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Experimental Studies |
Division of Reproductive Biology and Medicine, Department of Obstetrics and Gynecology (D.R.S., S.T.N., J.W.O.), Institute of Toxicology and Environmental Health, University of California (D.R.S., J.W.O., B.L.L.), Davis, California 95616; Connective Therapeutics, Inc. (M.S.E., M.E.E., E.P.A.), Palo Alto, California 94303; Department of Obstetrics and Gynecology, Helsinki University Central Hospital (M.S.), Helsinki, Finland
Address all correspondence and requests for reprints to: Dr. Dennis R. Stewart, Med:Reproductive Biology, Suber House, University of California, Davis, California 95616.
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Abstract |
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To investigate the relationship of relaxin and glycodelin, two studies were conducted. In the first study, relaxin, glycodelin, and ovarian steroids were measured in daily serum samples from nonconceptive and conceptive natural cycles. Profiles of relaxin and glycodelin were closely associated, with the onset of relaxin preceding glycodelin secretion by 1–2 days in nonconceptive cycles, and the pregnancy-associated increases in each hormone differing by about 2 days. The second study tested the hypothesis that relaxin stimulates glycodelin secretion. Samples were obtained from patients injected with human relaxin for 28 days. In subjects demonstrating ovarian cyclicity, glycodelin secretion was elevated, but it was not detected in subjects without ovarian cyclicity or in placebo-treated control subjects.
This study reveals a close temporal and quantitative relationship between relaxin and glycodelin profiles in the late luteal phase and early pregnancy. It also demonstrates that relaxin administration can stimulate glycodelin production from a developed endometrium. This is the first report of a nonsteroidal ovarian factor that controls glycodelin secretion, and these results suggest a function for relaxin during early pregnancy. Glycodelin is a potent inhibitor of sperm zona pellucida binding by virtue of its extensive carbohydrate structure, but it is normally at a nadir in the periovulatory period. The data demonstrate that relaxin can stimulate glycodelin secretion throughout the menstrual cycle, including the periovulatory period, when relaxin-induced glycodelin secretion could have a contraceptive effect.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Glycodelin has long been associated with progesterone (P) secretion (8). However, although P may be required for glycodelin synthesis, profiles of the two substances are quite disparate. P peaks early in the luteal phase and is declining as glycodelin begins to increase (4). In pregnancy, P plateaus in early pregnancy (9), whereas glycodelin continues to increase (6). Estradiol (E) is also believed to be required for optimal glycodelin secretion (10, 11), but the profiles of E and glycodelin are not synchronous.
P and E did not elevate glycodelin secretion in women without ovarian function who became pregnant through embryo transfer and exogenous steroid hormone support (12), although circulating levels of ovarian steroids were within normal ranges. Additionally, women whose anterior pituitaries were suppressed during luteal formation had no increase in glycodelin associated with the pregnancy, although they had low basal glycodelin concentrations (13). These observations indicate that either a pituitary or ovarian signal is involved in glycodelin secretion.
Our previously reported profiles of circulating relaxin (14, 15, 16) are similar to published glycodelin profiles (4, 6). Relaxin also is absent in the circulation of women without functional ovaries who become pregnant through embryo transfer (17, 18) (our unpublished observations), which supports the hypothesis that relaxin is the ovarian factor responsible for endometrial secretion of glycodelin. The effect of administering additional relaxin on circulating glycodelin concentrations was studied in late pregnancy, a time when relaxin and glycodelin are elevated (19). Although there was no observed effect on glycodelin concentrations, it was not reported whether the posttreatment relaxin concentrations were significantly elevated over pretreatment levels or compared with control values. These researchers also noted that studies in late pregnancy may not be a "true reflection of events in early pregnancy" (19).
A rigorous longitudinal comparison of circulating E, P, relaxin, and glycodelin in natural cycles has not been reported. To evaluate the relationship among relaxin, glycodelin, and ovarian steroids, daily serum samples were collected from women with natural cycles undergoing artificial insemination. To directly test the hypothesis that relaxin stimulates glycodelin secretion, serum samples were obtained from a study in which subjects were injected with relaxin for a 28-day interval.
