RNA-seq analysis identifies age-dependent changes in expression of mRNAs - encoding N-glycosylation pathway enzymes in mouse gonadotropes.

Follicle-stimulating hormone (FSH) is a glycoprotein that is assembled as a heterodimer of α/ß subunits in gonadotropes. Each subunit contains two N-glycan chains. Our previous in vivo genetic studies identified that at least one N-glycan chain must be present on the FSHß subunit for efficient FSH dimer assembly and secretion. Moreover, macroheterogeneity observed uniquely on human FSHß results in ratiometric changes in age-specific FSH glycoforms, particularly during menopausal transition. Despite the recognition of many prominent roles of sugars on FSH including dimer assembly and secretion, serum half-life, receptor binding and signal transduction, the N-glycosylation machinery in gonadotropes has never been defined. Here, we used a mouse model in which gonadotropes are GFP-labeled in vivo and achieved rapid purification of GFP+ gonadotropes from pituitaries of female mice at reproductively young, middle, and old ages. We identified by RNA-seq analysis 52 mRNAs encoding N-glycosylation pathway enzymes expressed in 3- and 8-10-month-old mouse gonadotropes. We hierarchically mapped and localized the enzymes to distinct subcellular organelles within the N-glycosylation biosynthetic pathway. Of the 52 mRNAs, we found 27 mRNAs are differentially expressed between the 3- and 8-10-month old mice. We subsequently selected 8 mRNAs which showed varying changes in expression for confirmation of abundance in vivo via qPCR analysis, using more expanded aging time points with distinct 8-month and 14-month age groups. Real time qPCR analysis indicated dynamic changes in expression of N-glycosylation pathway enzyme-encoding mRNAs across the life span. Notably, computational analysis predicted the promoters of genes encoding these 8 mRNAs contain multiple high probability binding sites for estrogen receptor-1 and progesterone receptor. Collectively, our studies define the N-glycome and identify age-specific dynamic changes in mRNAs encoding N-glycosylation pathway enzymes in mouse gonadotropes. Our studies suggest the age-related decline in ovarian steroids may regulate expression of N-glycosylation enzymes in mouse gonadotropes and explain the age-related N-glycosylation shift previously observed on human FSHß subunit in pituitaries of women.

Machine learning to improve false-positive results in the Dutch newborn screening for congenital hypothyroidism.

OBJECTIVE: The Dutch Congenital hypothyroidism (CH) Newborn Screening (NBS) algorithm for thyroidal and central congenital hypothyroidism (CH-T and CH-C, respectively) is primarily based on determination of thyroxine (T4) concentrations in dried blood spots, followed by thyroid-stimulating hormone (TSH) and thyroxine-binding globulin (TBG) measurements enabling detection of both CH-T and CH-C, with a positive predictive value (PPV) of 21%. A calculated T4/TBG ratio serves as an indirect measure for free T4. The aim of this study is to investigate whether machine learning techniques can help to improve the PPV of the algorithm without missing the positive cases that should have been detected with the current algorithm. DESIGN & METHODS: NBS data and parameters of CH patients and false-positive referrals in the period 2007-2017 and of a healthy reference population were included in the study. A random forest model was trained and tested using a stratified split and improved using synthetic minority oversampling technique (SMOTE). NBS data of 4668 newborns were included, containing 458 CH-T and 82 CH-C patients, 2332 false-positive referrals and 1670 healthy newborns. RESULTS: Variables determining identification of CH were (in order of importance) TSH, T4/TBG ratio, gestational age, TBG, T4 and age at NBS sampling. In a Receiver-Operating Characteristic (ROC) analysis on the test set, current sensitivity could be maintained, while increasing the PPV to 26%. CONCLUSIONS: Machine learning techniques have the potential to improve the PPV of the Dutch CH NBS. However, improved detection of currently missed cases is only possible with new, better predictors of especially CH-C and a better registration and inclusion of these cases in future models.

Ancestral glycoprotein hormone and its cognate receptor present in primitive chordate ascidian: Molecular identification and functional characterization.

