Single-Cell Transcriptome Analysis of Small Cell Neuroendocrine Carcinoma of the Endometrium Reveals ISL1 as a Potential Biomarker for Diagnosis and Treatment.

BACKGROUND: As a dedifferentiated tumor, small cell endometrial neuroendocrine tumors (NETs) are rare and frequently diagnosed at an advanced stage with a poor prognosis. Current treatment recommendations are often extrapolated from histologically similar tumors in other sites or based on retrospective studies. The exploration for diagnostic and therapeutic markers in small cell NETs is of great significance. METHODS: In this study, we conducted single-cell RNA sequencing on a specimen obtained from a patient diagnosed with small cell endometrial neuroendocrine carcinoma (SCNEC) based on pathology. We revealed the cell map and intratumoral heterogeneity of the cancer cells through data analysis. Further, we validated the function of ISL LIM Homeobox 1 (ISL1) in vitro in an established neuroendocrine cell line. Finally, we examined the association between ISL1 and tumor staging in small cell lung cancer (SCLC) patient samples. RESULTS: We observed the significant upregulation of ISL1 expression in tumor cells that showed high expression of the neuroepithelial markers. Additionally, in vitro cell function experiments demonstrated that the high ISL1 expression group exhibited markedly higher cell proliferation and migration abilities compared to the low expression group. Finally, we showed that the expression level of ISL1 was correlated with SCLC stages. CONCLUSIONS: ISL1 protein in NETs shows promise as a potential biomarker for diagnosis and treatment.

Identification and validation of serum metabolite biomarkers for endometrial cancer diagnosis.

Endometrial cancer (EC) stands as the most prevalent gynecological tumor in women worldwide. Notably, differentiation diagnosis of abnormity detected by ultrasound findings (e.g., thickened endometrium or mass in the uterine cavity) is essential and remains challenging in clinical practice. Herein, we identified a metabolic biomarker panel for differentiation diagnosis of EC using machine learning of high-performance serum metabolic fingerprints (SMFs) and validated the biological function. We first recorded the high-performance SMFs of 191 EC and 204 Non-EC subjects via particle-enhanced laser desorption/ionization mass spectrometry (PELDI-MS). Then, we achieved an area-under-the-curve (AUC) of 0.957-0.968 for EC diagnosis through machine learning of high-performance SMFs, outperforming the clinical biomarker of cancer antigen 125 (CA-125, AUC of 0.610-0.684, p < 0.05). Finally, we identified a metabolic biomarker panel of glutamine, glucose, and cholesterol linoleate with an AUC of 0.901-0.902 and validated the biological function in vitro. Therefore, our work would facilitate the development of novel diagnostic biomarkers for EC in clinics.

Understanding placentation in ruminants: a review focusing on cows and sheep.

Mammals differ regarding their placentae, but in all species placental trophoblasts interact intimately with the uterine endometrium to mediate the transfer of nutrients from the mother to the embryo/fetus through the closely juxtaposed microcirculatory systems of the uterus and placenta. Placentation in ruminants is intermediate between the non-invasive type, as observed in the epitheliochorial placenta of pigs, and the invasive type, as observed in the haemochorial placentae of mice and humans. In ruminants, placental trophoblast cells invade uterine endometrial tissue, but invasion is believed to be limited to the endometrial luminal epithelium (LE). In the LE there are varying degrees of syncytialisation among species, with syncytialisation being more extensive in sheep than cows. The hallmarks of placentation in ruminants include: (1) an extended period in which conceptuses (embryos and associated placental membranes) elongate and must be supported by secretions (histotroph) from the uterus; (2) a cascade involving an array of adhesion molecules that includes integrin-mediated attachment of the conceptus trophoblast to the endometrial LE for implantation; (3) syncytialisation of the developing early placenta, a process for which there is currently limited understanding; and (4) development of placentomes that define the cotyledonary placentae of cows and sheep, and provide haemotrophic support of fetal development.

