22. Sheldrick EL, Flick-Smith HC. Effect of ovarian honnones on oxytocin receptor concentrations in explants of uterus from ovariectomized ewes. ] Reprod FertiI1993;97:241-5. 23. Soloff MS, Fernstrom MA, Periyasami S, Soloff S, Baldwin S, Wieder M. Regulation of oxytocin receptor concentration in rat uterine explants by estrogen and progesterone. Can] Biochem Cell Bioi 1983;61 :625-30.
December 1994 Am J Obstet Gynecol
24. Sheldrick EL, Flint AP. Endocrine control of uterine oxytocin receptors in the ewe. ] Endocrinol 1985; 106:249-58. 25. Randolph GW, Fuchs A. Pulsatile administration enhances the effect and reduces the dose of oxytocin required for induction of labor. Am] Perinatol 1989;6: 159-66. 26. Cummiskey KC, Dawood MY. Induction of labor with pulsatile oxytocin. AM] OBSTET GYNECOL 1990;163:186874.
Receptors for and myometrial responses to oxytocin and vasopressin in preterm and term human pregnancy: Effects of the oxytocin antagonist atosiban Thomas Bossmar, MD," Mats Akerlu~d, MD, PhD," Guido Fantoni, MD; Jazec Szamatowicz, MD,b Per Melin, PhD,d and Mario Maggi, MD, PhDc
Lund and Malmo, Sweden, Florence, Italy, and Bialystok, Poland OBJECTIVE: Our purpose was to study myometrial oxytocin and type V1 vasopressin receptors, the in vitro contractile effects of these hormones, and the influence of an oxytocin antagonist. STUDY DESIGN: Women delivered by cesarean section preterm (n = 51) and at term (n = 71), with and without labor contractions, gave myometrium for the estimation of oxytocin and V1 vasopressin receptors. The in vitro myometrial effects of the peptides and the influence on these of the competitive oxytocin receptor blocking agent 1-deamino-2-D-Tyr(OEt)-4-Thr-8-0rn-oxytocin were also tested. RESULTS: The median concentration of oxytocin receptors was 116 fmol/mg protein (range 15 to 372 fmol/mg protein) in patients delivered preterm not in labor, 134 fmol/mg protein (27 to 1421 fmol/mg protein) in the beginning of labor, and 46 fmol/mg protein (9 to 140 fmol/mg protein) in advanced labor. At term the corresponding concentrations were 172 (25 to 629),223 (24 to 414), and 70 (21 to 92) fmol/mg protein. The concentration of V1 vasopressin receptors also decreased in advanced labor. In advanced labor after oxytocin infusion a reduction in the concentration of the receptor for this hormone was observed, which appeared to be related to the duration and dose of treatment. Oxytocin receptors did not vary between women with different indications for cesarean section. The oxytocin effects in vitro and the degree of inhibition by the antagonist of oxytocin responses correlated with the concentration of oxytocin receptors but not with that of V1 vasopressin receptors. No correlation was seen between the response to vasopressin and concentrations of oxytocin or V1 vasopressin receptors. CONCLUSIONS: The effect of oxytocin on the myometrium in pregnancy is mediated by an oxytocin receptor, whereas vasopressin acts on both oxytocin and vasopressin receptors. The initiation of labor both preterm and at term may be primarily related to increased release of oxytocin, which is locally produced in the uterus and not detectable in the plasma, but oxytocin and vasopressin receptors may playa role in the regulation of labOr. The analog 1-deamino-2-D-Tyr(OEt)-4-Thr-8-0rn-oxytocin, which blocks both the oxytocin and the V1 vasopressin receptor, should inhibit labor both preterm and at term, the former confirming results of recent clinical studies in Sweden and the United States. (AM J OBSTEr GVNECOL 1994;171:1634-42.)
Key words: Oxytocin and VI vasopressin receptors, myometrium, preterm pregnancy, term pregnancy
From the Departments of Obstetrics and Gynecology, University Hospitals of Lund" and Bialystok: the Endocrinology Unit, University of Florence,' and Ferring Pharmaceuticals." Supported by Swedish Medical Research Council grant No. B93-17X06571-11, Polish Research Council grant No. KBN-447299203, the University of Lund, Sweden, Ferring Pharmaceuticals, and R. W. Johnson Pharmaceutical Research Institute.
