Relaxin stimulates expression of vascular endothelial growth factor in normal human endometrial cells in vitro and is associated with menometrorrhagia in women

Elaine N. Unemori1, Mark E. Erikson, Susan E. Rocco, Kerri M. Sutherland, Dawn A. Parsell, John Mak and Beverly H. Grove

Connetics Corporation, 3400 W. Bayshore Rd., Palo Alto, California 94303, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although the role of the reproductive hormone, relaxin, in rodents is well documented, its potential contribution to human reproduction is less well defined. In this study, we examine the effects of relaxin on human endometrial cells in vitro and describe the clinical effects of relaxin on menstrual flow in women. In cultured endometrial cells, relaxin specifically induces the expression of an angiogenic agent, vascular endothelial growth factor (VEGF). cAMP is implicated as a second messenger involved in VEGF stimulation. VEGF expression is temporally regulated in the endometrium, and our results suggest that relaxin, which is secreted by the corpus luteum and is present in the endometrium during the menstrual cycle and pregnancy, may be involved in regulating endometrial VEGF expression. Relaxin was recently tested in a clinical trial for efficacy in the treatment of progressive systemic sclerosis, and was administered at levels up to 10 times higher than that measured during pregnancy. The most frequent relaxin-related adverse event reported during the course of the study was the onset of menometrorrhagia, defined in this study as heavier-than-usual or irregular menstrual bleeding. The intensification of menstrual flow observed in these patients is consistent with the hypothesis that relaxin mediates neovascularization of the endometrial lining.

Key words: endometrium/menometrorrhagia/menstrual cycle/relaxin/vascular endothelial growth factor


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Relaxin is a polypeptide hormone that is present in the circulation in humans during the menstrual cycle and throughout pregnancy (Sherwood, 1994Go). Relaxin is first detectable in the circulation approximately 8 days following the luteinizing hormone (LH) peak (Stewart et al., 1990Go). In non-conceptive cycles, the relaxin peak is relatively small and occurs over 3–4 days but, if pregnancy occurs, concentrations continue to rise to approximately 1 ng/ml during the first trimester, after which time concentrations level off or decrease slightly. Despite numerous reports of distinct and important functions in rodents and pigs, relaxin's role in human reproduction remains an important question (Goldsmith et al., 1995Go). Based on its importance in parturition in animal models, relaxin was evaluated in clinical trials for efficacy as a topically applied cervical ripening agent in women, but failed to show any effect on modified Bishop score, length of labour, or oxytocin requirement (Brennand et al., 1997Go).

In primates, however, there is evidence that relaxin, like other secretory products of the ovary, affects the morphology and secretory profile of the uterine lining. Relaxin binds specifically and with high affinity to human endometrial cells (Osheroff and King, 1995Go). In addition, levels of expression of glycodelin (Stewart et al., 1997Go), insulin-like growth factor binding protein-1, and prolactin (Tseng et al., 1992Go) in human endometrial cells increase in response to relaxin treatment in vitro. In-vivo studies testing relaxin in primates are limited in number; however, one such study examining the effect of a relatively crude preparation of relaxin on the uterus in immature or castrated macaque monkeys found that relaxin, in conjunction with oestrogen treatment, stimulated new blood vessel growth in the endometrium (Dallenbach-Hellwig et al., 1966; Hisaw et al., 1967Go).