A Comparative Proteomic Analysis to Explore the Influencing Factors on Endometritis Using LC-MS/MS.

The inflammatory system activated by uterine infection is associated with decreased fertility. Diseases can be detected in advance by identifying biomarkers of several uterine diseases. Escherichia coli is one of the most frequent bacteria that is involved in pathogenic processes in dairy goats. The purpose of this study was to investigate the effect of endotoxin on protein expression in goat endometrial epithelial cells. In this study, the LC-MS/MS approach was employed to investigate the proteome profile of goat endometrial epithelial cells. A total of 1180 proteins were identified in the goat Endometrial Epithelial Cells and LPS-treated goat Endometrial Epithelial Cell groups, of which, 313 differentially expressed proteins were accurately screened. The proteomic results were independently verified by WB, TEM and IF techniques, and the same conclusion was obtained. To conclude, this model is suitable for the further study of infertility caused by endometrial damage caused by endotoxin. These findings may provide useful information for the prevention and treatment of endometritis.

Association between redundant endometrium and endometrial polyps: a pilot study.

BACKGROUND: The aim of this study was to explore the organic features of redundant endometrium (RE), we examined the expression of different endometrial hormone receptors, oncogenes, and cell replication markers, in normal endometrium (NE), endometrial polyps (EP) and RE specimens. METHODS: This was an experimental study examining endometrial tissue expression of estrogen receptors (ER1 and 2), progesterone receptors (PR-A+B), androgen receptor (AR), insulin receptor (Insulin-R), insulin-like growth factor receptor 1 (IGFR-1), thyroid hormone receptor (TH-RB), B-cell lymphoma 2 (Bcl-2), Ki67, HOXA10, in women with NE, EP and RE, of women undergoing hysteroscopy for benign gynecologic pathology. Specimens were separated in 3 groups: NE, EP, RE. Endometrial samples were processed for real-time RT-PCR analyses. Main outcome measure was tissue expression of the markers in the three groups. RESULTS: Of the 16 patients, 2 had NE, 8 had RE, 5 had EP, 1 had both, RE and EP. Compared to NE, RE and EP showed significantly increased Bcl-2, Insulin-R, ER-ß, PR-A+B, and TRB expression (P<0.044), with EP showing significantly increased PR-A+B, compared to RE (3.29±0.47 fg/µg RNA versus 1.86±0.34 fg/µg RNA; P=0.023). The other markers were not significantly different across the three groups: Ki67 appeared non-significantly decreased, while HOXA10, IGF-R1, AR, and ER-α, were non-significantly increased. CONCLUSIONS: RE showed biochemical characteristics different from NE. Similar to endometrial polyps, RE showed enhanced cell differentiation, but not cell replication. These changes in RE could be detrimental for embryo implantation and should be of consideration in women undergoing fertility treatments.

Significance of multiple myeloma oncogene 1 immunohistochemistry in chronic endometritis detection in patients with recurrent pregnancy losses: an observational study.

The most reliable chronic endometritis diagnosis is based on immunohistochemistry plasma cell identification in endometrial samples. Our study aimed to compare multiple myeloma oncogene 1 (MUM1) and syndecan-1/CD138 immunohistochemistry staining for chronic endometritis diagnosis among patients with recurrent pregnancy loss. We evaluated the presence of endometrial stromal changes. Fifty-four patients with a history of at least two intrauterine pregnancy losses underwent diagnostic hysteroscopy in the follicular phase of the cycle with endometrial aspiration biopsy. In all 54 cases, three successive sections were cut from each paraffin-embedded tissue block for hematoxylin and eosin (H&E), CD138 and MUM1 staining. The goal was to evaluate the level of agreement between the MUM1 and CD138 results and plasma cell detection rate in assessing the endometrial stromal changes. The concordance analysis between CD138 and MUM1 immunohistochemistry staining showed consistent results in 43 of 54 (79.6%) cases. The level of agreement was moderate, based on a Kappa value of 0.60. MUM1 immunostaining was positive for CE in more cases than CD138 staining, and this difference was statistically significant, showing a higher sensitivity of MUM1 in plasma cell detection (p=0.01). Endometrial stromal changes were observed in the majority of cases - 49/54 (90%). Samples without stromal changes were consistently negative for plasma cells using both CD138 and MUM1 staining. We demonstrated that MUM1 staining, used in conjunction with assessing endometrial stromal changes, contributes to a more accurate and comprehensive diagnosis of chronic endometritis.

