bluuebunny/crossref_metadata_2025 huggingface dataset:
Topline Results of the CARE-PWS Phase 3 Study: Intranasal Carbetocin Improves Hyperphagia and Anxiety and Distress Symptoms in Prader-Willi Syndrome (PWS)
Authors: Ryman Davis , Deal Cheri L
Published: May 2021

Prader-Willi syndrome (PWS) is a complex genetic disorder associated with multiple neuroendocrine abnormalities including significantly decreased hypothalamic oxytocin levels, resulting in symptoms of severe hyperphagia (an unrelenting false sense of starvation) and multiple severe neuropsychiatric and behavioral issues. CARE-PWS, a multi-center, randomized, double-blind, placebo-controlled phase 3 study, has evaluated the efficacy, safety, and tolerability of intranasal carbetocin, a selective oxytocin receptor agonist, in participants with PWS.

Eligible participants aged 7 through 18 with genetically confirmed PWS were randomized in equal proportions to three treatment arms for the 8-week placebo-controlled period of the study: carbetocin 9.6 mg, carbetocin 3.2 mg, or a matching placebo, administered by nasal spray three times a day with meals. The primary endpoint assessed changes from baseline to week 8 in Hyperphagia Questionnaire for Clinical Trials (HQ-CT) or Children’s Yale-Brown Obsessive Compulsive Scale (CY-BOCS) scores for the carbetocin 9.6 mg arm vs placebo, and the first secondary endpoint assessed changes from baseline to week 8 in HQ-CT or CY-BOCS scores for the carbetocin 3.2 mg arm vs placebo. Additional secondary endpoints included changes from baseline to week 8 in PWS Anxiety and Distress Questionnaire (PADQ) scores, and Clinical Global Impression of Change (CGI-C) scores evaluating the overall change in severity of PWS symptoms at week 8.

Due to COVID-19, enrollment was closed early with 119 evaluable participants for the primary analysis. In the carbetocin 9.6 mg arm, trends toward numerically greater improvements in HQ-CT and CGI-C scores relative to placebo were observed but did not reach statistical significance; however, the carbetocin 3.2 mg arm demonstrated a significant improvement in HQ-CT scores (LS mean improvement vs placebo -3.14 points, p=0.016). In the 3.2 mg arm, additional consistent evidence of improvements versus placebo was seen in multiple secondary endpoints, including CGI-C (p=0.027) and PADQ (p=0.027). Numeric trends toward improvement in CY-BOCS scores were observed in each dose arm, but did not reach statistical significance versus placebo. During the subsequent long-term follow-up period of the study, both carbetocin arms have experienced continued numeric improvements from baseline across multiple endpoints. Intranasal carbetocin was generally well-tolerated; the most frequently reported adverse event was flushing, which was generally mild and transient.

In conclusion, results of the CARE-PWS study support that intranasal carbetocin appears to be safe and well tolerated, and reduces hyperphagia and anxiety and distress behaviors in PWS.
6-Year-Old Girl With a Luteinized Follicular Ovarian Cyst and an Estradiol Level > 1,000 PG/ML
Authors: Jacob Marianne , Poppas Dix , Lekarev Oksana
Published: May 2021

Background: Precocious puberty in girls is defined as onset of secondary sexual characteristics, such as breast development, before 8 years of age. To differentiate between central and peripheral precocious puberty, laboratory and imaging evaluation is helpful. When gonadotropins are low but estradiol is elevated, results may suggest a primary ovarian source of estrogen production. Small ovarian cysts are not uncommon, are benign and self-resolve. However, large ovarian cysts are rare, let alone ones requiring surgical removal.

Clinical Case: A 6 year 7 month old girl presented with several days of breast tenderness and palpable bilateral breast tissue noted by her mother. There was no history of vaginal bleeding. There were no reported exposures to estrogen-containing products. Her mother reached menarche at age 14 years. The patient was born full term and was otherwise healthy. On exam, her height was at the 90-95th %ile (mid-parental height at the 95th %ile) and her growth velocity was 10.9 cm/yr. She had Tanner 2 breasts (1 cm breast bud on the left and 1.5 cm on the right), Tanner 1 pubic hair and no axillary hair, body odor, acne or café-au-lait macules. A bone age was read as 6 years at a chronological age of 6 years 7 months. A laboratory evaluation revealed an estradiol of 1,029 pg/mL (<15 pg/mL), LH <0.02 mIU/mL, FSH <0.09 mIU/mL, 17-hydroxyprogesterone (17-OHP) 410 ng/dL (<91 ng/dL), AFP 2.3 ng/mL (<6.1 ng/mL), beta-hCG <2 mIU/mL, TSH 2.41 mIU/L (0.5-3.2 mIU/L), and free T4 0.9 ng/dL (0.9-1.4 ng/dL). Pelvic ultrasound revealed a large unilocular cystic structure measuring 6.5 x 4.1 x 6.1 cm in the left adnexal region with no left ovary visualized. The right ovary appeared prepubertal. The uterus was prepubertal in appearance with endometrial thickness of 2 mm. Abdominal ultrasound showed no evidence of a suprarenal mass. A laparoscopic cyst resection was completed, given the risk of left ovarian torsion. Cytology was negative; pathology revealed a luteinized follicular cyst. Repeat labs in one month showed a prepubertal estradiol level of 6.7 pg/mL with LH 0.02 mIU/mL and FSH 0.38 mIU/mL. 17-OHP normalized to 29 ng/dL. Breast tissue had regressed.

