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Reduced Serum Glycodelin and Insulin-Like Growth Factor-Binding Protein-1 in Women with Polycystic Ovary Syndrome during First Trimester of Pregnancy

Hospital de Clinicas Caracas (D.J.J.) and Central University of Venezuela (D.J.J., S.J.), Caracas 1040, Venezuela; Departments of Medicine (P.A.E., J.E.N.) and Obstetrics and Gynecology (J.E.N.), Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298; Department of Clinical Chemistry (M.S.), Biomedicum, University Central Hospital, and Department of Obstetrics and Gynecology (R.K.), Biomedicum, Helsinki University Central Hospital, Helsinki SF-00290, Finland; and Department of Medicine (J.-P.B.), Université de Sherbrooke, Sherbrooke, Canada J1H 5N4

Address all correspondence and requests for reprints to: John E. Nestler, M.D., Medical College of Virginia, P.O. Box 980111, Virginia Commonwealth University, Richmond, Virginia 23298-0111. E-mail: nestler{at}hsc.vcu.edu.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The polycystic ovary syndrome (PCOS) is associated with an increased rate of early pregnancy loss (EPL). Hyperinsulinemia is an independent risk factor for EPL and has been found to decrease levels of glycodelin and IGF binding protein-1 (IGFBP-1), two major endometrial proteins. We hypothesized that serum glycodelin IGFBP-1 concentrations would be reduced in women with PCOS during the first trimester of pregnancy.

Fasting serum insulin, glycodelin, and IGFBP-1 were measured, and oral glucose tolerance tests were performed in 72 women with PCOS and 62 normal women. Each woman was seen once and assigned to one of three gestational groups: wk 3–5, 6–8, and 9–11.

The insulin sensitivity index during oral glucose tolerance test was lower in women with PCOS compared with normal women throughout the first trimester (P < 0.0001). Both serum glycodelin and IGFBP-1 were markedly lower in women with PCOS (for glycodelin: wk 3–5, P < 0.0001; wk 6–8, P = 0.03; wk 9–11, P = 0.19; and for IGFBP-1: wk 3–5 and 6–8, P < 0.0001; wk 9–11, P = 0.0003). Comparing women with PCOS who experienced EPL with those who did not, serum glycodelin was significantly lower during wk 3–8 (P < 0.02) and serum IGFBP-1 during wk 9–11 (P = 0.003).

During the first trimester, serum glycodelin and IGFBP-1 concentrations are markedly decreased in PCOS, implicating endometrial epithelial and stromal dysfunction during periimplantation and early pregnancy as a possible mechanism for EPL in PCOS. These decreases are likely to be secondary to hyperinsulinemia and reduced insulin sensitivity.

	Introduction

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THE POLYCYSTIC OVARY syndrome (PCOS) is characterized by chronic anovulation and hyperandrogenism, and is the most common cause of anovulatory infertility in the United States (1). Affected women present not only with infertility, but also suffer from a high rate of early pregnancy loss (EPL), defined as miscarriage, during the first trimester. The syndrome is associated with a 30–50% rate of early loss of clinically recognized pregnancies after either spontaneous or assisted conception (2, 3, 4, 5, 6), a rate 3-fold higher than that reported for normal women (7, 8). In most cases, no apparent cause of EPL can be identified (9), but, in addition to defects in the developing embryo, adverse alterations in endometrial function likely play a role.

Glycodelin and IGF binding protein-1 (IGFBP-1) are major endometrial secretory proteins that may play important roles in endometrial receptivity during the implantation period and in the maintenance of pregnancy (10, 11). Glycodelin is a glycoprotein produced by secretory/decidualized endometrial glands during the luteal phase, and facilitates implantation by inhibiting the immune response of the endometrium to the embryo (10, 11, 12, 13, 14). Decreased serum concentrations of glycodelin are associated with retarded endometrial development, EPL, and recurrent miscarriage (15, 16). IGFBP-1 is a protein that facilitates adhesion processes at the feto-maternal interface during the periimplantation period (17, 18). In nonpregnant women, IGFBP-1 is produced primarily by the liver and is negatively regulated by insulin (19, 20). During pregnancy, IGFBP-1 is produced by the endometrium as well (21).

