Development of a long term, ex vivo, patient-derived explant model of endometrial cancer.

Incidence of endometrial cancer (EC) is rising in the developed world. The current standard of care, hysterectomy, is often infeasible for younger patients and those with high body mass index. There are limited non-surgical treatment options and a lack of biologically relevant research models to investigate novel alternatives to surgery for EC. The aim of the present study was to develop a long-term, patient-derived explant (PDE) model of early-stage EC and demonstrate its use for investigating predictive biomarkers for a current non-surgical treatment option, the levonorgestrel intra-uterine system (LNG-IUS). Fresh tumour specimens were obtained from patients with early-stage endometrioid EC. Tumours were cut into explants, cultured on media-soaked gelatin sponges for up to 21 days and treated with LNG. Formalin-fixed, paraffin embedded (FFPE) blocks were generated for each explant after 21 days in culture. Tumour architecture and integrity were assessed by haematoxylin and eosin (H&E) and immunohistochemistry (IHC). IHC was additionally performed for the expression of five candidate biomarkers of LNG resistance. The developed ex vivo PDE model is capable of culturing explants from early-stage EC tumours long-term (21 Days). This model can complement existing models and may serve as a tool to validate results obtained in higher-throughput in vitro studies. Our study provides the foundation to validate the extent to which EC PDEs reflect patient response in future research.

"I'm not getting paid to give you a TED talk on how my trans body works". Experiences of hysterectomy gender affirming surgery: A qualitative study.

OBJECTIVE: To explore the experiences of care surrounding hysterectomy as part of gender affirming surgery. METHODS: An in-depth reflexive thematic analysis from accounts by 10 out of 12 people was undertaken. Experiences were then mapped to the surgery journey as a template for developing system responsiveness. RESULTS: No one person's experience of the procedure was affirmed across the entire surgery journey. Transgender health literacy was central to inclusive practice as it mediated bodily autonomy being upheld. The physical care environment influenced the experience, for example, the waiting room was marginalizing (intimidating), with a gendered clinic name and toilets. Some participants took a female support person/partner so that "people looking would assume that I was there supporting her, not the other way around." Communication misalignments were evident around information provided/understood about fertility and ovarian preservation. Participants were also placed in the position of both receiving care and providing education: "I also shouldn't have to be going in there for treatment, and then being expected to educate the medical professional that's meant to be helping me… I'm not getting paid to give you a TED talk on how my trans body works." The experiences mapped across the surgery journey highlighted multiple levels of service provision development needed to foster inclusive practice, for example, from workforce education to healthcare policy. CONCLUSION: Healthcare for transgender people can be unsafe and inequitable. Increasing transgender health responsiveness across the surgery journey will facilitate better alignments in communication and uphold bodily autonomy, leading to safer and inclusive practice.

Investigational drugs for the treatment of dysmenorrhea.

INTRODUCTION: Dysmenorrhea is the most common cause of gynecological pain among women that has considerable impact on quality of life and psychosocial wellbeing. Non-steroidal anti-inflammatory drugs (NSAIDs) and hormonal therapies are most commonly used to treat dysmenorrhea. However, given these drugs are often associated with bothersome side effects and are less effective when there is an underlying cause contributing to dysmenorrhea (e.g. endometriosis), a patient-centered approach to managing dysmenorrhea is important. Various new drugs are currently being investigated for the treatment of primary and secondary dysmenorrhea. AREAS COVERED: This review provides an updated overview on new therapeutic targets and investigational drugs for the treatment of primary and secondary dysmenorrhea. The authors describe the clinical development and implications of these drugs. EXPERT OPINION: Among the investigative drugs discussed in this review, anti-inflammatories show the most promising results for the treatment of dysmenorrhea. However, given some trials have considerable methodological limitations, many drugs cannot be currently recommended. Research focused on understanding the mechanisms involved in menstruation and its associated symptoms will be important to identify new therapeutic targets for dysmenorrhea. Further robust clinical trials are required to better understand the efficacy and safety of investigational drugs for treating primary and secondary dysmenorrhea.

Increasing incidence of endometrial cancer in Aotearoa New Zealand: Health professionals' perspective.

