Deciphering the role of PGRMC2 in the human endometrium during the menstrual cycle and in vitro decidualization using an in vitro approach.

STUDY QUESTION: What is the human endometrial non-classical progesterone receptor (PGR) membrane component 2 (PGRMC2) expression pattern throughout the menstrual cycle and what role does it play during decidualization? SUMMARY ANSWER: Endometrial PGRMC2 expression fluctuates during the human menstrual cycle and is abundantly expressed in human endometrial stromal cells (hEnSCs) during in vitro decidualization, process where PGRMC2 is involved in embryo implantation-related pathways. WHAT IS KNOWN ALREADY: The endometrial response to progesterone is mediated by the classical and non-classical PGRs. We previously demonstrated that PGR membrane component 1 (PGRMC1) is critical for endometrial function, embryo implantation, and future placentation, however, the role(s) of PGRMC2, which is structurally similar to PGRMC1, have not been studied in the human endometrium. STUDY DESIGN, SIZE, DURATION: This prospective study comprehensively evaluated the endometrial expression of PGRMC2 throughout the human menstrual cycle and during in vitro decidualization of hEnSCs (isolated from 77 endometrial biopsies that were collected from 66 oocyte donors), using immunohistochemistry, RT-qPCR, western blot, transcriptomic, and proteomic analyses. In addition, functional analysis was carried out to validate the implication of PGRMC2 in hEnSCs during embryo invasion using an in vitro outgrowth model. PARTICIPANTS/MATERIALS, SETTING, METHODS: In vitro decidualization of hEnSCs was induced using co-treatment with cAMP and medroxyprogesterone 17-acetate progestin, and evaluated by measuring prolactin by ELISA and F-actin immunostaining. RT-qPCR was employed to compare expression with other PGRs. To reveal the function of PGRMC2 during the decidualization process, we specifically knocked down PGRMC2 with siRNAs and performed RNA-seq and quantitative proteomics techniques (SWATH-MS). The common differentially expressed genes (DEGs) and proteins (DEPs) were considered for downstream functional enrichment analysis. Finally, to verify its implication in the trophoblast invasion, an outgrowth model was carried out where hEnSCs with silenced PGRMC2 were co-cultured with human trophoblastic spheroids (JEG-3) following in vitro decidualization. MAIN RESULTS AND THE ROLE OF CHANCE: In contrast to PGRMC1 and classical PGRs, endometrial PGRMC2 gene expression was significantly lower during the late- versus mid-secretory phase (P < 0.05). Accordingly, the elevated PGRMC2 protein abundance observed in the endometrial epithelial glands throughout the menstrual cycle dropped in the late secretory phase, when abundance decreased in all endometrial compartments. Nevertheless, PGRMC2 protein increased during the mid-secretory phase in stromal and glandular cells, and PGRMC2 mRNA (P < 0.0001) and protein (P < 0.001) levels were significantly enhanced in the membranes/organelles of decidualized hEnSCs, compared to non-decidualized hEnSCs. Notably, PGRMC1 and PGRMC2 mRNA were significantly more abundant than classical PGRs throughout menstrual cycle phases and in decidualized and non-decidualized hEnSCs (P < 0.05). RNA-seq and proteomics data revealed 4687 DEGs and 28 DEPs, respectively, in decidualized hEnSCs after PGRMC2 silencing. While functional enrichment analysis showed that the 2420 upregulated genes were mainly associated with endoplasmic reticulum function, vesicular transport, morphogenesis, angiogenesis, cell migration, and cell adhesion, the 2267 downregulated genes were associated with aerobic respiration and protein biosynthesis. The protein enrichment analysis showed that 4 upregulated and 24 downregulated proteins were related to aerobic respiration, cellular response, metabolism, localization of endoplasmic reticulum proteins, and ribonucleoside biosynthesis routes. Finally, PGRMC2 knockdown significantly compromised the ability of the decidualized hEnSCs to support trophoblast expansion in an outgrowth model (P < 0.05). LARGE-SCALE DATA: Transcriptomic data are available via NCBI's Gene Expression Omnibus (GEO) under GEO Series accession number GSE251843 and proteomic data via ProteomeXchange with identifier PXD048494. LIMITATIONS, REASONS FOR CAUTION: The functional analyses were limited by the discrete number of human endometrial biopsies. A larger sample size is required to further investigate the potential role(s) of PGRMC2 during embryo implantation and maintenance of pregnancy. Further, the results obtained in the present work should be taken with caution, as the use of a pure primary endometrial stromal population differentiated in vitro does not fully represent the heterogeneity of the endometrium in vivo, nor the paracrine communications occurring between the distinct endometrial cell types. WIDER IMPLICATIONS OF THE FINDINGS: The repression of endometrial PGRMC2 during the late- versus mid-secretory phase, together with its overexpression during decidualization and multiple implications with embryo implantation not only highlighted the unknown roles of PGRMC2 in female reproduction but also the potential to exploit PGRMC2 signaling pathways to improve assisted reproduction treatments in the future. STUDY FUNDING/COMPETING INTEREST(S): This research was funded by Instituto de Salud Carlos III (ISCIII) granted to F.D. (PI20/00405 and PI23/00860), co-funded by the European Union. Y.M.-L. was supported by a predoctoral research grant from Generalitat Valenciana (ACIF/2019/262). R.G.-M. was supported by Generalitat Valenciana (CIAPOT/2022/15). P.d.C. was supported by a predoctoral grant for training in research into health (PFIS FI20/00086) from the Instituto de Salud Carlos III. I.D.-H. was supported by the Spanish Ministry of Science, Innovation and Universities (FPU18/01550). A.P. was supported by the Instituto de Salud Carlos III (PFIS FI18/00009). This research was also supported by IVI Foundation-RMA Global (1911-FIVI-103-FD). The authors declare no conflict of interest.