Angiogenesis in the endometrium, as in other sites, is stimulated by exposure of pre-existing endothelial cells to angiogenic agents. Studies have correlated an increase in vascular endothelial growth factor (VEGF) expression (Ferrara and Davis-Smyth, 1997Go) at both the protein and mRNA levels with new vessel growth in the endometrium in multiple species (Charnock-Jones et al., 1993Go; Cullinan-Bove and Koos, 1993Go; Schweik et al., 1993; Das et al., 1997Go; Torry and Torry, 1997Go). There is also evidence that inhibiting VEGF activity contributes to lack of endometrial maturation (Ferrara et al., 1998Go). For these reasons, we tested the effect of relaxin on expression of VEGF in human endometrial cells in vitro. We have found that relaxin treatment specifically increases the expression of VEGF but not other angiogenic agents, such as placental growth factor (PlGF), angiogenin, transforming growth factor-ß (TGF-ß), or basic fibroblast growth factor (bFGF). In a recently concluded clinical trial (Seibold et al., 1997Go), women receiving relaxin treatment reported menometrorrhagia significantly more often than women receiving a placebo. Menometrorrhagia was not accompanied by systemic elevations in VEGF. The increased frequency of menometrorrhagia in women receiving relaxin is consistent with the hypothesis that relaxin stimulates neovascularization in the endometrial lining of the uterus, and may reflect relaxin's normal physiological role in women.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell culture
Normal human endometrial (NHE) cells were derived by trypsinization of tissue obtained from a normal uterus, as previously described (Fei et al., 1990Go). Cells were passaged in Dulbecco's modified Eagle's medium (DMEM):F12 supplemented with 10% newborn calf serum or fetal bovine serum and 2 mmol/l L-glutamine. Cells were routinely used for experiments between passages 14–23. Cells were seeded at a density of 5x104 cells/cm2 in 24- or 48-well plates and used for experiments 24 h later. For experiments involving addition of steroids to cultures, phenol red-free DMEM:F12 was used. 17ß-oestradiol was used in doses ranging from 10–9–10–6 mol/l, and medroxyprogesterone at 10–6 mol/l.

Tissue culture reagents
Recombinant human relaxin-2 (Hudson et al., 1984Go) (Lot 63601, 1.5 mg/ml in 25 mmol/l acetate, pH 5.5) was manufactured by Connetics Corporation (Palo Alto, CA, USA). Forskolin, insulin, 17ß-oestradiol, and medroxyprogesterone were purchased from Sigma Chemical Company (St Louis, MO, USA). Insulin-like growth factor (IGF-1) was purchased from R&D Systems (Minneapolis, MN, USA). Dideoxyadenosine was purchased from Calbiochem (San Diego, CA, USA).

[32P]-relaxin binding assays
Relaxin was phosphorylated as previously described (Parsell et al., 1996Go). NHE cells were incubated at room temperature with [32P]-relaxin (20–400 pmol/l) in the presence or absence of 1.4 µmol/l unlabelled relaxin. The number of relaxin binding sites and their affinity for relaxin were determined using the LIGAND program (Munson and Rodbard, 1980Go).

Enzyme-linked immunosorbent assay (ELISA)
A quantitative sandwich ELISA specific for human VEGF was purchased from R&D Systems and performed according to manufacturer's specifications. The lower limit of detection of VEGF was 15.6 pg/ml. Immunoassays for TGF-ß, angiogenin, bFGF, and PlGF were also purchased from R&D Systems. Lower limits of detection in the respective assays were 31.2 pg/ml for TGF-ß, 78.1 pg/ml for angiogenin, 5 pg/ml for bFGF, and 15.6 pg/ml for PlGF. Serum relaxin levels were assayed in a quantitative sandwich ELISA immunoassay utilizing a goat affinity-purified anti-human relaxin polyclonal antibody and an affinity-purified, peroxidase conjugated anti-human relaxin polyclonal antibody, as previously described (Unemori et al., 1996Go). The assay has a lower limit of detection of 20 pg/ml.

Relaxin clinical trial
A randomized, placebo-controlled, double-blind clinical trial was conducted to test relaxin's safety and efficacy in patients with progressive systemic sclerosis (Seibold et al., 1997Go). Relaxin was formulated in a buffer of 20 mmol/l acetate, pH 5.5. The placebo was identical except for the active ingredient. Relaxin was delivered by continuous subcutaneous infusion at two doses, 25 µg/kg/day and 100 µg/kg/day, for 24 weeks. Patients were screened 4–8 weeks prior to initiation of treatment and came for a follow-up assessment 2 weeks after cessation of therapy. Patients ranged from 15–66 years in age and gave written informed consent. There were 22 women who received relaxin at 25 µg/kg/day, 20 women who received 100 µg/kg/day, and 16 women who received the placebo. Adverse events were defined as: (i) any sign or symptom observed by the investigator or reported by the subject which was not being assessed as an efficacy parameter; (ii) any efficacy parameter which required medical intervention; (iii) any clinically significant laboratory abnormality or any clinically significant abnormality of any other clinical test; or (iv) any abnormality detected on physical examination.

