Induction of labor with RU 486 (mifepristone) in relaxin-deficient rats: antepartum administration of relaxin facilitates delivery and increases pup survival

Induction of labor with RU 486 (mifepristone) in relaxin-deficient rats: antepartum administration of relaxin facilitates delivery and increases pup survival

American Journal of Obstetrics and Gynecology (2004) 190, 229e38 www.elsevier.com/locate/ajog Induction of labor with RU 486 (mifepristone) in relax...

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American Journal of Obstetrics and Gynecology (2004) 190, 229e38

www.elsevier.com/locate/ajog

Induction of labor with RU 486 (mifepristone) in relaxin-deficient rats: Antepartum administration of relaxin facilitates delivery and increases pup survival Shuangping Zhao, PhD,a O. David Sherwood, PhDa,b,* Department of Molecular and Integrative Physiology,a and the College of Medicine, University of Illinois at Urbana-Champaign,b Urbana, Ill

–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– KEY WORDS Cervix Induced delivery Rat Relaxin RU 486

Objective: This study was conducted to determine whether antepartum administration of relaxin improves RU 486einduced delivery at term in rats that lack circulating endogenous relaxin. Study design: Pregnant rats were modified two ways to obtain circulating levels of relaxin and progesterone that resemble those of pregnant humans: relaxin was immunoneutralized throughout the second half of the 23-day pregnancy and high progesterone levels were sustained until term by inserting progesterone implants on day 20. Porcine relaxin was administered subcutaneously from 8 AM on day 20 until delivery. Labor was induced by administering RU 486 subcutaneously at 4 AM on day 22. Results: After induction of labor with RU 486, labor and delivery were faster, and the incidence of live births was higher when rats were also administered relaxin during the antepartum period. Conclusion: Antepartum administration of relaxin in combination with RU 486 has beneficial effects on delivery in relaxin-deficient rats. Ó 2004 Elsevier Inc. All rights reserved.

––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Relaxin and progesterone are secreted by the corpora lutea throughout pregnancy in rats. Relaxin is detectable in the peripheral blood by day 10, and serum levels remain elevated (>40 ng/mL) from day 12 until delivery on day 23.1 At functional luteolysis during the 2 days

Supported by National Institutes of Health grant No. PHS 1 R01 HD40448 (O. D. S.) and National Institutes of Health postdoctoral fellowship from the University of Illinois Reproductive Biology Training grant No. PH5 5 T32 HD07028 and a Lalor Foundation postdoctoral fellowship (S. Z.). * Reprint requests: O. David Sherwood, PhD, Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, 524 Burrill Hall, 407 S Goodwin Ave, Urbana, IL 61801. E-mail: [email protected] 0002-9378/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/S0002-9378(03)00946-3

before birth, there is both a surge in serum relaxin levels (O100 ng/mL) that is attributable to the release of relaxin from luteal cell storage granules and a decline in serum progesterone levels that is necessary for birth to occur in this species.1 Two models were developed to determine the physiologic roles of relaxin during rat pregnancy. With the MCA1-treated pregnant rat model, a monoclonal antibody designated MCA1 that is specific for rat relaxin is administered intravenously daily from day 12 through day 22 to neutralize the biologic actions of circulating relaxin.2 With the ovariectomized pregnant rat model, rats are ovariectomized on day 9 and then given replacement therapy with progesterone and estrogen in subcutaneous Silastic silicone rubber (Dow Corning, Midland, Mich) implants.3 Relaxin is given subcutaneously by

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Figure 1 Diagram of the design of experiment 1. See Material and methods for details.