Identification of miR-30c-5p microRNA in Serum as a Candidate Biomarker to Diagnose Endometriosis.

The diagnosis of endometriosis by laparoscopy is delayed until advanced stages. In recent years, microRNAs have emerged as novel biomarkers for different diseases. These molecules are small non-coding RNA sequences involved in the regulation of gene expression and can be detected in peripheral blood. Our aim was to identify candidate serum microRNAs associated with endometriosis and their role as minimally invasive biomarkers. Serum samples were obtained from 159 women, of whom 77 were diagnosed with endometriosis by laparoscopy and 82 were healthy women. First, a preliminary study identified 29 differentially expressed microRNAs between the two study groups. Next, nine of the differentially expressed microRNAs in the preliminary analysis were evaluated in a new cohort of 67 women with endometriosis and 72 healthy women. Upon validation by quantitative real-time PCR technique, the circulating level of miR-30c-5p was significantly higher in the endometriosis group compared with the healthy women group. The area under the curve value of miR-30c-5p was 0.8437, demonstrating its diagnostic potential even when serum samples registered an acceptable limit of hemolysis. Dysregulation of this microRNA was associated with molecular pathways related to cancer and neuronal processes. We concluded that miR-30c-5p is a potential minimally invasive biomarker of endometriosis, with higher expression in the group of women with endometriosis diagnosed by laparoscopy.

Proteomic Profiling Identifies Candidate Diagnostic Biomarkers of Hydrosalpinx in Endometrial Fluid: A Pilot Study.

Hydrosalpinx is a fluid occlusion and distension of the fallopian tubes, often resulting from pelvic inflammatory disease, which reduces the success of artificial reproductive technologies (ARTs) by 50%. Tubal factors account for approximately 25% of infertility cases, but their underlying molecular mechanisms and functional impact on other reproductive tissues remain poorly understood. This proteomic profiling study applied sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) to study hydrosalpinx cyst fluid and pre- and post-salpingectomy endometrial fluid. Among the 967 proteins identified, we found 19 and 17 candidate biomarkers for hydrosalpinx in pre- and post-salpingectomy endometrial fluid, respectively. Salpingectomy significantly affected 76 endometrial proteins, providing insights into the enhanced immune response and inflammation present prior to intervention, and enhanced coagulation cascades and wound healing processes occurring one month after intervention. These findings confirmed that salpingectomy reverses the hydrosalpinx-related functional impairments in the endometrium and set a foundation for further biomarker validation and the development of less-invasive diagnostic strategies for hydrosalpinx.

