Lower E2 Levels Research Articles

Eicosatrienoic acid inhibits estradiol synthesis through the CD36/FOXO1/CYP19A1 signaling pathway to improve PCOS in mice

Polycystic ovary syndrome (PCOS) is a common metabolic and endocrine disorder characterized by abnormal elevation in hormone levels, with currently lacking effective treatment options. N-3 polyunsaturated fatty acids (PUFA) have broad pharmacological activity and play a beneficial role in the development of PCOS. In this study, we observed that n-3 PUFA-eicosatrienoic acid (ETA) improves the estrous cycle and ovarian morphology in dehydroepiandrosterone (DHEA)-induced PCOS mice, particularly serum hormone levels. Additionally, it suppresses the expression of CYP19A1 and E2 synthesis in human granulosa-like tumor cell line (KGN) cells. Further investigation revealed that ETA significantly upregulates the expression of CD36, cAMP, P-PKA, and FOXO1 in KGN cells and mouse ovaries to lower E2 levels. This conclusion was supported by inhibiting CD36 and FOXO1 at both the mouse and cellular levels. Additionally, ETA treatment decreased the expression of ESR1, Kiss1, Gnrh in the hypothalamus, and GnRHR, Lhβ, Egr1, Pitx1, Sf1 in the pituitary of PCOS mice. No differences were observed after ETA treatment in the CD36 and FOXO1 inhibitor groups, indicating that ETA improves PCOS mice by regulating the hypothalamic-pituitary axis through E2 synthesis inhibition. In summary, we have elucidated for the first time the mechanism by which CD36 regulates E2 synthesis in ovarian granulosa cells and demonstrated that ETA activates the CD36 receptor to inhibit E2 synthesis through the cAMP/PKA/FOXO1/CYP19A1 signaling pathway, thereby improving hormonal imbalance and treating PCOS. This provides a new strategy for the effective prevention and treatment of PCOS.

17β-Estradiol reduces inhibitory synaptic currents in entorhinal cortex neurons through G protein-coupled estrogen receptor-1 activation of extracellular signal-regulated kinase.

Estrogens are believed to modulate cognitive functions in part through the modulation of synaptic transmission in the cortex and hippocampus. Administration of 17β-estradiol (E2) can rapidly enhance excitatory synaptic transmission in the hippocampus and facilitate excitatory synaptic transmission in rat lateral entorhinal cortex via activation of the G protein-coupled estrogen receptor-1 (GPER1). To assess the mechanisms through which GPER1 activation facilitates synaptic transmission, we assessed the effects of acute 10 nM E2 administration on pharmacologically isolated evoked excitatory and inhibitory synaptic currents in layer II/III entorhinal neurons. Female Long-Evans rats were ovariectomized between postnatal day (PD) 63 and 74 and implanted with a subdermal E2 capsule to maintain continuous low levels of E2. Electrophysiological recordings were obtained between 7 and 20 days after ovariectomy. Application of E2 for 20 min did not significantly affect AMPA or NMDA receptor-mediated excitatory synaptic currents. However, GABA receptor-mediated inhibitory synaptic currents (IPSCs) were markedly reduced by E2 and returned towards baseline levels during the 20-min washout period. The inhibition of GABA-mediated IPSCs was blocked in the presence of the GPER1 receptor antagonist G15. GPER1 can modulate protein kinase A (PKA), but blocking PKA with intracellular KT5720 did not prevent the E2-induced reduction in IPSCs. GPER1 can also stimulate extracellular signal-regulated kinase (ERK), a negative modulator of GABAA receptors, and blocking activation of ERK with PD90859 prevented the E2-induced reduction of IPSCs. E2 can therefore result in a rapid GPER1 and ERK signaling-mediated reduction in GABA-mediated IPSCs. This provides a novel mechanism through which E2 can rapidly modulate synaptic excitability in entorhinal layer II/III neurons and may also contribute to E2 and ERK-dependent alterations in synaptic transmission in other brain areas.

Associations between estradiol and hyperuricemia and the mediating effects of TC, TG, and TyG: NHANES 2013-2016.

To explore the relationship between estradiol (E2) and the incidence of hyperuricemia (HUA) in adult women and to explore whether glucolipid metabolism disorders play a mediating role in mediating this relationship. A total of 2,941 participants aged 20-65 years were included in the National Health and Nutrition Examination Survey (NHANES) 2013-2016. Multivariate logistic regression analysis was performed to evaluate the correlations of E2 with HUA. Multivariate linear regression analysis was performed to evaluate the associations between E2 and triglyceride (TG), total cholesterol (TC), and the triglyceride-glucose index (TyG). The restricted cubic spline (RCS) model was used to further explore the association between E2 and HUA and between TG, TC, and TyG and HUA. Mediation analyses were performed to examine whether TC, TG, and TyG mediated the relationship between E2 and HUA. After adjusting for covariates, logistic regression revealed that ln(E2) was significantly associated with HUA in the female subgroup (p = 0.035) and that the incidence of HUA tended to increase with decreasing ln(E2) (p for trend = 0.026). Linear regression showed that E2 was significantly associated with TC (p = 0.032), TG (p = 0.019), and TyG (p = 0.048). The RCS model showed that ln(E2) was linearly correlated with the incidence of HUA (p-overall = 0.0106, p-non-linear = 0.3030). TC and TyG were linearly correlated with HUA (TC: p-overall = 0.0039, p-non-linear = 0.4774; TyG: p-overall = 0.0082, p-non-linear = 0.0663), whereas TG was non-linearly correlated with HUA. Mediation analyses revealed that TC, TG, and TyG significantly mediated the relationship between ln(E2) and HUA (TC, indirect effect: -0.00148, 7.5%, p = 0.008; TG, indirect effect: -0.00062, 3.1%, p = 0.004; TyG, indirect effect: -0.00113, 5.6%, p = 0.016). In conclusion, this study demonstrated that compared with women aged 20-45 years, women aged 45-55 years and 55-65 years had lower E2 levels and a greater incidence of HUA. E2 levels and the incidence of HUA were negatively associated in female individuals but not in male individuals. In addition, TC, TG, and TyG, which are markers of glucolipid metabolism, played a mediating role in the association between E2 and HUA.

