Molecular Human Reproduction Vol.10, No.1 pp. 65–70, 2004
`
`DOI: 10.1093/molehr/gah007
`
`Differential kinetics of serum and cervical insulin-like
`growth factor-binding protein-1 during mifepristone–
`misoprostol-induced medical termination of early
`pregnancy
`
`Helena Honkanen1, Eeva-Marja Rutanen1 and Oskari Heikinheimo1,2,3
`
`1Department of Obstetrics and Gynaecology, Helsinki University Central Hospital, Helsinki, and2Department of Biomedicine,
`University of Helsinki, Helsinki, Finland
`
`3To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, Helsinki University Central Hospital,
`P.O.Box 140, SF-00029, HUS Finland. E-mail: oskari.heikinheimo@helsinki.fi
`
`The kinetics of cervical and circulating phosphoisoforms of insulin-like growth factor-binding protein-1 (IGFBP-1) in normal
`and pathological early pregnancy are not well known. We investigated the profiles of IGFBP-1 in serum and in cervical secretion
`during medical termination of early pregnancy. Sixteen women requesting termination of pregnancy, with <63 days of amenor-
`rhoea, received 200 mg of mifepristone on day 0, followed by either oral or vaginal administration of 0.8 mg of misoprostol on
`day 2. Serum and cervical swab samples, collected up to 6 weeks following the beginning of the treatment, were analysed for
`IGFBP-1 using two immunoenzymometric assays recognizing different patterns of IGFBP-1 phosphoisoforms. Serum mifepris-
`tone was also assayed. In the cervical samples, IGFBP-1 concentration, measured with both assays, increased substantially 2
`days following administration of mifepristone. At 3 h after administration of misoprostol, IGFBP-1 had further increased sev-
`eral-fold in the cervix, but the increase was more pronounced as measured by the assay with preference for the amniotic fluid
`isoforms of IGFBP-1. A strong negative correlation was found between the time to abortion and the increase in cervical IGFBP-
`1 after administration of misoprostol, as measured by the assay preferring the phosphorylated isoforms of IGFBP-1. At 6 weeks,
`IGFBP-1 in the cervix had decreased to lower than pre-treatment levels, as measured by both assays. In serum, both assays
`showed a significant increase in IGFBP-1 concentrations after administration of mifepristone, and the highest values were meas-
`ured on day 2, already before misoprostol administration. Thus, the kinetics of circulating and cervical IGFBP-1 differed from
`each other, indicating different sources and regulation of serum and cervical IGFBP-1.
`
`Key words: early pregnancy/IGFBP-1/medical abortion/mifepristone/misoprostol
`
`Introduction
`
`Insulin-like growth factor-binding protein-1 (IGFBP-1) is produced
`mainly by the liver and decidualized endometrium (Rutanen et al.,
`1985; Julkunen et al., 1988) but high concentrations (100–1000-fold
`higher than those in serum) of IGFBP-1 are also found in amniotic
`fluid (Rutanen et al., 1985; Julkunen et al., 1988). IGFBP-1 in
`amniotic fluid differs from that in decidua in its phosphorylation status
`(Martina et al., 1997; Nuutila et al., 1999). Amniotic fluid contains
`non-phosphorylated and less phosphorylated isoforms of IGFBP-1,
`whereas decidua contains phosphorylated isoforms including a highly
`phosphorylated isoform not present in amniotic fluid (Rutanen et al.,
`1982; Martina et al., 1997). The phosphorylation status of IGFBP-1
`affects its ability to bind insulin-like growth factors (IGF);
`the
`phosphorylated forms have higher IGF-binding affinity compared with
`less and non-phosphorylated isoforms (Jones et al., 1991). Both in
`decidua and in amniotic fluid the degree of IGFBP-1 phosphorylation
`increases from early pregnancy to late pregnancy (Koistinen et al.,
`1993).
`Assessment of the different profiles of IGFBP-1 phosphoisoforms is
`utilized in clinical practice. The detection of amniotic fluid isoforms of
`IGFBP-1 in the cervix is used to diagnose rupture of fetal membranes
`
`(actim(cid:212) PROM test; Medix Biochemica, Finland) (Rutanen et al.,
`1996). Increased levels of decidua-derived, phosphorylated isoforms
`of IGFBP-1 in cervical secretion have been shown to predict cervical
`ripening in term pregnancy (Nuutila et al., 1999). In addition, an
`increase in the levels of phosphorylated isoforms of IGFBP-1 in the
`cervix indicates a risk of preterm delivery (Kekki et al., 2001; Lembet
`et al., 2002).