The glycoprotein hormone (GPH) system is fundamentally significant in regulating the physiology of chordates, such as thyroid activity and gonadal function. However, the knowledge of the GPH system in the primitive chordate ascidian species is largely lacking. Here, we reported an ancestral GPH system in the ascidian (Styela clava), which consists of GPH α subunit (Sc-GPA2), GPH ß subunit (Sc-GPB5), and the cognate leucine-rich repeat-containing G protein-coupled receptor (Sc-GPHR). Comparative structure analysis revealed that distinct from vertebrate GPH ß subunits, Sc-GPB5 was less conserved, showing an atypical N-terminal sequence with a type II transmembrane domain instead of a typical signal peptide. By investigating the presence of recombinant Sc-GPA2 and Sc-GPB5 in cell lysates and culture media of HEK293T cells, we confirmed that these two subunits could be secreted out of the cells via distinct secretory pathways. The deglycosylation experiments demonstrated that N-linked glycosylation only occurred on the conserved cysteine residue (N78) of Sc-GPA2, whereas Sc-GPB5 was non-glycosylated. Although Sc-GPB5 exhibited distinct topology and biochemical properties in contrast to its chordate counterparts, it could still interact with Sc-GPA2 to form a heterodimer. The Sc-GPHR was then confirmed to be activated by tethered Sc-GPA2/GPB5 heterodimer on the Gs-cAMP pathway, suggesting that Sc-GPA2/GPB5 heterodimer-initiated Gs-cAMP signaling pathway is evolutionarily conserved in chordates. Furthermore, in situ hybridization and RT-PCR results revealed the co-expression patterns of Sc-GPA2 and Sc-GPB5 with Sc-GPHR transcripts, respectively in ascidian larvae and adults, highlighting the potential functions of Sc-GPA2/GPB5 heterodimer as an autocrine/paracrine neurohormone in regulating metamorphosis of larvae and physiological functions of adults. Our study systematically investigated the GPA2/GPB5-GPHR system in ascidian for the first time, which offers insights into understanding the function and evolution of the GPH system within the chordate lineage.

Cloning of gonadotropin Gph-alpha, FSH-beta and LH-beta subunits and seasonal profiles of steroid hormones in wild-caught Nile perch, Lates niloticus.

The Nile perch (np; Lates niloticus) is a freshwater teleost species with a potential for aquaculture in freshwater surroundings. However, wild-caught breeders have persistently failed to spawn spontaneously in captivity. Cloning of the gonadotropin subunits and analysing seasonal variation in reproductive hormone levels for a 1-year period were done to gain knowledge on the physiological basis underlying the reproductive biology of np. The ß-follicle-stimulating hormone (FSH-ß) and ß-luteinizing hormone (LH-ß) subunits and their common α-glycoprotein (Gph-α) subunit were cloned using 3' and 5' RACE-PCR. The nucleotide sequences of the npgph-α, npfsh-ß, and nplh-ß subunits were 664, 580 and 675 nucleotides in length, encoding peptides of 124, 120 and 148 amino acids, respectively. The deduced amino acid sequence of each mature subunit showed high similarity with its counterparts in other teleost. Sequence analysis showed that npFSH-ß is more similar to higher vertebrate FSH-ßs than to higher vertebrate LH-ßs. Heterologous immunoassay was calibrated to analyse pituitary LH levels. While the LH immunoassay showed parallelism of npLH with that of tilapia (ta), no parallelism for FSH was found. Levels of pituitary LH were higher in females at gonadal stages of vitellogenic oocytes, mature secondary oocytes and mature tertiary oocytes with migrating nucleus than in pre-vitellogenic oocytes and early and late perinucleolus oocytes. Using competitive steroid ELISA, variations in the levels of the steroid hormones 11-ketotestosterone (11-KT) in males and E2 in females were characterized in relation to month and reproductive index of Nile perch. Our findings show that in females, gonadosomatic index and plasma E2 were highly correlated (R2 = 0.699, n = 172) and peaked from September to November while in males, the gonadosomatic index and plasma 11-KT peaked from October to November. In female fish, both steroid hormones were detected in the plasma but greatly varied in concentrations. E2 in particular, increased with the developmental stage of the gonads. The levels of steroid hormones, E2 and 11-KT in females and males respectively increased with fish size (total lengths) and suggest that females mature at a body length of 40-59 cm than their counter part males that mature at a total length of 60-70 cm. Taken together, we describe seasonal endocrine differences in wild-caught adult Nile perch which could potentially be exploited to manipulate the reproductive axis in cultured breeders.

Unconventional Actions of Glycoprotein Hormone Subunits: A Comprehensive Review.

