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Placenta (2001), 22, 591–596

doi:10.1053/plac.2001.0688, available online at http://www.idealibrary.com on

Placental Calcification: A Metastatic Process?

S. H. Poggia,d, K. I. Bostromb, L. L. Demerb, H. C. Skinnerc and B. J. Koosa


a
Nicholas S. Assali Perinatal Research Laboratory, Departments of Obstetrics and Gynecology, University of California,
Los Angeles, b Department of Medicine, University of California, Los Angeles and c Department of Geology and Geophysics,
Yale University, New Haven, CT 065113, USA
Paper accepted 7 March 2001

Placental calcification commonly increases with gestational age. The mechanism of apatite mineralization probably involves one of
three known mechanisms of tissue calcification: physiological (like bone), dystrophic (ischaemia-related) or metastatic (mineral-
ization in a supersaturated environment). This study was designed to determine the mechanism of calcification by examining
(1) the mineral content of placental calcifications in comparison to other physiological and pathological apatites, and (2) the
expression of bone morphogenetic proteins (BMPs), which are important in physiological calcification, across gestational age. By
energy-dispersive x-ray analysis (EDXA), the Ca/P weight ratio for apatitic mineral from mature calcifications was 2.000.05
(s.e.), which is similar to that for stones formed in a metastatic, supersaturated environment and lower than that observed in
physiological calcification. Biologically active BMP, which was determined by bioassay, was demonstrated in mature and
postmature placentae. The BMPs PLAB, PDF and related protein INSL-4 were identified by semiquantitative reverse
transcriptase polymerase chain reaction (RT-PCR), but their mRNA expression was independent of gestational age (7–41 weeks
of gestation). We conclude that (1) the identified BMPs were not related directly to placental calcification, which argues against
physiological calcification, and (2) the chemical composition of the apatitic mineral was suggestive of rapid formation in a
supersaturated environment, which is consistent with a metastatic mechanism of calcification.
Placenta (2001), 22, 591–596  2001 Harcourt Publishers Ltd

INTRODUCTION Three processes may lead to tissue calcification (Anderson,


The calcium content of placentas normally increases with 1983). In physiological calcification, as seen in bone and teeth,
gestational age, through a mineralization process that may be osteoblasts produce osteoid matrix, which permits hydroxy-
accelerated by some disorders of pregnancy (Jeacock et al., apatite (HA) formation in and on collagen fibres (Anderson,
1963). For example, calcifications are clearly visible in most 1989; Skinner, 1988). Secondly, dystrophic calcification occurs
post-term (>41 weeks gestation) placentae and are often in tissue necrosis. In this process, injured or dysfunctional
observed in placentas associated with pre-eclampsia and/or cellular membranes allow extracellular calcium to enter cells
fetal growth restriction (Jeacock et al., 1963; Quinlan et al., and combine with phosphate, producing apatitic mineral
1982). The mineral is found in the basal plate of the immature (Anderson, 1983). Finally, metastatic calcification develops in
placenta in association with extracellular fibrinoid (Benirschke an environment that is supersaturated with calcium and
and Kaufman, 2000) and, later in gestation, in the fibrinoid phosphate or oxalate, such as occurs with urolith formation
surrounding the basement membranes of villous endothelial (Anderson, 1983).
cells and in placental septae (Avery and Aterman, 1970; Varma The calcium/phosphate (Ca/P) weight ratio of the mineral
and Kim, 1985). Calcification occurs within extracellular varies according to mechanism of calcification. For example,
matrix vesicles of the placenta (Al-Zuhair et al., 1984; Varma the Ca/P weight ratio is greater in mature bone than in the less
and Kim, 1985), and in that respect the calcification process organized apatites that are associated with metastatic calcifica-
resembles the extracellular deposition associated with bone tion. Thus, the chemical composition of the mineral may help
formation. This provocative finding suggests that placenta and distinguish a physiological mechanism (e.g. bone formation)
bone may involve a similar mechanism of calcification. from a metastatic process.
Physiological and non-physiological calcification may also be
d
distinguished by determining the association of bone morpho-
To whom correspondence should be addressed at: 3PHC, genetic proteins (BMPs) and mineral. The formation and
Department of Obstetrics and Gynecology, Georgetown University
School of Medicine, 3800 Reservoir Road, Washington, DC, 20007. maintenance of bone tissues involves BMPs, a class of growth
Tel.: +1 202 687 8801; Fax: +1 202 687 1757; E-mail: factors that are defined by their ability to induce ectopic bone
poggis@gunsun.georgetown.edu formation via the induction of osteogenic cell differentiation
0143–4004/01/060591+06 $35.00/0  2001 Harcourt Publishers Ltd
592 Placenta (2001), Vol. 22