`The combination of bacterial vaginosis and increased levels of
`cervical phosphorylated isoforms of IGFBP-1—probably a marker of
`tissue damage at the chorio-decidual interface—at 10–17 weeks of
`gestation predicts an increased risk of infectious morbidity later in
`pregnancy (Kekki et al., 1999). However,
`to the best of our
`knowledge, the cervical release of IGFBP-1 in normal or pathological
`early pregnancy has not been studied elsewhere. Moreover,
`the
`regulation and source of circulating IGFBP-1 in early pregnancy is
`poorly understood.
`In medical termination of pregnancy, administration of mifepris-
`tone results in uterine contractions and decidual bleeding. Subsequent
`administration of prostaglandin enhances uterine contractions and
`results in expulsion of
`fetal material. Medical
`termination of
`pregnancy thus greatly resembles spontaneous miscarriage. We
`
`Molecular Human Reproduction vol. 10 no. 1 ª European Society of Human Reproduction and Embryology 2004; all rights reserved
`
`65
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`H.Honkanen, E.-M.Rutanen and O.Heikinheimo
`
`therefore evaluated the kinetics of IGFBP-1 both in serum and in
`cervical secretion during medical termination of early pregnancy with
`mifepristone and misoprostol.
`
`Materials and methods
`
`Subjects
`This study was carried out along with a World Health Organization double-
`blind multicentre trial (von Hertzen et al., 2003) comparing the efficacy of the
`route and duration of misoprostol administration in combination with
`mifepristone for medical abortion. The eligibility criteria used were the same
`as described in a previous study (World Health Organization, 2000). Only
`women with single intrauterine pregnancies, confirmed by ultrasonography,
`were included.
`The study group comprised 16 healthy women (age range 19–40, mean 31
`years) requesting medical termination of an unwanted pregnancy, with <63
`days of amenorrhoea, at the Department of Obstetrics and Gynaecology,
`Helsinki University Central Hospital, Helsinki, Finland. The study was
`approved by the local ethics committee. Each subject gave informed consent
`prior to participation in the study.
`In the efficacy study the women were randomized to one of three treatment
`groups. All women were given an oral dose of 200 mg mifepristone on day 0 of
`the study. The study was placebo-controlled regarding the route of
`administration of misoprostol on day 2, and the continuation of misoprostol
`treatment on days 3–9. On day 2 of the study (36–48 h after the administration
`of mifepristone), six women received 0.8 mg of misoprostol orally, while 10
`women received it vaginally. After administration of misoprostol, the women
`were observed for 3 h in the hospital. They were asked to note the onset of
`bleeding and the time of expulsion of fetal material.
`Starting on day 3, 11 women continued with 0.4 mg of oral misoprostol twice
`daily for 7 days.
`The women returned for follow-up visits 2 weeks and 6 weeks after
`beginning the treatment. Final assessment of complete abortion was carried out
`at the 6 week visit.
`Samples of serum and cervical secretion were obtained six times: sample I on
`day 0, just prior to administration of mifepristone; sample II on day 2, prior to
`administration of misoprostol; sample III on day 2, ~3 h after misoprostol;
`sample IV on day 3, prior to the first home misoprostol or placebo; sample V at
`2 weeks and sample VI at 6 weeks after beginning the treatment. The cervical
`samples were taken with Dacron swabs in speculum examination, as previously
`described (Nuutila et al., 1999; Kekki et al., 2001). A total of 12 women noted
`the exact time of abortion.
`Based partly on the same subject material, we have previously reported the
`effects of the route and duration of misoprostol administration on the
`disappearance of serum hCG and progesterone (Honkanen et al., 2002).
`
`Assays
`Concentrations of IGFBP-1 were measured by using two immunoenzymo-
`metric assays (Rutanen et al., 1993; Nuutila et al., 1999), based on monoclonal
`antibodies (Rutanen et al., 1988).
`Monoclonal antibody (Mab) 6303 (Medix Biochemica, Finland) detects all
`phosphorylated isoforms,
`including the highly phosphorylated isoform
`produced by decidua that is not present in amniotic fluid (Westwood et al.,
`1994; Martina et al., 1997). The assay using Mab 6303 in combination with
`Mab 6301 has been described previously (Nuutila et al., 1999). The detection
`limit of the assay was 0.25 mg/l and the intra- and inter-assay coefficients of
`variation (CV) were 4.6 and 6.4% respectively.