Endometrial glycogen metabolism during early pregnancy in mice.

Glucose is critical during early pregnancy. The uterus can store glucose as glycogen but uterine glycogen metabolism is poorly understood. This study analyzed glycogen storage and localization of glycogen metabolizing enzymes from proestrus until implantation in the murine uterus. Quantification of diastase-labile periodic acid-Schiff (PAS) staining showed glycogen in the glandular epithelium decreased 71.4% at 1.5 days postcoitum (DPC) and 62.13% at DPC 3.5 compared to proestrus. In the luminal epithelium, glycogen was the highest at proestrus, decreased 46.2% at DPC 1.5 and 63.2% at DPC 3.5. Immunostaining showed that before implantation, glycogen metabolizing enzymes were primarily localized to the glandular and luminal epithelium. Stromal glycogen was low from proestrus to DPC 3.5. However, at the DPC 5.5 implantation sites, stromal glycogen levels increased sevenfold. Similarly, artificial decidualization resulted in a fivefold increase in glycogen levels. In both models, decidualization increased expression of glycogen synthase as determine by immunohistochemistry and western blot. In conclusion, glycogen levels decreased in the uterine epithelium before implantation, indicating that it could be used to support preimplantation embryos. Decidualization resulted in a dramatic increase in stromal glycogen levels, suggesting it may have an important, but yet undefined, role in pregnancy.

B-cell lymphoma 6 expression is not associated with live birth in a normal responder in vitro fertilization population.

OBJECTIVE: To determine whether increased endometrial B-cell lymphoma 6 (BCL6) expression is associated with live birth in a normal responder in vitro fertilization (IVF) population. DESIGN: Case-control study. SETTING: University-affiliated infertility center. PATIENT(S): Two groups of women undergoing IVF with preimplantation genetic testing for aneuploidy followed by warmed, single, euploid embryo transfer. Group 1 consisted of women who failed to achieve live birth, and group 2 consisted of women who achieved live birth. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Endometrial BCL6 expression measured by immunohistochemistry in endometrial tissue samples. Overexpression was defined by mean HSCORE with a cutoff of positivity of >1.4, as previously described in the literature. RESULT(S): Twenty-seven patients who achieved live birth and 23 patients who failed to achieve live birth were included. B-cell lymphoma 6 expression/HSCORE and live birth rate were not associated (Odds ratio [OR], 0.78 [0.24-2.55]). Using a cutoff of >1.4 for positivity, 8 of 23 samples were positive for BCL6 in the no live birth group, whereas 7 of 27 were positive in the live birth group. There was no significant association between BCL6 positivity and live birth (OR, 0.66 [0.19-2.21]). CONCLUSION(S): The proportion of patients with BCL6 positivity did not significantly differ between those who achieved live birth and those who did not. In the population of patients at our center, who compromise of women who respond normally to IVF stimulation, BCL6 overexpression was not associated with IVF success. Physicians implementing BCL6 testing as a diagnostic tool for clinical decision making should counsel patients that results may have limited utility in predicting IVF outcomes in this population.

The morphofunctional pattern of neuronal biogenic amines in postpartum involution period-an in vivo study.

