Predicting Placental Pathologies by Ultrasound Imaging of the Human Placenta During Gestation
Conditions: Intrauterine Growth Restriction, Villitis, IUGR
Sponsor: Mayo Clinic

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Mayo Clinic 
IRB: 20-000991 
 
Predicting placental pathologies by ultrasound imaging of the human placenta during gestation 
 
Study Chair: 
Mauro Schenone, M.D. 
Obstetrics and Gynecology 
Mayo Clinic 
200 First Street, SW 
Rochester, MN 55905 
507-284-0210 
 
Co-Investigator: Shigao Chen PhD 
Professor of Radiology 
Mayo Clinic 
200 First Street, SW 
Rochester, MN 55905 
507-284-3703 
 
 
 
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MAYO CLINIC 
PARTICIPATING SITES 
Mayo Clinic Rochester Only 

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Protocol Resources 
 
 
Questions: 
Contact Name: 
Clinical data submission and record 
maintenance 
Protocol document, consent form, 
regulatory issues 
Emily N. Smith, R.N. 
Research Study Coordinator 
Phone:507-266-4813 
E-mail: smith.emily3@mayo.edu 
Adverse Events (AdEERS, MedWatch, Non- 
AER, AML/MDS) 
Emily N. Smith, R.N. 
Research Study Coordinator 
Phone:507-266-4813 
E-mail: smith.emily3@mayo.edu; 
lemens.maureen@mayo.edu 

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Index 
0.0 
Schema 
1.0 
Background 
2.0 
Goals 
3.0 
Patient Eligibility 
4.0 
Test Schedule 
5.0 
Grouping Factor 
6.0 
Registration/Randomization Procedures 
7.0 
Protocol Intervention 
8.0 
Intervention Modification Based on Adverse Events 
9.0 
Ancillary Treatment/Supportive Care 
10.0 
Adverse Event (AE) Reporting and Monitoring 
11.0 
Intervention Evaluation 
12.0 
Descriptive Factors 
13.0 
Follow–up Decision at Evaluation of Patient 
14.0 
Human Studies Evaluation 
15.0 
Statistical Considerations and Methodology 
16.0 
References 

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Schema 
 
 
 
 
 
 
 

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1.0 
Background 
 
Many pregnancy complications, including growth restriction, preterm birth and stillbirth, are 
associated with inflammation in the placenta, which is currently diagnosed after delivery. This non- 
infectious placental inflammation is known as villitis of unknown etiology (VUE) is characterized by 
maternal T cells infiltrating into the placenta and causing destruction of the villous architecture. 
However, it’s not known what leads to these maternal T cells becoming activated against fetal cells, 
nor it is understood whether VUE is a cause or an effect of the associated fetal complications. The 
main challenge faced by researchers studying VUE is that there are no strategies to predict VUE in 
utero. 
 
1.1 
Villitis of Unknown Etiology 
 
Intrauterine growth restriction (IUGR) appears to be associated with a placental pathology 
called villitis of unknown etiology (VUE). 1 During VUE, maternal T cells are found at high 
levels in the chorionic villi, causing destruction of placental architecture without clinical 
signs and symptoms of maternal (or fetal) infection. 2 VUE is diagnosed in 30% of all 
placentae. 3 However, exact prevalence and the significance of this diagnosis, along with its 
potential role in IUGR, remains unknown. As half of a fetus’ genetic material is paternally 
derived, VUE may represent a rejection response by the maternal immune system. Human 
leukocyte antigens (HLA), which are critical to the success of organ transplantation, may 
provide novel insights into VUE. Our published work identified that HLA class I and II 
receptors are upregulated in placentae with VUE compared to unaffected, gestational age- 
matched controls. 4 Additionally, HLA-typing of VUE affected mothers and their newborns 
demonstrated increased HLA-mismatch compared to unaffected pairs. 4 Our follow-up data 
(in review) utilizing T cell receptor spectratyping shows a significant expansion of T cell 
clones in placentae with VUE compared to controls; however, these clones are unique to each 
subject further indicating VUE is not a response to a shared antigen (as with an undetected 
infection). 
 
