bluuebunny/crossref_metadata_2025 huggingface dataset:
The Major Impact of Obesity on the Development of Type 2 Diabetes (T2D) in Women With PCOS: A Systematic Review and Meta-Analysis of Observational Studies
Authors: Anagnostis Panagiotis , Paparodis Rodis , Bosdou Julia , Bothou Christina , Goulis Dimitrios G , Macut Djuro P , Dunaif Andrea Elizabeth , Livadas Sarantis
Published: May 2021

Background/Aims: Polycystic ovary syndrome (PCOS) is associated with disordered carbohydrate metabolism and an increased risk for T2D. However, there are limited data on the magnitude of this risk. Furthermore, 50-80% of women with PCOS are obese and obesity is known to have a synergistic deleterious effect on glucose tolerance in affected women. We systematically reviewed the literature regarding the association between PCOS, obesity and T2D risk. Methods: A comprehensive search was conducted in PubMed, CENTRAL and Scopus databases. Data are expressed as relative risk (RR) with 95% confidence intervals (CI). The I2 index was employed for heterogeneity. The available data, did not allow us to analyze the impact of weight status as normal, overweight and obese and as a consequence the studied subjects were stratified as obese (BMI>30 kg/m2) and non-obese (BMI<30kg/m2). Results: Twelve studies fulfilled eligibility criteria, yielding a total of 224,284 participants (45,361 PCOS and 5,717 T2DM cases). Women with PCOS had a higher risk of T2D compared with to unaffected women (RR 3.13, 95% CI, 2.83-3.47, p<0.001; I2 40.1%). When women with PCOS were stratified according to the presence or absence of obesity, the RR for developing T2D in obese compared with non-obese women with PCOS was 4.20 (95% CI 1.97-9.10; p<0.001). Moreover, compared to control women, the RR for developing T2D was significantly increased only in obese PCOS, RR 4.06 (95% CI 2.75-5.98; p<0.001). There was a trend toward significantly increased risk in non-obese PCOS women [RR 2.68 (95% CI 0.97-7.49; p=0.06). Conclusion: Women with PCOS have a >3-fold increased risk of T2D compared to women without PCOS, but this risk is substantially increased by the presence of obesity. Accordingly, weight reduction should be pursued in these women. References: 1. Dunaif A, Segal KR, Futterweit W, Dobrjansky A. Profound peripheral insulin resistance, independent of obesity, in polycystic ovary syndrome. Diabetes. 1989;38(9):1165-1174.2. Legro RS, Kunselman AR, Dodson WC, Dunaif A. Prevalence and predictors of risk for type 2 diabetes mellitus and impaired glucose tolerance in polycystic ovary syndrome: a prospective, controlled study in 254 affected women. J Clin Endocrinol Metab. 1999;84(1):165-169.3. Ehrmann DA, Barnes RB, Rosenfield RL, Cavaghan MK, Imperial J. Prevalence of impaired glucose tolerance and diabetes in women with polycystic ovary syndrome. Diabetes Care. 1999;22(1):141-146.4. Rubin KH, Glintborg D, Nybo M, Abrahamsen B, Andersen M. Development and risk factors of type 2 diabetes in a nationwide population of women with polycystic ovary syndrome. J Clin Endocrinol Metab. 2017;102(10):3848-3857.
Differential Expression Changes in Human Decidua With Term and Preterm Labor: Role for Upstream Targets in the Prostaglandin Pathway
Authors: Hornaday Kylie K , Wood Stephen L , Anderson David , Slater Donna M
Published: May 2021

Introduction: Prostaglandins (PGs) are paracrine mediators thought to be important during pregnancy and labor. Synthesis of PGs is complex with three rate limiting steps, the second of which (PTGS2/cyclooxygenase) is a target for managing preterm labor, but this treatment is not always effective. It is suggested that alternate steps in the PG pathway may play distinct roles during labor and hence the potential as alternate targets for labor management. The first step of PG synthesis involves the action of phospholipase A2 (PLA2) enzymes, of which over a dozen isotypes are known. However, PLA2 expression and role in the decidua (maternal-fetal interface) is unknown. We hypothesize that distinct PLA2 isotypes are present within the decidua and play a role in term and preterm labor. To this end, we conducted an expression profile of PLA2 subtypes and related PG genes within human decidua and determined differential expression patterns with labor at term and preterm. Methods: Decidual samples from term (40wks) and preterm (26-37wks) Cesarean deliveries, were used to assess PG gene expression by RNAseq and qRT-PCR (n=7 preterm non-labor, n=11 preterm labor, n=31 term non-labor, n=31 term labor). Analyses were conducted using student’s t-test and one-way ANOVA to compare labor (non-labor vs labor) or with gestation (preterm vs term) and adjusted for multiple testing using the Benjamini-Hochberg method. Results: RNAseq analysis identified 30 highly expressed PG genes in decidua, of which 12 had not been previously reported in the uterus, including 7 PLA2 isotypes. Confirming previous work, expression of PTGS2 was higher with labor (p=0.011). In contrast, we demonstrate that PLA2 isotype expression is lower with labor, with further differences between term and preterm samples. With term labor, expression of PLA2 isotype 2D was lower compared to non-labour (p=0.047). Meanwhile, preterm labor was associated with lower expression of PLA2 isotype 16 (p=0.025) and 4C (p=0.026), compared to preterm non-labour. Epression of PLA2 subtypes 2A, 15 and 4A late was highest in late labor (p=0.016, 0.004, 0.03, respectively). Conclusion: The presence of multiple PLA2 subtypes within the decidua suggests the potential for fine tuning PG synthesis during pregnancy. Expression of PLA2 isotypes 16 and 4C was uniquely associated with preterm labor, suggesting these isotypes may play a role in the pathogenesis of preterm labor. Further investigation of the functional role of PLA2 isotypes may provide insight to a novel mechanism for preterm labor and identify potential targets for labor management.
Expression of Cannabinoid Receptors in Mouse and Human Intrauterine Tissues: Insights into the Role of Endocannabinoid Signaling in Late Pregnancy
Authors: Urrego Daniela , Hornaday Kylie Karmila , Wood Stephen L , Slater Donna Michelle
Published: May 2021