Postpartum uterine diseases are associated with significant imbalance in the levels of biogenic amines (BAs) in rat uterus. Mast cells (MCs) are the main suppliers of BAs such as serotonin, catecholamines, and histamine in uterus. There is limited evidence of the BA-positive elements involved in the physiological regulation of uterus during postpartum involution. The aim of present study is to determine the concentration and distribution of biogenic amines (BAs) such as histamine, serotonin, and catecholamines in the uterine endometrium, myometrium, and peritoneal fluid (PF) during the postpartum uterine involution. A total of 110 nulliparous outbred female nonpregnant Wistar rats of mature age were divided into eleven groups (n=10 per group) according to days of postpartum involution. Tissue specimens of uterine segments, PF were prepared. Serotonin, catecholamines, and histamine concentrations were examined by fluorescence-histochemical techniques. The fluorescence of the BA-positive elements was detected and analyzed by microspectrofluorimetry. Results were analyzed using the Kruskal-Wallis chi-squared test and pairwise Mann-Whitney-Wilcoxon tests with "Benjamini-Hochberg correction" in R 3.6.3. Mast cells in uterine segments, PF exhibited characteristic yellowish-green fluorescence. The highest MCs number was reported in corpus uteri on the 15th day of postpartum involution. Serotonin, catecholamines, and histamine levels were significantly higher in BA-positive elements in the initial days. BA content was dynamic and relies on the time elapsed after parturition. There was statistically significant difference in the levels of BAs in the cornu and cervix uteri. A single morphofunctional complex of BA supply was noticed in the reproductive system of the rats. The coupled interactions of intra- and extra-organic BA-positive elements was associated with anabolic/catabolic equilibrium in uterus through the metabolism of serotonin, catecholamines, and histamine during postpartum involution.

Low-dose aspirin can downregulate progesterone resistance and increase the expression of LIF in endometriosis during the implantation window.

AIM: Study the effect of low-dose aspirin on the endometrial receptivity in endometriosis rat models. MATERIALS AND METHODS: This study is to explore the expressions of progesterone receptor and LIF among three groups of endometriosis rat models: control group (n = 12), EMs group (n = 15), and aspirin group (n = 17). The expressions of progesterone receptor (PR), PRA, PRB, and leukemia inhibitory factor receptor (LIFR) in eutopic endometrium were determined using immunohistochemistry technology, western blot, and qRT-PCR. The levels of LIF in eutopic endometrium and serum were detected by western blot, qRT-PCR, and ELISA. RESULTS: The expressions of PR, PRA, and PRB protein were significantly increased in the eutopic endometrium after low-dose aspirin treatment, and the level of PRB mRNA was also increased while the ratio of PRA/PRB mRNA was decreased in the eutopic endometrium. The levels of LIF in eutopic endometrium and serum were increased compared with the untreated endometriosis rats. However, the expression of LIFR was not statistically different among the three groups. CONCLUSIONS: The results suggest that the low-dose aspirin treatment could downregulate progesterone resistance and increase the expression of LIF of endometriosis rats during the implantation window, which could improve endometrial receptivity and enhance the pregnant rate of endometriosis. It may provide a potential treatment method for endometriosis-related infertility.

Equine early pregnancy endocrine profiles and ipsilateral endometrial immune cell, gene expression and protein localisation response.

We investigated the early effects of the equine embryo on maternal serum concentrations of insulin-like growth factor 1 (IGF1), leptin and adiponectin, uterine immune cells and genes and proteins related to embryo development and the maintenance of pregnancy. Ipsilateral endometrial expression was assessed on Days 7 and 13 after ovulation for the following transcripts: oestrogen receptor ERα (ESR1), progesterone receptor (PGR), progestin and adipoQ receptor family member 5 (PAQR5), oxytocin receptor (OXTR), prostaglandin-endoperoxide synthase 2 (PTGS2), raf-1 proto-oncogene serine/threonine kinase (RAF1), p21-activated kinase 6 (PAK6), fibroblast growth factor family member 9 (FGF9), IGF1 and its receptor (IGF1R), mucin 1 (MUC1), osteopontin (OPN), leptin receptor (LEPR) and adiponectin receptors 1 and 2 (ADIPOR1 and ADIPOR2). Ipsilateral endometrial immunological cell infiltration and immunohistochemical protein localisation were evaluated on Days 7, 10 and 13 after ovulation for ERα, PGR, OXTR, PTGS2, IGF1, IGF1R, IGF2 and MUC1. Serum hormone concentrations were not affected by reproductive status. Pregnancy downregulated ESR1 and PGR mRNA levels, upregulated the expression of all other genes and affected the expression of all genes, except PGR, on Day 7 (compared with eight genes affected at Day 13). Proteins were affected by pregnancy or by its interaction with other variables (day of extraction and endometrial compartment). Pregnant mares had a higher lymphocyte count, which decreased towards Day 13. The effect of pregnancy on leucocytes and proteins was more evident in superficial endometrial compartments. The results of this study suggest that the equine embryo exerts prompt paracrine regulation of critical biological processes.