Conclusion: This case describes the rare finding of a large luteinized follicular ovarian cyst that required surgical removal in a 6-year-old girl in the setting of a significantly elevated estradiol level. Luteinized follicular cysts have been described in newborns, though rare. To our knowledge, this is the first described case of a luteinized follicular cyst in this patient’s age group. Laboratory and imaging evaluation should be considered in girls presenting with precocious puberty, despite the extent of thelarche, as the clinical examination does not always correlate with degree of estradiol elevation. This is especially important if clinical changes are acute and other features are consistent with puberty, such as rapid linear growth.
A Case of Severe MIS-C in Pediatric Diabetes: Complications of COVID-19
Authors: Dye Alyssa M , Arnold Sandra R , Finkel Terri H , Kim Ahlee
Published: May 2021

Background: Multisystem inflammatory syndrome in children (MIS-C) is a serious inflammatory response to a prior coronavirus disease of 2019 (COVID-19), characterized by fever, inflammation, and multiorgan dysfunction. Current literature does not indicate a relationship between pediatric diabetes and risk of developing MIS-C. Here, we report a case of pediatric type 2 diabetes (T2D) with diabetic ketoacidosis (DKA) and severe multi-organ dysfunction with SARS-CoV-2 serology positivity.

Clinical Case: A 13-year-old African American female with obesity (Body Mass Index >99th %) and poorly controlled T2D (HbA1c 12.8%) presented to the emergency department for one week of sore throat, headache, and abdominal pain. SARS-CoV-2 positivity was confirmed by PCR. The next day, she was found unconscious at home. She was diagnosed with DKA and was directly admitted to the pediatric intensive care unit. IV insulin and aggressive fluid resuscitation were initiated and her DKA resolved over the next 72 hrs. However, she exhibited continued altered mental status with slurred speech and significant respiratory distress requiring respiratory support. Due to the severe presentation and multi-organ involvement, a multi-disciplinary care team was formed. Further workup confirmed acute respiratory distress syndrome with pneumonia; severe acute kidney injury (AKI, creatinine of 4.56 mg/dL); presumed myocarditis (ST elevation on EKG, troponin 4.47 ng/mL, BNP 129.8 pg/mL); punctuate intraparenchymal hemorrhage in the splenium of the corpus callosum; transaminitis (AST 188 u/L, ALT 100 u/L); pancreatitis (amylase 651 u/L, lipase >9500 u/L); thrombocytopenia with consumptive coagulopathy (platelet 81 X 103/µL, d-dimer 5.91 mcg FEU/mL), increased inflammatory markers (ESR 53 mm/hr, ferritin 127 ng/mL), and positive SARS-CoV-2 serology. A presumed diagnosis of MIS-C was made per the Centers for Disease Control and Prevention definition and she was started on dexamethasone and intravenous immunoglobulin (IVIG). While consideration was given to the possibility of acute COVID-19 infection in combination with DKA, she was not a candidate for remdesivir due to AKI. On day 12, she developed new dysarthria, dyspraxia and behavioral changes. Encephalopathy workup was negative (CSF Encephalopathy autoimmune panel negative, NMDA receptor negative) and she was restarted on dexamethasone and IVIG. She was discharged on day 28.

Conclusion: There is a paucity of literature of MIS-C associated with COVID-19 in the pediatric diabetes. Our case highlights several novel aspects of MIS-C with concurrent poorly controlled diabetes and DKA, including severe central nervous system manifestations and prolonged hospitalization. Further studies are warranted to elucidate an association between pediatric diabetes and MIS-C and to develop guidelines for management of MIS-C in poorly controlled pediatric diabetes.
A Novel Mutation of the AR Androgen Receptor Gene
Authors: Aljasser Abdullah Abdulruhman
Published: May 2021

Androgen insensitivity syndrome (AIS), formerly known as testicular feminization, is an X-linked recessive condition resulting in a failure of normal masculinization of the external genitalia in chromosomally male individuals. The basic etiology of androgen insensitivity syndrome is a loss-of-function mutation in the androgen receptor (AR) gene. Loss of AR function means that, despite normal levels of androgen synthesis, the typical postreceptor events that mediate the effects of hormones on tissues do not occur. This results in the phenotype of prenatal undervirilization of external genitalia, absence of pubic and axillary hair, lack of acne, and absence of voice changes at puberty We present This baby reffered at age of 2 months from pediatric surgery as a case of bilateral inguinal hernia and chromosomes 46xy. Phenotypically female no male structures no phallus and single opening and visible labia, both test are in the inguinal canals. HCG stimulation test shows: Testosterone: the level at (0) time: 0.8 nmol/l then 3 days: 31.4 nmol/l. DHT dihydrotetostrerone: the level at (0) time: 13 NG/L then 3 days: 485 The baby was given 3 doses of Testosterone injections 150mg but no response at the genetalia. Radiological investigations shows ultrasound both tests at the inguinal canals and no uterus Also MRI of pelvis shows absence of uterus and both test at the inguinal canals Molecular genetics analysis for Androgen receptor gene: Exon 7 c.2512 G >A hem. P.Glu838lys missense, novel VUS,likely pathogenic. The segregations analysis test in the process. Her we present a novel mutation of the AR gene not reported yet in literature.
Are Current Normal Values of 11DOC Useful for Diagnosis of Non Classical Congenital Adrenal Hyperplasia Due to 11β- Hydroxylase Deficiency?
Authors: Barash Galia , Drach Lior , Naugolni Larisa , Yacoel Tamar , Bistritzer Tzvi Tzvi , Rachmiel Marianna
Published: May 2021