a miniature osmotic pump. Findings with both models are in close agreement, and they demonstrate that relaxin plays a major role in promoting marked extensibility (softening) and growth of the cervix, and thereby enables rapid and safe delivery of the young.1-3 The physiology of relaxin and progesterone in women differs from that in rats.1 In women, both relaxin and progesterone are secreted by the corpora lutea during the first trimester. Circulating relaxin levels are maximal during the first trimester and they do not exceed 2 ng/mL. Serum relaxin levels decline at the end of the first trimester and they are only approximately 0.5 ng/mL during late pregnancy. Serum relaxin levels do not surge before birth in humans as they do in rats. During late pregnancy, serum levels of relaxin in women are more than two orders of magnitude lower than they are in rats. There is currently no evidence that the low serum levels of relaxin contribute to cervical modifications during late pregnancy in women. The growing placenta is the predominant source of circulating progesterone after the first trimester. Serum levels of progesterone increase progressively during the second and third trimesters, and they do not drop markedly during the antepartum period, as is the case in rats. RU 486 (mifepristone) binds progesterone receptors (PR) with greater affinity than does progesterone,4 and the administration of this antiprogesterone during pregnancy promotes softening and dilatation of the cervix in rats and primates.5 Moreover, RU 486 induces labor when administered during late pregnancy in rats.6 Although RU 486 is used extensively to terminate human

Zhao and Sherwood pregnancy during the first trimester, its use to induce both labor and cervical ripening at term in the human has received only limited experimental attention. After the administration of RU 486 to humans at term, it was reported that delivery took place between 24 and 96 hours after treatment, and there was an improvement in the cervical score.7-11 However, the administration of RU 486 did little to facilitate the delivery process. The duration of labor was not shortened,9 the frequency of instrument deliveries was not reduced,9 and with one exception,11 the frequency of cesarean sections was not reduced.7-10 Thus, available evidence indicates that RU 486 induces delivery in humans at term, but it does not remodel the cervix to the extent that labor is shorter and delivery is easier than with a control. The current study was conducted to test the hypothesis that the induction of labor by administration of RU 486 in combination with relaxin during late pregnancy results in faster labor and delivery than does induction of delivery with RU 486 alone under conditions of endogenous relaxin deficiency. To test this hypothesis, the pregnant rat was modified in two ways to obtain serum levels of relaxin and progesterone that more nearly resemble those in late pregnancy in the human. First, rats were made relaxin deficient throughout the second half of pregnancy by neutralizing endogenous circulating relaxin with a monoclonal antibody for rat relaxin. Second, rats were given progesterone implants during late pregnancy to ensure that serum progesterone levels remain elevated at term pregnancy, as is the case in pregnant women. Finding that the administration of RU 486 and relaxin in combination was most beneficial at labor induction, the effect of each agent alone, and in combination, on the extensibility and growth of the cervix was determined in relaxin-deficient pregnant rats.

Material and methods Animals Primiparous Sprague-Dawleyederived rats (Harlan, Indianapolis, Ind) were bred at 70 to 84 days of age. The day that sperm was found in the vagina was designated day 1 of pregnancy. The animals arrived on day 3 and were housed individually. Rats had free access to water and Teklad 6% mouse/rat diet 8664 (Harlan/Teklad, Madison, Wis), and they were maintained in a light-controlled room with alternating 14 hours of light (7 AMe9 PM) and 10 hours of darkness at a temperature of 23(C to 25(C. The animal experimentation described in this study was approved by the University of Illinois Institutional Animal Care and Use Committee.

Effects of RU 486 and relaxin on delivery Figure 1 shows a diagram of experiment 1. From day 8 of pregnancy, the light schedule was shifted to 9 PM to