A key feature of PCOS is insulin resistance with compensatory hyperinsulinemia (22, 23, 24, 25, 26), and hyperinsulinemic insulin resistance has been implicated in both the anovulation and hyperandrogenism of the disorder (27, 28, 29, 30). Notably, hyperinsulinemia also has been identified as an independent risk factor for EPL in PCOS (27). This is consistent with two studies that reported marked reductions in the rates of first-trimester pregnancy loss in women with PCOS who were treated with the insulin-sensitizing drug metformin throughout pregnancy (28, 29).

The mechanism by which hyperinsulinemic insulin resistance contributes to miscarriage is unknown, but may involve suppression of endometrial glycodelin and IGFBP-1 production, resulting in an inhospitable endometrial milieu. In support of this idea, insulin reduction with metformin in nonpregnant women with PCOS resulted in a 3-fold increase in luteal-phase serum glycodelin concentrations and a 4-fold increase in serum IGFBP-1 concentrations (30). However, it is unknown whether serum glycodelin or IGFBP-1 levels during pregnancy are altered in PCOS.

We hypothesized that the hyperinsulinemic insulin resistance of PCOS would decrease circulating concentrations of glycodelin and IGFBP-1 during pregnancy in women with PCOS. As a corollary, we also hypothesized that serum concentrations of glycodelin and IGFBP-1 during the first trimester of pregnancy would correlate with insulin sensitivity. To test our hypotheses, we conducted a cross-sectional study of pregnant women with and without PCOS during the first trimester of pregnancy, and assessed weekly serum glycodelin, IGFBP-1, insulin, and insulin sensitivity.

	Subjects and Methods

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Study subjects

We studied 141 women during the first trimester of pregnancy, defined as up to 12 wk gestation. Seventy-seven women had PCOS, and 64 were randomly selected, healthy, ovulatory women. PCOS was diagnosed before pregnancy, and defined by oligomenorrhea (eight or fewer menstrual periods annually before pregnancy) and hyperandrogenism (elevated serum free testosterone concentration or hirsutism) (31). All women with PCOS had polycystic ovaries by ultrasonography, although this was not an inclusion criterion. They also had normal thyroid function tests, serum prolactin, and serum 17-hydroxyprogesterone. All the control subjects met the following criteria: documented history of regular monthly menses before pregnancy, normal serum total and free testosterone, and normal health based on interview, medical history, physical examination, and standard laboratory tests. In addition, none of the women, neither control subjects nor subjects with PCOS, had diabetes mellitus as determined by a 2-h oral glucose tolerance test (OGTT), and none were taking insulin-sensitizing agents during the study period. Although some of the women with PCOS had been on metformin previously, none were taking this medication at least 3 months before pregnancy. All of the women with PCOS studied achieved pregnancy spontaneously. None of the women with PCOS received clomiphene citrate or gonadotropin stimulation to achieve pregnancy. All women were recruited from the Hospital de Clinicas Caracas and an affiliated hospital in Caracas, Venezuela. Specifically, women with PCOS were recruited consecutively from a specialty PCOS clinic, whereas normal women were recruited from the general obstetrical clinic or responded to advertisements. All women who met the inclusion criteria and were willing to participate were entered into the study. The institutional review boards of the Hospital Clinicas de Caracas and Virginia Commonwealth University approved the study, and each woman gave written informed consent.

Protocol

Each woman was studied once during the first trimester of pregnancy, and they were divided into the following three groups based on fetal gestational age: 1) from the start of wk 3 through the end of wk 5 (wk 3–5), 2) from the start of wk 6 through the end of wk 8 (wk 6–8), and 3) from the start of wk 9 through the end of wk 11 (wk 9–11). Gestational age was defined as the number of weeks since the last menstrual period. Pregnancy was confirmed by a urine pregnancy test or serum human chorionic gonadotropin (hCG) concentration of more than 50 IU/liter and ultrasonographic evidence of a gestational sac within the uterine cavity. Some women were studied after biochemical confirmation of pregnancy, and the ultrasonographic confirmation of pregnancy was obtained several weeks later. Blood was drawn in the fasting state for determination of serum glycodelin, IGFBP-1, sex steroids, and SHBG. Subsequently, the women underwent a 75-g dextrose OGTT with determinations of serum insulin and glucose at 0, 60, and 120 min. Miscarriage was documented by a serial decline in serum hCG and progesterone, and confirmed by uterine ultrasonography.