Introduction: Endocannabinoid signaling (ECS), mediated primarily by cannabinoid receptors (CNR) 1 and 2, is implicated in embryo implantation, decidualization, and placentation, but less is known about its role in late pregnancy or labour. Reports of elevated serum endocannabinoid concentrations during labour suggest that ECS may modulate uterine function leading up to and during parturition. Effects on uterine function likely vary depending on: the type of cannabinoid present, the intrauterine tissue, and CNR1/2 expression. Study of ECS gene expression in late pregnancy is therefore important to determine the contribution of this pathway to the normal and pathologic physiology of labour, for instance in preterm labour. Examining ECS gene expression in pregnancy may also contribute insight into the effects of exogenous cannabinoid consumption (reported in ~5% of pregnancies) on the late-pregnant uterus. To understand when and where cannabinoid signaling may impact uterine functions, we conducted an observational study on pregnant human and mouse intrauterine tissues.

Methods: Human amnion, chorion, decidua, placenta, and upper/lower myometrium (n=6 participants) were biopsied at cesarean delivery (term, non-labour) to determine CNR expression by qPCR. Additional decidua (n=80) and upper/lower myometrium (n=82) samples were similarly obtained with and without labour, at term and preterm, for CNR expression analysis by qPCR. Mouse uteri for CNR expression analysis by qPCR were obtained from timed-mated C57BL6 mice at days 15-20 of pregnancy, in active labour, or post-partum. Human and mouse expression data were analyzed by Student’s t-test, one-way ANOVA (Bonferroni post-hoc), and Pearson’s correlation, as appropriate.

Results: Term non-labour chorion, placenta, myometrium and decidua express CNRs, with 2-fold higher expression of CNR1 versus CNR2. In myometrium and decidua, neither CNR1 or CNR2 expression differed with labour status. CNR1 expression correlated positively with gestational age in decidua (r=0.346, p=0.0266) and lower segment myometrium (r=0.3667, p=0.0270) with labour. In mouse uteri, Cnr1 and Cnr2 expression significantly increased post-partum compared to mid/late pregnancy (vs day 19, p<0.0001 Cnr1; vs days 15-18, p<0.05 Cnr2).

Conclusion: ECS may occur in the human decidua and myometrium throughout pregnancy and labour as CNR1 and CNR2 expression is maintained throughout. In our mouse model, higher Cnr1 and Cnr2 expression post-partum suggests that ECS may play a role in uterine resolution following delivery. Additional RNAseq analysis of the mouse tissues studied is underway to address whether expression of genes involved in production, transport or metabolism of cannabinoids are altered in late pregnancy or with the initiation of parturition.
Homeodomain Transcription Factors: Integral Modulators of Human Decidualization
Authors: Haller Meade , Yin Yan , Ma Liang
Published: May 2021

Ineffective embryo implantation accounts for a significant percentage of female infertility, and often renders IVF procedures unsuccessful. Decidualization, the dramatic uterine morphological response to ovarian hormone exposure, is a prerequisite for embryo implantation. Despite its significance in reproduction, the genetic framework of decidualization was not systematically studied until our recent development of a suitable high-throughput screening tool, immortalized human endometrial stromal cells (hESCs) that carry the yellow fluorescent protein gene under the control of the progesterone-sensitive prolactin promoter (PRL-Y cells). We recently used PRL-Y cells to perform a genome-wide siRNA functional screen and results revealed that 36 members of the homeodomain-containing family of transcription factors (HDTFs) are modulators of human decidualization. To determine which HDTFs are transcriptionally sensitive to ovarian hormone exposure, RT-PCR was performed on wildtype hESCs for the 36 HDTF hits over a 72-hour time course of E2/P4/cAMP exposure. Twenty HDTF hits (55%) were both detectable by PCR and showed variable expression in response to ovarian hormone treatment. Interestingly, all of these homeodomain factors, with a few distinct exceptions, exhibited decreased transcriptional expression in response to ovarian hormone treatment. This suggests that precious energy is used to transcribe these factors during the pre-decidualized phase, and that they may be required to maintain homeostasis during times of low hormone exposure. Because siRNA is not fully efficient, in order to confirm which HDTFs are required for normal decidualization, we generated a doxycyline-inducible Cas9-expressing hESC clone in order to subsequently generate individual knockout hESC lines for each HDTF hit. Cas9 expression was turned on 5 days prior to crRNA and trRNA transfection targeting the first exon of each HDTF. Five days after transfection, the cells were treated for 72 hours with ovarian hormone induction medium before RNA was isolated for gene expression analysis. As a pool of cells prior to any cloning (which likely includes knockout and wildtype cells in different ratios) the results indicate that several HDTFs are required for proper decidualization. The reporter transcripts of PRL and EREG are significantly abrogated or entirely undetectable in certain knockout lines. Interestingly these include some original siRNA HDTF hits whose expression is undetectably low by PCR. Sequencing validation will be necessary to confirm that knocking out such low levels of these transcripts genuinely has the robust effect on the human decidualization reaction that we are witnessing in these results. Together these findings comprise significant initial steps in characterizing the intricate upstream roles of HDTFs in human decidualization and female fertility.
Identification of Cellular Damage in Uteri From Hyperinsulinemic Mice Treated With Unopposed Estradiol
Authors: Kelly Katherine C , Huang Jane , Joshi Aditi A , Tal Aya , Wong Serena , Flannery Clare A
Published: May 2021

Unopposed estradiol and obesity are known risk factors for endometrial adenocarcinoma (EC). Endometrium from women with obesity was found to have an increase in mutations relative to tissue from normal weight women, indicating DNA damage may be accelerated in the setting of obesity. Since obesity is associated with high levels of insulin and anovulatory cycles, we sought to mimic these conditions in a mouse model. We previously found that hyperinsulinemic MKR mice, without the confounder of obesity, have an increased incidence of nuclear atypia in endometrial glands. We hypothesized that hyperinsulinemia and unopposed estradiol have a synergistic effect on inducing abnormal architecture and DNA damage in the endometrium, than either alone.

