bluuebunny/crossref_metadata_2025 huggingface dataset:
The Effects of Adiponectin on Infertile Women Undergoing IVF/ICSI Treatment and on Human Granulosa Cells
Authors: Qin Lixian , Sitticharoon Chantacha , Petyim Somsin , Keadkraichaiwat Issarawan , Sririwhitchai Rungnapa , Maikeaw Pailin , Churintaraphan Malika
Published: May 2021

Adiponectin, one of the most abundant adipocyte-secreted protein, has been involved in female reproductive regulation. This study aimed to 1) compare serum adiponectin levels in various phases of in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) treatment including Phase I (the beginning of gonadotropin stimulation), Phase II (around 8 days after gonadotropin stimulation), and Phase III (on the day of ovum pick-up) between success and unsuccess subjects; 2) compare follicular fluid (FF) adiponectin levels between success and unsuccess groups; 3) compare serum adiponectin levels among different phases of IVF/ICSI treatment in success and unsuccess groups; 4) compare the levels of adiponectin between serum and FF; and 5) investigate the effects of adiponectin on mRNA expressions of follicle stimulating hormone receptor (FSHR) and CYP19A1 (aromatase) in human granulosa-like tumor cell line (KGN) (n=3). In the human study, recruited participants (n=30) with age of 26-40 years were enrolled between April 2018 - May 2019. Blood samples were collected at Phases I, II, and III while FF samples were collected at Phase III. Adiponectin levels were comparable between success and unsuccess subjects in both serum (all phases) and FF (Phase III). Furthermore, serum adiponectin levels were comparable among Phase I, II, and III in success and unsuccess groups. In Phase III, serum adiponectin showed positive correlations with serum adiponectin in Phase I and II and serum FSH in Phase I. Interestingly, adiponectin levels in FF were significantly lower than serum at Phase III in unsuccessful pregnancies but were comparable in successful pregnancies. Moreover, FF adiponectin had a negative correlation with serum LH at Phase III in success subjects. In the KGN cell study, adiponectin had no effects on FSHR and CYP19A1 (aromatase) mRNA expression compared with control. In conclusion, high adiponectin levels in serum compared to FF might contribute to unsuccessful IVF/ICSI treatment.
The Hepatokine Adropin Is Regulated by Estrogen
Authors: Stokar Joshua , Gurt Irina , Yakubovsky Oran , Cohen-Kfir E Einav , Hallak Noa , Dresner-Pollak Rivka
Published: May 2021

Introduction: menopause is associated with weight gain, visceral adiposity and NAFLD. Rodent ovariectomy (OVX) is an accepted model for human menopause. Many OVX studies add high-fat diet or old-age to accentuate deranged phenotype. We have shown OVX alone induced weight gain and changes in liver transcriptome including downregulation of Enho, encoding for the hepatokine adropin (1). Here, we explore changes in VAT cytokine and adipokine genes, hepatic miRNA, and liver triglyceride content induced by OVX, in addition to estrogen’s role in regulation of adropin. Methods: 9-week-old C57BL/6J female mice underwent OVX or sham surgery. Groups of 10 mice were sacrificed at 6- and 12-weeks post-surgery and tissues harvested including mesenteric adipose tissue representing VAT. Liver TG was quantified using Cayman colorimetric assay. In-vitro studies performed in the murine hepatic cell-line, BNL1.ME. Adropin was measured using ELISA. Results: OVX induced adverse inflammatory cytokine & adipokine gene expression in VAT at 6-weeks post-surgery (Il18 1.1 p=0.01, Rares2 2.9, p=0.003, Retn 5.5, p=0.002) and 12-weeks post-surgery (Tnfa 2.3 p<0.001, Cxcl5 1.9 p=0.04). In the liver, OVX induced an increase in TG content at 12 weeks post-surgery (realtive increase vs sham 2.0 p=0.05). Hepatic Enho expression showed a strong inverse coorelation with total body weight gain (r= -0.7 p<0.001) and liver TG content (r=-0.4, p=0.04). In-vitro, estrogen induced an increase in Enho (relative mRNA change vs. growing medium 2.6, p=0.004); though protein level was unchanged, a trend for increased adropin was found in supernatant (relative change vs control 2.2 P=0.09). In-silico analysis of data from OVX mice treated with estrogen showed up-regulation of Enho (relative change vs vehicle, 6 p<0.001). At 6-weeks post-surgery OVX induced changes in hepatic miRNA profile with 48 miRNAs differentially expressed vs SHAM (24 up & 24 down). Integrating data from same sample RNA-SEQ and miRNA-SEQ created a network of differently expressed miRNA with oppositely differently expressed known specific mRNA targets. mIR-29, a known regulator of Enho in liver, was not found to be correlated with Enho expression in this context. Conclusions: OVX alone is sufficient to induce adverse changes in VAT gene expression and liver TG. Hepatic adropin gene expression is regulated by estrogen and its downregulation was strongly correlated to phenotypes relevant to menopause induced metabolic dysfunction, weight gain and increased liver fat. Thus, adropin should be further explored as a novel therapeutic and/or biomarker for menopause induced metabolic dysfunction. (1) Stokar, J., Gurt, I., Cohen-Kfir, E., Yakubovsky, O., Hanna, A., Assayag, E., & Dresner-Pollak, R. (2019). RNA-Seq Analysis of Ovariectomy-Induced Changes in Mouse Liver Reveals New Targets for Menopause-Associated Metabolic Derangement. Journal of the Endocrine Society, 3(Supplement_1), SUN-033.
Adult Onset Isolated Hypogonadotropic Hypogonadism- a Cause of Secondary Amenorrhea
Authors: Patel Shailendra B , Yada Srujana Kamala
Published: May 2021