Expression of nerve growth factor (NGF) in endometrium as a potential biomarker for endometriosis - Single tertiary care centre study.

OBJECTIVE: to identify novel biomarkers for peritoneal endometriosis in eutopic endometrium thus giving an oportunity for non-invasive diagnosis. DESIGN: A cross-sectional single-center study SETTING: tertiary care hospital PATIENTS: 49 patients subjected to laparoscopy because of suspected endometriosis, 33 patients out of the group qualified to the study had sufficient endometrial tissue taken and were in their follicular phase of menstrual cycle. INTERVENTIONS: biopsy sampling of eutopic endometrial tissue during diagnostic or diagnostic and terapeutic laparoscopy, questionaires, MAIN OUTCOME MEASURE(S): qRT-PCR to evaluate the mRNA expression of selected candidate marker genes in endometrium: ARO1 (aromatase), CXCL8 (interleukin 8), NGF (nerve growth factor), VEGF-A (vascular endothelial growth factor A), PDGF-A (platelet-derived growth factor A). RESULTS: mRNA expression of ARO1, CXCL8, VEGF-A and PDGF-A did not differ significantly between women with and without endometriosis. NGF mRNA expression was decreased in women with endometriosis. CONCLUSIONS: Observed preliminary results suggest a possible role of NGF in early diagnosis of peritoneal endometriosis. The role of NGF changes in eutopic endometrium of patients with peritoneal endometriosis needs further evaluation.

Dysmenorrhea in patients with adenomyosis: A clinical and demographic study.

OBJECTIVE: To identify the risk factors associated with dysmenorrhea in adenomyosis and to discuss the potential hormone-based understanding of pain mechanisms. STUDY DESIGN: Adenomyosis patients with mild or no dysmenorrhea (n = 40, Group 1) and moderate-to-severe dysmenorrhea (n = 80, Group 2) were recruited. Charts of all patients were recorded. An immunohistochemistry (IHC) analysis was performed to detect the cellular levels of estrogen receptor-α (ER-α), estrogen receptor-ß (ER-ß), gonadotropin-releasing hormone receptor (GnRH-R), and neurofilaments (NFs) in 60 cases. RESULTS: A history of cesarean section (CS) was positively related to the degree of dysmenorrhea in adenomyosis (OR (95 % CI): 4.397 (1.371-14.104)). The ER-α levels in the eutopic endometrium (EUE) of Group 2 were higher than those in the ectopic endometrium (ECE) of Group 1. Group 2 had higher NF levels in the ECE than in the EUE. CONCLUSION: A history of CS is a risk factor for adenomyosis with moderate-to-severe dysmenorrhea. For patients with adenomyosis, high ER-α levels in the EUE and high NF levels in the ECE may be related to moderate-to-severe dysmenorrhea. These hormone-based mechanisms may contribute to our understanding of the pathogenesis of dysmenorrhea in adenomyosis.

Altered N-linked glycosylation in endometrial cancer.