At 8-10 weeks old, cohorts of MKR (n=20) and WT (n=20) mice underwent ovariectomy and placement of either an estradiol (E2) or placebo (P) pellet. Metabolic profiling included insulin tolerance testing and MR for body composition. At 3 months post-implantation, mice received a partial hysterectomy and second pellet replacement. At 6 months, the remaining uterus was bisected into pieces. A blinded histological analysis was conducted by a gynecology pathologist. A marker of DNA damage due to oxidative stress, 8-oxoguanine-DNA-glycosylase (8-OHdG), was quantified by ELISA. Data was analyzed using Kruskal-Wallis test with multiple test correction, or Fischer’s exact test.

By 6 months, MKR-E2 treated mice had a 27% lower body weight than MKR-P mice (p<0.05), and 31% lower than WT-E2 mice (p<0.01). WT-E2 and WT-P had similar weight, and were similar to MKR-P (p=ns). Percent body fat was similar across all 4 cohorts of mice (p=ns). Since placebo-treated mice had small, atrophied uteri with minimal gland formation, E2 pellet failure was determined by the presence of small, atrophied uteri and occurred in 4 MKR and 3 WT mice at either 3 or 6 months. All other MKR and WT E2 treated mice had enlarged uteri. The frequency of endometrial gland dilation was similar in MKR-E2 and WT-E2 uteri (p=ns), but all MKR mice had moderate-severe dilation, whereas WT mice had 50% mild and 50% moderate-severe dilation (p=0.07). Focal hyperplasia was present in one MKR-E2 mouse, and nuclear atypia was present in one WT-E2 mouse. MKR-P uteri had a 7-fold higher mean 8-OHdG relative to MKR-E2 uteri (5.0±3.7 vs 0.7±1.6, p<0.002). WT-E2 and WT-P uteri had similar 8-OHdG (1.6±0.8 vs 1.8±0.6, p=ns), as did MKR-E2 and WT-E2 uteri (p=ns).

Our findings show that hyperinsulinemia exacerbates the cystic dilation induced by chronic unopposed estradiol, indicating a synergy of insulin and estradiol in promoting abnormal glandular growth in the endometrium. Surprisingly, uterine DNA damage was highest in the setting of hyperinsulinemia alone, in a hormonal state mimicking post-menopause. Further work is needed to understand the effect of estradiol on intrauterine oxidative stress-induced damage.
Long-Term Effects of Late Gestation in Utero Hypoxic Stress on Mood Disorders: Sex and Age Differences
Authors: Mabry Steve , Wilson E Nicole , Rybalchenko Nataliya , Engelland Rachel , Fadeyibi Oluwadarasimi , Osikoya Oluwatobiloba , Cushen Spencer , Goulopoulou Styliani , Cunningham Rebecca Lynn
Published: May 2021

Introduction: In utero insults have been linked with increased fear and anxiety in progeny. In utero hypoxic stress is associated with a multitude of gestational complications such as pregnancy-associated hypertensive disorders and intrauterine growth restriction. Maternal hypertension during pregnancy is also associated with increased mood and anxiety disorders in progeny. However, it is unknown if these associations are due to in utero hypoxic stress. We hypothesized that exposure to late gestational hypoxia will have a long-term impact on anxiety in progeny. Methods: Timed pregnant female Long-Evans rats were exposed to five days (gestational days: 15-20) of chronic intermittent hypoxia (CIH) or room air (normoxia - 21% O2) for 8 hours during their sleep phase. Each CIH cycle was 6 min of 3 min hypoxia (10% O2) and 3 min normoxia (21% O2) for a total of 10 CIH cycles/hour. At weaning (PND 28), progeny was pair-housed with a conspecific of same sex and similar weight. To examine mood and anxiety disorders, we quantified anxiety-related behaviors (time spent in the center of open field arena, marble burying test, social and anti-social behaviors with conspecifics) along with quantifying food intake and circulating sex hormone levels during puberty (postnatal day, PND 40-45) and young adulthood (PND 60-65) in male and female progeny. Results: Gestational CIH did not impact circulating sex hormones or food intake, regardless of sex or age of progeny. However, gestational CIH increased anxiety related behaviors in pubertal females. These effects of gestational CIH on anxiety in pubertal females were not maintained, as these behaviors resolved in young adulthood. Gestational CIH did not impact male progeny, regardless of age. Conclusion: Exposure to CIH during gestation resulted in increased anxiety related behaviors in pubertal female progeny. In utero hypoxia during late gestation may temporarily increase the risk for mood and anxiety disorders in pubertal females.
Long-Term Effects of Late Gestation in Utero Hypoxic Stress on Neurodegeneration: Sex and Age Differences
Authors: Wilson E Nicole , Mabry Steve , Rybalchenko Nataliya , Engelland Rachel , Fadeyibi Oluwadarasimi , Osikoya Oluwatobiloba , Cushen Spencer , Goulopoulou Styliani , Cunningham Rebecca Lynn
Published: May 2021