Patients were instructed prior to administration of relaxin to report any physical changes or new symptoms they noticed during the course of the study. They were not prospectively instructed to report incidents of menstrual irregularities using the specific terms menorrhagia or metrorrhagia. The information was recorded on case report forms regardless of whether they were believed to be associated with the study medication and entered verbatim into a database. As part of standard clinical trial safety data analysis, the verbatim terms were coded to preferred terms, which are standard terms assigned to adverse events and link an event to a body system for summarization of adverse events. The terms menorrhagia, metrorrhagia, and menstrual disorder were the preferred terms assigned to an event based on the verbatim adverse event description and the patient's menstrual history. Events describing heavy menses were coded to menorrhagia, and events describing prolonged or irregular menses were coded to metrorrhagia. Postmenopausal patients reporting any type of menstruation were coded to menstrual disorder. Date of first reported onset of events was recorded. All reports of reproductive system-related adverse events were retrieved from the database following completion of the study.

Serum samples that were collected from patients at screening, first day of dosing and at 1, 2, 4, 8, 12, 16, 20, and 24 weeks after initiation of relaxin treatment were assayed for relaxin concentrations as part of the clinical trial study design. Serum samples drawn at screening and at visits immediately prior to and at the first report of adverse event onset were assayed for serum VEGF concentrations.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Specific binding of relaxin to human endometrial cells
Relaxin bound specifically to NHE cells, which was consistent with previous reports (Osheroff and King, 1995Go) (data not shown). Scatchard analysis demonstrated 963 relaxin binding sites/cell and a Kd of 150 pmol/l.

Relaxin stimulation of VEGF expression in endometrial cells in vitro
NHE cells were treated with relaxin (0.001–100 ng/ml) for 24 h, then the conditioned media were collected and assayed for VEGF content by ELISA. The cells secreted a baseline level of VEGF of 875.0 ± 93.6 pg/ml, and the addition of relaxin resulted in an increase in secretion in a dose-dependent manner with an almost twofold increase detectable following addition of relaxin at 1 ng/ml (Figure 1aGo). Relaxin did not cause an alteration in cellular morphology or proliferation rate, measured by [3H]-thymidine incorporation (data not shown). Following the addition of 100 ng/ml of relaxin to NHE cells, elevated levels of VEGF protein expression were detectable by 24 h and secretion persisted at a constant rate over 96 h (Figure 1bGo). Pretreatment of NHE cells with concomitant addition of oestrogen and/or progesterone did not alter their production of VEGF in response to relaxin, nor did it alter [32P]-relaxin binding from control levels (data not shown). Other members of the relaxin gene family, insulin and IGF-1, were tested for their ability to induce VEGF expression in NHE cells (Figure 2Go). IGF-1 was a weak inducer of VEGF protein expression in comparison to relaxin. Insulin was unable to induce expression at the doses tested (0.1–100 ng/ml).




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Figure 1. Induction of vascular endothelial growth factor (VEGF) in normal human endometrial (NHE) cells by relaxin in a dose- and time-dependent fashion. (A) Relaxin (RLX) was added to endometrial cells at doses ranging from 0.001–100 ng/ml, and conditioned media collected at 24 h. VEGF was quantified in the media by enzyme-linked immunosorbent assay (ELISA). (B) Endometrial cell cultures were untreated or treated with relaxin (100 ng/ml), and conditioned media were harvested at 24, 48, 72, 96, and 120 h. Accumulation of VEGF in the media was measured by ELISA. Mean VEGF concentrations detected in conditioned media harvested at each time point from untreated cells were subtracted from mean VEGF concentrations found in relaxin-treated cells at the same time point, to yield net VEGF for each time point. Bars represent means of net VEGF ± SD, n = 3. *P < 0.05 compared with the untreated group (Student Newman–Keul's test).