O-213 Analysis of risk factors for monozygotic twins following in vitro fertilization-embryo transfer

Abstract Study question To investigate the risk factors associated with the incidence of monozygotic twinning（MZT）following in vitro fertilization and embryo transfer (IVF-ET). Summary answer MZT following IVF-ET may be associated with a reduced endometrial thickness on the day of embryo transfer and a decrease in estradiol levels post-transfer. What is known already MZT pregnancy arises from the division of a single fertilized ovum, resulting in offspring with identical genetic material. These twins exhibit markedly higher rates of complications compared to dizygotic counterparts, including increased incidences of miscarriage, congenital anomalies, intrauterine fetal demise, growth restriction, and preterm delivery. The underlying biological processes leading to MZT remain elusive. Presently, scholarly discourse centers around two predominant models and one hypothesis: the zygote division model, the embryonic membrane fusion model, and the over-mature gametes hypothesis, respectively. IVF-ET has been implicated in the heightened occurrence of MZT, suggesting a potential iatrogenic element to this phenomenon. Study design, size, duration A retrospective cohort study has been conducted. We analyzed 582 IVF-ET cycles at a large teaching hospital from January 2007 to December 2022, including 291 clinically confirmed MZTs, and 291 matched non-MZTs. Participants/materials, setting, methods A total of 582 participants, 291 in both MZT group and non-MZT group. There were 146 fresh embryo transfer cycles and 145 frozen-thawed cycles in the MZT group. Each cycle type was matched on a 1:1 basis by age and embryo transfer date with a control group that did not result in MZT following IVF-ET. We examined the disparities between the MZT group and the non-MZT group, and performed the Logistics regression analysis. Main results and the role of chance Significant differences were observed in the MZT group compared to the non-MZT group in the fresh embryo transfer cycle, particularly regarding estradiol (E2) and progesterone (P) levels on day 7 post-transfer, E2/P ratios on day 14 post-transfer, and endometrial thickness on the day of transfer (p<0.05). Logistic regression analysis indicated a correlation between reduced endometrial thickness on the transfer day and the occurrence of MZT. During the frozen-thawed cycle, significant differences were noted in E2/P ratios, E2 levels on day 7 post-transfer, and endometrial thickness on embryo transfer day (p<0.05). Logistic regression analysis suggested that lower E2 levels on day 7 post-transfer and reduced endometrial thickness on embryo transfer day were predictive of MZT. Additional potential influencing factors included the duration of the participants’ menstrual cycle, the protocol of ovulation induction, and the duration of embryo culture in vitro. Limitations, reasons for caution Primarily, the retrospective design may introduce bias, notably due to the potential absence of comprehensive data. Secondly, the study is constrained by the sample size, with 146 fresh cycles and 145 frozen cycles, which may not provide a fully representative statistical power. Wider implications of the findings The incidence of MZT may implicate signaling pathways at the maternal-fetal interface, with early embryonic split potentially serving as a compensatory mechanism in response to suboptimal estrogen levels or diminished endometrial thickness, thus enhancing embryonic viability. Trial registration number not applicable

Plasma Homocysteine Levels as Cardiovascular Disease Risk vis-a-vis Estrogen Levels in Pre and Postmenopausal Women

Objectives: The objective of the study is to evaluate plasma homocysteine level and its relation with serum estrogen in pre and postmenopausal women as a risk factor in coronary artery disease (CAD). Materials and Methods: A cross-sectional study was conducted among premenopausal (n = 50) and postmenopausal women (n = 50) with an estimation of plasma homocysteine (Hcy) and serum estradiol (E2). Postmenopausal women based on their angiographic status were sub-grouped into A (with normal coronaries) and B (with CAD). Results: The levels of Hcy, 21.98 (20.89–24.05) μmol/L, increased significantly (P < 0.0001) and that of E2, 3.05 (2.32–3.6) pg/mL, decreased significantly (P = 0.0001) in the postmenopausal group when compared to Hcy, 6.11 (4.94–8.27) μmol/L and E2, 26.37 (25.3–29) pg/mL in premenopausal group. The levels of Hcy were elevated (24.7 ± 3.92 μmol/L) in postmenopausal Group B in comparison with postmenopausal Group A (21.37 ± 3.31 μmol/L), P = 0.002. Logistic regression analysis showed Hcy and body mass index to be independent predictors of CAD. Plasma Hcy showed an area under curve (AUC) of 1.00 with sensitivity and specificity of 100% at cutoff >12.6 μmol/L between pre and postmenopausal groups and AUC of 0.722 with 60% sensitivity and 84% specificity at cutoff >23.7 μmol/L between postmenopausal Groups A and B. Conclusion: We observed low E2 and high Hcy levels in postmenopausal women in comparison with premenopausal women. Among postmenopausal women, those with CAD had higher Hcy levels. Assessing Hcy levels routinely in postmenopausal women will help in better risk prediction of CAD and may also help in conjunction with other risk factors to decide the initiation of hormone replacement therapy.