Introduction: In utero insults have been proposed to lead to the onset of neurodegenerative diseases later in life, such as Parkinson’s disease (PD). In utero hypoxia is associated with a multitude of conditions, such as maternal sleep apnea, preeclampsia, gestational diabetes, and maternal hypertension. Exposure to in utero hypoxia may impact male progeny more than female progeny, which may underlie the male biased sex differences in PD. It is currently unknown whether late gestational hypoxic stress has a long-term effect on brain regions associated with PD, such as the nigrostriatal pathway. We hypothesized that exposure to late gestational hypoxia will result in nigrostriatal impairment in adult male progeny compared to adult female progeny. Methods: Timed pregnant female Long-Evans rats were exposed to five days (gestational days: 15-20) of chronic intermittent hypoxia (CIH) or room air (normoxia - 21% O2) for 8 hours during their sleep phase. Each CIH cycle was 6 min of 3 min hypoxia (10% O2) and 3 min normoxia (21% O2) for a total of 10 CIH cycles/hour. Gestational age at delivery was recorded and neonate’s body weights were measured within 12-16 hours from birth. At weaning (postnatal day, PND 28), progeny was pair-housed with a conspecific of the same sex and similar weight. To examine PD, we focused on PD associated characteristics of oxidative stress in the nigrostriatal pathway and behavioral impairments of motor (open field activity and ultrasonic vocalizations) and cognitive (spatial memory) function during puberty (PND 40-45) and young adulthood (PND 60-65). Results: Gestational CIH had no effect on the duration of gestation, litter size, and neonatal weight at birth. Gestational CIH did not impact circulating oxidative stress, regardless of sex or age of progeny. Offspring gross motor function (open field activity) and cognitive (Morris Water maze) function were unaffected by gestational CIH. In contrast, gestational CIH impaired ultrasonic vocalizations in adult male progeny. Gestational CIH increased the latency to vocalize and decreased the loudness of the vocalizations in adult male progeny. Conclusion: Exposure to CIH during gestation resulted in nigrostriatal impairment in adult male progeny, as evidenced by impaired ultrasonic vocalizations that require a functional nigrostriatal pathway. In utero hypoxia during late gestation may increase the risk for PD in males.
Adrenocortical Carcinoma Treatment in the Netherlands: An Analysis From the Netherlands Cancer Registry From 2014 to 2019
Authors: Steenaard Rebecca , Rutjens Marieke , Haak Harm Reinout
Published: May 2021

Background: Adrenocortical carcinoma (ACC) is a rare disease with often poor prognosis. Previous research has shown that surgery in a Dutch Adrenal Network (DAN) center increases the chance of survival. We aim to explore the determinants and survival of patients with ACC recently treated in the Netherlands both within and outside DAN centers. Methods: We analyzed retrospectively collected data from 172 adult patients with newly diagnosed ACC and 97 patients with recurrence or new metastases, registered between 2014 and 2019 in the Netherlands Cancer Registry. Differences in survival were analyzed with cox-regression analysis. Results: More than half of the new cases presented with advance disease (25.7% stage III, 34.6% stage IV) and the median survival was 29 months. The majority of treatments occurred within a DAN center (87.2% of surgery, compared to 56.4% between 1999 and 2009; and 94.5% of medical treatment). There were no differences in patient characteristics between the centers apart from a relatively high number of patients with stage IV disease outside DAN centers (47.2% vs. 28.7%). Adrenal resection and mitotane therapy both resulted in a significant survival benefit (resection HR 0.29, CI95%[0.17–0.49]; mitotane HR 0.61, CI95%[0.37–0.99], corrected for stage). Still, a remarkable proportion of patients with advanced disease received no mitotane treatment (39.8%). Due to the small number of patients treated outside DAN centers, survival benefits could not be tested. Conclusions: Centralization of ACC care in the Netherlands has improved since the previous report, but a further improvement in centralization of surgery can be made. Adrenal resection and mitotane treatment remain the main form of treatment, with a clear survival benefit. Further research is necessary to determine why mitotane treatment is withheld in a large proportion of patients with advance disease.
Systemic Inflammation among Pregnant African American Women Is Related to Depressive Symptoms and Intimate Partner Violence
Authors: Saadat Nadia , Zhang Liying , Padmanabhan Vasantha , Engeland Christopher G , Misra Dawn P , Giurgescu Carmen
Published: May 2021

Systemic inflammation (e.g., higher levels of pro-inflammatory cytokine interleukin [IL]-6 and C-reactive protein [CRP]) during pregnancy increases risk for both the mother and the infant. For the mother, systemic inflammation is associated with an increased risk of pregnancy complications (e.g., preeclampsia). For the infant, cytokine dysregulation may lead to long term immune dysregulation and susceptibility to metabolic diseases. Pregnant African American women are more likely to have greater systemic inflammation compared with pregnant non-Hispanic White women. They also experience a higher prevalence of chronic conditions (e.g., asthma), have higher levels of depressive symptoms, and report more incidents of intimate partner violence (IPV). We hypothesize that maternal systemic inflammation is related to chronic disease, depressive symptoms, and IPV among pregnant African American women. Pregnant African American women (n=180) were recruited at prenatal clinics in the Midwest. Women completed a questionnaire and had blood drawn at a prenatal visit. Plasma cytokine levels (interferon [IFN]-γ, tumor necrosis factor [TNF]-α, IL-6, IL8 and IL10) were measured by multiplex array and CRP was measured by ELISA. Data were log (x+1) transformed and analyzed using descriptive statistics and correlational analysis (Pearson and point-biserial correlations). Women had a mean age of 27±5.5 years (range 18-41) and a mean gestational age at data collection of 16±5.8 weeks (range 8-29). Fifty-five percent of women were employed, 42% had annual household income < $10,000, and 16% smoked cigarettes during pregnancy. Twenty-five percent of women reported asthma, 8% hypertension, 6% diabetes, and 4% thyroid disease. Twenty-seven percent of women had Center for Epidemiological Studies-Depression (CES-D) scores ≥ 23, which have been correlated with depression diagnosis. IPV during the year before pregnancy was more frequent (8%) than IPV during pregnancy (5.1%). Women who reported hypertension had higher levels of TNF-α (r= 0.151, p< 0.05); and women with asthma had higher levels of CRP (r= 0.20, p< 0.01). Women who reported higher CES-D scores (r= 0.19, p< 0.05) had higher CRP levels. Women who reported IPV during pregnancy had higher IL-8 levels (r= 0.17, p< 0.05). Women who were employed had lower levels of IL-6 compared to women who were not employed (r= -0.180, p< 0.05). These results indicate that systemic inflammation is related to chronic health conditions (hypertension, asthma), depressive symptoms, IPV and employment status in pregnant African American women. The relationships between systemic inflammation, depressive symptoms and IPV, with a focus on metabolic and molecular pathways should be investigated to better understand the mechanisms by which maternal and fetal health are affected.
The Circadian Clock Gene Bmal1 Modulates Myometrium Contractile Function in Pregnant Mice
Authors: Van Quynh Duong Thu , Yaw Alexandra , Nguyen Duong , Hoffmann Hanne Mette
Published: May 2021