and the subsequent formation of lamellar bone and marrow


(Urist, 1965). In particular, BMP-2 appears to be essential for
the normal formation of bone and teeth (Riley et al., 1996). In
addition to bone, this physiological process involving BMPs
has been observed in atherosclerotic plaque (Bostrom et al.,
1993).
Several BMPs have been detected in human placentae,
including prostate-derived factor (PDF) (Hromas et al., 1997)
and placenta BMP (PLAB) (Paralkar et al., 1998). Insulin-like
growth factor (INSL-4), an inductive protein that is important
in bone and cartilage formation, has been also been detected in
immature placentae (Laurent et al., 1998). These proteins have
been implicated in embryogenesis, but they may also be
involved in other processes, including placental calcification. If
these BMPs are involved in placental calcification, their
expression might be expected to increase with gestational age
as the placentae become more calcified.
This study was designed to determine (1) the mineral
content of placental calcifications, and (2) the expression of
bone morphogenetic proteins across gestational age. The
results provide information on whether placental calcification
is formed passively in a metastatic or dystrophic environment Figure 1. Scanning photomicrograph showing calcified villi from a post-date
or actively propagated like the physiological process involved placenta. The apatitic mineral (white material) consists of a porous aggregate
in the formation of bone, teeth or atherosclerotic plaque. of tiny (1–5 ) lamellae (magnification bar=200 m).

left and right vastus lateralis muscle of two nude mice and in
METHODS
the left and right rectus abdominus muscle of three Sprague-
The following procedures were approved by the UCLA Dawley rats. Empty capsules were placed in two animals (one
Medical Institutional Board and the Chancellor’s Animal mouse and one rat) as a control. The animals were euthanized
Research Committee. when radiographs showed radioopacities consistent with calci-
fication. The tissue was fixed with 4 per cent paraformalde-
hyde and stained with haematoxylin and eosin. Histologic
examination was performed using light microscopy (100 and
Chemical analysis of placental mineral
400 magnification). A pathologist, who was blinded to the
Five heavily calcified term (37–41 weeks gestation) and post- origin of the material, examined the slides for the presence
dates placentas were fixed in 10 per cent zinc formalin solution, of bone.
dehydrated using serial ethanol solutions, cleared with xylene,
and infiltrated with paraffin. Sections (6 ) were mounted on
Thermanox coverslips (Nunc, Naperville, IL, USA) to avoid RT-PCR
any chemical artifacts, especially silica (Poggi et al., 1998). The
samples on the coverslips were treated with xylene to extract Placentae from 7–42 weeks’ gestation were snap-frozen
paraffin. After carbon-coating, the energy dispersive JEOL (80C) immediately after delivery. Total ribonucleic acid
JXA-8600 electron microprobe was used to make the chemical (RNA) was extracted using an RNA isolation kit (Statagene,
analyses using settings that have been previously described La Jolla, CA, USA). Reverse transcription of 3 mg total RNA
(Poggi et al., 1998). Five to 10 replicate analyses were per- was carried out for 90 min at 37C in 50 T buffer, supple-
formed on each of five placentae. The mineral weight ratios are mented with 0.5  of each d NTP (Pharmacia, Piscataway, NJ,
expressed as meanss.e. USA), 50–80 units RNase Block (Stratagene), and 50 U of
M-MuLV reverse transcriptase (Stratagene).
Polymerase chain reaction (PCR) was performed using
Detection of BMP activity in vivo specific primers for BMP-2, BMP-4, PLAB, PDF, and
INSL-4 (Gibco, Grand Island, NY, USA) in a volume of 10 ,
BMP was isolated from heavily calcified term and postdate using Pfu buffer, 7 U of native Pfu polymerase (Stratagene),
placentas using guanidine hydrochloride extraction followed 0.1 l [P32]--dCTP (Amersham), 25 ng of each primer, and
by lyophilization (Kawamura and Urist, 1988). Gelatin cap- 1 l of template (from a 50-l RT reaction). Thermal cycling
sules containing the isolate (25–100 mg) were implanted in the was performed as follows: (1) initial denaturation at 96C for
Poggi et al.: Placental Calcification 593