Low Estradiol Level and Endometrial Thickness on the Day of Endometrial Transformation Influence Clinical Pregnancy After Intrauterine Insemination.

The causes of pregnancy failure after intrauterine insemination (IUI) are controversial. The purpose of this study was to investigate the influencing factors on clinical pregnancy after IUI. This study retrospectively analyzed 1464 cycles of IUI performed at the Meizhou People's Hospital between March 2014 and June 2023. The χ2 test and logistic regression analysis was applied to assess the associations between the some factors (maternal age, paternal age, cycle type (natural cycle or ovulation induction cycle), hormone level on the day of endometrial transformation (estradiol (E2), luteinizing hormone (LH), and progesterone (P)), endometrial thickness on the day of endometrial transformation, and forward motile sperm concentration after treatment) and pregnancy failure. Among the 1464 IUI cycles in this study, 268 cycles of assisted reproduction resulted in clinical pregnancy, with a clinical pregnancy rate of 18.3%. During the cycles with clinical pregnancy, there were 25 (12.9%) preterm births and 169 (87.1%) full-term births. The E2 level on the day of endometrial transformation in clinical pregnancy group was higher than that in the pregnancy failure group (658.79±656.02 vs 561.21±558.83 pg/mL)(P=0.025). The clinical pregnancy group had a higher percentage of endometrial thickness between 8 and 13mm on the day of endometrial transformation than the pregnancy failure group (83.2% vs 75.0%)(P=0.002). The results of regressions analysis showed that low E2 level on the day of endometrial transformation (<238.3 pg/mL vs ≥238.3 pg/mL: OR 1.493, 95% CI: 1.086-2.052, P=0.014), and endometrial thickness <8mm on the day of endometrial transformation (<8mm vs 8-13mm: OR 1.886, 95% CI: 1.284-2.771, P=0.001) may increase risk of pregnancy failure performed IUI. Low estradiol level, and endometrial thickness on the day of endometrial transformation may increase risk of pregnancy failure performed intrauterine insemination.

Production of fertile YY pseudofemales in Nile tilapia with LG23 as the sex chromosome

Nile tilapia display conspicuous differences in growth rates between genders, as males tend to outpace and outgrow females. The production of all male Nile tilapia populations using genetically male tilapia (GMT) technology represents the most efficient and scientifically grounded approach. Most of the Nile tilapia stocks in aquaculture use LG23 as sex chromosome. The crux of GMT lies in the acquisition of YY supermales and YY pseudofemales (pF). Nonetheless, the utilization of GMT is impeded by the lower fertility and even infertility of feminized YY-pF. In this study, we found that YY-pF exhibited male-like genital papillae, and exhibited significantly higher androgen levels and lower E2 levels compared with that of fertile XY-pF. YY-pF carries two copies of Amhy but lacks Amh, and XY-pF possesses one Amhy and one Amh. In XY-pF, the expression of Amh/Amhy was only observed in granulosa cells of phase I and II follicles, while in YY-pF, Amhy expression persisted from phase I to IV. On the contrary, Cyp19a1a was found to be highly expressed in granulosa cells of phase III and IV follicles in XY-pF, but significantly reduced in granulosa cells of phase III and IV follicles in YY-pF, exhibiting a reciprocal expression pattern with Amhy. Consistently, YY-pF with one copy of Amhy mutation (YY-Amhy+/−-pF) were fertile. These results indicated that prolonged expression of Amhy led to a decrease in the expression of Cyp19a1a, followed by a decrease in serum E2 levels and an increase in androgen levels. Based on these data, we treated YY-pF with both E2 and trilostane (androgen synthetase inhibitors) simultaneously at the vitellogenic stage to make it fertile. The acquisition of fertile YY-pF laid the foundation for the widespread application of GMT.

Association between serum estradiol level and appendicular lean mass index in middle-aged postmenopausal women.

Previous studies investigating the association between loss of estrogen at menopause and skeletal muscle mass came to contradictory conclusions. To evaluate the association between serum estradiol level and appendicular lean mass index in middle-aged postmenopausal women using population-based data. This study included 673 postmenopausal women, aged 40-59 years, from the National Health and Nutrition Examination Survey between 2013 and 2016. Weighted multivariable linear regression models were used to evaluate the association between serum E2 Level and appendicular lean mass index (ALMI). When non-linear associations were found by using weighted generalized additive model and smooth curve fitting, two-piecewise linear regression models were further applied to examine the threshold effects. There was a positive association between serum E2 level and ALMI. Compared to individuals in quartile 1 group, those in other quartiles had higher ALMI levels. An inverted U-shaped curve relationship between serum E2 Level and ALMI was found on performing weighted generalized additive model and smooth curve fitting, and the inflection point was identified as a serum E2 level of 85 pg/mL. Our results demonstrated an inverted U-shaped curve relationship between serum E2 levels and ALMI in middle-aged postmenopausal women, suggesting that low serum E2 levels play an important in the loss of muscle mass in middle-aged postmenopausal women.

