Inorganic Phosphate in the Pathogenesis of Pregnancy-Related Complications

Inorganic phosphate (Pi) is an essential nutrient that fulfills critical roles in human health. It enables skeletal ossification, supports cellular structure and organelle function, and serves key biochemical roles in energetics and molecular signaling. Pi homeostasis is modulated through diet, intestinal uptake, renal reabsorption, and mobilization of stores in bone and extracellular compartments. Disrupted Pi homeostasis is associated with phosphate wasting, mineral and bone disorders, and vascular calcification. Mechanisms of Pi homeostasis in pregnancy remain incompletely understood. The study presented herein examined biological fluid Pi characteristics over the course of gestation. Correlations with gestation age, pregnancy number, preterm birth, preeclampsia, diabetes mellitus, and placental calcification were evaluated during the last trimester. The results support that maternal urinary Pi levels increased during the third trimester of pregnancy. Reduced levels were observed with previous pregnancy. Amniotic fluid Pi levels decreased with gestation while low second trimester levels associated with preterm birth. No significant difference in urinary Pi levels was observed between preeclampsia and controls (8.50 ± 2.74 vs. 11.52 ± 2.90 mmol/L). Moreover, increased maternal urinary Pi was associated with preexisting diabetes mellitus in preeclampsia. Potential confounding factors in this study are maternal age at delivery and body mass index (BMI)—information which we do not have access to for this cohort. In conclusion, Pi levels provide clinical information regarding the pathogenesis of pregnancy-related complications, supporting that phosphate should be examined more closely and in larger populations.


Introduction
Phosphorus is an essential micronutrient that serves as key biochemical roles in cellular structure, organelle function, energetics and molecular signaling, and skeletal ossification. Phosphorus is a primary component of hydroxyapatite crystals, which are minerals that reinforce bone strength [1,2]. Elemental phosphorus circulates in the blood in the form of phosphoric acid (PO 4 ), also termed inorganic phosphate or phosphate (P i ). P i homeostasis is modulated through diet, intestinal uptake, renal reabsorption, and mobilization of stores in bone and extracellular compartments. Disrupted P i homeostasis is associated with P i wasting, refeeding syndrome, chronic kidney disease (CKD) mineral and bone mineralization disorders (BMD), and medial vascular calcification. P i transport into cells (symport) occurs against a 100-fold concentration gradient via transmembrane phosphate transporter proteins, such as the type III sodium-dependent phosphate transporters Slc20a1 (PiT-1) and Slc20a2 (PiT-2). In early preeclampsia (PE), placental expression of the sodium-dependent phosphate transporters Slc20a1 and Slc20a2 is highly reduced; yet in late PE, Slc20a2 is significantly

OHSU Amniotic Fluid Samples
De-identified amniotic fluid samples were obtained from OHSU (MTA-OUT16-095). A total of 11 s trimester and 9 third trimester samples were obtained. Gestation age of delivery (GAD) ranged between 25 and 39 weeks on average. Gestation age of sample collection (GASC) ranged between 17 and 35 weeks on average. We tested whether amniotic fluid phosphate levels were an indicator for preterm or early pre-term birth for second and third trimester of pregnancy.

P i Quantification
Maternal urine P i levels were determined with the Phosphate Assay Kit (Sigma Aldrich, St. Louis, MO, USA; MAK308) and amniotic fluid P i levels were determined with the QuantiChrom Phosphate Assay Kit (Bioassays Systems, Hayward, CA, USA; DIPI500) according to the manufacturer's instructions.

Methods for Von Kossa Staining and Imaging of Placenta Tissue
The von Kossa staining was performed as previously described in Speer et al. 2009 [31] with the inclusion of an optimally reduced 22 min von Kossa treatment. Placental tissue section from 8 normotensive pregnant women and 8 preeclamptic women were stained, and images of mounted sections were acquired on a Keyence BZ-X800 microscope using the Keyence BZ-X software (Keyence, Ozaka, Japan) at 4× magnification. File names were coded. Individual calcified lesions were imaged on a Nikon E800 Upright Microscope (Nikon Corp., Tokyo, Japan) at 2.5 and 10× magnification and file names were coded. Semi-quantitative analysis of calcified lesions was performed on all tissue sections using ImageJ/Fiji (NIH, Bethesda, MD, USA) histomorphometry software.

