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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 11 5361-5365
Copyright © 2003 by The Endocrine Society

Serum Osteoprotegerin as a Determinant of Bone Metabolism in a Longitudinal Study of Human Pregnancy and Lactation

K. E. Naylor, A. Rogers, R. B. Fraser, V. Hall, R. Eastell and A. Blumsohn

Bone Metabolism Group (K.E.N., A.R., R.E., A.B.) and Department of Obstetrics and Gynecology (R.B.F., V.H.), University of Sheffield, Sheffield, United Kingdom S5 7AU

Address all correspondence and requests for reprints to: Dr. A. Blumsohn, University of Sheffield, Bone Metabolism Group, Clinical Sciences Center (North), Northern General Hospital, Herries Road, Sheffield, United Kingdom S5 7AU. E-mail: ablumsohn{at}sheffield.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Osteoprotegerin (OPG) is a soluble decoy receptor that inhibits bone resorption by binding to receptor activator of nuclear factor {kappa}B ligand. Murine studies suggest that OPG is elevated in pregnancy, but its role in human pregnancy is unknown. We evaluated the relationship among OPG, bone turnover, and bone density in a longitudinal study of planned human pregnancy and lactation (n = 17; age, 20–36 yr). Samples were collected before conception; at 16, 26, and 36 wk gestation; and at 2 and 12 wk postpartum. Indexes of bone resorption included serum ß C-terminal and urinary N-terminal (uNTX) telopeptides of type I collagen. OPG increased by 110 ± 16% (mean ± SEM) at 36 wk (P < 0.001), followed by a rapid postpartum decline in both lactating and nonlactating women. Bone resorption was elevated at 36 wk (serum ß C-terminal telopeptides by 76 ± 17%; urinary N-terminal telopeptides by 219 ± 41%; P < 0.001). The tissue source of OPG in pregnancy is unknown. Human breast milk contains large amounts of OPG (162 ± 58 ng/ml in milk vs. 0.42 ± 0.03 ng/ml in nonpregnant serum). However, the rapid postpartum decline in serum OPG and the low serum OPG in neonates suggest a placental source. There was no correlation between change in OPG and bone turnover or bone mineral density (P > 0.05), and the physiological importance of elevated OPG in human pregnancy remains uncertain.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
OSTEOPROTEGERIN (OPG) IS an osteoblast-derived protein that binds to receptor activator of nuclear factor {kappa}B (RANK) ligand (RANKL), blocking the RANK-RANKL signaling pathway that regulates osteoclastogenesis and osteoclast activation (1, 2, 3). It has been reported that a single sc dose of OPG in postmenopausal women decreases bone resorption (4). OPG, however, is expressed in several tissues other than bone, including lung, heart, kidney, and placenta (5). The RANK-RANKL signaling pathway also appears to be involved in the development of lactating mammary tissue (6).

Changes in calcium metabolism during pregnancy and lactation are well described (7, 8, 9, 10, 11). During pregnancy, maternal calcium homeostasis adapts to provide calcium for the growing fetus. We and others have reported that trabecular bone mineral content is lower postpartum compared with preconception values (12, 13, 14), but that bone mineral content at cortical sites is higher postpartum (12, 15). There is an increase in fractional absorption of calcium from the gut (16) and an increase in the rate of bone formation as well as bone resorption (12, 13, 17). However, the mechanisms regulating bone turnover during pregnancy are unknown.

Serum OPG is elevated in murine pregnancy, and it has been speculated that the increase in OPG during pregnancy may prevent excessive maternal bone resorption and protect the maternal skeleton (18). The expression of OPG is increased by estradiol (E2) in vitro (19), and it has been speculated that the rise in OPG during murine pregnancy could relate to increased E2 production (18). However, little is known about changes in OPG in human pregnancy. Longitudinal studies of skeletal metabolism during human pregnancy are complicated by the lack of preconception sampling or by the use of volunteers receiving therapy for assisted conception. Uemura et al. (20) measured serum OPG in human pregnancy and reported no significant longitudinal change, although OPG increased significantly during labor. However, there was no baseline assessment before conception in this study. The results may also reflect the performance of the particular OPG assay used. As OPG is present in placental tissue (5, 21), the increase in OPG during labor may simply reflect placental separation. The well-described increase in bone resorption during pregnancy was also not observed in this study (20), although the marker of bone resorption used [serum C-terminal telopeptide of type I collagen (sCTX)-matrix metalloprotease] is an imperfect indicator of mature bone collagen resorption (22).