1634
Received for publication December 7,1993; accepted May 13,1994. Reprint requests: Mats Akerlund, MD, PhD, Department of Obstetrics and Gynecology, University Hospital, S-221 85 Lund, Sweden. Copyright © 1994 by Mosby-Year Book, Inc. 0002-9378194 $3.00 + 0 6/1/57532
Volume 171, Number 6 Am J Obstet Gynecol
The human myometrium in pregnancy possesses oxytocin and type VI vasopressin receptors on which these peptides may act, I but data are conflicting regarding whether there is a significant increase in the plasma level of oxytocin or vasopressin before the onset of labor. 2. 3 However, oxytocin originates not only in the hypothalamus-posterior pituitary, but also in the decidua, amnion, chorion, and placenta:' 5 and hormone released from the latter sources may not necessarily be reflected in an increased plasma level. An increased myometrial oxytocin receptor concentration, resulting in a more pronounced effect of the oxytocin being available, has also been suggested as mechanism for labor induction, I. 6. 7 but it is not completely clear how receptors will change during the process of labor, vary between different obstetric complications, and be influenced by oxytocin treatment. In women in preterm labor the amount of data supporting the theory of an elevated receptor concentration as a common cause of contractions was very limited and estimations were performed for only a few of the different causes of this syndrome. I. 6. 7 Furthermore, the regulation of these receptors during different stages of preterm labor is virtually unknown. The therapeutic effect in this condition of the competitive oxytocin receptor blocking agent I-deamino-2-D-Tyr(OEt)-4Thr-8-0rn-oxytocin S - 1O is in agreement with a role of these receptors in the initiation of preterm labor contractions. The compound should be effective in the presence of both unchanged and elevated concentrations of oxytocin at the receptor. In this investigation we studied myometrial oxytocin and VI vasopressin receptors extensively in pregnant women undergoing cesarean section for a wide variety of indications, both preterm and at term and both before and during labor. These results were correlated to clinical data and findings in studies of contractile effects of the posterior pituitary hormones and the inhibition by I-deamino-2-D-Tyr(OEt)-4-Thr-8-0rnoxytocin of oxytocin responses of isolated myometrium from the same women. Material and methods
Subjects. Myometrial tissue samples were obtained from 122 white women who were undergoing cesarean section. They were all well informed of the purpose and procedure of the investigation and gave consent for participation. The study was approved by the local ethics committee. In 51 women the cesarean section was performed before the end of the thirty-sixth week of pregnancy. Twenty-seven were not in labor, with a cervical dilatation of 0 to 1.5 cm and zero to one contractions per 10 minutes, 18 in the beginning of labor with a cervical dilatation of 1.5 to 3 cm and one to two contractions per 10 minutes, and six were in advanced labor with a
Bossmar et al.
1635
more pronounced cervical change or higher frequency of contractions. The number of women delivered at term in the corresponding groups were 45, 9, and 17. At operation a specimen of myometrial tissue with a length of approximately 3 cm, a width of 0.7 x 0.5 cm, and a weight of about 2 gm was obtained from the upper rim of the transverse isthmic incision, this being located at the same part of the uterus irrespective of the presence of labor contractions. The material was immediately divided in two parts, one of which was placed in oxygenated Krebs-Ringer solution at 0° C and the other deep-frozen at - 80° C. The former part was used for myometrial contractility studies within 12 hours of operation. Membrane preparations. Details of the membrane preparation techniques were given previously. I. Jl In short, the previously frozen uterine specimens were first put in one type of buffer (10 mmoVL Tris-hydrochloric acid, pH 7.4, containing 1.5 mmoVL ethylenediaminetetraacetic acid, 0.5 mmoVL dithiothreitol, 1 mmoVL benzamidine, 0.01% bacitracin, and 0.002% soybean trypsin inhibitor) at 4° C and homogenized. The homogenate was centrifuged at 1000g for 10 minutes and the resulting supernatant centrifuged at 160,000g for 30 minutes at 4° C. The resulting pellet was dispersed in a second type of buffer (buffer II; 50 mmoVL Tris maleate, pH 7.6, containing 10 mmoVL magnesium sulfate, 1 mmoVL benzamidine, 0.01% bacitracin, and 0.002% soybean trypsin inhibitor) and then spun again at 160,000g for 30 minutes. The final pellet was then resuspended in buffer II and divided