Our results indicate a close temporal relationship between relaxin and glycodelin in the late luteal phase and early pregnancy. We also show that relaxin can stimulate glycodelin production at any time in the menstrual cycle. Through stimulation of glycodelin during the periovulatory period, relaxin could have a potential contraceptive action.
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Subjects and Methods |
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All subjects in study 1 supplied daily serum samples beginning with the first detection of the midcycle LH rise in urine (Ovuquick, Monoclonal Antibodies, Mountain View, CA) and continuing through the ensuing menses. The women ranged in age from 21–39 yr, had a history of regular menses, had normal reproductive status, and ovulated spontaneously. The women were recruited to participate during cycles of artificial insemination with donor semen. The women either had no sexual partner or had an azoospermic partner. This study was approved by the University of California-Davis human subjects committee, and all subjects gave written informed consent. This group has been described previously (9).
Serum LH, 17ß-estradiol, and P were assayed using commercial kits (Diagnostic Products Corp., Los Angeles, CA). All cycles in study 1 were analyzed for evidence of hCG by a double antibody immunoradiometric assay (20) with a sensitivity of 0.65 IU/L. Relaxin was determined by enzyme immunoassay (14). Relaxin, E, and P data from some of these cycles have been previously reported (9, 15). Ten nonconceptive cycles, 10 conceptive cycles, and 1 early spontaneous abortion cycle were selected for glycodelin determinations as previously described (21). Data were aligned either to the midcycle LH surge or to the ensuing menses. To normalize the endocrine data for statistical comparisons, hormone values were transformed by taking the logarithm. Data were converted to an arithmetic scale for graphics (geometric mean).
Study 2
Eighteen patients (16 women and 2 men) with late (>2 yr), stable, diffuse scleroderma received recombinant human relaxin (n = 12) or placebo (n = 6) for 28 days. The patients ranged in age from 18–61 yr and gave written informed consent. The study was approved by the human subjects committee at each participating institution. All patients had adequate renal, pulmonary, cardiovascular, and hematological function and were not receiving other experimental treatment during this study. The scleroderma patients were not selected for this study by any particular reproductive criteria.
The study was a double blind design with relaxin or vehicle administered by continuous sc infusion with dose groups of 50, 100, and 200 µg/kg·day. In each dose group, four subjects received relaxin, and two received placebo. Infusion began at time zero of treatment day 1 and continued until day 29. All subjects were sampled at 0, 1, 2, 4, and 6 h on day 1 and then on days 4, 8, 15, 22, 29, 36, and 43. Serum relaxin, glycodelin, and P were determined in all samples using the same methods as in study 1. Serum P values were used to determine the stage of menstrual cycles. The relaxin group was retrospectively divided by P values into subjects showing evidence of menstrual cycles (n = 6) and those not showing cycles (n = 6) regardless of relaxin dose group.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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P concentrations in nonconceptive cycles (Fig. 1
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increased immediately after the LH peak, plateaued on days 5–9 after
the LH peak, and then declined until the end of the cycle. Serum E rose
in the early luteal phase and then peaked and declined at approximately
the same time as P. Serum relaxin began to rise by day 9 after the LH
peak. Relaxin increased to peak values about 12 days after the LH peak
and then declined. Glycodelin concentrations began to rise on day 10
after the LH peak, increased over the next 2 days, and then plateaued
through the end of that menstrual cycle. The rise in glycodelin was
detected about 8 days after the increase in P and 1 day after the first
increase in relaxin.
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) to show the relationships of hormones in
the late luteal phase in cycles of different lengths. P and E levels
were maximal from 6–10 days before menses, and both then began to
decline until menses. Relaxin began to increase in the last 7 days of
the luteal phase and peaked 2–3 days before menses. Glycodelin was
elevated in the last 5 days of the luteal phase, whereas most of the
glycodelin was found during the menses of the ensuing cycle.
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). P increased by approximately 60% from the peak
nonconceptive concentrations of 15 ng/mL to plateau around 25 ng/mL
during days 12–16 after the LH peak, after which there was a slight
decline. Relaxin, E, and glycodelin concentrations increased linearly
during early gestation.
