Scientific Editing

Hannah L. Dimmick, PhD

Scientific editing services allow you to ensure your written documents are as well-communicated as possible. The scientific editor is able to assist on a variety of types of documents – manuscripts, funding applications, conference abstracts, etc. Any stage of the process is welcome. If you need assistance brainstorming, organizing, or a final set of eyes on a draft prior to submission, you are welcome to submit. Writing is about communication, and you want to communicate your research and/or your ideas in an effective way. Scientific editing can ensure your documents are as polished and professional as possible. It may detect logical jumps, correct grammatical errors, or suggest room for improvement in organization or flow. Additionally, journal- and funding source-specific formatting help is available.  

Results from a Human Centered Design Process to Co-create a Peer-Led Sexual and Reproductive mHealth Intervention among Female Students in Zambia

Karen Hampanda,¹ Evans Nkole,² Julia Thorne,¹ Madeleine Sehrt,¹ Lillian Nayame,³ Inyambo Mumbula,³ J. Anitha Menon,³ Mulanda J. Mulawa,³ Twaambo Hamoonga,³ Alain Amstutz,⁴,⁵ Lisa L. Abuogi L,¹ Oliver Mweemba³

^{1 Department of Obstetrics and Gynecology, University of Colorado 2 Centre for Infectious Disease Research Zambia (CIDRZ) 3 University of Zambia 4 University of Basel 5 University of Oslo}

Background

HIV and pregnancy-related complications are leading causes of death for adolescent girls and young women (AGYW) in sub-Saharan Africa. In Zambia, oral pre-exposure prophylaxis (PrEP) and modern contraception are freely available; however, uptake is low among AGYW. Using Human Centered Design (HCD) workshops, this study sought to co-design a peer-led mhealth intervention to prevent HIV and unintended pregnancy among female university students – a neglected, high-risk subpopulation. 

Methods

We recruited 154 stakeholders to participate in seven HCD workshops, including female and male students, student HIV peer educators, sexual and reproductive health (SRH) service providers, and student group leaders. Through participatory HCD activities, we collected qualitative data and artifacts (e.g. photographs of output from activities) to inform student SRH needs and co-design key intervention components and the content and functionality of a mobile application to facilitate the intervention.

Results

Participants in the HCD workshops highlighted their interest in using a peer-based messenger app to disseminate SRH information and facilitate health service navigation among university students. Key barriers to intervention efficacy included access to mobile phones and internet to use the app, lack of cultural and religious acceptance of an SRH intervention for young women, and fears around health provider attitudes. Peer navigator characteristics considered to be important included the ability to make students feel comfortable and accepted, thorough SRH training, and being similar age as students. Facilitators for effective intervention included addressing social barriers and improving accessibility to youth friendly SRH services. Recommended content of the mobile application included SRH information and local SRH resources, and private, secure communication between users of the app. “TwySHE” was recommended as the name of the intervention and app, meaning in local dialects “she needs to know.”     

Conclusions

Through the HCD workshops, we gained significant insight into the design and implementation of an acceptable peer navigator mhealth intervention aimed at increasing modern contraception and PrEP use among at-risk female university students. Peer navigators with lived experience, stigma reduction efforts, and youth friendly SRH services were identified as key components needed within the proposed intervention. Participants universally felt that a mobile app was an acceptable way to facilitate the peer navigator intervention. Results from these HCD workshops will be useful in implementing the proposed intervention among university students in Zambia and could be applied to other similar settings across sub-Saharan Africa.

Intention to use PrEP among Sexually Active Female University Students in Zambia: a Cross Sectional Survey to Understand Influential Characteristics and Behaviors

Hampanda K, Bolt M, Nayame L, Sehrt M, Thorne J, Harrison M, Pitney J, Amstuz A, Abuogi L, Mweemba O

Background

Adolescent girls and young women (AGYW) in sub-Saharan Africa are one of the most at-risk populations for HIV acquisition. In Zambia, HIV prevalence is four times higher for young women (9%) compared to young men (2%) aged 20-24. While oral pre-exposure prophylaxis (PrEP) is widely available in Zambia, uptake is slow among AGYW. Female university students are a neglected high-risk sub-population for HIV infection due to demographic and behavioral risk factors. This study sought to establish predictors of intention to use PrEP among sexually active female Zambian university students.

Methods

We recruited female-identifying students at least 18 years of age at a public university in Lusaka, Zambia to complete an online REDCap survey. The questionnaire included intention to use PrEP in the next year, knowledge, perceptions, and stigma of PrEP, HIV risk perception, and epidemiologic HIV risk factors, including sexual behaviors, pregnancies, and recent STI symptoms. Missing data was addressed with multiple imputation, and analyses were conducted with descriptive statistics, regression analysis, and mediation analysis. 

Results

Among sexually active survey participants with complete outcome data (n=454), 118 (26%) indicated intention to use PrEP in the next year. Any prior or current use of PrEP was extremely rare (< 5%). Odds of intention to use PrEP were higher among those with self-perceived high HIV risk (aOR 3.08); lower with more negative PrEP stigma scores (higher score equals more stigma, aOR 0.91); lower with more negative PrEP perceptions (higher score equals greater negative perception, aOR 0.91); and higher with more years at university (aOR 1.47)(all p < .01). The number of epidemiologic HIV risk factors was associated with greater PrEP intention in a model adjusting for covariates but not for self-perceived HIV risk; after adjustment for self-perceived HIV risk, this relationship weakened substantially. Further analyses estimated that self-perceived HIV risk mediates 69% of the relationship between epidemiologic HIV risk factors and PrEP intention. Participants with a high epidemiological HIV risk did not rank themselves as such, with only 29% of them correctly assessing their high risk (p<0.001).

Conclusions

While intention to use PrEP does not equate to actual PrEP use, it is a necessary step in the cascade. This study found relatively high interest in PrEP among sexually active female university students in Zambia. Our findings underscore the importance of reducing PrEP stigma among AGYW to increase PrEP demand. Perceived high HIV risk emerged as a significant predictor of intention to use PrEP in the next year. Yet, the women in this study often underestimated their HIV risk. Along with education and stigma reduction, there is a need for approaches that help young women accurately assess their risk of HIV and make informed decisions about PrEP uptake.

Integrated Mental Health Services in a Fetal Care Center

McKenzie Hanigan, BS; Kori A. Baker, BS; Heather Buxton, MD, MEd; Virginia Lijewski, MPH; Henry L. Galan, MD; Teresa Harper, MD; Erin McNulty, MD; Katherine Arora-Frank, MSW; Cristol Keenan, RN; Cristina Wood, MD, MS; Jamie Jamison, RN; Katherine Cox

Objective

The Colorado Fetal Care Center (CFCC) identified the need for mental health services for high-risk pregnancies and established an integrated mental health program in September 2023. The objective of this study was to compare Edinburgh Postpartum Depression Scale (EPDS) scores in CFCC patients with and without enrollment in the mental health program and to examine the utilization and impact of these services.

Study Design

Utilization of mental health services, diagnoses, and therapeutic interventions were collected on all patients seen by the CFCC mental health team between 9/05/2023 and 7/16/2024. Descriptive statistics were used to define the patient population and services, and a t-test was performed to compare mean EPDS scores of patients with and without mental health services.

Results

In 10 months, 49 patients (21% of the CFCC’s delivery population) were seen by the mental health program. Most patients were white (75.5%), non-Hispanic (71.4%), married (67.3%), and spoke English as a primary language (98%). The majority of patients had a fetal diagnosis associated with cardiac anomalies (28.6%) and were referred for psychiatric evaluation in the postpartum period by OB hospitalists. Patients received a median of 4 psychiatric visits and 11 social work visits. Most patients engaged in supportive psychotherapy and approximately 61% utilized medication management. Depression (26.5%), anxiety (12.2%), and trauma related disorders (57.1%) were the most common diagnoses identified in this patient cohort. The mean EPDS score within 6 weeks of delivery was higher for patients referred to the program (12.1 vs. 4.0, p <0.01).

Conclusions

The integrated mental health program at CFCC has significantly improved identification of and support for mental health in high-risk pregnancies. Current program engagement aligns with national prevalence of perinatal mood and anxiety disorders (PMADs). The high utilization of therapy and medication management within this program validates the need for integrated mental health programming. Future efforts will focus on expanding these services to better support patients, with a particular emphasis on NICU families, and measurement of patient outcomes to address remaining gaps in care.

Cheap, convenient, and discrete: why young women in a high HIV prevalence setting of Zambia overuse emergency contraception

Sehrt M, Nayame L, Thorne J, Amstutz A, Harrison M, Mweemba O, Hampanda, K.

Background

Unintended pregnancies among adolescent girls and young women (AGYW) are common worldwide but rates in Sub-Saharan Africa are especially high. In Zambia, the study setting, one in three women have been pregnant by age 19. The objective of this study is to investigate female university students use and perceptions of modern contraception methods in Lusaka, Zambia. 

Methods

Female university students over the age of 18 who participated in an online survey during the parent study were invited to opt-in to participate in a semi-structured focus group discussion (FGD). Discussion topics included sexual and reproductive health perceptions and needs, focused on unintended pregnancy and modern contraception perceptions and behaviors. FGDs were audio-recorded, transcribed verbatim, and thematically analyzed using constant comparison methods. 

Results

A total of 34 female students participated in five FGDs. Overarching themes included a consistent pattern of emergency contraception (i.e., “the morning after pill”) overuse among female university students, which was the preferred option for pregnancy prevention because it is convenient, cheap, and discrete. Emergency contraception overuse occurs in the context of numerous social and health system barriers to accessing long-term, more reliable methods of contraception, including stigma surrounding premarital sex, particularly for young women, fear of poor treatment from nurses or being denied products, and fears of community or family finding out they were accessing contraception, as well as many misconceptions surrounding the side effects of more reliable contraception methods, such as the intrauterine device.

Conclusions

Female university students frequently use emergency contraception to avoid unintended pregnancy despite the availability of other more reliable methods at public health clinics. This study highlights why emergency contraception is the preferred pregnancy prevention methods in a sub-population of young women. Along with improving sexual reproductive health education, efforts to reduce unintended pregnancy in such settings should consider differentiated service delivery models that would enable women to access more reliable contraception methods in an easier and discrete way.

Integration of Multidisciplinary Substance Use Treatment into Routine Prenatal and Postpartum Care 

Kylie Culp, MD; Rachael Duncan, PharmD, BCPS, BCCCP; Stefka Fabbri, MD, MPH

Introduction

There has been a startling 190% increase in pregnancy-associated deaths due to drug-related causes over the past decade. Enhanced screening and treatment for substance and opioid use disorders (SUD/OUD) during pregnancy and the postpartum period are urgently needed, as all of these deaths are preventable. 

Methods

Denver Health, an urban safety net institution, is collaborating with Maternal Overdose Matters (MOMs+) and the Colorado Perinatal Care Quality Collaborative (CPCQC)  to improve the screening, treatment and connection to resources for pregnant and postpartum patients affected by substance use. Involving a multidisciplinary approach across obstetrics, midwifery,  family medicine, anesthesia, nursing, lactation, social work, neonatology, and addiction medicine, the program spans the entire DH healthcare system reaching across 15 outpatient prenatal clinics and multiple inpatient units. The scope of the initiative spans prenatal care, labor and delivery, postpartum care, obstetric triage, and non-obstetric inpatient units. The aim of the program is to integrate multidisciplinary SUD/OUD care into routine perinatal care such that there is standardized universal screening throughout pregnancy and postpartum; robust access to addiction and behavioral health providers, along with peer support services; standardized checklist embedded in the medical record that promotes best practices; decrease of stigmatization through staff education; equitable care by creating standardized guidelines for toxicology testing, social services reporting, and treatment of SUD/OUD.