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Zhao and Sherwood 11 AM to synchronize more precisely the time of delivery among rats.1 On day 9, the rats were laparotomized under ether anesthesia, and the number of implantation sites was determined. Only rats with seven or more implantation sites were used.1 The pregnant rats were divided into seven groups: phosphate-buffered saline solution (PBS), monoclonal antibody for fluorescein, monoclonal antibody control (MCAF), PBS + progesterone (P), PBS + P + antiprogesterone RU 486 (AP), monoclonal antibody specific for rat relaxin (MCA1), MCA1 + P + AP, and MCA1 + P + porcine relaxin (R) + AP. Three treatment groups were made relaxin deficient by following a previously described procedure for immunoneutralization of endogenous circulating relaxin with monoclonal antibody MCA1.2 In brief, unanesthetized rats were placed in a restraining device and injected in the tail vein once daily between 9 and 10 AM with 5 mg of MCA1 from day 12 through day 19 and with 10 mg MCA1 from day 20 through day 22. Control rats were injected with the same amount of MCAF or 0.5 mL of PBS vehicle. During the antepartum period (3 days before delivery), control groups PBS and MCAF received empty progesterone implants, relaxin vehicle, and RU 486 vehicle. Both groups, which had endogenous circulating relaxin and gave birth spontaneously early on day 23 of pregnancy, were used to obtain normal delivery data.2 Group PBS + P was used to determine how effectively the administration of progesterone blocked delivery in rats. At 8 AM on day 20, rats were anesthetized with methoxyflurane (Metofane, Schering-Plough, Union, NJ), and two silicon tubing implants that contained progesterone were inserted subcutaneously along each flank as previously described.3 Group PBS + P + AP was used to determine how effectively RU 486 induced delivery in progesterone-blocked rats. Because this group had endogenous circulating relaxin throughout the second half of pregnancy, it also served as a positive control for endogenous circulating relaxin. This group was fitted with progesterone implants on day 20 as previously described, and delivery was induced by subcutaneous administration of 8 mg of RU 486 (Sigma Chemical Co, St Louis, Mo) at 4 AM on day 22 in 0.4 mL of vehicle (ethanol/sesame oil, 1:7). Group MCA1 was a negative control for relaxin. These rats were fitted with empty Silastic silicone rubber implants on day 20 rather than with progesterone implants so that they would give birth spontaneously at term in the absence of the effects of either endogenous relaxin or RU 486. Group MCA1 + P + AP determined the influence of RU 486 alone on delivery in relaxin-deficient rats. Group MCA1 + P + R + AP determined the influence of RU 486 in combination with antepartum treatment with porcine relaxin on delivery in relaxin-deficient rats. Porcine relaxin was used during the antepartum period for two reasons. First, there is only 42% amino acid se-

Figure 2 Diagram of the design of Experiment 2. See Material and methods for details.

quence identity between rat and porcine relaxin and porcine relaxin is not bound by antibody MCA1.1 Second, porcine relaxin is highly bioactive in rats.1,3 Immediately after the progesterone implants were inserted at 8 AM on day 20, an osmotic pump (model 2001, Alza Corp, Palo Alto, Calif) that released porcine relaxin12 at a rate of 5 mg per hour was inserted subcutaneously over the spine and caudal to the scapula. This rate of infusion of porcine relaxin was five times higher than a dose found to restore cervical weights and delivery in ovariectomized pregnant rats.3 Rats in the other six groups were fitted with osmotic pumps containing saline solution vehicle. Animals were observed continuously for several birth parameters from 9 PM on day 22 until noon on day 25 according to previously used procedures.2,3 The day of delivery was designated day 1 post partum. Both pup weights and the incidence of live pups were determined at 11 AM on days 1 to 3 post partum. At noon on day 25 animals were autopsied to determine whether fetuses or placentas were retained in utero.

Effects of RU 486 and relaxin on extensibility and growth of the cervix The ovariectomized pregnant rat model3 was used to compare the effect of progesterone withdrawal with that of relaxin administration during the antepartum period on cervical extensibility and growth. An advantage of this model over the MCA1-treated pregnant rat model is that it enables the comparison of the effects of two

232 Table I

Zhao and Sherwood Mean (GSEM) pregnancy and labor induction parameters for experiment 1

PBS MCAF PBS + P PBS + P + AP MCA1 MCA1 + P + AP MCA1 + P + R + AP

Rats (No.)

Implantation sites (No.)

Fetuses at term (No.)