BACKGROUND: The incidence of endometrial cancer is globally increasing. Aotearoa New Zealand is no exception with a 59% increase in cases over that last ten years. AIMS: We report a sub-set of themes which pertain to provider reflections of rising endometrioid-type endometrial cancer incidence in individuals with high weight. MATERIALS AND METHODS: Fifteen semi-structured interviews with healthcare professionals experienced in providing care to women with endometrial cancer were audio-recorded and transcribed. Interviews were analysed using reflexive thematic analysis. RESULTS: Two main themes emerged: (1) concerns for the future; and (2) impact on fertility and treatment options. Healthcare professionals discussed rising incidence in younger people and a need for increased awareness about the association of excess weight as a risk factor for developing the disease. The concern extended to workforce and equipment shortfalls of meeting the needs of individuals with higher weight, which subsequently influenced treatment options, health outcomes and survivorship. CONCLUSIONS: Rising incidence of endometrial cancer in individuals with high weight presents multiple chances for inequitable access and health outcomes over the care continuum for endometrial cancer. Action is required to address incidence, awareness, access to equitable and inclusive treatment, and survivorship.

Can storytelling of women's lived experience enhance empathy in medical students? A pilot intervention study.

PURPOSE: This pilot study aimed to investigate the acceptability and efficacy of a patient storytelling intervention (live and recorded) on empathy levels of medical students. MATERIALS AND METHODS: Medical students participated in a storytelling intervention that had three components: listening to live or recorded stories from women with abnormal uterine bleeding, reflective writing, and a debriefing session. Empathy scores of students pre- and post-intervention were measured using the Jefferson Scale of Empathy-student version (JSE-S). Students also completed a feedback survey. Descriptive and inferential statistics were used to analyse quantitative data and content analysis was used for text comments. RESULTS: Both live and recorded storytelling interventions had positive effects on student's empathy scores post intervention. Overall, students were satisfied with the intervention and reported that it improved their understanding of life experiences of women. Suggestions were made for an in-person storytelling session and interactive discussion after listening to each story. CONCLUSION: A storytelling intervention has the potential to improve medical students' empathy and understanding of lived experience of women with health conditions. This could be valuable when student-patient interactions are limited in healthcare settings, or to enable stories of small numbers of patient volunteers to reach students.

Non-invasive tests for endometriosis are here; how reliable are they, and what should we do with the results?

A reliable non-invasive biomarker for endometriosis is highly likely in the coming years. In the lead-up to this, clinicians need to be aware of commercially available tests as they become accessible, be aware of the level of evidence to support them and be prepared to counsel and manage patients who present with the results of such tests. One such test gaining popularity in Europe was developed using a machine-based learning algorithm to analyse thousands of microRNAs based on a 200-patient cohort with suspected endometriosis in France. We explore the background science for this commercially available test; outline the questions that remain to be answered; and caution against its use outside of a research setting.

Exploring health professionals' viewpoint of provision of nutrition advice for women with endometrial cancer.

AIMS: The aim of this study was to explore barriers and facilitators to delivery and uptake of nutrition advice to women diagnosed with endometrial cancer from a health professionals' viewpoint. METHODS: Fifteen semi-structured interviews with health professionals with experience in providing healthcare to women diagnosed with endometrial cancer were audio-recorded and transcribed. Interviews were analysed using reflexive thematic analysis. Topics included high weight as a risk factor for endometrial cancer, nutrition information sources, and barriers and facilitators to delivering nutrition advice in clinical care. RESULTS: Four themes were identified. The first three exist as barriers to women receiving nutrition advice-how to navigate conversations about high weight, access to limited resourcing and health professionals feeling powerless to overcome system influences. The fourth theme identified a community approach need to facilitate a supportive environment and share knowledge. CONCLUSIONS: This study, through the lens of health professionals, highlights barriers to the delivery and uptake of nutrition advice at the patient, community and system levels. Enhancing survivorship for women after the diagnosis of endometrial cancer may be enabled by further understanding of how to overcome barriers and promote facilitators. Communication and partnership with women are imperative to achieving this.

Retrospective review of endometriosis surgery at Te Whatu Ora - Capital and Coast.