Myelodysplastic syndromes mortality in Spain: a comprehensive age-period-cohort and joinpoint analysis.

OBJECTIVE: The present study aims to assess the mortality trends in myelodysplastic syndromes (MDS) in Spain from 1980 to 2021. METHODS: Deaths and mid-year population data were collected from the National Institute of Statistics. We estimated age-standardised mortality rates (ASMRs) per 100,000 person-years for all ages and ages 35-64. Joinpoint regression identified significant changes in mortality trends. The independent effects of age, period and birth cohort on MDS mortality were also examined. RESULTS: MDS-related deaths gradually increased from 36 in 1980 to 1118 in 2021, with an overall increase of 6.6% in age-standardised mortality rates (ASMRs) for both men and women. Joinpoint analysis identified four periods for both men and women: 1980-1987 (stable rates), 1987-1990 (sharp increase), 1990-1999 (slower increase) and 1999-2021 (stable rates). ASMRs (35-64 years) increased by 2.5% over the study period, with a turning point identified in 1996 when rates decreased. Mortality from MDS increases with age and is higher in men. The cohort's relative risk increased until the mid-1950s and then stabilised, whilst the period relative risk increased between 1982 and 1996 and then stabilised. CONCLUSION: The results of this study indicate a progressive increase in MDS-related deaths in Spain between 1980 and 2021. Notably, this increase was more pronounced in men than in women. Analysis of birth cohort trends revealed shifts in MDS risk, characterised by an increase until the mid-twentieth century, followed by a stabilisation. Using joinpoint analysis, four distinct periods were identified, shedding light on the changing patterns of mortality over time. These findings help to shape future research directions and inform public health strategies. They also provide optimism for advances in MDS treatment and potential reductions in mortality.

Suboptimal use of ovarian function suppression in very young women with early breast cancer: a real-world data study.

PURPOSE: The incidence of breast cancer in young women (BCYW) has increased in recent decades. Malignant disease in this subset is characterized by its aggressiveness and poor prognosis. Ovarian function suppression (OFS) in these patients improves survival especially in hormone receptor-positive (HR +) cases. The Regan Composite Risk (RCR) is a prognostic tool to identify high-risk HR + BC candidates for OFS. Our study sought to characterize a Chilean cohort of early HR + BCYW assessing the use of OFS and its related prognosis and the utility of RCR in our patients. METHODS: This was a retrospective population cohort study that included ≤ 35-year-old early HR + /human epidermal growth factor receptor 2 -negative (HER2-) BC patients treated between 2001 and 2021. Analysis included clinical-pathological characteristics, treatment strategies, and survival. Also, we evaluated the association between RCR and survival. RESULTS: A total of 143 patients were included into our study, representing 2.9% of all early BC cases in our registry. Median age was 31 years old (range: 19-35). Most patients (93%) received endocrine therapy (ET). Of these, 18% received OFS. No survival differences were observed among treatment strategies. Median RCR score for patients treated with CT plus ET was significantly higher vs. ET alone (2.95 vs. 1.91; p = 0.0001). Conversely, patients treated with tamoxifen alone had significantly lower RCR scores vs. OFS (2.72 vs. 3.14; p = 0.04). Higher RCR scores were associated with poorer overall survival. CONCLUSION: Less than 20% of very young women with early HR + /HER2-BC in our cohort received OFS, in most cases, this involved surgical oophorectomy. RCR score was higher in patients that underwent CT and OFS and was associated with survival, regardless of treatment. We confirm the RCR score as a valuable prognostic tool to identify high-risk BC patients who could benefit from OFS.

Involvement and Autonomy of Minors in Medical Settings: Perceptions of Children Undergoing Surgery and Parents.