Results

The initiative will be evaluated based on workflow processes such as checklist completion, access to care, and connection to community resources. Clinical outcomes such as frequency of screening, initiation of treatment, and distribution of naloxone will also be measured.  In initial data collection, we have improved screening rates by over 20% and have eliminated racial and ethnic biases in urine toxicology testing. 

Implications

By identifying and providing timely access to care for individuals affected by substance use, there is potential to significantly decrease perinatal mortality. With appropriate training and support, obstetric providers can play a pivotal role in delivering comprehensive care for all medical disorders, including substance use.

The Problem of Laparoscopic Salpingectomy Cancellation: Patient Reported Reasons and Areas for Improvement

Megan Masten¹, Sofie Rosenberg², Nicole Larrea³, Claire Schultz³

1. Department of Obstetrics and Gynecology, Division of Family Planning, University of Colorado Anschutz Medical Campus, Aurora, CO, United States.
2. University of Colorado School of Medicine, Aurora, CO
3. Department of Obstetrics and Gynecology, Denver Health and Hospital Authority, Denver, CO, United States.

Objectives 

To identify patient-reported reasons for canceling laparoscopic salpingectomies for permanent contraception via a semi-structured phone interview and survey.

Methods

Patients who were 18-50 years old and English or Spanish speaking who canceled a laparoscopic salpingectomy for permanent contraception at our urban county hospital were offered participation. A survey was sent and semi-structured phone interview utilized with prompts to explore hospital-related, insurance-related, and personal reasons for cancellation, as well as future contraceptive plans. A qualitative review of responses was performed via inductive coding. Codes were reviewed and edited for consensus. 

Results

Fifteen phone interviews were conducted between January 2023 - February 2024. The majority of participants identified as white/Caucasian (73.3%) and Hispanic/Latinx (60%). The majority (80%) had public insurance. The most common factor contributing to surgery cancellation was financial concerns, with insurance issues being the frequently mentioned within this category. Other barriers included lack of childcare and transportation issues. Most participants were confident about desiring permanent contraception (9/15) and still desired laparoscopic salpingectomy (8/15). A minority (2/15) of participants felt sure they did not want laparoscopic salpingectomy, and 4/15 were unsure due to fertility concerns, changes in relationship status, and other issues. Payment plans, cost transparency, and a set start time were reported as ways the hospital could help patients receive laparoscopic salpingectomy. 

Conclusions 

Financial concerns are a major barrier to receiving laparoscopic salpingectomy for permanent contraception despite steeply discounted services at our institution and public insurance coverage. Most participants in our study still desired laparoscopic salpingectomy. 

Mifepristone Initiation for Management of Early Pregnancy Loss in the Emergency Department: A Quality Improvement Study

Sofie Rosenberg, BS², Nancy Fang, MD MS^1,2

1. Department of Obstetrics and Gynecology, Division of Family Planning, University of Colorado Anschutz Medical Campus, Aurora, CO, United States.
2. University of Colorado School of Medicine, Aurora, CO

Objective

Early Pregnancy Loss (EPL) refers to nonviable intrauterine pregnancy containing either an empty gestational sac or a gestational sac containing an embryo/fetus with no fetal cardiac activity within the first 12 weeks of gestation.¹ Approximately 18% of EPLs are diagnosed in the emergency department (ED).² We conducted a quality improvement initiative to improve access to mifepristone in the ED and aimed to characterize changes in EPL management before and after implementation.

Methods

A multi-disciplinary team from the ED and OBGYN revised a care pathway for vaginal bleeding in the ED to include the addition of mifepristone for medical management of EPL (November 2022).  We conducted a retrospective chart review (December 2021 - December 2023) to measure uptake of the new protocol. Charts were selected based on ICD-10 coding for vaginal bleeding during pregnancy, miscarriage, missed abortion, incomplete abortion. We performed descriptive analyses using chi-squared tests (IBM SPSS Version 29). This study was deemed exempt by the Colorado Multiple Institutional Review Board.

Results

Of the 718 charts reviewed, 16.7% met criteria for EPL and were eligible for medical management at time of presentation.  The median age was 29.4 and the majority of patients were publicly insured (62.4%). The addition of mifepristone to our ED was associated with fewer patients electing expectant management (68.6% v 32.5%, p=0.002) and fewer additional ED visits (54.1% v 31.7%, p=0.020). Mifepristone administration for management of EPL increased significantly (27.3% vs 82.4%, p=0.001).

Conclusions

The ED is a common place for diagnosis and management of EPL. After the initiation of the new protocol, patients were more likely to elect medication management and less likely to return to the ED. Our study shows the ED is a safe and effective clinical space to provide medication management for EPL.

Citations

1. American College of O, Gynecologists' Committee on Practice B-G: ACOG Practice Bulletin No. 200: Early Pregnancy Loss. Obstet Gynecol 2018, 132(5):e197-e207. 
2. Benson LS, Holt SK, Gore JL, Callegari LS, Chipman AK, Kessler L, Dalton VK: Early Pregnancy Loss Management in the Emergency Department vs Outpatient Setting. JAMA Netw Open 2023, 6(3):e232639.
   

Induction of the RAS signaling network following neoadjuvant chemotherapy predicts shorter disease-free intervals

Benjamin G. Bitler¹, Matthew J. Sikora², Kian Behbakht³, Lucy Van Kleunen⁴, Rebecca J. Wolsky², Aaron Clauset⁴

1. Department of Obstetrics and Gynecology, Division of Reproductive Sciences, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.
2. Department of Pathology, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.
3. Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA. 
4. Department of Computer Science, University of Colorado Boulder, Boulder, Colorado, USA

A significant challenge in clinically managing high grade serous carcinoma (HGSC) of tubo-ovarian origin is the high rate of disease recurrence, as recurrent disease is often less responsive to chemotherapy. However, the neoadjuvant chemotherapy (NACT) treatment paradigm represents a unique opportunity to define chemo-response, e.g. chemotherapy-induced tumor microenvironment remodeling. Our study of NACT-treated HGSC uncovered that HGSC tumors with an increased chemo-induced stress response (e.g., IL-6, WNT4, ATF3, or CEPBbeta induction) have an increased risk of recurrence and shorter disease-free intervals. In addition, we and others have described that immune cell tumor infiltration, including CD8 T cells, natural killer cells, and B cells, are positively correlated with improved survival outcomes in patients with HGSC. In this study, we expanded our NACT cohort of matched pre- and post-chemotherapy tumors. We hypothesized that this larger cohort would reveal novel signatures of immune responses and immune infiltration following chemotherapy, predicting delayed disease recurrence. The primary objective is to identify targetable approaches to prevent HGSC disease recurrence following chemotherapy. 

We examined 65 matched pre- and post-chemotherapy primary HGSC tumors with the Nanostring Immune Oncology 360 platform (University of Colorado Cohort n=34 and James NE GSE201600 Cohort n=31). A board-certified gynecologic oncology pathologist (RJW) confirmed the presence of tumor cells, and RNA was collected from formalin-fixed paraffin-embedded tumors. The Nanostring assay was completed using the nCounter technology and the analysis was performed using nSolver software. The cohort's average age at diagnosis was 62.3 years old, and the average disease-free interval (DFI = date of first recurrence—date of chemotherapy completion) was 11 months, with 53 of 65 patients having a recurrence. There were 25 chemosensitive (>12 months DFI), 22 chemoresistant (6 to 12 months DFI), and 18 chemorefractory samples (<6 months DFI). The primary focus was identifying associations between disease-free interval and fold change in gene expression between pre- and post-chemotherapy tumor samples. 

Based on the changes in gene expression signatures, we observed a significant association between elevated RAS signaling and disease-free intervals. Specifically, chemotherapy-induced NRAS expression significantly correlated to shorter DFI (High NRAS induction, DFI=7 months; Low NRAS induction, DFI=14 months; Hazard Ratio: 0.488, 95% CI 0.26-0.90, p=0.0098). Tumors with induction of NF1, a tumor suppressor and negative regulator of RAS, were more often defined as chemosensitive compared to chemorefractory (p=0.0091). 

Our study provides insights into the management of HGSC. HGSC tumors with chemotherapy-induced activation of the RAS signaling network (elevated NRAS or depleted NF1 expression) are associated with a worse prognosis. These findings are in line with previous research in NACT. The RAS signaling network, therefore, represents a potential therapeutic approach to extend disease-free intervals, a crucial step in improving patient outcomes. 

Alisertib, an Aurora Kinase A inhibitor, works as CBX2 inhibitor and decreases tumor progression in HGSC.  

Ritsuko Iwanaga, Tomomi Yamamoto, Elizabeth R. Woodruff, Benjamin G. Bitler, and Lindsay W. Brubaker

Objectives

Polycomb repressor complex 1 (PRC1) is a key epigenetic regulator that is often linked with transcriptional repression of target genes. PRC1 plays critical roles during embryonic development, stem cell renewal, and cancer development. Chromobox 2 (CBX2), a subunit of PRC1, is aberrantly upregulated in multiple cancers including high grade serous carcinoma (HGSC) of tubo-ovarian origin. In patients with HGSC, increased CBX2 expression in tumors is significantly associated with worse disease-free and poor survival outcomes. Our work and others have shown that knocking down CBX2 in HGSC models decreases stemness, increases cisplatin chemosensitivity and delayed tumor progression in an immune competent mouse model. We developed CBX2 inhibitory peptide (CBX2i) to demonstrate pre-clinical efficacy. To accelerate pharmacological development process, we have performed molecular similarity-based screening within FDA approved drugs, finding CBX2 targeted therapy agents.

Methods

We used several HGSC models – OVCAR4, COV504, and PEO1. We performed bio-similarity screening and identified 10 small molecules that are significantly biosimilar to the CBX2i structure. We evaluated their ability to 1) Inhibit HGSC cell proliferation, 2) Downregulate a CBX2 target gene, TMEFF1, and 3) Attenuate stemness via ALDEFLUOR, ALDH assay. We also performed ultralow dilution assay (ULDA) in 3D culture conditions and evaluated stem cell frequency when the cells were treated with cisplatin and Alisertib. Next, we used an immune-competent ID8 ovarian cancer mouse model and treated tumor burdened mice with combination of cisplatin and Alisertib, to assess Alisertib have an additive effect on tumor dissemination and tumor weight. 

Results

Among 10 molecules identified by bio-similarity screening, four molecules including Raltitrexed, GTX-007, LY315920, and Alisertib decreased TMEFF1 expression, decreased cell proliferation, reduced ULDA colony formation, and limited ALDH activity. Alisertib was identified as the top candidate based on its ability to inhibit proliferation and stemness and improve response to chemotherapy. We found that Alisertib is less effective in CBX2 knockout HGSC cells. An ULDA showed that compared to vehicle control, Alisertib significantly reduced the frequency of stem cells (1:48 versus 1:1022). Consistent with prior work, cisplatin increased the stem cell frequency (1:41) and this response was blunted when combined with Alisertib (1:804). Next, we evaluated the anti-tumor efficacy of the combination of cisplatin and Alisertib in a syngeneic ID8 mouse model. Compared to controls, Alisertib decreased average tumor weight (0.0917g vs. 0.0544g, p=0.0396) and average number of dissemination sites (27.2 vs. 8.7, p=0.0028). Compared to Cisplatin alone, we observed that the combination has an additive effect on decreasing the dissemination site (p=0.0265).