INTRODUCTION: Endometriosis is an under-researched disease, with Aotearoa-specific data severely lacking. Current estimates of parameters such as rates of endometriosis diagnosis, indication for surgery and sites of disease are based on international data. There is currently no published data on endometriosis surgeries in Aotearoa New Zealand. AIMS: We aimed to describe the laparoscopic surgeries conducted for suspected endometriosis at Te Whatu Ora - Capital and Coast, including the prevalence of endometriosis in this cohort, indication for surgery, symptoms experienced, endometriosis stage and sites involved, number of repeat laparoscopies, and prevalence of endometriosis at repeat surgery. MATERIALS AND METHODS: To conduct this retrospective cross-sectional study, data were extracted from Te Whatu Ora - Capital and Coast systems to identify all records indicating surgery for suspected endometriosis during 2018 and 2019. Variables investigated included age, ethnicity, endometriosis diagnosis (International Classification of Diseases-10 Clinical Modification coding), stage of endometriosis, histological report and endometriosis symptoms (pain and/or fertility). RESULTS: There were 436 surgeries for suspected endometriosis performed during 2018 and 2019, and endometriosis was diagnosed in 68.3% of these surgeries. Pacific and Asian people were under-represented in the study cohort compared to the demographics of the hospital catchment area (Pacific: 3.0% vs 8.4%, Asian: 9.9% vs 12.9%). The most common indication for surgery was pain. There were 76 surgeries performed for suspected recurrence of endometriosis, and endometriosis was identified in 55.6% of these. CONCLUSIONS: Endometriosis surgeries in this hospital in Aotearoa show similar presentations and surgical findings to international data. Our findings highlight areas requiring more research in an Aotearoa-specific context.

Reflections on access to care for heavy menstrual bleeding: Past, present, and in times of the COVID-19 pandemic.

The symptom of heavy menstrual bleeding (HMB) affects at least a quarter of reproductive-age menstruators. However, given the variance in diagnosing the underlying causes, barriers, and inequity in access to care for HMB, and therefore reporting of HMB, this figure is likely to be a gross underestimate. HMB can have a detrimental impact on quality of life. From the limited reports available it is estimated that around 50%-80% of people with HMB do not seek care for this debilitating symptom, and for those that do, it may take years until the first treatment action. Almost half of those with HMB believe their condition is unworthy of treatment, that there is no treatment available, or that it is "normal". The term "medical gaslighting" has recently been used to describe how healthcare professionals disregard people's symptoms. This has the ensuing effect of negatively compounding self-doubt and embarrassment around menstrual bleeding, creating a cycle of diminished experience inhibiting further seeking of care. There is a scarcity of intervention studies informing increased access to care. Multilevel approaches are needed to increase access to care and support for those with HMB. During the COVID-19 global pandemic, barriers to care access and support for HMB have been exacerbated and, given the significant pressures that this pandemic has placed on healthcare services worldwide, it will take many years to recover. In the meantime, it is crucial to ensure that medically "benign" conditions such as the symptom of HMB are not overlooked.

Ambitions for palliative and end of life care: mapping examples of use of the framework across England.

BACKGROUND: Since 2015, the Ambitions for Palliative and End of Life Care: a national framework for local action has provided guidance for care within England and beyond. Relaunched in 2021, the Framework sets out six Ambitions which, collectively, provide a vision to improve how death, dying and bereavement are experienced and managed. However, to date, there has been no central evaluation of how the Framework and its Ambitions have been implemented within service development and provision. To address this evidence gap, we investigated understanding and use of the Framework. METHODS: An online questionnaire survey was conducted to identify where the Framework has been used; examples of how it has been used; which Ambitions are being addressed; which foundations are being used; understanding of the utility of the Framework; and understanding of the opportunities and challenges involved in its use. The survey was open between 30 November 2021-31 January 2022, promoted via email, social media, professional newsletter and snowball sampling. Survey responses were analysed both descriptively, using frequency and cross-tabulations, and exploratively, using content and thematic analysis. RESULTS: 45 respondents submitted data; 86% were from England. Findings indicate that the Framework is particularly relevant to service commissioning and development across wider palliative and end of life care, with most respondents reporting a focus on Ambition 1 (Each person is seen as an individual) and Ambition 3 (Maximising comfort and wellbeing). Ambition 6 (Each community is prepared to help) was least likely to be prioritised, despite people welcoming the focus on community in national guidance. Of the Framework foundations, 'Education and training' was seen as most necessary to develop and/or sustain reported services. The provision of a shared language and collaborative work across sectors and partners were also deemed important. However, there is some indication that the Framework must give more prioritisation to carer and/or bereavement support, have greater scope to enhance shared practice and mutual learning, and be more easily accessible to non-NHS partners. CONCLUSIONS: The survey generated valuable summary level evidence on uptake of the Framework across England, offering important insights into current and past work, the factors impacting on this work and the implications for future development of the Framework. Our findings suggest considerable positive potential of the Framework to generate local action as intended, although difficulties remain concerning the mechanisms and resources necessary to enact this action. They also offer a valuable steer for research to further understand the issues raised, as well as scope for additional policy and implementation activity.

