The uterine secretome initiates growth of gynecologic tissues in ectopic locations.

Endosalpingiosis (ES) and endometriosis (EM) refer to the growth of tubal and endometrial epithelium respectively, outside of their site of origin. We hypothesize that uterine secretome factors drive ectopic growth. To test this, we developed a mouse model of ES and EM using tdTomato (tdT) transgenic fluorescent mice as donors. To block implantation factors, progesterone knockout (PKO) tdT mice were created. Fluorescent lesions were present after oviduct implantation with and without WT endometrium. Implantation was increased (p<0.05) when tdt oviductal tissue was implanted with endometrium compared to oviductal tissue alone. Implantation was reduced (p<0.0005) in animals implanted with minced tdT oviductal tissue with PKO tdT endometrium compared to WT endometrium. Finally, oviductal tissues was incubated with and without a known implantation factor, leukemia inhibitory factor (LIF) prior to and during implantation. LIF promoted lesion implantation. In conclusion, endometrial derived implantation factors, such as LIF, are necessary to initiate ectopic tissue growth. We have developed an animal model of ectopic growth of gynecologic tissues in a WT mouse which will potentially allow for development of new prevention and treatment modalities.

A Randomized Controlled Trial of Precision Nutrition Counseling for Service Members at Risk for Metabolic Syndrome.

INTRODUCTION: Metabolic syndrome (MetS) is a threat to the active component military as it impacts health, readiness, retention, and cost to the Military Health System. The most prevalent risk factors documented in service members' health records are high blood pressure (BP), low high-density lipoprotein cholesterol, and elevated triglycerides. Other risk factors include abdominal obesity and elevated fasting blood glucose. Precision nutrition counseling and wellness software applications have demonstrated positive results for weight management when coupled with high levels of participant engagement and motivation. MATERIALS AND METHODS: In this prospective randomized controlled trial, trained registered dietitians conducted nutrition counseling using results of targeted sequencing, biomarkers, and expert recommendations to reduce the risk for MetS. Upon randomization, the treatment arm initiated six weekly sessions and the control arm received educational pamphlets. An eHealth application captured diet and physical activity. Anthropometrics and BP were measured at baseline, 6 weeks, and 12 weeks, and biomarkers were measured at baseline and 12 weeks. The primary outcome was a change in weight at 12 weeks. Statistical analysis included descriptive statistics and t-tests or analysis of variance with significance set at P < .05. RESULTS: Overall, 138 subjects enrolled from November 2019 to February 2021 between two military bases; 107 completed the study. Demographics were as follows: 66% male, mean age 31 years, 66% married, and 49% Caucasian and non-Hispanic. Weight loss was not significant between groups or sites at 12 weeks. Overall, 27% of subjects met the diagnostic criteria for MetS on enrollment and 17.8% upon study completion. High deleterious variant prevalence was identified for genes with single-nucleotide polymorphisms linked to obesity (40%), cholesterol (38%), and BP (58%). Overall, 65% of subjects had low 25(OH)D upon enrollment; 45% remained insufficient at study completion. eHealth app had low adherence yet sufficient correlation with a valid reference. CONCLUSIONS: Early signs of progress with weight loss at 6 weeks were not sustained at 12 weeks. DNA-based nutrition counseling was not efficacious for weight loss.

Rapid cold sterilization of 3D printed surgical instruments for the austere environment.

INTRODUCTION: Medical operations are vulnerable to global supply chain fluctuations. The ability to locally produce and reliably sterilize medical equipment may mitigate this risk. This project developed a reliable high-level disinfection process for 3D printed surgical tools. METHODS: Surgical instruments and consumables were designed and printed from various materials. Devices contaminated with known and unknown bacteria underwent one of three cleaning methods followed by high-level disinfection using submersion in a Cidex OPA Solution. Devices were then cultured on blood agar plates and incubated for 48 h. Positive and negative controls were performed. RESULTS: The results of control experiments showed no growth on negative controls and significant growth on all positive control plates. Of the three cleaning methods tested, one showed no growth: cleaning with isopropyl alcohol and chlorhexidine followed by Cidex bath. DISCUSSION: This project successfully developed a rapid high-level disinfection process for 3D printed surgical instruments made from two different types of 3D printing material.

Cellular junction and mesenchymal factors delineate an endometriosis-specific response of endometrial stromal cells to the mesothelium.