Interval from AP administration to straining (h)

9 9 7 9 10 12 11

13.6 G 0.5 13.4 G 0.6 13.7 G 1.6 13.9 G 0.6 13.4 G 0.6 13.2 G 0.5 13.2 G 0.5

12.7 G 0.7 13.1 G 0.5 12.1 G 2.3 12.6 G 0.8 11.2 G 1.0 11.9 G 0.6 12.6 G 0.5

NAP NAP NAP 22.2 G 0.3 NAP 22.8 G 0.4 22.2 G 0.4

Time of onset of straining (h) 526.0 G 0.3 527.0 G 0.5 NL 527.4 G 0.2 527.1 G 0.4 527.8 G 0.4 527.2 G 0.4

AP, Antiprogesterone RU 486; NAP, no AP; NL, no labor.

means of withdrawal of progesterone on the cervix: administration of the antiprogesterone RU 486 and removal of the source of progesterone. Figure 2 shows a diagram for experiment 2. At 9 AM on day 8 of pregnancy, the rats were anesthetized with ether and two progesterone implants were inserted subcutaneously along each flank as described in experiment 1. Also, on day 8 a Silastic silicone rubber tubing implant containing 2 mg of 17b-estradiol dissolved in sesame oil (Sigma Chemical Co) was inserted subcutaneously just anterior to the scapula as previously described.3 The pregnant rats were divided into six groups. Group ovariectomized + progesterone + estrogen (OPE) was the negative control. The influence of progesterone withdrawal on the cervix in relaxin-deficient rats was examined by either administering RU 486 at 4 AM on day 22 as described in experiment 1 (group OPE + AP) or by removing the progesterone implants (group OPE-P). The influence of relaxin alone on the cervix was examined by subcutaneous insertion of osmotic pumps containing porcine relaxin into group OPER rats at 8 AM on day 20 as described with experiment 1. The influence of progesterone withdrawal on the cervix in relaxin-treated rats was examined by either administering RU 486 (group OPER + AP) or by removing the progesterone implants (group OPER-P) at 4 AM on day 22. At midnight on day 22, animals were killed. Cervices were removed, weighed, placed in Krebs-Ringer bicarbonate buffer, pH 7.5, and maintained at 4(C until their extensibility was determined immediately after tissue collection. Cervical extensibility was determined as previously described.13 An additional experiment (experiment 3) was conducted to compare the effects of the administration of RU 486 with that of relaxin during the antepartum period on cervical wet weight, lumen circumference, and cell proliferation. Groups OPE, OPE + AP, OPER, and OPER + AP were treated as in experiment 2 with the following modifications. To compare the effects of treatment on cell proliferation, the thymidine analog 5-bromo-2[prime]-deoxyuridine (BrdU) was administered subcutaneously at a rate of 10 mg per hour with

the same osmotic minipump (model 2ML1, Alza Corp) used to administer porcine relaxin (5 mg/h). Both BrdU and porcine relaxin were administered continuously from 8 AM on day 20 until the cervices were collected at midnight on day 22. Cervices were removed, weighed, bisected in cross section to obtain cephalic and caudal halves, and fixed in neutral-buffered formalin for 24 hours. Subsequent procedures for morphometric immunohistochemical analysis of BrdU incorporation into cells was conducted as previously described.14 Between 30 and 60 fields were analyzed per section and four sections were used per animal. Therefore, a total of at least 120 fields of analysis were evaluated for each animal. Both epithelial cells and stromal cells were analyzed. The stromal cells analyzed were fibroblasts, cells associated with blood vessels (endothelial cells and smooth muscle cells), and cells within both the inner circular and outer longitudinal smooth muscle bundles. Cervical lumen circumference was determined by morphometric analysis using a public domain National Institutes of Health (NIH) image program developed at the US NIH that is available on the Internet at http://rsb.info.nih.gov/nih-image/. Because each cervix has two lumina and four sections were used per animal (two sections from each cervical half), a total of eight measurements were made for each rat.

Statistical analysis All data were analyzed by one-way analysis of variance (ANOVA) and the Tukey test.

