Conditions: Uterine Atony Uterine Atony With Hemorrhage Postpartum Hemorrhage Cesarean Section Complications
Keywords: calcium chloride uterine atony postpartum hemorrhage cesarean delivery intrapartum cesarean
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 120
Healthy volunteers: no

Brief summary:
Postpartum hemorrhage (PPH) is the leading cause of maternal morbidity and mortality worldwide. Up to 80% of PPH is caused by uterine atony, the failure of the uterine smooth muscle to contract and compress the uterine vasculature after delivery. Laboratory and epidemiological studies show that low extracellular and serum calcium levels, respectively, decrease uterine contractility. A pilot study performed by the investigators supports the hypothesis that intravenous calcium chloride is well tolerated and may have utility in preventing uterine atony. The proposed research will establish the relationship between uterine tone and calcium through a clinical trial with an incorporated pharmacokinetic and pharmacodynamic (PK/PD) study. In a randomized, placebo-controlled, double-blind trial, investigators will establish the effect of 1 gram of intravenous calcium chloride upon quantitative blood loss and uterine tone during cesarean delivery in parturients with high risk of uterine atony. Investigators will concurrently collect serial venous blood samples to measure calcium for PK/PD modeling in this pregnant study cohort. High-quality clinical research and development of novel therapeutics to manage uterine atony are critical to reduce the high maternal morbidity and mortality from PPH.

Detailed description:

Arms:
arm 1: 1 gram of calcium chloride in total volume 60mL with sterile saline, delivered over 10 minute controlled infusion at a constant rate (360mL/hour) beginning 2 minutes after fetal delivery and 1 minute after delayed cord clamp.

This intervention occurs IN ADDITION TO standard care with oxytocin 2 unit bolus and infusion at 7.5 units per hour which begins immediately after fetal delivery. arm 2: 60mL sterile saline, delivered over 10 minute controlled infusion at a constant rate (360mL/hour) beginning 2 minutes after fetal delivery and 1 minute after delayed cord clamp.

This intervention occurs IN ADDITION TO standard care with oxytocin 2 unit bolus and infusion at 7.5 units per hour which begins immediately after fetal delivery.

Interventions: intervention 1: Calcium chloride intervention 2: Saline placebo
Intervention descriptions: intervention 1: See arm description above intervention 2: see arm description above

Protocol document:
Prot_SAP_000.pdf: 
 
 
 
 
Calcium Chloride for prevention of blood loss from uterine atony during 
intrapartum cesarean delivery 
Study Protocol and Statistical Analysis Plan 
NCT05027048 
August 24, 2021 


1 
 
Calcium Chloride for prevention of blood loss 
from uterine atony during intrapartum 
cesarean delivery 
 
A placebo controlled, double blind randomized controlled trial and 
pharmacokinetic pharmacodynamic study 
 
PI: Jessica Ansari, MD 
Senior Faculty Advisors: Dr. 
, Dr. 
 
Data Safety Monitoring Committee: Dr. 
, Dr. 
 
 
Grant Support: SOAP Young Investigator Grant 2020 
 
 
 
 FAER Mentored Research Training grant (pending) 
 
 
 


2 
 
SPECIFIC AIMS:  
We propose to perform a randomized controlled trial (RCT) assessing calcium chloride (CaCl2) as 
a novel intervention to prevent hemorrhage from uterine atony with nested PK/PD analysis. 
PPH is the leading cause of maternal morbidity and mortality worldwide1,2. Concerningly, the incidence 
of PPH has been rising3. Uterine atony, defined as failure of the uterus to adequately contract after placental 
delivery, causes approximately 80% of PPH2. Current medications used to prevent or treat uterine atony are 
limited by poor efficacy, adverse side effects, expense, and contraindications to use2. As such, there is a critical 
need for effective, safe, and inexpensive pharmacologic measures for uterine atony. 
Intravenous CaCl2 is an inexpensive, shelf-stable drug with good biologic and epidemiologic plausibility 
to treat and/or prevent uterine atony. Calcium is already frequently utilized in obstetric anesthesia for indications 
such as hypocalcemia from massive transfusion and overcoming magnesium toxicity. Multiple in vitro studies 
have demonstrated that adequate extracellular calcium levels are necessary for forceful myometrial contraction 
in response to oxytocin5-7. Epidemiologic studies show that low ionized calcium levels predict the development 
of severe postpartum hemorrhage in a dose-response fashion8.  
We recently conducted a 40-patient placebo-controlled, double-blind pilot RCT assessing a one-gram 
dose of intravenous CaCl2 as a novel agent to prevent uterine atony during cesarean delivery. This pilot 
demonstrated the feasibility of enrollment and good patient tolerance of the intervention. Moreover, we found a 
trend toward efficacy in prevention of uterine atony in women who received calcium as compared to placebo 
infusion (p=0.07, relative risk 0.38, 95% CI 0.15-1.07, number needed to treat 3.3). 
Pharmaceutical development and pharmacokinetic characterization in pregnancy has become a National 
Institute of Health priority9. The NICHD has established an Obstetric and Pediatric Pharmacology and 
Therapeutics Branch dedicated to advancing knowledge of drug PK/PD in pregnancy and childhood given critical 
knowledge gaps in these areas9. We will collect and analyze PK/PD for intravenous CaCl2 within our trial. Building 
upon the promising results from our pilot study, we aim to: 
AIM 1: Establish the effect of a CaCl2 infusion on blood loss and uterine tone during cesarean 
delivery in parturients at heightened risk for uterine atony in a double-blind, randomized, placebo-
controlled trial. We hypothesize that, as suggested by our pilot study, CaCl2 infusion will reduce quantitative 
blood loss (QBL, primary outcome) and improve a numerical rating score for uterine tone (NRSUT, secondary 
outcome) compared to placebo without significant safety or tolerability concerns.  
AIM 2: Measure PK/PD of intravenous CaCl2 in pregnant patients. Establishing the PK of intravenous 
CaCl2 in parturients is of great relevance whether or not CaCl2 is shown to be effective in preventing atony in Aim 
1, given the multiple indications for calcium administration in obstetric patients. We will generate a PK/PD model 
for calcium in pregnancy that includes estimates of covariate effects and intra-and interindividual variability using 
non-linear mixed effects modeling. Meanwhile, PD modeling of calcium effect upon uterine tone will allow us to 
quantify calcium’s effect on myometrial function.  
Significance: If CaCl2, an inexpensive, widely available drug, has efficacy in the prevention or treatment 
of uterine atony, it has the potential to change the maternal standard of care and decrease the leading cause of 
maternal morbidity and mortality.  We will leverage the intervention tested in the RCT, the infusion of CaCl2, to 
define the PK/PD of calcium in pregnant women at term. This has potential relevance in defining approaches for 
administering calcium in various obstetrical contexts and will facilitate dose optimization. 
 