Assays

Blood samples were centrifuged immediately after collection, and serum was stored at -20 C until assayed. Serum glycodelin and IGFBP-1 were assayed in Helsinki by sandwich-type immunofluorometry, as previously described (32, 33). Serum insulin was measured by a highly specific RIA (Diagnostic Products, Los Angeles, CA) that had cross-reactivity with proinsulin of approximately 40% at midcurve. Serum hormones and SHBG, measured as protein, were assayed in Richmond, Virginia, using commercial kits as previously described (34, 35). Serum free testosterone was calculated by the method of Sodergard et al. (36) assuming a serum albumin of 4.0 g/dl. Insulin sensitivity index (ISI) during OGTT was calculated by the Matsuda method (37), using the following equation: [10,000/square root of (fasting glucose x fasting insulin) x (mean glucose x mean insulin during OGTT)], which is highly correlated with the rate of whole-body glucose disposal during the euglycemic insulin clamp (r = 0.73; P < 0.0001). All samples from an individual subject were analyzed in duplicate. Serum hCG was measured by the IMX microparticles immunoassay (Abbott Laboratories, Abbott Park, IL). The intraassay coefficient of variation was 8% for glycodelin and IGFBP-1, 5.5% for insulin, and less than 10% for all serum steroid hormones. The lower limit of detection for insulin was 1.2 IU/ml (8.6 pmol/liter).

Statistical analysis

Results are reported as means ± SE. The significance of differences between the two groups of women (PCOS and normal) was tested using Student’s two-tailed unpaired t test. Strength of association between serum glycodelin, IGFBP-1, fasting insulin, and free testosterone was determined using Pearson correlation coefficient. Logarithmic transformations were used for variables not normally distributed (fasting insulin, ISI, IGFBP-1, total and free testosterone, estradiol, and 17-OH progesterone) in all statistical analysis. Responses of serum glucose and insulin to oral dextrose administration were analyzed by calculating the areas under the curves by the trapezoidal rule (area under the curve of glucose and area under the curve of insulin) using absolute values (the total area from zero concentration). Statistical significance was defined as P < 0.05, with Bonferroni’s correction for multiple comparisons. Statistical analysis was performed with JMP version 4.0.4 (SAS Institute, Inc., Cary, NC).

	Results

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Clinical characteristics

Of the 141 pregnant women who entered the study, 7 were withdrawn because they received hormonal therapy with hCG. This left 134 women, 72 with PCOS and 62 normal women. Clinical characteristics of the study populations are given in Table 1. By chance, there were no significant differences in age and body mass index between the women with PCOS and normal women. Women with PCOS had significantly younger ages at menarche compared with normal women, and tended to have higher rates of past EPL than normal women, although not achieving significance.

Insulin and glucose (Table 1). Fasting serum insulin levels were up to 3- to 7-fold higher in the women with PCOS during wk 3–5 and 9–11 (P < 0.0001) compared with normal women, but did not differ between groups during wk 6–8 (P = 0.27). Conversely, ISI was markedly decreased during all the weeks studied (P < 0.0001). Likewise, area under the serum insulin curve was greater in women with PCOS throughout the first trimester (P < 0.0001 for all weeks).

Fasting serum glucose was higher in the PCOS group during wk 3–5 (P = 0.001), but thereafter was comparable between groups. However, the area under the serum glucose curve was higher in the PCOS group throughout the first trimester (P < 0.0001 for all weeks).

Glycodelin and IGFBP-1 (Fig. 1). Both serum glycodelin and IGFBP-1 concentrations were markedly lower in women with PCOS compared with normal women. Serum glycodelin was 56% lower in women with PCOS during wk 3–5 (P < 0.0001) and 23% lower during wk 6–8 (P = 0.03), but by wk 9–11, was similar between the two groups (P = 0.19). Serum IGFBP-1 was 60–70% lower in women with PCOS during wk 3–5 and 6–8 (P < 0.0001 for both) and 39% lower during wk 9–11 (P = 0.003).

View larger version (20K): [in this window] [in a new window]   FIG. 1. Comparison of mean serum glycodelin (A) and mean serum IGFBP-1 (B) in the two groups of women during the first trimester. In normal women, n = 18 for wk 3–5, n = 23 for wk 6–8, and n = 21 for wk 9–11. In women with PCOS, n = 21 for wk 3–5, n = 26 for wk 6–8, and n = 25 for wk 9–11. To convert glycodelin from nanograms per milliliter to picomoles per liter, multiply by 35.7, and to convert IGFBP-1 from nanograms per milliliter to picomoles per liter, multiply by 40. Bar graphs represent means + SE.

Sex steroids (Table 1).