It is well established that cell surface glycans play a vital role in biological processes and their altered form can lead to carcinogenesis. Mass spectrometry-based techniques have become prominent for analysing N-linked glycans, for example using matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Additionally, MALDI MS can be used to spatially map N-linked glycans directly from cancer tissue using a technique termed MALDI MS imaging (MALDI MSI). This powerful technique combines mass spectrometry and histology to visualise the spatial distribution of N-linked glycans on a single tissue section. Here, we performed N-glycan MALDI MSI on six endometrial cancer (EC) formalin-fixed paraffin-embedded (FFPE) tissue sections and tissue microarrays (TMA) consisting of eight EC patients with lymph node metastasis (LNM) and twenty without LNM. By doing so, several putative N-linked glycan compositions were detected that could significantly distinguish normal from cancerous endometrium. Furthermore, a complex core-fucosylated N-linked glycan was detected that could discriminate a primary tumour with and without LNM. Structural identification of these putative N-linked glycans was performed using porous graphitized carbon liquid chromatography tandem mass spectrometry (PGC-LC-MS/MS). Overall, we observed higher abundance of oligomannose glycans in tumour compared to normal regions with AUC ranging from 0.85-0.99, and lower abundance of complex N-linked glycans with AUC ranges from 0.03-0.28. A comparison of N-linked glycans between primary tumours with and without LNM indicated a reduced abundance of a complex core-fucosylated N-glycan (Hex)2(HexNAc)2(Deoxyhexose)1+(Man)3(GlcNAc)2, in primary tumour with associated lymph node metastasis. In summary, N-linked glycan MALDI MSI can be used to differentiate cancerous endometrium from normal, and endometrial cancer with LNM from endometrial cancer without.

The role of prokineticins in recurrent implantation failure.

The aim of the present study was to investigate the expression patterns of prokineticins (PROK) and prokineticin receptors (PROKR) in the endometrium of women with recurrent implantation failure (RIF). Fifteen (15) women with RIF and 15 fertile controls were enrolled in this study. Endometrial samples were taken from study participants with an endometrial biopsy cannula during the implantation window. Real time polymerase chain reaction and immunohistochemistry were used to determine PROK/PROKR mRNA expression and protein localization, respectively. PROK1 mRNA levels were 6.09 times higher compared to endometrial samples obtained from women with RIF than in samples obtained from fertile controls, whereas PROKR1 mRNA levels were 2.46 times lower in endometrial samples obtained from women with RIF than in samples from fertile controls. In addition, decreased PROKR1 was supported by immunohistochemistry analysis at protein level. There was no statistically significant difference between women with RIF and fertile controls regarding PROK2 and PROKR2 levels. Altered expression of the PROK1/PROKR1 system could be one of the numerous abnormalities in the endometrium of women with RIF.

LPS-treatment of bovine endometrial epithelial cells causes differential DNA methylation of genes associated with inflammation and endometrial function.

BACKGROUND: Lipopolysaccharide (LPS) endotoxin stimulates pro-inflammatory pathways and is a key player in the pathological mechanisms involved in the development of endometritis. This study aimed to investigate LPS-induced DNA methylation changes in bovine endometrial epithelial cells (bEECs), which may affect endometrial function. Following in vitro culture, bEECs from three cows were either untreated (0) or exposed to 2 and 8 µg/mL LPS for 24 h. RESULTS: DNA samples extracted at 0 h and 24 h were sequenced using reduced representation bisulfite sequencing (RRBS). When comparing DNA methylation results at 24 h to time 0 h, a larger proportion of hypomethylated regions were identified in the LPS-treated groups, whereas the trend was opposite in controls. When comparing LPS groups to controls at 24 h, a total of 1291 differentially methylated regions (DMRs) were identified (55% hypomethylated and 45% hypermethylated). Integration of DNA methylation data obtained here with our previously published gene expression data obtained from the same samples showed a negative correlation (r = - 0.41 for gene promoter, r = - 0.22 for gene body regions, p < 0.05). Differential methylation analysis revealed that effects of LPS treatment were associated with methylation changes for genes involved in regulation of immune and inflammatory responses, cell adhesion, and external stimuli. Gene ontology and pathway analyses showed that most of the differentially methylated genes (DMGs) were associated with cell proliferation and apoptotic processes; and pathways such as calcium-, oxytocin- and MAPK-signaling pathways with recognized roles in innate immunity. Several DMGs were related to systemic inflammation and tissue re-modelling including HDAC4, IRAK1, AKT1, MAP3K6, Wnt7A and ADAMTS17. CONCLUSIONS: The present results show that LPS altered the DNA methylation patterns of bovine endometrial epithelial cells. This information, combined with our previously reported changes in gene expression related to endometrial function, confirm that LPS activates pro-inflammatory mechanisms leading to perturbed immune balance and cell adhesion processes in the endometrium.