Conclusions

CBX2 is an attractive therapeutic target in the HGSC chemotherapy resistant context. We discovered that Alisertib reduces stemness and improves chemotherapy anti-tumor responses. Future work will interrogate Alisertib-dependent immune responses and survival outcomes.  

Integrated Ethics Curriculum Meets OB/GYN Resident Reported Needs 

Jennifer Braverman, MD, MA, Virginia Lijewski, MPH, and Heather Straub, MD

Introduction

Ethics education is an important part of OB/GYN training. The baseline survey showed that OB/GYN residents and faculty desired more formal ethics education. 

Methods

From 2020-2023, existing MFM Division “perinatal ethics grand rounds” were incorporated with other lectures on non-MFM ethical topics in the resident curriculum. Surveys were given at baseline, midpoint, and after 2 years regarding perception of volume, importance, content of ethical material and comfort with ethically challenging situations. This was considered IRB exempt.

Results

13 lectures/activities were presented by 6 fellows, 6 residents, and 5 faculty/staff from 7 OB/GYN Divisions. Overall, 29 residents and 44 faculty completed the baseline survey. Most residents (95.7%) and all faculty reported ethics education should be offered and/or required to complete residency.  Prior to the program, a large majority (81.3%) of residents wanted more ethics education while after the 2-year mark 57% residents wanted more while 42.9% wanted the same as current (P <0.01). No resident replied they wanted less ethics education. More residents felt “somewhat prepared” to deal with ethically challenging situations after the program (71.4% vs 56.3%, p <0.01). There were significant differences between resident and faculty preferred topics. The top three topics for faculty were: 1. Informed consent, 2. Doctor-Patient relationship, 3. Abortion, while the top three topics for residents were: 1. Doctor-Patient relationship, 2. Forgoing life support, and 3. Access to Healthcare (p<0.01)

Conclusions/Implications

An integrated ethics curriculum can meet the perceived needs of OB/GYN residents and faculty by having them involved in activity/lecture development. 

Learning objectives (min 3)

1. Understand gaps in ethics education 
2. Learn about an integrated ethics curriculum 
3. Consider whether this curriculum could meet your educational needs 
   

Disparities in Child Protective Services reporting among pregnant individuals with positive urine toxicology at delivery hospitalization

Chase Hopkins, MPH¹, Virginia Lijewski, MPH¹, Victoria Cates, MD¹, Jeanelle Sheeder, PhD¹, Kaylin Klie, MD², Heather Straub, MD¹

1. University of Colorado, Department of Obstetrics and Gynecology
2. University of Colorado, Department of Family Medicine

Objective

This study aims to assess differences in Child Protective Services (CPS) reporting among individuals with a positive urine toxicology (uTox) test during delivery hospitalization. We hypothesized that individuals reported to CPS are more likely to identify as non-White race.

Methods

Individuals receiving prenatal care and delivering at any UCHealth location between 2017 and 2022 with a positive uTox test at delivery were included. Demographics, medical history, uTox results, infant toxicology, and CPS reporting were collected from patients' medical records. Mann-Whitney U tests for continuous variables and chi-squared or Fisher's exact tests for categorical variables were used to compare patients with and without CPS reporting. Multivariable logistic regression was used to determine independent predictors of reporting.

Results

There were 819 individuals with a positive uTox during their delivery hospitalization identified, with CPS reporting documented for 74%. Race was not associated with increases in CPS reporting. Residing in an urban area (aOR 4.2, 95% CI 2.4-7.3), testing positive for illicit substance use (including cannabis) at delivery (aOR 6.0, 95% CI 4.0-9.1), infant receipt of testing (aOR 2.2, 95% CI 1.1-4.4), and infant positivity for any substance (aOR 3.8, 95% CI 2.4-6.0) increased the odds of CPS reporting.

Conclusions

Race did not affect CPS reporting rates. However, other demographic, medical, and substance use factors were associated with the likelihood of reporting. Toxicology testing patterns and CPS reporting varied depending on the delivery hospital. Our study results highlight the need for standardized and equitable substance use detection and reporting policies.

Retrospective evaluation of patient characteristics and pregnancy status in a large cohort receiving preconception counseling

Rabbia Imran¹, Virginia Lijewski¹, Lamia Alamri¹, Teresa Harper¹

1. University of Colorado School of Medicine, Aurora, CO 

Keywords: preconception counseling, MFM, maternal-fetal-medicine, conception, prenatal, recommendation adherence

Objective 

To investigate demographic and clinical characteristics among patients seeking preconception counseling from MFM specialists. Our primary objective was to determine whether patients who adhered to all recommendations were more likely to become pregnant within 12 months of preconception care. Our secondary objective was to explore differences between the pregnant group vs. the non-pregnant group regarding recommendations.

Study Design 

This is a retrospective study of patients ≥18 receiving preconception counseling at the University of Colorado between 1/2020 and 12/2022. Patients who were pregnant at the time of the preconception visit were excluded. Patient demographics, referral reason, provider recommendations, adherence to recommendations, and pregnancy status at 12 months post-appointment were collected by reviewing electronic medical records. Chi-square or Fisher’s exact tests were used to compare categorical variables.

Results

Of the 563 study participants,184 (32.7%) became pregnant within 12 months of preconception care. Overall, most patients were non-Hispanic White (76.7%), married/partnered (90.8%), ≥35 years old (51.5%), and had an advanced degree (32.7%). Patients were commonly referred to preconception counseling by their primary obstetric provider (89.9%) due to existing medical conditions (46.9%) and/or IVF planning (29.1%). Patients who became pregnant were more likely to be < 35 years old and married/partnered (p < 0.01). The types of recommendations given between pregnant vs. non-pregnant patients were not significantly different, including general preconception (p = 0.30), weight management (p = 0.12), disease management (p = 0.23), medication management (p = 0.78), lab tests (p = 0.18), other/future pregnancy recommendations (p = 0.18), and recommendations to proceed or delay conception (p = 0.10). A larger proportion of pregnant patients followed all recommendations (p < 0.01).

Conclusion

The types of recommendations given by MFM specialists were consistent, and patients who fully adhered to preconception recommendations were more likely to become pregnant. Further analysis is needed to investigate the impact of specific recommendations on adherence. Our data also highlights the need to engage a more diverse population in preconception care. We hope to use these data to develop consistent clinical care recommendations and optimize preconception services.

Teens with Diabetes Receive Low Rates of Contraception and Preconception Counseling

Resident: McKenna Kelly MD
Primary Mentor: Shannon Son MD, MSC      
Additional Collaborators: Lauren Sayres MD, Erin Finn MD, Kristen J. Nadeau MD, MS, Layla Abushamat MD, Adnin Zaman MD, Linda Barbour MD, MSPH

Objective

In 2022, US females aged 15-19 years had a birth rate of 13.5 per 1000. Pregnancy in adolescents with type 2 diabetes mellitus (T2DM) is associated with poor obstetric outcomes, including high rates of major fetal malformations. While contraceptive and preconception counseling at puberty is strongly recommended by the American Diabetes Association and American College of Obstetricians and Gynecologists, a recent study demonstrated low rates of counseling in adult reproductive age females with diabetes. We aimed to assess contraceptive and preconception counseling rates in an adolescent population with T2DM. We hypothesized that counseling rates would be higher in adolescents than adults with diabetes.

Methods

In this retrospective cohort, adolescents 12-21 years old, assigned female at birth, and seen in a pediatric academic T2DM clinic between July 1, 2021 and December 31, 2021 were evaluated. The primary outcome was a composite rate of contraceptive counseling or prescription in the last year, preconception counseling, or gynecology referral. Patients taking hormone therapy for other indications were excluded. Adolescent data were compared to a previously collected cohort of 50 adult females with diabetes.

Results

Seventy adolescents met entry criteria and 21 (30%) achieved the primary composite outcome as compared to 18% in a previously collected adult cohort (p=0.2). Contraception counseling was documented for 19 (27.1%) adolescent patients and two (4.0%) adult patients (p=0.001). Preconception counseling was documented for one (1.4%) adolescent patient and seven (14%) adult patients (p=0.01). There was no evidence of endocrinologist-prescribed contraception during the chart review period. Two (2.9%) adolescents were referred to gynecology providers and no gynecology referrals were placed for the adult patients.  Contraception was less commonly utilized by adolescents compared to adults (25.7% vs 62.0%; p=0.0001). Adolescents most commonly utilized combined oral contraceptive pills (cOCPs) (10%), implants (7%), and abstinence (4%).      

Conclusions

Despite robust recommendations for preconception/contraceptive counseling in patients with diabetes, implementation remains grossly inadequate in academic endocrinology clinic settings for both adolescent and adult patients. Our findings call for intensive efforts to improve these practices with a goal of minimizing unplanned pregnancies as well as associated poor obstetric outcomes.

The social vulnerability (SV) index is related to toxicology testing during pregnancy

Virginia Lijewski, MPH¹, Jeanelle Sheeder, MSPH, PhD¹, Heather Straub, MD¹

1. University of Colorado, Department of Obstetrics and Gynecology

Objective

To compare the demographic profiles and SV of pregnant patients who did and did not receive urine toxicology (uTox) testing. We also wished to determine if the declaration of the opioid epidemic as a National Public Health Emergency (PHE) on 10/27/17 led to increased testing rates. We hypothesized that patients receiving uTox testing were more likely to be SV than those who did not and that testing rates would increase post-PHE declaration.

Study Design

Patients receiving prenatal care and delivered within a multihospital academic healthcare system with urban, suburban, and rural hospitals from 8/23/2016-12/31/2018 were included. Patient addresses were matched to the CDC Social Vulnerability Index to obtain overall and domain-specific (Household Composition, Socioeconomic Status, Racial/Ethnic Minority Status, and Housing/Transportation) SV scores. Scores ≥0.75 indicate high SV. Chi-squared or Fisher’s exact tests were used to compare categorical variables, and Mann-Whitney U tests were used to compare continuous variables.

Results

3,938 patients were included; 3,405 (86%) did not receive uTox testing, and 533 (14%) did. Testing rates did not differ pre- and post-PHE (14% vs. 13%; p=0.25). Patients with uTox testing were more likely to be non-Hispanic Black (15% vs. 11%; p<0.01) or Hispanic (28% vs. 22%; p<0.01), older (27 yr vs. 20 yr; p<0.01), unmarried (53% vs. 78%; p<0.01), and without private insurance (22% vs. 61%; p<0.01). Overall SV scores were significantly higher among those who received uTox testing (29% vs. 22%; p<0.01). All domain-specific SV scores were higher among those who received uTox testing, except for racial/ethnic minority status (26% vs. 23%; p=0.25). Patients who received uTox testing were more likely to have inadequate prenatal care (37% vs. 24%; p<0.01), fetal or neonatal demise (3% vs. 1%; p<0.01), abruption (4% vs. 2%; p=0.01), PPROM (8% vs. 6%; p=0.01), and preterm delivery <37 weeks (27% vs. 12%; p<0.01).   

Conclusions

The PHE declaration did not impact uTox testing rates. Demographic and other clinical differences in uTox testing may highlight provider biases in substance use detection practices. A high SV score may be a helpful tool for investigating other ways to make substance use testing in pregnancy more reliable.

Reducing the Burden of Genetic Variants of Uncertain Significance: The value of a multidisciplinary approach to clinical interpretation.

