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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
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
DISCUSSION
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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