Caesarian section should be avoided unless medically required. Caesarian section is often a result of failed labor enhancement. While most natural births occur at night, labor enhancement is often scheduled during the day. We propose that the disparity between the commonly used timing of labor enhancement in the clinic, from the timing of natural birth, could contribute to the high rate of labor induction failure. Oxytocin receptor agonists are the most used labor enhancing agents. In pregnant non-human primates, oxytocin enhances uterine contractions more efficiently at night than during the day, suggesting a daily change in uterine sensitivity to oxytocin in pregnancy. To identify the molecular mechanisms generating daily changes in uterine function, we here explore the role of the molecular clock gene, Bmal1 (Brain and muscle ARNTL1-like), in the pregnant mouse myometrium. BMAL1 is a transcription factor required to generated circadian rhythms at the cellular level. We hypothesize that Bmal1 in uterine myometrial cells generates circadian rhythms and establishes the daily change in uterine contractile response to oxytocin. To evaluate circadian rhythms ex vivo, we collected myometrium samples from the validated circadian Per2:luciferase reporter mice at gestation day 17-18. We found that the pregnant mouse myometrium possesses circadian rhythms, which are generated by the molecular clock, as triple transgenic Per2:luciferase mice with Bmal1 conditionally deleted in the myometrium (cKO) do not have rhythmic expression of the Per2:luciferase reporter. To determine if BMAL1 is required to establish uterine contractions, we used a myograph to measure ex vivo uterine contractions at gestation day 17-18. In controls, uterine contraction force was significantly higher at ZT15 (3h after lights OFF) versus ZT3 (3h after lights ON). Interestingly, our preliminary data show increased basal contractile force at ZT15 in cKO as compared to controls. In addition, the cKO uterus contracted stronger to oxytocin than controls. Our findings identify Bmal1 as a clock gene modulating basal contractions in the mouse uterus and indicate Bmal1 might be a regulator of uterine sensitivity to oxytocin. Future work will focus on identifying the molecular mechanisms driven by BMAL1 to regulate uterine function in pregnancy. This work has the potential to provide insights into how we can improve labor enhancing treatment strategies in the clinic in the future.
Impact of Testosterone Therapy on Hematocrit and Polycythemia: Evaluation of Data From Two Ongoing Open-Label Randomized Single-Center Clinical Trials
Authors: Gonzalez Daniel , Kresch Eliyahu , Ory Jesse , Nackeeran Sirpi , Blachman-Braun Ruben , Molina Manuel , Ramasamy Ranjith
Published: May 2021

Introduction & Objective: Testosterone (T) replacement therapy (TRT) is the mainstay treatment for male hypogonadism. The most commonly reported adverse event among men using TRT is polycythemia. What is unknown is whether the short-acting vs. long-acting testosterone preparations have different effects on hematocrit. We hypothesized that short-acting testosterone therapy will be physiologic and have lesser effect on hematocrit compared to long-acting TRT. We evaluated data from two simultaneous ongoing open-label, randomized, two-arm clinical trials to evaluate the impact of TRT on Hematocrit and compared prevalence rates of polycythemia among subcutaneous T pellets (long-acting) and Intranasal Testosterone (Natesto) or Intramuscular Testosterone cypionate (TC) (short-acting).

Subject and Methods: Hypogonadal men (2 AM serum T < 300 ng/dL assayed by LC-MS/MS) aged 18-65 years were randomized into open-label randomized clinical trials. Eligible subjects received: Trial 1: 800mg subcutaneous Testopel T pellets; Trial 2: 11mg TID Intranasal testosterone (Natesto) or 200mg x 2 weeks TC for 2 months. Serum T, Hematocrit (HCT), and prevalence of polycythemia (as defined as HCT >50%) were collected at baseline and after 2 months of therapy. Data are presented as a post-hoc analysis of the two randomized clinical trials and reported as median and interquartile range [25th-75th], paired sample analysis (baseline versus follow-up) was performed with the Wilcoxon rank test to determine change during time within the different TRT modalities, with p<0.05 considered significant.

Results: Median change for serum T between baseline and 2mo follow-up to subcutaneous T pellets was 542 [454-757] ng/dL, Intranasal Testosterone 706 [517-1010] ng/dL, and TC 525 [280-712]ng/dL. T pellets showed a statistically significant increase in HCT from 44.6 [42.0-46.6] to 46.7 [42.6-48.9] (p<0.001), with a prevalence of 7/47 (14%) men developing polycythemia. A safety trigger for HCT greater than 54% occurred in 2/47 (4%). The treatment effect was independent of baseline serum testosterone. TRT with Natesto decreased HCT, from 43.4 [41.6-46.1] to 43.4 [40.6-46.5], however not statistically significant (p=0.262). TC statistically increased HCT from 41.6 [40.3-43.1] to 43.8 [43.5-47.4] (p=0.018), with 0% of men developing polycythemia in both groups.