The glycoprotein hormones (GPH) are heterodimers composed of a common α subunit and a specific ß subunit. They act by activating specific leucine-rich repeat G protein-coupled receptors. However, individual subunits have been shown to elicit responses in cells devoid of the receptor for the dimeric hormones. The α subunit is involved in prolactin production from different tissues. The human chorionic gonadotropin ß subunit (ßhCG) plays determinant roles in placentation and in cancer development and metastasis. A truncated form of the thyrotropin (TSH) ß subunit is also reported to have biological effects. The GPH α- and ß subunits are derived from precursor genes (gpa and gpb, respectively), which are expressed in most invertebrate species and are still represented in vertebrates as GPH subunit paralogs (gpa2 and gpb5, respectively). No specific receptor has been found for the vertebrate GPA2 and GPB5 even if their heterodimeric form is able to activate the TSH receptor in mammals. Interestingly, GPA and GPB are phylogenetically and structurally related to cysteine-knot growth factors (CKGF) and particularly to a group of antagonists that act independently on any receptor. This review article summarizes the observed actions of individual GPH subunits and presents the current hypotheses of how these actions might be induced. New approaches are also proposed in light of the evolutionary relatedness with antagonists of the CKGF family of proteins.

Comparative effectiveness of recombinant human follicle-stimulating hormone alfa (r-hFSH-alfa) versus highly purified urinary human menopausal gonadotropin (hMG HP) in assisted reproductive technology (ART) treatments: a non-interventional study in Germany.

BACKGROUND: This study compared the effectiveness of recombinant human follicle-stimulating hormone alfa (r-hFSH-alfa; GONAL-f®) with urinary highly purified human menopausal gonadotropin (hMG HP; Menogon HP®), during assisted reproductive technology (ART) treatments in Germany. METHODS: Data were collected from 71 German fertility centres between 01 January 2007 and 31 December 2012, for women undergoing a first stimulation cycle of ART treatment with r-hFSH-alfa or hMG HP. Primary outcomes were live birth, ongoing pregnancy and clinical pregnancy, based on cumulative data (fresh and frozen-thawed embryo transfers), analysed per patient (pP), per complete cycle (pCC) and per first complete cycle (pFC). Secondary outcomes were pregnancy loss (analysed per clinical pregnancy), cancelled cycles (analysed pCC), total drug usage per oocyte retrieved and time-to-live birth (TTLB; per calendar week and per cycle). RESULTS: Twenty-eight thousand six hundred forty-one women initiated a first treatment cycle (r-hFSH-alfa: 17,725 [61.9%]; hMG HP: 10,916 [38.1%]). After adjustment for confounding variables, treatment with r-hFSH-alfa versus hMG HP was associated with a significantly higher probability of live birth (hazard ratio [HR]-pP [95% confidence interval (CI)]: 1.10 [1.04, 1.16]; HR-pCC [95% CI]: 1.13 [1.08, 1.19]; relative risk [RR]-pFC [95% CI]: 1.09 [1.05, 1.15], ongoing pregnancy (HR-pP [95% CI]: 1.10 [1.04, 1.16]; HR-pCC [95% CI]: 1.13 [1.08, 1.19]; RR-pFC [95% CI]: 1.10 [1.05, 1.15]) and clinical pregnancy (HR-pP [95% CI]: 1.10 [1.05, 1.14]; HR-pCC [95% CI]: 1.14 [1.10, 1.19]; RR-pFC [95% CI]: 1.10 [1.06, 1.14]). Women treated with r-hFSH-alfa versus hMG HP had no statistically significant difference in pregnancy loss (HR [95% CI]: 1.07 [0.98, 1.17], were less likely to have a cycle cancellation (HR [95% CI]: 0.91 [0.84, 0.99]) and had no statistically significant difference in TTLB when measured in weeks (HR [95% CI]: 1.02 [0.97, 1.07]; p = 0.548); however, r-hFSH-alfa was associated with a significantly shorter TTLB when measured in cycles versus hMG HP (HR [95% CI]: 1.07 [1.02, 1.13]; p = 0.003). There was an average of 47% less drug used per oocyte retrieved with r-hFSH-alfa versus hMG HP. CONCLUSIONS: This large (> 28,000 women), real-world study demonstrated significantly higher rates of cumulative live birth, cumulative ongoing pregnancy and cumulative clinical pregnancy with r-hFSH-alfa versus hMG HP.

Development and characterization of in vitro self-assembled recombinant human follicle stimulating hormone originated from goat mammary epithelial cells.