Background: A non-classic form of 11β-hydroxylase deficiency (NC 11β-OHD) has been reported to cause mild androgen excess, with a clinical presentation of precocious puberty, menstrual cycle abnormalities, or hirsutism during adolescence. Since genetic diagnosis of NC 11βOHD is yet not routinely available, the current gold standard for biochemical diagnosis is elevated 11 DOC levels after corticotropin stimulation test (ACTHstimT). However, there are no clear hormone level cutoffs. One of the accepted references for basal and stimulated levels for the pediatric population was published in 1991 by Lashansky et al1. Aim: To determine the correlation between 11DOC levels measured during ACTHstimT, clinical symptoms attributed to NC11βOHD and androgen levels at presentation, and long-term follow-up among children and adolescents with hyperandrogenism. Methods: a retrospective study including all patients who underwent ACTHstimT between 20072015, in one center, during which 11 DOC levels were routinely measured as part of the test. Clinical data was collected from the patients’ medical files and, by telephone calls for complete long-term follow-up. 11DOC levels before and after ACTHstimT were categorized as elevated according to both pre-defined cut-offs; greater than 1.5 times the 95th percentile according to Lashansky1 normal level for sex and age, and greater than 1.5 times the upper limit of the normal level of the commercial kit. Results: Data were complete at presentation for 136 patients, 92 females, and for long for 98 patients, 68 females, mean follow up duration of 3.1 years (1.37,5.09). There was no statistically significant difference in the number of cases with elevated 11DOC according to both cut-offs, among patients with precocious and early puberty, premature adrenarche nor acne. Higher baseline and stimulated 11 DOC levels were demonstrated in females who presented with mild hirsutism and regular menses. Long term data demonstrated no statistically significant difference in the number of cases with elevated 11DOC levels among patients with compromised final adult height, PCOS or hyperandrogenism. There was negative correlation between stimulated 11 DOC levels and basal levels of testosterone, androstenedione and DHEAS levels. Conclusions: This report demonstrates that the current interpretation of 11DOC levels, basal and ACTHstimulated in children, according to 1.5 times the highest range, of both, the Lashansky1 acceptable norms for children, and some of the laboratory’s kit, are not clinically applicable.1Lashansky G, Saenger P, Fishman K, Gautier T, Mayes D, Berg G and Reiter E. Normative data for adrenal steroidogenesis in a healthy pediatric population: Age- and sex-related changes after adrenocorticotropin stimulation. J. Clin. Endocrinol. Metab. 1991; 73(3): 674-686.
Food Perception and Differences in Dietary Decision-Making in Youth With Congenital Adrenal Hyperplasia
Authors: Luo Shan , Overholtzer Lloyd N , Lim Seung-Lark , Kim Elaine , Fraga Nicole , Herting Megan M , Tanawattanacharoen Veeraya K , Geffner Mitchell E , Kim Mimi S
Published: May 2021

Background: Youth with congenital adrenal hyperplasia (CAH) have a higher prevalence of obesity, early adiposity rebound, and increased fat mass. Unsuccessful dietary self-control could contribute to obesity, and understanding food-seeking behavior could therefore guide prevention. Dietary decision-making involves key brain regions such as the limbic system and the prefrontal cortex, which are associated with choice and reward. These regions (i.e., prefrontal cortex, amygdala, hippocampus) can be smaller in volume in CAH patients. However, little is known about dietary decision-making in CAH. We hypothesized that CAH youth would exhibit differences in dietary decision-making and aimed to study food choices in CAH youth compared to controls.

Methods: 37 CAH youth (12.2 ± 3.1 y, 60% male, BMIz 1.6 ± 0.8) and 100 controls (11.7 ± 2.4 y, 57% male, BMIz 0.9 ± 1.2) completed a behavioral computer-based food choice task. They rated 30 high- and 30 low-calorie food cues for tastiness, healthiness, and liking. Food pairs discordant for taste and health ratings were generated, and youth were asked to choose the item they wanted to eat. Cursor-trajectory analyses measured area under the curve (AUC) and maximum deviation time (MDT), with successful choice trials evident when the healthier food was chosen. Based on individual ratings for food cues, β-coefficients for ratings predicting food preference were generated.

Results: CAH youth and controls did not show differences in food ratings (P > 0.30 for all) or in the percentage of successful trials of total choice trials (P = 0.16). However, CAH youth showed larger mean AUCs compared to controls [T(135) = 2.15; P = 0.03] suggesting that they may experience more conflict and exert more cognitive effort in decision-making. CAH youth also had longer mean MDTs [T(102.3) = 2.59; P = 0.01] in successful choice trials, indicating a later time at which a final decision was made. β-health and β-taste predicting food preference did not differ between groups, and β-health was correlated with successful choice trials in both CAH (r = 0.38, P = 0.02) and controls (r = 0.48, P < 0.01). However, β-taste was negatively correlated with successful choice trials in controls only (r = -0.42, P < 0.01; CAH r = -0.22, P = 0.18).

Conclusion: Although youth with and without CAH had similar perceptions of food, CAH youth may exert greater cognitive effort and experience more conflict in dietary decision-making. This could suggest that factors inherent to CAH such as abnormal neural pathways, or disease treatment, could affect the cognitive control of food choices in CAH youth.
Growth Hormone Therapy for Children With Duchenne Muscular Dystrophy and Glucocorticoid Induced Short Stature
Authors: Lavi Eran , Cohen Amitay , Dor Talya , Tsabari Reuven , Zangen David
Published: May 2021