1.2 
Ultrasound Imaging 
The scientific premise for this project is supported by strong animal data out of John 
Hopkins. This group utilized in utero Doppler ultrasound to identify intrauterine 
inflammation in a mouse model. Pregnant mice injected with lipopolysaccharide into the 
uterine cavity have intrauterine inflammation, which researchers found to cause elevated 
resistance indices, pulsatility indices and a greater occurrence of absent end-diastolic flow in 
the umbilical and uterine arteries.5 Women diagnosed with fetal IUGR during pregnancy will 
undergo Doppler ultrasound to monitor signs of fetal health. Inflammation is also associated 
with abnormal growth of microvessels. 6,7 Current Doppler technology is not sensitive enough 
to detect these microvessels, whereas the new microvessel imaging technology can detect 
these small changes in blood flow.8,9 Therefore, as microvessel change is associated with 
inflammation, and inflammation is related to VUE and its associated fetal complications, we 
believe combining Doppler imaging with microvessel imaging will provide more power to 
identify placental villitis, possibly even those cases of mild villitis. Additionally, we will 
measure the ultrasound attenuation coefficient (UAC) in the placenta 11, which would provide 
information on fat fraction that may be a useful evaluation of placenta health. 
 
 
Study Plan: This study will collect: 
1. 
Ultrasound measures during the third trimester (>28 weeks) 
2. 
Clinical placental specimen after birth 
3. 
Patient demographics from the electronic medical record 
 

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Studies utilizing these three things will be performed as described in the sections that follow. 
 
2.0 
Goals 
 
We aim to develop an ultrasound method to identify VUE induced inflammation in the human 
placenta during gestation. We hypothesize that hemodynamic changes can also be observed in the 
human placenta diagnosed with VUE by conventional Doppler ultrasound and microvessel imaging 
(also called 3D imaging in the consent form). 
 
2.1 
Primary 
 
3.11 
Our goal is to determine whether Doppler ultrasound measures of blood flow 
can predict VUE during gestation in a cohort of growth restricted fetuses 
compared to normal controls. 
 
2.2 
Secondary 
 
2.12 
We aim to measure differences in microvessel imaging to determine if it can 
provide stronger evidence for VUE diagnosis. 
 
 
3.0 
Patient Eligibility 
 
3.1 
Registration – Inclusion criteria IUGR cohort: 
 
3.11 
Age ≥18 and ≤45 years at study entry. 
 
3.12 
Diagnosis of IUGR before admission to labor and delivery. 
 
3.14 
Ability to provide written informed consent. 
 
3.16 
Weight greater than 110 pounds (50 kilograms, a standard requirement in 
obstetrics studies that include blood draws). 
 
3.17 
>28 weeks gestation 
 
3.2 
Registration – Inclusion criteria control cohort: 
 
3.21 
Age ≥18 and ≤45 years at study entry. 
 
3.22 
No known pregnancy complications at obstetrics visit (+/- 1 week gestational 
age of IUGR cohort) 
 
3.23 
Ability to provide written informed consent. 
 
3.24 
Weight greater than 110 pounds (50 kilograms, a standard requirement in 
obstetrics studies that include blood draws). 
 
3.25 
>28 weeks gestation 
 
3.3 
Registration – Exclusion criteria 
 
3.31 
Known immunodeficiency 
 
3.32 
Chronic, active viral infections, including HIV-1/2, HTLV-1/2, hepatitis B/C. 

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3.33 
Known autoimmune disease (e.g., rheumatoid arthritis or systemic lupus 
erythematosus) 
 
3.34 
Solid organ or transplant recipient. 
 
3.35 
Multiple gestations. 
 
3.36 
Not planning on delivering at Mayo Clinic 
 
3.37 
Ruptured membranes 
 
3.38 
<28 weeks gestation 
 
4.0 
Test Schedule 
 
Tests and procedures 
At registration (3rd trimester diagnosis 
of IUGR 
+\- 1 week for all controls 
Maternal and fetal 
demographics1 
X 
Ultrasound imaging 
X 
Clinical placenta specimen 
collection2 
X 
1. Abstracted from the medical record. 
2. Placenta pathologic sampling and review per clinical protocol 
 
5.0 
Grouping Factor: None. 
 
6.0 
Registration/Randomization Procedures 
 
6.1 
Participants will be recruited from patients obtaining antenatal care for pregnancy in the 
obstetrics clinic at Mayo Clinic in Rochester, Minnesota. Women who meet the 
eligibility criteria (see above) will be offered the opportunity to participate in the study. 
Informed consent will be obtained prior to enrollment. 
 
