The timing for initiating estrogen stimulation in artificial cycle for frozen-thawed embryo transfer can be flexible

Background There remains a lack of evidence to demonstrate whether the initiation time of estrogen stimulation is flexible in the proliferative endometrial phase during the artificial cycle for frozen-thawed embryo transfer (AC-FET). Methods FET records were retrospectively reviewed from a large university-affiliated reproductive medicine center. Only the patients who were undergoing their first embryo transfer with a single blastocyst in the AC-FET cycles were included: thereby 660 cycles were recruited, and the patients were grouped according to their day of estrogen usage initiation as early initiation group (estrogen stimulation initiated during days 2–5 of menses, n = 128) and the late initiation group (estrogen stimulation initiated on or after the 6th day of menses, n = 532). The primary outcome was the ongoing pregnancy rates (OPR). Results The rates of biochemical and clinical pregnancies were significantly higher in the late initiation group relative to those in the early initiation group, however, no significant differences were noted between the two groups for OPR. Furthermore, after adjusting for the results of the potential confounders, no impact was observed in the initiation time of estrogen stimulation on the OPR. Conclusions This study provides evidence that initiating the estrogen stimulation on after days 2–5 of menses do not exert adverse effects on the OPR in AC-FETs. Thus, AC-FET can be scheduled in a flexible manner without compromising on the pregnancy outcomes.

The Timing for Initiating Estrogen Stimulation in Artificial Cycle for Frozen-Thawed Embryo Transfer can be Flexible

Background: There remains a lack of evidence to demonstrate whether the initiation time of estrogen stimulation is flexible in the proliferative endometrial phase during the artificial cycle for frozen-thawed embryo transfer (AC-FET).Methods: FET records were retrospectively reviewed from a large university-affiliated reproductive medicine center. Only the patients who were undergoing their first embryo transfer with a single blastocyst in the AC-FET cycles were included: thereby 660 cycles were recruited, and the patients were grouped according to their day of estrogen usage initiation as early initiation group (estrogen stimulation initiated during days 2–5 of menses, n = 128) and the late initiation group (estrogen stimulation initiated on or after the sixth day of menses, n = 532). The primary outcome was the ongoing pregnancy rates (OPR). Results: The rates of biochemical and clinical pregnancies were significantly higher in the late initiation group relative to those in the early initiation group, however, no significant differences were noted between the 2 groups for OPR. Furthermore, after adjusting for the results of the potential confounders, no impact was observed in the initiation time of estrogen stimulation on the OPR.Conclusions: This study provides evidence that initiating the estrogen stimulation on after days 2–5 of menses do not exert adverse effects on the OPR in AC-FETs. Thus, FET can be scheduled in a flexible manner, according to the ovulatory and endometrial statuses and patient and/or clinic preference, without compromising on the clinical outcomes.

ALDH1 expression predicts progression of premalignant lesions to cancer in Type I endometrial carcinomas

In type 1 endometrial cancer, unopposed estrogen stimulation is thought to lead to endometrial hyperplasia which precedes malignant progression. Recent data from our group and others suggest that ALDH activity mediates stemness in endometrial cancer, but while aldehyde dehydrogenase 1 (ALDH1) has been suggested as a putative cancer stem cell marker in several cancer types, its clinical and prognostic value in endometrial cancer remains debated. The aim of this study was to investigate the clinical value of ALDH1 expression in endometrial hyperplasia and to determine its ability to predict progression to endometrial cancer. Interrogation of the TCGA database revealed upregulation of several isoforms in endometrial cancer, of which the ALDH1 isoforms collectively constituted the largest group. To translate its expression, a tissue microarray was previously constructed which contained a wide sampling of benign and malignant endometrial samples. The array contained a metachronous cohort of samples from individuals who either developed or did not develop endometrial cancer. Immunohistochemical staining was used to determine the intensity and frequency of ALDH1 expression. While benign proliferative and secretory endometrium showed very low levels of ALDH1, slightly higher expression was observed within the stratum basalis. In disease progression, cytoplasmic ALDH1 expression showed a step-wise increase between endometrial hyperplasia, atypical hyperplasia, and endometrial cancer. ALDH1 was also shown to be an early predictor of EC development, suggesting that it can serve as an independent prognostic indicator of patients with endometrial hyperplasia with or without atypia who would progress to cancer (p = 0.012).