Background: Duchenne Muscular Dystrophy (DMD) is the most common form of inherited muscle disease in childhood. DMD patients have severe growth retardation due to several reasons including chronic treatment with glucocorticoid (GC). Data regarding the efficacy and safety of Recombinant Growth Hormone (rGH) treatment in DMD patients is very limited. The aim of this study is to evaluate efficacy and safety of growth hormone treatment in 4 DMD boys with glucocorticoid induced growth failure. Methods and Results: 4 prepubertal patients with DMD on high dose of GC (Deflazecort or Prednisone) at an age range between 12-13.6 years and significantly delayed bone age (8.5-11 years) were studied. rhGH was subcutaneously administered at a dose of 0.033 mg/kg/d for a duration of 12-18 months. Pretreatment annual growth rate of the 4 patients (0, 3,2.5, 1.5 cm/yr) improved during treatment (3.8, 7.8, 7.2, 3.3 cm/yr respectively). While height SD improved in 2 patients: -2.74 and -2.96 height SD to -2.52 and -2.64 height SD on rGH therapy, In the other two patients pretreatment continuous decline in height SD was arrested. Motor function decline was similar pre-growth hormone and during treatment. Cardiopulmonary function measured by ejection fraction and forced vital capacity was unchanged during the treatment period. Conclusions: This report of growth hormone therapy in DMD patients revealed an improved prepubertal growth velocity without detrimental effects observed on neuromuscular and cardiopulmonary function. Larger randomized control studies are required to prove safety and efficacy of this treatment in DMD patients.
Gut Microbiota Assessment in Obese Children and Adolescents by Machine Learning Algorithms
Authors: d’Annunzio Giuseppe , Biassoni Roberto , Marco Eddi Di , Valle Alberto La , Piccolo Gianluca , Bonaretti Carola , Napoli Flavia , Patti Giuseppa , Minuto Nicola , Maghnie Mohamad
Published: May 2021

Gut microbiota has been recently established to play a contributory role in the development and progression of obesity, a multifactorial disease predisposing to several comorbidities. Our aim was to evaluate the gut microbiota composition by machine learning algorithms in 33 Italian obese children and adolescents. Patients were divided in 3 groups: simple obesity (n=10, mean age 11.6 +3.0, median 10.8), metabolic syndrome (n=16, mean age 13.3+3.0, median 13.5) or Prader Willi syndrome (n=7, mean age 8.3+5.3, median 8.7). Inclusion criteria were living in Northern Italy, born singleton birth, personal history negative for acute or chronic gastrointestinal diseases and/or antibiotic or probiotics administration in the previous month. As controls 17 healthy control (mean age 12.0+2.4 median 10.6) were analyzed using the same approach. Statistical analysis for sparse high-throughput sequencing data algorithm (metagenomeSeq) using cumulative sum scaling for data normalization was performed. False discovery rate adjusted p-value using zero-inflated Gaussian fit statistical model (indicated with q). Over all analyses Parasutterella resulted with a q=0.014424, the comparison between obese patients and controls was q=0.021194. In the overall analysis Acidaminococcus intestini showed q=0.039528 while the comparison in pairs of two between metabolic syndrome and controls was q=0.03979. Using the EdgeR algorithm Clostridium bartlettii abundance between Prader Willi patients and controls resulted in q=0.02189. In overall analysis Ruminococcus flavefaciens resulted q=6.1528E-17 (using the DESeq2 algorithm); in pairs analysis Ruminococcus flavefaciens showed significant difference in Prader Willi patients as compared to obese (q=0.013755) and metabolic syndrome ones (q=0.021898). In overall analysis Veillonellaceae showed a FDR q=0.029303. while its richness resulted more than 150 times higher in metabolic syndrome patients than in controls (q=0.039793 evaluated with DESeq2 algorithm). Among Veillonellaceae descendants, the Veillonella rogosae showed, on the contrary, the lowest abundance in metabolic syndrome patients as compared to other groups. In detail, Veillonella rogosae abundances were 13 (FDR q=0.014566), around 20 times (FDR q=0.010646) and >20 (FDR q=0.0025008) less abundant in metabolic syndrome patients than obese, Prader Willi patients and controls, respectively. Significant differences in gut microbiota composition was found among patients with different degrees of obesity and controls. Further, Prader Willi patients showed a peculiar microbiota assessment.
Hormonal Replacement Therapy in Premenopausal Women With Hypogonadism: An Analysis of Prescriber Practices
Authors: Martirossian Alexandra Nicole , Williams Kelley , Silverstein Julie Martha
Published: May 2021

Introduction: Estrogen replacement therapy in premenopausal women with hypogonadism is important for reducing risk of osteoporosis, cardiovascular disease, and urogenital atrophy. Numerous formulations of estrogen are available and there is limited evidence to guide management. We conducted a preliminary retrospective study to determine prescribing practices of hormonal replacement therapy (HRT) in premenopausal women with hypogonadism and frequency of DEXA scan screening.

Methods: Using ICD 10 and billing codes, females ages 18-51 with a diagnosis of hypogonadism were identified. Patients with a diagnosis of prolactinoma, breast or endometrial cancer, and significant thrombotic disease as well as patients without clinical data were excluded. Information regarding etiology of hypogonadism, age of diagnosis, type/dose of estrogen and progesterone prescribed, prescriber specialty [obstetrics and gynecology (OB/GYN), endocrinology (ENDO), primary care (PCP), or other], and DEXA results was recorded. Prescriptions for estrogen and progesterone were compared in women with primary vs secondary hypogonadism and among specialties. For bone density, we analyzed the frequency of DEXA scan ordering. Statistical analysis was performed using Fisher’s Exact Test.

Results: Out of 1,306 patients identified, 150 met criteria for analysis. 99 (66%) had primary hypogonadism, 47 (31%) had secondary hypogonadism, and 4 (3%) had mixed or unknown type. OB/GYN was the most common prescriber (n=88, 59%) followed by ENDO (n=39, 26%) and PCP/other (n=23, 15%). For all patients, type of estrogen prescribed differed by specialty (p=0.041) with ENDO most commonly prescribing transdermal estradiol (47%), OB/GYN either transdermal estradiol (30%) or oral ethinyl estradiol (30%), and PCP/other oral estradiol (45%). Patients with primary, but not secondary hypogonadism, were prescribed more transdermal estradiol by ENDO as compared to OB/GYN (68% vs 27%, p=0.039). In patients with secondary hypogonadism on oral ethinyl estradiol, mean daily dose (mcg) differed among providers ((ENDO 27 ± 6, OB/GYN 35 ± 8, PCP/other 10 ± 0, p=0.04). There was no difference in dosing of other types of estrogen, prescribing practices for progesterone, or frequency of DEXA scans among providers. DEXA scans were performed at least once in 59 (39%) and more than once in 20 (13%) patients.