Results Effects of RU 486 and relaxin on delivery Pregnancy and labor induction parameters for experiment 1 are given in Table I. There was no difference among the seven treatment groups in either the number of implantation sites on day 9 or the number of fetuses at term. Rats in group PBS + P did not go into labor by noon on day 25. The administration of RU 486 effectively induced labor and delivery. The mean interval

Zhao and Sherwood

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Figure 3 Influence of RU 486 and relaxin, alone and in combination, on delivery in experiment 1. A, Duration of straining. B, Duration of delivery. C, Incidence of live pups on day 1 post partum. D, Numbers of rats that did not complete delivery. Treatment groups are indicated at the base of the Figure. Each column in A to C represents the mean (+SEM). For statistical analysis, logarithmic transformation was done with duration of (A) straining and (B) delivery data in an attempt to equate variance among treatment groups. Values with different superscript letters differ (P ! .05). The number of animals per treatment group is indicated at the base of each column.

from the administration of RU 486 until labor (straining) did not differ among the three groups that received AP. Moreover, the time of onset of straining of the groups induced to deliver with RU 486 did not differ from that of the groups that underwent spontaneous luteolysis and labor (PBS, MCAF, MCA1). Delivery parameters for experiment 1 are shown in Figure 3. In groups PBS and MCAF, which had endogenous relaxin and underwent spontaneous delivery, the durations of straining and delivery were rapid; nearly 100% of pups were alive on day 1 post partum and delivery was completed. After the induction of labor with RU 486 in group PBS + P + AP, a group in which endogenous relaxin was not neutralized, delivery parameters did not differ from the PBS and MCAF control groups. When endogenous relaxin was neutralized with MCA1 and animals underwent spontaneous delivery (group MCA1), the durations of straining and delivery

were longer and the incidence of live pups on day 1 post partum was lower than in the group PBS and group MCAF controls (P % .05). At autopsy on day 25, two rats in group MCA1 had retained fetuses and placentas in utero. Delivery was not improved when labor was induced in relaxin-deficient MCA1-treated rats by administering only RU 486. No delivery parameter differed from that in relaxin-deficient group MCA1. Also, similar to group MCA1, three rats in group MCA1 + P + AP had retained fetuses and placentas in utero at autopsy on day 25. In contrast, when labor was induced with RU 486 in relaxin-deficient MCA1-treated rats that were administered relaxin during the antepartum period (group MCA1 + P + R + AP), the durations of labor and delivery were shorter and the incidence of live pups on day 1 post partum was greater than in group MCA1 + P + AP (P%.05). Moreover, delivery parameters for group MCA1 + P + R + AP did not differ from

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Figure 4 Lactational performance over the first 3 days post partum in experiment 1. Values are mean (CSEM). A, Pup weights. B, Incidence of live pups. a, Pup weights and incidence of live pups in the groups that were replete with endogenous circulating relaxin (groups PBS, MCAF, and PBS + P + AP) were greater than in the three groups where circulating endogenous relaxin was neutralized with monoclonal antibody MCA1 (P % .01).

those in the relaxin-replete control groups PBS and MCAF that were delivered spontaneously. Additionally, this group gave birth to all fetuses and placentas. Mammary nipples fail to develop during the second half of pregnancy in MCA1-treated rats, and pups cannot grasp them to obtain milk.1 Figure 4 demonstrates that pups born of mother rats that were replete with endogenous-circulating relaxin (groups PBS, MCAF, and PBS + P + AP) gained weight and maintained a high incidence of live pups throughout the first 3 days post partum. In contrast, none of the pups born of endogenous relaxin-deficient mother rats that were not administered relaxin during the antepartum period (groups MCA1

Zhao and Sherwood

Figure 5 Influence of RU 486 and relaxin, alone and in combination, on cervical extensibility at (A) extension and (B) 20 minutes after extension. Each column represents the mean (+SEM). Values with different superscript letters differ (P ! .05). The number of animals per treatment group is indicated at the base of each column.

and MCA1 + P + AP) either gained weight or remained alive on day 3 post partum. These findings provide additional evidence that the administration of MCA1 effectively neutralized endogenous relaxin activity. The antepartum administration of relaxin in MCA1-treated rats improved pup survival to a modest degree. Although most pups in group MCA1 + P + R + AP died by day 3 post partum, 17% of the pups (born of 4 of the 11 mother rats) remained alive. The survival of pups appeared to be attributable to sufficient porcine relaxineinduced growth of a few nipples during the antepartum period to enable a portion of the litter to obtain milk.