`Mab 6305 (Medix Biochemica) detects the non-phosphorylated and the less
`phosphorylated isoforms present in amniotic fluid (Westwood et al., 1994;
`Martina et al., 1997), but does not recognize the highly phosphorylated isoform
`of IGFBP-1 (Westwood et al., 1994). The assay using Mab 6305 has been
`described elsewhere (Rutanen et al., 1993). The detection limit of the assay was
`0.4 mg/l and the intra- and inter-assay coefficients of variation were 3.4 and
`7.4% respectively. All samples from an individual subject were measured in the
`same assay.
`Serum mifepristone was assayed as described previously (Heikinheimo et al.,
`1986).
`
`66
`
`Figure 1. (A) Concentrations (minimum, 25th percentile, median, 75th
`percentile and maximum) of insulin-like growth factor-binding protein-1
`(IGFBP-1)
`in cervical secretion measured by the assay preferring the
`phosphorylated isoforms. (B) Concentrations (minimum, 25th percentile,
`median, 75th percentile and maximum) of IGFBP-1 in cervical secretion
`measured by the assay preferring the amniotic fluid isoforms.
`
`Statistical methods
`Skewness of the IGFBP-1 values was corrected by logarithmic transformation
`before analysis of variance for repeated measures to analyse the longitudinal
`changes in IGFBP-1 levels in the treatment groups. Data on IGFBP-1
`concentrations are presented as medians, and as 25th and 75th percentiles.
`Comparison of two groups was carried out by the Mann–Whitney U-test.
`Comparison of concentrations before treatment and at 6 weeks was carried out
`by Wilcoxon’s signed rank test. Correlations between variables were assessed
`by calculating Spearman’s coefficient of correlation (r). These analyses were
`performed using SPSS 8.0 for Windows. P < 0.05 was considered statistically
`significant.
`
`Results
`Clinical course
`
`All 16 women had complete abortion. A total of nine women started to
`bleed before administration of misoprostol. The median time to
`
`NEPTUNE GENERICS – Ex. 1019
`Page 2
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`

`

`abortion after administration of misoprostol in the 12 subjects who
`noticed the expulsion of fetal material was 210 min (range 135–719).
`At 2 weeks, 12 women were still bleeding.
`
`Cervical IGFBP-1
`
`The profiles of IGFBP-1 in cervical secretion were similar in the three
`treatment groups (vaginal or oral misoprostol on day 2, continuation of
`oral misoprostol for 7 days or not); thus the data on all subjects were
`combined for the analyses. The data on these 16 subjects are presented
`in Figure 1A and B.
`the
`IGFBP-1 in cervical secretion at
`The median levels of
`consecutive time-points were 27.0, 73.0, 196.0, 118.0, 17.5 and 5.2
`mg/l, as measured by the assay preferring the phosphorylated isoforms
`(Figure 1A). On day 2, prior to administration of misoprostol, the
`median level in cervical secretion was 2.73pre-treatment median
`(PTM) (P < 0.05). At 3 h after administration of misoprostol, it had
`further increased, being 7.33PTM. At day 3, the median level of
`cervical IGFBP-1 had decreased to 4.43PTM. Two weeks later, it was
`0.63PTM, and at 6 weeks, it was 0.23PTM (P < 0.05 versus PTM).
`The median levels of IGFBP-1 in cervical secretion were lower as
`measured by the assay preferring the amniotic fluid isoforms at each
`time-point, except on day 2, 3 h after administration of misoprostol,
`when the median level was 4.6 times higher by the assay preferring the
`amniotic fluid isoforms. The median levels of IGFBP-1 in cervical
`secretion at the consecutive time-points were 10.0, 45.0, 911.0, 94.5,
`8.3 and 2.4 mg/l (Figure 1B). On day 2, prior to administration of
`misoprostol, the median level of cervical IGFBP-1 was 4.53PTM (P <
`0.05), and at 3 h after administration of misoprostol it was at its
`highest, 91.13PTM. By day 3, the median level of cervical IGFBP-1
`had fallen to 9.53PTM. At 2 weeks, the median level was 0.83PTM,
`and at 6 weeks, 0.23PTM (P < 0.05 versus PTM).