Conclusions: Overall, premenopausal women with hypogonadism seeing ENDO as compared to other providers were more frequently prescribed transdermal estradiol as compared to oral estradiol and/or other types of estrogen. Only dosages of oral ethinyl estradiol differed by provider. Despite an increased risk of osteoporosis, a minority of patients underwent DEXA scans. This study highlights heterogeneity in prescribing practices and underscores the need for further research to guide management of premenopausal women with hypogonadism.
Bone Morphogenetic Protein 2 Increases Human Trophoblast Invasion by Up-Regulating Integrin Beta3
Authors: Hu Cuiping , Yan Junhao
Published: May 2021

The adequate invasion of extravillous trophoblast cells (EVTs) is indispensable for the implantation of embryos and subsequent remodeling of uterine spiral arteries in early human gestation. Bone morphogenetic protein 2 (BMP2), which is abundantly expressed at the maternal-fetal interface, has been shown to promote the human EVT invasion process (1). Integrin switching (i.e., a switch from α6β4 to αvβ3) plays essential roles in cell-extracellular matrix adhesion and has been reported to influence EVT migration and invasion (2). Moreover, integrin β3 has been found to promote the adhesion, invasion, and migration abilities of embryonic trophoblasts (3). However, whether integrin β3 participates in BMP2 signaling and mediates BMP2-increased-human trophoblast invasion remains unknown. The purpose of our study was to explore the effects of BMP2 on integrin αvβ3 expression and the possible mediation role of integrin β3 in BMP2-regulated human trophoblast invasion. We used immortalized human trophoblast cell line (HTR8/SVneo) and primary human extravillous trophoblast cells (EVTs) isolated from first-trimester villi as study models. RT-qPCR and Western blot assay were respectively utilized to detect the messenger RNA and protein levels of intergrin αv and β3. The function of the target protein was studied by siRNA knockdown, and the trophoblast invasion ability was checked by Matrigel-coated transwell invasion assays. Our results demonstrated that the mRNA and protein levels of integrin β3, rather than integrin αv, were up-regulated after BMP2 treatment in HTR8/SVneo and primary EVT cells. Importantly, siRNA-mediated down-regulation of integrin β3 significantly inhibited basal and BMP2-induced HTR8/SVneo cell invasionas measured by transwell invasion assay. In conclusion, we findings support that BMP2 promotes human trophoblast cell invasion by up-regulating integrin β3 expression, benefiting the in-depth understanding of the pro-invasive effect of BMP2 on human trophoblasts during early pregnancy. Reference: (1) Hong-Jin Zhao et al., Cell Death Dis 2018;9:174. (2) Damsky, C.H. et al, Development 1994; 120, 3657-3666. (3) Dong-Mei He et al., Reproduction 2019;157:423-430.
Sex Differences in the Human Placenta MicroRNA Transcriptome
Authors: Flowers Amy E , Gonzalez Tania L , Eisman Laura E , Joshi Nikhil , Wu Di , Zhang Yizhou , Santiskulvong Chintda , Tang Jie , Buttle Rae , Sauro Erica , Clark Ekaterina L , Jefferies Caroline , Chan Jessica , Lin Yayu , Williams John , Pisarska Margareta Danuta
Published: May 2021

Maternal and fetal pregnancy outcomes vary based on fetal sex likely due to differences in placental function, reflected by sex differences in RNA expression. RNA transcripts are subject to fine-tuning control by post-transcriptional regulation including miRNAs binding to target RNAs and altering gene expression. Here we identify sexually dimorphic miRNA expression throughout gestation in the human placenta. Next-generation sequencing was used to identify human placenta miRNA expression profiles in first and third trimester uncomplicated pregnancies using discarded tissue obtained after chorionic villous sampling (n=113) and placenta (n=47). Differential expression analysis and mRNA target analysis were also examined. Sequencing identified 2,503 unique mature miRNAs expressed in each trimester. Of these, 13 significantly sexually dimorphic (FDR<0.05) miRNAs were identified in the first trimester and 4 significantly sexually dimorphic miRNAs were identified in the third trimester, including one miRNA, hsa-miR-361-5p, expressed across gestation. All of these sexually dimorphic miRNAs were significantly upregulated in females compared to males. Pathways analysis with predicted targets suggests sex differences in cancer and inflammation-related pathways in the first trimester and inflammation and growth-related pathways in the third trimester. Differential expression analysis on sex-segregated data identified 613 miRNAs upregulated in female placentas and 636 miRNAs upregulated in male placentas across gestation (FDR<0.05). In conclusion, fetal sex affects placental miRNA expression profiles and differentially expressed miRNAs may affect relevant downstream pathways, which may account for differences in pregnancy outcomes due to fetal sex. This work provides an expression atlas to direct functional studies investigating placental sex differences.
Single-Cell RNA-Seq of Mouse Decidual Leukocytes Reveals Intriguing Gestational Changes in the Immune Cell Landscape and Effects of SRC-1/-2 Double-Deficiency
Authors: Chen Jingfei , Duan Jialei , Montalbano Alina , Li Boxun , Hon Gary , Mendelson Carole R
Published: May 2021