Associations between serum estradiol and IL-6/sIL-6R/sgp130 complex in female patients with major depressive disorder

BackgroundIt has been hypothesized that the IL-6/sIL-6R/sgp130 complex, an inflammatory complex, plays a critical role in the pathogenesis of major depressive disorder (MDD). Estradiol (E2) is a sex steroid hormone involved in emotional regulation and MDD. This study aimed to investigate the relationship between E2 and IL-6/sIL-6R/sgp130 complex in patients with MDD.MethodsUsing enzyme-linked immunosorbent assay, the levels of IL-6, sIL-6Rα, and sgp130 were compared between 117 female patients with MDD and 122 healthy controls.The serum concentrations of E2 and other biomarkers were also measured.Results(1) The serum levels of IL-6 and sIL-6Rα in patients with MDD were significantly higher than those in the control group, while the serum levels of sgp130 and E2 were significantly lower (all P < 0.05). (2) Low levels of E2 were associated with high levels of IL-6 and low levels of sgp130 (all P < 0.01). (3) HAMD-24 score was positively correlated with the serum level of IL-6, but negatively correlated with the serum levels of sgp130 and E2(all P < 0.05). (4) IL-6 and sgp130 had certain prognostic values in MDD, and the combination of various indicators showed a significantly superior prognostic value.ConclusionsThe IL6/sIL-6R/sgp130 complex in female patients with MDD was closely related to E2 level. In addition, IL-6 and sgp130 may be valuable serum biomarkers for the diagnosis and prognosis of MDD in women.

Lower serum 15-HETE level predicts nasal ILC2 accumulation during COX-1 inhibition in AERD

Aspirin-exacerbated respiratory disease (AERD) is associated with high levels of cysteinyl leukotrienes, prostaglandin D2, and low levels of prostaglandin E2. Further, 15-hydroxyeicosatetraenoic acid (15-HETE) levels may have predictive value in therapeutic outcomes of aspirin desensitization. Accumulation of nasal group 2 innate lymphoid cells (ILC2s) has been demonstrated during COX-1 inhibition in AERD, although the relationships between tissue ILC2 accumulation, reaction symptom severity, and novel lipid biomarkers are unknown. We sought to determine whether novel lipid mediators are predictive of nasal ILC2 accumulation and symptom scores during COX-1 inhibitor challenge in patients with AERD. Blood and nasal scraping samples from patients with AERD were collected at baseline and COX-1 inhibitor reaction and then processed for flow cytometry for nasal ILC2s and serum for lipidomic analysis. Eight patients with AERD who were undergoing aspirin desensitization were recruited. Of the 161 eicosanoids tested, 42 serum mediators were detected. Baseline levels of 15-HETE were negatively correlated with the change in numbers of airway ILC2s (r= -0.6667; P= .0428). Docosahexaenoic acid epoxygenase metabolite 19,20-dihydroxy-4Z,7Z,10Z,13Z,16Z-docosapentaenoic acid (19,20-diHDPA) was positively correlated with both changes in airway ILC2s (r= 0.7143; P= .0305) and clinical symptom scores (r= 0.5000; P= .0081). Low levels of baseline 15-HETE predicted a greater accumulation of airway ILC2s in patients with AERD who were receiving COX-1 inhibition. Further, increases in the cytochrome P pathway metabolite 19,20-dihydroxy-4Z,7Z,10Z,13Z,16Z-docosapentaenoic acid (19,20-diHDPA) were associated with increased symptoms and nasal ILC2 accumulation. Future studies to assess how these mediators might control ILC2s may improve the understanding of AERD pathogenesis.

Bioactive compounds from four Indian medicinal plants have different potency to induce sex reversal in Nile tilapia: A chromatographic, molecular docking and in silico analysis.

Herbal plants such as Basella alba (Family: Basellaceae), Tribulus terrestris (Family: Zygophyllaceae), Asparagus racemosus (Family: Asparagaceae) and Mucuna pruriens (Family: Fabaceae) are mentioned in Indian traditional system of medicine Ayurveda to possess androgenic activity and increase male virility. The plants have been reported to improve testosterone level and sperm production in experimental male rodents as well. Male Nile tilapias grow more quickly than females and hence are preferred for monosex Nile tilapia culture. Ethanol extracts of B. alba leaves (EB) and T. terrestris seeds (ET), and methanol extract of A. racemosus roots (MA) and M. pruriens seeds (MM) were found effective to induce masculinization in Nile tilapia. The present study intends to evaluate the anti-aromatase activity of EB, ET, MA and MM, to identify the androgenic bioactive compounds in the extracts, and to determine their pharmacokinetics. The study may validate the use of those plant extracts and their major bioactive phytoconstituents in the field of aquaculture and pharmaceuticals. The four crude plant extracts were first fractioned through column and thin layer chromatography (TLC). Three days old Nile tilapia juveniles (mean weight 0.025±0.009g; mean length 12.50±0.12mm; n=50 fish/replicate, 3 replicates/treatment) were then fed diets fortified with the obtained fractions for 30 days. After 30 days, fish were sacrificed and gonad aromatase mRNA expression, and 11-ketotestosterone (11-KT) and estradiol (E2) levels were measured. Fractions yielding the highest male percentage for each plant were subject to gas chromatography-mass spectrometry (GC-MS) analysis. The in silico docking and SwissADME study were conducted with the components showing higher peak percentage in chromatogram. After column chromatography and TLC analysis, EB, ET, MM and MA yielded 6 (EB1 - EB6), 8 (ET1- ET8), 14 (MM1-MM14) and 5 (MA1- MA5) fractions, respectively. Fish fed EB2, ET2, MA2 and MM13 fraction fortified diets showed significantly (p<0.05) higher male percentage (92.32%-98.39%) compared to other treatment groups. EB2, ET2, MA2 and MM13 fed fish showed significantly (p<0.05) higher 11-KT level compared to control male (+247.52 - +397.76%) and lower E2 level compared to control female (-95.92% to -90.65%). Aromatase mRNA expression was significantly (p<0.05) down-regulated by all these four fractions (-1.32 to -5.65 fold) with respect to control female. GC-MS analysis revealed the presence of 1-Octadecene (OD) in EB2, Phenol, 2,4-bis(1,1-dimethylethyl) (PD) in ET2 and MA2, 9,12-Octadecadienoic acid (Z,Z)- (ODDA) in MM13. In silico molecular docking indicated that PD is more effective than ODDA and OD to inhibit aromatase. In addition, PD showed better pharmacokinetics and more drug-likeness compared to OD and ODDA in SwissADME analysis. The present results indicate that ET and MA are more potent to produce all-male tilapia by means of aromatase inhibition. PD can be an ideal compound to achieve masculinization in Nile tilapia through dietary administration, but further investigation is required.