 


3 
 
 
BACKROUND AND SIGNIFICANCE 
Uterine atony and postpartum hemorrhage: PPH, defined by the American College of Obstetricians and 
Gynecologists as a cumulative blood loss of >1000mL within 24 hours of birth, is the leading cause of maternal 
major morbidity and mortality worldwide.1,2 Uterine atony, defined as failure of the uterus to adequately contract 
after placental delivery, causes about 80% of PPH.3 Rates of PPH requiring blood transfusion in the United 
States increased 5-fold from 1993 to 2014, an increase largely attributed to rising incidence of uterine atony4.  
 
Current clinical management of uterine atony involves prophylaxis with oxytocin and treatment with second-line 
uterotonics including methylergonovine, carboprost, and misoprostol2. However, the latter medications all carry 
significant limitations of poor efficacy, adverse side effect profiles, expense, or contraindications to use.2 For 
example, methylergonovine is contraindicated in women with preeclampsia or hypertensive diseases. 
Carboprost, which may cost up to $1000 per dose, is contraindicated in women with asthma and requires 
refrigeration, significantly limiting its utility in poor resourced settings. Misoprostol causes fever and rigors and 
its efficacy for treating uterine atony has been called into question.2 As such, additional prevention or treatment 
modalities for uterine atony--especially inexpensive, shelf-stable, and well-tolerated drugs—are essential.  
 
Calcium: Intravenous CaCl2 is an inexpensive, widely-available, shelf-stable drug which is familiar to 
anesthesiologists in a variety of contexts. On obstetric units, calcium is utilized for indications including 
hypocalcemia from rapid transfusion of citrate-containing blood products and overcoming magnesium toxicity. 
 
Biological plausibility: calcium and uterine contractility: In vitro studies have established calcium’s important 
physiological role in uterine contractility. Uterine myometrial contractility is dependent upon an influx of calcium 
from intracellular stores in the sarcoplasmic reticulum as well as extracellular calcium.5-7 Myometrial contraction 
amplitude diminishes in the setting of low extracellular calcium.5-7 Oxytocin’s efficacy in inducing myometrial 
contraction diminishes by 60-75% in the setting of low or absent extracellular calcium levels in in vitro studies 
with human uterine strips.7  
 
Clinical and epidemiologic evidence: serum calcium levels impact myometrial function: In a large 
retrospective cohort study, low ionized calcium levels predicted severity of PPH in a dose-response relationship, 
with an odds ratio of 1.97 for each 0.1mmoL/L decrease in ionized calcium level8. A prospective observational 
study also linked serum calcium levels with spontaneous labor onset, finding higher serum calcium in laboring 
than matched non-laboring patients at the same gestational age10. Finally, calcium channel blockers are regularly 
utilized to prevent uterine contractility in the setting of preterm labor11.  
 
In one prior small RCT, Farber and colleagues examined the effect of low dose CaCl2 administered during 
scheduled cesarean delivery in 60 patients upon hemodynamics (primary outcome). No differences in 
development of uterine atony (secondary outcome) were noted among the study groups who received 0, 200mg, 
or 400mg of CaCl2; however, there were only 4 total cases of uterine atony and none in the 400mg group, such 
that the study was grossly underpowered with respect to this outcome11.  
 