Figure 2. Photomicrograph of a section of rat rectus muscle 42 days after implantation of placental extract. Osteoid, lacunae, osteoblasts (OB) and osteoclasts
(OC), which are consistent with immature bone, are visible next to normal muscle (M) (magnification bar=10 m).

one min, (2) cycling for cDNA-specific number of cycles placentae. The mineral, which was very porous in appearance
between 96C for 1 min, at 65C for 2 min and at 72C for (Figure 1), consisted of small (1–5  in diameter) lamellae that
1 min, (3) final extension at 72C for 5 min. For semiquanti- formed larger mineral composites that ranged to over 200  in
tative PCR, multiple PCR cycles were performed and the diameter. In five term and post-term placentae, the Ca/P
number of cycles was chosen so that amplification remained weight ratio was 2.000.05. Mg +2, Na + , K + , and Cl  were
well within the linear range, as assessed by densitometry (NIH present, but in very low concentrations (0.140.04,
Image J, version 1.08I, public domain program, http:// 0.460.17, 0.040.01, 0.030.01 weight per cents,
rsb.info.nih.gov/nih-image). An equal volume from each PCR respectively).
reaction was analyzed by 6 per cent non-denaturing poly- The presence of biologically active BMP in placental tissue
acrylamide gel electrophoresis and dried gels were examined was determined by bioassay that involved placing encapsulated
using autoradiography. GuHCl-extracted placental fragments in the musculature of
RT-PCR trends were confirmed by Northern analysis, rodents. Subsequent microscopic examination of the tissue
which was performed as generally described by Tintut et al. (35–42 days after implantation) surrounding the site of implan-
(1998). Ten g of total RNA from placental tissues was used tation showed osteoid, lacunae, osteoblasts and osteoclasts
for analysis that involved the use of the 5 -ended-labelled PCR (Figure 2). These findings, which are consistent with early
primers (non-coding sequences) as probes. Hybridization was bone formation, indicate that biologically active BMPs are
performed overnight at 42C, and the membranes were washed present in term and postdate placentas. Implantation of gelatin
twice at RT for 10 min with 5SSPE and once at 37C capsules alone failed to induce calcification.
for 10 min with 5SSPE with 0.1 per cent SDS before RT-PCR determined whether the biologically-active BMPs
autoradiography. corresponded to the expression of previously described placen-
tal BMPs. The results show that PDF and PLAB mRNA is
expressed in all placentae analysed from 7 to 41 weeks of
RESULTS gestation. The expression of these BMPs appeared to be
Energy dispersive spectroscopic analysis detected calcium and relatively constant over this broad range of gestational ages
phosphate as the only significant elements in the mineral of the (Figure 3). Genetic expression of INSL-4 tended to increase
594 Placenta (2001), Vol. 22

with gestational age. BMP-2 and the closely related BMP-4


were not detected in embryonic, preterm, term or post-term
placentas. The relatively constant expression of PLAB and
PDF, as well as the increasing trend in INSL-4, was confirmed
by Northern blot analysis.