Conclusions: Long acting TRT appears to increase hematocrit compared to short-acting testosterone therapies. Treatment of hypogonadal men with Intranasal T Natesto and testosterone cypionate successfully achieved target serum T level and maintained HCT levels. Longer-term durability and safety effects of the intervention remain to be further investigated.
Safety and Efficacy of Clomiphene Citrate in the Treatment of Secondary Hypogonadism. A Retrospective Study
Authors: Garcia-Touza Mariana
Published: May 2021

“Introduction: Clomiphene (Clomid) has an off-label indication to treat hypogonadism in select populations. “Based on clinical practice, this medication in certain types of hypogonadism is equally effective as testosterone in treating hypogonadism. It will lead to less side effects, lower treatment cost, and will decrease rates of infertility in the male veteran population.” Secondary hypogonadism can be most often caused by opioid use, obesity, sleep apnea, and diabetes.” “Aim of Study: To evaluate the safety and efficacy of Clomiphene Citrate in Treatment of Secondary Hypogonadism in comparison with testosterone. “Study Population: Data was obtained from the Veterans Administration Data Warehouse through the Veterans Administration Informatics and Computing Infrastructure. Data was extracted using SQL. There were 405,824 male patients with a diagnosis of hypogonadism (87.1%)and infertility (12.9%). nationally at the VA. Of these, 9566 patients have been treated with clomiphene citrate and 232,123 with various testosterone therapy. “ The two groups were then matched by propensity method to controls at a ratio of about 1:1 for Age, race, BMI and time for follow-up as potential confounding factors hat could have affected inclusion in the study controls. Patients without either Clomiphene or testosterone treatment were excluded. Statistical Analysis: SAS was used for propensity matching (PSM, greedy near) Categorical variables were evaluated as frequency counts with percentage within group, as well as ODDS ratio (OR) and differences were evaluated by a chi-square method. Comparisons of continuous variables were done were done by simple and paired t-test. Kaplan Meier plots and Cox Hazard ratio calculations were used to examine time dependent risk between treatments. Actual p-values are shown and p-values lower than 0.0001 are shown as such. All comparisons used a two-sided assumption. Measurements: Testosterone laboratory measures were recorded for start and end of trial. Survival was taken as the difference in days between start date and date of death. New diagnosis of Osteoporosis and Polycythemia was that which occurred after initiation of therapy. “Results: Clomid treatment normalized testosterone levels in 53.2% versus 46.8% in the testosterone group (OR 1.32 P<0.005). All-cause mortality was in the clomid group 0.16% and 1.62%in the testosterone group (OR 0.16 P<0.001). The incidence of new Osteoporosis for clomid was 3.9 % versus 5.9% for testosterone (OR 0.65 P<0.001)” Conclusion: This is a retrospective study comparing the efficacy and side effects of clomiphene versus testosterone for treatment of hypogonadism. The study showed that clomiphene is more effective than testosterone to treat secondary hypogonadism. We also found decreased overall mortality and incidence of polycythemia and osteoporosis.
Assessment of Chronic Supraphysiological Glucocorticoid Dosing Needs of Patients With Adrenal Insufficiency
Authors: Mun Jeff , McGlotten Raven , Nieman Lynnette K
Published: May 2021

Background: Patients who require higher than replacement glucocorticoid doses to avoid symptoms of adrenal insufficiency (AI) may have inadequate adherence or abnormal drug absorption or metabolism. The goal of this study was to identify why excessive glucocorticoid doses were needed in patients with ongoing AI symptoms. Methods: We performed pharmacokinetic (PK) analysis of glucocorticoid plasma concentrations (prednisone, prednisolone and dexamethasone measured by tandem mass spectrometry, Mayo Laboratories, and cortisol by immunoassay, Clinical Center) in 8 AI patients after weight-based oral hydrocortisone (HC) dose (1), IV HC 20 mg and /or prednisone 5 mg PO. The time (Tmax) to maximal plasma concentrations (Cmax), time to a 50% decrease in concentration (T1/2), elimination rate (ER) and area under the concentration curve (AUC) were determined using MATLAB and SimBiology, and compared to literature reference ranges (RR) (1,2). Results: Patients included one man; six had secondary AI due to previous supraphysiologic hydrocortisone or prednisone treatment and two had primary autoimmune AI. One of the latter was appropriately replaced with thyroid hormone. No patient was taking any medication known to be a strong inhibitor or inducer of CYP3A4 and none were taking oral estrogens. To study the potential contribution of intestinal absorption to abnormal pharmacokinetics, serum cortisol values were compared to expected values at 3.5 or 4 hours after a weight-based oral dose of HC in eight patients (1); 7 patients had values at the 50 - 80th centile of expected values. The eighth patient’s cortisol level at 4 hours after 5 mg HC was 30.3 nmol/L, below the 10th centile. She then underwent the same sampling with a 15 mg dose, with values also at the 10th centile. To uncover any discrepancy between PK oral and IV HC administration, four patients, including the patient with a low 4 hour value (LowHC4h) underwent sampling after 20 mg hydrocortisone, IV (2). Tmax and Cmax were within the RR in all four patients, while one patient had a faster T1/2 but an AUC similar to others. The LowHC4h patient had a dexamethasone level 8 hours after a 1 mg dose that was also within the RR and was maintained on dexamethasone. All others were eventually able to be weaned to a conventional glucocorticoid replacement dose. Conclusions: Evaluation of oral and IV HC PK may be useful in patients suspected of having abnormal absorption of oral glucocorticoids. Ref: 1. Mah PM et al. Clin Endo 61:367,20042. Thomson AH et al. Clin Endo 66:789,2007
Dietary Coconut Oil Mitigates Hyperandrogenemia in Obese Female Pigs Due to Suppression of Androgen Steroidogenesis in the Adrenal Cortex and Theca Externa
Authors: Shah Payal , Kothmann Kadden Heinrich , Skenandore Cassandra Suzanne , Browning Luke , Goblet Camille , Chau Laura , Grizzaffi Matthew , Newell-Fugate Anne Elizabeth
Published: May 2021