Informed consent presupposes competence and represents a formal decision by an informed person who has the legal capacity to accept medical action or participate in research. Our aim was to analyze the perceptions of minors and their parents about the age at which they consider that a minor is competent for making health decisions. A descriptive observational study was carried out in 302 minors between 12 and 17 years of age undergoing elective surgery, and 302 parents (range 30 to 62 years). Two semistructured questionnaires were designed, one for the minors and the other, for the parents. A total of 20.1% of minors and 31.1% of parents believe that patients should not make decisions related to their health until they are 18 years old. A total of 74.9% of the minors surveyed consider that from 16 years of age, the minor is empowered to make decisions. In parents, this percentage is 60%. In the pediatric setting, each case and situation must be examined individually to determine if the minor meets the condition of maturity to decide. The ideal is to promote the minor's participation in decision-making, giving them the opportunity to participate in the process in a manner appropriate to their capacity.

The effect of vitrification on blastocyst mitochondrial DNA dynamics and gene expression profiles.

PURPOSE: Does vitrification/warming affect the mitochondrial DNA (mtDNA) content and the gene expression profile of blastocysts? METHODS: Prospective cohort study in which 89 blastocysts were obtained from 50 patients between July 2017 and August 2018. mtDNA was measured in a total of 71 aneuploid blastocysts by means of real-time polymerase chain reaction (RT-PCR). Transcriptomic analysis was performed by RNA sequencing (RNA-seq) in an additional 8 aneuploid blastocysts cultured for 0 h after warming, and 10 aneuploid blastocysts cultured for 4-5 h after warming. RESULTS: A significant decrease in mtDNA content just during the first hour after the warming process in blastocysts was found (P < 0.05). However, mtDNA content experimented a significantly increased along the later culture hours achieving the original mtDNA levels before vitrification after 4-5 h of culture (P < 0.05). Gene expression analysis and functional enrichment analysis revealed that such recovery was accompanied by upregulation of pathways associated with embryo developmental capacity and uterine embryo development. Interestingly, the significant increase in mtDNA content observed in blastocysts just after warming also coincided with the differential expression of several cellular stress response-related pathways, such as apoptosis, DNA damage, humoral immune responses, and cancer. CONCLUSION: To our knowledge, this is the first study demonstrating in humans, a modulation in blastocysts mtDNA content in response to vitrification and warming. These results will be useful in understanding which pathways and mechanisms may be activated in human blastocysts following vitrification and warming before a transfer.

The NR2F2-HAND2 signaling axis regulates progesterone actions in the uterus at early pregnancy.

Endometrial function is dependent on a tight crosstalk between the epithelial and stromal cells of the endometrium. This communication is critical to ensure a fertile uterus and relies on progesterone and estrogen signaling to prepare a receptive uterus for embryo implantation in early pregnancy. One of the key mediators of this crosstalk is the orphan nuclear receptor NR2F2, which regulates uterine epithelial receptivity and stromal cell differentiation. In order to determine the molecular mechanism regulated by NR2F2, RNAseq analysis was conducted on the uterus of PgrCre;Nr2f2f/f mice at Day 3.5 of pregnancy. This transcriptomic analysis demonstrated Nr2f2 ablation in Pgr-expressing cells leads to a reduction of Hand2 expression, increased levels of Hand2 downstream effectors Fgf1 and Fgf18, and a transcriptome manifesting suppressed progesterone signaling with an altered immune baseline. ChIPseq analysis conducted on the Day 3.5 pregnant mouse uterus for NR2F2 demonstrated the majority of NR2F2 occupies genomic regions that have H3K27ac and H3K4me1 histone modifications, including the loci of major uterine transcription regulators Hand2, Egr1, and Zbtb16. Furthermore, functional analysis of an NR2F2 occupying site that is conserved between human and mouse was capable to enhance endogenous HAND2 mRNA expression with the CRISPR activator in human endometrial stroma cells. These data establish the NR2F2 dependent regulation of Hand2 in the stroma and identify a cis-acting element for this action. In summary, our findings reveal a role of the NR2F2-HAND2 regulatory axis that determines the uterine transcriptomic pattern in preparation for the endometrial receptivity.

Deciphering a shared transcriptomic regulation and the relative contribution of each regulator type through endometrial gene expression signatures.