Transcriptomic identification of differentially expressed genes in Levonorgestrel resistant endometrial cancer cell lines.

Endometrial cancer (EC) is the most common gynecologic malignancy in the world and incidence is steadily increasing. The Levonorgestrel Intrauterine System (LNG-IUS) is an alternative conservative treatment for early-stage EC, however, Levonorgestrel (LNG) resistance occurs for 1 in 3 people. This study aimed to present potential LNG resistance mechanisms and identify differentially expressed genes (DEGs) in EC cell lines. Two LNG resistant cell lines were developed through long term culture in LNG (MFE296R and MFE319R ). Whole transcriptome sequencing was carried out on triplicate RNA samples. EdgeR v3.32.1 was used to identify differentially DEGs. Blast2go V6.0 (BioBam software) was used for functional annotation and analysis of genomic datasets. Protein interactions were investigated using the STRING database, including the identification of genes with high levels of interaction (HUB genes). Select DEGs and HUB genes were validated by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Fifteen DEGs were identified according to FDR < 0.05 and logFC < 2. Protein analysis identified six HUB genes with a degree of connectivity > 10. Relative mRNA expression of MAOA, MAOB, THRSP, CD80, NDP, LINC01474, DUSP2 and CXCL8 was significantly upregulated in both LNGR cell lines. Relative protein expression of GNAO1 and MAOA were significantly upregulated in both LNGR cell lines. This research identified novel markers of resistance in LNGR cell lines. We discussed potential mechanisms of LNG resistance including dedifferentiation and immunostimulation. The next step for this research is to validate these findings further in both translational and clinical settings.

Interventions to improve access to care for abnormal uterine bleeding: A systematic scoping review.

BACKGROUND: Women with abnormal uterine bleeding (AUB) experience barriers to accessing healthcare services. OBJECTIVES: To identify and describe the evidence on interventions to improve healthcare access of women with AUB. SEARCH STRATEGY: A systematic search of databases including Medline, CINAHL, EMBASE, Scopus, and Cochrane register for clinical trials on February 26, 2021. SELECTION CRITERIA: Studies including women with AUB and investigating an intervention to improve access at the levels of individual patient, community, organization, health system, or medical education. DATA COLLECTION AND ANALYSIS: Data extraction and descriptive analysis of the country, study design, settings, participant characteristics, intervention, outcome measures, and key findings. MAIN RESULTS: We identified 20 studies and most interventions (13 studies) targeted organizational changes. Creating a multidisciplinary team, bringing services together and developing a care pathway improved the availability of services. Management of AUB in an outpatient setting improved the affordability. The use of decision aids improved patient engagement in consultations. There is a lack of interventions at an individual or community level targeting health literacy, health beliefs, social acceptability, and opportunity to reach and pay for services. CONCLUSIONS: Community-based culturally-adapted interventions focusing on access to women with different socio-economic and cultural backgrounds should be investigated.

A promising future for endometriosis diagnosis and therapy: extracellular vesicles - a systematic review.

Endometriosis is a chronic, inflammatory gynaecological disease that can have severe negative impacts on quality of life and fertility, placing burden on patients and the healthcare system. Due to the heterogeneous nature of endometriosis, and the lack of correlation between symptom and surgical disease severity, diagnosis and treatment remain a significant clinical challenge. Extracellular vesicles (EVs) are biologically active particles containing molecular cargo involved in intercellular communication, that can be exploited for diagnostic and therapeutic purposes.We systematically reviewed studies exploring EVs and their role in endometriosis, specifically addressing diagnostic and therapeutic potential and current understanding of pathophysiology. Five databases (Pubmed, Embase, Medline, Web of Science, Google Scholar) were searched for keywords 'endometriosis' and either 'extracellular vesicles' or 'exosomes'.There were 28 studies included in the review. Endometrium derived EVs contribute to the development of endometriosis. EVs derived from endometriosis lesions contribute to angiogenesis, immunomodulation and fibrosis. Such EVs can be detected in blood, with early data demonstrating utility in diagnosis and recurrence detection. EV isolation techniques varied between studies and only eight of twenty-eight studies fully characterised EVs according to current recommended standards. Reporting/type of endometriosis was limited across studies. Varied patient population, type of sample and isolation techniques created bias and difficulty in comparing studies.EVs hold promise for improving care for symptomatic patients who have never had surgery, as well as those with recurrent symptoms after previous surgery. We encourage further EV research in endometriosis with the inclusion of rigorous reporting of both the patient population and technical methodology used, with the ultimate goal of achieving clinical utility for diagnosis, prognosis and eventually treatment.