 


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Figure 2. Vascular endothelial growth factor (VEGF) stimulation by relaxin and family members, insulin and insulin-like growth factor (IGF-1). Relaxin (RLX), insulin, and IGF-1 were added to normal human endometrial cells at 0.1, 1, and 10 ng/ml (molar concentrations of 0.016, 0.16, and 1.6 µmol/l for all cytokines). Media were collected at 24 h and assayed for VEGF by enzyme-linked immunosorbent assay. Bars represent means ± SD, n = 3. *P < 0.05 compared with the untreated control group (Student Newman–Keul's test).

 
To ascertain the specificity of the VEGF response to relaxin, the expression of other angiogenic proteins by NHE cells was measured. PlGF, TGF-ß, or bFGF were below the level of assay detection in conditioned media following the addition of relaxin (0.01–100 ng/ml) to NHE cells. Angiogenin was expressed at concentrations approximating 400 pg/ml, as measured by ELISA, and its levels were not altered by relaxin treatment (Table IGo).


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Table I. Expression of angiogenic cytokines by relaxin-stimulated normal human endometrial (NHE) cells
 
Relaxin signalling
Because relaxin is known to induce the expression of cAMP in NHE cells (Fei et al., 1990Go), the adenyl cyclase activator forskolin was tested for its ability to stimulate VEGF production. Forskolin was added to cultures at doses ranging from 160 nmol/l–4 µmol/l, and VEGF protein was measured in conditioned media 24 h later (Table IIGo). Forskolin increased the expression of VEGF in a dose-dependent manner, with maximum stimulation of approximately threefold over control cells. The addition of the phosphodiesterase inhibitor IBMX (1 mmol/l) to NHE cells increased basal levels of VEGF expression, presumably by increasing intracellular levels of cAMP, while the addition of the adenyl cyclase inhibitor dideoxyadenosine (1.2 mmol/l) inhibited the relaxin effect.


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Table II. Induction of vascular endothelial growth factor (VEGF) in normal human endometrial (NHE) cells cells by forskolin and the phosphodiesterase inhibitor IBMX, and inhibition of induction by dideoxyadenosine
 
Menometrorrhagia association with relaxin administration
For the purposes of this analysis, heavier menstrual bleeding (menorrhagia), irregular or prolonged menstrual bleeding (metrorrhagia), and postmenopausal menstrual bleeding (menstrual disorder) were regarded as one adverse event, menometrorrhagia. Menometrorrhagia was the reproductive system-related adverse event most commonly reported by systemic sclerosis patients being administered relaxin in the clinical study. Relaxin-related effects on the mammary gland consistent with those reported in rats and pigs, i.e. nipple enlargement, were not reported by patients in this study (data not shown).

Nine of the 41 women receiving relaxin and three of the women on the placebo had previously had a hysterectomy and were excluded from the analysis on menometrorrhagia. Of the remaining 32 women in the relaxin treatment groups, 23 (72%) reported experiencing menometrorrhagia, compared with two of the 13 women (15%) on the placebo (P = 0.0003 by Fisher's exact test). Patients reporting menometrorrhagia were about equally represented in both relaxin dose groups, 13 in the 25 µg/kg/day group and 10 in the 100 µg/kg/day group. Their circulating relaxin concentrations at the time of adverse event reporting ranged from 2.00–83.31 ng/ml (Table IIIGo). The ages of the women experiencing menometrorrhagia ranged from 15–60 years. Although complete reproductive histories were not always taken for the purposes of this trial, it is likely that 17 of the 23 relaxin-treated women who reported menometrorrhagia were cycling. These included all of those who were recorded in their charts as being of childbearing potential (ages 27 to 44 years), and, judging by patient age, also those that were of non-childbearing potential due to tubal ligation (ages 30 to 42 years). Four other women were recorded as postmenopausal and were on hormone replacement therapy. One patient (no.7) was postmenopausal and was not reported to be taking hormone supplements. Another woman (patient no. 14) was listed as of non-childbearing potential, but was only 31 years old at the start of the trial; she reportedly had experienced pelvic inflammatory disease in her history but no further information was detailed regarding her reproductive status. The nine relaxin-treated women who did not report menometrorrhagia ranged in age from 27–62 years old. Five of them were of childbearing age. The other four were postmenopausal and two of them were on hormone replacement therapy.