into 0.5 ml aliquots, frozen in solid carbon dioxide, and stored at - 80° C until assayed. Concentration of protein was determined with Bio-Rad protein reagent (Bio-Rad Laboratories, Munchen, Germany). Binding assays. As previously described, I, II aliquots of membranes (0.3 mg/ml) were incubated with ligands in buffer II in the presence of 0.1% bovine serum albumin at 22° C in tubes containing tritiated d(CH 2 )s Tyr-Me-arginine vasopressin (50.7 Cilmmol, New England Nuclear; Boston) for 60 minutes. This arginine vasopressin analog binds specifically to the VI vasopressin receptor. Aliquots were also incubated in tubes containing tritiated oxytocin (36 Cilmmol, New England Nuclear) for 180 minutes. Tritiated d(CH 2 )sTyr-Me-arginine vasopressin and tritiated oxytocin were present at 0.7 nmoVL in tubes containing increasing concentrations (0.1 to lOOOO nmoVL) of the corresponding unlabeled peptides and at 0.03 to 0.7 nmoVL in tubes without unlabeled ligands (final volume 0.25 ml). All measurements were performed in triplicate. Mter incubation membranes were filtered through Whatman GF/B filters (Clifton, N.J.) that had been presoaked in ice-cold 50 mmoVL Tris with a pH of 7.4 and in 0.1 % bovine serum albumin with the Brandel M-48R 48-well cell harvester (Gaithersburg, Md.). Fil-
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December 1994 Am J Ob,tet Gynecol
OTreceptor (fmol/mg protein)
.1421
600
400
200
I
--+--
-.
i
O~-----r--------r--------r--------r-------~----
before
beginning noOT
beginning OT
advanced noOT
advanced OT
State of labor
Fig. 1. Concentrations of the oxytocin (OT) receptor (median is indicated) of women delivered by cesarean section preterm while not in labor with cervical dilatation of 0 to 1.5 cm and zero to one contraction per 10 minutes, in beginning of labor with cervical dilatation of 1.5 to 3 cm and one to two contractions per 10 minutes, and in advanced labor with more pronounced cervical change or higher frequency of contractions. Subjects in latter two groups who had or had not received oxytocin (OT) infusion for inducing or stimulating labor contractions are accounted for separately. In women who had not received oxytocin, concentrations in beginning of labor and in advanced labor differed significantly.
ters were washed twice with 3 ml of ice-cold 50 mmoVL Tris with a pH of 7.4 and placed in liquid scintillation vials. Radioactivity retained by filters was measured in a liquid scintillation counter after overnight incubation in 10 ml of scintillation fluid. Oxytocin receptors were studied with homologous competition curves for oxytocin in the presence of 2 nmoVL d(CH2)5Tyr-Me-arginine vasopressin to mask the VI vasopressin site. Similarly, the density of VI arginine vasopressin receptors was investigated with homologous displacement curves for d(CH2)5Tyr-Mearginine vasopressin in the presence of 5 nmol!L oxytocin to block the oxytocin receptors. 1. 11 In 42 uterine specimens receptor density was evaluated with two- or three-point Scatchard plots, freezing the dissociation constant values at 0.5 nmol!L for oxytocin curves and at 0.16 nmoVL for d(CH2)5Tyr-Me-arginine vasopressin curves. Myometrial contractility studies. Myometrial segments with a standardized size of 2 x 2 x 10 mm, all with the same muscle fiber direction, were prepared for final dissection under a microscope at tenfold magnification. "Six strips from each patient were prepared. They were mounted in organ baths containing 10 ml
of Krebs-Ringer solution at pH 7.4 at a temperature of 37° C and aerated with carbogen. After an adaptation period of at least 30 minutes isometric contractions at a resting tension of 2 mN were recorded with a Grass force transducer (model IT 03). The effect of 30 nmol!L oxytocin and arginine vasopressin and the inhibitory effect of the same concentration of I-deamino- 2 -D-Tyr( 0 Et )-4-Thr-8-0rn -oxytocin on the responses of oxytocin were tested on the preparations. 12 The oxytocin and vasopressin responses were measured as the area under the curve during a 10minute period after administration. The antagonist was added to the tissue bath 1 minute before the oxytocin administration and the effect expressed as percent inhibition of the oxytocin response. Repeated washing was used between each drug administration. Statistical methods. Results obtained in different groups of women were compared by Mann-Whitney testing. Spearman's rank test was used for analysis of correlations.
Results Receptor estimations. Oxytocin bound with a dissociation constant of 0.60 ± 0.39 nmol!L (~ean ± SD,
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OTreceptor (fmollmg protein)
600
400
y
200
..:!.....