The aims of our study were 1) to evaluate the change in serum OPG in a longitudinal study of planned human pregnancy and lactation using three different OPG assays, 2) to evaluate the change in RANKL during pregnancy, 3) to examine the relationship between the change in OPG and the change in bone resorption and bone mineral content as a consequence of pregnancy, and 4) to evaluate the potential tissue source of OPG during pregnancy and lactation.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Participants and sample collection

The study design for the change in bone turnover markers and bone density has been described previously (12). Women planning a pregnancy (n = 35) were recruited by advertisement. Exclusion criteria included assisted conception or any disease or use of medication known to affect bone metabolism. Seventeen women became pregnant and completed the study (mean age, 29 yr; range, 20–36 yr; all Caucasian). Baseline samples were collected at 6-month intervals before conception. The interval between baseline sampling and conception was 3.0 ± 0.6 (mean ± SEM) months. None was breast-feeding at the time of entry into the study, and 10 were primigravida. All were singleton pregnancies and delivered full-term (mean gestation, 40 wk; range, 36–42).

Serum and 24-h urine samples were collected before pregnancy (mean ± SEM, -12 ± 2.8 wk); at 16 (16 ± 0.3), 26 (26 ± 0.3), and 36 (36 ± 0.3) wk gestation; and at 2 (1.9 ± 0.3) and 12 (12.7 ± 0.3) wk postpartum. At 2 wk postpartum, 15 women were breast-feeding, and at 12 wk postpartum 9 were breast-feeding. Samples were also collected from 36 additional premenopausal control women who were not pregnant and not lactating (mean age, 31 yr; range, 20–39 yr). Urinary data are expressed as 24-h output, as creatinine output may be variable during pregnancy. All blood samples were collected between 0900 and 0930 h after an overnight fast. Samples were left to clot for 30 min before centrifugation at 2000 x g for 10 min and were stored at -80 C until assay. Samples from individual subjects were measured in one analytical batch.

Blood samples were also collected from 22 premature neonates [mean age, 12 d; mean gestational age, 32 ± 2 wk (±SD)] and 9 umbilical cords (paired arterial and venous samples). Samples of human breast milk (n = 5; collected within 7 d postpartum, mid/hind collection) were collected for determination of OPG.

Study protocols conformed to the Revised Helsinki Declaration of 1983 and were approved by the North Sheffield local research ethics committee. All patients gave written informed consent.

Biochemical measurements

Serum OPG was measured using three different assays: an in-house paired antibody immunometric ELISA and two commercially available assays [OPG ELISA, Biomedica Gruppe, Vienna, Austria; coefficient of variation (CV), <10%, LOD, 0.14 pmol/liter; OPG ELISA, Biovendor Laboratory Medicine, Inc., Palackeho, Czech Republic; CV, 4%; LOD, 0.5 U/liter]. The in-house assay is an immunometric ELISA (mouse monoclonal capture antibody MAB8051, secondary goat polyclonal antibody BAF805, R&D Systems, Minneapolis, MN) with recombinant human OPG as standard (R&D Systems). Incubation times were adjusted to optimize sensitivity and minimize matrix effects. Irradiated microtiter plates were coated overnight with capture antibody and blocked with 1% BSA. Serum was diluted 1:5 with assay buffer and incubated for 2 h. Detection was with biotinylated second antibody, streptavidin-horseradish peroxidase, and tetramethylbenzidine. Recovery of recombinant OPG from serum was between 100.1 and 109.6% (mean ± SEM, 105.4 ± 1.9; n = 5). Within-batch imprecision based on replicate analysis was 3.4% (CV) between 100 and 700 pg/ml (3.5%, between 100 and 200 pg/ml). Between-batch imprecision was less than 10% at 98 and 255 pg/ml. The detection limit was 7 pg/ml based on replicate assay of zero standard, and the assay was linear on serial dilution. For measurement of OPG in breast milk, the milk was diluted 200-fold to obtain data within the analytical range.