Importance of the endometrial immune environment in endometrial cancer and associated therapies.

Endometrial cancer is rising in prevalence. The standard treatment modality of hysterectomy is becoming increasingly inadequate due primarily to the direct link between endometrial cancer and high BMI which increases surgical risks. This is an immunogenic cancer, with unique molecular subtypes associated with differential immune infiltration. Despite the immunogenicity of endometrial cancer, there is limited pre-clinical and clinical evidence of the function of immune cells in both the normal and cancerous endometrium. Immune checkpoint inhibitors for endometrial cancer are the most well studied type of immune therapy but these are not currently used as standard-of-care and importantly, they represent only one method of immune manipulation. There is limited evidence regarding the use of other immunotherapies as surgical adjuvants or alternatives. Levonorgestrel-loaded intra-uterine systems can also be effective for early-stage disease, but with varying success. There is currently no known reason as to what predisposes some patients to respond while others do not. As hormones can directly influence immune cell function, it is worth investigating the immune compartment in this context. This review assesses the immunological components of the endometrium and describes how the immune microenvironment changes with hormones, obesity, and in progression to malignancy. It also describes the importance of investigating novel pathways for immunotherapy.

Recommendations for extracellular vesicle miRNA biomarker research in the endometrial cancer context.

Endometrial cancer (EC) is the most common gynaecological malignancy in the developed world, and concerningly incidence is rising, particularly in younger people. Therefore, there is increased interest in novel diagnostic and prognostic biomarkers. Extracellular vesicles (EVs) are membrane-bound particles present in bodily fluids that have the potential to facilitate non-invasive, early diagnosis of EC and could aid with monitoring of recurrence and treatment response. EV cargo provides molecular insight into the tumor, with the lipid bilayer providing stability for RNA species usually prone to degradation. miRNAs have recently become a focus for EV biomarker research due to their ability to regulate cancer related pathways and influence cancer development and progression. This review evaluates the current literature on EV miRNA biomarkers with a focus on EC, and discusses the challenges facing this research. This review finally highlights areas of focus for EV miRNA biomarker research going forward, such as standardization of normalization approaches, sample storage and processing, extensive reporting of methodologies and moving away from single miRNA biomarkers.

Circulating cell-free DNA undergoes significant decline in yield after prolonged storage time in both plasma and purified form.

OBJECTIVES: Circulating DNA (cirDNA) is generally purified from plasma that has been biobanked for variable lengths of time. In long-term experiments or clinical trials, the plasma can be stored frozen for up to several years. Therefore, it is crucial to determine the stability of cirDNA to ensure confidence in sample quality upon analysis. Our main objective was to determine the effect of storage for up to 2 years on cirDNA yield and fragmentation. METHODS: We stored frozen EDTA plasma and purified cirDNA from 10 healthy female donors, then quantified cirDNA yield at baseline, and at regular intervals for up to 2 years, by qPCR and Qubit. We also compared cirDNA levels in non-haemolysed and haemolysed blood samples after 16 months of storage and tested the effect of varying DNA extraction protocol parameters. RESULTS: Storage up to two years caused an annual cirDNA yield decline of 25.5% when stored as plasma and 23% when stored as purified DNA, with short fragments lost more rapidly than long fragments. Additionally, cirDNA yield was impacted by plasma input and cirDNA elution volumes, but not by haemolysis. CONCLUSIONS: The design of long-term cirDNA-based studies and clinical trials should factor in the deterioration of cirDNA during storage.

Molecular Profiling of Endometrial Cancer: An Exploratory Study in Aotearoa, New Zealand.