..·
·
-....· y
.
.
-,I
O~-----T--------~-------'--------~--------~--beginning beginning advanced advanced before OT no OT noOT OT
State of labor
Fig. 2. Concentrations of oxytocin (OT) receptor (median is indicated) of women delivered by cesarean section at term while not in labor with cervical dilatation of 0 to 1.5 cm and zero to one contraction per 10 minutes, in beginning of labor with cervical dilatation of 1.5 to 3 cm and one to two contractions per 10 minutes, and in advanced labor with more pronounced cervical change or higher frequency of contractions. Subjects in latter two groups who had or had not received oxytocin infusion for inducing or stimulating labor contractions are accounted for separately. In women who had not received oxytocin, concentrations in beginning of labor and in advanced labor differed significantly.
n = 81) to the oxytocin site, whereas d(CH2)2Tyr-Mearginine vasopressin showed a dissociation constant of 0.34 ± 0.18 nmoVL (n = 46) for the VI vasopressin site. No differences were seen between the various groups of patients. Concentrations of the oxytocin receptor in different stages of labor in women delivered preterm are shown in Fig. 1 and those in term-delivered women in Fig. 2, and the statistical significances of observed differences are shown in Table I. The interindividual variation was large, particularly in women in the beginning of labor both preterm and at term. In patients delivered preterm who had not received oxytocin treatment the median concentration of the oxytocin receptor of those not in labor was 116 fmoVmg protein (range 15 to 372 fmoVmg protein), in the beginning of labor 134 fmoVmg protein (27 to 1421 fmoVmg protein), and in advanced labor it had decreased significantly to 46 fmoVmg protein (9 to 140 fmoVmg protein). In women delivered at term and not having received oxytocin treatment the receptor concentration in the group not in labor was 172 fmoVmg protein (25 to 629 fmoVmg protein) and in the beginning of labor 223 fmoVmg
protein (24 to 414 fmoVmg protein). In the advanced labor it had again decreased to a median of 70 fmoVmg protein (range 21 to 92 fmoVmg protein), a decrease that was also observed for preterm and term patients taken together (Table I). There were no statistically significant differences in oxytocin receptor concentration between women in preterm and term labor in corresponding stages of labor (Figs. 1 and 2). In none of the groups with indications for cesarean section was the concentration of oxytocin receptors significantly different from that in other women at the same stage of labor (Tables II and III). The concentrations of the oxytocin receptor did not correlate significantly with the pregnancy weeks (Ts = 0.11) or the age of the patients (Ts = 0.08). Concentrations of the VI vasopressin receptor in women who had not received oxytocin treatment did not vary significantly between those in pre term and in term labor (Table IV), between different stages of labor (Fig. 3), or between women with different indications for cesarean section (not shown). Concentrations of oxytocin and VI vasopressin receptors in women who had been treated by oxytocin
1638 Bossmar at al.
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Am J Obstet Gynecol
Table I. Significance of differences
Preterm Term All
Beginning vs advanced
Before vs advanced
Before vs beginning
p= P=
p = 0.043
0.065 0.026 P = 0.002
NS NS NS
P= P=
0.086 0.001
NS, Not significant.
Table II. Myometrial oxytocin receptor concentrations (femtomoles per milligram of protein, median values with number of subjects in parentheses) in women with various indications for cesarean section before term and in different stages of labor State of labor Advanced
Beginning Indication for cesarean section
Before
Preeclampsia Placenta previa Abruptio placentae Disproportion-breech presentation Diabetes mellitus Fetal distress Poor obstetric history Fetal growth retardation Multipregnancy-polyhydramnios Premature rupture of membranes Previous cesarean section Other
127 (7) 163 (1) 108 50 15 132 167 124 213 116
No oxytocin
(1) (3) (1) (3) (2) (3) (1) (5)
1
Oxytocin
No oxytocin
I
Oxytocin
88 (2) 45 (1) 92 (3) 163 (5) 65 (2) 93 (1)
74 (2) 288 (3)
28 (1) 76 (1) 46 (1)
94 (1)
478 (1)
No significant difference was seen between any of the groups.