Endometriosis is a debilitating gynecologic disorder that affects ∼10% of women of reproductive age. Endometriosis is characterized by growth of endometriosis lesions within the abdominal cavity, generally thought to arise from retrograde menstruation of shed endometrial tissue. While the pathophysiology underlying peritoneal endometriosis lesion formation is still unclear, the interaction between invading endometrial tissue and the peritoneal mesothelial lining is an essential step in lesion formation. In this study, we assessed proteomic differences between eutopic endometrial stromal cells (ESCs) from women with and without endometriosis in response to peritoneal mesothelial cell (PMC) exposure, using single-cell cytometry by time-of-flight (CyTOF). Co-cultured primary eutopic ESCs from women with and without endometriosis with an established PMC line were subjected to immunostaining with a panel of Maxpar CyTOF metal-conjugated antibodies (n = 28) targeting cell junction and mesenchymal markers, which are involved in cell-cell adhesions and epithelial-mesenchymal transition. Exposure of the ESCs to PMCs resulted in a drastic shift in cellular expression profiles in ESCs derived from endometriosis, whereas little effect by PMCs was observed in ESCs from non-endometriosis subjects. The transcription factor SNAI1 was consistently repressed by PMC interactions. ESCs from endometriosis patients are unique in that they respond to PMCs by undergoing changes in adhesive properties and mesenchymal characteristics that would facilitate lesion formation.

An intrauterine genomic classifier reliably delineates the location of nonviable pregnancies.

OBJECTIVE: To compare the intrauterine gene expression signatures of women with surgically confirmed ectopic pregnancy (ECT) and those of women with miscarriage to inform the development of a genomic classifier for the reliable delineation of pregnancy location in women with clinically nonviable pregnancies of unknown location (NV-PULs). DESIGN: Discovery-based prospective cohort study. SETTING: Academic medical center. PATIENT(S): Women with clinically nonviable early pregnancy to include abnormal intrauterine pregnancy (AIUP), ECT, or NV-PUL. INTERVENTION(S): Endometrial (EM) pipelle sampling of the uterus was conducted at the time of scheduled surgery for clinically nonviable early pregnancy (dilation and curettage, manual vacuum aspiration, or laparoscopy). All pregnancy locations were surgically and/or histologically confirmed as intrauterine or ectopic. MAIN OUTCOME MEASURE(S): Gene expression profiles as determined by array hybridization, quantitative real-time polymerase chain reaction, and nCounter technology. RESULT(S): Intrauterine samples were obtained by EM pipelle from 27 women undergoing surgery for a clinically nonviable early pregnancy. Comparison of array-based global gene expression signatures from women with histologically confirmed ECT versus AIUP revealed 61 differentially expressed genes from which the 5 most informative were included in the pregnancy location classifier. All 5 genes (C20orf85, LRRC46, RSPH4A, WDR49, and ZBBX) were cilia-associated and showed increased expression in pipelle samples from women with ECT relative to expression in samples from women with AIUP. The 5-gene classifier demonstrated an average area under the receiver operator characteristic curve of 0.97 for the detection of ECT. In an external test set composed of publicly available EM pipelle-based gene expression data from a study with similar ECT and AIUP cohorts (n = 19), the classifier revealed an average area under the receiver operator characteristic curve of 0.84. CONCLUSION(S): Consistently increased expression of cilia-associated genes in the uterine cavity of women with ECT provides a reliable molecular signal for the delineation of pregnancy location in women with clinically assessed NV-PUL. A classifier consisting of the 5 most informative cilia-associated genes demonstrated 91% (42/46) accuracy in predicting the pregnancy location.

DRB4*01:01 Has a Distinct Motif and Presents a Proinsulin Epitope That Is Recognized in Subjects with Type 1 Diabetes.

DRB4*01:01 (DRB4) is a secondary HLA-DR product that is part of the high-risk DR4/DQ8 haplotype that is associated with type 1 diabetes (T1D). DRB4 shares considerable homology with HLA-DR4 alleles that predispose to autoimmunity, including DRB1*04:01 and DRB1*04:04. However, the DRB4 protein sequence includes distinct residues that would be expected to alter the characteristics of its binding pockets. To identify high-affinity peptides that are recognized in the context of DRB4, we used an HLA class II tetramer-based approach to identify epitopes within multiple viral Ags. We applied a similar approach to identify antigenic sequences within glutamic acid decarboxylase 65 and pre-proinsulin that are recognized in the context of DRB4. Seven sequences were immunogenic, eliciting high-affinity T cell responses in DRB4+ subjects. DRB1*04:01-restricted responses toward many of these peptides have been previously described, but responses to a novel pre-proinsulin 9-28 peptide were commonly observed in subjects with T1D. Furthermore, T cells that recognized this peptide in the context of DRB4 were present at significantly higher frequencies in patients with T1D than in healthy controls, implicating this as a disease-relevant specificity that may contribute to the breakdown of ß cell tolerance in genetically susceptible individuals. We then deduced a DRB4 motif and confirmed its key features through structural modeling. This modeling suggested that the core epitope within the pre-proinsulin 9-28 peptide has a somewhat unusual binding motif, with tryptophan in the fourth binding pocket of DRB4, perhaps influencing the availability of this complex for T cell selection.