Serum estradiol, on the other hand, was significantly lower in women with PCOS compared with normal women during wk 6–8 and 9–11. In contrast to the normal women, in whom serum estradiol levels increased as pregnancy progressed, serum estradiol did not change in the PCOS group. Serum dehydroepiandrosterone sulfate was significantly higher in women with PCOS during wk 3–5 and 6–8, but became similar by wk 9–11.

Correlations with insulin sensitivity (Fig. 2). When the entire sample of women was analyzed together, a significant positive correlation was observed between serum glycodelin and the ISI obtained during the OGTT (ISI) during wk 3–5 only. In contrast, serum IGFBP-1 correlated positively with the ISI during all the weeks studied. However, it should be noted that the possibility that these correlations resulted from the clustering of ISI within normal women and PCOS women rather than from a true correlation cannot be excluded. In this regard, when women with PCOS were analyzed separately, no significant correlations were observed between serum glycodelin and ISI or between IGFBP-1 and ISI except for during wk 9–11, in which a significant positive correlation between IGFBP-1 and ISI was observed (r = 0.42; P = 0.04; n = 25).

View larger version (22K): [in this window] [in a new window]   FIG. 2. Correlations between serum glycodelin and insulin sensitivity during oral glucose tolerance [ISI (OGTT)] (upper graphs) and between serum IGFBP-1 and ISI (OGTT) (lower graphs) for all women. In women with PCOS (•), n = 21 for wk 3–5, n = 26 for wk 6–8, and n = 25 for wk 9–11. In normal women (), n = 18 for wk 3–5, n = 23 for wk 6–8, and n = 21 for wk 9–11. Glycodelin and IGFBP-1 units are nanograms per milliliter. To convert glycodelin from nanograms per milliliter to picomoles per liter, multiply by 35.7, and to convert IGFBP-1 from nanograms per milliliter to picomoles per liter, multiply by 40.

Of the 134 women studied, EPL occurred subsequent to the study in 12 women. Ten were women with PCOS, and two were normal women. Fifty percent of the miscarriages occurred during wk 3–5, 30% during wk 6–8, and 20% during wk 9–11. Women with PCOS had significantly more miscarriages compared with normal women (14 vs. 3%, respectively; P = 0.02). There were no significant clinical and metabolic differences between the women with PCOS and normal women who miscarried.

In the PCOS group, there were marked differences in serum glycodelin and IGFBP-1 between women with EPL and women without EPL. Specifically, serum glycodelin was decreased by 48 and 78% in the women with PCOS with EPL during wk 3–5 (P = 0.02) and 6–8 (P = 0.01), respectively. Serum IGFBP-1 during wk 9–11 was 60% lower in women with PCOS with EPL (P = 0.003). Apart from lower SHBG concentrations during wk 3–5 (P = 0.004) and higher serum progesterone during wk 9–11 (P = 0.03) in women with EPL, other parameters did not differ between the groups.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The aim of this study was to test the hypothesis that serum glycodelin and IGFBP-1 concentrations would be decreased in pregnant women with PCOS during the first trimester. Indeed, during the first 2 months (wk 3–8) of pregnancy, both serum glycodelin and IGFBP-1 levels were strikingly lower in women with PCOS compared with normal women. During the last weeks of the first trimester (wk 9–11), serum IGFBP-1 remained suppressed in women with PCOS, whereas serum glycodelin became comparable to that of normal women.

These findings are significant in that they may provide a mechanism for first-trimester miscarriage in PCOS. Because glycodelin inhibits mixed lymphocyte reaction and natural killer cell activity (12, 14), impaired production of glycodelin presumably allows a maternal immune response against the embryo. Therefore, low concentrations of serum glycodelin suggest a deficient endometrial environment for implantation and maintenance of pregnancy. Likewise, given that IGFBP-1 exerts paracrine actions and is correlated with adhesion processes at the feto-maternal interface, low concentrations of endometrial IGFBP-1 would adversely affect embryo implantation. Although the liver is the main source of circulating IGFBP-1 in the nonpregnant state, it seems likely that inhibition of IGFBP-1 production by insulin would occur both in the liver and the endometrium. Therefore, we postulate that the low concentrations of IGFBP-1 noted in pregnant women with PCOS likely reflect decreased endometrial as well as hepatic synthesis and secretion.