Effects of RU 486 and relaxin on extensibility and growth of the cervix Pregnancy parameters for experiments 2 and 3 are given in Table II. There was no difference among the treat-

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Zhao and Sherwood Table II

Mean (GSEM) pregnancy parameters, cervical weight, and luminal circumference for experiments 2 and 3

Experiment 2 OPE OPE + AP OPE-P OPER OPER + AP OPER-P Experiment 3 OPE OPE + AP OPER OPER + AP a,b

Rats (No.)

Implantation sites (No.)

Fetuses at autopsy (No.)

Cervical weight (mg)

17 17 10 5 15 7

14.5 G 0.6 15.4 G 0.5 13.7 G 0.5 16.0 G 0.7 15.4 G 0.4 15.7 G 0.8

9.6 G 0.8 11.3 G 0.8 10.0 G 1.1 12.2 G 1.7 11.2 G 0.7 12.7 G 1.5

80.7 G 4.0a 92.5 G 6.2a 91.9 G 3.6a 140.2 G 4.0b 147.9 G 9.5b 141.7 G 5.0b

6 8 6 6

13.9 G 0.8 12.6 G 0.8 13.3 G 0.8 14.0 G 0.6

10.6 G 1.0 10.2 G 0.8 10.3 G 1.6 11.9 G 0.9

92.0 G 4.2a 104.7 G 7.0a 153.8 G 9.5b 139.0 G 3.9b

Cervical lumen circumference (mm)

8.3 G 0.7a 8.9 G 0.4a 13.3 G 1.0b 14.2 G 0.5b

Different superscript letters indicate significant difference (P!.01).

ment groups in either the number of implantation sites on day 9 or the number of fetuses at midnight on day 22. The effects of RU 486 and relaxin on the extensibility of the cervix are shown in Figure 5. In the relatively inextensible (hard) control group OPE, the mean slope (grams of tension/millimeter of extension) was highest. In cervices obtained from rats treated with only RU 486 (group OPE + AP), the slope was lower than in group OPE control, both at extension and 20 minutes after extension. Withdrawal of progesterone implants also increased cervical extensibility. Twenty minutes after extension (final extensibility) the mean slope was lower in group OPE-P than in control group OPE. However, it was not as low as in the group administered RU 486 alone. Relaxin markedly increased cervical extensibility. Mean slope was lowest after administration of relaxin during the antepartum period (group OPER). The slope in group OPER was lower than in group OPE-P, but it did not differ from group OPE + AP. Neither coadministration of RU 486 (group OPER + AP) nor withdrawal of progesterone implants (group OPER-P) influenced cervical extensibility in relaxin-treated rats. Three parameters were used to examine the effects of RU 486 and relaxin on growth of the cervix: wet weight, lumen circumference, and cell proliferation. Neither cervical weight nor lumen circumference differed from control group OPE after the administration of RU 486 alone (group OPE + AP) (Table II). In contrast, both cervical weight and lumen circumference were markedly greater than those in the control group after administration of relaxin during the antepartum period (group OPER), and they were not influenced by the coadministration of RU 486 (group OPER + AP). Consistent with these findings, the administration of RU 486 alone had no effect on proliferation of cervical epithelial cells, stromal fibroblasts, or cells associated with blood vessels. In contrast, antepartum administration of relaxin dramatically increased cell proliferation in all three cel-

lular compartments (Figure 6, A to C). The rate of cell proliferation was extremely low in the circular and longitudinal smooth muscle cell bundles, and neither RU 486 nor relaxin influenced the rate of cell proliferation in these cells (Figure 6, D).