A 23-year-old African American female was referred for secondary amenorrhea evaluation. She attained menarche at 12 years and had regular menses. At 18 years, she used OCPs for few months, and used plan B, after which her menses stopped. She had hot flashes and sweating. She was placed on progesterone, but never had withdrawal bleeding. Review of systems was positive for intentional weight loss after her menses stopped, hair loss and nipple discharge. She denied any loss of sensation of smell. She has a PMH of asthma, anxiety and OSA. Family history was not significant for any fertility issues. She smoked cannabis after menses stopped. On physical examination, vitals were stable, BMI of 35 kg/meter2, well-developed secondary sexual characteristics, no thyromegaly, acne or hirsutism. Upon work up, CBC, CMP were normal, Urine pregnancy test was negative, gonadotropins were undetectable (FSH- <0.7mIU/ml, LH- <0.2mIU/ml), anti-mullerian hormone was 3.82ng/ml (WNL), Estradiol was also absent (<15pg/ml), with a low Total testosterone (11ng/dl), TSH was 1.17uIU/ml, Free T4 was 1.1ng/dl, ACTH was 9.58pg/ml, Cortisol was 14.8mcg/dl, and Prolactin was 1.5ng/ml. MRI brain was normal with normal pituitary gland, no focal lesion visualized. Pelvic ultrasound showed ovaries 5.9mL and 4.6mL with multiple follicles present bilaterally. Diagnosis of adult-onset isolated hypogonadotropic hypogonadism (IHH) was made. Patient was started on estradiol patches and progesterone. IHH is a genetic disorder of defective production or action of GnRH. IHH when associated with anosmia is called Kallmann syndrome. It was first described by German American geneticist Joseph Kallmann in 1944. GnRH is a decapeptide, produced in arcuate nucleus and pre-optic nucleus of hypothalamus. GnRH stimulates anterior pituitary to secrete FSH and LH. IHH is caused due to impaired migration of GnRH neurons to brain during embryogenesis. It is inherited as autosomal or X-linked dominant or recessive. Gene mutations associated are ANOS-1, FGFR, PROK-2. It is rare in females. IHH has a broad spectrum of clinical presentation from complete absence of sexual development to partial completion of puberty. It presents with microphallus, cryptorchidism, cleft lip/palate, syndactyly, renal aplasia. In childhood presents with anosmia, hearing deficits, dental agenesis, mirror movements, short stature. During puberty, absent pubertal growth spurt, amenorrhea, lack of virilization, no secondary sexual characteristics, infertility. In partial forms, known as Adult onset or acquired form of IHH, patients have slight testicular growth, thelarche, menarche. Goals of treatment are- pubertal induction, maintenance of sexual maturation and restoration of fertility. In females, pre-puberty only estrogen is given, after puberty both estrogen and progesterone are used, for fertility, pulsatile gonadotropins or GnRH analogues are used.
To Be or Not to Be: Pregnant or Not
Authors: Patel Bijal , Kamalimotlagh Sharifeh , Patel Nilesh , Pinsker Richard W
Published: May 2021

Background: Beta Human chorionic gonadotropin (β-hCG) is used for diagnosing pregnancy. Differential diagnosis for elevated β-hCG includes false positive test due to antibody interference, gestational trophoblastic disease (GTD), pituitary production of β-hCG or non-gestational malignancies including hepatic, neuroendocrine, breast, ovarian, pancreatic, cervical, and gastric cancers. We are presenting a case of a menopausal woman noted to have persistently positive serum pregnancy test.

Case: 42-year-old female (G3 P2 A1) with a history of thyroid nodules and hypothyroidism was referred to endocrinology clinic for persistently positive serum pregnancy test without being pregnant. Last C-section delivery was 13 yrs ago. Last menstrual period was more than one year ago. Prior to that, her menstruation had always been irregular. Patient denied any spotting, pelvic discomfort/pain or weight loss in last few months. Seven months ago, POCT urine UCG test was done as per radiology protocol prior to CT abdomen for flank pain, and came back positive. Serum β-hCG was mildly positive at 4.3 (0.0 - 2.4 mIU/ml). Pelvic ultrasound did not show presence of a gestational sac. Repeat serum β-hCG level a week later was 3.8 mIU/ml. Patient was told she had early miscarriage. However, about 6 months later, PCP repeated serum β-hCG level and was elevated at 6.2 mIU/ml and thus patient was referred to endocrinology. Further work up showed FSH 61.3 (post-menopausal: 13.1-86.5 mIU/ml), prolactin 12.6 (3.0-18.6 ng/mL), TSH 9.41(0.47 - 4.70 uIU/ml) and estradiol level <15 pg/ml. β-hCG by dilution was ordered but was cancelled by lab for unknown reason. Patient was then also seen by OB-GYN who advised patient that she is menopausal and started her on estrogen-progesterone (E/P) therapy. About 4 weeks after starting E/P therapy, urine UCG came back negative. Hyper- glycosylated hCG (H-hCG) was not elevated. Patient was explained that false positive pregnancy test was due to pituitary production of β-hCG.

Discussion: We propose considering following aspects when approaching a case of elevated β-hCG in absence of pregnancy. 1) Hook effect can mask very high level of β-hCG, repeating test after dilution will rule out hook effect. 2) Many case studies have shown pituitary production of β-hCG in perimenopausal women. Perimenopausal women 41-55 years of age with mildly elevated serum β-hCG levels (5.0-14.0 mIU/ml) and concomitant FSH level over 45.0 mIU/ml are likely to have pituitary production of β-hCG(1). If we can suppress β-hCG with two weeks of E/P therapy, this supports pituitary production of β-hCG. 3) If above testing is equivocal then one can consider checking H-hCG which is predominately secreted by the more invasive cytotrophoblastic cells, and supports diagnosis of GTD.