BACKGORUND: While various endometrial biomarkers have been characterized at the transcriptomic and functional level, there is generally a poor overlap among studies, making it unclear to what extent their upstream regulators (e.g., ovarian hormones, transcription factors (TFs) and microRNAs (miRNAs)) realistically contribute to menstrual cycle progression and function. Unmasking the intricacies of the molecular interactions in the endometrium from a novel systemic point of view will help gain a more accurate perspective of endometrial regulation and a better explanation the molecular etiology of endometrial-factor infertility. METHODS: An in-silico analysis was carried out to identify which regulators consistently target the gene biomarkers proposed in studies related to endometrial progression and implantation failure (19 gene lists/signatures were included). The roles of these regulators, and of genes related to progesterone and estrogens, were then analysed in transcriptomic datasets compiled from samples collected throughout the menstrual cycle (n = 129), and the expression of selected TFs were prospectively validated in an independent cohort of healthy participants (n = 19). RESULTS: A total of 3,608 distinct genes from the 19 gene lists were associated with endometrial progression and implantation failure. The lists' regulation was significantly favoured by TFs (89% (17/19) of gene lists) and progesterone (47% (8 /19) of gene lists), rather than miRNAs (5% (1/19) of gene lists) or estrogen (0% (0/19) of gene lists), respectively (FDR < 0.05). Exceptionally, two gene lists that were previously associated with implantation failure and unexplained infertility were less hormone-dependent, but primarily regulated by estrogen. Although endometrial progression genes were mainly targeted by hormones rather than non-hormonal contributors (odds ratio = 91.94, FDR < 0.05), we identified 311 TFs and 595 miRNAs not previously associated with ovarian hormones. We highlight CTCF, GATA6, hsa-miR-15a-5p, hsa-miR-218-5p, hsa-miR-107, hsa-miR-103a-3p, and hsa-miR-128-3p, as overlapping novel master regulators of endometrial function. The gene expression changes of selected regulators throughout the menstrual cycle (FDR < 0.05), dually validated in-silico and through endometrial biopsies, corroborated their potential regulatory roles in the endometrium. CONCLUSIONS: This study revealed novel hormonal and non-hormonal regulators and their relative contributions to endometrial progression and pathology, providing new leads for the potential causes of endometrial-factor infertility.

ThyroidPrint®: clinical utility for indeterminate thyroid cytology.

Molecular testing contributes to improving the diagnosis of indeterminate thyroid nodules (ITNs). ThyroidPrint® is a ten-gene classifier aimed to rule out malignancy in ITN. Post-validation studies are necessary to determine the real-world clinical benefit of ThyroidPrint® in patients with ITN. A single-center, prospective, noninterventional clinical utility study was performed, analyzing the impact of ThyroidPrint® in the physicians' clinical decisions for ITN. Demographics, nodule characteristics, benign call rates (BCRs), and surgical outcomes were measured. Histopathological data were collected from surgical biopsies of resected nodules. Of 1272 fine-needle aspirations, 109 (8.6%) were Bethesda III and 135 (10.6%) were Bethesda IV. Molecular testing was performed in 155 of 244 ITN (63.5%), of which 104 were classified as benign (BCR of 67.1%). After a median follow-up of 15 months, 103 of 104 (99.0%) patients with a benign ThyroidPrint® remained under surveillance and one patient underwent surgery which was a follicular adenoma. Surgery was performed in all 51 patients with a suspicious for malignancy as per ThyroidPrint® result and in 56 patients who did not undergo testing, with a rate of malignancy of 70.6% and 32.1%, respectively. A higher BCR was observed in follicular lesion of undetermined significance (87%) compared to atypia of undetermined significance (58%) (P < 0.05). False-positive cases included four benign follicular nodules and six follicular and four oncocytic adenomas. Our results show that, physicians chose active surveillance instead of diagnostic surgery in all patients with a benign ThyroidPrint® result, reducing the need for diagnostic surgery in 67% of patients with preoperative diagnosis of ITN.

Association of blood cadmium and lead levels with self-reported reproductive lifespan and pregnancy loss: The national health and nutrition examination survey 1999-2018.