Maternal hemorrhage and pharmacokinetic drug studies and maternal hemorrhage are National 
Institutes of Health (NIH) priorities: The National Institute of Child Health and Human Development (NICHD) 
specifically calls for investigations dedicated to “improv[ing] the prevention of and response to labor and delivery 
complications that lead to maternal morbidity and mortality and extend[ing] studies of key adverse events to the 
postpartum period (“the fourth trimester”) to include hemorrhage”9. In addition, pregnant women are historically 
excluded from drug trials, and despite physiologic changes including larger plasma and adipose volumes, 
increased glomerular filtration rate, and bidirectional changes in hepatic enzyme function, PK/PD drug studies 
in this population are often lacking13. The NICHD 2020 strategic plan includes themes centered upon funding 
PK/PD studies in pregnant women and children and improving pregnancy outcomes9. The proposed work for 
this FAER award meets both of these NICHD strategic goals by addressing maternal hemorrhage and PK/PD. 
 
 
 


4 
 
PRELIMINARY STUDIES 
We performed a 40-patient double-blind pilot RCT examining the impact of 1 gram of CaCl2 or saline placebo in 
addition to standard oxytocin during cesarean delivery in parturients with heightened risk of uterine atony. In 
the pilot study, we demonstrated feasibility by successfully enrolling all 40 patients in a one-year timeframe 
despite a lack of dedicated funding or research staff. The drug infusion was well-tolerated, with no adverse 
hemodynamic consequences. An equal number of patients in the calcium and placebo groups reported 
possible drug side effects including flushing, nausea, or vomiting (30% in each arm). Obtaining and measuring 
venous blood specimens for PK analysis also proved feasible. 
 
Although inferring intervention efficacy is not the aim of a pilot trial, there were several trends noted within our 
pilot trial data that suggest CaCl2 warrants a larger, definitive study as a potential intervention for uterine atony 
(Table 1).  Only 20% of patients who received calcium had uterine atony, pre-defined as requirement for a 
second line uterotonic agent, placement of an intrauterine balloon for tamponade, or hemorrhage with blood loss 
>1000mL, compared to 50% of patients who received the saline placebo infusion (relative risk 0.38, P=0.07, 
95% CI 0.15-1.07, NNT 3.3). Blood loss, uterine tone score, total oxytocin dose, and intraoperative fluid 
requirements all showed similar promising trends (Table 1).  
 
As part of the pilot RCT, we also 
gathered serial serum venous blood 
samples from a subset of participants. 
This was done for two main purposes: 1) 
to ensure a reliable but safe rise in ionized calcium from the 1-gram dose of CaCl2, and 2) to generate sample 
size calculations for a definitive study of CaCl2 PK in a similar pregnant patient cohort. Data and a 1-compartment 
PK model are shown in Figure 1. As anticipated, a rapid and reliable and safe-level peak in ionized calcium was 
obtained in subjects who received CaCl2. 
 
 
Table 1: Outcomes related to atony and hemorrhage, pilot RCT. 
Values are provided as median [interquartile range] 
 
Calcium (n=20) 
Placebo (n=20) 
P value 
Uterine atony 
incidence 
20% (n=4) 
50% (n=10) 
0.07 
Blood Loss (mL) 
750 [600-800] 
850 [650-1000] 
0.15 
Uterine tone NRS (0-10) 
9 [8-9] 
8 [7-9] 
0.13 
Oxytocin  
 
 
 
Total bolus dose 
(units) 
2 [2-2] 
2 [2-4] 
0.14 
Maximal infusion rate 
(units/hr) 
7.5 [7.5-15] 
15 [7.5-22.5] 
0.41 
Fluids and transfusion 
 
 
 
Crystalloid (mL) 
1200 [1000-
2000] 
1750 [1500-
2000] 
0.03 
Colloid (mL) 
0 [0-0] range [0-
500] 
0 [0-0] range [0-
1000] 
0.08 
Figure 1: pilot data: ionized calcium concentrations 
over time. Blue data points represent ionized 
calcium venous blood concentrations from 13 
patients who received calcium, and red data points 
represent ionized calcium in 11 patients who 
received a saline placebo infusion. 


5 
 
 
 
EXPERIMENTAL DESIGN AND METHODS 
SPECIFIC AIM 1 OVERVIEW: Based on our preliminary data, we propose a double-blind, placebo-controlled, 
120-patient RCT to assess the effect of prophylactic CaCl2 infusion upon blood loss and uterine tone at the time 
of intrapartum cesarean delivery. Patients will be randomly assigned to receive a prophylactic infusion containing 
one gram of dilute CaCl2 or saline placebo in addition to standard of care oxytocin after fetal delivery. We will 
assess quantitative blood loss and serial measurements of uterine tone.  
 
Study population: The study will enroll adult pregnant patients age 18-45 years of age at >34 weeks gestation 
who require intrapartum cesarean delivery and have received an exogenous oxytocin infusion for labor induction 
or augmentation, as these are known risk factors for uterine atony1,2. Exclusions include emergent surgery, 
known cardiac dysfunction, renal dysfunction with serum Cr >1.0, hypertension necessitating intravenous 
hydralazine or labetalol during labor, and treatment with calcium channel blockers or digoxin. 
 
Subject enrollment: All laboring patients on the unit receive a brief informational handout about this study upon 
admission (Appendix 1). Research staff visit all laboring patients twice daily after permission to approach and 
introduction by the bedside nurse, once in the morning and once in the afternoon, to obtain informed consent 
from all willing laboring patients if they require cesarean delivery. Consent and study inclusions/exclusions are 
confirmed by the clinical anesthesiologist at the time of decision by the obstetrics team to proceed with an 
intrapartum cesarean delivery. 
 