infusion before cesarean section and who were in the beginning of or in advanced labor are shown in Figs. 1 through 3 and in Tables II through IV. There were no statistically significant differences in oxytocin receptor concentrations between oxytocin-treated women preterm or at term or within these groups between those who had or had not received oxytocin treatment but who were in corresponding stages of labor (Figs. 1 and 2). For oxytocin-treated, pre term- and term-delivered patients taken together there was a significant difference between the oxytocin receptor concentrations in the beginning of labor (median) 119 fmoVmg protein (46 to 643 fmoVmg protein) and in advanced labor of 43 fmoVmg protein (17 to 105 fmoVmg protein)
total dose, duration of infusion, and interval between stopped infusion and operation are shown in Table V. The difference between the groups of shorter and longer durations of oxytocin infusion and groups of lower and higher total doses of oxytocin was not statistically significant. The cervical status did not differ between these groups. The total dose and duration of oxytocin treatment did not influence the concentration of VI vasopressin receptors, and no influence of the interval between stopped infusion and operation on either receptor was seen. The correlation between the concentration of oxytocin receptors and the durati{)n of oxytocin infusion had a coefficient, T., of -0.54
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Table III. Myometrial oxytocin receptor concentrations (femtomoles per milligram protein, median values with number of subjects in parentheses) in women with various indications for cesarean section at term and in different stages of labor State of labor Beginning Indication for cesarean section
Before
Preeclampsia Placenta previa Abruptio placentae Disproportion-breech presentation Diabetes mellitus Fetal distress Poor obstetric history Fetal growth retardation Primary uterine inertia Labor dystocia Multipregnancy-polyhydramnios Previous cesarean section Other
629 (1) 108 (3)
No oxytocin
143 (10) 99 (2)
I
Advanced Oxytocin
126 (3) 414 (I)
189 (7) 240 (2)
119 (3)
No oxytocin
I
92 (1) 57 (1)
39 (3)
52 (2)
34 (5)
93 (1) 167 (2) 187 (13) 160 (5)
Oxytocin
61 (I) 39 (2) 81 (I)
223 (I)
No significant difference was seen between any of the groups.
Table IV. Concentration of vasopressin VI receptors (femtomoles per milligram protein; median values, range, and number of observations are indicated) of women delivered by cesarean section pre term or at term while not in labor with cervical dilatation of 0 to 1.5 cm and zero to one contraction per 10 minutes, in beginning oflabor with cervical dilatation of 1.5 to 3 cmand one to two contractions per 10 minutes, and in advanced labor with more pronounced cervical change or higher frequency of contractions* State of labor
Pre term delivery
Term delivery
Before Beginning, no oxytocin Beginning, oxytocin Advanced, no oxytocin Advanced, oxytocin
116 (57-252, n = 16) 116 (41-207, n = 6)
105 (33-291, n = 24) 108 (34-135, n = 4) 148 (103-193, n = 2) 65 (n = I) 72 (5-111, n = 9)
115 (94-139, n = 3) 91 (n = I)
No significant differences were seen between preterm~ and term-delivered patients. *Subjects in·the latter group who had or had not received oxytocin infusion for inducing or stimulating labor contractions are accounted for separately.
correlation was seen between the responses to vasopressin and concentrations of oxytocin receptor (rs = 0.03) or the VI vasopressin receptor (rs = 0.07). The inhibitory effect of l-deamino-2-D-Tyr(OEt)-4Thr-8-0rn-oxytocin on oxytocin responses correlated with concentrations of the oxytocin receptor (rs = 0.39, P = 0.007) but not with those of the VI vasopressin receptor (rs = 0.40). Comment The lack of difference in oxytocin or VI vasopressin receptor affinity among different states of labor in this investigation is in agreement with a previous report l and supports the concept that an up regulation in such affinity is not involved in mechanisms oflabor induction. In the current study the concentration of oxytocin receptors correlated with the in vitro effects of oxytocin on uterine strips from the same women. This confirms the validity of the techniques used.
The fact that the in vitro effects of oxytocin correlated with concentrations of the oxytocin but not the VI vasopressin receptor confirms the previous finding that oxytocin stimulates uterine contractile activity in pregnant women by a specific effect on the oxytocin receptor.1 The lack of correlation between the in vitro effects of vasopressin and concentrations of the oxytocin or VI vasopressin receptor is also in agreement with previous data indicating that the action of this peptide is not confined to only one receptor but involves both the oxytocin and VI vasopressin sites. I The concentrations of the oxytocin receptor in different stages of term labor were generally in agreement with those found previously, I but in spite of a numeric difference between women before and in the beginning of labor and a material that was in all about 10 times larger than that of the previous studies we could not confirm statistically the hypothesis that an increase in oxytocin receptors is involved in the initiation of labor contrac-
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Am J Obstet Gyneco1
V1 VPreceptor (fmol/mg protein) 300
.' 200
...