Cadmium and lead are known to interfere with the endocrine function. Thus, hormonally regulated processes such as menarche, menopause and pregnancy are likely influenced by chronic exposure to these metals. In US post-menopausal women, who already completed their reproductive lifespan, we evaluated the association between blood cadmium and lead levels with self-reported reproductive lifespan and personal history of pregnancy loss. We selected 5317 post-menopausal women participating in the National Health and Nutrition Examination Survey (NHANES), 1999-2018. Blood cadmium and lead levels were measured by inductively coupled plasma mass spectrometry. Reproductive lifespan was defined as the number of years between self-reported age at menarche and menopause. Personal history of pregnancy loss was defined as number of self-reported pregnancy losses out of the self-reported number of pregnancies. The fully adjusted mean difference in reproductive lifespan (95% confidence interval [CI]) comparing the 80th to the 20th percentiles of blood cadmium and lead distributions was, respectively, 0.50 (0.10, 0.91) and 0.72 (0.41, 1.03) years. Ever smoker showed stronger association of blood lead with reproductive lifespan. For self-reported pregnancy loss, the corresponding fully adjusted relative prevalence (95% CI) was 1.10 (0.93, 1.31) for cadmium and 1.10 (1.00, 1.21) for lead, and remained similar after additional adjustment for reproductive lifespan. In never smokers, the relative prevalence was 1.07 (1.04, 1.11) and 1.16 (1.05, 1.28) for blood cadmium and lead, respectively. These findings suggest that blood cadmium and lead exposures increase reproductive lifespan and prevalence of pregnancy loss in the general population. Additional studies are needed to improve the understanding of mechanisms and prevention potential of metals-related pregnancy outcomes.

Drug burden index in people living with HIV over 50 years of age in a real clinical practice cohort.

OBJECTIVES: To determine DBI and its relationship with polypharmacy and pharmacotherapeutic complexity (PC) in a cohort of PLWH over 50 years of age at follow-up of pharmacotherapy in a tertiary hospital. METHODS: Observational and retrospective study that included PLWH in active antiretroviral treatment over 50 years of age who have been followed up in outpatient pharmacy services. Pharmacotherapeutic complexity was estimated through Medication Regimen Complexity Index (MRCI). Collected variables included comorbidities, current prescriptions and its classification according to anticholinergic and sedative activity and associated risk of falls. RESULTS: Studied population included 251 patients (85.7% men; median age: 58 years, interquartile range: 54-61). There was a high prevalence of high DBI scores (49.2%). High DBI was significantly correlated with a high PC, polypharmacy, psychiatric comorbidity and substances abuse (p<0.05). Among sedative drugs, the most prescribed were anxiolytic drugs (N05B) (n=85), antidepressant drugs (N06A) (n=41) and antiepileptic drugs (N03A) (n=29). For anticholinergic drugs, alpha-adrenergic antagonist drugs (G04C) were the most prescribed (n=18). Most frequent drugs associated with risk of falls were anxiolytics (N05B) (n=85), angiotensin-converting enzyme inhibitors (C09A) (n=61) and antidepressants (N06A) (n=41). CONCLUSION: The DBI score in older PLWH is high and it is related to PC, polypharmacy, mental diseases and substance abuse as is the prevalence of fall-related drugs. Control of these parameters as well as the reduction of the sedative and anticholinergic load should be included in the lines of work in the pharmaceutical care of people living with HIV+.

Phytoestrogens Present in Follicular Fluid and Urine Are Positively Associated with IVF Outcomes following Single Euploid Embryo Transfer.