Western-style diets (WSD) cause cardiometabolic disease and hyperandrogenemia in reproductive-age women. A promising alternative for improved fertility in obese women may derive from the nutraceutical, coconut oil. We hypothesized that dietary coconut oil included in a high fat diet would mitigate hyperandrogenemia due to obesity through depression of androgen steroidogenesis in theca externa and adrenal cortex. Seventeen sexually mature female Ossabaw pigs were divided into 3 diet groups: control (n=6; C; 2200 kcal/pig/day), WSD (n=5; WSD; 5000 kcal/pig/day), or high fat coconut oil diet (n=6; COC; 5000 kcal/pig/day). Ingredients in WSD and COC diets were the same except 9% of calories in WSD were from lard and 9% of calories in COC were from coconut oil. Pigs were fed for 9 cycles (~ 8 mos) and fasting blood was collected at cycle 0 (baseline), 7, and 9. After cycle 7, ovarian steroidogenesis was suppressed by oral progestogen for 18 days. On day 18 while ovarian steroidogenesis was still suppressed, an ACTH stimulation was performed. On day 19, progestogen was withdrawn and 7.5 µg/kg IM dexamethasone was given every 12 hours to suppress adrenal steroidogenesis. On day 20, 3000 IU/m2 human chorionic gonadotropin (hCG) was given IV. Pigs underwent a washout cycle post-stimulations and were euthanized when they had dominant ovarian follicles in cycle 9 for collection of follicular fluid (FF), theca externa, and adrenal cortex. FF was assessed for steroid hormones by LC-MS/MS. Adrenal cortex and theca cells were cultured as follows for 48 hours after which cells and media were collected: 1) theca: control, insulin (I; 100 ng/ml), LH (10 ng/ml), or LH+I (10 ng/ml + 100 ng/ml); 2) adrenal: control or 1 µM ACTH. WSD pigs had a protracted estrous cycle length and increased FF testosterone, dehydroepiandrosterone sulfate, androstenedione (A4), androstenediol, and allo-pregnanolone (Allo-Preg) compared to C pigs. Both serum and cell culture media A4 concentrations were higher in response to ACTH in WSD pigs. By 72 hours post-hCG, COC pigs had higher serum A4 than C or WSD pigs, but in vitro LH yielded decreased cell culture media A4 in both WSD and COC pigs. COC pigs had increased FF dihydrotestosterone and Allo-Preg compared to C pigs. These results suggest that dietary coconut oil administration in obese females may depress excessive androgen steroidogenesis by adrenal cortex but may only partially mitigate excessive androgen production by theca externa.
Audit on Steroid Replacement in Confirmed or Suspected COVID-19 Patients With Adrenal Insufficiency or Adrenal Suppression in a District General Hospital
Authors: Haddad Aiman
Published: May 2021

Aim: In response to new updated guidance from Society For Endocrinology April 2020, this Audit was conduct to assess the adequacy of steroid replacement in confirmed or suspected COVID-19 patients with adrenal insufficiency or adrenal suppression admitted to Bedford District General Hospital. In steroid dependent patients (Addison’s disease, adrenal insufficiency, pituitary steroid insufficiency, use of long term steroids for other conditions - 5mg or more of prednisolone (or equivalent doses) daily for longer than 4 weeks) admitted to hospital very unwell with confirmed or suspected COVID recommendations is to start on Hydrocortisone 100 mg per IV injection followed by continuous IV infusion of 200 mg hydrocortisone/24h (alternatively 50 mg every 6 h per intravenous or IM bolus injection). Method: Retrospect data collection on Patients admitted in May 2020 to Bedford Hospital with suspected or confirmed COVID 19 disease with adrenal insufficiency or on long term steroid use. Those patients should be started on Hydrocortisone 100 mg per IV injection followed by continuous IV infusion of 200 mg hydrocortisone/24h (alternatively 50 mg every 6 h per intravenous or IM bolus injection).

Results: In May 2020, 295 patients admitted under the medical team in Bedford Hospital with confirmed or suspected COVID-19. Only 12 patients met the inclusion criteria, one patient with a diagnosis of Addison disease and the remaining 11 patients on long term steroids. None of these patients were managed as per updated guidelines. 6 patients had less than the adequate dose, they were started on prednisolone 30-40mg. 4 patients dose of oral steroids was only doubled, 1 patient received the same dose of oral steroid and the only confirmed Addison had higher dose of hydrocortisone. Moreover, In June 2020, The RECOVERY Outcome trial results showed that Dexamethasone 6mg for 10 days reduces the death by one third in hospitalised patient with severe respiratory complications of COVID-19. Dexamethasone 6mg is 12 times the physiological required steroid dose, this is equivalent to 240mg hydrocortisone, which is adequate for steroid replacement in patients with adrenal insufficiency or suppression.