Follicle stimulating hormone (FSH), composed of FSHα and FSHß subunits, is essential for female follicle development and male spermatogenesis. The recombinant human FSH (rhFSH) products on the market are mainly generated from mammalian cells and are expensive. Large animal mammary gland bioreactors are urgently needed to produce large amounts of rhFSH. However, there are currently no effective methods to prepare rhFSH by large animals mainly due to the fact that excessive accumulation of FSH might cause many adverse effects in animals. We herein report the development and characterization of functional self-assembled rhFSH produced in goat mammary epithelial cells (GMECs). FSHα and FSHß stably expressed in Chinese hamster ovary (CHO) cell lines were secreted into culture medium and well glycosylated. Importantly, FSHα and FSHß expressed apart were able to assemble into functional FSH. We next inserted human FSHα or FSHß gene separately into goat ß-Lactoglobulin locus in GMECs by CRISPR/Cas9. Inactive FSHα and FSHß subunits expressed from GMECs assembled into rhFSH as analyzed by His-tag pull down assay. Functional assessment of rhFSH by cAMP induction assay, mouse ovulation induction and rat ovarian weight gain experiments showed that the bioactivity of self-assembled rhFSH expressed by GMECs was comparable to that of Gonal-F both in vitro and in vivo. Our study demonstrated that FSHα and FSHß can be separately expressed and assembled into functional rhFSH, and provided the basis for future preparing FSH by goat mammary gland bioreactor with less health problems on the producing animals.

Identification and semi-relative quantification of intact glycoforms of human chorionic gonadotropin alpha and beta subunits by nano liquid chromatography-Orbitrap mass spectrometry.

The human chorionic gonadotropin (hCG) protein belongs to a family of glycoprotein hormones called gonadotropins. It is a heterodimer made of two non-covalently linked subunits. The α-subunit structure, hCGα, has 2 N-glycosylation sites, while the beta subunit, hCGß, has 2 N- and 4 O-glycosylation sites. This leads to numerous glycoforms. A method based on the analysis of hCG glycoforms at the intact level by nano-reversed phase liquid chromatography coupled to high resolution mass spectrometry (nanoLC-HRMS) with an Orbitrap analyzer was previously developed using a recombinant hCG-based drug, Ovitrelle®, as standard. It allowed the detection of about 30 hCGα glycoforms, but didn't allow the detection of hCGß glycoforms. This method was thus here significantly modified (addition of a pre-concentration step of the sample to increase the sample volume from 70 nl to 1 µl, optimization of the gradient slope and the nature and content of the acidic additive in the mobile phase). It led to an improvement of the separation of hCGα and hCGß glycoforms, which allowed for the first time the detection of 33 hCGß glycoforms at intact level. In addition, a higher number of hCGα glycoforms (42 in total, i.e. a 40% increase) was detected. The figures of merit of this new method were next assessed. The relative standard deviations (RSDs) of the retention time ranged between 0.02 and 0.95% (n = 3), with an average value of 0.36% for the alpha glycoforms and between 0.01 and 1.08% (n = 3) with an average value of 0.23% for the beta glycoforms. The RSDs of the relative peak area measured on the extracted ion chromatogram of each glycoform were below 20% (n = 3), with an average value of 9.8%, thus allowing semi-relative quantification. Therefore, this method has a high potential for rapid quality control aiming for the detection and comparison of glycoforms present in glycoprotein-based pharmaceutical preparations.

Insulinoma-associated Protein 1 Expression and Its Diagnostic Significance in Female Genital Tract Neuroendocrine Carcinomas.

Neuroendocrine carcinomas (NECs) are rare, but aggressive malignant tumors of the female genital tract, especially in the uterine the cervix. Beside histologic morphology, positivity of neuroendocrine markers with immunohistochemistry plays an important role in diagnosis of NECs. Insulinoma-associated protein 1 (INSM1) is a novel marker reported to be widely expressed in a variety of neuroendocrine tumors. A previous study also suggested INSM1 has superior performance to conventional neuroendocrine markers in cervical NECs. In our present study, comparison between immunomarkers was performed in female genital tract NECs. Forty-nine patients with gynecologic NECs (4 vagina, 39 cervix, 5 endometrium, 1 ovary) were included from 1993 to 2019 at our center. Immunohistochemistry was performed with INSM1, CD56, synaptophysin (SYN), chromogranin-A (CgA), and thyroid transcription factor 1 (TTF1). The results show INSM1 has superior sensitivity and intensity compared with CD56, SYN, CgA, and TTF1 in cervical small cell NECs, but not in large cell NECs. In contrast to cervical NECs, INSM1 immunohistochemistry shows only focal and weak staining in endometrial NECs. Our result suggested INSM1 is a sensitive marker which can be used as first-line test in histologic suspicious cervical cases, especially small cell NECs. However, negative INSM1 stain does not exclude the possibility of NECs. In endometrial NECs, conventional panel with CD56, SYN, CgA has better diagnostic performance than INSM1 alone.