Single-cell RNA-seq of Mouse Decidual Leukocytes Reveals Intriguing Gestational Changes in the Immune Cell Landscape and Effects of SRC-1/-2 Double-DeficiencyOur previous findings suggest that the fetus signals the mother when it is ready to be born through secretion of surfactant components/immune modulators, surfactant protein A (SP-A) and platelet-activating factor (PAF) by the fetal lung into amniotic fluid (AF). This occurs with increased proinflammatory cytokine expression by fetal AF macrophages (Mϕ), increased myometrial NF-κB activation and contractile (CAP) gene expression. Steroid receptor coactivator (Src)-1 and Src-2 are critical for developmental induction SP-A and PAF by the fetal lung. The finding that pregnant wild-type (WT) mice carrying Src-1 and Src-2 double-deficient fetuses (Src-1/-2d/d) manifested a marked delay (~38h) in parturition, further suggests that signals for parturition arise from the fetus and that Src-1 and Src-2 serve a critical role. Infiltrating leukocytes at the maternal-fetal interface (MFI)/decidua are known to play a central role in pregnancy maintenance and parturition timing. However, there is limited knowledge regarding gestational changes in immune cell composition toward term. To analyze gestational changes in the composition of immune cells within decidua and effects of Src-1/-2d/d, we conducted single-cell RNA-seq of ~17,000 decidual leukocytes (CD45+) from pregnant mice at 15.5 and 18.5 dpc carrying WT or Src-1/-2d/d fetuses. Unsupervised clustering identified 22 distinct decidual immune cell clusters, comprised of Mϕ, B cells, natural killer cells, neutrophils, dendritic cells and monocytes. Significant differences in cell type composition and transcriptional profiles were found across study groups (WT @ 15.5 dpc vs. 18.5 dpc; Src-1/-2d/dvs. WT @15.5 dpc and 18.5 dpc). Interestingly, in deciduas of pregnant mice carrying WT fetuses, Mϕ and B cell clusters markedly increased between 15.5 and 18.5 dpc, whereas, neutrophils declined; however, these gestational changes did not occur in pregnant mice carrying Src-1/-2d/d fetuses. Differential gene expression and gene ontology enrichment analyses revealed specific gene expression patterns distinguishing these immune cell subtypes to uncover their putative functions. These findings highlight the complexity and dynamics of the decidual immune cell landscape during the transition from myometrial quiescence to contractility, and the fetal-maternal interactions leading to parturition. Support: NIH grants R01-HL050022 (CRM) and P01-HD087150 (CRM), Burroughs Wellcome Preterm Birth Grant #1019823 (CRM).
DHT Differentially Regulates T Helper Cell Related Cytokines and MicroRNAs In Visceral and Subcutaneous Adipose Tissue of Female Mice
Authors: Kothmann Kadden H , Kay Matthew , Romney Sherdina E , Acebo Sarah , Reyna Andrea J , Choudhury Mahua , Rutkowski Joseph M , Newell-Fugate Annie E
Published: May 2021

Hyperandrogenemic, insulin resistant polycystic ovarian syndrome (PCOS) patients often have low-grade inflammation due to elevated circulating pro-inflammatory markers. As up to 60% of PCOS patients are obese, whether this low-grade inflammatory state is due to increased adiposity or other factors such as hyperandrogenemia is unknown. Moreover, the systemic inflammation of obesity is correlated with recruitment of pro-inflammatory immune cell populations to WAT. We hypothesized that short-term administration of the potent androgen, dihydrotestosterone (DHT), to female mice would increase pro-inflammatory cytokines and microRNA (miR) associated with pro-inflammatory cytokines and immune cell populations in WAT. Sexually mature, normally-cycling female C57/Bl6 mice received a daily sc injection of oil (0 g; n=7) or DHT (27.5 g; n=7) beginning at estrus. Females had vaginal cytology daily. After three cycles or 12-16 days if mice became acyclic, mice were euthanized for collection of blood and WAT. Serum was analyzed for DHT and testosterone (TEST) by LC-MS/MS. TaqManTM Array Mouse Immune Response PCR assays (Thermofisher Scientific) were used to measure transcript expression levels in vWAT and scWAT. Ingenuity Pathway Analysis (IPA) (Qiagen) was used to analyze relationships between different transcript levels in each treatment group for each tissue. DHT mice had 17 fold higher serum DHT levels than oil mice but there was no difference in serum TEST between treatment groups. DHT mice had a significantly longer estrous cycle length then oil mice. Short-term administration of DHT significantly upregulated 23% (21 of 92) of transcripts in scWAT and downregulated 49% (45 of 92) of transcripts in vWAT. The top four canonical pathways identified by IPA in WAT were: T helper cell 1 (Th1), Th1 & T helper 2 activation, Helper T cell differentiation, and Altered B & T cell signaling. Based on the Th1 pathway derived from IPA, the following miRs (both -3p and 5p) downstream of Th1 activation targets were selected for qPCR in vWAT and scWAT: miR21, miR146a, miR29a, and miR155. Interestingly, miR-21a-5p, miR-146a-5p, and miR-155-5p were significantly upregulated in scWAT from DHT mice. No miRs were different between treatment groups in vWAT. We demonstrate for the first time that short-term DHT administration may cause immunosuppression in vWAT and inflammation, possibly mediated by miRs, in scWAT of female mice.
Prolactin Receptor (PRLR) Isoform Expression in the Murine Oviduct
Authors: Radecki Kelly C , Lorenson Mary Y , Walker Ameae M
Published: May 2021