Serum RANKL was measured by ELISA (Biomedica Gruppe; intraassay CV, 7%). Baseline RANKL was undetectable in seven of the samples from the pregnancy study, and a value equal to the detection limit of the assay (0.4 pmol/liter) was assigned to these samples. Serum total E2 was measured using an immunochemiluminescent assay (Roche Elecsys, Roche, Mannheim, Germany; detection limit, 5 pg/ml; CV, <3.5% over the premenopausal reference interval).

The serum ß form of the C-terminal telopeptide of type I collagen (sßCTX), a marker of bone collagen degradation (22, 23), was measured by immunoassay (Roche; CV, 10.6% at concentrations <0.1 ng/ml and <4.0% at higher concentrations). Urinary N-terminal telopeptide of type I collagen (uNTX) was measured by ELISA (Osteomark, Ostex International, Seattle, WA; CV, 5%). Serum bone-specific alkaline phosphatase was measured using a wheat-germ lectin precipitation assay (Sigma-Aldrich Corp., Poole, UK; Roche; CV, 6%) that does not cross-react with placental alkaline phosphatase (24).

Bone density measurements

Total body bone mineral density (BMD) was measured before pregnancy and at 2 wk postpartum by dual energy x-ray absorptiometry (Lunar DPX, software version 3.6, Lunar Corp., Madison, WI). The total body scan was divided into separate regions of interest (upper and lower limbs, and spine) to determine changes in predominantly trabecular or cortical bone. Total body BMD was analyzed after exclusion of the head region (CV, 0.9%). The precision of regional measurements (CV) in healthy volunteers were: arms, 3.3%; legs, 3.6%; and spine, 1.4%.

Statistical analysis

Longitudinal changes were tested using repeated measures ANOVA with time as the within-subject factor. Data were log-transformed before analysis. The significance of the main effect of measurement time was further analyzed by single degree of freedom contrasts to compare the measurement at each time with preconception values using the Bonferroni adjustment for multiple comparisons. The significance of any postpartum change was determined by performing contrasts relative to values at 36 wk gestation. At the spine (measurement CV, 3.6%) 11 subjects would be required to detect a change of 4.5% (the observed change) at P < 0.05 and 80% power. Other comparisons between groups were performed using t tests or ANOVA with post hoc Scheffé comparisons as appropriate. Statistical analyses were performed using Statgraphics Plus version 4 (Manugistics, Inc., Rockville, MD) and the Statistical Package for Social Sciences version 10.0 (SPSS, Inc., Chicago, IL). All tests were two-sided, and P < 0.05 was regarded as significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Changes in all analytes during pregnancy and the postpartum period are shown in Fig. 1Go and Table 1Go. Serum OPG increased during pregnancy, with maximum values in the third trimester (Fig. 1Go). At 26 wk gestation OPG was increased by 51 ± 13% (mean ± SEM) compared with preconception values (P < 0.01), and at 36 wk OPG had increased by 110 ± 16% compared with preconception values (P < 0.001). Serum OPG decreased rapidly and significantly postpartum (-31 ± 5% by 2 wk postpartum; P < 0.001). The magnitude of decline in OPG from 36 wk gestation to 12 wk postpartum was significantly slower in breast-feeding women (n = 9; -27 ± 7%) compared with that in women who were not breast-feeding at 12 wk postpartum (n = 8; -50 ± 5% decline; P = 0.017). However, at 12 wk postpartum serum OPG was not significantly different from preconception values in either breast-feeding or non-breast-feeding women.



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  		FIG. 1. Time course of change in OPG (A; antibodies MAB8051 and BAF805) and sßCTX (B) during pregnancy, 2 wk postpartum (pp2), and 12 wk postpartum (pp12). The mean ± SE are shown together with the results for individual participants. Overall significance of time by repeated measures ANOVA, P < 0.0001 for both analytes. Asterisks indicate post hoc contrast comparisons with the preconception value after Bonferroni correction for multiple comparisons: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 

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  		TABLE 1. Time course of change in one OPG assay (antibodies MAB8051 and BAF805), E2, and bone turnover markers before conception, during pregnancy, 2 wk postpartum (pp2), and 12 wk postpartum (pp12)

 
The increase in serum OPG at 36 wk of pregnancy was similar using the three different OPG assays (Table 2Go), although the Biovendor assay showed a greater relative increase than the Biomedica ELISA (by ANOVA, P = 0.02; by Scheffé post hoc test, P < 0.5). The correlations among the three OPG assays at 36 wk were: in-house vs. Biovendor: r = 0.69, P = 0.004; in-house vs. Biomedica: r = 0.49, P = 0.05; and Biomedica vs. Biovendor: r = 0.88, P < 0.00001. Serum RANKL was lower in late pregnancy (mean, 0.77 ± 0.08 pmol/liter at wk 36 compared with baseline, 1.03 ± 0.14 pmol/liter; P = 0.03, by paired t test).