Abstract 1726: Loss of NF1 drives hormone dependent mammary carcinogenesis in a rat model with intact immune system

Abstract Loss of NF1 plays a major role as an oncogenic driver in up to 33% of all breast cancers (BC). Loss of NF1 is also a prognostic indicator for increased cancer risk at an earlier age, poorer outcomes, and therapeutic resistance. NF1 has recently been shown to bind the estrogen receptor alpha (ER) and act as a transcriptional corepressor. This helps explain findings in ER+ BC. Changes to NF1 that abrogate ER signaling thereby lead to Ras driven tumor resistance to endocrine therapy as cells are able to grow in low levels of E2 (and tamoxifen). Hence, NF1-mutant tumors represent a distinct molecular class in need of new therapeutics targeting both Ras and ER pathways. We have generated preclinical mammalian models of NF1 loss and BC that can be used to evaluate the role of NF1/ER transcriptional signaling in BC, the role of immune cells in BC, and therapeutics. Our models include a pathogenic patient missense allele c.3827G&amp;gt;A, p.R1276Q (knockin or KI) as well as a 14 base pair deletion c.3661_3674del, p.P1220fs*1223 (knockout or KO) model. Heterozygous (het) Nf1 female rats develop mammary gland adenocarcinoma spontaneously, but het KO rats develop multiple tumors with earlier onset. Tumors are generally Grade 2 and do not differ by genotype. By 16 weeks, 70% of KO females have developed at least one tumor whereas only 20% of KI rats and 2% of WT rats have developed tumors. This impacts survival as 76% of KI and 58% of KO females survive to one year. By 2 years, both alleles have 54% survival. The divergence in phenotype between KI and KO alleles may be due to residual function of R1276Q missense NF1 protein. A more in-depth analysis indicates that mammary tumor formation likely begins relatively early, as we find evidence of aberrant morphology and hyperplasia prior to the formation of palpable tumors. Interestingly, we find histological evidence of lung metastases and expression of breast markers GCDFP15, MGA, and CEA in the lung. Again, we see allele-specific effects in that KO rats develop lung tumors earlier than KI rats. Using single nuclei RNASeq to characterize the transcriptional profile of the mammary tumors, we find allele-specific effects beyond repression of Ras activity that drive aggressive tumor development. We identified different tumor cell populations (2 epithelial cell populations, Myeloid cells, B cells, T cells, Basal mammary cells, and WT specific cells) and identified different pathways altered due to the loss of Nf1 including Ephrin B signaling, Cyclin and Cell Cycle signaling, and Glycolysis signaling. Our overall goal is to characterize the phenotype of these rat models in terms of histopathology, Ras signaling, hormone signaling, immune components, and targeted drug response; which can then be compared with what is known regarding patients with somatic or germline inactivation of NF1 and breast cancer. Ultimately, this will provide better prognostic predictions for patients and better therapeutic options. Citation Format: Deeann Wallis, Christian Fay, Kelley Bradley, Erik Westin, William Bradley, Hui Liu, David Crossman, Jeremy B. Foote, Robert Kesterson. Loss of NF1 drives hormone dependent mammary carcinogenesis in a rat model with intact immune system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1726.

Abstract P3-08-01: Loss of NF1 drives hormone dependent mammary carcinogenesis in a rat model with intact immune system