Kestutis Micke, MS, CGC; Hannah Elfman, MS, CGC; Patrick McGrath, PhD; David Clouthier, PhD; Katherine Fantauzzo, PhD; Charu Kaiway, MD; Desiree, Demille, MD; Sasha Parets, MD; Paul Vrana, MD; Nicole Burns, MD; Hannah Cox, MD; Manesha Putra, MD; Shawn McCandless, MD; Austin Larson, MD

Background

Genetic testing during pregnancy is a powerful tool for diagnosing genetic syndromes, however, results are not always straightforward. A variant of uncertain significance (VUS) may be reported in up to 42% of whole exome sequencing (WES) results. A VUS is a genetic variant that has unclear connections to a health condition. As a VUS is not clearly benign or pathogenic, the American College of Medical Genetics does not recommend utilizing a VUS in clinical decision making, particularly in irreversible decisions such as termination of pregnancy. The inclusion of a VUS can induce significance anxiety or complicate a family’s decision-making during pregnancy. We formed a multidisciplinary team to review all prenatal WES results and clarify interpretation of any reported variants. We hypothesized that multidisciplinary review would reduce the number of VUSs and increase the diagnostic rate of genetic testing.

Methods

A clinical database of prenatal WES cases performed through the Children’s Hospital Colorado, UCHealth, and University of Colorado has been maintained and cases were analyzed from this dataset. WES results for an individual were categorized as diagnostic, non-diagnostic, or uncertain. We analyzed the frequency of each result at the time of test report, following multidisciplinary review, and postnatally. Additionally, multiple genetic variants may be reported for a single case. Thus, we also analyzed the frequency of diagnostic, non-diagnostic, and uncertain results for each individual variant at the same time points. 

Results

We identified 118 cases in which prenatal WES was completed and for which postnatal outcomes were available. Laboratory reported test results demonstrated 26 (22%) cases with diagnostic results, 53 (45%) with non-diagnostic results, and 39 (33%) with uncertain results. Following review by the multidisciplinary team, the number of uncertain results was reduced to 10 (8%), while the number of diagnostic and non-diagnostic results increased to 42 (36%) and 66 (56%), respectively. Similar numbers were seen postnatally.
Fifty-two (44.1%) individuals had zero variants reported. Of the remaining 66 cases, there were 109 total variants, with 58 variants classified as VUSs. There were 9 remaining VUSs following multidisciplinary review. Twenty-one variants were upgraded to diagnostic, while 28 were downgraded to non-diagnostic. 

Discussion

Due to multidisciplinary review of prenatal WES results, we have demonstrated the ability to reduce the number of uncertain variants in our patient population by 85%. These efforts may reduce patient anxiety and uncertainty during pregnancy. Further, clinical reclassification of VUSs allows the medical team to confidently make management recommendations prenatally and postnatally. Our data demonstrates how advanced diagnostic genetic testing can be successfully integrated into practice and reduce uncertainty for patients and care teams.

Testing the Clinical Significance of Third Trimester Uterine Artery Dopplers in Pregnancies Complicated by Fetal Growth Restriction

Roopjit K. Sahi¹ MBBS, Manesha Putra¹ MD, John C. Hobbins¹ MD

1. Division of Maternal Fetal Medicine, Department of Obstetrics and Gynecology, University of Colorado Anschutz Medical Campus, Aurora, CO

Objectives

To analyze the third trimester uterine artery Dopplers in severe and non-severe fetal growth restricted (FGR) pregnancies and to evaluate any association of abnormal Doppler values with fetal cardiac parameters and adverse neonatal outcomes.

Methods

Pregnancies were analyzed from an ongoing observational study at one of the high-risk pregnancy clinics, and classified as either severe or non-severe FGR (per SMFM guidelines, either estimated fetal weight, EFW or abdominal circumference, AC less than 3rd percentile or abnormal Dopplers with EFW or AC between 3rd – 10th percentile was categorized as ‘severe’; all other FGRs with EFW or AC less than 10th percentile were termed ‘non-severe’). Fetal biometric (EFW; AC), Doppler values (umbilical artery pulsatility index, UA PI; uterine artery pulsatility index, Ut A PI; cerebroplacental ratio, CPR), and cardiac variables (cardiac area, CA; global sphericity index, GSI; transverse width, TW; left ventricular cardiac output, LVCO) were analyzed in both groups. Different Doppler combinations were tested to assess any improvement in the predictive performance for adverse fetal cardiac and neonatal outcomes.

Results

In a cohort of 102 pregnancies, 62 (60.8%) were categorized as severe FGR and 40 (39.2%) were sub-grouped as non-severe FGR. The incidence of abnormal Ut A PI was 25.8% in severe FGR group and 27.5% in the non-severe group. Abnormal UA PI had a strong association with abnormal Ut A PI – odds ratio (OR) 4.51 (1.32 – 16.2; p 0.01), and CPR had an inverse association with Ut A PI - OR 0.11 (0.01 – 0.69; p 0.03). Odds of Fenton birthweight being less than 10th percentile with abnormal Ut A PI was 3.33 (0.99 – 11.34; p 0.05) in severe FGR group; and 9.2 (1.93 – 43.6; p 0.005) in non-severe group. Severe FGR fetuses with abnormal Ut A PI had a higher incidence of hearts with GSI less than 5th percentile (OR 4.42, p 0.03). However, multivariate analysis of Ut A PI along with other Doppler indices to predict adverse fetal cardiac or neonatal outcomes did not show any significant improvement. 

Conclusion

Elevated Ut A. Doppler values are significantly associated with preterm delivery and smaller neonates with rounder hearts in fetal growth restricted pregnancies. Thus, the role of maternal hemodynamics in the pathophysiology of FGR may need reassessment.

Does integration of diabetic educators into pregnancy care improve glycemic control?  

Heather Straub, MD, Virginia Lijewski, MPH, Amy Wehbe, MS, Jenifer Kruse, MS, David Duarte-Corado, BA

Objective

To determine whether integrating diabetic educators into pregnancy care improves glycemic control.

Study Design

A formalized diabetes in pregnancy program was developed in 2019 involving 2 diabetic educators supervised by MFM provider & secure electronic messaging. We hypothesize that integration of diabetes educators improves glycemic control, & tested this using a pre-post study design. Prior to establishment of the program (10/1/2018-9/30/2019; PRE), communication was through telephone messaging with the MFM provider. The POST period was a year after the program began (10/1/2021-9/30/2022). Variables of interest were volume of patients seen, # of ultrasounds per patient, demographics, medication initiation, percentage of time of logs in goal, delivery medication advice & documentation of compliance. This study was IRB exempt. 

Results

In the PRE period, 87 women were seen for diabetes vs. 271 in the POST. There were no significant differences regarding age (32 vs 33, P=0.88), race/ethnicity (58.6% vs 59.8%, Non-Hispanic white), gestational age (weeks) of diagnosis (28 vs 29, P-0.09) or public insurance status (17.2% vs 17.7 %, P=0.85). (Table) In the POST group, there were significantly more women who were pre-gestational diabetic (14.8% vs 4.6%, P=0.01) & on continuous glucose monitoring (0% vs 6.3%, P=0.017). There was no difference in medication initiation (50.6% vs 56.8%), but a significant shift from Glyburide to Insulin (Table). Average number of scans per patient was not different (2.5 vs 2.1, P=0.37), but more patients only had 1 scan POST (52% vs 34%, P<0.01). There was better compliance in the POST group with more uploads of glucose logs (average 5 vs 7, P<0.01), higher percentage of glucose values in range (81.8% vs 90.9%, P<0.01) & documented delivery recommendations (29.9% vs 95.3%, P<0.01).  Documented reasons for non-compliance were stopping uploading logs, declining insulin, & cancelling appointments. 

Conclusion

A formalized diabetes in pregnancy program improved glycemic control parameters & resulted in a 3-fold increase in number of patients seen without increasing number of scans per patient. 

Table: Comparison of selected maternal demographics & outcomes pre/post program initiation 

Novel role of the mechanosensor channel Piezo1 in the vasodilatory responses of human myometrial and chorionic plate arteries.

Germán A. Arenas, Aya Kondo, Christine Formento, Ramón A. Lorca

Introduction

During gestation, the uteroplacental vasculature undergoes substantial
adaptations to ensure proper blood flow from the mother to the fetus. Increased hemodynamic forces are detected by mechanosensory proteins within the endothelium and vascular smooth muscle. Piezo1, a non-selective cation channel, is a mediator of shear stress-induced vasodilation. Although Piezo1 has been described in murine models of pregnancy, its role in the human uteroplacental circulation remains unknown. Recently, Piezo1-evoked vasodilation in resistance arteries has been described to involve activation
of the large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel, a known regulator of uterine perfusion during pregnancy. We hypothesize that Piezo1-induced vasodilation is mediated by large-conductance calcium-activated potassium (BK_Ca) channels in the human myometrial (MyoA) and chorionic plate artery (CPA).

Methods

All participants signed written consent forms approved by COMIRB. Inclusion
criteria considered maternal age 18-45 years, pregnancy body mass index <30 kg/m² and newborns with birthweight between 10^th and 90^th percentile. MyoA and CPA were isolated from myometrium and placenta, respectively. Arteries segments (~2 mm) were mounted in wire myograph chambers containing Krebs buffer (37°C). To assess Piezo1-mediated vasoreactivity, increasing concentrations (1 nM – 30 mM) of Yoda1, a selective Piezo1
agonist, were added to the bath after pre-constricting the vessels. Dooku1 (Yoda1- induced Piezo1 activation antagonist), ODQ (soluble guanylyl cyclase inhibitor), paxilline and iberiotoxin (both BK_Ca channel inhibitors) were added individually as a pre-treatment to further dissect Piezo1-mediated vasodilation signaling. Immunofluorescence assays
were performed to localize Piezo1 in endothelium and vascular smooth muscle in myometrium and placenta.

Results

Piezo1 activation induced a decrease in vasoconstriction in both MyoA (~50%) and CPA (~30%). Piezo1-mediated vasodilation is decreased in presence of BK_Ca channel blockers in MyoA and CPA. Immunofluorescence experiments showed that Piezo1 is localized in both endothelial and vascular smooth muscle cells.

Conclusions

Piezo1 channel activation induce vasodilatory responses in human MyoA and CPA from healthy pregnancies. BK_Ca channel activation is a mediator of Piezo1- induced vasodilation in uteroplacental vasculature. Future studies will address the role of Piezo1/BK_Ca channel signaling in pregnancy complications such as fetal growth restriction.

Acceptability And Feasibility of Initiating a Low-Fat Eating Plan in Reproductive Aged Women with Obesity

Katelyn Duffy BA, ¹ Katherine Kuhn MS,¹ Asma Giornazi MS, ¹ Nicola Hendricks MD,¹ Lauren Gibbs MD,² Andrew P Bradford PhD,¹ Dorothy Mitchell-Leef MD,² Nanette F. Santoro MD.¹ 

1. Department of Obstetrics and Gynecology, University of Colorado School of Medicine, Aurora, CO, United States;
2. Morehouse School of Medicine, Atlanta, GA, United States.