Comment The current study demonstrates that the induction of labor in rats at term by administration of RU 486 in combination with relaxin enables faster labor, faster delivery, and higher survival of the pups than does induction of labor with RU 486 alone under conditions of endogenous relaxin deficiency. After their administration during the antepartum period, both relaxin and RU 486 increase cervical extensibility, but only relaxin promotes dramatic growth of the cervix. We do not know with certainty how antepartum administration of relaxin in combination with RU 486 facilitates delivery. Although there was a tendency for cervical softening to be greater after the administration of relaxin than after the administration of RU 486, significant differences between the two treatments were not detected at the P % .05 level. Only relaxin promoted marked increases in cervical wet weight, lumen circumference, and cell proliferation. Therefore, it seems likely that it is the combination of relaxin’s marked effects on both extensibility and growth of the cervix that enable more rapid, safe, and complete delivery of the pups than occurs when labor is induced by administration of RU 486 alone to relaxin-deficient MCA1-treated rats. The finding that antepartum administration of relaxin promoted marked increases in cervical lumen circumference and also proliferation of cervical epithelial cells, fibroblasts, and cells associated with blood vessels is in agreement with a previous study that examined the influence of relaxin on the cervix when the hormone was administered throughout the second half of rat pregnancy.14

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Zhao and Sherwood

Figure 6 Influence of RU 486 and relaxin, alone and in combination, on cervical cell proliferation in (A) epithelium, (B) stromal fibroblasts, (C) blood vessel cells, and (D) both circular and longitudinal smooth muscle bundle cells. Each column represents the mean (+SEM). Values with different superscript letters differ (P ! .01). The number of animals per treatment group is indicated at the base of (or above) each column.

Available evidence indicates that there are two general mechanisms whereby RU 486 may contribute to the cervical softening observed in this study. First, it may act as an antiprogesterone.4 Second, it is possible that RU 486 induces transdominant negative activity toward estrogen receptors.4 During late pregnancy, estrogen decreases cervical extensibility in rats.15 Therefore, it is possible that the RU 486einduced increase in cervical extensibility is attributable, at least in part, to transdominant negative suppression of estrogen’s effects on cervical extensibility. The data in this report do not support this second mechanism. The observation that cervical extensibility increased after removal of progesterone implants, as well as after administration of RU 486, supports the view that it is through the removal of progesterone’s actions, rather than the blocking of estrogen’s effects, that RU 486 brings about cervical softening in the rat. Why administration of RU 486 was more effective in promoting cervical softening within 20 hours of its administration than was the removal of progesterone im-

plants for the same period cannot be explained with certainty. RU 486 is rapidly absorbed but slowly metabolized.16 Accordingly, a possible explanation is that the administration of the large dose of RU 486 provides high cellular levels of the antiprogesterone that displace the progesterone in active progesterone-PR complexes within minutes. In contrast, after the removal of progesterone implants, progesterone is metabolized and cleared from the serum and cells over a more prolonged period.17 Although it is well documented that RU 486 promotes softening of the cervix in rats and primates,4 the influence of RU 486 on growth of the cervix has received little experimental attention. One group reported that when primiparous Wistar rats were given a single subcutaneous injection of RU 486 on day 18 of pregnancy at a dose (15 mg/kg) similar to that used on day 20 of pregnancy in the current report, cervical wet weight increased significantly by 18 hours after the administration of RU 486.6 We cannot account for the difference between the two studies. It may be attributable to either the use of different strains of rats or different