'1' 100
.I. I
.,' I
o~----~------~--------~------~------~---advanced beginning beginning advanced before OT noOT noOT OT
State of labor
Fig. 3. Concentrations of VI vasopressin (VP) receptor (median is indicated) of women delivered by cesarean section preterm and at term, taken together, while not in labor with cervix dilatation of 0 to 1.5 cm and zero to one contraction per 10 minutes, in beginning of labor with cervical dilatation of 1.5 to 3 cm and one to two contractions per 10 minutes, and in advanced labor with more pronounced cervical change or higher frequency of contractions. Subjects in latter two groups who had or had not received oxytocin (OT) infusion for inducing or stimulating labor contractions are accounted for separately. Concentration in advanced labor in oxytocin-treated patients differed significantly from that of women before or in beginning of labor who had not received oxytocin treatment.
tions.l. 6, 7 A significant increase between concentrations before and in the beginning of labor did not even occur when values from pre term- and term-delivered patients were pooled in such a comparison. One explanation for this lack of difference is the wide interindividual variation in our results, something that may have been related to the fact that the material of the current study was obtained from patients with the full spectrum of indications for cesarean section, whereas the women giving myometrium for the previous studies had only a limited number of diagnoses. 1. 6. 7 In the preterm condition there was also a numeric but insigificant increase in oxytocin receptor concentrations when women not in labor were compared with subjects in the beginning of labor. Previous assumptions that an elevated concentration of this receptor is an important factor in the cause of preterm labor was supported by results from a small patient material, limited to only a few causes and not confirmed statistically, 1,6,7 whereas again the myometrial samples for the current study were obtained from about 10 times more patients who represented the full spectrum of indications for preterm cesarean section. In our material very high concentrations of the oxytocin receptor were seen
in some women in the beginning of labor, suggesting a role of those receptors in the initiation of labor in some cases. However, in view of the widespread interindividual variation in concentrations it is inconceivable that an up-regulation of receptors is a common mechanism for induction of the uterine activity in preterm labor, a condition seen in conjunction with some very different obstetric diseases. A more general factor in the initiation of labor than an increased level of the myometrial oxytocin receptors may instead be a raised release of oxytocin of uterine-fetoplacental origin not detectable in the plasma." 5 The lower oxytocin receptor concentration in advanced labor than in the beginning of labor found in the current study is in agreement with previous reports in the human. 1 , 6, 7 This finding suggests that oxytocin is a less important uterine stimulator in advanced labor than in early stages of the delivery process. The decrease in receptor concentration may be related to a down-regulating effect of oxytocin on its own receptor. An increase in circulating oxytocin during labor, if existing, is certainly limited,2, 3 but a more substantial release of oxytocin from uterine and fetoplacental sources 4 , 5 may be the cause of lowered oxytocin recep-
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Table V. Concentration of oxytocin and vasopressin VI receptors (femtomoles per milligram protein; median values, range, and number of observations are indicated) of women delivered by cesarean section while in advanced labor with cervix dilated > 3 cm or with more than two contractions per IO minutes who had received oxytocin infusion for inducing or stimulating labor contractions; degree of cervical dilatation in different groups is also indicated Dose, duration, and interval between stopped infosion and operation
Total oxytocin dose s5 nmol >5 nmol Duration of oxytocin infusion s3 hr >3 hr Interval between stopped infusion and operation s30 min >30 min
Cervical dilatation
Oxytocin
Vasopressin Vl
6 (5-lO, n 9 (4-lO, n
= 6) = 6)
81 (17-105,n = 7) 34 (21-57, n = 6)
36 (5-9, n = 5) 80 (56-111, n = 5)
6 (5-10, n 9 (4-10, n
= 6) = 6)
81 (17-105, n = 7) 34 (21-57, n = 6)
36 (5-91, n = 5) 80 (56-111, n = 5)
7 (4-lO, n 8 (5-lO, n
= 8) = 4)
57 (21-94, n = 9) 32 (17-lO5, n = 4)
56 (5-111, n 80 (72-95, n
= 7) = 3)