Introduction

Obesity exerts adverse effects on the reproductive axis in women, resulting in relative hypogonadotropic hypogonadism, reduced fertility, adverse pregnancy outcomes, increased birth defects and transgenerational metabolic dysfunction in their offspring. This is believed to be partly due to diet related factors leading to hyperlipidemia and hyperinsulinemia. It is unknown whether administration of a eucaloric low-fat eating plan will improve reproductive axis function in women with obesity. In the US, 40% of reproductive aged women have obesity. Furthermore, the non-Hispanic Black population has a greater prevalence of obesity (57%), as well as poorer fertility treatment outcomes compared to White women. A better understanding of the causes and possible treatments for this severe degree of reproductive axis dysfunction is crucial. Our goal is to develop a low-fat eating plan that would be acceptable and feasible for a diverse population of reproductive aged women with obesity. The current project sought to understand preferences and motivations for women to participate in a research study that would provide them with low fat foods for one menstrual cycle while monitoring their hormones.

Methods

Eighteen women enrolled into 4 different focus groups. To assure we included the perspectives of a diverse population, but importantly Black women, the focus groups were conducted at two locations: the University of Colorado, which serves a predominantly White and Hispanic population and Morehouse School of Medicine, which serves a predominantly Black population. Trained facilitators conducted in-person and video focus groups. The enrolled women were asked by the facilitators about aspects of a planned dietary intervention that would provide a very low fat (22% daily calories from fat) eucaloric eating plan with the intention to control fat exposure but not to promote weight loss. Past experiences with dieting, acceptability of low-fat foods, potential barriers to adherence, and overall logistics of the intervention were discussed. Suggestions for maximizing adherence were solicited. Transcripts of the sessions were reviewed for thematic consistency.

Results

Women with obesity expressed interest in implementing a low-fat dietary intervention and receiving instruction on how to maintain eating habits they viewed beneficial for health and future fertility. Adequate provision of ethnically appropriate foods, social support to help avoid lapses, and tasty alternatives to high fat foods were considered ideal aspects of a feasible intervention. Incentives and graduated compensation for adherence were desirable features.

Conclusion

A low-fat, eucaloric dietary intervention that aims to investigate the reproductive axis in women with obesity is appealing to a diverse population of potential participants. Our participants demonstrated clear interest in learning about and implementing dietary modifications that could improve their health as well as their future fertility. These insights will be incorporated into a study design to incentivize and optimize enrollment and aid in protocol adherence.

Refinements in Understanding Human Milk-Derived Components in Their Use as “Liquid Breast Biopsies”

Kara Fusco, Department of OBGYN, Division of Reproductive Sciences

Jenifer Monks, Department of OBGYN, Division of Reproductive Sciences

Jayne Martin Carli, Department of OBGYN, Division of Reproductive Sciences

Introduction

Current understanding of the biological process of human lactation is limited due to the inability to obtain lactating breast tissue. Our group uses components of human milk as “liquid breast biopsies” to identify the unique biological signatures present in the lactating mammary gland. Specifically, we defined the transcriptomes of human milk-derived mammary epithelial cells (MECs) by single cell RNA sequencing. Here, we aim to determine how similar MECs which are lost into the milk are to those which remain in the epithelial layer of the mammary gland.

Methods

In an ancillary study of breastfeeding mothers after pregnancies affected by gestational diabetes, we collected human milk samples and separated milk components by centrifugation. The dense, pelleted components contain MECs, and the buoyant, floating layer contains milk fat globules (MFGs). MFGs are secreted from MECs in the epithelium and retain cytoplasmic fragments of those cells. MFGs are currently considered the benchmark for examining the transcriptomes of the lactating mammary gland.
We conducted RNA sequencing of MFGs to define the mammary signature of these individuals. In comparison, we sequenced pelleted MECs from the same set of milk samples, using fluorescence activated cell sorting to isolate viable, CD45- MECs. This allows us to identify the relevance of these cells to those actively secreting milk components.

Results

We first examined genes specific to the pregnancy and lactation stage of mammary gland development (WikiPathways #WP2817). We did not identify substantial differences in these genes between MFGs and milk-derived MECs, except for TPM3 (p= 0.047), MYC (p= 0.029), ELF5 (p= 0.029), and ERBB3 (p= 0.00007). We also investigated apoptosis and anoikis-related genes and did not identify any differential gene expression. Intriguingly, pathway analysis of genes differentially expressed in MFGs identified a robust ribosomal signature. This suggests trafficking and inclusion of ribosomes into the MFG upon secretion from the epithelium. Removal of this ribosomal signature identified elevated expression of immune function components in MFGs. In addition, pelleted cells express higher levels of genes that we have previously identified as markers of progenitor-like MECs including JUN (p=0.0047), KLF6 (p= 0.0047), MT2A (p= 0.011), MT1E (p= 0.039), and JUND (p = 0.022). Pelleted cells also preferentially retained cytoskeletal components, identified through pathway analysis of genes differentially expressed in these cells.

Conclusion

Our current work demonstrates that the human milk-derived MEC signature is remarkably similar to the “gold standard” MFG signature of cells residing in the mammary epithelium. Milk-derived MECs also provide more information about cells earlier in the differentiation process than those represented by MFGs. Taken together, we provide further validation for the use of human milk components as “liquid breast biopsies” for mammary gland research. 

The placental microenvironment: How villous stroma impacts endothelial cell mechanosensation

Stefano Ginocchio, Amanda Flockton, Emily Su

Background

In severe, early-onset fetal growth restriction with absent or reversed umbilical artery Doppler indices (FGR), placentas exhibit sparse vasculature due to impaired angiogenesis. Extracellular matrix (ECM), a fundamental component of tissue microenvironment, is a key regulator of angiogenesis. We previously reported that cell-derived matrices (CDM) generated from FGR or control placental stromal fibroblasts (FBs) impact placental endothelial cell (EC) angiogenic capacity. We sought to determine how placental stromal ECM-derived cues impact placental angiogenesis, hypothesizing that stromal derangements in FGR result in aberrant EC transcriptional responses mediating angiogenic properties.

Methods

Human villous tissue (VT), placental ECs, and FBs were obtained from FGR or control (uncomplicated, term) placentas. To generate CDM, FBs were seeded to confluence, subjected to ascorbic acid, then decellularized. Control (N=6) and FGR (N=6) VT and CDM underwent ECM-optimized extraction followed by mass spectrometry (MS) with an Orbitrap Fusion Lumos Tribrid spectrometer. VT was also stained with picrosirius red (PSR) normalized to terminal villous area (PSR ratio) and analyzed with AI-driven Visiopharm platform. Control or FGR ECs were plated on either control or FGR CDM, fixed, followed by immunofluorescent analysis for nuclear and cytoplasmic localization of YAP, a key transcriptional co-activator of cell proliferation and migration that is regulated by ECM cues. For proteomic data, results were filtered to 1% FDR. One-way ANOVA or t-tests were utilized for other comparisons.  

Results

FGR villous tissue exhibited significantly more fibrotic villi as demonstrated by PSR ratio (0.122 vs 0.047, p<0.0001). MS analyses showed no significant differences in absolute peak intensities among core matrisome proteins between control or FGR VT. However, although both groups had equal amounts of total matrisome proteins, the FGR matrisome exhibited a significantly increased proportion of collagens 1A1 (COL1A1), 1A2 (COL1A2), and fibronectin (FN1) (34.87% vs 23.64%, p=0.032). Within CDM, 55 of 96 core matrisome proteins were differentially expressed, with FGR CDM demonstrating significant increases in absolute counts of COL1A1 (4.90E6 vs 2.31E6, p=0.0036), COL1A2 (1.72E6 vs 0.96E6, p=0.043), and FN1 (11.6E6 vs 3.73E6, p=0.0002). Furthermore, COL1A1 and FN1 were both significantly more insoluble in FGR than control CDM (COL1A1: 78.6% vs 61.6%, p=0.0043; FN1: 83.7% vs 37.6%, p<0.0001). Culturing control or FGR ECs on either control or FGR CDM derived showed significant differences (overall p<0.0001) in YAP nuclear-to-cytoplasmic ratio, with both control and FGR ECs exhibiting increased nuclear translocation when cultured on FGR CDM. 

Conclusion

FGR placental villous stroma exhibit ECM compositional properties characteristic of fibrosis. These derangements are recapitulated in our CDM model and contribute to impaired cellular mechanosensing as observed by differential subcellular EC YAP localization in response to either control or FGR CDM.

Placental endothelial cell mechanosensation in severe, early-onset fetal growth restriction

Stefano Ginocchio, Taylor Hord, Amanda Flockton, Emily Su

Background

Placentas from pregnancies with severe, early-onset fetal growth restriction and absent or reversed umbilical artery Doppler indices (FGR) exhibit sparse vasculature due to impaired angiogenesis. Stiffness of tissue stromal microenvironment is one factor that regulates angiogenesis. We previously reported that placental stromal fibroblast (FB) cell-derived matrices (CDM) impact fetoplacental endothelial cell (EC) angiogenic potential, where CDM generated from uncomplicated pregnancies partially rescues proliferative and migratory deficiencies in FGR ECs, while CDM from FGR pregnancies impairs angiogenesis in control ECs. Furthermore, we have preliminarily found that FGR placental stromal ECM exhibits compositional properties characteristic of fibrosis that is recapitulated in our CDM model. These data support shear wave elastography studies that have also found that FGR placentas exhibit increased stiffness. Our objective was to determine how fetoplacental ECs respond to varying stiffnesses, hypothesizing that FGR EC mechanosensing capacity of biophysical stiffness cues is attenuated. 

Methods

Human fetoplacental ECs were isolated from FGR or control (uncomplicated, term) placentas. Silicone hydrogel plates with elastic moduli of 0.5 kPa or 8 kPa were utilized to represent stiffness of normal or FGR placentas, respectively. Equal numbers of cells were plated on fibronectin-coated hydrogels or plastic tissue culture plates. EC proliferation was assessed over 4 days via time-lapsed, live-cell imaging. Angiogenic gene expression of ECs plated on 0.5 and 8 kPa hydrogels was analyzed with qPCR. Two-way ANOVA or t-tests were utilized to assess statistical significance.

Results

Overall, significant differences in control and FGR EC proliferation on 0.5 kPa (physiologic), 8 kPa (pathologic), and plastic conditions (p=0.02) were seen. Specifically, control ECs exhibited differential responses to substrate stiffness (p=0.049), which were not evident in FGR ECs. When subjected to pathologic stiffness, control ECs expressed significantly less vascular endothelial growth factor (VEGFA; p=0.048), placental growth factor (PLGF; p=0.03), and vascular endothelial growth factor receptor 2 (VEGFR2; p=0.01) than when plated on 0.5 kPa hydrogels, whereas stiffness did not impact FGR EC expression of these genes. In contrast, angiopoietin 2 (Ang2; p=0.03) and angiopoietin receptor Tie2 (TEK; p=0.01) transcripts were significantly reduced solely in FGR ECs exposed to pathologic stiffness, with no effect of stiffness on these genes in control ECs. Furthermore, regardless of substrate, pro-angiogenic metalloprotease (MMP)-2 (p<0.001) and MMP-14 (p<0.001) mRNA expression was significantly decreased in FGR ECs as compared to controls, while anti-angiogenic MMP-7 (p<0.001) expression was significantly increased. 

Conclusion

FGR ECs exhibit impaired mechanosensing capacity of biophysical stiffness cues, resulting in decreased proliferative capacity and aberrant expression of angiogenic genes and MMPs capable of remodeling the stromal microenvironment.

An Eucaloric High-Fat Diet Suppresses Urinary Estrogen and Progesterone Metabolites, Mimicking Reprometabolic Syndrome in Normal Weight Women

Asma Giornazi MS¹, Katherine Kuhn, MS¹, Katelyn Duffy, BA¹, Andrew P. Bradford, PhD¹, Nanette F. Santoro, MD¹.