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  		TABLE 2. Concentration of OPG before conception and percentage increase at 36 wk gestation

 
Serum OPG in neonates was significantly lower than that in premenopausal nonpregnant nonlactating women and pregnant women (Fig. 2Go). OPG in venous umbilical cord blood was significantly higher than that in premenopausal serum (n = 9; P < 0.001), but was not significantly different from maternal serum at 36 wk gestation (P > 0.05). There was also no significant difference between paired arterial and venous umbilical cord OPG (n = 6; by paired t test). Human breast milk contained very large quantities of OPG, approximately 200 times the concentration in maternal serum (n = 5; 162,000 ± 58,000 pg/ml; in-house assay).



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  		FIG. 2. Serum OPG (antibodies MAB8051 and BAF805) in neonates, premenopausal nonpregnant nonlactating women, and at 36 wk gestation. By ANOVA, P < 0.0001, all groups significantly different from each other (post hoc Scheffé test, P < 0.05). Enclosed boxes indicate the interquartile range of the data, and horizontal lines indicate the median. The whiskers indicate the range between highest and lowest values.

 
Serum E2 was increased approximately 150-fold throughout pregnancy and declined to below preconception values by 2 wk postpartum (Table 1Go). Serum ßCTX increased by 76 ± 17% at 36 wk compared with preconception values (Fig. 1Go; P < 0.001). uNTX was significantly elevated by 16 wk of pregnancy (Table 1Go; increase, 48 ± 13%; P < 0.01) and was maximal at 36 wk (219 ± 41%; P < 0.001), followed by a postpartum decline. Bone-specific alkaline phosphatase increased during pregnancy (106 ± 18% increase at 36 wk; P < 0.001) and remained elevated by 12 wk postpartum (58 ± 15% increase; P < 0.001).

There was no significant change in total body BMD postpartum (1.051 ± 0.018 g/cm2) compared with preconception (1.046 ± 0.018 g/cm2). Bone density of the spine decreased by 4.5 ± 1.5% between the preconception and 2 wk postpartum measurement (P < 0.01). In contrast, BMD of the arms and legs (predominantly cortical bone) increased by 2.8 ± 1.0% and 1.8 ± 0.5% between the preconception and the 2 wk postpartum measurement, respectively (both P < 0.01). Correlations between change in OPG at 36 wk and change in E2, bone turnover, and BMD at 2 wk postpartum are shown in Table 3Go. There was no relationship between change in OPG at 36 wk and change in any other analyte or BMD at any regional site. There was, however, a significant negative correlation between the change in serum ßCTX and the pregnancy-related decrease in BMD at the spine. There was no correlation between the change in E2 and either OPG or RANKL. There were also no significant relationships between change in BMD at any skeletal site and absolute change in OPG (data not shown).


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  		TABLE 3. Correlations between percentage change from preconception values at 36 wk gestation for OPG (MAB8051 and BAF805), E2 markers of bone turnover, and bone density (BMD 2 wk postpartum)

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
We observed a significant increase in OPG during pregnancy, with a rapid decline toward to preconception values postpartum. This is consistent with previous observations in a murine study (18). One previous study in human pregnancy did demonstrate an increase in maternal serum OPG during labor, but no significant increase during earlier pregnancy (20). However, preconception measurements were not available in this study. We also observed an increase in both bone formation and bone resorption during pregnancy, consistent with previous studies (13, 25).

The relative contributions of different tissues to the increase in maternal serum OPG during pregnancy is uncertain. Osteoblastic expression of OPG is increased by estradiol in vitro (19). However, although we observed a marked increase in serum E2 during pregnancy, this did not correlate with change in OPG. The tissue source of serum OPG in nonpregnant individuals is also not known. OPG is expressed in several tissues apart from bone (5), and administration of E2 or bisphosphonates to humans does not result in a significant decline in serum OPG despite a change in bone turnover (26). This suggests a substantial nonskeletal contribution to circulating OPG.