Analysis of the human chorionic gonadotropin protein at the intact level by HILIC-MS and comparison with RPLC-MS.

In the present work, the human chorionic gonadotropin (hCG) hormone was characterized for the first time by hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution (HR) quadrupole/time-of-flight (qTOF) mass spectrometry (MS) at the intact level. This heterodimeric protein, consisting of two subunits (hCGα and hCGß), possesses 8 potential glycosylation sites leading to a high number of glycoforms and has a molecular weight of about 35 kDa. The HILIC conditions optimized in a first paper but using UV detection were applied here with MS for the analysis of two hCG-based drugs, a recombinant hCG and a hCG isolated from the urine of pregnant women. An amide column (150 × 2.1 mm, 2.6 µm, 150 Å), a mobile phase composed of acetonitrile and water both containing 0.1% of trifluoroacetic acid, and a temperature of 60 °C were used. The gradient was from 85 to 40% ACN in 30 min. The use of TFA that had been shown to be necessary for the separation of glycoforms caused, as expected, an ion suppression effect in MS that was partially overcome by increasing the amount of protein injected (2 µL at 1 mg mL-1) and reducing the detection m/z range (from 1500 to 300). These conditions allowed the detection of different glycoforms of hCGα. The performance of the HILIC-HRMS method was compared with that previously obtained in RPLC-HRMS in terms of the number of detected glycoforms, selectivity, and sensitivity. The complementarity and orthogonality of the HILIC and RP modes for the analysis of hCG at the intact level were demonstrated.

Pituitary Glycoprotein Hormones in Human Milk before and after Pasteurization or Refrigeration.

Our aims were to investigate the presence of pituitary glycoprotein hormones in preterm and donor milk, and to examine the effects of Holder pasteurization and refrigeration on the levels of these hormones. We measured follicle-stimulating hormone (FSH), luteinizing hormone (LH), and thyroid-stimulating hormone (TSH) in milk samples from mothers who delivered prematurely (n = 27) and in samples of mothers who delivered at term and donated milk to the Mother's Milk Bank of Iowa (n = 30). The gonadotropins and TSH were present in similar amounts within human milk produced for preterm and term infants. FSH increased 21% after refrigeration (p < 0.05), while LH declined by 39% (p < 0.05). Holder pasteurization decreased LH by 24% (p < 0.05) and increased TSH by 17% (p < 0.05). Holder pasteurization followed by refrigeration resulted in a 21% increase in FSH and a 41% decrease in LH (both p < 0.05), resulting in more than a 3-fold increase in donor milk FSH:LH ratios (p < 0.05 versus fresh donor milk). Despite structural similarities, the gonadotropins are differentially impacted by Holder pasteurization and refrigeration, and this results in marked alterations in the relative amount of FSH and LH that may be administered to preterm infants, potentially swinging hormonal balance towards ovarian hyperstimulation in females and hypogonadism in males.

CD57 as a routine neuroendocrine marker for liver metastasis.

BACKGROUND: The characterization of hepatic metastases as having neuroendocrine origins is essential and the main markers currently used are chromogranin A (CgA) and synaptophysin (Syn). However, these markers may exhibit certain limitations, and the use of CD56 and CD57 can also be considered, although, due to low specificity, their use is discouraged. AIM: This study sought to compare the immunohistochemical expression of these markers in hepatic metastases of neuroendocrine neoplasms (NEN). MATERIALS AND METHODS: Eighteen samples, were used for immunohistochemical staining with CgA, Syn, CD56, and CD57 antibodies. The immunostaining reactions were compared according to its intensity (I), the percentage of labeled cells (P), and a final score (I × P). Statistical agreement between the markers was also evaluated. RESULTS: CD57 was expressed in the highest number of cases and also showed the most intense expression. CgA showed the highest number of cases with more than 80% positively stained area (72.2%), followed by CD57 (61.1%). The highest average score (I × P) was obtained for CD57 (9.1 ± 4.1). The best indices of agreement were between CgA and CD57 with respect to positivity (P = 0.021) and score (P = 0.014). According to the primary site, stomach/duodenum, lungs, and undetermined subgroups showed the highest average scores for CD57, followed by CgA. For the small bowel subgroup, the highest average score was obtained for CgA, followed by CD57. CONCLUSION: Our results highlight the importance of CD57 in the evaluation of hepatic metastases of NEN and indicate that this marker should be included with CgA and Syn in routine diagnostic panels.