1. Department of Obstetrics and Gynecology, University of Colorado School of Medicine, Aurora, CO, United States

Introduction

Reprometabolic syndrome is associated with relative hypogonadotropic hypogonadism, reduced fecundability, and increased pregnancy loss in women with obesity. We employed a eucaloric, high-fat diet (HFD) to attempt to reproduce the decreased urinary estrogen and progesterone metabolites, characteristic of this phenotype, in healthy women of normal BMI.

Materials and Methods

18 women with normal BMI (18-24.9kg/m²), mean age 29.1±6.3, were recruited for a four-month study including a prescribed 30-day, eucaloric, high-fat, dietary intervention (48% calories from fat). Women collected daily morning urine for 4 menstrual cycles. Daily urinary estrone conjugate (E1c) and pregnanediol-3-glucuronide (Pdg) levels were measured by immunoassay before, during and after the HFD. Paired t tests were used to compare average urinary E1c and Pdg levels and area under the curve (AUC) to determine the impact of the HFD. Cycles were aligned by LH peak (day 0).

Results

Participants were weight stable throughout the 4-month study period with no change in BMI (pre-diet BMI 21.46±1.95 kg/m2 and post-diet BMI 21.35±1.81 kg/m2). Average mid cycle E1c peak was significantly lower during (32.4 ng/mg creatinine) and after the HFD (26.5 ng/mg creatinine), compared to pre-diet levels (43.2 ng/mg creatinine; p< 0.001). Post-diet urinary Pdg (AUC) was also significantly lower than pre-diet (p< 0.01). Follicular (days -14 to 0) and luteal phase (days 1-14) E1c AUC was significantly decreased post HFD (p< 0.01). Luteal phase Pdg levels were also significantly decreased post HFD (p<0.001). No significant changes in menstrual cycle length were observed throughout the study.

Conclusion

Consumption of a high-fat diet for one month was sufficient to induce a significant reduction in E1c and Pdg, characteristic of Reprometabolic Syndrome of obesity, in normal weight, eumenorrheic women. The HFD diet had prolonged effects on all hormones that lasted throughout the two-cycle post diet period. These findings provide important insights regarding the mechanisms underlying reproductive dysfunction in obesity and indicates that a dietary intervention may be an effective strategy to mitigate sub-optimal reproductive hormone levels in women with obesity-related subfertility. Future studies will elucidate the impacts of a low-fat diet on reproductive hormones and investigate the rebound period of this phenomenon.

Placental ceramide content is decreased in maternal obesity and GDM, independent of fetal sex. 

Marta Hita Hernandez,¹ Karin A Zemski Berry,³ Thomas Jansson,¹ and Theresa L. Powell^1,2

1. Division of Reproductive Sciences, Departments of Obstetrics and Gynecology
2. Pediatrics, Neonatology
3. Division of Endocrinology, Metabolism, and Diabetes, School of Medicine, University of Colorado Anschutz Medical Campus

Objectives

Ceramides inhibit insulin signaling in many tissues, including the placenta. We have previously shown that adiponectin inhibits insulin signaling and insulin-stimulated amino acid transport by increasing ceramide synthesis. This has been proposed to be a mechanism by which maternal adiponectin controls placental function, including nutrient transfer to the fetus, and fetal growth.  In pregnancies complicated by obesity or gestational diabetes (GDM) maternal plasma adiponectin is typically decreased. Moreover, maternal adiponectin levels are inversely correlated with birth weight. The aim of this study was to determine placental ceramide content in maternal obesity with or without GDM. 

Methods

Placental sphingolipid content was assessed by LC/MSMS in female and male placentas collected at term from normal weight women (n=10), obese women (n=10), and obese women with GDM (n=10). The average placental weight was 579.4 ± 161.9, 661.7 ± 117 and 698.4 ± 136.4 grams for the normal weight, obese and obese with GDM group respectively. The average birth weight was 3.3 ± 0.199, 3.46 ± 0.27 and 3.66 ± 0.47 kilograms for the normal weight, obese and obese with GDM group respectively. The results were compared using one or two-way ANOVA and expressed as the mean ± SD. 

Result

Total ceramides were decreased in placentas from obese and obese women with GDM (p<0.02). In both obese and obese women with GDM, ceramides 16:0 were decreased only in male placentas (p<0.05), while ceramides 22:0 (p<0.02) and 24:0 (p<0.04) were lower in female placentas only.  

Conclusion

Our data suggest that obesity and GDM decreases placental ceramide content, independent of fetal sex. These changes may be due to lower circulating maternal adiponectin, which has been shown to decrease placental ceramide synthesis leading to activation of insulin and mTOR signaling. The lower placental ceramide content in obesity and GDM may contribute to fetal overgrowth in these pregnancy complications through the activation of placental nutrient transport by mTOR.

Harnessing the potential of stem cells to model placental and embryonic development.

Rowan M. Karvas

Naïve embryonic stem cells (hESCs) transcriptionally and epigenetically resemble epiblast from pre-implantation human blastocysts, giving rise more efficiently to trophoblast and primitive endoderm lineages. This enhanced potency was harnessed to derive human trophoblast stem cells (hTSCs) from hESC sources. 3D modeling of hTSCs as organoids allows for the study of multiple cell lineages of a tissue, encapsulating multi-functional cell types. Stem cell-derived trophoblast organoids (SC-TOs) transcriptionally and spatially represent trophoblast organoids from primary sourced hTSCs. SC-TOs exhibit multiple cell types, including two cytotrophoblast populations, 2 syncytiotrophoblast populations, and an early extravillous population. These populations closely match the same cell types in vivo. The model can be utilized to test susceptibility to emerging viral pathogens ZIKV and SARS-CoV-2. Additionally, given a reduction in WNT signals, SC-TOs can be differentiated to extravillous trophoblast organoids (SC-EVTOs). When in contact with cell types of the human endometrium (uterine stromal and epithelial glandular cells), SC-EVTOs display heightened invasive activity when cultured upon endometrial cells in vitro. 

Given the potency for all lineages of the early blastocyst (epiblast, primitive endoderm, and trophectoderm), we endeavored to generate a model of the human blastocyst in 3D, termed “blastoids”. We were able to enhance blastoid efficiency to 80-90% with our improved conditions from previous publications. We extended culture of the blastoids to post-implantation timepoints via attachment to 3D gels in vitro. To understand cellular diversity of our blastoids, we used single cell RNA sequencing and characterized three distinct timepoints- day 7, day 14, and day 21 of blastoid culture. Day 7 resembles the same approximate timepoint compared to in vivo human datasets. Day 14 represents a timepoint shortly before primitive streak formation in the epiblast, with mass diversification occurring in the trophectoderm to give rise to all post implantation trophoblast types. At day 21, the trophoblast populations remain fairly constant with improved invasion, while the epiblast and hypoblast population proceed to transcriptional profiles resembling cell populations arising in early gastrulation of the CS7 human embryo. Utilizing immunofluorescence, we were able to spatially locate anteriorizing signals such as CER1 and LEFTY being expressed in locations opposed to posterior-expressed T (BRACHYURY). By 21 days, primary villi made up entirely of invasive cytotrophoblasts were well developed, while secondary villi were beginning to form- a process involving the incorporation of extraembryonic mesoderm within the invasive trophoblast strands. 

Moving forward, my lab will utilize both models to understand the development of early human placental villi and the process of implantation. 

Administration of Proteins Secreted by the Placenta Markedly Improves Liver Outcomes in Premature Guinea Pigs

Avery C Kramer¹, Kelsey J Faer¹, Rebecca M Dyson², Alice Freeman², Theresa L Powell^1,3, Mary J Berry², Thomas Jansson¹

^{1 Departments of OB/GYN and 3 Pediatrics University of Colorado, Aurora, CO, USA; 2 Department of Paediatrics and Child Health and Biomedical Research Unit, University of Otago, Wellington, New Zealand}

Email: avery.kramer@cuanschutz.edu

Background

Extremely premature infants (born <28 week’s gestation) are at increased risk of early morbidity and mortality and long-term sequela, including hepatic steatosis.  Emerging evidence indicates that the placenta secretes factors critical for normal fetal organ development. We have identified 142 proteins secreted by the preterm placenta into fetal circulation in humans. Placental proteins may support organ development and prematurity deprives the neonate of these factors. We hypothesized that administration of candidate placental proteins improves liver development of premature guinea pigs (GPs). 

Methods

Preterm delivery was induced in pregnant GPs at gestational day 62 (term 69-70) and all pups received postnatal thermal, respiratory, and nutritional support in a GP Neonatal Intensive Care Unit. Pups received either 5 candidate proteins (PROT; Activated Protein C, Fibroblast Growth Factor 1, Annexin A1, Ephrin B1, and R-spondin 3, n=15) or saline (control, CON, n=18) via a subcutaneous mini-osmotic pump for ~7days until term equivalent age (TEA). Animals were sacrificed at TEA+7 and tissues collected.

Results

PROT pups had improved survival and greater liver weight as a proportion to total body weight compared to CON pups (p<0.05). Male CON pups had greater mRNA expression of type I (p<0.002) and III (p<0.005) collagen compared to female CON and PROT pups. PROT pups had reduced hepatic lipid accumulation compared to CON pups (p<0.002). 

Conclusions

Prematurely delivered GP pups that received placental proteins had improved survival and liver development. We speculate that human placental proteins may represent a novel intervention to improve organ development in extremely premature infants.

Obesity, rather than insulin resistance, impairs breastmilk production during early lactation. 

Name & affiliation
Jayne Martin Carli 
University of Colorado School of Medicine
Department of Obstetrics and Gynecology 
Division of Reproductive Sciences

Co-authors
James McManaman and Jenifer Monks

Introduction/Background 

Fewer than 25% of American mothers breastfeed to the extent recommended by the WHO and CDC. This results in an estimated $167.9B of morbidity- and mortality-related excess costs. Women with obesity and diabetes are at elevated risk of early termination of breastfeeding. Societal factors drive these outcomes, however there are likely also biological causes of low milk supply in this population. Milk production is rarely quantified in research studies, limiting our understanding of causal factors.

Methods 

We conducted a prospective cohort study to investigate hourly milk production and its drivers in women with normal weight and glycemia (NW/NG, BMI <25kg/m²), overweight/obesity and normal glycemia (OB/NG, BMI 27-39 kg/m²), or overweight/obesity and gestational diabetes, requiring medication (OB/GDM, BMI 27-39 kg/m²), using an hourly breastpumping protocol (n=10/group). Patients of UCHealth Obstetrics and Midwifery clinics were recruited after routine glucose tolerance testing and enrolled after 36wks gestation, when they were provided lactation counseling. Participants were parity matched across all groups, and BMI matched between the OB/NG and OB/GDM groups. Blood and milk samples were collected to identify underlying relationships between systemic and mammary factors and lactation outcomes. 

Results 

Between 2-4 weeks postpartum, hourly milk production was substantially different between participants in the NW/NG (41.3 ±2.3 g/hr) and OB/NG (34.6 ±3.2 g/hr; p=0.05) groups but was not different between participants in the OB/NG and OB/GDM groups (33.9 ±4.6 g/hr, p=0.8; all avg ±SEM, Mann-Whitney U). Ongoing studies are focused on determining whether hourly milk production is correlated with mammary-specific factors or circulating lactogenic and/or metabolic hormones. The functional state of the mammary gland is being assessed by using milk fat globule and milk-derived mammary epithelial cell transcriptomes.