Difference in oxytocin receptor concentration between patients with smaller or higher total doses of oxytocin and shorter or longer durations of oxytocin infusion was not statistically significant (p = 0.086 in both cases), nor were any other significant differences observed among various groups. tor concentration as delivery progresses. A further confirmation of the existence of such a mechanism of down-regulation is the finding of a tendency for reduced oxytocin receptor concentration in women who had received oxytocin infusion for ~ 3 hours or of a total dose > 5 nmol in comparison with those having had shorter treatment or lower total dose. A timerelated magnitude of down-regulation of oxytocin receptors is in agreement with some animal data. I' The vasopressin receptor concentration did not differ between women before and in the beginning of labor, but the vasopressin VI receptor may still playa role for the induction of uterine activity in pregnant women, the fetus secreting substantial amounts of vasopressin during stressful situations such as labor. 14 A down-regulating effect of vasopressin, presumably of fetoplacental origin, on the VI vasopressin site may also have caused the lowered concentration of this receptor in advanced labor after oxytocin treatment. The group in advanced labor without oxytocin treatment was too small to enable any statistical comparison, but the mean value in this group was in accord with that in the women having had such treatment, which supports this theory. The analog I-deamino-2-D-Tyr(OEt)-4-Thr-8-0rnoxytocin inhibited the effects of oxytocin in vitro to a degree that perfectly correlated with the concentration of oxytocin receptors. This again confirms that oxytocin stimulates uterine contractile activity in pregnant women by a specific effect on the oxytocin receptor. I The fact that inhibition of the in vitro effects of oxytocin was seen on myometrium from women in preterm labor with a variety of different causes supports the observation of a consistent therapeutic effect of the analog in this condition. s,1O However, although not demonstrated in the current study, this analog also blocks the VI vasopressin receptor l2 • 15 and in primary dysmenorrhea, a
condition in which an increased circulating level of va sopr~ssin plays a causal role,16 I-deamino-2-D-Tyr(OEt)-4Thr-8-0rn-oxytocin inhibits contractions, increases uterine blood flow, and alleviates the pain. 17 If vasopressin effects on the uterus contribute to the contractile activity in preterm labor, any stimulating action of this hormone on the uterus should consequently also be blocked by the analog. This again is in agreement with the inhibition by I-deamino-2-D-Tyr(OEt)-4-Thr-8Orn-oxytocin in pre term labor.solO REFERENCES 1. Maggi M, Del Carlo P, Fantoni G, et al. Human myome-
2.
3. 4. 5. 6.
7.
trium during pregnancy contains and responds to VI VP receptors as well as oxytocin receptors. J Clin Endocrinol Metab 1990;70: 1142-54. Fuchs AR, Romero R, Keefe 0, et al. Oxytocin secretion and human parturition: pulse frequency and duration increase during spontaneous labor in women. AM J OBSTET GYNECOL 1991;165:1515-23. Thornton SS, Davison JM, Baylis PH. Plasma oxytocin during the first and second stages of spontaneous human labour. Acta Endocrinol 1992;126:425-9. Lefebvre. DL, Giaid A, Bennett H, et al. Oxytocin gene expression in rat uterus. Science 1992;256:1553-5. Chibbar R, Miller FD, Mitchell BF. Synthesis of oxytocin in amnion, chorion and decidua may influence the timing of human parturition. J Clin Invest 1993;91:185-92. Fuchs AR, Fuchs F, Husslein P, Soloff MS. Oxytocin receptors and human parturition: a dual role for oxytocin in the initiation of labour. Science 1982;215:1396-8. Fuchs AR, Fuchs F, Husslein P, Soloff MS. Oxytocin receptors in pregnant human uterus. AM J OBSTET GYNECOL 1984; 150:734-41.
8. Akerlund M, Stromberg P, Hauksson A, et al. Inhibition of uterine contractions of premature labour with an oxytocin analogue. Results from a pilot study. Br J Obstet Gynaecol 1987;94: lO40-4.
.
9. Andersen LF, Lyndrup J, Akerlund M, Melin P. OT receptor blockade: a new principle in the treatment of the preterm labour? Am J Perinatol 1989;6: 196-9. 10. Goodwin TM, Paul R, Silver H, et al. The effect of the
Castro, Hobel, and Gornbein
oxytocin antagonist atosiban on preterm uterine activity in the human. AM] OBSTET GYNECOL 1994;170:474-8. 11. Maggi M, Magini A, Fiscella A, et al. Sex steroid modulation of neurohypophysial hormone receptors in human nonpregnant myometrium. ] Clin Endocrinol Metab 1992;74:385-92. 12. Melin P, Trojnar ], Johansson B, et al. Synthetic antagonists of the myometrial response to oxytocin and vasopressin. ] Endocrinol 1986; 111: 125-31. 13. Engstrom T, Atke A, Vilhardt H. Oxytocin receptors and contractile response of the myometrium after long term infusion of prostaglandin F2"" indomethacin, oxytocin and an oxytocin antagonist in rats. Regul Pept 1988;20:65-72.
