Transcriptional profiling of mucus production in rhesus macaque endocervical cells under hormonal regulation.

OBJECTIVE: Endocervical mucus production is a key regulator of fertility throughout the menstrual cycle. With cycle-dependent variability in mucus quality and quantity, cervical mucus can either facilitate or block sperm ascension into the upper female reproductive tract. This study seeks to identify genes involved in the hormonal regulation of mucus production, modification, and regulation through profiling the transcriptome of endocervical cells from the non-human primate, the rhesus macaque (Macaca mulatta). INTERVENTION: We treated differentiated primary endocervical cultures with estradiol (E2) and progesterone (P4) to mimic peri-ovulatory and luteal-phase hormonal changes. Using RNA-sequencing, we identified differential expression of gene pathways and mucus producing and modifying genes in cells treated with E2 compared to hormone-free conditions and E2 compared to E2-primed cells treated with P4. MAIN OUTCOME MEASURES: We pursued differential gene expression analysis on RNA-sequenced cells. Sequence validation was done using qPCR. RESULTS: Our study identified 158 genes that show significant differential expression in E2-only conditions compared to hormone-free control, and 250 genes that show significant differential expression in P4-treated conditions compared to E2-only conditions. From this list, we found hormone-induced changes in transcriptional profiles for genes across several classes of mucus production, including ion channels and enzymes involved in post-translational mucin modification that have not previously been described as hormonally regulated. CONCLUSION: Our study is the first to use an in vitro culture system to create an epithelial-cell specific transcriptome of the endocervix. As a result, our study identifies new genes and pathways altered by sex-steroids in cervical mucus production.

Addressing the Gray Zone in Affirmative Mastectomy: An Analysis of Fischer 2 Patients.

BACKGROUND: Surgical decision making in gender-affirming mastectomy (GAM) is based on a patient's classification using the Fischer scale. Fischer 1 patients are excellent candidates for periareolar (PA) approach and Fischer 3 patients almost exclusively undergo double incision with free nipple grafting (DIFNG). Fischer 2 patients are in a gray zone in which decision making is more challenging. In this patient population, periareolar approaches can lead to increased complication and revision rates but free grafting procedures seem excessive. We have created a treatment algorithm to address Fischer 2 patients and additionally developed a novel technique, the batwing, to provide patients with more options. METHODS: A retrospective chart review was undertaken to analyze the Fischer classification of all patients undergoing top surgery by a single surgeon at an academic institution from 2014 to 2021. The choice of surgical technique used as well as the outcomes of GAM among Fischer 2 patients was analyzed. RESULTS: Four hundred four patients underwent GAM, and 51 (11%) had Fischer 2 classification. The surgical techniques used were PA (27%), batwing (39%), nipple-sparing double incision (NSDI, 24%), and DIFNG (10%). Of those, 10% had major complications and 20% requested revision for contour irregularities. Major complication rates for PA, batwing, NSDI, and DIFNG were as follows: 2 of 14 patients (14%), 1 of 20 patients (5%), 1 of 12 patients (8%), and 1 of 5 patients (20%), respectively. The revision rate by technique was PA (36%), batwing (15%), NSDI (17%), and DIFNG (0%). CONCLUSIONS: For Fischer 2 patients, batwing and NSDI techniques avoid the need for free nipple graft while providing better exposure, improved control of nipple-areolar complex position, and decreased rate of revision as compared with the PA technique. The complication rate was not significantly different. We present an algorithm accounting for Fischer grade, unique patient characteristics, and patient desires.

Impact of State Laws on Dispensing Opioid Prescriptions Following Posterior Lumbar Interbody Fusion Procedures: A Retrospective Large National Database Study.

STUDY DESIGN: Retrospective Cohort Study. OBJECTIVES: This study aimed to examine the effect of state legislation on prescribing behavior after a commonly performed spinal procedure, posterior lumbar interbody fusion (PLIF). METHODS: Two cohorts of patients from the Pearl Diver Database were created based on patients who underwent PLIF surgery in 2014-15 and 2018-19. We compared opioid prescription rates and morphine-milli-equivalent (MME) between states with and without prescription legislation. RESULTS: We analyzed 50 958 PLIF patients from 2014-15 and 46 751 patients from 2018-19. Among them, 38 states passed opioid prescription laws in 2016-2017, while 12 states did not. The percentage of patients receiving opioid prescriptions within 365 days post-surgery remained similar in both time periods (49% in 2014-15 and 48% in 2018-2019). This trend was consistent across states with and without prescription legislation (50% vs 48% in 2014-2015, and similar in 2018-19). Opioid prescription quantity significantly decreased in all states between 2014-15 and 2018-19. In states with legislation, average MME dropped from 9198 ± 21 002 to 4932 ± 13 213 (46.4% decrease), and in states without legislation, it decreased from 9175 ± 21 032 to 4994 ± 11 687 (45.6% decrease). However, these differences were not statistically significant (P = .7985). CONCLUSION: From 2014 to 2018, there was a significant decrease in the number of opioids prescribed after PLIF. However, this decrease occurred irrespective of state legislation on prescribing practices being passed. We believe the reduction in opioids prescribed was due to increased awareness surrounding the dangers of opioids among physicians.