Conclusion 

Recent epidemiological studies have suggested that body weight is a stronger biological predictor of low milk production than insulin resistance. Our findings align with this assessment and will point toward the biological underpinnings of this relationship. This pilot work will inform a larger scale study to identify potential avenues for intervention to increase milk supply in mothers with impaired lactation.

Funding sources 

National Institute of Child Health & Human Development K99/R00 Pathway to Independence Award (K99 HD107496) and Ludeman Family Center for Women’s Health Research Early Career Faculty Research Development Award

Sexual dimorphism in human placental signaling

Shimada H^1,2, Erickson K^1,3, Powell TL^1,3, and Jansson T¹

1. Department of Obstetrics and Gynecology University of Colorado, Anschutz Medical Campus, Aurora, CO, US
2. Departments of Obstetrics & Gynecology, Sapporo Medical University, Sapporo, Japan,
3. Department of Pediatrics, University of Colorado, Anschutz Medical Campus, Aurora, CO, US

Objectives

Male fetuses generally grow faster in utero and are heavier at birth than females, whereas the rates of perinatal mortality and morbidity in boys often are higher than in girls. It is widely believed that sexual dimorphism in placental function contributes to these differences. However, although there is ample evidence that placental gene expression varies with fetal sex, detailed information on fetal sex differences in placental signaling activity is lacking. Placental mechanistic target of rapamycin (mTOR) signaling is a critical hub linking maternal metabolism, placental function and fetal growth. Placental mTOR is regulated by an array of upstream pathways including AMP-activated kinase (AMPK) and insulin/IGF-1 signaling and has multiple downstream targets, including O-linked N-acetylglucosamine (GlcNAc) transferase (OGT). We hypothesized that placental signaling pathway that promote fetal growth, such as mTOR, is activated in male as compared to in female term placenta.

Methods

After informed written consent, we collected placentas from 46 women (n=23; female, n=23; male) with uncomplicated pregnancies at term (gestational age 37-41 weeks) and delivering infants with normal birth weight (appropriate-for-gestational-age). Study subjects had either normal body mass index (BMI) or were overweight (BMI, 18.5-29.9kg/m²). Western blotting was used to measure the expression and phosphorylation of components of mTOR, AMPK and insulin/IGF-1 signaling and the expression of OGT. Statistical significances of differences were determined by unpaired t-test. P values < 0.05 were considered significant. Liner regression analysis was also used to determine correlations between birth weight z-score and the density of protein expression, and the coefficient of determination (R-squared; r²) was calculated.

Results

Maternal age, maternal BMI, gestational age at delivery and placental weights were not statistically different between women delivering male and women delivering female infants. The mode of delivery was similar between groups. Although birth weight was higher in male infants (p=0.034), birth weight z-score was not statistically different between groups (p>0.05). In male placentas, S6RP phosphorylation (Ser-235/236) was statistically higher than in female placentas (p=0.006). In contrast, there were no differences between male and female placentas in any of the other measured placental signaling targets (p>0.05). Phosphorylation of AKT (Thr-308) (r²=0.30, p=0.007) was significantly positive correlated with birth weight z-score in male and 4EBP1 phosphorylation (Thr-37/46) (r²=0.187, p=0.039) was significantly negatively correlated with birth weight z-score in male, but not in female placentas.

Conclusion

Overall, few fetal sex differences in placental signaling activity were observed. However, our data suggest that the activity of placental mTORC1 signaling is higher in male placenta than in female, which may contribute to the difference in fetal growth.

Claudin-4 remodeling of  nucleus-cell cycle crosstalk maintains ovarian tumor genome stability and drives resistance to genomic instability-inducing agents.

Fabian R. Villagomez,¹ Julie Lang,² Daniel Nunez-Avellaneda,³ Kian Behbakht,⁴ Hannah L. Dimmick, ¹ Patricia Webb¹, Kenneth P Nephew^5,6,7, Margaret Neville¹, Elizabeth R. Woodruff¹, Benjamin G. Bitler.¹,* 

1. Division of Reproductive Sciences, Department of Obstetrics and Gynecology, School of Medicine, University of Colorado, Anschutz Medical Campus, Aurora, Colorado.
2. Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus, Aurora, USA.
3. Deputy Directorate of Technological Development, Linkage, and Innovation, National Council of Humanities, Sciences, and Technologies, Mexico City, Mexico.
4. Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, The University of Colorado Anschutz Medical Campus, Aurora, Colorado.
5. Medical Sciences, Indiana University School of Medicine, Bloomington, Indiana.
6. Melvin and Bren Simon Comprehensive Cancer Center, Indiana University, Indianapolis, Indiana.
7. Department of Anatomy, Cell Biology & Physiology, Indiana University, Indianapolis, Indiana. 

Objectives

Cell cycle dysregulation is a fundamental hallmark of cancer that drives both altered cell proliferation and genome instability (GI). Significant alterations in nuclear morphology are closely linked to genome instability, and tumor cells can either prevent or eliminate it to maintain optimal tumor growth. Claudin-4 is aberrantly overexpressed in most epithelial ovarian carcinomas (EOC) and is associated with the development of therapy resistance and poor patient survival—a phenomenon closely related to the regulation of genetic alterations. Thus, claudin-4 overexpression has potential clinical significance in the treatment and prognosis of ovarian cancer. We reported that claudin-4 forms a biological axis with the amino acid transporters SLC1A5 and LAT1 to control GI through autophagy, indicating that claudin-4 participates in the elimination of GI once it is formed. However, the molecular mechanisms underlying the relationship of claudin-4 with the regulation of genome instability are still poorly understood. This study aimed to determine claudin-4’s effect on cell cycle progression and nuclear architecture and how its regulation may lead to changes in GI and therapy resistance in ovarian cancer cells.

Methods

We employed authenticated, mycoplasma-free EOC cells (OVCA429, OVCAR3, OVCAR8) to investigate claudin-4 elimination via CRISPR interference and overexpression through lentiviral transduction. Additionally, we disrupted claudin-4 function using a unique claudin mimic peptide (CMP). To characterize nuclear architecture and the cytoskeleton of ovarian cancer cells, we utilized immunofluorescence, live-cell imaging, and confocal microscopy, along with staining of nuclear (DAPI, Hoechst, lamin B1, lamin A/C) and cytoskeletal markers (phalloidin and LifeAct). We analyzed the cell cycle using propidium iodide staining and flow cytometry. Moreover, we assessed survival of ovarian cancer cells during treatment with olaparib, both alone and in combination with CMP and forskolin via colony formation assay. To explore oxidative stress response, these cell lines were engineered to express HIF-1α, allowing for the measurement of reactive oxygen species (ROS) during the same treatments. Data analysis and statistical evaluations were conducted using ImageJ and Prism software, employing unpaired t-tests and one-way ANOVA with Tukey's multiple comparisons test (p<0.05).

Results

We found claudin-4 facilitates genome maintenance by linking the nuclear envelope and cytoskeleton dynamics with cell cycle progression. Claudin-4 caused nuclei constriction by excluding lamin B1 and promoting perinuclear F-actin accumulation, associated with remodeling nuclear architecture, thus altering nuclear envelope dynamics. Consequently, cell cycle modifications due to claudin-4 overexpression resulted in fewer cells entering the S-phase and reduced genomic instability. Importantly, disrupting biological interactions of claudin-4 using CMP and forskolin altered oxidative stress cellular response and increased the efficacy of PARP inhibitor treatment.

Conclusions

Our data indicate claudin-4 protects tumor genome integrity by remodeling the crosstalk between the nuclei and the cell cycle, leading to resistance to genomic instability formation and the effects of genomic instability-inducing agents. 

Bibliographic References

1. Hanahan D. Hallmarks of Cancer: New Dimensions. Cancer Discov 2022;12:31-46
2. Singh I, Lele TP. Nuclear Morphological Abnormalities in Cancer: A Search for Unifying Mechanisms. Results Probl Cell Differ 2022;70:443-67
3. Zeimet AG, Fiegl H, Goebel G, Kopp F, Allasia C, Reimer D, et al. DNA ploidy, nuclear size, proliferation index and DNA-hypomethylation in ovarian cancer. Gynecol Oncol 2011;121:24-31
4. Andor N, Graham TA, Jansen M, Xia LC, Aktipis CA, Petritsch C, et al. Pan-cancer analysis of the extent and consequences of intratumor heterogeneity. Nat Med 2016;22:105-13
5. Andor N, Maley CC, Ji HP. Genomic Instability in Cancer: Teetering on the Limit of Tolerance. Cancer Res 2017;77:2179-85
6. Villagomez FR, Lang J, Rosario FJ, Nunez-Avellaneda D, Webb P, Neville M, et al. Claudin-4 Modulates Autophagy via SLC1A5/LAT1 as a Mechanism to Regulate Micronuclei. Cancer Res Commun 2024;4:1625-42
7. Hicks DA, Galimanis CE, Webb PG, Spillman MA, Behbakht K, Neville MC, et al. Claudin-4 activity in ovarian tumor cell apoptosis resistance and migration. BMC Cancer 2016;16:788
8. Breed C, Hicks DA, Webb PG, Galimanis CE, Bitler BG, Behbakht K, et al. Ovarian Tumor Cell Expression of Claudin-4 Reduces Apoptotic Response to Paclitaxel. Mol Cancer Res 2019;17:741-50
9. Boylan KL, Misemer B, De Rycke MS, Andersen JD, Harrington KM, Kalloger SE, et al. Claudin 4 Is differentially expressed between ovarian cancer subtypes and plays a role in spheroid formation. Int J Mol Sci 2011;12:1334-58
10. Agarwal R, D'Souza T, Morin PJ. Claudin-3 and claudin-4 expression in ovarian epithelial cells enhances invasion and is associated with increased matrix metalloproteinase-2 activity. Cancer Res 2005;65:7378-85
11. Yamamoto TM, Webb PG, Davis DM, Baumgartner HK, Woodruff ER, Guntupalli SR, et al. Loss of Claudin-4 Reduces DNA Damage Repair and Increases Sensitivity to PARP Inhibitors. Molecular cancer therapeutics 2022;21:647-57
    

Ablation of hematopoietic stem cell derived adipocytes reduces tumor burden in syngeneic mouse models of high-grade serous carcinoma

Elizabeth R Woodruff¹, Courtney A Bailey^1,2, Francis To¹, Vyshnavi Manda³, Joanne K Maltzahn⁴, Timothy M Sullivan⁴, Meher P Boorgula^5,6, Maria Sol Recouvreux⁷, Ruby Vianzon⁸, Bogi Conrad⁸, Kathleen M Gavin⁸, Kimberly R Jordan⁹, Dwight J Klemm^4,10, Sandra Orsulic⁷, Benjamin G Bitler¹, Zachary L Watson¹

1. Division of Reproductive Sciences, Department of Obstetrics & Gynecology, University of Colorado School of Medicine, Aurora, CO 80045, USA
2. Biomedical Engineering Program, University of Colorado Boulder, Boulder, CO 80309, USA
3. Department of Medicine, University of Colorado School of Medicine, Aurora, CO 80045, USA
4. Department of Pulmonary Sciences and Critical Care, University of Colorado School of Medicine, Aurora, CO 80045, USA
5. University of Colorado Cancer Center, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
6. Department of Biomedical Informatics, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
7. Departments of Obstetrics & Gynecology and Pathology, and the Jonsson Comprehensive Cancer Center, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA
8. Division of Geriatric Medicine, Department of Medicine, University of Colorado School of Medicine, Aurora, CO 80045, USA
9. Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, CO 80045, USA
10. Geriatric Research, Education and Clinical Center, Rocky Mountain Regional Veterans Administration (VA) Medical Center, Aurora, CO 80045, USA

In this study we examine the influence of hematopoietic stem cell derived adipocytes (HSCDAs) on the proliferation and metastasis of high-grade serous carcinoma (HGSC), the most common type of ovarian cancer. HSCDAs are a subtype of adipocytes which begin as myeloid cells that traffic from bone marrow to adipose tissue, particularly visceral depots, and then differentiate and accumulate therein. We hypothesize that HSCDAs promote HGSC progression and establish a pro-tumoral niche within peritoneal adipose tissues such as the omentum.