7.0 
Protocol Intervention 
 
This study will take place in the Rochester Methodist Hospital and will enroll women 
attending antenatal care through the Department of Obstetrics and Gynecology. Annually, 
2500 women receive care and deliver at the Mayo Clinic Family Birth Center. This study will 
consist of two cohorts: 1) women diagnosed with intrauterine growth restriction (IUGR) and 
2) women having uncomplicated pregnancies. Forty women will be enrolled after being 
diagnosed with IUGR. Based on our clinical data we expect 50% to have a VUE diagnosis. 
Another cohort of 20 women having uncomplicated pregnancies will be enrolled, matched on 
gestational age at ultrasound and fetal sex. 
 
7.1 
Study Methodology 
 
Following consent, women will undergo a comprehensive Doppler ultrasound of the 
placenta. Prenatal ultrasound is routinely used in clinical practice and considered 
safe. The ultrasound output level used in this study will be below the FDA limit for 
fetal imaging. Ultrasound measures to be collected include uterine artery (UtA) and 

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umbilical artery (UA), systolic/diastolic (S/D) ratio, resistance index (RI), pulsatility 
index (PI) and presence of early diastolic notch will be collected for each subject. 
 
Additionally, we will explore the benefit of novel ultra-sensitive Doppler technology 
for microvessel imaging of the placenta. This technology can substantially improve 
small vessel imaging on ultrasound. A Verasonics ultrasound scanner will be used for 
ultra-sensitive Doppler imaging. We will collect data on vessel density, periphery-to- 
center vessel density ratio (VDR), microvessel morphology and the ultrasound 
attenuation coefficient (UAC). Relevant information about the Verasonics scanner 
can be found in the Device section.  
 
Maternal and fetal demographics will be collected for all study subjects. Statistical 
differences between variables in our three groups will be determined ANOVA by 
Kruskal-Wallis tests if normality is not met. Significance will be defined as a p-value 
≤0.05. 
 
7.2 
Pathologic Review 
 
In order to confirm VUE diagnosis, placentae will be collected after delivery and 
placed in formalin. A minimum of 6 samples will be obtained, 2 from cord and 
membranes and 4 from parenchyma. Placental tissue from the control cohort will 
then be processed and embedded into paraffin blocks (FFPE) for pathological review 
for the research study. Placental tissue from the IUGR cohort will be processed and 
embedded into paraffin blocks (FFPE) for pathological review as per standard 
clinical pathology protocols. Blocks will be prepped and stained with hematoxylin 
and eosin for histologic examination by our expert pathologist, Dr. Reade Quinton. 
All diagnoses will be based on established and published criteria from the 
Amsterdam Placental Workshop Group and Society for Pediatric Pathology.10 
Severity, based on immune cell infiltration into the placenta, will be correlated back 
to ultrasound findings and pregnancy outcome. We will also share de-identified 
FFPE blocks collected for research only (not clinical residual tissues) with 
Alpenglow. Alpenglow Biosciences is located in One Discovery Square – Advanced 
Diagnostics Laboratory. The team will test whether their 3D histology technique can 
work for placental vascular evaluation (feasibility study). 
7.3 
Collection of Clinical Data 
 
Study participants will provide consent for study personnel to access their obstetrical 
record for information about age, fetal sex, potential confounders (see exclusion 
criteria) and gestational age at sample collection. 
 