References: 1) Gronowski AM, et al. Use of serum FSH to identify perimenopausal women with pituitary hCG. Clinical Chemistry. 2008. 54:4. 652-6.
A Retrospective Analysis of Progestogen Use in Transgender Women
Authors: Chang Julia J , Zhang Phoebe , Ariel Danit
Published: May 2021

Background: Many transgender women seek progestogens as part of their feminizing gender-affirming hormone treatment (GAHT), particularly to address breast development, body composition, mood stabilization, and changes in sexual desire and sleep. However, there are few studies that evaluate the role of progestogen use in feminizing GAHT. Reasons for initiating and continuing progestogen therapy have been mostly anecdotal to date, dependent on patient preference and provider practice style, and data about effects remain scarce.

Objective: To characterize progestogen use patterns in transgender women at one academic center; identify reasons for initiation and continuation of its use; and determine perceived related benefits and adverse events.

Methods: Retrospective analysis of transgender women prescribed feminizing GAHT at Stanford Health Care (SHC) between 2014-2020. Those using either combination estrogen + micronized progesterone or combination estrogen + medroxyprogesterone acetate (MPA) were included in our sample. Twenty-five consecutive charts were reviewed in detail for patterns of progestogen use, reasons for initiation and continuation, and its perceived effects.

Results: 410 transgender women sought care at SHC between 2014-2020. Ninety-six patients (23%) were prescribed micronized progesterone and 15 patients (4%) MPA at some point in their care, in addition to estrogen use. Of the 25 patient charts reviewed in detail (median age 30 years, IQR 25-42), 32% (n = 8) had clear documentation in the medical record of why progestogen was started and 25% (n = 6) included a description of the patient’s perceived effects with progestogen therapy. The most common reason to start progestogen was improved breast development (20%, n = 5). Three individuals reported improved nipple/breast growth. Median duration of progestogen use was 34.4 months (IQR 22.3-47.4) with 22 out of 25 patients still on progestogen therapy at time of chart review. Median duration of estrogen use prior to initiation of progestogen use was 14.2 months (IQR 7.1-35.8). No cardiovascular or thromboembolic events were noted in the reviewed patients on progestogen therapy.

Discussion: Over one in four transgender women seeking feminizing GAHT received a form of progestogen therapy as part of their overall GAHT. Most patients on progestogens lack documentation of why it was started or whether the patient seems to benefit from its addition. Given the continued paucity of data supporting or against progestogen use in feminizing GAHT, gender-affirming hormone providers may benefit from further education on how to counsel patients about progestogen use and how to assess for potential progestogen-associated effects over time. Finally, more consistent documentation in the electronic medical record will allow for more definitive conclusions regarding the benefits and risks of progestogen therapy in the future.
Development of Repository From a Pediatric Gender Clinic
Authors: Gortakowski Michele , Gordner Chelsea C
Published: May 2021

Objectives: With the publication of updated guidelines for care of transgender and gender non-conforming individuals, there has been an increase in the presence of gender diversity in both mainstream media and medical literature. Several gaps currently exist in medical knowledge regarding long term effects of gender-affirming therapies. There is a lack of standardization in study design, patient sampling, and outcome measures, and most studies are retrospective. Here we describe the creation of both a retrospective and prospective repository of patients who presented to the Massachusetts Medical School-Baystate Medical Center (UMass-Baystate) pediatric gender program.

Methods: Baystate Medical Center is located in western MA and is a tertiary referral center. A pediatric gender clinic was created in 2014. A repository containing both retrospective and prospective data was approved by the UMass-Baystate IRB to include patients ages 2 to 24 years of age who presented to our gender clinic. Retrospective data was obtained using the McKesson billing database. Sociodemographic, clinical and behavioral health data were collected. We are consenting individuals as they present to the clinic for the prospective component. Those that have consented fill out a survey at each visit. The repository has been approved to follow outcome data for 25 years.

Results: To date, we have 218 individuals in the repository, 75 of which are in the prospective component. Age of presentation ranged from 6 yrs to 24 yrs with an average age of 15 yrs. 62% identified as transmale, 31% as transfemale and the remainder as gender fluid or other. 75% have been prescribed gender affirming hormone therapy (56% GnRH agonist therapy, 20% estrogen, 58% testosterone). Of those being followed prospectively, 76% identified as white, 19% Hispanic. 79% were satisfied or very satisfied with their care.

Conclusions: Here we describe the demographic and clinical characteristics of patients that have presented to our gender clinic since 2014. The creation of our gender repository will allow us to assess sociodemographic, clinical and behavioral health outcomes of treatment, including metabolic parameters, bone health, and mental health outcomes in our pediatric population. Future projects include assessment of the change in cardiovascular risk in individuals on gender-affirming hormone therapy.
Patient and Parent Reported Psychosocial Concerns in Children and Adolescents With Turner syndrome
Authors: Fimmel Fiona , Clary Lauren E , Shankar Roopa Kanakatti
Published: May 2021