Design and evaluation of a novel nanodrug delivery system for reducing the side effects of clomiphene citrate on endometrium.

BACKGROUND: Stimulation of ovulation with clomiphene citrate can cause side effects on endometrial receptivity. Formulation with nano-size may be an alternative therapy for women with ovulatory disorders. In this study, we investigated sustained-release clomiphene citrate by using Phosal-based formulation (PBF) and evaluate its decreased side effect on the endometrial receptivity. METHODS: In the in-vitro study, CC loaded PBF was analyzed using Zetasizer, Fourier-transform infrared spectroscopy (FTIR), and Transmission electron microscopy (TEM). In the in-vivo study, 24 female mice were randomly divided into three groups: CC (5 mg/kg), CC/PBF (5 mg/kg) and SS (1 ml) daily administered and injected with 5 IU HCG and mated after two days. At day 4.5, pregnant mice were euthanized and endometrial tissue was extracted for quantitative polymerase chain reaction (Q-PCR) analysis. RESULTS: The optimized PBF contained Phosal 50PG/glycerol in a 2:8 ratios (w/w) and the particle size of optimum formulation was 67 ± 0.30551 nm and the release of CC from CC-containing PBF was slightly faster in the first 24 h; wherein, 29% of CC was released, and 76% of CC was released up to 120 h. The mRNA levels of leukemia inhibitory factor (LIF), leukemia inhibitory factor receptor alpha (LIFR), HOXA10, Heparin-binding epidermal growth factor (HB-EGF), and epidermal growth factor (EGF) were significantly upregulated and MUC1 and PGR mRNA levels were significantly downregulated in the CC-containing PBF-treated animals compared with only CC group (P < 0.05). CONCLUSION: Sustained release formulation of clomiphene citrate increased its targeting efficiency and improved the impact of the CC on implantation. Graphical abstract A new Phosal Based Formulation (PBF) was designed to decrease the side effects of Clomiphene citrate (CC) on endometrium. This drug formulation could react better during implantation by increasing the expression of genes involved in implantation. The in vivo study demonstrated that the CC-containing PBF in mice has a significantly higher endometrial receptivity, compared with the suspension.

Weighted Gene Correlation Network Meta-Analysis Reveals Functional Candidate Genes Associated with High- and Sub-Fertile Reproductive Performance in Beef Cattle.

Improved reproductive efficiency could lead to economic benefits for the beef industry, once the intensive selection pressure has led to a decreased fertility. However, several factors limit our understanding of fertility traits, including genetic differences between populations and statistical limitations. In the present study, the RNA-sequencing data from uterine samples of high-fertile (HF) and sub-fertile (SF) animals was integrated using co-expression network meta-analysis, weighted gene correlation network analysis, identification of upstream regulators, variant calling, and network topology approaches. Using this pipeline, top hub-genes harboring fixed variants (HF × SF) were identified in differentially co-expressed gene modules (DcoExp). The functional prioritization analysis identified the genes with highest potential to be key-regulators of the DcoExp modules between HF and SF animals. Consequently, 32 functional candidate genes (10 upstream regulators and 22 top hub-genes of DcoExp modules) were identified. These genes were associated with the regulation of relevant biological processes for fertility, such as embryonic development, germ cell proliferation, and ovarian hormone regulation. Additionally, 100 candidate variants (single nucleotide polymorphisms (SNPs) and insertions and deletions (INDELs)) were identified within those genes. In the long-term, the results obtained here may help to reduce the frequency of subfertility in beef herds, reducing the associated economic losses caused by this condition.