Subject exposures: Sequential subject study ID numbers will be randomized in 1:1 ratio to one of two 
experimental groups. Study drugs will be prepared daily by the research pharmacy and labeled only with the 
subject ID # and “study drug”. The patient, anesthesiologist, obstetricians, and research staff will remain blinded 
to assignment. All study subjects will receive standard care with prophylactic oxytocin bolus and infusion, as well 
as second-line uterotonics on an as-needed basis per institutional protocol. (Appendix 2)  
• Calcium: 1 gram CaCl2 in total volume 60mL delivered over 10 minutes beginning 2 minutes after fetal delivery. 
• Placebo: 60mL sterile saline delivered over 10 minutes beginning 2 minutes after fetal delivery 
 
Outcomes and Measurement: The primary outcome will be quantitative blood loss (QBL) in mL, which is 
calculated by standardized methodology including volumetric (suction cannister contents after subtracting 
amniotic fluid volume) and gravimetric (weighing soiled surgical sponges, drapes, and pads) assessment. 
Secondary outcomes will include the following: uterine tone assessments by the obstetric attending on a 
validated 10-point numerical rating scale14 (NRSUT) at study drug initiation 2 minutes after fetal delivery and 
reassessed every 5 minutes thereafter until fascial closure. Additionally, total oxytocin bolus and infusion doses, 
second line uterotonics, change in hematocrit from preoperative to postoperative day one values, total 
intravenous fluids, and a composite outcome indicating hemorrhage morbidity including transfusion during the 
hospital stay, hysterectomy, uterine artery embolization, intensive care admission, and death will be recorded. 
Please see Appendix 3 for forms to be filled out by research staff.  
 
Data Analysis: Analyses will be conducted using R and supervised by a biostatistical consultant from the 
Stanford Quantitative Sciences unit. Continuous data will be represented using mean (± SD) or median 
(interquartile range) for variables not normally distributed and compared using parametric or non-parametric t-
tests as appropriate.  Categorical data will be presented using proportions and compared using a chi-square (or 
Fishers Exact) test. Two-sided p-values less than 0.05 will be considered statistically significant. 
 
Analysis of Primary Aim: The primary outcome of the study is the difference in quantitative blood loss between 
the two groups on an intention-to-treat basis. Patients who are found to have non-atonic reasons for blood loss 
including morbidly adherent placenta, hysterotomy extension, cervical, or vaginal lacerations will be included in 
primary outcome assessment. The trial is also powered for planned subgroup analysis excluding these cases. 
In addition, subgroup analyses based upon risk factors for atony including chorioamnionitis, total oxytocin 
exposure, indication for cesarean, magnesium infusion, and preeclampsia will be performed. 
 


6 
 
Sample size: Power analysis of the data from our pilot RCT revealed that a sample size of 31 patients per arm 
will have 90% power to detect a difference of 200mL in quantitative blood loss between groups at the p<0.05 
significance level. The sample size was increased to 60 patients per arm to allow sufficient power for the planned 
subgroup analysis excluding patients with non-atonic etiologies of blood loss which may occur in up to 40% of 
cases. This same sample size will also generate 86% power to detect a rank test difference of 2 points in the 
ordinal secondary outcome measure of NRSUT. We anticipate signing consent documents with approximately 
1200 laboring patients to obtain the 120 subjects who meet enrollment criteria, as most of these patients will 
have normal spontaneous vaginal delivery, some may later decline to participate, some may develop exclusions 
such as renal dysfunction, and some may require stat delivery without time to safely administer the study 
medications in addition to standard care. 
 
Patient safety: All potential side effects of calcium will be recorded as part of this study, including flushing, arm 
discomfort, nausea, vomiting, bradycardia, tachycardia, arrhythmia, and hypertension. A single dose of dilute 
CaCl2, infused slowly through an intravenous (IV) line in the sensate upper extremity of an un-sedated patient, 
is unlikely to cause severe adverse effects. The major concern with CaCl2 is tissue necrosis from extravasation. 
Any discomfort in the IV site will warrant immediate study drug infusion discontinuation with careful assessment 
of the extremity. The clinical anesthesiologist caring for the patient will also monitor for potential hemodynamic 
consequences of calcium infusion including bradycardia, hypotension, or hypertension. The anesthesiologist 
providing care to the patient will be instructed to assume the study drug contains 1 gram of CaCl2 for all cases, 
and to immediately discontinue the infusion if they believe any the patient may be having a severe adverse effect. 
A data safety monitoring committee of two attending anesthesiologists with no involvement in the trial and with 
a track record of data safety monitoring has been created for this trial. Any potential adverse events including 
patient hemodynamic compromise or concern for drug extravasation will be reviewed immediately ad hoc, and 
they will determine whether unblinding is required and when the study may resume.  
 
Management of hypercalcemia: Acute hypercalcemia may manifest as altered mental status, bradycardia, or 
hypotension. We do not anticipate clinical hypercalcemia given the patient population (young, healthy, renal 
dysfunction and digoxin use are contraindications), the dilute and slow infusion, the single dose of calcium, and 
the excellent patient tolerance and peak ionized calcium levels obtained during our pilot study. However, 
management of acute hypercalcemia would include immediately pausing the infusion if still running, 
administering a fluid bolus of 500mL to 1000mL isotonic fluid, and obtaining a serum calcium level at the 
discretion of the clinical anesthesia team. If necessary, the team could consider additional administration of 10-
20mg intravenous furosemide to lower serum calcium further if needed. 
 