Statistical Analysis of Experimental Data
The following statistical tests were used to analyze quantitative data in GraphPad Prism 6 software for Windows (GraphPad Software Inc., La Jolla, CA, USA). For comparison of two groups, a p-value was determined by a two-tailed Student's T-test with unequal variance. For comparison of three or more groups, a p-value was determined by a Welch's T-test with unequal variance. Slope calculations were obtained by Deming (model II) linear regression.

Figure 1.
Clinical characteristics of cohort and association with preeclampsia. Clinical characteristics of cohort (control, n = 9; preeclampsia, n = 7). We observed that gestational age at delivery (GAD) was increased in the Preeclampsia group (A). We did not observe differences on the preexistence of pregnancies (B) and diabetes incidence (C) in both Control and Preeclampsia groups. Symbols: • Control; ▪ Preeclampsia; * p≤0.05. Abbreviations: GAD: Gestational Age at Delivery; NGT: normal glucose tolerance.

Increased Maternal Urine Pi Levels are Associated with Preeclampsia
Decreased levels of Pi have been described in the maternal urine of preeclampsia patients [32]. We quantified Pi excretion in maternal urine from normotensive and preeclampsia groups. The average Pi maternal urine level was higher in the preeclampsia group (8.50 ± 2.74 vs. 11.52 ± 2.90 mmol/L; p = 0.4653), but this difference was not statistically significant ( Figure 2A). Ectopic vascular calcification has been previously associated with preeclampsia [22,33]. von Kossa staining was used to detect calcified lesions on placental tissue slides of the preeclampsia group studied ( Figure 2B-E). The average % Area Calcified was higher in the preeclampsia group (0.0028 ± 0.0014 vs. 0.0048 ± 0.0034; p = 0.6117), but this difference was not statistically significant ( Figure 2B). The distribution of the crystal deposits (black deposits) suggested a formation of calciumphosphate deposits in chorionic villi ( Figure 2D-E). Clinical characteristics of cohort and association with preeclampsia. Clinical characteristics of cohort (control, n = 9; preeclampsia, n = 7). We observed that gestational age at delivery (GAD) was increased in the Preeclampsia group (A). We did not observe differences on the preexistence of pregnancies (B) and diabetes incidence (C) in both Control and Preeclampsia groups. Symbols: • Control;

Clinical Characteristics
Participant characteristics are shown in Figure 1. The gestational ages at delivery (GAD) is statistically significantly higher in the preeclampsia group compared to the normotensive group (32.08 ± 0.27 and 30.68 ± 0.49 weeks; p = 0.0375; Figure 1A). A statistically significant association was not observed between preexisting pregnancies and normotensive or preeclampsia groups (0.56 ± 0.18 and 0.43 ± 0.20; p = 0.6420; Figure 1B), or preexisting diabetes mellitus (100% ± 40% and 129% ± 45%; p = 0.6420; Figure 1C). Figure 1. Clinical characteristics of cohort and association with preeclampsia. Clinical characteristics of cohort (control, n = 9; preeclampsia, n = 7). We observed that gestational age at delivery (GAD) was increased in the Preeclampsia group (A). We did not observe differences on the preexistence of pregnancies (B) and diabetes incidence (C) in both Control and Preeclampsia groups. Symbols: • Control; ▪ Preeclampsia; * p≤0.05. Abbreviations: GAD: Gestational Age at Delivery; NGT: normal glucose tolerance.

Increased Maternal Urine Pi Levels are Associated with Preeclampsia
Decreased levels of Pi have been described in the maternal urine of preeclampsia patients [32]. We quantified Pi excretion in maternal urine from normotensive and preeclampsia groups. The average Pi maternal urine level was higher in the preeclampsia group (8.50 ± 2.74 vs. 11.52 ± 2.90 mmol/L; p = 0.4653), but this difference was not statistically significant ( Figure 2A). Ectopic vascular calcification has been previously associated with preeclampsia [22,33]. von Kossa staining was used to detect calcified lesions on placental tissue slides of the preeclampsia group studied ( Figure 2B-E). The average % Area Calcified was higher in the preeclampsia group (0.0028 ± 0.0014 vs. 0.0048 ± 0.0034; p = 0.6117), but this difference was not statistically significant ( Figure 2B). The distribution of the crystal deposits (black deposits) suggested a formation of calcium-phosphate deposits in chorionic villi ( Figure 2D-E).