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Table III. Relaxin-treated patients reporting menometrorrhagia
 
There was no apparent correlation between time of onset of menometrorrhagia and circulating relaxin levels measured at the time of reporting (Table IIIGo). Nine of the 23 women reported first onset within 2 weeks of initiating treatment, and eight more experienced first onset within the next 6 weeks. First reported onset in the rest of the women occurred up to 20 weeks after the start of relaxin treatment. The two women on the placebo who reported experiencing menometrorrhagia did so at 1 and 4 weeks after the start of the study.

Because of the finding that relaxin stimulates VEGF production by endometrial cells in vitro, and because we postulate that this is the mechanism by which menometrorrhagia is stimulated in relaxin-treated women, we looked for changes in serum VEGF concentrations in response to relaxin administration. VEGF content of serum drawn at screening was compared with VEGF concentrations demonstrable in serum drawn at the visit preceding first report of menometrorrhagia onset, as well as at the visit at which the report was made. Baseline VEGF concentrations proved to be highly variable, ranging from 71 to 1337 pg/ml prior to initiation of drug treatment (Figure 3Go). There were no consistent patterns in VEGF concentrations observed when comparing the three sampling dates that would indicate a positive, or negative, response to relaxin. Generally, VEGF concentrations remained within the range of individual baseline readings. The two placebo patients who reported menometrorrhagia had VEGF concentrations of 710 and 469 ng/ml at screening and demonstrated VEGF patterns that were not inconsistent with those seen in relaxin-treated patients (data not shown).



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Figure 3. Serum vascular endothelial growth factor (VEGF) concentrations in 23 relaxin-treated patients at screening, at the visit preceding first reported onset, and at the visit of the first report. VEGF was assayed in a commercially available enzyme-linked immunosorbent assay (ELISA) according to manufacturer's instructions. Each serum sample was assayed in duplicate in the ELISA. Patient numbers correspond to those assignments made in Table IIGo. Patients are grouped according to time of reported onset. Data on patients no. 7 and 21 were not available.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
During every menstrual cycle, relaxin is secreted by the corpus luteum as a small pulse of relatively short duration (Stewart et al., 1990Go). If pregnancy and rescue of the corpus luteum occur, relaxin secretion continues and is amplified so that levels of approximately 1 ng/ml are achieved during the first trimester and are maintained throughout pregnancy. Relaxin's appearance in the circulation early in human pregnancy is suggestive of a role during a period when systemic relaxin exposure in other species does not occur. For example, in rodents and in pigs, circulating relaxin does not appear until mid-pregnancy and subsequently displays a large prepartum surge (Sherwood, 1994Go). These species have provided ample evidence that relaxin is relevant during pregnancy or for parturition. For example, relaxin causes pubic ligament lengthening in the mouse, cervical ripening in the rat (Huang et al., 1989Go; Cheah et al., 1995Go), uterine relaxation in the rat and pig, and mammary gland development in the rat and pig (Hurley et al., 1991Go; Kuenzi et al., 1995Go). However, because of the obvious differences in temporal patterns of relaxin expression, as well as the results of some in-vitro comparative analyses (Petersen et al., 1991Go), it is difficult to extend the relevance of these biological activities to the human situation.

In the present study, we examine relaxin's potential effects on the human endometrium and demonstrate that one of relaxin's specific effects on isolated human endometrial cells is the stimulation of VEGF expression. In vitro, VEGF is stimulated by relaxin in a dose-dependent manner, with the threshold of response occurring between 0.01 and 0.1 ng/ml relaxin, roughly equivalent to circulating levels present in women during the menstrual cycle and early pregnancy (Stewart et al., 1990Go). VEGF is present in the endometrium in a variety of species (Shweiki et al., 1993Go; Das et al., 1997Go), including the human (Charnock-Jones et al., 1993Go; Shifren et al., 1996Go) during periods of active blood vessel proliferation, leading to the inference that VEGF may be an important mediator regulating cycle-specific vessel growth in the endometrial lining of the uterus. Baseline levels of VEGF secretion by the NHE cells are significant, perhaps consistent with its expression observed in situ in the endometrium during periods of relative quiescence (Torry and Torry, 1997Go). It has been postulated that VEGF may be necessary for maintenance of the differentiated phenotype of endothelial cells, as well as for stimulating proliferation.