Feasibility: Stanford Children’s Hospital performs 4600-4700 deliveries per year, of which 1600-1950 are 
cesarean. More than 400 cesarean deliveries per year meet study inclusion criteria. Without dedicated funding 
or research staff, we were easily able to enroll 40 patients in the pilot study in a 1-year time period. Plans for the 
study have been accepted by the group of 15 fellowship trained obstetric anesthesiologists who cover labor and 
delivery, all of whom participate in monthly division research meetings and are well-versed in study protocol for 
any actively recruiting studies. In addition, we have the support and collaboration of an excellent obstetric 
division. There are no concurrent competing studies in this study cohort.  
 
Potential difficulties: Given our successful pilot, we believe this study will proceed smoothly but are ready for 
unanticipated problems.  We have received support from obstetricians, the quantitative sciences unit (statistics), 
and labor and delivery nursing for the study.  The greatest source of potential difficulties involves enrollment of 
120 patients at a single site during a pandemic. We have infrastructure in place to allow research assistants 
obtain consent remotely utilizing iPads if necessary. In addition, we have obtained a letter of commitment from 
a second site at Brigham and Women’s Hospital if needed to facilitate enrollment. 
 
Weaknesses: The primary outcome (QBL) and secondary outcome of NRSUT are surrogate outcomes for clinical 
endpoints of major morbidity or mortality but allow for a more reasonable study sample size for a single center.  
 
 


7 
 
 
]SPECIFIC AIM 2 OVERVIEW: Serial venous blood sampling for ionized calcium level in willing study participants 
will allow us to also establish the PK/PD of CaCl2 in term parturients. PK findings have applicability to a variety 
of clinical indications for calcium in pregnant patients, while PD will enhance mechanistic understanding of the 
impact of serum ionized calcium level upon uterine contractility. 
 
Venous blood samples for pharmacokinetic analysis: 0.5 mL venous blood will be collected at up to 6 
timepoints for willing study participants at each of the following time intervals relative to study drug infusion: 
baseline (prior to fetal delivery), 0-5 minutes, 5-10 minutes, 10-30 minutes, 30-60 minutes, and 60-240 minutes. 
Ionized calcium level and pH of blood specimens will immediately be analyzed using an iSTAT machine with a 
EG7+ cartridge (Abbott Core Laboratory Systems, Lake Forest, IL) in an adjacent operating room by research 
staff or obstetric anesthesia fellows uninvolved in patient care or data analysis. Venous blood pH and ionized 
calcium values will be recorded directly into the secure research database but not entered into the patient’s 
medical record as this would compromise blinding. Blood specimens will not be stored.  
 
As a second peripheral IV is frequently placed in the opposite arm from the primary peripheral IV for patients 
undergoing intrapartum cesarean delivery as part of standard care in case of hemorrhage, this IV will be used 
for blood collection whenever possible. If not possible to draw blood or this IV is not placed, the patient will be 
asked for permission to obtain blood specimens using a 22g winged blood collection needle. If possible, these 
specimens will be collected from the lower extremity (saphenous vein or foot), as these veins are dilated and 
insensate in patients undergoing cesarean delivery under neuraxial anesthesia. Sterile technique will be utilized 
for all blood collections. 
 
 
Pharmacokinetic and pharmacodynamic analysis: We will assess the data with graphical validation to assure 
data validity. General analyses will be conducted with R statistical programming language (R Foundation for 
Statistical Computing, Vienna, Austria). PK and PD models will be characterized using nonlinear mixed effects 
modeling with the program NONMEM 7.416. 
 
We anticipate a two-compartment mammillary model with an effect site. Calcium is administered into the central 
compartment, also the site of drug sampling. Calcium is also eliminated from the plasma compartment. Calcium 
may distribute from the plasma compartment to one or more compartments. Drug effect is mediated by the 
concentration in a hypothetical “effect site”, in this case the uterus17. The effect site permits modeling of 
hysteresis between the time course of plasma calcium concentration increase and increase in uterine tone 
measured in Aim 1. PK modeling will be done in the volume and clearance domain and include the influence (if 
any) of age, weight, gestational age, pH, starting heamtocrit, and blood loss. 
 
Equation 1 is an equation for the sigmoidal PD model. Because the 
measure of drug effect is the NRSUT is an ordinal measure, it will be 
dichotomized into 4 categories, 0-3, 4-10, 7-10, and 9-10, and 
modeled as the probability of being in each category as shown in Figure 2. PD   models will be estimate p0, 
pMAX, Ce50, and  γ (the baseline probability of uterine tone X, the 
maximum probability of uterine tone X, the concentration associated 
with 50% of the change in probability, and the steepness of the 
concentration versus probability relationship). The effect of pH and uterotonics including oxytocin, 
methylergonovine, and carboprost on the PD parameters will be explored. 
 