Anti-Müllerian hormone receptor type 2 (AMHR2) expression in bovine oviducts and endometria: comparison of AMHR2 mRNA and protein abundance between old Holstein and young and old Wagyu females.

Anti-Müllerian hormone (AMH) is a glycoprotein produced by granulosa cells of preantral and small antral follicles that has multiple important roles in the ovaries. Recent studies have revealed extragonadal AMH regulation of gonadotrophin secretion from bovine gonadotrophs. In this study we investigated whether the primary receptor for AMH, AMH receptor type 2 (AMHR2), is expressed in bovine oviducts and endometria. Reverse transcription-polymerase chain reaction detected expression of AMHR2 mRNA in oviductal and endometrial specimens. Western blotting and immunohistochemistry were performed to analyse AMHR2 protein expression using anti-bovine AMHR2 antibody. Immunohistochemistry revealed robust AMHR2 expression in the tunica mucosa of the ampulla and isthmus, as well as in the glandular and luminal epithelium of the endometrium. AMHR2 mRNA (measured by real-time polymerase chain reaction) and AMHR2 protein expression in these layers did not significantly differ among oestrous phases in adult Wagyu cows (P>0.1). In addition, AMHR2 mRNA and protein expression in these layers did not differ among old Holsteins (mean (±s.e.m.) age 91.9±6.4 months) and young (26.6±0.8 months) and old (98.8±10.2 months) Wagyu cows. Therefore, AMHR2 is expressed in bovine oviducts and endometria.

Comparison of endometrial prostanoid profiles in three infertile subgroups: the missing part of receptivity?

OBJECTIVE: To study the prostanoid profile of the endometria of patients with recurrent implantation failure (RIF), unexplained infertility (UIF), and recurrent miscarriages (RM), and to compare them with the endometria of healthy fertile controls. DESIGN: Prospective cohort study. SETTING: University hospital. PATIENT(S): Fifteen patients with RIF, 18 patients with UIF, 16 patients with RM, and 23 fertile controls were recruited. INTERVENTION(S): Endometrial samples were taken during the window of implantation. After tissue homogenization and extraction, analysis with ultra-performance liquid chromatography diode array detector electrospray ionisation tandem mass spectrometry was performed. MAIN OUTCOME MEASURES: Concentrations of prostaglandin (PG) D1, PGE1, PGF1α, 6-ketoPGF1α, PGD2, PGE2, PGF2α, 15-deoxy-Δ12,14-PGJ2, PGD3, PGE3, PGF3α, thromboxane B2, 13,14-dihydro-PGE1, 13,14-dihydro-PGF1α, 13,14-dihydro-PGF2α, 13,14-dihydro-15-keto-PGE1, 13,14-dihydro-15-keto-PGE2, and 13,14-dihydro-15-keto-PGF2α were assessed. RESULT(S): Comparison of the endometria of patients with UIF and the controls showed no statistically significant differences. When the endometria of patients with RIF were compared with the controls, thromboxane B2 (TXB2) was found significantly higher (843.1 pg/mg vs. 133.5 pg/mg). When the endometria of patients with RM were compared with controls, 13,14-dihydro-15-keto PGF2α and TXB2 were found significantly higher (3907.30 pg/mg vs. 17.80 pg/mg and 858.7 pg/mg vs. 133.5 pg/mg respectively). CONCLUSION(S): We identified increased endometrial presence of TXB2 in patients with RM and RIF, and 13,14-dihydro-15-keto PGF2α in patients with RM. Although common ground is observed for RM and RIF, prostanoids, on the other hand, might make their own contribution to endometrial receptivity as important as genes and proteins. Attempts to normalize the prostaglandin profile of the endometrium via enzymatic activity can open new therapeutic options.