`A strong correlation was found between the IGFBP-1 values
`measured by the two assays in cervical secretion before any
`medication (r = 0.98, P < 0.01).
`No difference was found between subjects receiving misoprostol
`orally and those receiving it vaginally in the percentage increase in
`IGFBP-1 levels at day 2 measured by either assay.
`Women who started to bleed before misoprostol administration
`did not differ from those who did not, as regards the levels of
`cervical
`IGFBP-1 on day 2, and the percentage changes in
`IGFBP-1 (day 0 versus day 2), measured by either assay. Similarly,
`at 2 weeks, the cervical secretion levels of IGFBP-1 in the women
`who were still bleeding did not differ from those who had stopped
`bleeding.
`The time to abortion correlated inversely with the increase in
`cervical IGFBP-1 after administration of misoprostol on day 2,
`measured by the assay preferring the phosphorylated isoforms
`(r = –0.73, P < 0.05), whereas it did not correlate with the
`concentration measured by the assay preferring the amniotic fluid
`isoforms.
`
`Serum IGFBP-1
`
`The profiles of IGFBP-1 in serum, as measured by the two assays,
`were similar in the three treatment groups, thus the data on all subjects
`were combined for the analyses. The data on these 16 subjects are
`presented in Figure 2A and B.
`The median levels of IGFBP-1 in serum at the consecutive time-
`points were 86.0, 232.5, 175.0, 147.0, 71.0 and 64.0 mg/l (Figure 2A),
`as measured by the assay preferring the phosphorylated isoforms. On
`day 2, prior to administration of misoprostol, the median level of
`serum IGFBP-1 was 2.73PTM (P < 0.001). At 3 h after administration
`of misoprostol on day 2, it had slightly decreased, being 2.03PTM. By
`
`IGFBP-1 during medically induced abortion
`
`(A) Serum levels (minimum, 25th percentile, median, 75th
`Figure 2.
`percentile and maximum) of insulin-like growth factor-binding protein-1
`(IGFBP-1) measured by the assay preferring the phosphorylated isoforms.
`(B) Serum levels (minimum, 25th percentile, median, 75th percentile and
`maximum) of IGFBP-1 measured by the assay preferring the amniotic fluid
`isoforms.
`
`the median level of IGFBP-1 had further decreased to
`day 3,
`1.73PTM. At 2 weeks, the median had decreased to 0.83PTM, and at
`6 weeks, to 0.73PTM (NS versus PTM).
`A similar trend was noted in the serum kinetics of IGFBP-1 as
`measured by the assay preferring the amniotic fluid isoforms
`(Figure 2B). The median levels at consecutive time-points were
`10.3, 29.0, 23.0, 15.3, 3.1 and 3.3 mg/l. On day 2, prior
`to
`administration of misoprostol, the median serum IGFBP-1 level was
`2.83PTM (P < 0.001). At 3 h after administration of misoprostol on
`day 2, it had declined slightly, to 2.23pre-treatment median. By day 3,
`the median level had decreased to 1.53PTM. At 2 and 6 weeks, it was
`0.33PTM (P < 0.05 versus PTM).
`There was a significant correlation between serum IGFBP-1
`measured by the two assays before any medication (r = 0.61, P =
`0.013), and 6 weeks later (r = 0.86, P < 0.01).
`
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`H.Honkanen, E.-M.Rutanen and O.Heikinheimo
`
`Figure 3. Serum levels (minimum, 25th percentile, median, 75th percentile
`and maximum) of mifepristone.
`
`Serum mifepristone
`
`Figure 3 shows the serum levels of mifepristone. There was a
`significant negative correlation between serum IGFBP-1 level on day
`2, 3 h after misoprostol, measured by the assay preferring the amniotic
`fluid isoforms, and the peak mifepristone level on day 2 (r = –0.58, P =
`0.02).
`The percentage changes in serum and cervical IGFBP-1 from day 0
`to day 2, measured by either assay, did not correlate with the peak
`mifepristone level measured on day 2. Moreover, the percentage
`changes in serum IGFBP-1, measured by either assay, and in serum
`mifepristone from day 2 (before misoprostol, or 3 h after misoprostol)
`to day 3 did not correlate.