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Zhao and Sherwood days of pregnancy when the RU 486 was administered. In any event, the lack of increase in cervical wet weight, lumen circumference, and cell proliferation observed 20 hours after RU 486 in the current study provides solid support for the view that RU 486 did not promote growth of the cervix. At the current time, dinoprostone (prostaglandin E2) is used for cervical ripening at term in women. Although dinoprostone promotes cervical effacement and dilation, it also causes uterine hyperstimulation in sufficiently high frequency to make it recommended that it be administered at or near the delivery suite by an attendant skilled in the management of labor, as well as the diagnosis of both uterine activity patterns and fetal distress.18 Relaxin has potential advantages over dinoprostone as a cervical softening agent at term. First, it promotes both cervical softening and growth within a period as brief as 8 hours.19 Second, relaxin inhibits, rather than stimulates, uterine contractility during the second half of pregnancy in rats.20 Moreover, there is evidence that the quiescent effects of relaxin on myometrial activity do not interfere with delivery. In rats, delivery occurs rapidly, in spite of elevated serum levels of endogenous rat relaxin1 or the continuous administration of porcine relaxin throughout late pregnancy and delivery.3 The findings in this report encourage the view that administration of RU 486 in combination with sufficient recombinant human relaxin (rhRlx) to provide pharmacologic serum levels of the hormone may offer a relatively natural and effective method for the induction of labor at term in the human. Two clinical trials in the mid-1990s failed to demonstrate an effect of rhRlx on birth in pregnant women. Therefore, it is important to understand how the rhRlx was administered and to take note of conclusions reached by the investigators who reported the trials. The clinical trials21,22 were conducted with the expectation that when rhRlx is placed in the vagina the hormone effectively passes through the squamous epithelium, enters the blood, and thereby reaches relaxin receptors within the cervix in amounts sufficient to be effective. That does not appear to happen. The reports of both clinical trials indicated that circulating blood levels of relaxin were not elevated by treatment with up to 4 mg of rhRlx. Moreover, both reports postulated that the lack of absorption of relaxin was the reason that the treatment failed to influence birth.21,22 It remains to be demonstrated that relaxin promotes softening and/or growth of the cervix in humans. However, there are reasons to think that it may do so. In rats and pigs, in which the effects of relaxin on the cervix have been examined most extensively, the hormone has dramatic effects on cervical softening and/or growth.1 There is evidence that the human reproductive tract is also responsive to relaxin. During the course of a recent 24-week

clinical trial, which examined the use of rhRlx for the treatment of systemic sclerosis, heavy and irregular menstrual bleeding occurred more frequently in rhRlx-treated subjects than in placebo-treated control subjects, and these adverse effects were postulated to be attributable to the influence of rhRlx on the uterine vasculature.23 Additionally, when human lower uterine segment fibroblasts were used as an in vitro model for human pregnancy cervix, rhRlx stimulated the expression of the matrix metalloproteinases procollagenase and prostromelysin.24 At the current time, there are no reports of the effects of systemic administration of rhRlx on growth and/or softening of the cervix in humans or other primates. There is need for such studies. Should human relaxin prove to be an effective cervical softening agent at term, possible drawbacks to its use will require consideration. Currently, there are no known risks associated with the prepartum administration of relaxin. Nevertheless, in view of the marked proliferative effect of the hormone on cervical cells, attention should be given to the possibility that pharmacologic levels of relaxin promote target tissue oncogene potential. In conclusion, the current study demonstrates that, under conditions of endogenous relaxin deficiency, the induction of labor in rats at term by administration of RU 486 in combination with relaxin enables faster labor, faster delivery, and higher survival of the pups than does induction of labor with RU 486 alone. Findings in this report support the view that it is by promoting not only softening but also growth of the cervix that relaxin facilitates delivery. The physiologic mechanisms of relaxin differs in rats and humans. Therefore, to further explore relaxin’s potential as a therapeutic agent at the induction of labor in women, there is need for investigation of the effects of systemic administration of relaxin on softening and growth of the cervix in humans or other primates.

Acknowledgments We thank H. Lee, D. LoTurco, and J. Kumar for assistance with observation of rats during delivery, B. Sylavong for supervision of animal care, the School of Life Sciences Artist Service for preparation of figures, and the College of Medicine Document Management Center for assistance with preparation of the manuscript.

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