Liganded T3 receptor ß2 inhibits the positive feedback autoregulation of the gene for GATA2, a transcription factor critical for thyrotropin production.

The serum concentration of thyrotropin (thyroid stimulating hormone, TSH) is drastically reduced by small increase in the levels of thyroid hormones (T3 and its prohormone, T4); however, the mechanism underlying this relationship is unknown. TSH consists of the chorionic gonadotropin α (CGA) and the ß chain (TSHß). The expression of both peptides is induced by the transcription factor GATA2, a determinant of the thyrotroph and gonadotroph differentiation in the pituitary. We previously reported that the liganded T3 receptor (TR) inhibits transactivation activity of GATA2 via a tethering mechanism and proposed that this mechanism, but not binding of TR with a negative T3-responsive element, is the basis for the T3-dependent inhibition of the TSHß and CGA genes. Multiple GATA-responsive elements (GATA-REs) also exist within the GATA2 gene itself and mediate the positive feedback autoregulation of this gene. To elucidate the effect of T3 on this non-linear regulation, we fused the GATA-REs at -3.9 kb or +9.5 kb of the GATA2 gene with the chloramphenicol acetyltransferase reporter gene harbored in its 1S-promoter. These constructs were co-transfected with the expression plasmids for GATA2 and the pituitary specific TR, TRß2, into kidney-derived CV1 cells. We found that liganded TRß2 represses the GATA2-induced transactivation of these reporter genes. Multi-dimensional input function theory revealed that liganded TRß2 functions as a classical transcriptional repressor. Then, we investigated the effect of T3 on the endogenous expression of GATA2 protein and mRNA in the gonadotroph-derived LßT2 cells. In this cell line, T3 reduced GATA2 protein independently of the ubiquitin proteasome system. GATA2 mRNA was drastically suppressed by T3, the concentration of which corresponds to moderate hypothyroidism and euthyroidism. These results suggest that liganded TRß2 inhibits the positive feedback autoregulation of the GATA2 gene; moreover this mechanism plays an important role in the potent reduction of TSH production by T3.

Eritropoyetina (EPO) / Erythropoietin (EPO)

La eritropoyetina (EPO) es una hormona glucoproteica producida principalmente en las células peritubulares de los riñones, que regula la producción y diferenciación eritrocitaria. La síntesis de eritropoyetina está regulada por cambios en la disponibilidad del oxígeno, es decir, a mayor cantidad de oxígeno, menor producción de EPO, y a la inversa. La EPO se une a los receptores en las células madre hematopoyéticas CD34+, promoviendo la activación de los genes para estimular su proliferación y diferenciación hasta eritrocitos maduros [1]. La medición de eritropoyetina en suero es de gran utilidad para el diagnóstico de eritrocitosis y el establecimiento de su etiología. Su determinación en suero permite identificar si la producción eritrocitaria es autónoma (primaria), como en el caso de la producida en la policitemia vera, o secundaria, mediada por la eritropoyetina. En pacientes con eritrocitosis secundaria, los niveles de eritropoyetina están aumentados como un mecanismo compensatorio cuando es por hipoxia [2], o pueden ser el resultado de una patología que estimula su producción, como ocurre con ciertos tumores, entre otros factores. De acuerdo con la clasificación vigente de neoplasias hematolinfoides de la Organización Mundial de la Salud, los niveles disminuidos de eritropoyetina son un criterio menor para el diagnóstico de policitemia vera [2], y los niveles normales o elevados de eritropoyetina descartan que se trate de esta entidad e indican al médico tratante que debe explorar otras causas de eritrocitosis [2].

Proteomic identification of predictive biomarkers for malignant transformation in complete hydatidiform moles.

INTRODUCTION: Protein expression in cells are associated with oncogenesis. This study aims to explore proteomic profiles and discover potential biomarkers that can predict malignant transformation of hydatidiform mole. METHODS: Retrospective analysis was done in 14 cases of remission hydatidiform mole and 14 cases of hydatidiform mole who later developed malignancy (GTN group). Molar tissues were retrieved from -70 °C frozen tissue. Subsequently, a large-scale proteomic analysis was performed to identify proteins and compare their abundance levels in the preserved molar tissues from these two groups using a dimethyl-labeling technique coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: A total of 2,153 proteins were identified from all samples. 22 and 10 proteins were significantly up-regulated and down-regulated, respectively, in the GTN group compared with the mole group. These altered proteins were found in several biological groups such as cell-cell adhesion, secreted proteins, and ribonucleoproteins. Several hormone-related proteins were among the most up-regulated proteins in the GTN group including choriogonadotropin subunit beta (ß-hCG) and alpha (α-hCG), growth/differentiation factor 15, as well as both pregnancy-specific beta-1-glycoproteins 2 and 3. In contrast, protein S100-A11 and l-lactate dehydrogenase A chain, were down-regulated in molar tissue from most patients in the GTN group. DISCUSSION: This study identified a set of differentially expressed proteins in molar tissues that could potentially be further examined as predictive biomarkers for the malignant transformation of CHMs. A molar proteome database was constructed and can be accessible online at http://sysbio.chula.ac.th/Database/GTD_DB/Supplementary_Data.xlsx.