Background: Pediatric patients with Turner syndrome (TS) are reported to have a higher incidence of anxiety, depression and social isolation but there is minimal data on self-reported measures. Methods: We utilized the validated PROMIS (Patient Reported Outcomes Measurement Information System) question bank to develop 4-question short forms to assess patient and parent self-reported scores in the domains of depression, anxiety, and peer relationships in our TS cohort. Completed PROMIS short forms from TS patients seen in the multidisciplinary TS clinic between 1/1/2019 and 6/1/2020 were retrospectively analyzed. Clinical data were also abstracted from medical records and correlated with the measures of depression, anxiety, and peer relationships. Descriptive analyses of the T-scores were completed using a non-parametric Wilcoxon rank-sum test and the dyad results were analyzed for agreement between parent and patient reporting. Results: Data from 26 patients (mean age 13y, range 4.6-20.6y) were included in the analysis: 3.9% were clinically referred for depression/anxiety after psychology assessment, 15% had developmental delays and 15% had an individualized education plan. Nearly 77% had short stature (<5%ile), 58% had received growth hormone and 54% were on estrogen therapy. Median scores were not statistically different from population norms for patient (N=22) or parent (N=25) reports in the 3 domains and did not vary by age, estrogen supplementation or stature. Internal reliability of the questionnaires measured by overall Cronbach alpha for each domain were acceptable (0.73-0.93). While parent and patient reported anxiety scores were correlated (R=.452, p=.040.), the depression and peer relationship scores were not correlated. Parent-reported peer relationship scores were negatively correlated with patient-reported anxiety (R=-0.46, p=0.04) and parent reported anxiety (R=-0.43, p=0.04) scores. Parent perceived depression and anxiety measures were also correlated (R=0.77, p< 0.001) as were patient reported scores in these domains (R=0.6, p=0.003). Conclusions: In contrast to existing literature in TS indicating a higher incidence of psychosocial concerns, the patient and parent self-reported scores on depression, anxiety and peer relationship were not statistically different from the population normative data, and parent and patient perceived scores were not correlated in all domains. While the small number of patients in this pilot study may be insufficiently powered to detect a small difference in scores in these domains, the data underscores the need to develop a TS-specific validated psychosocial questionnaire that more adequately assesses the psychosocial concerns in this population.
A Novel Role of Nuclear and Membrane Receptor on Isoflavone-Induced Neuritogenesis and Synaptogenesis
Authors: Ariyani Winda , Miyazaki Wataru , Koibuchi Noriyuki
Published: May 2021

Thyroid hormone (TH) receptor (TR) and estrogen receptor (ER) play crucial roles in brain development. TR and ER are involved in dendrite growth, spines, and synapse formation in neurons. Soybean isoflavones, such as genistein, daidzein, and daidzein metabolite, S-equol are known to exert their action through TR, ER, and GPER1, a G-protein-coupled ER. However, the mechanisms of isoflavones action on brain development, especially during neuritogenesis and synaptogenesis, have not yet been extensively studied. We evaluated the effects of isoflavones using mouse primary cerebellar culture, astrocyte-enriched culture, Neuro-2A clonal cells, and co-culture with neurons and astrocytes. Soybean isoflavone augmented TH- or estradiol (E2)-mediated dendrite arborization of Purkinje cells. Such augmentation was suppressed by G15, a selective GPER1 antagonist, and ICI 182.780, an antagonist for ERs in both cultures. The knockdown of nuclear TRs or ERs also significantly reduced the dendrite arborization of Purkinje cells. It also increased the mRNA levels of TH-responsive genes, including Mbp, Bdnf, Rc3, Ntf3, Camk2b, Hr, and also Syn1, Syp, and Psd95 that are involved in synaptic plasticity. Isoflavones also increased the protein levels of synapsin-1, synaptophysin, and PSD95 in dendrite and membrane fraction of the cerebellar culture. To study further the molecular mechanism, we used Neuro-2A clonal cells. Isoflavones also induced neurite growth of Neuro-2A. The knockdown of TRs, ERs, and GPR30 by RNAi reduced isoflavones-induced neurite growth. Moreover, the co-culture study of Neuro-2A and astrocytes also showed an increase in isoflavones-induces neurite growth. In addition, isoflavones increased the localization of synapsin-1 or synaptophysin and F-actin in filopodia tips during Neuro-2A differentiation. The knockdown of nuclear ERs or GPR30 significantly reduced the number of filopodia and synapsin-1 or synaptophysin expression levels in neurite and membrane fractions. However, there are no significant effects of filopodia formation after co-culture with astrocytes. These results indicate that nuclear ERs and TRs play an essential role in isoflavones-induces neuritogenesis. Non-genomics signaling through membrane receptor and F-actin are necessary for the isoflavones-induces synaptogenesis. Astrocytes-neurons communication also increased isoflavones-induced neuritogenesis, but not synaptogenesis.
Androgen Receptor Blocker Improves the Cardiometabolic Profile in a Rat Model of Polycystic Ovary Syndrome, but at What Cost?
Authors: Pruett Jacob E , Everman Steven , Fernandez Edgar David Torres , Davenport Kacey , Romero Damian G , Cardozo Licy L Yanes
Published: May 2021