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). It should be
noted that the scales for E and P are the same as those in
nonconceptive cycles (Fig. 1
). In contrast, relaxin concentrations rose
as they did in early gestation to peak about 8-fold higher than
nonconceptive concentrations. Likewise, glycodelin concentrations
showed dramatic increases over nonconceptive concentrations, with peak
premenstrual concentrations (
400 µg/L) about 13-fold higher than
premenstrual concentrations in nonconceptive cycles (30 µg/L from day
-1; Fig. 2).
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Adverse effects of relaxin treatment were limited to infusion site reactions or minor bleeding. Relaxin concentrations achieved were at or above normal pregnancy levels. The patients were not grouped by dose of relaxin, as all achieved circulating concentrations at or above normal pregnancy concentrations (1–2 ng/mL).
Two subjects in the placebo-treated group (n = 6) demonstrated
ovarian cyclicity (Fig. 5) and had a total of four
samples showing glycodelin (highest value, 7.4 µg/L) in the late
luteal phase. Glycodelin was not found in the other samples from these
or the other four subjects in this group. In the relaxin-treated
subjects not showing cycles (n = 6), glycodelin was not detected
in any sample (Fig. 6). This group consisted of two men
and four women, two of whom were hysterectomized. When relaxin was
administered to subjects with evidence of ovarian cycles (n = 6),
all had glycodelin production during parts of the cycle when glycodelin
is not normally produced (Fig. 7). All subjects except
no. 505 had continuously elevated glycodelin during relaxin treatment.
Subject 505 had elevated glycodelin for a period of at least 14 days
(days 14–29) during relaxin treatment.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The onset and pattern of glycodelin concentrations during gestation are closely aligned to those of relaxin concentrations, with the pregnancy-associated increase occurring about 2 days after the pregnancy-associated rise in relaxin. The patterns of P and glycodelin appear unrelated in early pregnancy. E begins to increase 2 days after the midcycle LH peak, and its profile also appears unrelated to that of glycodelin. The relaxin and glycodelin profiles are similar during early pregnancy, with linear increases beginning about 10 days after the LH peak.
In the early transient pregnancy, the concentrations of E and P did not exceed those observed during a normal luteal phase. However, the relaxin and glycodelin concentrations were both substantially elevated, like those in early pregnancy. This observation provides strong circumstantial evidence that relaxin is the ovarian signal responsible for glycodelin secretion. Although E and P appear to be required for glycodelin production, their roles may involve the proper development of a secretory endometrium, which then requires the presence of relaxin for initiating or stimulating glycodelin secretion.
The absence of circulating glycodelin in men and women with no evidence of ovarian cycles demonstrates the importance of a properly developed endometrium for the production of circulating glycodelin. Small amounts of glycodelin were measured in two of the placebo-treated subjects, but these subjects were cycling women, and glycodelin was detected at the secretory phase of the cycle, a time when it is normally present. Relaxin did not stimulate glycodelin secretion in either men or women without ovarian cycles.
All subjects with ovarian cyclicity who were administered relaxin had circulating glycodelin at times of the cycle when it is not normally produced, and five of the six subjects had detectable glycodelin throughout their cycles. The stimulation of glycodelin by relaxin during the nonluteal phase of the cycle is remarkable when one considers the small amount of glandular tissue remaining after menstruation. The deep basal glands are not shed and may be the site of glycodelin production during the follicular phase (22). This may be the reason why glycodelin concentrations in these subjects are lower than those observed during pregnancy. Although these glands were not stimulated by E and P to maintain a secretory state, relaxin was still capable of stimulating glycodelin secretion.
We have presented evidence that relaxin is most likely the ovarian factor responsible for glycodelin secretion. Studies in which glycodelin is suppressed when the ovary is nonfunctional (12) might be explained by the lack of adequate circulating relaxin, as relaxin is absent in the circulation of women without functional ovaries who become pregnant through embryo transfer (17, 18) (our unpublished observations). Conversely, when the ovary is stimulated with hCG on days 5, 7, or 9 after the LH surge (23), the resulting glycodelin concentrations were highly variable, but tended to be higher. This could be the result of enhanced relaxin secretion in patients treated later in the luteal phase, as relaxin is only stimulated by hCG if hCG is given on or after day 8 of the luteal phase (24).