The most common and most deadly form of “ovarian”cancer is High-Grade Serous Carcinoma (HGSC), now appreciated to arise from epithelial cells of the fallopian tube/oviduct. The serum analyte with the greatest specificity and sensitivity that correlates with the presence or absence of HGSC is prolactin (PRL). However, unknown is whether elevated PRL initiates disease or is the result of disease, although once cancer is present,it is clear that PRL promotes disease. In order to examine the potential for disease initiation in normal animals, it was first important to determine which cells in the mouse oviduct respond to PRL. In many cell types, the long form of the Prlr mediates increased survival, proliferation and migration, whereas the short forms have the opposite effect. After establishing equivalent efficiencies of primers for the long form (LF) and 3 short forms (SF1-3) of the murine Prlr and the endogenous control gene, Gapdh, the mRNA expression profiles of these forms of the receptor were followed by RT-qPCR as a function of stage of the estrous cycle (proestrus, estrus, metestrus, diestrus - determined by vaginal cytology) and region of the oviduct (isthmus, ampulla and fimbria). Expression of SF1 was not detected. SF2 was essentially consistently expressed throughout the oviduct and at all stages of the estrous cycle. By contrast, the ratio of LF to SF3 varied by region of the tube, with more SF3 towards the fimbria and more LF towards the isthmus. The epithelium of the oviduct is primarily composed of multi-ciliated cells and secretory cells, with more ciliated cells towards the fimbria and more secretory cells towards the isthmus. The RT-qPCR results therefore suggested the possibility that a greater proportion of LF Prlr was present on secretory cells and a greater proportion of SF3 Prlr on ciliated cells. Using antibodies raised against intracellular peptide regions specificto the LF (aa 309-325) and SF3 (aa 281-296), both receptor isoforms were localized by immunofluorescence to apical regions of both epithelial cell types,but the presence of receptors on cilia (clearly demonstrated by 3Dreconstruction and rotation) complicated analysis of relative fluorescence by microscopy. Only the LF Prlr signals via Stat5 and so it was anticipated thatStat5 activation could serve as a substitute marker of the relative presence ofLF Prlr. Following in vivo intraperitoneal injection of PRL (5μg/g, 30 min),activated Stat5 was localized to epithelial cells at the base of, and in between, mucosal folds, thereby suggesting a further regionality to receptor distribution. For the fimbrial region only, which is where HGSC is thought to arise, expression of both the LF and SF3 Prlr changed as a function of the stage of the estrous cycle, with highest mRNA expression at diestrus/proestrus. Ongoing work includes flow cytometry of epithelial subpopulations and spatialanalysis of gene expression.
Heterozygous Eif4nif1 Stop Gain Mice Replicate the Primary Ovarian Insufficiency Phenotype
Authors: Moriwaki Mika , Welt Corrine Kolka
Published: May 2021

We identified a stop-gain mutation in eIF4ENIF1 in a family in which multiple women developed primary ovarian insufficiency (POI) at approximately age 30 years. We hypothesized that the same mutation in a mouse model would replicate POI. Methods: The Eif4enif1 C57/Bl6 transgenic mouse model contains a floxed exon 10-19 cassette and a conditional knock-in cassette containing exon 10 with the c.1286C>G stop-gain mutation causing familial POI and WT exons 11-19 (Eif4enif1WT/flx). The hybrid offspring of CMV-Cre mice with Eif4enif1WT/flx mice were designated Eif4enif1WT/Δ for simplicity. Follicles were counted in fixed H&E stained ovaries from mice age days 1-5 (primordial and primary follicles), day 10, day 22 (first wave of growing follicles from small preantral to small antral follicles), week 20 (peak fertility), then every 2 months from 10 months to 26 months (follicle exhaustion). Litter frequency, pup number and genotype were recorded. Serum FSH levels were measured by the University of Virginia Ligand Assay and Analysis Core. Results: The heterozygotes have no outward or internal phenotypic differences compared to WT (Eif4enif1WT/flx), with the exception of reproductive organs in females and males. A subset of female heterozygotes (Eif4enif1WT/Δ) had no litters for 20 weeks (2 of 18; 11%). In those with litters, the average length of time between litters was not different but the final litter was earlier (5.6±2.7 vs. 10.5±0.7 months; p=0.02). Heterozygous breeding pair (Eif4enif1WT/Δx Eif4enif1WT/Δ) litter size was 60% of WT litter size (3.9±2.3 vs. 7.2±2.1 pups/litter; 0<0.001). The genotypes were 35% Eif4enif1WT/flx and 65% Eif4enif1WT/Δ, with no homozygotes. The number of follicles in ovaries from Eif4enif1WT/Δ mice was lower starting at the primordial (499±290 vs. 1445±381) and primary follicle stage (1069±346 vs. 1450±193) on day 10 (p<0.05). The preantral follicle number was lower starting on day 21 (213±86 vs. 522±227; p<0.01) and the antral follicle count was lower starting on week 20 (78±38 vs. 119±18; p<0.01). The FSH level in 12-month old mice during estrus was higher in a heterozygote compared to WT (25.0 vs. 12.1 ng/mL). Conclusions: Heterozygous Eif4enif1 stop-gain mutants have follicle loss documented by day 10, decreased pup number with no homozygotes, earlier end of reproductive function and elevated FSH levels. These mice replicate the POI phenotype in women. eIF4ENIF1 regulates protein translation by binding and storing eIF4E bound mRNA. Therefore, the unique mouse model provides a platform to study temporal and spatial regulation of protein translation across oocyte and embryo development in mammals. Further studies will determine whether follicle loss results from premature protein translation in oocytes.
A Case of Hyperreactio Luteinalis Complicated With Biochemical Hyperandrogenism, Symptomatic Hyperthyroidism and Preeclampsia
Authors: Wong K Y , Mak M W H , Lee K M , Lee K F
Published: May 2021