Catestatin regulates vesicular quanta through modulation of cholinergic and peptidergic (PACAPergic) stimulation in PC12 cells.

We have previously shown that the chromogranin A (CgA)-derived peptide catestatin (CST: hCgA352-372) inhibits nicotine-induced secretion of catecholamines from the adrenal medulla and chromaffin cells. In the present study, we seek to determine whether CST regulates dense core (DC) vesicle (DCV) quanta (catecholamine and chromogranin/secretogranin proteins) during acute (0.5-h treatment) or chronic (24-h treatment) cholinergic (nicotine) or peptidergic (PACAP, pituitary adenylyl cyclase activating polypeptide) stimulation of PC12 cells. In acute experiments, we found that both nicotine (60 µM) and PACAP (0.1 µM) decreased intracellular norepinephrine (NE) content and increased 3H-NE secretion, with both effects markedly inhibited by co-treatment with CST (2 µM). In chronic experiments, we found that nicotine and PACAP both reduced DCV and DC diameters and that this effect was likewise prevented by CST. Nicotine or CST alone increased expression of CgA protein and together elicited an additional increase in CgA protein, implying that nicotine and CST utilize separate signaling pathways to activate CgA expression. In contrast, PACAP increased expression of CgB and SgII proteins, with a further potentiation by CST. CST augmented the expression of tyrosine hydroxylase (TH) but did not increase intracellular NE levels, presumably due to its inability to cause post-translational activation of TH through serine phosphorylation. Co-treatment of CST with nicotine or PACAP increased quantal size, plausibly due to increased synthesis of CgA, CgB and SgII by CST. We conclude that CST regulates DCV quanta by acutely inhibiting catecholamine secretion and chronically increasing expression of CgA after nicotinic stimulation and CgB and SgII after PACAPergic stimulation.

Safety of Follitropin Alfa/Lutropin Alfa for Stimulation of Follicular Development.

INTRODUCTION: Recombinant human luteinizing hormone (r-hLH) is used in a fixed-ratio combination with recombinant human follicle-stimulating hormone (r-hFSH) for the stimulation of follicular development. OBJECTIVE: The objective of this article was to conduct a review of safety data to evaluate the risks of r-hFSH/r-hLH treatment. METHODS: Data were retrieved from the Global Safety Database (Merck KGaA, Darmstadt, Germany) including reports from healthcare professionals, patients, health authorities, clinical trials, non-interventional studies, and the literature. Reports of important risks (identified and potential) as per the risk management plan applicable at the time of data retrieval were obtained up to December 2017. The estimated patient exposure to r-hFSH/r-hLH in the post-marketing setting was 427,012 treatment cycles. Nine hundred patients received r-hFSH/r-hLH during company-sponsored clinical trials (pre- and post-marketing). RESULTS: We identified 72 case reports describing important risks related to r-hFSH/r-hLH use, including 46 cases of ovarian hyperstimulation syndrome (10.8 per 100,000 treatment cycles) and 24 of hypersensitivity reaction (5.6 per 100,000 treatment cycles). No thromboembolic events were reported. One congenital anomaly, not suspected to be related to r-hFSH/r-hLH use, was reported during a clinical trial; the event was resolved by corrective surgery. Two fatal cases were identified; one case of recurrent malignant melanoma (suspected to be related to r-hFSH/r-hLH use) and one case resulting from complications of ovarian hyperstimulation syndrome. CONCLUSION: Cumulative reporting rates of important identified and potential risks of r-hFSH/r-hLH during a 10-year surveillance period demonstrate the benefit-risk balance is positive. This post-marketing surveillance and continued surveillance of safety events should provide reassurance about the use of r-hFSH/r-hLH in clinical practice.

[Gangliocytic paragangliomas: a clinicopathologic study].