Increased Maternal Urine P i Levels are Associated with Preeclampsia
Decreased levels of P i have been described in the maternal urine of preeclampsia patients [32]. We quantified P i excretion in maternal urine from normotensive and preeclampsia groups. The average P i maternal urine level was higher in the preeclampsia group (8.50 ± 2.74 vs. 11.52 ± 2.90 mmol/L; p = 0.4653), but this difference was not statistically significant ( Figure 2A). Ectopic vascular calcification has been previously associated with preeclampsia [22,33]. von Kossa staining was used to detect calcified lesions on placental tissue slides of the preeclampsia group studied ( Figure 2B-E). The average % Area Calcified was higher in the preeclampsia group (0.0028 ± 0.0014 vs. 0.0048 ± 0.0034; p = 0.6117), but this difference was not statistically significant ( Figure 2B). The distribution of the crystal deposits (black deposits) suggested a formation of calcium-phosphate deposits in chorionic villi ( Figure 2D-E).

Maternal Urine P i Levels Increase during Gestational Age
Maternal urine P i levels were determined to assess P i variations over the course of human gestation. We observed that P i excretion increased between weeks 28-34 during third trimester of pregnancy in all cohorts ( Figure 3A), and in both control and preeclampsia groups ( Figure 3B).

Figure 2.
Maternal urine inorganic phosphate (Pi) levels association with preeclampsia and placental calcification. Phosphate levels were determined for maternal urine (control, n = 9; preeclampsia, n = 7), and we observed a trend of increased phosphate levels with preeclampsia (A). urine (control, n = 9; preeclampsia, n = 7), and we observed a trend of increased phosphate levels with preeclampsia (A). Placental calcification (% Area Calcified) was determined for placental tissue sections (control, n = 8; preeclampsia, n = 8), and we observed a trend of increased placental calcification with preeclampsia (B). Images of von Kossa staining of a placental tissue section of preeclampsia donor identify negative (C) and positive regions (D, E) (black deposits; C and D magnification ×2.5, E magnification ×10). Box in (D) correspond to region imaged in (E). The distribution of the crystal deposits suggested a formation of calciumphosphate mineral deposition at chorionic villi. Symbols: • Control; ▪ Preeclampsia; * p≤0.05.

Maternal Urine Pi Levels Increase during Gestational Age
Maternal urine Pi levels were determined to assess Pi variations over the course of human gestation. We observed that Pi excretion increased between weeks 28-34 during third trimester of pregnancy in all cohorts ( Figure 3A), and in both control and preeclampsia groups ( Figure 3B). Maternal urine inorganic phosphate (Pi) levels association with gestational age at delivery (GAD). Phosphate levels were determined for maternal urine (control, n = 9; preeclampsia, n = 7). We observed that phosphate levels increase with time during third trimester of pregnancy, in all cohort

Increased Maternal Urine Pi Levels are Associated with Pre
Decreased levels of Pi have been described in the ma We quantified Pi excretion in maternal urine from norm average Pi maternal urine level was higher in the preecla mmol/L; p = 0.4653), but this difference was not statisticall calcification has been previously associated with preeclam to detect calcified lesions on placental tissue slides of the p The average % Area Calcified was higher in the preeclam 0.0034; p = 0.6117), but this difference was not statistically the crystal deposits (black deposits) suggested a form chorionic villi ( Figure 2D-E).

Maternal Urine Pi Levels Increase during Gestational Age
Maternal urine Pi levels were determined to assess Pi variations over the course of human gestation. We observed that Pi excretion increased between weeks 28-34 during third trimester of pregnancy in all cohorts ( Figure 3A), and in both control and preeclampsia groups ( Figure 3B). Maternal urine inorganic phosphate (Pi) levels association with gestational age at delivery (GAD). Phosphate levels were determined for maternal urine (control, n = 9; preeclampsia, n = 7). We observed that phosphate levels increase with time during third trimester of pregnancy, in all cohort

Maternal Urine P i Levels Altered with Number of Pregnancies and with Diabetes and Preeclampsia
We observed that P i levels decreased significantly on maternal urine with increased number of pregnancies (14.92 ± 2.57 vs. 4.72 ± 1.59 mmol/L; p = 0.0045; Figure 4A). This trend of decreased P i excretion in a second pregnancy for both normotensive and preeclampsia groups ( Figure 4B), points to a putative decrease in bioavailability of phosphate with a second pregnancy. Furthermore, we observe a trend of increased average % Area Calcified in second pregnancies for both normotensive and preeclampsia groups (0.0007 ± 0.0004 vs. 0.0069 ± 0.0033; p = 0.0805; Figure 4C). Association of maternal urinary P i levels with preexisting diabetes mellitus (100% ± 35% vs. 141% ± 34% mmol/L; p = 0.4180; Figure 4D) and preeclampsia ( Figure 4E) was evaluated, and indicated that diabetes dysregulates Pi excretion during pregnancy, and this is exacerbated with superimposed preeclampsia. In contrast, a modest trend of decreased average % Area Calcified was observed with preexisting diabetes for both normotensive and preeclampsia groups (0.0051 ± 0.0033 vs. 0.0025 ± 0.0016; p = 0.5005; Figure 4F).