8.0 
Intervention Modification Based on Adverse Events: Not applicable 
 
 
9.0 
Ancillary Treatment/Supportive Care: Not applicable. 
 
 
10.0 
Adverse Event (AE) Reporting and Monitoring 
 
10.11 Adverse events will be assessed as a routine part of every clinic visit during 
pregnancy. These will be reported to the principal investigator. The principal 
investigator will make a decision on whether the adverse event is expected or 
unexpected, and if the adverse event is related to the procedure. Based on this 
information, the PI will determine whether the adverse event should be reported as an 
expedited report or as part of routine clinical data. 

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There are no known risks of ultrasound imaging to either the mother or fetus during 
pregnancy. Therefore, we expect no study related adverse events to occur. 

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10.12 Expected vs. Unexpected 
• 
The determination of whether an AE is expected is based on whether 
the event is generally associated with the condition being studied (i.e. 
IUGR), whether in the research study or in the setting of standard clinical 
care. 
• 
Unexpected AEs are those events that are generally not associated 
with the condition being studied and may have a relationship to the 
procedure or device (i.e. ultrasound) being studied. 
 
 
10.13 Assessment of Attribution 
When assessing whether an adverse event is related to a medical treatment or 
procedure, the following attribution categories are utilized: 
 
Definite - The adverse event is clearly related to the specimen collection. 
Probable - The adverse event is likely related to the specimen collection. 
Possible - The adverse event may be related to the specimen collection. 
Unlikely - The adverse event is doubtfully related to the specimen collection. 
Unrelated - The adverse event is clearly NOT related to the specimen 
collection. 
 
11.0 
Intervention Evaluation: Not applicable. 
 
12.0 
Descriptive Factors: None. 
 
13.0 
Follow–up Decision at Evaluation of Patient 
 
13.1 
Women who have either (a) developed NO pregnancy complications, or (b) 
developed IUGR will be eligible for the research ultrasound and placental pathology 
review. 
 
13.2 
Women who have developed pregnancy complications including but not limited to 
those complications listed below will be ineligible if: 
 
13.21 They develop a severe infection 
 
13.22 If they have any other complication which is felt by the study participant’s 
obstetrician to preclude her from further participation in the study 
 
13.3 
If an eligible participant after signing a consent form refuses to undergo the research 
collections required, no further data submission is necessary. 
 
13.4 
When a participant is found to be ineligible (that is, the patient is determined not to 
have satisfied each and every eligibility criteria for study entry at the time of 
registration), the participant will discontinue further study participation. 
 
13.5 
A participant may discontinue study participation at any time. 
 
 
14.0 
Human Studies Evaluation 
 
14.1 
Vulnerable population 

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Our protocol is in accord with the Code of Federal Regulations, Part 46: Protection of 
Human Subjects (section 46.204), which states that pregnant women may be involved in 
research if the following conditions are met: (1) there is a prospect of direct benefit to 
pregnant women and their fetuses, (2) the risk to the fetus is not greater than minimal, 
and (3) the purpose of the research is the development of important biomedical 
knowledge that cannot be obtained by other means. 
 
Fetuses diagnosed with IUGR and/or preterm labor/birth face a significant risk of 
morbidity and mortality before their first birthday. Currently, we don’t know what causes 
IUGR or spontaneous preterm birth, but many studies indicate the immune system is a 
significant contributor to their pathobiology. VUE is strongly associated with these 
diagnoses and has a significant risk of recurrence in a subsequent pregnancy. New animal 
data suggests that prenatal ultrasound may be a real-time clinical tool for assessing fetal 
risk of adverse outcomes. Identifying placental inflammation early may allow for 
effective intervention to minimize placental damage and therefore improve fetal and 
neonatal outcomes. 
 
15.0 
Statistical Considerations and Methodology 
 
15.1 
This is a feasibility study; therefore, no formal power analysis was completed 
 
A total of 61women will be accrued; 40 with a diagnosis of IUGR and 20 gestational 
age matched controls (+/- 1 week) will be analyzed. These women must be 
undergoing obstetric care for their pregnancy at Mayo Clinic. We are not offering 
remuneration; however, based on our previous studies we had a high recruitment and 
retention rate. 
 