Abstract Loss of NF1 plays a major role as an oncogenic driver in many cancer types and can be found in up to 33% of all breast cancers (BC). Loss of NF1 is also a prognostic indicator for increased cancer risk at an earlier age, poorer outcomes, and therapeutic resistance. In addition, certain NF1 genotypes may increase cancer risks, while others do not. NF1 is largely perceived as a classic Ras-opathy syndrome due to inactivating mutations in neurofibromin affecting RAS-MAPK signaling. However, recently it has been shown that NF1 binds estrogen receptor alpha (ER) and acts as a transcriptional corepressor. This helps explain some BC findings specifically in ER+ BC. In this model, specific changes to NF1 that abrogate ER signaling lead to Ras driven tumor resistance to endocrine therapy as cells are able to grow in low levels of E2 (and tamoxifen). Hence, NF1-mutant tumors represent a distinct molecular class in need of new therapeutics. A preclinical mammalian model of NF1 loss and BC would be helpful in both evaluating the role of NF1/ER transcriptional signaling in BC, evaluating the role of immune cells in BC, and testing therapeutics. Novel Nf1 rat models have a very robust ER+ BC phenotype, therefore more closely recapitulating clinical tumors compared to other preclinical models. Our models include a pathogenic patient missense allele c.3827G&amp;gt;A, p.R1276Q (knockin or KI), associated in humans with familial spinal NF1 and malignancy, as well as a 14 base pair deletion c.3661_3674del, p.P1220fs*1223 (knockout or KO) model. Heterozygous (het) Nf1 females develop mammary gland adenocarcinoma spontaneously, but het KO rats develop multiple tumors with earlier onset while het KI rats tend to develop fewer tumors with later onset. Tumors are generally Grade 2 and do not differ by genotype. By 16 weeks, 70% of KO females have developed at least one tumor whereas only 20% of KI rats and 2% of WT rats have developed tumors. This impacts survival as by 1 year 76% of KI females survive yet only 58% of KO females survive. However, by 2 years, both alleles have 54% survival. The divergence in phenotype between patient and null alleles may be due to residual function of R1276Q missense NF1 protein. A more in-depth analysis indicates that mammary tumor formation likely begins relatively early, as we find evidence of aberrant morphology and hyperplasia prior to the formation of palpable tumors. Interestingly, we find histological evidence of lung metastases and expression of breast markers GCDFP15, MGA, and CEA in the lung. Again, we see allele-specific effects in that KO rats develop lung tumors earlier than KI rats. While het male rats also develop mammary tumors at low rates, they experience longer survival times (76% of KO males survive 1 year and 98% of KI males survive 1 year) and males also develop tumors in other locations. Using single nuclei RNASeq to characterize the transcriptional profile of the mammary tumors, we find allele-specific effects beyond repression of Ras activity that drive aggressive tumor development. We identified different tumor cell populations (2 epithelial cell populations, Myeloid cells, B cells, T cells, Basal mammary cells, and WT specific cells) and identified different pathways altered due to the loss of Nf1 including Ephrin B signaling, Cyclin and Cell Cycle signaling, and Glycolysis signaling. Our overall goal is to characterize the phenotype of these rat models in terms of histopathology, Ras signaling, hormone signaling, immune components, and targeted drug response and compare/contrast them with what is known regarding patients with somatic or germline inactivation of NF1 and breast cancer. Ultimately, this will provide better prognostic predictions for patients and better therapeutic options for treatment. Citation Format: Deeann Wallis, Christian Fay, Kelley Bradley, Erik Westin, William Bradley, Hui Liu, Laura Lambert, David Crossman, Jeremy Foote, Robert Kesterson. Loss of NF1 drives hormone dependent mammary carcinogenesis in a rat model with intact immune system [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-08-01.

Maximize the safety and efficacy of fertility preservation by random start/dual ovarian stimulation for early breast cancer patients.

What is the more efficient and safer protocol during controlled ovarian hyperstimulation (COH) for early-stage breast cancer patients seeking emergency fertility preservation before adjuvant chemo/radiotherapy? This retrospective, case-series study involved two early-stage (Ia) breast cancer patients that requested for fertility preservation within 3 weeks. Random start/dual stimulation protocols with aromatase inhibitor (AI) were used to maximize oocyte yield and suppress serum estradiol (E2) level. E2 levels on trigger day during dual COH were between 112.0 and 407.0pg/mL. Duration of COH could be shortened to only 17 days, and up to 41 oocytes were successfully retrieved with two retrievals. This remarkable efficient and safe protocol involves the combination of random start/dual stimulation with step-up AI dosage beforehand which not only maximize oocyte yield within the shortest possible timeframe, but also to maintain the low level of E2 to avoid over-stimulating estrogen-sensitive cancer cells and to decrease the risk of developing ovarian hyperstimulation syndrome (OHSS).

Apolipoprotein C3 is negatively associated with estrogen and mediates the protective effect of estrogen on hypertriglyceridemia in obese adults

BackgroundBoth estrogen and apolipoprotein C3 (ApoC3) play crucial roles in lipid metabolism. But the link between them remains unclear, and it is unknown whether estrogen regulates triglyceride (TG) levels via ApoC3. Researchers hypothesized that estrogen exerts a regulatory effect on ApoC3 metabolism, and that this regulation could play a significant role in lipid metabolism. To explore this potential link, the present investigation aimed to examine the associations between estradiol (E2), ApoC3, and TG levels in both males and females.MethodsA total of 519 obese people (133 males and 386 premenopausal females) were recruited. Based on their TG levels, the participants were split into two groups [hypertriglyceridemia (HTG) group: TG ≥ 1.7 mmol/L; control group: TG < 1.7 mmol/L]. Serum ApoC3, E2, and TG levels were measured and compared in those two groups for both sexes separately. To ascertain the connection among E2, ApoC3, and TG, linear regression and mediation analysis were used.ResultsParticipants in the HTG group presented higher levels of ApoC3 (P < 0.001). In contrast, they tend to have lower E2 levels than the control. Linear regression analysis proposed that in both sexes, E2 was negatively associated with ApoC3 levels. The relationship remained significant after adjustment for confounding factors (male: standardized β = -0.144, t = -2.392, P < 0.05; female: standardized β = -0.077, t = -2.360, P < 0.001). Furthermore, mediation analysis revealed the relationship between reduced E2 levels and elevated TG levels is directly mediated by ApoC3.ConclusionsIn obese men and premenopausal women, ApoC3 was negatively and linearly correlated with serum E2 levels. The findings showed that estrogen may suppress ApoC3 expression and thus lower TG levels.