Maternal Urine Pi Levels Altered with Number of Pregnancies and with Diabetes and Preeclampsia
We observed that Pi levels decreased significantly on maternal urine with increased number of pregnancies (14.92 ± 2.57 vs. 4.72 ± 1.59 mmol/L; p = 0.0045; Figure 4A). This trend of decreased Pi excretion in a second pregnancy for both normotensive and preeclampsia groups ( Figure 4B), points to a putative decrease in bioavailability of phosphate with a second pregnancy. Furthermore, we observe a trend of increased average % Area Calcified in second pregnancies for both normotensive and preeclampsia groups (0.0007 ± 0.0004 vs. 0.0069 ± 0.0033; p = 0.0805; Figure 4C). Association of maternal urinary Pi levels with preexisting diabetes mellitus (100% ± 35% vs. 141% ± 34% mmol/L; p = 0.4180; Figure 4D) and preeclampsia ( Figure 4E) was evaluated, and indicated that diabetes dysregulates Pi excretion during pregnancy, and this is exacerbated with superimposed preeclampsia. In contrast, a modest trend of decreased average % Area Calcified was observed with preexisting diabetes for both normotensive and preeclampsia groups (0.0051 ± 0.0033 vs. 0.0025 ± 0.0016; p = 0.5005; Figure 4F). . Maternal urine inorganic phosphate (Pi) levels and placental calcification association with preexisting pregnancies, diabetes and preeclampsia. Phosphate levels were determined for maternal urine (control, n = 9; preeclampsia, n = 7). We observed that phosphate levels decrease with preexisting pregnancies in all cohorts (A). We observed a trend of reduced phosphate levels with preexisting pregnancies in both the Control and Preeclampsia groups (B). We observed a trend of increased phosphate levels with preexistence of diabetes in all cohorts (D) and Preeclampsia groups (E). Placental calcification (% Area Calcified) was determined for placental tissue sections (control, n = 8; preeclampsia, n = 8), and we observed a trend of increased placental calcification with preexistence of previous pregnancies in all cohorts (C) and decreased with preexistence of diabetes (F). Symbols: * p≤0.05. Abbreviations: diab: diabetes; NGT: normal glucose tolerance; PE: preeclampsia; preg: pregnancy.

Amniotic Fluid Pi Levels Decrease during Gestational Age
Amniotic fluid Pi levels were determined to assess Pi variations over the course of human gestation. The gestational age at sample collection (GASC), between 17 and 35 weeks of pregnancy, overlaps the gestational age at delivery (GAD) 25 and 39 weeks ( Figure 5A), which includes both second and third trimester of pregnancy. We observed that Pi levels decrease with time during pregnancy ( Figure 5C-D), in accordance with a previous study ran between 16-26 weeks [34]. Lastly, we tested whether Pi was an indicator of preterm birth and observed an association between low . Maternal urine inorganic phosphate (P i ) levels and placental calcification association with preexisting pregnancies, diabetes and preeclampsia. Phosphate levels were determined for maternal urine (control, n = 9; preeclampsia, n = 7). We observed that phosphate levels decrease with preexisting pregnancies in all cohorts (A). We observed a trend of reduced phosphate levels with preexisting pregnancies in both the Control and Preeclampsia groups (B). We observed a trend of increased phosphate levels with preexistence of diabetes in all cohorts (D) and Preeclampsia groups (E). Placental calcification (% Area Calcified) was determined for placental tissue sections (control, n = 8; preeclampsia, n = 8), and we observed a trend of increased placental calcification with preexistence of previous pregnancies in all cohorts (C) and decreased with preexistence of diabetes (F). Symbols: * p ≤ 0.05. Abbreviations: diab: diabetes; NGT: normal glucose tolerance; PE: preeclampsia; preg: pregnancy.