Equation 1: Pharmacodynamic model for the 
probability of uterine NRS and calcium concentration 


8 
 
Inter-subject random effects (e.g., subject to subject differences) on model parameters will be estimated and 
applied using additive, proportional, and exponential models as appropriate. Residual intrasubject variability 
(e.g., noise) will typically require an additive and multiplicative error model. The influence of covariates effects 
(e.g., age, weight, gestational age, pH, uterotonics including oxytocin, methylergonovine, and carboprost) on 
model parameters will be analyzed by serial inclusion/exclusion with significance determined by the likelihood 
ratio test. Covariates on the PK and PD parameters deemed significant by likelihood ratio test will be validated 
using jackknife cross-validation as described by Sheiner17 and only included in the final model if validated.  
 
 
 
Table 2 shows the results of 1000 bootstrap replications of data from the original 24 individuals in the pilot PK 
study, resampled with N=60 (half of the proposed study population), and N=120 (the entire proposed study 
population)18. As expected, the estimates are more precise with increasing study size. As seen in the table, the 
parameters estimated for the initial 24 subjects had considerable uncertainty for clearance and the volume of 
distribution. If the study gathers PK samples in half of the patients (N=60), V1 will be estimated within less than 
a 40% range (Table 2, numbers in red). This should be sufficient to predict the dose of calcium required to reach 
a target calcium concentration in parturients. Our study will have greater power than predicted by the bootstrap 
as we will collect 5-6 samples per patient rather than the 2-3 samples obtained in the pilot study. 
 
 
 
Figure 2: Ordinal model of uterine NRS showing hypothetical 
probability of NRS>3, >6, and >8 as a function of calcium 
Table 2: Bootstrap analysis using pilot ionized calcium values. The mean and 95% 
confidence intervals are shown for each parameter. 


9 
 
Rigor and Transparency: Prior research that serves as the key support for the proposed study is rigorous as 
there is basic science and epidemiologic plausibility for this intervention. Sample size and statistical power for 
both aims of this research proposal build upon our own pilot clinical trial data. Weaknesses of the only prior 
clinical trial to investigate calcium’s relationship to uterine atony included low calcium dose, small sample size, 
and a study population with a very low baseline incidence of uterine atony12. We have addressed all of these 
weaknesses with our clinical trial design by using a higher dose of calcium that we have shown in our pilot study 
accomplishes a measurable peak in ionized calcium levels shortly after infusion, a larger sample size based on 
rigorous estimation techniques, and a study population with very high baseline incidence of uterine atony. Our 
blinding and randomization should ensure a robust and unbiased approach. Finally, the proposed study takes 
into account sex as a biological variable by studying a condition unique to pregnant women. It also promises to 
bridge gaps in pharmacokinetic and pharmacodynamic studies in pregnant women. 
 
Implications: If CaCl2 shows efficacy in reducing blood loss from atony, it has potential to change the maternal 
standard of care worldwide and directly impact the leading cause of morbidity and mortality in pregnant women. 
 
Future Directions: The likely next step will include obtaining NIH funding to execute a larger, multicenter RCT 
with expanded inclusion criteria.  
 
 
 


10 
 
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Quantitative Blood Loss in Obstetric Hemorrhage. Obstet Gynecol. 2019 Dec;134(6):e150-e156. 
15. Cole NM, Abushoshah I, Fields KG, Carusi DA, Robinson JN, Bateman BT, Farber MK. The interrater 
reliability and agreement of a 0 to 10 uterine tone score in cesarean delivery. Am J Obstet Gynecol 
MFM. 2021 May;3(3):100342. 
16. NONMEM 7.4, Icon Development Solutions, Dublin, Ireland 
17. Sheiner LB, Stanski DR, Vozeh S, Miller RD, Ham J. Simultaneous modeling of pharmacokinetics and 
pharmacodynamics: application to d-tubocurarine. Clin Pharmacol Ther 1979;25(3):358-71. 
18. Browne RH. On the use of a pilot sample for sample size determination. Stat Med 1995;14(7):1933-40. 
 
 


11 
 
 
Appendix 1: Patient informational handout 
 
] 
 
Stanford OB Anesthesia Study Information Sheet for Patients 
Trial Investigating Calcium to Prevent Bleeding during Cesarean 
 
This information sheet is provided to laboring patients to provide some information in case you might 
later qualify for one of our studies. At some point, an anesthesiologist or research assistant might 
approach you about this study.  They would at that point go through a much more detailed 
explanation and consent with your permission.  However, you are welcome to ask questions any time! 
 
What is the study?  In this study, Stanford OB Anesthesiologists are investigating whether giving 
some extra calcium through a patient’s IV after the baby is delivered during a cesarean can help the 
uterus to contract and prevent blood loss.   
 
I am in labor, not having a Cesarean. Why am I receiving this information? Until babies are born, 
we never know for sure that cesarean delivery will not be required.  In this study, we are particularly 
trying to enroll patients who require cesarean delivery after laboring, as these patients have a higher 
risk of blood loss from poor uterine contraction after delivery. 
 
How would this study change my care? If you were to qualify and consent for this study, you would 
receive all of the same medications and doses that any other patient receives.  If you took part in this 
study, after your baby was delivered, an anesthesiologist would ALSO give a dose of extra calcium or 
some plain saline (known as a placebo) through your IV.  Your anesthesiologist and you would not 
know whether you received calcium or the saline placebo. With your permission, the anesthesiologist 
may also collect several small blood samples from your IV during your cesarean to measure your 
calcium levels. You would still be welcome to participate without giving blood samples. 
 