The impact and safety of phytoestrogens, plant-derived isoflavones with estrogenic activity predominantly present in soy, on female reproductive health and IVF outcomes continues to be hotly debated. In this prospective cohort study, 60 women attending IVI-RMA New Jersey undergoing IVF with single frozen embryo transfer (SET/FET) of good-quality euploid blastocyst after PGT-A analysis were recruited. Concentrations of two phytoestrogens (daidzein and genistein) in follicular fluid (FF) and urine (U) were measured by UPLC-MSMS, both collected on vaginal oocyte retrieval day. These measurements correlated with IVF clinical outcomes. In models adjusted for age, BMI, race/ethnicity, and smoking status, higher FF phytoestrogen concentrations were significantly associated with higher serum estradiol, enhanced probability of implantation, clinical pregnancy, and live birth. Moreover, higher urine phytoestrogen concentrations were significantly associated with improved oocyte maturation and fertilization potential and increased probability of clinical pregnancy and live birth. Finally, higher FF and urine phytoestrogen concentrations were associated with a higher probability of live birth from a given IVF cycle. Our results suggest that dietary phytoestrogens improved reproductive outcomes of women undergoing IVF treatment. However, additional prospective studies are needed to optimize the use of phytoestrogens to further enhance reproductive outcomes and/or protect against reproductive insults.

Antioxidant Supplementation Alleviates Mercury-Induced Cytotoxicity and Restores the Implantation-Related Functions of Primary Human Endometrial Cells.

Mercury (Hg) cytotoxicity, which is largely mediated through oxidative stress (OS), can be relieved with antioxidants. Thus, we aimed to study the effects of Hg alone or in combination with 5 nM N-Acetyl-L-cysteine (NAC) on the primary endometrial cells' viability and function. Primary human endometrial epithelial cells (hEnEC) and stromal cells (hEnSC) were isolated from 44 endometrial biopsies obtained from healthy donors. The viability of treated endometrial and JEG-3 trophoblast cells was evaluated via tetrazolium salt metabolism. Cell death and DNA integrity were quantified following annexin V and TUNEL staining, while the reactive oxygen species (ROS) levels were quantified following DCFDA staining. Decidualization was assessed through secreted prolactin and the insulin-like growth factor-binding protein 1 (IGFBP1) in cultured media. JEG-3 spheroids were co-cultured with the hEnEC and decidual hEnSC to assess trophoblast adhesion and outgrowth on the decidual stroma, respectively. Hg compromised cell viability and amplified ROS production in trophoblast and endometrial cells and exacerbated cell death and DNA damage in trophoblast cells, impairing trophoblast adhesion and outgrowth. NAC supplementation significantly restored cell viability, trophoblast adhesion, and outgrowth. As these effects were accompanied by the significant decline in ROS production, our findings originally describe how implantation-related endometrial cell functions are restored in Hg-treated primary human endometrial co-cultures by antioxidant supplementation.

PI3K/AKT/mTOR Signaling Pathway in HPV-Driven Head and Neck Carcinogenesis: Therapeutic Implications.

High-risk human papillomaviruses (HR-HPVs) are the causal agents of cervical, anogenital and a subset of head and neck carcinomas (HNCs). Indeed, oropharyngeal cancers are a type of HNC highly associated with HR-HPV infections and constitute a specific clinical entity. The oncogenic mechanism of HR-HPV involves E6/E7 oncoprotein overexpression for promoting cell immortalization and transformation, through the downregulation of p53 and pRB tumor suppressor proteins, among other cellular targets. Additionally, E6/E7 proteins are involved in promoting PI3K/AKT/mTOR signaling pathway alterations. In this review, we address the relationship between HR-HPV and PI3K/AKT/mTOR signaling pathway activation in HNC with an emphasis on its therapeutic importance.

Access disparities and underutilization of germline genetic testing in Chilean breast cancer patients.

PURPOSE: Latin American reports on genetic cancer risk assessments are scarce. In Chile, current breast cancer (BC) guidelines do not define strategies for germline genetic testing. Our study sought to quantify the disparities in access to genetic testing in Chilean BC patients, according to international standards and their clinical characteristics to explore improvement strategies. METHODS: Retrospective analysis of invasive BC databases including patients treated in a Public Hospital (PH) and in an Academic Private Center (AC) in Santiago, Chile between 2012 and 2021. RESULTS: Of 5438 BC patients, 3955 had enough data for National Comprehensive Cancer Network (NCCN) categorization. From these, 1911 (48.3%) fulfilled NCCN criteria for germline testing, of whom, 300 were tested for germline mutations and 268 with multigene panels. A total of 65 pathogenic variants were found in this subset. As expected, BRCA1/2 mutations were the most frequent (17.7%). Access to genetic testing was higher in AC versus PH (19.6% vs. 10.3%, p = 0.0001). Other variables associated with germline genetic testing were BC diagnosis after 2018, being 45 years old or younger at diagnosis, BC family history (FH), FH of ovarian cancer, non-metastatic disease, and triple-negative subtype. CONCLUSION: In our cohort, 15% of BC patients who met NCCN criteria for germline testing were effectively tested. This percentage was even lower at the PH. Current recommendations encourage universal genetic testing for BC patients; however, our findings suggest that Chile is far from reaching such a goal and national guidelines in this regard are urgently needed. To our knowledge, this is the first study of its kind in Chile and Latin America.