DISCUSSION

The Ca/P weight ratio of 2.000.05 observed in human


placental calcification resembles that seen in human stones
(uroliths, sialoliths) that are formed in supersaturated environ-
ments (Table 1). The very rapid crystallization that occurs
under these conditions results in a less organized, porous
apatitic lattice consisting of small lamellar crystals with a lower
Ca/P weight ratio. In contrast, bone and atherosclerotic lesions
typically have Ca/P weight ratios that approximate 2.15, the
value for stoichiometrically ideal hydroxyapatite (Table 1).
The physiological calcification process, in which osteoid
mineralizes about 10 days after its initial secretion, allows the
crystal lattice to be propagated in an organized manner,
allowing formation of mineral in equilibrium (Skinner, 1988).
This finding contrasts with metastatic calcifications, where
very rapid mineralization results in a less organized and less
mature mineral. Less is known about the ‘typical’ calcification
in dystrophic calcifications, which appear to have a Ca/P
weight ratio above 2.15, which suggests that another phase
besides apatite may be present (Poggi et al., 1998). The lower
Ca/P ratios and porous, lamellar aggregates observed support
a non-physiological mechanism of placental calcification that
most closely resembles metastatic calcification.
Bone morphogenetic proteins, which are biologically active
even after extraction with the strong denaturant GuHCl, were
detected in heavily calcified, term placentae using the classic
in-vivo assay. RT-PCR and Northern analysis showed PLAB
and PDF, as well as INSL-4 expression of the previously
described placental BMPs. The relative mRNA expression of
the placental BMPs occurred virtually independent of gesta-
tional age, but BMP-2 and BMP-4, which have been suggested
to be involved in both bone and atherosclerotic plaque calcifi-
cation, were not detected. INSL-4 showed a slight trend
towards increased expression. These results do not support
the hypothesis that the increased placental calcification with
gestational age depended critically on greater expression of
BMPs. Thus, placental calcification does not appear to be a
physiological process.
These results do not exclude the involvement of a dys-
trophic process. Ca/P weight ratios in dystrophic calcification
have been reported only for calcified necrotic breast carcinoma.
Thus, the limited information concerning the mineral com-
Figure 3. Upper panel. Placental expression of PDF, PLAB, and INSL-4 position of dystrophic calcifications prevents firm comparative
after indicated number of weeks. Total RNA and cDNA was prepared and conclusions.
PCR was performed using specific primers for each growth factor. GAPDH is The results must be interpreted with caution for other
shown for comparison. Lower panel. Relative signal intensity of the three
growth factors after normalization to GAPDH. The results are expressed as reasons. For example, non-identified BMPs may be present
fold increases of expression. The signal corresponding to 7 weeks was in each in placenta which promote calcification. In addition, BMP
case regarded as one-fold. inhibitors which have been previously described for other
Poggi et al.: Placental Calcification 595

Table 1. Calcium/Phosphate (Ca/P) weight ratios associated with tissue calcification

Tissue Mature Bonea Arteryb Necrotic Tumourc Urolithd Placenta


Ca/P ratio 2.15 2.16 2.440.08 1.980.04 2.000.05
Mechanism Physiological Physiological Dystrophic Metastatic Metastatic?

a
Skinner, 1988; b Schmid et al., 1980; c Poggi et al., 1998; d Kodaka et al., 1994.

BMPs (e.g., chordin, noggin and Gremlin) may be present observed in delayed appearance of ossification sites (Zilanti
(Piccolo et al., 1996; Brunet et al., 1998; Hsu et al., 1998). et al., 1987). In such fetuses, calcium, which would otherwise
Altered expression of similar inhibitors to PLAB or PDF be used for bone formation, would likely pass back to the
would alter their biological activity, but would be removed by maternal circulation via passive placental diffusion. Such a
the strong denaturant used prior to the in-vivo assay. Thus, blunting of net calcium flow to the fetus may create the
the results of the in-vivo assay may not reflect the true calcium-enriched environment that promotes the precipitation
biological activity of placental BMPs. of calcium and phosphate to form highly insoluble apatite in
Calcium is actively transported across the placenta through- the ‘placental sink’. An alteration in fetal calcium utilization
out gestation, making the fetus relatively hypercalcemic may also contribute to calcium deposition in mature placentae,
(Husain and Mughal, 1992; Pitkin, 1985). This action, which although it does not exclude other possibilities.
supports the growing fetal skeleton, is mediated primarily by In summary, the mechanism of placental calcification
fetal parathyroid related protein, which increases greatly appears to be most consistent with a process that occurs in an
through pregnancy, rather than maternal parathyroid hormone environment that is supersaturated with calcium and phos-
(Hosking, 1996). Calcium binding proteins are thought to phate. A physiological, bone-like mechanism and a dystrophic
buffer the process, preventing disruption of intracellular pro- process appear less likely to be involved.
cesses (Tuan, 1985). Growth-restricted fetuses often have
abnormalities in mineralization of bone, which may be

ACKNOWLEDGEMENTS

We wish to thank Dr Alan DeCherney for his generous support and Dr Scott Nelson for his review of the histology. This work was supported in part by National
Institutes of Health Grants HD-18478 and HL-30568.

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