Why calcium?  Why is this being studied?  Calcium is present in all of our bodies and is essential for 
normal functions like blood clotting, heart, bone, and muscle function.  Research shows that it is 
possible that extra calcium may also help the uterus muscle contract, which is important to help prevent 
bleeding after delivery.  We are investigating whether women who require Cesarean have less blood 
loss and better uterine contraction after delivery if they receive some extra calcium. This study will 
help us to answer that important question. 
 
For Participant's rights questions, contact 1-866-680-2906. 
 
 


12 
 
Appendix 2: Stanford Labor and Delivery protocol for intrapartum cesarean delivery 
 
Monitoring: All patients undergoing intrapartum cesarean delivery are monitored by continuous pulse oximetry, 3-lead 
EKG, and noninvasive blood pressure oscillometry. Blood pressures are obtained every 1 minutes prior to fetal delivery. 
For clinically stable patients at the discretion of the anesthesia team, blood pressure frequency may be decreased to 
every 2.5-5 minutes after fetal delivery. 
 
Antibiotics: 
• 
Patients without antibiotic allergy or chorioamnionitis receive 2 grams intravenous cefazolin (3 grams if >120 kg) 
prior to incision and 500 mg of intravenous azithromycin over a one-hour infusion for endometritis prophylaxis. 
• 
Patients with a clinical diagnosis of chorioamnionitis instead receive intravenous ampicillin 2g, gentamycin 5 
mg/kg over a one-hour infusion, and clindamycin 900 mg intravenously  
 
Neuraxial anesthesia: 
• 
Patients with a labor epidural in situ receive epidural top-up with divided doses of 2% lidocaine with epinephrine 
1:200,000 to achieve a T4 dermatomal level to pinprick bilaterally (generally 15-25 mL total volume). 100 mcg 
epidural fentanyl is added at the discretion of the attending anesthesiologist. All patients receive 3 mg epidural 
morphine prior to case conclusion for postoperative analgesia. 
• 
Patients without a labor epidural in situ receive a single-shot spinal or combined spinal-epidural anesthetic, with a 
standard intrathecal medication cocktail of 1.6 mL 0.75% hyperbaric bupivacaine, 15 mcg fentanyl, and 100 mcg 
morphine. 
 
Uterine tone assessment and uterotonic medications: 
• 
At the time of fetal delivery, 2 units of intravenous oxytocin are administered, and an oxytocin infusion is initiated 
at 7.5 units/hr 
• 
2 minutes after fetal delivery, the uterine tone is assessed by palpation of the uterine fundus and rated on a scale 
from 0-10, with 0 representing a completely atonic uterus and 10 representing a firm and completely contracted 
uterus. This score is recorded. For uterine tone score <8/10, an additional 2 units oxytocin are administered, and 
oxytocin infusion increases to 15 units per hour 
• 
7 minutes after fetal delivery, uterine tone is again assessed by palpation and score recorded. For scores <8/10, 
oxytocin 2 units are administered for a 3rd time, oxytocin infusion increases to 30 units/hr, and the anesthesia and 
obstetric teams discuss whether a second line uterotonic may be warranted. If so, the anesthesia team begins 
preparation of the second line uterotonic agent. This agent is administered if uterine tone is still deemed 
inadequate at the discretion of the anesthesia and obstetric teams. 
• 
12 minutes after delivery, uterine tone is again assessed by palpation and score recorded. For scores <8/10, the 
obstetric and anesthesia teams will discuss whether a second line uterotonic may be clinically indicated. 
• 
The uterotonic protocol outlined can be changed based on clinical picture or obstetrician request. 
 
Quantitative blood loss: 
Quantitative blood loss (QBL) is measured for all cesarean deliveries by a standardized protocol. The protocol follows 
nationally recommended methodology that combines gravimetric (measurement of suction cannister contents after 
subtraction of the amniotic fluid volume) and volumetric (weighing all surgical sponge and drape and subtracting dry 
weights) QBL.1,2 This process is performed by the clinical labor and delivery nurses, all of whom receive standardized 
training in performing the technique. 
 
1. 
Quantitative Blood Loss in Obstetric Hemorrhage: ACOG COMMITTEE OPINION, Number 794. Obstetrics and gynecology 
2019;134(6):e150-e156.  
2. Lyndon A, Lagrew D, Shields L, Main E, Cape V: Improving Health Care Response to Obstetric Hemorrhage.(California 
Maternal Quality Care Collaborative Toolkit to Transform Maternity Care). Published by the California Maternal Quality Care 
Collaborative. 2015. 
 
 
 
 
 


13 
 
Appendix 3 Protocol Sheet:  CALCIUM CHLORIDE FOR PREVENTION AND TREATMENT OF BLOOD 
LOSS FROM UTERINE ATONY DURING INTRAPARTUM CESAREAN DELIVERY 
 
 
 
 
STUDY ID NUMBER (on study drug syringe): ______________________ 
 
Reminder: This study investigates whether one gram of calcium chloride IV, administered over 10 minutes 
starting 2 minutes after fetal delivery, decreases blood loss from uterine atony during intrapartum cesarean. 
• 
Primary outcome: Quantitative Blood Loss 
• 
Secondary outcomes: Uterine tone scores, uterotonics, side effects 
 
Please confirm inclusions with a checkmark or X: 
 
Patient is 18-55 years old 
 
            Patient is having an intrapartum cesarean delivery 
 
Patient received oxytocin infusion for labor induction or augmentation. 
 