Artificial Intelligence-Augmented Electrocardiogram in Determining Sex: Correlation with Sex Hormone Levels

ObjectiveTo study the relationship between the sex probability derived from the artificial intelligence (AI)-augmented electrocardiogram (ECG) and sex hormone levels. Patients and MethodsAdult patients with total testosterone (TT; ng/dL) or estradiol (E2; pg/mL) levels (January 1, 2000, to December 31, 2020) with ECGs obtained within 6 months of the blood sample were identified. The closest ECG to the blood test was used. The AI-ECG model output ranges from 0.0 to 1.0, with higher numbers indicating high probability of being male. Low male probability was defined as ≤0.3, intermediate as 0.31 to 0.69, and high as ≥0.7. Continuous variables are expressed as median (interquartile range). ResultsPaired TT-ECGs were available in 58,084 male subjects and 11,190 female subjects. Paired E2-ECGs were available in 2835 male patients and 18,228 female patients. TT levels had moderate positive correlation with AI-ECG male sex probability (r=0.46, P<.001). Male subjects with low AI-ECG male sex probability had lower TT and higher E2 levels compared with men with high probability (TT: 303 [129-474] vs 381 [264-523], P <.001; E2: 35 [21-49] vs 32 [22-38], P=.05). Female subjects with high AI-ECG male sex probability had higher TT and lower E2 levels compared with those who had low male probability (TT: ≤50 years of age: 31 [18-55] vs 26 [16-39], P<.001; >50 years of age: 27 [12-68] vs 20 [12-34], P<.001; E2: ≤50 years of age: 58 [30-124] vs 47 [25-87], P=.001; >50 years of age: 30 [10-55] vs 21 [10-41], P=.006). ConclusionIn this study, TT levels were lower and E2 levels higher with decreasing AI-ECG male probability in both sexes. Male and female patients with discordant AI-ECG sex probability had significantly different TT or E2 levels. This suggests that the ECG could be used as a biomarker of hormone status.

Live birth associated with peak serum estradiol levels in letrozole intrauterine insemination cycles

To identify whether the serum estradiol (E2) level on the day of human chorionic gonadotropin (hCG) trigger or luteinizing hormone (LH) surge (hCG-LH) was associated with the live birth rate (LBR) during ovulation induction (OI) or controlled ovarian hyperstimulation with letrozole followed by intrauterine insemination (IUI). Retrospective cohort study. Large, multicenter private practice. A total of 2,368 OI-IUI cycles in patients treated with letrozole followed by IUI were evaluated from January 1, 2014, to July 31,2019. Ovulation induction with letrozole, followed by autologous IUI. The primary outcome measure was the LBR as a function of the serum E2 level at the time of hCG administration or LH surge, adjusting for age, body mass index, the largest follicle diameter, and the number of follicles ≥14 mm in diameter. The clinical pregnancy rate as a function of the E2 level, pregnancy rate as a function of the lead follicle diameter, and pregnancy loss rates were the secondary outcome variables. A total of 2,368 cycles met the inclusion criteria. Outcomes were evaluated at the 25th (E2 level, 110 pg/mL), 50th (157 pg/mL), 75th (225 pg/mL), and 90th (319 pg/mL) percentiles. The LBRs ranged from 9.4% to 11.1% in the lower E2 cohorts and from 12.5% to 13.5% in the higher E2 cohorts. The LBR was significantly greater in the cohort of women with higher E2 levels in all percentile comparisons except for the 90th percentile. The mean periovulatory follicle diameter of ≥20 or <20 mm was not independently associated with the LBR or clinical pregnancy rate, despite a significantly higher mean E2 level in the larger follicle group. In letrozole OI cycles followed by IUI, lower LBRs and clinical pregnancy rates were found in women with lower E2 levels than in those with higher E2 levels at the 25th, 50th, and 75th percentile E2 level quartiles. Where possible, delaying hCG trigger until the E2 level increases after aromatase inhibition and approaches the physiologic periovulatory level may improve the pregnancy rates with letrozole followed by IUI.

PS-C28-6: HIGH TESTOSTERONE AND LOW ESTRADIOL SERA LEVELS ARE ASSOCIATED WITH UNSTABLE PLAQUE COMPOSITION IN MEN WITH SEVERE CAROTID ATHEROSCLEROSIS