We took primary human adipose biopsies and flow sorted the myeloid and mesenchymal populations. We differentiated these adipose precursors in vitro into mature CMAs and HSCDAs, respectively. RNA-Seq shows that HSCDAs have a distinct transcriptional profile from CMAs. Using ELISA, we found that HSCDAs secreted greater amounts of inflammatory cytokines IL-6 and IL-8 than CMAs. We incubated HGSC cell lines in conditioned media from HSCDAs and CMAs and performed reverse phase protein array (RPPA) and proliferation assays. HGSC cells showed elevated protein markers of epithelial to mesenchymal plasticity, including fibronectin, as well as increased serine phosphorylation of pro-survival AKT1/2. Conversely, HGSC cells in HSCDA media downregulated tumor suppressors including Wnt regulator GSK3β. Depending on the cell line and adipose donor, HGSC cells also showed altered growth rates in conditioned media.

We next investigated the role of HSCDAs in HGSC progression and metastasis in vivo. We generated immunocompetent mice that were either HSCDA Proficient (can make both adipocyte subtypes) or Deficient (can only make CMAs). We performed two independent tumor studies using the ID8 (Tp53-/-, Brca2-/-) and SO (Tp53-/-, Brca1/2 wild-type, Hras and Myc amplified) syngeneic models. In both models, overall tumor burden was lower in HSCDA Deficient mice. In the ID8 model, omentum tumors from HSCDA Deficient mice, relative to Proficient mice, showed reduced proliferation (Ki67) and apoptosis (cleaved caspase 3).

In agreement with reduced tumor size and Ki67, bulk RNA-Seq of omentum tumors from HSCDA Deficient mice showed downregulation of multiple metabolic pathways. We also observed reduced inflammatory pathways in ID8 tumors from HSCDA Deficient mice, so we interrogated immune cell infiltration into omentum tumors. Compared to HSCDA Proficient mice, tumors from HSCDA Deficient mice showed reduced densities of dendritic cells (DC) and natural killer (NK) cells. By spatial analysis we found fewer DCs, NKs, and B-cells near tumor cells. Overall, our data suggest that HSCDAs can promote HGSC survival and plasticity while downregulating tumor suppressors and also alter the peritoneal immune and metabolic environment to promote HGSC progression.

Effects of Menopause on Innate Immune Defense Mechanisms in Urinary Cells

Ariel Y. Chen, BA; Lina M. Loza, MD; Fernando Pereira Beserra, PhD; Humberto D. Escobedo, PhD; Annika Smith, PhD; Kathleen A. Connell, MD; Michael Schurr, PhD; Joshua Johnson, PhD; Marsha K. Guess, MD, MS

Introduction

Urinary tract infections (UTIs) are the most common bacterial infection worldwide with recurrent infections (rUTI) reported following more than 25% of initial infections.

Postmenopausal people have increased susceptibility to rUTI and hypoestrogenism is thought to play a crucial role. To date, the underlying mechanisms influencing the predisposition for rUTI in postmenopausal people are relatively unknown. We hypothesize that postmenopausal people have downregulated expression of antimicrobial peptides (AMPs) at baseline which predisposes them to developing UTIs.

Methods

Female-identifying people >18 years old were recruited from the University of Colorado Urogynecology clinics at the Anschutz Medical and Lone Tree campuses. People with an active infection, bladder pathology, a genitourinary malignancy, or fecal incontinence as well as those receiving antibiotics, hormone therapy, or supplements to prevent UTI <1 month prior to presentation were excluded. Urine samples from mid-stream clean catch were obtained from all participants and stratified based on menopausal status. Urinary cells were isolated from urine samples, mRNA was extracted, and AMP expression was quantified via quantitative reverse- transcriptase polymerase chain reaction (qRT-PCR). Target AMPs were secretory leukocyte peptidase inhibitor (SLPI) and psoriasin (S100A7). Pro-inflammatory cytokine Interleukin-1 beta (IL-1β) was also assessed and normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Unpaired two-tailed student’s t-tests were used to compare gene expression mean fold change for the three AMPs between pre- and post-menopausal people. A p- value <0.05 was deemed significant.

Results

Urinary cells from 14 premenopausal and 36 postmenopausal people were obtained. Compared to premenopausal people, postmenopausal people had significantly lower expression of SLPI (0.8674 vs 2.407, p = 0.0013) and S100A7 (0.7388 vs 9.296, p < 0.0001). Cells from
postmenopausal people were shown to have a twelve-fold reduction in S100A7 compared to cells from premenopausal people. IL-1β was significantly increased in postmenopausal people, (4.383 vs 0.1558, p = 0.0036).

Conclusion

Postmenopausal people have significantly lower expression of the AMPs and increased levels of the proinflammatory cytokine, IL-1β compared to premenopausal people. AMPs serve as an important first response in innate immunity. A loss of innate immune defense and a predisposition to inflammation may play an important role in increased rUTI incidence in postmenopausal people.

Extracellular Matrix Remodeling in Uterosacral Ligaments: A Pilot Study Utilizing Raman Spectroscopy

Ashley E. Hilton, Dylan M. Asmar, Lea Savard, Juana Hutchinson-Colas, David Orlicky, Jamie Arruda, Lauren Rascoff, Marsha Guess, Joshua Johnson, Virginia Ferguson, Kathleen Connell

Introduction

Pelvic organ prolapse (POP) is a prevalent condition among women, characterized by descent of pelvic organs. Uterosacral ligaments (USLs) play a critical role in pelvic floor support. Alterations in extracellular matrix (ECM) are implicated in POP pathogenesis. Investigative studies using Raman spectroscopy on cervical tissue have been performed. This pilot study demonstrates a novel application of this method to elucidate USL ECM in POP and identify potential therapeutic targets.

Objectives

This study aims to investigate ECM remodeling in USLs of women with POP compared to healthy controls using Raman spectroscopy.

Methods

USL samples were collected during hysterectomy and were flash frozen until Raman analysis was performed. High signal-to-noise ratio Raman spectra were collected and processed from hydrated USL biopsies. Spectra were measured from three regions within each sample to ensure representative data acquisition. Data analysis was performed, statistical significance was determined using Mann-Whitney U. Spectra were evaluated for prominent peaks in ranges indicating collagen prevalence (855, 938 cm^-1) and regions indicative of collagen structure and organization such as amide I (1600-1700 cm^-1) and amide III (1200-1330 cm^-1). Additional peaks correlating with ECM components such as lipids and glycosaminoglycans were evaluated. 

Results

Analyses were performed on 18 POP and 12 control. Compared to controls, women with POP were significantly older, had higher vaginal parity, and were less likely to use hormones. There was no difference in body mass index, menopausal status, or smoking status between groups (Table. 1). Overall, similar spectra and peaks for ECM components were noted between POP and control, indicating similar ECM content of the ligament and published spectra for collagen. Compared to controls, POP USLs demonstrate an increase in peak height specific to proline (1640 cm^-1; +8.76%; p=0.026), a key precursor component of collagen (Fig. 2). However, POP USLs did not show similar increases in hydroxyproline (1665 cm^-1; N.S.) which is associated with collagen structure stabilization. The ratio corresponding to hydroxyproline to proline (1665/1640) displayed a decreasing trend in POP (-6.69%; p=0.066) which is associated with poor wound healing. POP USL also demonstrated less disordered collagen (Fig.3), detected via reduced 1245 cm^-1 peak height (-7.06%; p=0.044) and 1245/1270 ratio (+5.16%; p=0.021), suggesting altered organization of excess collagen fibers. 

Conclusions

These preliminary pilot study findings highlight the importance of ECM remodeling in USLs and implications for POP pathogenesis. Changes in load structures with POP may explain observed collagen prevalence and organization, which may be deposited to stiffen supporting ligaments. These differences were observed across all stages of POP, suggesting alterations in collagen secondary structure occur even with early stages of this condition. Further studies into understanding ECM alterations and mechanical testing may elucidate targeted interventions to preserve USL integrity and mitigate risk of POP development and progression.

Impact of early stage ER+ breast cancer on innate immunity in postmenopausal patients

Lina M. Loza, MD, Ariel Y. Chen, BA , Fernando Pereira Beserra, PhD, Annika Smith, PhD, Humberto D. Escobedo, PhD, Kathleen A. Connell, MD, Radhika Acharya-Leon, MD, Michael Schurr, PhD, Joshua Johnson, PhD, Marsha K. Guess, MD, MS 

Background

Urinary tract infections (UTIs) are a major global issue, affecting over 150 million people and costing over $2.5 billion annually in the US ^1-4. Up to 30% of patients have a recurrence within six months ^5,6. Innate immunity plays a crucial role in defending against uropathogens in a hormone-dependent manner ⁷.

Breast cancer, the most common cancer globally, often involves estrogen receptor-positive cases treated with anti-estrogen therapy to reduce cancer recurrence. Recent studies have identified UTIs as a frequent grade 4 complication in these patients^11-12. Our current understanding of the role of estrogen in UTIs is limited. Here, we aim to determine the impact of estrogen receptor positive (ER+) breast cancer on the expression of psoriasin (S100A7), secretory leukocyte peptidase inhibitor (SLPI), and interleukin-1 beta (IL-1B).

Materials and Methods

Postmenopausal women >18 years old were recruited from the University of Colorado Urogynecology clinics at Anschutz Medical and Lone Tree campuses and Highlands Ranch Hospital Oncology Center. Exclusion criteria included active infection, antibiotic use or UTI diagnosis within the last month, hormone therapy, genitourinary malignancy, bladder pathology, and fecal incontinence. Mid-stream, clean-catch urine samples were collected, urinary cells were isolated and mRNA was extracted. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) was performed to evaluate gene expression of S100A7, SLPI, and IL-1B.  Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as house-keeping gene. Relative mean fold change in gene expression was evaluated using unpaired student’s t-test with statistical significance set at p>0.005. 

Results

We enrolled 45 women. Fourteen post-menopausal women had a diagnosis of ER+ breast cancer with low oncotype or ductal carcinoma in-situ (DCIS) and thirty-one post-menopausal women without breast disease were included as our control group. Post-menopausal breast cancer patients had significantly higher expression of S100A7 compared to non-breast cancer patients (1.767 vs 0.738, p=0.033). SLPI gene expression was also higher in breast cancer patients but did not achieve statistical significance (1.392 vs 0.867, p= 0.0687). In contrast, breast cancer patients had lower expression of IL-1B compared to non-breast cancer post-menopausal patients (3.533 vs 4.383, p=0.549). 

Discussion

Our study shows distinct variations in urinary AMP gene expression between post-menopausal patients with ER+ breast cancer compared to postmenopausal women without a diagnosis of breast cancer. Notably, our data indicates significantly elevated levels of S100A7, an AMP with high antibacterial activity against E.coli. Our findings may help elucidate differential pathways and specific uropathogens that contribute to UTI in women with breast cancer.