Patient still wants to participate and has signed consents 
 
Please confirm this patient has NO EXCLUSIONS: 
 
Patient does not have renal dysfunction with serum Cr>1.0 mg/dL 
 
Patient does not have any known cardiac conditions 
 
Patient is not receiving a calcium channel blocker (none in last 24 hours) 
 
Patient is not receiving digoxin for a maternal or fetal indication 
 
This is not an emergency in which participation in the study could hinder or delay care 
 
Study drug:  
• 
The syringe contains 60mL saline with 1-gram Calcium Chloride OR 60mL saline placebo 
 
• 
Administer via syringe pump and microbore tubing over 10 minutes 
o Program a basic infusion on Alaris pump for basic infusion, 360mL/hr 
o Attach microbore tubing to injection port closest to the patient 
o Start infusion 2 minutes after fetal delivery (1 minute after cord clamp) 
 
• 
Assume that every infusion contains 1 gram of calcium and discontinue if concerns for extravasation 
(arm pain and redness or signs of infiltration), hemodynamic compromise (bradycardia and hypotension 
most common). Contact Dr. Ansari via email or cell (760) 845-0328 for any concerns and document on 
next page. 
 
PLEASE FILL OUT ALL INFORMATION ON THE NEXT PAGE. 
 
 
v


14 
 
STUDY ID NUMBER (on study drug syringe): ______________________ 
 
USE EPIC COMPUTER CLOCK FOR ALL TIMES 
Fetal Delivery and oxytocin 2 unit bolus :   
 
Time: ____________________________ 
 
2 minutes after fetal delivery: 
• 
Ask obstetrician to rate uterine tone 0-10:   Tone: ____________________________ 
 
• 
Start study drug infusion:   
 
 
Time Started: ______________________ 
 
• 
If infusion was paused or stopped 
 
Time paused: _______________________ 
 
 
 
 
 
 
 
 
Time restarted: ______________________ 
 
7 minutes after fetal delivery: 
• 
Ask obstetrician to rate uterine tone 0-10: 
Tone: ____________________________ 
 
12 minutes after fetal delivery: 
• 
Ask obstetrician to rate uterine tone 0-10: 
Tone: ____________________________ 
 
19 minutes after fetal delivery: 
• 
Ask obstetrician to rate uterine tone 0-10: 
Tone: ____________________________ 
 
Side effects (did the patient experience any of the following possibly related to the study drug? Circle Yes/No) 
 
 
 
• 
Arm or IV site discomfort?  
 
 
YES     /    NO 
 
• 
Concern for extravasation? 
 
 
YES     /    NO  
 
 
• 
Heart rate change or arrhythmia? 
 
YES     /    NO     Describe: ___________________ 
 
• 
Hypertension or hypotension  
 
YES     /    NO     Describe: ___________________ 
 
• 
Flushing 
 
 
 
 
YES     /    NO 
 
• 
Abnormal sensations or tastes 
 
YES     /    NO     Describe: ___________________ 
 
• 
Nausea  
 
 
 
 
YES     /    NO      
 
• 
Vomiting 
 
 
 
 
YES     /    NO      
 
• 
Other   
 
 
 
 
YES     /    NO     Describe: ___________________ 
 
Was the infusion started exactly 2 minutes after fetal delivery? 
YES     /    NO 
 
 
If not, please explain why: ____________________________________________________________ 
 
Was the whole infusion given? If not or if paused, please explain why below 
 
_______________________________________________________________________________________ 


15 
 
STUDY ID NUMBER (on study drug syringe): ______________________ 
 
 
If patient consents to up to 6 lab draws of 0.5mL venous blood: 
• 
Research staff (fellow or research assistant) to analyze samples in a different operating room.  
• 
DO NOT participate in patient care or converse with staff or the patient in the delivery room other than 
to obtain permission to obtain blood samples, as your knowledge of these lab values could compromise 
blinding of the patient, anesthesiologist, or obstetrician. 
 
Times to target for blood samples relative to the start of study drug infusion: 
• 
Baseline (prior to study drug start) 
• 
10-30 minutes 
• 
0-5 minutes 
• 
30-60 minutes 
• 
5-10 minutes 
• 
60-240 minutes 
 
Carefully document the time that the blood was drawn using EPIC computer clock for each sample. 
Use the research iSTAT machine and research EG7+ cartridges. 
For patient ID on the iSTAT, please enter the study ID number and not the MRN 
 
Time study drug started:  
_______________________ 
 
 
Baseline: 
 
Time: _______________________  
pH: 
_______________________ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
iCa:  
_______________________ 
 
 
Sample 2: 
 
Time: _______________________  
pH: 
_______________________ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
iCa:  
_______________________ 
 
 
Sample 3: 
 
Time: _______________________  
pH: 
_______________________ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
iCa:  
_______________________ 
 
 
Sample 4: 
 
Time: _______________________  
pH: 
_______________________ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
iCa:  
_______________________ 
 
 
Sample 5: 
 
Time: _______________________  
pH: 
_______________________ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
iCa:  
_______________________ 
 
 
Sample 6: 
 
Time: _______________________  
pH: 
_______________________ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
iCa:  
_______________________