Amniotic Fluid P i Levels Decrease during Gestational Age
Amniotic fluid Pi levels were determined to assess P i variations over the course of human gestation. The gestational age at sample collection (GASC), between 17 and 35 weeks of pregnancy, overlaps the gestational age at delivery (GAD) 25 and 39 weeks ( Figure 5A), which includes both second and third trimester of pregnancy. We observed that P i levels decrease with time during pregnancy ( Figure 5C-D), in accordance with a previous study ran between 16-26 weeks [34]. Lastly, we tested whether P i was an indicator of preterm birth and observed an association between low second trimester phosphate amniotic fluid levels and preterm birth, supporting that P i should be examined more closely and in a larger population ( Figure 5D). second trimester phosphate amniotic fluid levels and preterm birth, supporting that Pi should be examined more closely and in a larger population ( Figure 5D).

Discussion
Phosphorus is an essential micronutrient involved in a number of required biological processes. The purpose of this study was to characterize phosphate level dynamics over the course of gestation in maternal and fetal biological fluids. Hypophosphaturia is an important feature of severe preeclampsia that may be indirectly related to altered renal function [32]. The correlation with gestational age at delivery (GAD), pregnancy number, preterm birth, preeclampsia, diabetes mellitus, and placental calcification was evaluated. Potential confounding factors include maternal age at delivery and body mass index (BMI)-information which we do not have access to. Furthermore, the average GAD is higher for the preeclampsia group compared to the normotensive group, whilst no difference was observed regarding preexisting pregnancies and diabetes mellitus.
Maternal phosphate excretion increases during pregnancy, with maximum phosphate urine levels observed during the third trimester [35]. Similar to these findings, our study shows that maternal urine Pi levels increased linearly between weeks 28-34 during the third trimester of pregnancy in all cohorts, including normotensive and preeclampsia groups. This supports that during pregnancy, decreased vascular resistance may induce adaptions, sustained by nitric oxide production, which results in the expansion of plasma volume by stimulating renal sodium and water retention and contributes to higher glomerular filtration rate (GFR) [36]. Renal excretion of both calcium and phosphate is decreased in preeclamptic women compared to normotensive women [32,[37][38][39], as a possible result of a compensatory mechanism of decreased renal filtration rate and Figure 5. Amniotic fluid (AF) inorganic phosphate (P i ) as an indicator of gestational age. Phosphate levels were determined with amniotic fluid collected between 17 and 35 weeks of pregnancy and we tested whether phosphate was an indicator for preterm birth (A). We observed that phosphate levels decrease with time during pregnancy (B, C) and an association between low second trimester levels and preterm birth (D). Symbols: * p ≤ 0.05. Abbreviations: AF: amniotic fluid; GAD: gestational age at delivery; GASC: gestational age at sample collection; Pi: inorganic phosphate.