Background: Hyperreactio luteinalis (HL) describes the development of multiple large ovarian cysts during pregnancy, which regress post-partum. We report a case of HL complicated with preeclampsia, biochemical hyperandrogenism and hyperthyroidism. Clinical Case: A 31-year-old non-obese Chinese woman presented at 14-week gestation for lower abdominal pain. USG showed a single fetus, multiple ovarian cysts with largest measured 39.5ml. She complained of hand tremor, palpitation but no vomiting. She had no goiter, orbitopathy or family history of thyroid disease. fT4 was 23.1pmol/L (normal: 9.8-19.8pmol/L) and TSH was <0.01mIU/L. Anti-TG, anti-TPO and anti-TSHR antibodies were negative. She had history of silent miscarriage at 6-week gestation in her first pregnancy 2 years ago, USG showed normal ovaries at that time. Carbimazole was started at 16-week gestation for fT4 26.6pmol/L (normal: 9.4-18.5pmol/L). The largest ovarian cyst increased to 130ml at 19-week gestation. Serum β-hCG was 251926IU/L (normal: 4060-165400IU/L). HL with hCG-mediated hyperthyroidism was suspected. Serum total testosterone was 22.9nmol/L (normal: 2.2-10.7nmol/L) and serum androstenedione was 70.5nmol/L (normal: 0.28-9.81nmol/L). Ferriham Gallwey score was 4. fT4 fell to 13.8pmol/L (normal: 8.8-17.0pmol/L) but TSH remained suppressed. Carbimazole was stopped at 22-week gestation with no rebound in fT4 level. She developed preeclampsia and GDM at 27-week gestation. IUGR was evident despite decreasing β-hCG level and ovarian cyst shrinkage. She had emergency LSCS for severe preeclampsia at 33-week gestation. A 1510g female baby with normal genitalia was delivered. Placenta pathology was normal. 2 days after delivery, β-hCG fell to 7081IU/L; fT4 was 9.9pmol/L (normal: 9-19pmol/L) and TSH was 0.25mIU/L (normal: 0.35-4.5mIU/L). Clinical Lessons: 1) hCG stimulates growth of ovarian stroma and androgen secretion, results in virilization in 30% of HL patients. However, only 5% of patients had hyperthyroidism. LH and hCG are structurally similar and bind to the same receptor. In contrast, hCG is a weak agonist of TSH receptor: a hCG level of more than 100000IU/L is required to cause clinical thyrotoxicosis. Since 30% of HL patients have normal hCG level, this may explain the lower incidence of hyperthyroidism than hyperandrogenism. 2) Degree of maternal virilization does not correlate with testosterone level. Study by Condic et al. found significant overlap of testosterone levels in women with (13.7-197.5nmol/l) and without (6.2-37.3nmol/l) virilization. Genetic polymorphism of androgen receptor may account for the different clinical manifestation. Fetal virilization is rare, due to protective role of placental aromatase. 3) Elevated hCG in apparently “normal” singleton pregnancy may be due to poor placentation in early gestation and is a risk factor for preeclampsia and IUGR in HL patients.
An Unusual Cause of Primary Amenorrhea
Authors: Marenych Nadiia , Patel Sabah , Gilden Janice L
Published: May 2021

Background: Müllerian agenesis is the most common cause of primary amenorrhea in patients with typical thelarche and adrenarche. Most of the time, this diagnosis is made at the age of 10-13 years. Clinical Case: We present a case of a 18 year-old Vietnamese female who was referred to the endocrine clinic for primary amenorrhea. She stated that pubic hair developed at age 15 years. Patient is sexually active and uses condoms. She has 10 siblings (7 sisters, 3 brothers), 6 older sisters with menarche at ages 12-16 and a younger sister of 10 years old who has not yet had menarche. In addition, her older sister has 3 biological children and there was no reported infertility in her family. She stated that a pelvic ultrasound had been done at age of 17 years that showed no uterus. Oral contraceptives had been previously trialed and failed to induce withdrawal bleeding. HT 157.48 cm, WT 55.34 kg. The patient had a normal female phenotype with normal bilateral breast development, and no hirsutism. She was not concerned with this issue, which questioned her personality and coping skills. Laboratory testing-cortisol 13.0 ug/dL (5.3- 2.5), ACTH 29 pg/mL (0-47), estradiol 46.04 pg/mL), total testosterone 39 ng/dL (2-45), free testosterone 4.2 pg/mL (0.1-6.4), TSH 1.80 uIU/mL (0.358- .74), free T4 0.99 ng/dL(0.76- .46), FSH 4.4 mIU/mL (Follicular Phase:2.3 - 12.6, Midcycle Peak:5.2-17.5 mIU/mL, Luteal Phase:1.7-12.9 mIU/mL), Progesterone <0.5 ng/mL (follicular -less than 0.8 ng/ml, luteal=4.1- 3.7 ng/ml, mid-luteal= 4.5-25.2 ng/ml), LH 3.7 mIU/mL (follicular phase = 1.9-26.2 mIU/mL, Midcycle = 22.8 - 6.1 mIU/mL, Luteal phase = 0.6-16.6 mIU/mL). Transvaginal ultrasound-uterus not visualized; ovaries were unremarkable. DXA scan-normal Z scores. She refused to have a karyotype analysis. Differential diagnoses included müllerian agenesis, 5-alpha-reductase deficiency and complete androgen insensitivity syndrome. Unfortunately, our patient declined karyotype testing. Based on clinical presentation, which showed normal female genitalia, absence of uterus and normal laboratory finding, the most likely diagnosis was müllerian agenesis (Mayesr-Rokitansky-Kuster-Hauser syndrome). This syndrome has an incidence of 1/4,500-5,000 females and is caused is caused by embryologic underdevelopment of the müllerian duct, with resultant agenesis or atresia of the vagina, uterus, or both. Patients with müllerian agenesis usually are identified when they are evaluated for primary amenorrhea with otherwise typical growth and pubertal development, as in our patient. Psychosocial and genetic counseling, as well as offering options for pregnancy are important. In addition, certain personality traits, such as higher neuroticism, depression, and decreased coping styles may be observed. References: Obstetrics and Gynecology, Müllerian agenesis: Diagnosis, management, and treatment. Vol.131, NO.1, January 2018