Estrogen-induced transcription at individual alleles is independent of receptor level and active conformation but can be modulated by coactivators activity

Steroid hormones are pivotal modulators of pathophysiological processes in many organs, where they interact with nuclear receptors to regulate gene transcription. However, our understanding of hormone action at the single cell level remains incomplete. Here, we focused on estrogen stimulation of the well-characterized GREB1 and MYC target genes that revealed large differences in cell-by-cell responses, and, more interestingly, between alleles within the same cell, both over time and hormone concentration. We specifically analyzed the role of receptor level and activity state during allele-by-allele regulation and found that neither receptor level nor activation status are the determinant of maximal hormonal response, indicating that additional pathways are potentially in place to modulate cell- and allele-specific responses. Interestingly, we found that a small molecule inhibitor of the arginine methyltransferases CARM1 and PRMT6 was able to increase, in a gene specific manner, the number of active alleles/cell before and after hormonal stimulation, suggesting that mechanisms do indeed exist to modulate hormone receptor responses at the single cell and allele level.

Estrogen receptor signaling is reprogrammed during breast tumorigenesis

Limited knowledge of the changes in estrogen receptor (ER) signaling during the transformation of the normal mammary gland to breast cancer hinders the development of effective prevention and treatment strategies. Differences in estrogen signaling between normal human primary breast epithelial cells and primary breast tumors obtained immediately following surgical excision were explored. Transcriptional profiling of normal ER+ mature luminal mammary epithelial cells and ER+ breast tumors revealed significant difference in the response to estrogen stimulation. Consistent with these differences in gene expression, the normal and tumor ER cistromes were distinct and sufficient to segregate normal breast tissues from breast tumors. The selective enrichment of the DNA binding motif GRHL2 in the breast cancer-specific ER cistrome suggests that it may play a role in the differential function of ER in breast cancer. Depletion of GRHL2 resulted in altered ER binding and differential transcriptional responses to estrogen stimulation. Furthermore, GRHL2 was demonstrated to be essential for estrogen-stimulated proliferation of ER+ breast cancer cells. DLC1 was also identified as an estrogen-induced tumor suppressor in the normal mammary gland with decreased expression in breast cancer. In clinical cohorts, loss of DLC1 and gain of GRHL2 expression are associated with ER+ breast cancer and are independently predictive for worse survival. This study suggests that normal ER signaling is lost and tumor-specific ER signaling is gained during breast tumorigenesis. Unraveling these changes in ER signaling during breast cancer progression should aid the development of more effective prevention strategies and targeted therapeutics.

Estrogen Regulation of T-Cell Function and Its Impact on the Tumor Microenvironment

Epidemiologic studies demonstrate significant gender-specific differences in immune system function. Males are more prone to infection and malignancies, while females are more vulnerable to autoimmune diseases. These differences are thought to be due to the action of gonadal hormones: Estrogen increases the inflammatory response and testosterone dampens it. More specifically, estrogen stimulation induces inflammatory cytokine production including interferon γ, interleukin (IL) 6, and tumor necrosis factor α, while testosterone induces IL-10, IL-4, and transforming growth factor β. More recent studies demonstrate threshold effects of estrogen stimulation on immune cell function: physiologic doses of estrogen (approximately 0.5 nmol/L) stimulate inflammatory cytokine production, but superphysiologic dosages (above 50 nmol/L) can result in decreased inflammatory cytokine production. This review reports findings concerning the impact of estrogen on CD8+ cytotoxic T cells and the overall immune response in the tumor microenvironment. Variables examined include dosage of hormone, the diversity of immune cells involved, and the nature of the immune response in cancer. Collective review of these points may assist in future hypotheses and studies to determine sex-specific differences in immune responses that may be used as targets in disease prevention and treatment.