Introduction: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women of reproductive age. PCOS is characterized by androgen excess and ovulatory dysfunction high prevalence of cardiovascular risk factors such as increased blood pressure (BP), insulin resistance (IR), and obesity. We have demonstrated previously that exposing prepubertal female rats to dihydrotestosterone (DHT) leads to increase in food intake (FI), body weight (BW), BP, and IR. We tested the hypothesis that administration of the AR blocker bicalutamide (BICA) would decrease BP, IR, and obesity in PCOS model. As there are previous reports of severe hepatotoxicity with the AR blocker flutamide, we also examined BICA effects in the liver. Methods: Four-week old female Sprague Dawley rats implanted with DHT pellets (7.5mg/90 days) or placebo (PBO) were randomized to standard chow diet with or without the AR blocker bicalutamide (BICA) at a dose of 250 mg/kg/day throughout the study (n=10/group). BW and FI were measured weekly. BP and heart rate (HR) were measured by radiotelemetry. Fasting plasma was collected for IR (Homeostatic model assessment for IR, HOMA-IR). At euthanasia, the liver was collected, as well as plasma for gamma glutamyl transferase (GGT), alanine transaminase (ALT), and aspartate transaminase (AST) quantification. Results: PCOS rats had increased BW, FI, IR, and BP compared to PBO. BICA treatment had no impact on BW (285.3 ± 7.0 vs 270 ± 8.2 g, P=0.2) as well as FI and HR in PCOS. However, in PCOS, BICA decreased HOMA-IR (5.10 ± 0.40 vs 3.33 ± 0.31, P<0.05) and BP (115.4 ± 0.7 vs 105.3 ± 0.2 mmHg, P<0.01). Compared to PBO, PCOS+BICA rats had similar IR (3.83 ± 0.28 vs 3.33 ± 0.31, P=0.7) and BP (107.4 ± 0.8 vs 105.3 ± 0.2 mmHg, P=0.9). In addition, the liver weight to tibia length ratio was drastically increased by BICA in PCOS (222.9 ± 9.5 vs 360.4 ± 16.9 mg/mm, P<0.0001) as well as GGT (0.88 ± 0.88 vs 11.67 ± 0.58 U/L, P<0.0001), though it decreased AST (60.2 ± 6.9 vs 42.4 ± 1.9 U/L, P<0.05) and had no impact on ALT. Conclusion: In summary, in a model of PCOS, BICA treatment abolished IR and BP, independent of FI, BW and HR. Prompt treatment with an AR blocker can normalize increased IR and BP triggered by androgen excess in females. Further studies need to be done to fully understand the effect of BICA in the liver in PCOS. The beneficial effect of AR blockers as a therapeutic option to improve the cardiometabolic profile in PCOS may be hampered by its liver toxicity.
DHT Causes Liver Steatosis via Transcriptional Regulation of SCAP in Lean female Mice
Authors: Andrisse Stanley , Myer Jessie , Bogle Dilip “Bobby” , Eregha Nicole , Lofton Taylor
Published: May 2021

Hyperandrogenemia (HA) and insulin resistance are hallmarks of polycystic ovary syndrome (PCOS). These hallmarks are also integral elements of non-alcoholic fatty liver disease (NALFD). Administering low dose dihydrotestosterone (DHT) induced a lean PCOS-like female mouse model. The molecular mechanism of HA-induced NAFLD has not been determined. We hypothesized that low dose DHT would interrupt hepatic lipid metabolism leading to NAFLD. We extracted white adipose tissue (WAT), liver, and skeletal muscle from control and low dose DHT female mice; and performed histological and biochemical lipid profiles, Western blot, immunoprecipitation, chromatin immunoprecipitation, and real-time quantitative PCR analyses. DHT lowered the 65 kD form of cytosolic SREBP1 in the liver and WAT compared to controls. However, DHT did not alter the levels of the active and inactive forms of SREBP2 in the liver and WAT. DHT increased SCAP protein expression and SCAP-SREBP1 binding via AR binding to intron-8 of SCAP leading to increased SCAP mRNA. FAS mRNA and protein expression was increased in liver of DHT mice. p-ACC levels were unaltered in liver but decreased in WAT. Other lipid metabolism pathways were examined in liver and WAT, but no changes were observed. Our findings suggest that DHT increased de novo lipogenic proteins resulting in increased NAFLD via regulation of SREBP1 in liver. We show that in the presence of DHT the SCAP-SREBP1 interaction is elevated leading to increased nuclear SREBP1 resulting in increased de novo lipogenesis. We propose that the mechanism of action is increased AR binding to an ARE in SCAP intron-8.
Epitranscriptomic Reader HNRNPA2B1 Confers Endocrine Resistance to Breast Cancer Cells
Authors: Klinge Carolyn M , Petri Belinda J , Piell Kellianne M
Published: May 2021

Despite new combination therapies improving survival of breast cancer patients with estrogen receptor α (ER+) tumors, the molecular mechanisms for endocrine-resistant metastatic disease remain unresolved. HNRNPA2B1 (Heterogeneous Nuclear Ribonucleoprotein A2/B1), an RNA binding protein that functions as reader of the N(6)-methyladenosine (m6A) mark in transcribed RNA, is upregualted in tamoxifen- and fulvestrant-resistant, estrogen receptor (ERα)+ LCC9 and LY2 cells derived from MCF-7 endocrine-sensitive luminal A breast cancer cells (1). The miRNA-seq transcriptome of MCF-7 cells transiently overexpressing HNRNPA2B1 (A2B1) identified gene ontology (GO) pathways including “cellular response to steroid hormone signaling and estradiol” and “positive regulation of protein ser/thr kinase activity”. Modest (~ 4.5-fold) stable HNRNPA2B1 overexpression in MCF-7 cells (MCF-7-A2B1) results in ablation of growth inhibition by 4-hydroxytamoxifen (4-OHT) and fulvestrant. This was not due to loss or decrease of ERα; in fact, ERα was increased. Conversely, transient knockdown of HNRNPA2B1 in LCC9 and LY2 cells sensitized the cells to growth inhibition by 4-OHT and fulvestrant while reducing ERα. MCF-7-A2B1 cells showed increased migration, invasion, clonogenicity, soft agar colony size, and markers of epithelial-to-mesenchymal transition. Like LCC9 cells, MCF-7-A2B1 cells showed activation of AKT and MAPK and high androgen receptor (AR). Treatment of MCF-7-A2B1 cells with either PI3K inhibitor Wortmannin or MEK inhibitor PD98059 inhibited soft agar colony formation and reduced colony size. Knockdown of HNRNPA2B1 in MCF-7-A2B1 reduces clonogenicity, but had no effect on the clonogenicity of either LCC9 or LY2 cells. These data suggest a role for HNRNPA2B1 in promoting the initiation of acquired endocrine-resistance by activating ser/thr kinase growth factor signaling pathways. Selective inhibition of HNRNPA2B1 may be a target to prevent acquistion of endocrine therapy resistance, but not treat established metastatic disease. Reference: (1) Klinge CM, Piell KM, Tooley CS, Rouchka EC. HNRNPA2/B1 is upregulated in endocrine-resistant LCC9 breast cancer cells and alters the miRNA transcriptome when overexpressed in MCF-7 cells. Scientific reports 2019; 9:9430
Estrogen Receptor Alpha Is Required to Protect Daily Metabolic Rhythms From Disruption by High-Fat Feeding in Female Mice
Authors: Omotola Oluwabukola B , Pendergast Julie S
Published: May 2021