Discussion
Phosphorus is an essential micronutrient involved in a number of required biological processes. The purpose of this study was to characterize phosphate level dynamics over the course of gestation in maternal and fetal biological fluids. Hypophosphaturia is an important feature of severe preeclampsia that may be indirectly related to altered renal function [32]. The correlation with gestational age at delivery (GAD), pregnancy number, preterm birth, preeclampsia, diabetes mellitus, and placental calcification was evaluated. Potential confounding factors include maternal age at delivery and body mass index (BMI)-information which we do not have access to. Furthermore, the average GAD is higher for the preeclampsia group compared to the normotensive group, whilst no difference was observed regarding preexisting pregnancies and diabetes mellitus.
Maternal phosphate excretion increases during pregnancy, with maximum phosphate urine levels observed during the third trimester [35]. Similar to these findings, our study shows that maternal urine P i levels increased linearly between weeks 28-34 during the third trimester of pregnancy in all cohorts, including normotensive and preeclampsia groups. This supports that during pregnancy, decreased vascular resistance may induce adaptions, sustained by nitric oxide production, which results in the expansion of plasma volume by stimulating renal sodium and water retention and contributes to higher glomerular filtration rate (GFR) [36]. Renal excretion of both calcium and phosphate is decreased in preeclamptic women compared to normotensive women [32,[37][38][39], as a possible result of a compensatory mechanism of decreased renal filtration rate and increased tubular reabsorption of calcium and phosphate in toxemia [32,39]. However, we observed a tendency of increased maternal urinary phosphate excretion in the preeclampsia group. The GAD in the preeclampsia is modestly higher in the preeclampsia group, which may contribute to this difference. Ectopic vascular calcification has also been previously associated with preeclampsia [22,33]. Indeed, we were able to detect calcified lesions on placental chorionic villi tissue slides of the preeclampsia group. We observed a trend of increased placental calcification on the preeclampsia group studied; however, by this method, there was no significant association in this cohort.
Phosphate metabolism is regulated by bone-derived FGF-23 and PTH, which influence the renal production and circulating concentrations of the active metabolite of VD, 1,25-hydroxyvitamin D [1,25(OH)D], which affects bone metabolism, intestinal absorption of calcium and phosphorus, and hypertension and vascular calcification [40]. Elevated serum phosphate has an inhibitory effect on the renal activation of 25-hydroxyvitamin D [25(OH)D] and 1,25-hydroxyvitamin D [1,25(OH)D], and lower concentrations of 1,25(OH)D have been associated with adverse cardiovascular outcomes [40]. During pregnancy, maternal serum levels of 1,25(OH)D increase up to twofold starting at 10-12 weeks of gestation and reaching a maximum in the third trimester [41,42]. As reviewed elsewhere [42], preeclamptic women are described to present lower circulating 25(OH)D3 levels than normotensive pregnant women, and VD supplementation showed promise for ameliorating the risk of PE. Contrary to the expected, we report an increase in maternal phosphaturia associated with preexisting diabetes mellitus alone. We also found that maternal urine P i levels are higher in association with preexisting diabetes and preeclampsia compared to diabetes and normotensive participants. Moreover, placental calcification was shown to decrease with preexisting diabetes. Dietary intake may influence these differences, as Mancini FR et al. [43] reported a linear association between increasing dietary phosphorus intake, and the risk for type 2 diabetes in women.
To the best of our knowledge, the present study is the first to demonstrate an association between the number of pregnancies and maternal urine phosphate levels. Indeed, we observed that maternal urine P i levels decreased with a second pregnancy, which was consistent for both normotensive and preeclampsia groups, pointing to a putative decrease in the bioavailability of phosphate with a second pregnancy. However, mothers with preexisting pregnancies may present higher maternal age at delivery. It is well accepted that advanced maternal age is a risk factor for perinatal and neonatal outcomes, gestational diabetes mellitus, gestational hypertension, preeclampsia, small for gestational age infants, spontaneous late preterm delivery, and cesarean section [44]. Furthermore, we observed a trend of increased placental calcification with the number of pregnancies; however, there was no significant association in this cohort.
Lastly, we tested whether phosphate in amniotic fluid was an indicator of preterm birth and its variation over the course of gestation. We observed that amniotic fluid phosphate levels decreased with gestation while low second trimester levels were associated with preterm birth, which suggested that amniotic fluid may be useful as a marker of gestational age. These observations are in conformity with results previously reported [34,45,46], that demonstrate that, during pregnancy, concentrations of phosphate [34,45,46] and calcium [45] decreased, and levels of uric acid [34,45] and creatinine [45,46] increased. This is consistent with the increasing fetal glomerular filtration rate and progressive maturation of tubular function, thus more micronutrients are resorbed at fetal tubules and, consequently, less will be excreted to the amniotic fluid [34,45,46].
During pregnancy, maternal bone mineral density (BMD) and bone resorption markers are modestly increased [21,22,42,47]. With increased number of pregnancies, we hypothesize that the BMD and phosphorus and calcium bioavailability is reduced. However, the information regarding maternal pre-pregnancy BMI as an indicator of maternal nutritional status was not available. Potential limitations to this study should be considered and addressed through future studies that build on these findings to evaluate candidate modifiers, such as maternal age at delivery, maternal body mass index (BMI), biological levels of calcium, VD, PTH, genetics and lifestyle. Future work will evaluate the relationship between phosphate dysregulation in preeclampsia, diabetes, maternal age, and number of pregnancies through the analysis of the tissue-specific and cell-specific nature of calcified lesions; expression of placental phosphate transporter expression levels (Slc20a1 and Slc20a2) and osteochondrogenic factors; maternal urinary calcium and phosphate excretion; and maternal age. In conclusion, phosphate levels provide clinical information regarding temporal fluctuation of maternal-fetal phosphate homeostasis in pregnancy-related complications, supporting that phosphate should be examined more closely and in larger populations.
Author Contributions: A.C.-B. performed most of the experiments and data analysis, and formal A.C.-B. contributor roles include data curation, formal analysis, investigation, validation, visualization, and writing (original draft, review, and editing). M.P.R. and L.M.P. contributor roles include data curation, project administration, resources, and writing (review & editing). M.C.W. contributor roles include conceptualization, data curation, formal analysis, funding acquisition, investigation, project administration, supervision, and writing (original draft, review, and editing). All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest:
The authors have no financial relationships, patents, or copyrights to declare. No payment or services from a third party were received for any aspect of the submitted work.