`
`Discussion
`
`In the present study we investigated the kinetics of IGFBP-1 both in
`serum and in cervical swab samples during medical termination of
`early pregnancy, using mifepristone followed by oral or vaginal
`misoprostol. The IGFBP-1 was quantified using two immunoenzymo-
`metric assays based on different monoclonal antibodies preferring
`different patterns of IGFBP-1 phosphoisoforms (Rutanen et al., 1993;
`Nuutila et al., 1999). As shown previously (Nuutila et al., 1999), one
`of the assays gives higher values at instances where non- and less
`phosphorylated isoforms of IGFBP-1—such as in the presence of
`amniotic fluid—predominate. In contrast, the other assay (Rutanen
`et al., 1993) prefers phosphorylated isoforms of IGFBP-1 present in
`decidua (Westwood et al., 1994; Martina et al., 1997).
`In agreement with the results of previous studies in women with
`term pregnancy (Nuutila et al., 1999), the levels of IGFBP-1 measured
`by the assay preferring the phosphorylated isoforms exceeded those of
`IGFBP-1 measured by the assay preferring the amniotic fluid
`isoforms, in samples of cervical secretion of early first trimester
`pregnancy. The median concentrations of
`IGFBP-1 in cervical
`samples measured by both assays increased following mifepristone
`administration. This increase was rapidly and significantly augmented
`by administration of misoprostol, but no such change was noted in
`serum. The median values of IGFBP-1 in cervical secretion measured
`by both assays also declined rapidly, reaching pre-treatment levels at 2
`weeks, and had decreased to below pre-treatment levels by 6 weeks.
`The kinetics of IGFBP-1 in serum differed from that in cervical
`
`68
`
`secretion, and mirrored that of serum mifepristone. Thus, in serum, the
`highest concentrations of IGFBP-1 measured by both assays were
`reached after mifepristone but before misoprostol administration.
`The clinical course of medical termination of early pregnancy with
`mifepristone and misoprostol
`is well known. Administration of
`mifepristone results in uterine contractions, and bleeding in 22–41%
`of women within 2 days (De Nonno et al., 2000; World Health
`Organization, 2001). Expulsion of the products of conception occurs
`in almost 50% of subjects within the first 3–4 h after misoprostol
`administration (Spitz et al., 1998; World Health Organization, 2000).
`In our study, 56% of
`the women started to bleed following
`mifepristone, and the median time to expulsion of the products of
`conception was 3.5 h after misoprostol administration.
`The rapid increase in cervical IGFBP-1 after administration of
`mifepristone is likely to originate from detachment of the decidua. In
`vitro, in cultures of endometrial stromal cells, progestins stimulate
`IGFBP-1 production (Bell et al., 1991; Tseng et al., 1992; Gao et al.,
`1994). However, in long-term cultures, progestin withdrawal further
`increases production of IGFBP-1 (Bell et al., 1991), and IGFBP-1
`mRNA levels have been observed to be transiently increased in 2–3
`days after replacing progestin with the antiprogestin mifepristone
`(Tseng et al., 1992). Also, in vivo, administration of mifepristone in
`the early luteal phase increases immunostaining of IGFBP-1 in human
`endometrium (Qiu et al., 2002). Thus, the observed increase of
`IGFBP-1 in cervical secretion might also reflect increased decidual
`synthesis of IGFBP-1 as a response to functional progesterone
`withdrawal caused by mifepristone. This is supported by the fact that
`the concentrations of IGFBP-1 were not different in subjects who
`reported uterine bleeding prior to misoprostol administration, and
`those who did not.
`On day 2, following administration of misoprostol, IGFBP-1 levels
`increased markedly in cervical secretion. A significant
`inverse
`correlation was found between the time to abortion and the increase
`in cervical IGFBP-1 on day 2 measured by the assay preferring the
`phosphorylated isoforms. This is likely to reflect the increased uterine
`sensitivity to exogenous prostaglandin—extensive decidual damage as
`evidenced by the increase in cervical IGFBP-1 correlates with the
`increased uterine contractility and rapid expulsion of fetal material.
`However, at this time-point, the increase in IGFBP-1 measured by the
`assay preferring the amniotic fluid isoforms was more pronounced,
`most likely reflecting amniotic fluid leakage (Nuutila et al., 1999). By
`day 3 the concentrations of IGFBP-1 had decreased significantly, and
`at 6 weeks, to less than pre-treatment levels. We thus conclude that the
`kinetics of IGFBP-1 isoforms in cervical secretion are in accordance
`with the well-characterized clinical course of medical abortion.