Transcriptional profiling of mucus production and modification in rhesus macaque endocervical cells under hormonal regulation.

Objective: Endocervical mucus production is a key regulator of fertility throughout the menstrual cycle. With cycle-dependent variability in mucus quality and quantity, cervical mucus can either facilitate or block sperm ascension into the upper female reproductive tract. This study seeks to identify genes involved in the hormonal regulation of mucus production, modification, and regulation through profiling the transcriptome of endocervical cells from the non-human primate, the Rhesus Macaque (Macaca mulatta). Design: Experimental. Setting: Translational science laboratory. Intervention: We treated differentiated primary endocervical cultures with estradiol (E2) and progesterone (P4) to mimic peri-ovulatory and luteal-phase hormonal changes. Using RNA-sequencing, we identified differential expression of gene pathways and mucus producing and modifying genes in cells treated with E2 compared to hormone-free conditions and E2 compared to E2-primed cells treated with P4. Main Outcome Measures: We pursued differential gene expression analysis on RNA-sequenced cells. Sequence validation was done using qPCR. Results: Our study identified 158 genes that show significant differential expression in E2-only conditions compared to hormone-free control, and 250 genes that show significant differential expression in P4-treated conditions compared to E2-only conditions. From this list, we found hormone-induced changes in transcriptional profiles for genes across several classes of mucus production, including ion channels and enzymes involved in post-translational mucin modification that have not previously been described as hormonally regulated. Conclusion: Our study is the first to use an in vitro culture system to create an epithelial-cell specific transcriptome of the endocervix. As a result, our study identifies new genes and pathways that are altered by sex-steroids in cervical mucus production.

Induction of developmental hematopoiesis mediated by transcription factors and the hematopoietic microenvironment.

The induction of hematopoiesis in various cell types via transcription factor (TF) reprogramming has been demonstrated by several strategies. The eventual goal of these approaches is to generate a product for unmet needs in hematopoietic cell transplantation therapies. The most successful strategies hew closely to clues provided from developmental hematopoiesis in terms of factor expression and environmental cues. In this review, we aim to summarize the TFs that play important roles in developmental hematopoiesis primarily and to also touch on adult hematopoiesis. Several aspects of cellular and molecular biology coalesce in this process, with TFs and surrounding cellular signals playing a major role in the overall development of the hematopoietic lineage. We attempt to put these elements into the context of reprogramming and highlight their roles.

Memory of Divisional History Directs the Continuous Process of Primitive Hematopoietic Lineage Commitment.

Hematopoietic stem cells (HSCs) exist in a dormant state and progressively lose regenerative potency as they undergo successive divisions. Why this functional decline occurs and how this information is encoded is unclear. To better understand how this information is stored, we performed RNA sequencing on HSC populations differing only in their divisional history. Comparative analysis revealed that genes upregulated with divisions are enriched for lineage genes and regulated by cell-cycle-associated transcription factors, suggesting that proliferation itself drives lineage priming. Downregulated genes are, however, associated with an HSC signature and targeted by the Polycomb Repressive Complex 2 (PRC2). The PRC2 catalytic subunits Ezh1 and Ezh2 promote and suppress the HSC state, respectively, and successive divisions cause a switch from Ezh1 to Ezh2 dominance. We propose that cell divisions drive lineage priming and Ezh2 accumulation, which represses HSC signature genes to consolidate information on divisional history into memory.

Induction of human hemogenesis in adult fibroblasts by defined factors and hematopoietic coculture.

Transcription factor (TF)-based reprogramming of somatic tissues holds great promise for regenerative medicine. Previously, we demonstrated that the TFs GATA2, GFI1B, and FOS convert mouse and human fibroblasts to hemogenic endothelial-like precursors that generate hematopoietic stem progenitor (HSPC)-like cells over time. This conversion is lacking in robustness both in yield and biological function. Herein, we show that inclusion of GFI1 to the reprogramming cocktail significantly expands the HSPC-like population. AFT024 coculture imparts functional potential to these cells and allows quantification of stem cell frequency. Altogether, we demonstrate an improved human hemogenic induction protocol that could provide a valuable human in vitro model of hematopoiesis for disease modeling and a platform for cell-based therapeutics. DATABASE: Gene expression data are available in the Gene Expression Omnibus (GEO) database under the accession number GSE130361.