BACKGROUND: Aotearoa, New Zealand, has one of the fastest-rising rates of endometrial cancer (EC) worldwide, increasing particularly in younger Maori and Pasifika women. There is a move towards using molecular profiling to direct treatment for each EC subtype. AIM: This study aimed to explore the molecular profiling of primary EC tissue in Aotearoa. METHODS: We used the PORTEC guidelines for the molecular subtyping of 90 patients' samples into four categories: POLE-mutated, p53 abnormal, mismatch repair deficient (MMRd) and no specific molecular profile (NSMP). The CTNNB1 mutation and L1CAM expression were also included in the analysis. POLE and CTNNB1 mutations were analysed using targeted next-generation sequencing (NGS). Novel mutations were assessed using VarSome. MMRd, L1CAM and p53 abnormalities were analysed using immunohistochemistry. RESULTS: In total, 15 samples were MMRd, 9 were p53 abnormal, 8 were POLE-mutated and the rest (56) were NSMP. Eleven samples had exon 3 CTNNB1 mutations and eleven novel POLE mutations were described. CONCLUSION: Surrogate markers for POLE mutations should be investigated. The validation of POLE variants and CTNNB1 mutations as part of an Aotearoa-based molecular panel is warranted.

Characterisation of Levonorgestrel-Resistant Endometrial Cancer Cells.

BACKGROUND: Endometrial cancer (EC) is the most common gynaecologic malignancy in the developed world, and incidence is increasing in premenopausal women. The levonorgestrel intrauterine system (LNG-IUS) is gaining traction as an alternative treatment for hyperplasia and early-stage EC for women who are unable to undergo surgery. Thirty to 60% of the women do not respond to this treatment, making the unknown mechanisms of levonorgestrel (LNG) resistance a critical obstacle for the conservative management of EC. This study aimed to characterise LNG-IUS treatment resistance in early-stage endometrial cancer in cell-line models. METHODS: LNG-resistant endometrial cancer cell lines (MFE296R and MFE319R) and cultures from three early stage endometrial cancer patients were developed. The behavioural profile of MFE296R and MFE319R was analysed using proliferation, adhesion, migration (wound healing and transwell) and invasion (spheroid) assays. LNG-sensitive cell lines (MFE296S and MFE319S) were compared to LNGR cell lines (MFE296R and MFE319R). A literature search was conducted to identify possible candidate biomarkers of LNG resistance. RT-qPCR was used to analyse the mRNA expression of 17 candidate biomarkers in MFE296R and MFE319R. mRNA expression of the top differentially expressed genes was measured using RT-qPCR in primary cultures. RESULTS: LNG resistance did not affect proliferation or invasion in immortalised endometrial cancer cells. Transwell migration was significantly increased in MFE319R cells (p=0.03). Cellular adhesion significantly decreased in both MFE296R cells (p=0.012) and MFE319R cells (p=0.04). mRNA expression of KLF4 and SATB2 was significantly amplified in MFE296R and MFE319R cells. mRNA expression of KLF4 was significantly upregulated LNG-resistant primary cell lines. CONCLUSION: LNG-resistant cells may have more oncogenic potential than their LNG-sensitive counterparts. Significant changes in the mRNA expression of KLF4 and SATB2 of LNG-resistant cells is a promising preliminary result in biomarker discovery for guiding LNG-IUS treatment of early stage endometrial cancer.

Total and endothelial cell-derived cell-free DNA in blood plasma does not change during menstruation.

Assays measuring cell-free DNA (cfDNA) in blood have widespread potential in modern medicine. However, a comprehensive understanding of cfDNA dynamics in healthy individuals is required to assist in the design of assays that maximise the signal driven by pathological changes, while excluding fluctuations that are part of healthy physiological processes. The menstrual cycle involves major remodelling of endometrial tissue and associated apoptosis, yet there has been little investigation of the impact of the menstrual cycle on cfDNA levels. Paired plasma samples were collected from 40 healthy women on menstruating (M) and non-menstruating (NM) days of their cycle. We measured total cfDNA by targeting ALU repetitive sequences and measured endothelial-derived cfDNA by methylation-specific qPCR targeting an endothelium-unique unmethylated CDH5 DNA region. CfDNA integrity and endothelial cfDNA concentration, but not total cfDNA, are consistent across time between NM and M. No significant changes in total (ALU-115 p = 0.273; ALU-247 p = 0.385) or endothelial cell specific (p = 0.301) cfDNA were observed, leading to the conclusion that menstrual status at the time of diagnostic blood collection should not have a significant impact on the quantitation of total cfDNA and methylation-based cancer assays.