Background and Objectives: Ischemic strokes caused by the rupture of unstable atherosclerotic plaques in the carotid arteries, are leading causes of mortality and disability worldwide. Sex differences exist in plaque composition, where men are more likely to develop unstable plaques than women. Sex hormones affect vascular reactivity and inflammation, but it remains unclear if they are involved in plaque instability and how their role differs in men and women. Herein, we investigated the association between circulating endogenous sex hormone levels and plaque composition/instability in men and women with severe carotid atherosclerosis. Design and method: Pre-operative fasting blood samples and plaque specimens were collected from post-menopausal women and men greater than 55 years old with severe carotid atherosclerosis who underwent a carotid endarterectomy at McGill-affiliated hospitals in Montreal, Canada. Plaque composition and stability were examined by experienced vascular pathologists according to gold-standard histological classifications. Liquid chromatography-tandem mass spectrometry-based methods were developed to quantify endogenous sex hormones, 17ß-estradiol (E2), testosterone, androstenedione, and dehydroepiandrosterone, in sera samples. Results: Subjects (n = 460) had a mean age of 71.0 ± 9.0, were 68.9% male, and had various comorbidities (hypercholesterolemia [82.6%], hypertension [84.6%], type 2 diabetes [34.5%]). Clinical characteristics were similar among post-menopausal women (n = 143) and men (n = 317) with stable versus unstable plaques. Men had a significantly greater proportion of unstable versus stable plaques compared to women (men unstable: 61.5%, women unstable: 41.3%; P &lt; 0.001), characterized predominantly by a large lipid-rich core, hemorrhage, and high presence of inflammatory cell infiltration. Men with unstable plaques had significantly higher levels of testosterone than men with stable plaques (P = 0.004). In men, testosterone levels were inversely associated with fibrosis (P &lt; 0.05), while directly associated with unstable plaque features, including greater lipid core size (P &lt; 0.05), and a greater presence of foam cells (P &lt; 0.05), neovascularization (P &lt; 0.01), and plaque (P &lt; 0.01) and cap inflammation (P &lt; 0.05). No associations were observed between testosterone levels and plaque features in women. Endogenous E2 levels were inversely associated with neovascularization (P &lt; 0.05) and plaque and cap inflammation (P &lt; 0.001) in men, while inversely associated with fibrosis levels in women (P &lt; 0.05). Conclusion: Our findings suggest that higher testosterone and lower E2 levels are associated with unstable plaque composition in men with carotid atherosclerotic disease and may partly explain the higher proportion of unstable plaques observed among men than women of similar age and comorbidity status. Instead, in women, estradiol appears to play a detrimental role in plaque instability by inversely affecting fibrous tissue levels (a key stabilizing feature of plaques).

Transcriptomic analysis reveals the molecular mechanisms underlying osteoclast differentiation in the estrogen-deficient pullets

Several previous reports have suggested that estrogen (E2) is a vital signal responsible for the regulation of skeletal homeostasis and bone remodeling in mammals. E2 could efficiently accelerate the growth of medullary bone in pullets during sexual maturity. Furthermore, the low E2 level can strengthen the mechanical bone functions in female hens. However, mechanistic studies to describe the effects of E2 on bone in pullets during the initiation of the puberty period are remaining elusive. Therefore, the aim of this study was to explore the effect of inhibiting E2 biosynthesis on the biomechanical properties and its molecular mechanism during sexual maturity of pullets. In this study, a total of 90 Hy-line Sonia pullets with comparable body weight at 13 wk of age were selected and categorized into 2 separate groups. Daily, 0.5 mg/4 mL of letrozole (LZ) was orally administered to the treatment (TRT) group and 4 mL of saline to the control (CON) group of pullets for 6 wk. Compared with the CON group, a lower plasma E2 level was observed in the TRT group. Furthermore, plasma P, Gla protein (BGP), and 1,25-dihydroxy vitamin D3 (1,25-(OH)2D3) levels were markedly suppressed, whereas the plasma alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) levels were significantly elevated. Moreover, the cortical bone thickness and breaking strength of the tibia and femur, the bone mineral density of the humerus, and the bone mineral content of the humerus as well as the femur were increased significantly. The expression levels of 340 differentially expressed genes (DEGs) differed significantly between the CON and TRT group in the tibia at 19 wk of age. Among them, 32 genes were up-regulated, whereas 308 were down-regulated in the TRT group. The variations in candidate genes associated with osteoclast differentiation and cell adhesion may indicate that LZ inhibits E2 biosynthesis, consequently, reduces osteoclast differentiation by suppressing inter-cellular communication and cells attaching to extracellular matrix components. Taken together, the present study demonstrated that inhibiting E2 synthesis during sexual maturity of pullets decreased osteoclast differentiation and considerably enhanced bone quality.

Exposure to heavy metals and hormone levels in midlife women: The Study of Women's Health Across the Nation (SWAN)

Exposure to heavy metals may alter the circulating levels of sex hormones. However, epidemiologic studies on heavy metals and sex hormones have been limited, and results have been inconsistent. We assessed the associations of heavy metals assayed in urine, including arsenic, cadmium, lead, and mercury, with repeated measures of serum estradiol (E2), follicle-stimulating hormone (FSH), testosterone, and sex hormone-binding globulin (SHBG) levels in the Study of Women's Health Across the Nation Multi-Pollutant Study. The sample included 1355 White, Black, Chinese, and Japanese women, aged 45–56 years at baseline (1999–2000), whose serum hormone levels were repeatedly measured through 2017. Urinary metal concentrations were measured at baseline. Linear mixed effect models were used to calculate percent changes in serum hormone levels per doubling of urinary metal concentrations, adjusting for demographics, socioeconomic status, lifestyle, health-related factors, and urinary creatinine. After multivariable adjustment, a doubling of urinary metal concentration was associated with lower E2 levels by 2.2% (95% CI: 4.0%, −0.3%) for mercury and 3.6% (95% CI: 5.7%, −1.6%) for lead; higher FSH levels by 3.4% (95% CI: 0.9%, 5.9%) for lead; and higher SHBG levels by 3.6% (95% CI: 1.3%, 5.9%) for cadmium. The overall joint effect using the Bayesian kernel machine regression showed that metal mixtures were inversely associated with E2 and positively associated with FSH levels. No association was found between metals and testosterone levels. Results from this prospective cohort study demonstrate that environmental heavy metal exposures, including cadmium, mercury, and lead, may disturb circulating levels of E2, FSH, and SHBG in midlife women.