The circadian system is a critical regulator of obesity in male mice, but its role in females is poorly understood. In our previous studies we found that estrogen regulates daily rhythms in female mice to confer resistance to diet-induced obesity, but the mechanism is unknown. Estrogen signals via the classical estrogen receptor alpha (ERα) to regulate metabolism and obesity. Therefore, in this study we tested the hypothesis that estrogen regulates daily metabolic rhythms in females via ERα. To do so, we studied daily rhythms in female global ERα knockout (ERα KO) with the circadian reporter, PERIOD2::LUCIFERASE, mice fed high-fat diet for 6 weeks. ERα KO female mice became obese and hyperglycemic when fed high-fat diet, while wild-type females were resistant to diet-induced obesity. Chronic high-fat diet feeding also reduced the amplitude of the daily rhythm of eating behavior in ERα KO, but not wild-type, female mice. In wild-type females, the amplitude of the locomotor activity rhythm increased during high-fat feeding. In contrast, high-fat feeding decreased the amplitude of the activity rhythm in ERα KO females. The temporal relationship between central and peripheral circadian tissue clocks was disrupted by high-fat feeding in ERα KO females since the phase of the liver PERIOD2::LUCIFERASE rhythm was advanced 4 hours by high-fat feeding in ERα KO mice compared to wild-type females. Taken together these results show that estrogen signals via ERα to protect daily metabolic rhythms from disruption by high-fat feeding in female mice.
Glucocorticoid Mediated Transcriptional Activity in Human Corneal Epithelial Cells Lacking the Glucocorticoid Receptor
Authors: Jonsson Andrea Joyce , Ennis Lydia , Cidlowski John A , Kadmiel Mahita
Published: May 2021

The cornea is the dome-shaped transparent outermost layer of the eye, forming a physical barrier to protect the internal structures of the eye. Glucocorticoids are a mainstay in the treatment of ophthalmic diseases for their anti-inflammatory and anti-angiogenic properties. However, high doses or chronic use of glucocorticoid therapy can lead to vision-impairing effects such as increase in intraocular pressure and the formation of cataracts. The exact signaling pathways responsible for these undesirable effects of glucocorticoid use is poorly understood. One of the major molecular actions of glucocorticoids is to regulate transcription through its cognate nuclear receptor, the glucocorticoid receptor. We have previously reported the effect of glucocorticoids on global gene expression and their role in wound healing and barrier function in immortalized human corneal epithelial cells (HCE-T). In the current study, we knocked down glucocorticoid receptor using siRNA (GRKD) to determine the function of the glucocorticoid receptor in HCE-T cells. Successful knockdown of glucocorticoid receptor was confirmed by RT-PCR and immunoblot experiments. Genome-wide microarray analysis was performed and an FDR adjusted p value less than 0.01 was considered the cut off to create the list of differentially expressed genes (DEGs). Comparison of GRKD cells to HCE-T cells expressing endogenous glucocorticoid receptor (referred as NTC for No Target Control siRNA) revealed that expression of 2150 genes was altered in HCE-T cells when glucocorticoid receptor was knocked down, indicating that glucocorticoid receptor in corneal epithelial cells regulates a large cohort of genes. Inhibition of matrix metalloproteases, granulocyte adhesion and diapedesis, cyclins and cell cycle regulation were the top canonical pathways predicted by Ingenuity Pathway Analysis (IPA) to be altered in GRKD cells. In a 6-hour treatment with dexamethasone (Dex), a synthetic glucocorticoid, or with vehicle as a control, NTC cells had 2039 Dex-regulated genes, while Dex was still able to regulate 1087 genes in GRKD cells. Of these 1087 genes, 895 genes were uniquely regulated by Dex in GRKD cells suggesting that glucocorticoids might be signaling through another receptor in corneal epithelial cells. The top canonical pathways predicted to be altered by Dex in GRKD cells included PI3K/ATK Signaling, ERK5 Signaling, Prostrate Cancer Signaling, Aldosterone Signaling in Epithelial Cells, and PPAR signaling. These findings suggest that Dex could regulate large cohorts of genes through other nuclear receptors in corneal epithelial cells. Given the wide use of ophthalmic Dex in forms including eyedrops, ointments, gels, and implants, it is of clinical significance to understand the molecular actions of synthetic glucocorticoids since they appear to be ligands for multiple nuclear receptors in ocular cells and tissues.
Inhibition of Estrogen Signaling Reverses Established Inguinal Hernias
Authors: Potluri Tanvi , Taylor Matthew Joseph , Zhao Hong , Bulun Serdar Ekrem
Published: May 2021