Conclusion: In view of these results and the outcome of the RECOVERY Trial, Local trust guidelines updated, indicated that any patient with Adrenal insufficiency or suppression including those on long term steroids very unwell admitted to the hospital should receive Dexamethasone if requiring oxygen or Hydrocortisone if not requiring oxygen. Recommendation of changes included teaching sessions delivered to doctors, posters on updated guidelines distributed in major areas in hospital and trust guidelines updated on the intranet.
Hepatic Dysregulation of Bile Acid Homeostasis in Hyperandrogenemic Female Mouse Model of Polycystic Ovary Syndrome
Authors: Huffman Alexandra M , Rezq Samar , Basnet Jelina , Cardozo Licy L Yanes , Romero Damian G
Published: May 2021

Introduction and Purpose: Polycystic Ovary Syndrome (PCOS) is recognized as the most common endocrine disorder in women of reproductive age. Notably, PCOS women with hyperandrogenism have a pronounced increased risk for cardio-metabolic comorbidities compared with healthy individuals. Bile acids are endocrine signaling molecules that modulate hepatic lipid, glucose, and energy metabolism by aiding in absorption of lipids. Alteration of bile acid homeostasis affects overall metabolic homeostasis and contributes to pathogenesis of an array of metabolic diseases, although the molecular mechanisms of this have not been studied in PCOS. Methods: Four-week old C57BL/6N female mice were implanted subcutaneously with dihydrotestosterone (DHT, 8.0 mg) or vehicle silastic tubes (n=8/grp). Weekly body weight, food intake, and body composition was assessed. Fasting serum was obtained and the oral glucose tolerance test (OGTT) was performed in the last week of treatment. Animals were euthanized on treatment day 90 and livers were harvested. Expression levels of mRNA were assessed using RT-qPCR. Results: DHT treated females had significantly higher liver mass (1,387 ± 51 vs 1,197 ± 29 g, p<0.05), increased lean mass (21.25 ± 0.27 vs 19.58 ± 0.23 g, p<0.05) and increased fat mass (4.83 ± 0.47 vs 3.59 ± 0.36 g, p<0.05) compared to the vehicle counterparts. These hyperandrogenemic females additionally showed altered glucose homeostasis, having increased fasting glucose (201.10 ± 11.11 vs 152.80 ± 9.23 mg/dL, p<0.05) and an increased area under the curve (209.2± 11.0 vs 160.8± 3.5 mg.min/dL, p<0.05) following OGTT. Hepatic expression of both classic (Cyp8b1, 1.4 ± 0.1-fold, p<0.05) and alternative (Cyp7b1, 2.0 ± 0.3-fold, p<0.05) bile acid synthesis cytochrome P450 enzyme genes were significantly upregulated in DHT treated animals. Additionally, expression of sulfotransferase Sult2a2 was completely abolished in DHT treated animals compared with vehicle animals, indicating the possibility of androgen regulation of the sulfonation of bile acids marked for elimination. Liver expression of both the bile acid receptor G-protein coupled bile acid receptor 1 and the androgen receptor were both significantly downregulated (Gpbar1: 0.68 ± 0.08-fold, AR: 0.46 ± 0.04-fold, p<0.05) in DHT treated animals. Conclusions: Bile acid synthesis, transport, and elimination are tightly controlled processes in the liver to maintain a constant bile acid pool and limit reabsorption. Together, our results highlight the potential role of androgens in DHT-treated female mice in the dysregulation of bile acid homeostasis and its potential contribution to influence metabolic dysfunction. (Supported by NIH grants NIGMS P20GM-121334 to LLYC and DGR, and NIH NIDDK R21DK-113500 to DGR and the Mississippi Center of Excellence in Perinatal Research.)
Metabolic PCOS Features Are Ameliorated by Mitochondrial Uncoupler BAM15 in a PCOS Mouse Model
Authors: Paris Valentina Rodriguez , Alexopoulos Stephanie J , Hu Ying , Shah Divya P , Aflatounian Ali , Edwards Melissa C , Bertoldo Michael , Kyle Hoehn L , Walters Kirsty A
Published: May 2021

Polycystic ovary syndrome (PCOS) is a prevalent endocrine condition characterized by endocrine, reproductive and metabolic dysfunction. At present, there is no cure for PCOS and current treatments are suboptimal. Obesity and adverse metabolic features are prevalent in women with PCOS, with weight loss having a beneficial effect on PCOS features. The use of dietary interventions aimed at weight loss have low long-term compliance in women suffering from PCOS. Recent data from animal studies has shown that a small molecule mitochondrial uncoupler, BAM15, is an effective method to pharmacologically treat obesity and metabolic diseases. Therefore, the aim of this study was to investigate the efficacy of BAM15 to ameliorate PCOS-traits in a hyperandrogenic PCOS mouse model. As expected, exposure of female mice to dihydrotestosterone (DHT) induced the PCOS metabolic features of increased body weight (P<0.05), lean mass (P<0.001), increased parametrial and mesenteric fat pad weights (both P<0.05) and adipocyte hypertrophy (P<0.05). Additionally, DHT-induced PCOS mice exhibited insulin resistance measured by HOMA-IR, increased cholesterol and fasting triglyceride levels and hepatic steatosis (all P<0.05). In contrast, DHT-induced PCOS females treated with BAM15 displayed body weights which were comparable with controls, a significant decrease in parametrial and mesenteric fat depot weights (P<0.05) and reduced adipocyte hypertrophy. Furthermore, BAM15 treatment decreased insulin resistance, cholesterol and fasting triglyceride levels, as well as the degree of hepatic steatosis observed in PCOS females, to levels comparable with controls. PCOS mice presented the reproductive PCOS traits of irregular cycles and ovulatory dysfunction, however BAM15 did not improve these PCOS traits. These findings demonstrate that the pharmacologic mitochondrial uncoupler BAM15 is able to ameliorate metabolic PCOS features in a hyperandrogenic PCOS mouse model. These data provide compelling evidence to support BAM15 as a potential innovative and viable therapeutic approach to manage metabolic traits associated with PCOS.