Notably, these results are consistent with studies reporting decreased concentrations of glycodelin in either luteal-phase serum or uterine flushings of women with unexplained infertility and recurrent miscarriages (15, 16, 38). The findings are also consistent with the reported effect of the insulin-sensitizing drug metformin to increase luteal-phase serum glycodelin and IGFBP-1 in nonpregnant women with PCOS (30), and provide a mechanism for the markedly decreased rate of EPL reported for women with PCOS who conceived while taking metformin and remained on the drug throughout the first trimester (28).

We further hypothesized that positive correlations would exist between serum levels of glycodelin and IGFBP-1 with insulin sensitivity. These relationships were observed when results from all women were analyzed together. However, when analyzed within groups (i.e. women with PCOS only or normal women only), the relationships did not hold. This strongly suggests the possibility of a group effect (i.e. the correlation was observed simply because of the difference in ISI between women with PCOS and normal women) or insufficient statistical power because of the limited number of women in each individual group.

There is still a possibility that insulin, either directly or indirectly, affects glycodelin secretion. Within the endometrium, glycodelin is produced by the epithelium, and IGFBP-1, which is insulin-regulated (20), by the stroma (39). Studies have shown that epithelial function is often regulated by stromal factors (40, 41, 42). Therefore, hyperinsulinemia may either directly or indirectly inhibit the production of stromal signals to the epithelium and thereby cause inadequate differentiation of the epithelium, as reflected by lower serum glycodelin concentrations.

In addition to evaluation of insulin, this study assessed EPL. We found that 14% of women with PCOS had EPL compared with 3% of normal subjects. Although this finding varies from the rates of 30–50% in PCOS (2, 3, 4, 5, 6) and 10–15% in normal women reported in other studies (7, 8), it is consistent with the 3-fold increased rate of EPL in PCOS that has been described in the literature (7, 8). Furthermore, although this study was not specifically designed to examine differences in EPL, women with PCOS who miscarried tended to have lower serum glycodelin and IGFBP-1 concentrations compared with those women with PCOS who did not miscarry, suggesting that low serum glycodelin and IGFBP-1 levels may prove useful in identifying women with PCOS who are at increased risk for EPL.

There are limitations to our study. We studied women in a cross-sectional rather than longitudinal cohort fashion, thereby making the sequence of events and a cause-effect relationship difficult to determine. For example, this study cannot determine whether decreased glycodelin is simply a biomarker for women at high risk of EPL due to other factors or whether deficient glycodelin production by the endometrium is itself involved in the pathologic process of miscarriage. In addition, although control subjects were randomly selected, the potential for selection bias still exists. Furthermore, we did not power the study to observe significant differences in rates of EPL or to determine subgroup correlations of insulin sensitivity with serum glycodelin and IGFBP-1 concentrations. However, neither of these focuses was the primary aim of the study. Lastly, we did not, for ethical reasons, obtain endometrial tissue or uterine flushings for direct measurement of glycodelin expression. Although these methods would have provided more accurate measurements of endometrial glycodelin expression, serum glycodelin has been shown to correlate well with endometrial glycodelin concentrations, as reported in a study that demonstrated that hormone replacement therapy causes a much greater elevation in serum glycodelin concentrations in women with intact uteri compared with hysterectomized women (10).

In summary, during the first trimester of pregnancy, serum concentrations of glycodelin and IGFBP-1 are markedly reduced in women with PCOS, implicating endometrial dysfunction at both epithelial and stromal levels during the periimplantation period as mechanisms for EPL in women with PCOS. Collectively, the results, coupled with previous observations (28, 30), are also consonant with inhibitory regulation of these proteins by insulin during early pregnancy. These findings raise the possibility, which needs to be explored by prospective studies, that 1) serum glycodelin and IGFBP-1 may prove useful in the identification of women at increased risk of miscarriage, and 2) interventions that increase serum glycodelin and IGFBP-1 during pregnancy, for example by improving insulin sensitivity, may prove useful in prevention of miscarriage in women with PCOS and perhaps other types of hyperinsulinemia-related infertility.

	Footnotes

 
This work was supported in part by National Institutes of Health Grants K12DA 14041-03 (to P.A.E.) and K24HD40237 (to J.E.N.) and grants from the Finnish Cancer Center Foundation (to M.S.), the University of Helsinki (to R.K.), and the Helsinki University Central Hospital Research Fund (to R.K.).

Abbreviations: EPL, Early pregnancy loss; hCG, human chorionic gonadotropin; IGFBP-1, IGF binding protein-1; ISI, insulin sensitivity index; OGTT, oral glucose tolerance test; PCOS, polycystic ovary syndrome.

Received June 9, 2003.

Accepted October 31, 2003.

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