Background: An inguinal hernia occurs when an intestinal loop or fat pushes through a weak spot in the lower abdominal muscle (LAM), causing a painful bulge that has the potential to cause bowel obstruction. Despite a high prevalence in men (~25%), non-surgical approaches are not available to treat this disease. We recently found a critical role of estrogen and estrogen receptor alpha (ERα) in inguinal hernia formation. To examine this further, we use a humanized aromatase mouse model (Aromhum) where all of the male mice develop scrotal hernias as a pre-clinical model to test the first pharmacological intervention for inguinal hernias. These mice are utilized because their skeletal muscle tissue contains aromatase and produces estradiol (E2), which acts via ERα in the LAM stromal fibroblasts and leads to fibrosis and muscle atrophy. Hypothesis: E2-ERα modulation can inhibit and reverse the formation of inguinal hernias in Aromhum mice by reducing LAM fibrosis and atrophy. Results: We tested three types of treatments to inhibit E2-ERα signaling: letrozole, fulvestrant, and raloxifene. Letrozole, an aromatase inhibitor, was shown to inhibit hernia formation and reversed small (150-175 mm2) scrotal hernias (n = 10-15/group, p<0.0001). The LAM tissues also showed a reduction in fibrosis (n = 5-8/group, p = 0.0004) and a concurrent increase in myofiber cross-sectional area (n = 5-8, p=0.0356) compared to placebo-treated mice. Similarly, fulvestrant and raloxifene, E2-ERα antagonists, also inhibited hernia formation (n = 10-15/group). Most interestingly, both drugs reversed large and severe hernias (>200 mm2, n = 10-15/group), accompanied by a decrease in muscle fibrosis and increase in myofiber cross-sectional area (ongoing study, n = 10-11, p<0.0001) compared to placebo mice. The drug-treated mice had lower expression of pro-fibrotic genes such as Mmp3, Emb, Spon2, Timp1, and Tgfb1 in the LAM tissues compared to placebo-treated LAM. Furthermore, we analyzed the differences in extracellular matrix producing genes and muscle regeneration markers between the placebo and drug-treated muscle tissues. Conclusion: We find that inhibition of the E2-ERα signaling pathway can reverse mild or severe inguinal hernias. Successful treatment is accompanied by decreased skeletal muscle fibrosis and reversal of myocyte atrophy. These interventions are promising non-surgical treatment options for patients suffering from severe and recurrent inguinal hernias.
Maternal Total Cortisol Levels in Early Pregnancy Depends on Fetal Sexual Dimorphism. But Finally No Association With Birth Weight
Authors: Twickler Theodorus Bartholomeus , Vrijkotte Tanja
Published: May 2021

Enhanced maternal cortisol levels may have a negative impact on fetal development with a higher risk for diseases later in life, e.g. premature cardiovascular disease and type 2 diabetes. Prior studies do assume even a sex specific impact. Currently, it is unknown whether sexual dimorphism in the fetus could display a different maternal cortisol level that is associated with intra uterine growth. In the present study (performed in the Amsterdam Born Children and their Development (ABCD) –cohort), we evaluated in 3049 pregnant women (in early pregnancy) whether fetal sex is related to the level of maternal serum total cortisol and whether this contributes to fetal growth. Maternal serum total cortisol levels increased along early pregnancy from on average 390±22 nmol/L (at 5th week of pregnancy) to 589±15 nmol/L (at 20th week of pregnancy). The presence of a female fetus was associated with higher maternal total cortisol level in a distinctive time interval along early pregnancy; before 11th week of pregnancy, no difference, and from the 12th week of pregnancy a difference of 15 (SE:7) nmol/L between mothers carrying a male vs female fetus was found and that difference increased to 45 (22) nmol/L at 20th week of pregnancy (p-for-interaction=0.05). Maternal serum total cortisol levels were negatively associated with maternal age, pBMI, smoking and parity, the last one also increasing with pregnancy duration. After adjusting for these factors, the association between fetal sex and maternal cortisol remained Maternal serum total cortisol levels were significantly associated with birth weight, standardized for pregnancy duration (ß -.22; SE:0.06; P < 0.001). Girls had a significantly lower birth weight (-132 SE:16 gram) compared to males, however, maternal cortisol did not alter the association between fetal sex and birth weight to a relevant degree indicating no mediation by maternal cortisol. In early pregnancy, the maternal total cortisol levels are related to fetal sex. However this difference in maternal total cortisol level was finally not related to birth weight.
Persistent COUP-TFII Expression Underlies the Myopathy and Impaired Muscle Regeneration Observed in Resistance to Thyroid Hormone-Alpha
Authors: Aguiari Paola , Liu Yan-Yun , Petrosyan Astgik , Cheng Sheue-Yann , Brent Gregory A , Perin Laura , Milanesi Anna
Published: May 2021

Myopathic changes, including muscular dystrophy and weakness, are commonly described in hypothyroid and hyperthyroid patients. Thyroid hormone signaling, via activation of thyroid nuclear receptor alpha (THRA), plays an essential role in maintaining muscle mass, function, and regeneration. A mouse model of resistance to thyroid hormone carrying a frameshift mutation in the THRA gene (THRA-PV) is associated with accelerated skeletal muscle loss with aging and impaired regeneration after injury(1,2). We previously demonstrated that the expression of nuclear orphan receptor chicken ovalbumin upstream promoter-factor II (COUP-TFII, or Nr2f2) persists during myogenic differentiation in THRA-PV myoblasts and skeletal muscle of aged THRA- PV mice. COUP-TFII is known to regulate myogenesis negatively and has a role in Duchenne-like Muscular Dystrophies(3). COUP-TFII physically and functionally interacts with THRA in primary myoblasts isolated from WT and THRA-PV mice, as demonstrated via co-immunoprecipitation and chromatin-immunoprecipitation. We observed that satellite cells from THRA-PV mice display impaired myoblast proliferation and in vitro myogenic differentiation compared to WT cells. However, the silencing of COUP-TFII expression using siRNA probes restores in vitro myogenic potential of THRA-PV myoblasts and shifts the mRNA expression profile closer to WT myoblasts, with a higher proliferation of myoblasts and a higher number of fully differentiated myotubes after 5 days of myogenic induction. Moreover, RNAseq analysis on myoblasts from THRA-PV mice after COUP-TFII knockdown shows that COUP-TFII silencing reverses the transcriptomic profile of THRA-PV myoblasts and results in reactivation of pathways involved in muscle function and extracellular matrix remodeling/deposition. These findings indicate that the persistent COUP-TFII expression in THRA-PV mice is responsible for the abnormal muscle phenotype. In conclusion, COUP-TFII and THRA cooperate during murine post-natal myogenesis, and COUP-TFII is critical for the accelerated skeletal muscle loss with aging and impaired muscle regeneration after injury in THRA-PV mice. These studies can help increase our knowledge of the mechanisms involved in thyroid hormone signaling during skeletal muscle regeneration, ultimately increasing the possibility of designing more specific treatments for patients with thyroid hormone-induced myopathies. References: 1. Milanesi, A., et al., Endocrinology 2016; 2. Kaneshige, M. et al., Proc Natl Acad Sci U S 2001; 3. Lee HJ, et al, Sci Rep. 2017.
Production of 11-Oxygenated Androgens by Testicular Adrenal Rest Tumors
Authors: Schröder Mariska A M , Turcu Adina F , O’Day Patrick , van Herwaarden Antonius E , Span Paul N , Auchus Richard J , Sweep Fred C G J , Claahsen-van der Grinten Hedi L
Published: May 2021