Although the source of circulating relaxin is the corpus luteum (25), there are alternate sources of relaxin that might also be important for endometrial glycodelin secretion. Relaxin is produced in endometrial glands during the mid- and late secretory phases (26, 27, 28) and early pregnancy (28). Additionally, the endometrial stroma in the mid- and late secretory phases and the parietal and basal decidua of early pregnancy produce relaxin (28, 29). The amounts of endometrial relaxin made during the cycle and pregnancy, how they correlate to serum levels, and the mechanisms controlling its synthesis are unclear. The relative amount of endometrial glandular relaxin is constant in the mid/late luteal phase and early pregnancy, whereas stromal/decidual relaxin appears to increase in early pregnancy (28), as assessed by immunocytochemistry. There is limited evidence that relaxin promotes the synthesis and secretion of relaxin from decidual cells, and it was suggested that decidual relaxin has an autocrine effect on its own production (30). Alternatively, it could be proposed that circulating relaxin has a regulatory effect on endometrial relaxin production. However, when we examined endometrial biopsies from luteal phases with lower than normal circulating relaxin, we did not find less glandular relaxin (27).
In pregnancies established by embryo transfer in women without functioning ovaries because of premature ovarian failure, the normal, pregnancy-associated increase in glycodelin to concentrations over 1000 µg/L was absent (12). However, a low basal (<125 µg/L) amount of glycodelin could be measured in the circulation of these women. Relaxin is also absent in circulation in women without functional ovaries, and this absence of relaxin could account for the failure of glycodelin to reach normal pregnancy concentrations (>1000 µg/L). The basal secretion of glycodelin in the absence of circulating relaxin may be the result of small amounts of locally produced relaxin in the endometrium. Although it is possible that some glycodelin secretion is the result of relaxin produced in the endometrium, the importance of this locally produced relaxin for glycodelin secretion could not be assessed in this study. These basal circulating levels of glycodelin may be sufficient to achieve its effects on immune suppression, especially if local uterine concentrations remain high. Glycodelin levels are particularly high at the feto-maternal interface throughout pregnancy (6).
Basal glycodelin secretion might also be derived from hemopoietic cells in bone marrow, another site of its synthesis (31). However, it should be noted that relaxin did not stimulate detectable amounts of glycodelin in the circulation of subjects without a developed endometrium, indicating that the endometrium is the major source of relaxin-induced glycodelin secretion.
The function of relaxin to stimulate glycodelin secretion in early pregnancy may explain why relaxin is found in high concentrations during early human pregnancy. Glycodelin may be important for local immunosuppression in early pregnancy (3), and relaxin may be the physiological stimulus for its secretion. Glycodelin suppresses natural killer cell activity in a potent and dose-dependent manner, with maximal activity at 50 µg/mL (7). Other functions of relaxin in early pregnancy may be to stimulate the production of decidual PRL (32, 33), aromatase (34), and insulin-like growth factor-binding protein-1 (35, 36). It must also be considered that the effects of relaxin on glycodelin secretion may not be direct, but mediated through one of these factors or other uterine products under the control of relaxin. However, there is no evidence at this time to suggest anything other than a direct effect of relaxin on glycodelin secretion.
The ability of relaxin to stimulate glycodelin throughout the menstrual cycle, including the periovulatory period, suggests that this natural hormone could have contraceptive effects. To be effective in the prevention of human spermatozoa binding to the zona pellucida, sufficient quantities of glycodelin would have to be present in the uterine fluid that the sperm must pass on their way to fertilization in the oviduct. It is known than glycodelin is found in uterine flushings during the mid- and late secretory phases of the cycle (37, 38). The glycodelin levels in the reproductive tract can be considerable, as in the late luteal phase "the concentration in uterine flushings were over a hundred times higher than the corresponding plasma samples" (39). Additionally, the human Fallopian tube produces glycodelin in vivo (40, 41) and in vitro (42), but the type of glycosylation of this substance has not been determined. Glycodelin concentrations as low as 0.01 µg/mL cause a significant reduction of sperm zona binding, whereas concentrations greater than 50 µg/mL cause more than 95% inhibition of sperm zona binding (5). Thus, the periovulatory glycodelin concentrations achieved with relaxin treatment are in a range that is likely to cause significant inhibition of sperm egg interaction.
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Footnotes |
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Received August 15, 1996.
Revised November 1, 1996.
Accepted December 2, 1996.
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