Clinical characteristics, risk factors, and outcomes in Chilean triple negative breast cancer patients: a real-world study.

BACKGROUND: Latin American (LA) studies on triple-negative breast cancer (TNBC) and their characteristics are scarce. This forces physicians to make clinical decisions based on data obtained from studies that include non-Hispanic patients. Our study sought to obtain local epidemiological data, including risk factors and clinical outcomes from a Chilean BC registry. METHODS: This was a retrospective population-cohort study that included patients treated at a community hospital (mid-low income) or an academic private center (high income), in the 2010-2021 period. Univariate and multivariate analyses were performed to identify prognostic factors associated with survival. RESULTS: 647 out of 5,806 BC patients (11.1%) were TNBC. These patients were younger (p = 0.0001) and displayed lower rates of screening-detected cases (p = 0.0001) compared to non-TNBC counterparts. Among TNBC patients, lower income (i. e., receiving treatment at a community hospital) was associated with poorer overall survival (HR: 1.53; p = 0.0001) and poorer BC specific survival (HR: 1.29; p = 0.004). Other risk factors showed no significant differences between TNBC and non-TNBC. As expected, 5-year OS was significantly shorter on TNBC versus non-TNBC patients (p = 0.00001). In our multivariate analyses TNBC subtype (HR: 2.30), locally advanced stage (HR: 7.04 for stage III), lower income (HR: 1.64), or non-screening detected BC (HR: 1.32) were associated with poorer OS. CONCLUSION: To the best of our knowledge, this is the largest LA cohort of TNBC patients. Interestingly, the proportion of TNBC among Chileans was smaller compared to similar studies within LA. As expected, TNBC patients had poorer survival and higher risk for early recurrence versus non-TNBC. Other relevant findings include a higher proportion of premenopausal patients among TNBC. Also, mid/low-income patients that received medical attention at a community hospital displayed lower survival versus private health center counterparts.

Endometrial Cells Acutely Exposed to Phthalates In Vitro Do Not Phenocopy Endometriosis.

Environmental factors that have been linked to an increased endometriosis risk include exposure to di-(2-ethylhexyl)-phthalate (DEHP), an endocrine disruptor. This study aims to investigate whether DEHP in vitro exposure in primary endometrial stromal cells (EnSC), primary endometrial epithelial cells (EnEC), and the human endometrial adenocarcinoma cell line Ishikawa properly mimics alterations described in the eutopic endometrium of women with endometriosis. Primary EnSC and EnEC, isolated from six fertile egg donors, and Ishikawa cells were exposed to DEHP (0.1, 1, and 10 µM) and were assessed for viability, endometriosis markers (IL-6, VEGF-A, HOXA10, EZH2, and LSD1), steroid receptor gene expressions (ER-1, ER-2, PR-T, PR-B, and PGRMC1), and invasive capacity. Viability after 72 h of DEHP exposure was not significantly affected. None of the endometriosis markers studied were altered after acute DEHP exposure, nor was the expression of steroid receptors. The invasive capacity of EnSC was significantly increased after 10 µM of DEHP exposure. In conclusion, acute DEHP exposure in primary endometrial cells does not fully phenocopy the changes in the viability, expression of markers, or steroidal receptors described in endometriosis. However, the significant increase in EnSC invasiveness observed after DEHP exposure could be a link between DEHP exposure and increased endometriosis likelihood.