Testicular adrenal rest tumors (TART) are a common complication in male patients with classic 21-hydroxylase deficiency (21OHD). TART are considered to have steroid-producing properties and may contribute to the androgen excess in 21OHD patients. This study aims to define the production of 11-oxygenated 19-carbon (11oxC19) steroids by TART. Steroids were measured in left (n=7) and right (n=4) spermatic vein- and simultaneously taken peripheral plasma (n=7) samples from seven men with 21OHD and TART using liquid chromatography-tandem mass spectrometry. In addition, steroids were quantified in TART cell- and adrenal cell-conditioned medium, with and without adrenocorticotropic hormone (ACTH) stimulation. Compared to peripheral blood of 21OHD patients with TART, the spermatic vein samples displayed the highest gradient for 11-hydroxytestosterone (11OHT; 96-fold) of the 11oxC19 steroids, followed by 11-ketotestosterone (47-fold) and 11-hydroxyandrostenedione (11OHA4; 29-fold), suggesting production of these steroids in TART. TART cell-conditioned medium contained higher levels of testosterone, and lower levels of androstenedione and 11OHA4 after ACTH stimulation compared to adrenal cell-conditioned medium, indicating ACTH-induced production of testosterone in TART. TART cells also produced 11OHT after 48 h of ACTH stimulation. Thus, in patients with 21OHD, TART produce 11oxC19 steroids, but in different proportions than the adrenals. The very high ratio of 11OHT in spermatic vein- versus peripheral vein blood suggests the 11-hydroxylation of testosterone by TART, and the in-vitro results indicate that this metabolism is ACTH-sensitive.
Development of Deep Learning-Based Automatic Detection Algorithm for Adrenal Nodules on Contrast-Enhanced Abdominal CT Scans
Authors: Ahn Chang Ho , Kim Tae Woo , Jo Kyungmin , Kong Sung Hye , Kim Jinhee , Kim Taesung , Shin Chan Soo , Choo Jaegul , Kim Jung Hee
Published: May 2021

Objective: Adrenal nodules are often incidentally detected on abdominal computed tomography (CT) scans due to their asymptomatic nature. We aimed to develop an automatic detection program for adrenal nodules on abdominal CT scans using deep learning algorithms. Methods: We retrospectively analyzed abdominal CT scans performed at two university-affiliated hospitals (n = 483 and n = 514, respectively) from 2006 to 2019. This dataset was randomly divided into training set (181 CTs without adrenal nodule and 362 CTs with adrenal nodule) and test set (291 CTs without adrenal nodule and 163 CTs with adrenal nodule). All CT scans were contrast-enhanced and the phase with the highest contrast between adrenal gland and adjacent normal tissues was selected for multi-phase CT. The core algorithm of our deep learning algorithm for adrenal nodule (DLAAN) was MULAN (Multitask Universal Lesion Analysis Network) algorithm whose backbone was a convolutional neural network. DLAAN was composed of two stages. The first stage was to detect the CT slice where normal adrenal gland or adrenal nodule were located. The second stage was for fine localization of adrenal nodule on the corresponding CT slice. The performance of DLAAN was evaluated using the area under the receiver operating characteristic curve (AUROC) for patient-level classification and free-response ROC for nodule-level localization. The figure of merit for free-response ROC was calculated as an average sensitivity when 0.5, 1, 2, and 4 false positives per slice were allowed. Results: The AUROC of DLAAN was 0.927 (95% confidence interval: 0.900–0.955). With a threshold probability of 0.9, the sensitivity and specificity were 86.5% and 89.0%, respectively. When left and right adrenal nodules were analyzed separately, the AUROC was 0.910 for left adrenal nodule and 0.957 for right adrenal nodule, respectively. The accuracy of DLAAN according to the size of adrenal nodule was 0.890, 0.734, 0.981, 1.00 and 1.00 for no adrenal nodule, adrenal nodule sized 1–2 cm, 2–3 cm, 3–4 cm and > 4 cm, respectively. The performance of DLAAN for the localization of adrenal nodule which was estimated by average sensitivity was 0.812. The number of CTs with at least one false positive nodule was 93/454 (20.5%). Conclusion: Our proof of concept study of deep learning-based automatic detection of adrenal nodule on contrast-enhanced abdominal CT scans showed high accuracy for both the classification of patients with or without adrenal nodule and the localization of adrenal nodule, although the performance of the algorithm decreased for small sized adrenal nodules. External validation with different CT settings and patient population is needed to assess the generalizability of our algorithm.