Objective: To investigate the clinicopathological features of gangliocytic paraganglioma(GP). Methods: Clinical data and pathological diagnosis of the 4 cases of GP were obtained through the medical record inquiry from January 2011 to December 2017 at the First Affiliated Hospital of Zhengzhou University. Routine HE staining and immunohistochemistry of CKpan, Syn, CgA, CD56, NSE and NF were performed. Clinical follow-up of the patients was obtained through telephone communication. Results: All 4 patients, including 2 male and 2 female patients, presented with intermittent abdominal pain and distention. The median age was 56 years. Preoperative CT showed local thickening of the duodenum wall with slight enhancement in all four cases. Endoscopic ultrasonography showed low level echo in the mucous layer and submucosa involved by the tumor in 3 of 4 cases. The maximal diameter of the tumor ranged from 0.6 to 1.8 cm with an average of 1.2 cm. Microscopically, the tumors consisted of epithelioid, spindle and ganglion-like cells, and the proportion of the three cell types was different among cases. Epithelioid cells expressed CKpan, Syn, CgA and CD56. Spindle cells expressed S-100 protein and SOX-10 and ganglion-like cells expressed NF, Syn, CgA and CD56.All tumour cells expressed NSE. All 4 patients had no recurrence a post-surgery follow-up period of 3 to 30 months. Conclusions: GP of the duodenum is a benign tumor with excellent prognosis after endoscopic excision. Although its incidence is very low, its diagnosis should be considered for any mass lesion of the duodenum, especially involving mucosa and submucosa of the second dudenal segment.

Plasma androgen receptor and serum chromogranin A in advanced prostate cancer.

Recently, mixed forms between adenocarcinoma and neuroendocrine prostate cancer (NEPC) have emerged that are characterized by persistent androgen receptor (AR)-signalling and elevated chromogranin A (CgA) levels. The main aim of this study was to analyze castration-resistant prostate cancer (CRPC) patients treated with abiraterone or enzalutamide, assessing progression-free/overall survival (PFS/OS) in association with circulating AR and CgA. AR aberrations were analyzed by droplet digital PCR in pre-treatment plasma samples collected from two biomarker protocols [197 patients from a retrospective study (REC 2192/2013) and 59 from a prospective trial (REC 6798/2015)]. We subdivided patients into three groups according to CgA by receiver-operating characteristic (ROC) curves. In the primary cohort, plasma AR gain and mutations (p.L702H/p.T878A) were detected in 78 (39.6%) and 16 (8.1%) patients, respectively. We observed a significantly worse PFS/OS in patients with higher-CgA than in patients with normal-CgA, especially those with no AR-aberrations. Multivariable analysis showed AR gain, higher-CgA and LDH levels as independent predictors of PFS [hazard ratio (HR) = 2.16, 95% confidence interval (95% CI) 1.50-3.12, p < 0.0001, HR = 1.73, 95% CI 1.06-2.84, p = 0.026, and HR = 2.13, 95% CI 1.45-3.13, p = 0.0001, respectively) and OS (HR = 1.72, 95% CI 1.15-2.57, p = 0.008, HR = 3.63, 95% CI 2.13-6.20, p < 0.0001, and HR = 2.31, 95% CI 1.54-3.48, p < 0.0001, respectively). These data were confirmed in the secondary cohort. Pre-treatment CgA detection could be useful to identify these mixed tumors and would seem to have a prognostic role, especially in AR-normal patients. This association needs further evaluation in larger prospective cohorts.

Photoperiodic regulation of plasma gonadotropin levels in previtellogenic greater amberjack (Seriola dumerili).

In Seriola species, exposure to a long photoperiod regime is known to induce ovarian development. This study examined photoperiodic effects on pituitary gene expression and plasma levels of follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) in previtellogenic greater amberjack (Seriola dumerili). The fish were exposed to short (8L:16D) or long (18L:6D) photoperiod. The water temperature was maintained at 22 °C. Compared with the short-photoperiod group, plasma Fsh levels were higher on days 10 and 30 in the long-photoperiod group, but plasma Lh levels did not significantly differ. On day 30, pituitary Fsh- and Lh-ß subunit gene expressions were also higher in the long-photoperiod group than the short-photoperiod group, whereas α-subunit gene expressions were higher on days 20 and 30. Throughout the experiment, average gonadosomatic index and plasma E2 levels did not significantly differ between the two groups. This study clearly demonstrated that a long photoperiod induced Fsh release in the previtellogenic fish followed by upregulation of pituitary Fsh and Lh subunit gene expressions. An increase in plasma Fsh levels may be a key factor that mediates the photoperiodic effect on the initiation of ovarian development.