It is possible that the increase in OPG during pregnancy is of placental origin. OPG is known to be expressed in placental tissue (5, 21). In addition, OPG binds to the antiapoptotic cytokine TNF-related apoptosis-inducing ligand, which may be involved in the maintenance of placental integrity (27, 28). The rapid postpartum decline in maternal OPG is consistent with a placental source. OPG is also likely to exist in different forms in serum, including both monomers and dimers (18, 29, 30), and the relative cross-reactivity of available immunoassays to these components is unknown. However, we observed a similar pregnancy-related increase in OPG using three different assays.

OPG in umbilical cord serum was not markedly elevated compared with that in maternal serum, and there was no arteriovenous discrepancy. This suggests that the fetus is not a source of OPG in the maternal circulation. However, it is not inconsistent with a placental source; syncytiotrophoblast cells are polarized cells, which are in direct contact with maternal blood. The placenta is generally impermeable to proteins, and this allows independent control of fetal and maternal plasma protein concentrations. Structural features of the placenta explain why the majority of secreted placental proteins enter the maternal, but not the fetal, compartment. Numerous placental-derived proteins, such as placental alkaline phosphatase and human chorionic gonadotropin, produced by the trophoblast are present in the maternal circulation, but not in the fetal circulation (31). Neonatal serum OPG (n = 22) was also lower than that observed in maternal serum. These data suggest that a fetal contribution to maternal OPG is unlikely.

The breast is also a potential source of maternal serum OPG. The RANK-RANKL signaling pathway appears to be involved in the development of lactating mammary tissue (6, 29). We have also found that breast milk contains a substantial amount of OPG. The presence of OPG in human breast milk has previously been demonstrated in a preliminary study using a qualitative assay (32). However, the rapid postpartum decline in maternal OPG toward preconception values in both breast-feeding and non-breast-feeding women suggests that the breast is not the primary contributor to maternal serum OPG during pregnancy. However, the significantly slower decline in breast-feeding women suggests that serum OPG might be in some part breast derived.

The physiological relevance of OPG during pregnancy is unknown. It has been suggested that the increase in serum OPG in a murine model may occur in response to increased bone turnover, and that OPG acts as a protective factor, preventing excessive bone loss during pregnancy (18). However, we observed no significant correlation between change in bone turnover or BMD and change in OPG during pregnancy. Bone turnover remained elevated at 12 wk postpartum, whereas OPG returned to near baseline values at 12 wk postpartum. Although we have explored the possible role of serum OPG in regulating bone metabolism during pregnancy, serum OPG may not reflect OPG activity in the bone microenvironment. Rogers et al. (33) reported a weak, but significant, positive relationship between OPG and both BMD and E2 in postmenopausal women. Szulc et al. (34) found a negative correlation between serum OPG and urinary deoxypyridinoline, a marker of bone resorption, in men over 40 yr of age. However, there was no correlation between OPG and BMD or bone formation markers.

In conclusion, we have demonstrated a progressive increase in serum OPG during human pregnancy, with a decline in serum RANKL. The tissue source of OPG in pregnancy is unknown, but the rapid postpartum decline in serum OPG, the low serum OPG in neonates, and the presence of OPG in placental tissue suggest a placental source. There was no correlation between change in OPG and bone turnover or BMD, and the physiological importance of elevated OPG in human pregnancy remains uncertain.


   Acknowledgments
 
This study could not have been conducted without the help of the patients who devoted time and effort to collect samples before, during, and after pregnancy. We thank laboratory staff, R. Beaumont, N. Wylde, R. Raynor, S. Clow, and the staff of the Osteoporosis Center, Northern General Hospital, Sheffield for help with various aspects of the study. We thank Dr. C. Smith and Mr. P. Iqbal for their contributions to the sample collection for this study.


   Footnotes
 
This work was supported in part by a research grant from WellBeing.

Abbreviations: BMD, Bone mineral density; CV, coefficient of variation; E2, estradiol; OPG, osteoprotegerin; RANK, receptor activator of nuclear factor {kappa}B; RANKL, RANK ligand; sßCTX, serum ß C-terminal telopeptides of type I collagen; uNTX, urinary N-terminal telopeptides of type I collagen.

Received March 19, 2003.

Accepted July 26, 2003.


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 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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