Single-cell transcriptome analysis reveals estrogen signaling augments the mitochondrial folate pathway to coordinately fuel purine and polyamine synthesis in breast cancer cells

Estrogen regulates diverse physiological effects and drives breast tumor progression by directly activating estrogen receptor α (ERα). However, due to the stochastic nature of gene transcription and the resulting heterogeneous cellular response, it is important to investigate estrogen-stimulated gene expression profiles at the single-cell level in order to fully understand how ERα regulates transcription in breast cancer cells. In this study, we performed single-cell transcriptome analysis on ERα-positive breast cancer cell lines following 17β-estradiol stimulation. Overall, we observed robust gene expression diversity between individual cells. Moreover, we found over two thirds of the genes in breast cancer cells displayed a bimodal expression pattern, which caused averaging artifacts and masked the identification of potential estrogen-regulated genes. We overcame this issue by reconstructing a dynamic estrogen-responsive transcriptional network from discrete time points into a pseudotemporal continuum. Pathway analysis of the differentially expressed genes derived from the pseudotemporal analysis showed an estrogen-stimulated metabolic switch that favored biosynthesis and cell proliferation but reduced estrogen degradation. In addition, we identified folate-mediated one-carbon metabolism as a novel estrogen-regulated pathway in breast cancer cells. Notably, estrogen stimulation reprogramed this pathway through the mitochondrial folate pathway to coordinately fuel polyamine and de novo purine synthesis. Finally, we showed AZIN1 and PPAT, key regulators in the above pathways, are direct ERα target genes and essential for breast cancer cell survival and growth. In summary, our single-cell study illustrated a dynamic transcriptional heterogeneity in ERα-positive breast cancer cells in response to estrogen stimulation and uncovered a novel mechanism of an estrogen-mediated metabolic switch.

Estrogen and high-fat diet induced alterations in C57BL/6 mice endometrial transcriptome profile

Unopposed estrogen stimulation and insulin resistance are known to play important roles in endometrial cancer (EC), but the interaction between these two factors and how they contribute to endometrial lesions are not completely elucidated. To investigate the endometrial transcriptome profile and the associated molecular pathway alterations, we established an ovariectomized C57BL/6 mouse model treated with subcutaneous implantation of 17-β estradiol (E2) pellet and/or high-fat diet (HFD) for 12 weeks to mimic sustained estrogen stimulation and insulin resistance. Histomorphologically, we found that both E2 and E2 + HFD groups showed markedly enlarged uterus and increased number of endometrial glands. The endometrium samples were collected for microarray assay. GO and KEGG analysis showed that genes regulated by E2 and/or HFD are mainly responsible for immune response, inflammatory response and metabolic pathways. Further IPA analysis demonstrated that the acute phase response signaling, NF-κB signaling, leukocyte extravasation signaling, PPAR signaling and LXR/RXR activation pathways are mainly involved in the pathways above. In addition, the genes modulated reciprocally by E2 and/or HFD were also analyzed, and their crosstalk mainly focuses on enhancing one another’s activity. The combination analysis of microarray data and TCGA database provided potential diagnostic or therapeutic targets for EC. Further validation was performed in mice endometrium and human EC cell lines. In conclusion, this study unraveled the endometrial transcriptome profile alterations affected by E2 and/or HFD that may disturb endometrial homeostasis and contribute to the development of endometrial hyperplasia.

SRC IncreasesMYCmRNA Expression in Estrogen Receptor-Positive Breast Cancer via mRNA Stabilization and Inhibition of p53 Function

ABSTRACTThe transcription factor geneMYCis important in breast cancer, and its mRNA is maintained at a high level even in the absence of gene amplification. The mechanism(s) underlying increasedMYCmRNA expression is unknown. Here, we demonstrate thatMYCmRNA was stabilized upon estrogen stimulation of estrogen receptor-positive breast cancer cells via SRC-dependent effects on a recently described RNA-binding protein, IMP1 with an N-terminal deletion (ΔN-IMP1). We also show that loss of the tumor suppressor p53 increasedMYCmRNA levels even in the absence of estrogen stimulation. However, in cells with wild-type p53, SRC acted to overcome p53-mediated inhibition of estrogen-stimulated cell cycle entry and progression. SRC thus promotes cell proliferation in two ways: by stabilizingMYCmRNA and by inhibiting p53 function. Since estrogen receptor-positive breast cancers typically express wild-type p53, these studies establish a rationale for p53 status to be predictive for effective SRC inhibitor treatment in this subtype of breast cancer.