All statistical analyses will be performed using JMP version 10.0 (SAS Institute). Data 
will be examined for normality using the Shapiro-Wilk test, and between-group 
differences in expression for each ultrasound measure in our three groups will be 
analyzed with the nonparametric Kruskal-Wallis tests (non-parametric) or ANOVA 
(parametric). Dunn’s method will be utilized to correct for multiple comparison testing. 
P-values<0.05 will be considered statistically significant. 
 
Principal component analysis (PCA) will be used to assess the ability of the biomarker 
panel to separate individual data points (score plot) by projection of the original data on 
the first two principal components (PC1 and PC2), as well as correlations among 
ultrasound measures (loading plot). This will reduce the complexity of multidimensional 
data models by linearly combining multiple factors and downsizing them into new 
variables or principal components while retaining the original information. 
 
15.2 
Inclusion of Women and Minorities 
 
This study will be available to all eligible women regardless of race or ethnic group. 
Based upon the most recent birth data at Rochester Methodist Hospital, where 
approximately 2,500 babies are born each year, 5% of mothers categorized 
themselves as Hispanic/Latina, 5% as Asian, 6% as Black/African American, 83% 
White, and 2% other. In 2019, 235 diagnoses of IUGR were made in Rochester, 
making this study highly feasible. 

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16.0 
References 
 
 
1 
Becroft, D. M., Thompson, J. M. & Mitchell, E. A. Placental villitis of unknown 
origin: epidemiologic associations. Am J Obstet Gynecol 192, 264-271, 
doi:10.1016/j.ajog.2004.06.062 (2005). 
2 
Redline, R. W. Villitis of unknown etiology: noninfectious chronic villitis in the 
placenta. Hum Pathol 38, 1439-1446, doi:10.1016/j.humpath.2007.05.025 
(2007). 
3 
Boog, G. Chronic villitis of unknown etiology. Eur J Obstet Gynecol Reprod 
Biol 136, 9-15, doi:10.1016/j.ejogrb.2007.06.018 (2008). 
4 
Enninga, E. A. L. et al. Upregulation of HLA-Class I and II in Placentas 
Diagnosed with Villitis of Unknown Etiology. Reproductive Sciences, 
doi:10.1007/s43032-019-00101-9 (2020). 
5 
Eloundou, S. N. et al. Placental malperfusion in response to intrauterine 
inflammation and its connection to fetal sequelae. PLoS One 14, e0214951, 
doi:10.1371/journal.pone.0214951 (2019). 
6 
Huang, C. et al. Noninvasive Contrast-Free 3D Evaluation of Tumor 
Angiogenesis with Ultrasensitive Ultrasound Microvessel Imaging. Sci Rep 9, 
4907, doi:10.1038/s41598-019-41373-0 (2019). 
7 
Gong, P. et al. Ultrasensitive Ultrasound Microvessel Imaging for Characterizing 
Benign and Malignant Breast Tumors. Ultrasound Med Biol 45, 3128-3136, 
doi:10.1016/j.ultrasmedbio.2019.08.009 (2019). 
8 
Song, P., Manduca, A., Trzasko, J. D. & Chen, S. Ultrasound Small Vessel 
Imaging With Block-Wise Adaptive Local Clutter Filtering. IEEE Trans Med 
Imaging 36, 251-262, doi:10.1109/TMI.2016.2605819 (2017). 
9 
Huang, C. et al. Debiasing-Based Noise Suppression for Ultrafast Ultrasound 
Microvessel Imaging. IEEE Trans Ultrason Ferroelectr Freq Control 66, 1281- 
1291, doi:10.1109/TUFFC.2019.2918180 (2019). 
10 
Khong, T. Y. et al. Sampling and Definitions of Placental Lesions: Amsterdam 
Placental Workshop Group Consensus Statement. Arch Pathol Lab Med 140, 
698-713, doi:10.5858/arpa.2015-0225-CC (2016). 
11 
Deeba F et al. Attenuation coefficient estimation of normal placentas. 
Ultrasound in Med. & Biol 45, 1081-1093, 
doi:10.1016/j.ultrasmedbio.2018.10.015 (2018).