The endometrial cells used in this study are most likely stromal, since IGF-BP1 is induced in these cells by relaxin, consistent with a stromal cell phenotype (data not shown). Neither oestrogen nor progesterone had any discernible effect on these cells in vitro. Others have reported that endometrial stromal cells are stimulated to produce VEGF in vitro by treatment with oestradiol (Shifren et al., 1996Go). However, it has also been reported that these cells have the potential to synthesize oestrogen in culture (Huang et al., 1989Go), suggesting the possibility of endogenous stimulation of a VEGF response, producing the baseline levels that we observe. Our data suggest that induction of VEGF over baseline is mediated, at least in part, by cAMP; the involvement of more than one second messenger system is consistent with VEGF regulation in other systems (Claffey et al., 1992Go). It is possible that relaxin stimulates transcriptional activation of the VEGF gene via cAMP, as the promoter contains four AP-1 and two AP-2 binding sites, which are believed to be able to transduce cAMP-mediated transcriptional activation (Tischer et al., 1991Go).

The results of our findings in vitro allow that, in women, relaxin expressed during the luteal phase of the menstrual cycle and early in pregnancy may be directly involved in inducing VEGF expression, and consequently neovascularization, in the endometrium. This is consistent with an analysis of factors contributing to uteroplacental circulation, which found that circulating relaxin levels were positively correlated with uterine flow in early pregnancy (Jauniaux et al., 1994Go). Our observation that exogenous relaxin administration was associated with menometrorrhagia in women supports this hypothesis, as does the previous finding that a crude preparation of relaxin administered to monkeys stimulated blood vessel proliferation in the endometrium (Dallenbach-Hellwig et al., 1966; Hisaw et al., 1967Go). Because relaxin administration in the clinical trial resulted in supraphysiological circulating levels (~3 ng/ml and ~10 ng/ml in the lower and higher dose groups respectively), it is perhaps not surprising that we observed not only excess menstrual bleeding, but also bleeding at irregular times during the menstrual cycle. All of the relaxin-treated women, who reported menometrorrhagia, except one, were likely either cycling or postmenopausal and on hormone replacement therapy. This suggests that oestrogen may be required for endometrial stimulation by relaxin, which is consistent with results observed in a study of the endometrium following steroid and relaxin supplementation in castrated or juvenile primates (Dallenbach-Hellwig et al., 1966; Hisaw et al., 1967Go).

Although we have shown here that exogenous, systemically administered relaxin can cause heavier than usual menstrual bleeding, the endometrium itself is also normally a source of relaxin (Yki-Jarvinen et al., 1985Go; Bryant-Greenwood et al., 1993Go). Endometrial relaxin is first immunocytochemically detectable 4 days following ovulation in the early secretory phase of the cycle and is inducible with exogenous progesterone administration in anovulatory and postmenopausal women. The timing of appearance of relaxin in the endometrium is also consistent with its potential role in neovascularization.

Circulating VEGF levels were not altered by relaxin treatment, which is consistent with the hypothesis that relaxin stimulates the expression and distribution of VEGF only in certain sites, such as in the endometrium. The lack of a measurable elevation in VEGF in the serum during early pregnancy has been previously reported and is contrasted with an increase in circulating VEGF that occurs during follicular luteinization in women undergoing in-vitro fertilization (Lee et al., 1997Go).

In conclusion, one of relaxin's physiological roles in women may be to promote the VEGF-mediated stimulation of endometrial neovascularization that occurs during the luteal phase of the menstrual cycle and in early pregnancy.


    Acknowledgments
 
The authors would like to thank Drs Woodruff Emlen, Martin Sanders, and Scott Harkonen for critically reading the manuscript. We would also like to acknowledge assistance with statistical analysis of the data by Dr Ananda Gubbi. All authors are employees of Connetics Corporation, Inc., a company which is developing relaxin for commercial purposes.


    Notes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on June 19, 1998; accepted on November 23, 1998.