December 1994 Am J Obstet Gynecol
14. Chard T, Hudson CN, Edwards CRW, Boyd NHR. Release of oxytocin and vasopressin by the human foetus during labour. Nature 1971;234:352-4. 15. Hauksson A, Akerlund M, Melin P. Uterine blood flow and myometrial activity at menstruation, and the action of vasopressin and a synthetic antagonist. Br] Obstet Gynaecol 1988;95:898-904. 16. Akerlund M, Stromberg P, Forsling ML. Primary dysmenorrhoea and vasopressin. Br ] Obstet Gynaecol 1979;86: 484-7. 17. Akerlund M. Can primary dysmenorrhoea be alleviated by a vasopressin antagonist? Acta Obstet Gynaecol Scand 1987;66:459-61.
Plasma levels of atrial natriuretic peptide in normal and hypertensive pregnancies: A meta-analysis Lony C. Castro, MD,' Calvin J. Hobel, MD,' and Jeffrey Gornbein, DrPhb
Los Angeles, California OBJECTIVE: Our goals were (1) to use meta-analysis to determine whether pregnancy and the puerperium are accompanied by alterations in plasma atrial natriuretic peptide levels when compared with the nonpregnant state and (2) to evaluate the additional effects of hypertensive disease during pregnancy on plasma atrial natriuretic peptide levels. STUDY DESIGN: Articles measuring atrial natriuretic peptide levels during pregnancy were reviewed. Data from articles meeting inclusion criteria were abstracted, and a meta-analysis was performed with the use of the maximum likelihood methods of Jennrich and Schluchter (Biometrics 1986;42:805-20). RESULTS: The mean atrial natriuretic peptide level in nonpregnant control subjects was 28.7 pg/ml (95% confidence interval 22.5 to 36.7). The mean plasma atrial natriuretic peptide level rose 41% to 40.5 pg/ml (95% confidence interval 31.7 to 51.8) in the third trimester (p < 0.0001). It was 71.1 pg/ml (95% confidence interval 51.2 to 98.7) or 148% greater than the mean nonpregnant level during the first week post partum (p < 0.0001). Compared with levels in pregnant control subjects, plasma atrial natriuretic peptide levels increased 52% to 52.1 pg/ml (95% confidence interval 32.9 to 82.5) in women with gestational hypertension (p < 0.005) and 130% to 78.8 pg/ml (95% confidence interval 52.3 to 118.8) in women with preeclampsia (p < 0.0001). Chronic hypertension did not significantly alter atrial natriuretic peptide levels. CONCLUSIONS: The 41 % increase in atrial natriuretic peptide levels in the third trimester suggests that atrial stretch receptors sense the expanded blood volume as normal to moderately increased. The rise in atrial natriuretic peptide during the first week post partum is consistent with known hemodynamic changes and suggests that atrial natriuretic peptide may be involved in the postpartum diuresis. The marked increase in plasma atrial natriuretic peptide levels observed in preeclampsia is not likely to result from elevated arterial pressures alone but may reflect underlying factors unique to this disease process. (AM J OBSTET GVNECOL 1994;171 :1642-51.)
Key words: Atrial natriuretic peptide, pregnancy, preeclampsia, hypertension, blood volume From the Division of Maternal-Fetal-Medicine, Department of Obstetrics and Gynecology, Cedars-Sinai Medical Center, a and the Department· of Biomathematics, b The University of California, Los Angeles, School of Medicine. Supported IJy United Cerebral Palsy Association grant No. R-40790 and IJy the Cigarette and Tobacco Surtax Fund of the State of California through the Tobacco Related Disease Research Program, grant No. 1KT96. Presented in part at the Eighth World Congress of Hypertension ill Pregnancy, Buenos Aires, Argentina, November 9, 1992.
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Received for publication January 12, 1994; revised July 26, 1994; accepted August 9, 1994. Reprint requests: Lony C. Castro, MD, Department of Obstetrics and Gynecology, Room 1738, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Los Angeles, CA 90048. Copyright © 1994 by MoslJy-Year Book, Inc. 0002-9378/94 $3.00 + 0 6/1/59680