`The regulation of circulating IGFBP-1 during pregnancy is poorly
`understood. Depending on the assay, either an increase or unchanged
`levels of
`IGFBP-1 throughout pregnancy have been reported
`(Westwood et al., 1994; Hills et al., 1996). The highly phosphorylated
`isoform of IGFBP-1 predominates throughout pregnancy (Westwood
`et al., 1994). However,
`the overall phosphorylation status of
`circulating IGFBP-1 changes during pregnancy, with the appearance
`of non- and less phosphorylated isoforms of IGFBP-1 (Westwood
`et al., 1994). Similarly, in our study, the serum concentrations of
`IGFBP-1, as measured by the assay preferring the phosphorylated
`isoforms of IGFBP-1, were 8-fold higher before treatment than those
`measured by the assay preferring the amniotic fluid isoforms.
`Following termination of pregnancy, the decrease in the non- and
`less phosphorylated isoforms of IGFBP-1 was more pronounced, thus
`at 2 and 6 weeks the corresponding ratios were 23:1 and 20:1,
`respectively. Roughly similar ratios of 2:1 in late first trimester and
`11:1 in non-pregnant women have been reported previously
`(Westwood et al., 1994).
`
`NEPTUNE GENERICS – Ex. 1019
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`

`A substantial increase in IGFBP-1 in serum was evident 2 days after
`administration of mifepristone. The kinetics of IGFBP-1 mirrored the
`circulating levels of mifepristone, yet
`the increase in IGFBP-1
`measured by either assay did not correlate with the peak level of serum
`mifepristone. Serum IGFBP-1 levels have been shown to fall
`immediately after administration of mifepristone in first trimester
`medical abortion, and to rise to higher than pre-treatment levels in 2
`days (Olajide et al., 1989). Due to a paucity of initial sampling after
`administration of mifepristone, we have possibly missed an initial
`decrease in serum levels of IGFBP-1.
`The mechanism by which mifepristone increases circulating
`IGFBP-1 levels remains enigmatic. Regeneration of decidual cells
`has previously been proposed as a source of increased serum IGFBP-1
`(Olajide et al., 1989). However, regeneration of decidua in the
`presence of high levels of antiprogestin is unlikely. Moreover, the
`serum levels of IGFBP-1, measured by the assay preferring the
`phosphorylated isoforms, exceeded those in cervical secretion on the
`morning of day 2, suggesting that decidual synthesis is an unlikely
`source of increased serum IGFBP-1.
`The effect of progesterone on circulating concentrations of IGFBP-
`1 is poorly understood; thus the effects of mifepristone on serum
`IGFBP-1 may not necessarily represent antiprogestagenic actions of
`mifepristone. In vitro, glucocorticoids stimulate IGFBP-1 mRNA
`expression in hepatoma cells (Orlowski et al., 1990). However, in vivo
`administration of dexamethasone to male volunteers suppresses serum
`levels of IGFBP-1 (Miell et al., 1993). As mifepristone is also an
`antiglucocorticoid, the increase in circulating IGFBP-1 might also
`reflect systemic antiglucocorticoid effects of mifepristone.
`We conclude that the kinetics of IGFBP-1 in cervical secretion are
`in line with the clinical course of medical abortion. Moreover, the
`increase in IGFBP-1 measured by the assay preferring the phos-
`phorylated isoforms was inversely and significantly correlated with
`the time to abortion. The kinetics of circulating IGFBP-1 differed from
`those in the cervix, indicating different regulation and most likely
`different sources of serum and cervical IGFBP-1. We thus speculate
`that the increase in serum IGFBP-1 is a result of a systemic action of
`mifepristone—possibly an antiprogestin and/or antiglucocorticoid
`action.
`
`Acknowledgements
`We wish to thank Ms Pirkko Timonen for her professional handling of the
`volunteers, and Ms Kristiina Nokelainen and Ms Marjatta Tevilin for their
`expert laboratory work. Financial support from The Population Council (New
`York, NY, USA) and Helsinki University Central Hospital Research Funds is
`gratefully acknowledged. Dr Heikinheimo is a recipient of a Finnish Medical
`Foundation Clinical Fellowship grant. The content of the present manuscript
`does not necessarily reflect the policy of any of the funding sources.
`
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`Submitted on May 15, 2003; resubmitted on September 16, 2003; accepted on
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