Autocrine Factor Research Articles

Endocannabinoid system and epigenetics in spermatogenesis and testicular cancer.

In mammals, male germ cell development starts during fetal life and is carried out in postnatal life with the formation of sperms. Spermatogenesis is the complex and highly orderly process during which a group of germ stem cells is set at birth, starts to differentiate at puberty. It proceeds through several stages: proliferation, differentiation, and morphogenesis and it is strictly regulated by a complex network of hormonal, autocrine and paracrine factors and it is associated with a unique epigenetic program. Altered epigenetic mechanisms or inability to respond to these factors can impair the correct process of germ development leading to reproductive disorders and/or testicular germ cell cancer. Among factors regulating spermatogenesis an emerging role is played by the endocannabinoid system (ECS). ECS is a complex system comprising endogenous cannabinoids (eCBs), their synthetic and degrading enzymes, and cannabinoid receptors. Mammalian male germ cells have a complete and active ECS which is modulated during spermatogenesis and that crucially regulates processes such as germ cell differentiation and sperm functions. Recently, cannabinoid receptor signaling has been reported to induce epigenetic modifications such as DNA methylation, histone modifications and miRNA expression. Epigenetic modifications may also affect the expression and function of ECS elements, highlighting the establishment of a complex mutual interaction. Here, we describe the developmental origin and differentiation of male germ cells and testicular germ cell tumors (TGCTs) focusing on the interplay between ECS and epigenetic mechanisms involved in these processes.

Expression of autocrine motility factor receptor (AMFR) in human breast and lung invasive micropapillary carcinomas.

The aim of this study was to evaluate the clinicopathological significance of autocrine motility factor receptor (AMFR) expression in a variety of human invasive micropapillary carcinomas (IMPC). AMFR expression was compared in 111 samples of a variety of human IMPCs which had intrinsic non-micropapillary components and with 26 cases of control pulmonary adenocarcinoma (CPA, carcinoma without an IMPC component) by immunohistochemistry (IHC). In the 137 cases analysed, AMFR expression was significantly elevated in the IMPC components compared to the non-IMPC components (p=.005) and normal tissues (p < .001). AMFR expression was also higher in the IMPC samples compared to their intrinsic non-IMPC components (p=.0234). Between the 69 cases of lung IMPC and 26 cases of CPA, AMFR expression was notably higher in the IMPC components than in the CPA components (p=.0455). However, there was no significant difference between the non-IMPC components in the lung and the CPA components (p=.4584). Moreover, in breast cancer, elevated AMFR expression was not significantly correlated with mixed type or pure type IMPC (p=.5969) or with age, gender, T stage, or lymph node metastasis (LNM). Between IMPC and CPA of the lung, there was no statistical significance in age, T stage, and LNM, where AMFR expression was higher in IMPC (p=.0071). Thus this study demonstrated that AMFR was overexpressed in a variety of human IMPC components compared with non-micropapillary components. This suggests that AMFR expression is a potential new prognostic indicator for different types of human IMPC, which might thus be a new therapeutic target.

THE EFFECT OF DIFFERENT CULTURE VOLUME OF SIBLING HUMAN EMBRYOS ON CLINICAL OUTCOMES

In order to select viable human embryos for transfer, single embryo culture has been adapted. Many efforts have been made to complement the drawbacks of individual culture. Previous studies demonstrated that culturing embryos in small volume improves embryonic development by enhancing autocrine factor. However, embryo-toxic factor may impair the embryo development. Since most studies were done by mouse, little work has been done in human. Earlier study showed that there is no correlation between reduced culture volume and human blastocyst formation.

IVF culture media refresh in a reduced volume on day4 aimed at improving non-invasive embryo selection does not affect embryo competence: a prospective analysis of 2605 embryos

Background and Aim: Omic analyses on spent-culture-media are emerging as non-invasive schemes for embryo selection. Media refresh and reduced culture volume are critical to improve their specificity and sensitivity. However, limited evidence exists to support embryo culture in reduced volumes and some authors claimed this practice might influence developmental and reproductive potential altering the concentration of autocrine factors and/or of potentially-toxic metabolites. It is therefore critical to outline suitable protocols to preserve embryonic competence while improving non-invasive embryo-selection strategies.Methods: Between March2018 and December2019, 1357 oocyte-pick-ups for PGT-A were conducted. All couples could choose to participate to a pilot non-invasive PGT-A study (ClinicalTrials.gov:NCT03520933) whose design involved culture media refresh the morning of day4 from 30 µl single drops of continuous-single-culture-media (CSCM) to 10 µl for ≥40hr until blastocyst full-expansion or developmental arrest. If not consenting to the study, no refresh was performed, and embryo culture was concluded in 30 µl of CSCM. ICSI, blastocyst-culture under mineral-oil (5%O2/6%CO2), grading according to Capalbo et al (2014), trophectoderm-biopsy, NGS-based analysis and vitrified-warmed euploid single-embryo-transfer (SET) were performed. Overall, 389 and 151 cycles met the inclusion criteria (maternal age <44yr, own gametes, ejaculated sperm, no severe-oligoasthenoteratozoospermia) in the control and study group. Only viable embryos in day4 were included: 1873 and 732 in the control and study group. The primary outcome was the blastulation-rate per day4 viable embryos. Secondary outcomes were blastocyst quality, euploidy, clinical-pregnancy-, miscarriage- and sustained-implantation-rate per euploid single-embryo-transfer. All transfers performed up to August 2021, and issuing from these cycles, were included (N=378 and 137, respectively). Fisher's exact tests and logistic-regression-analyses adjusted for confounders were conducted.Results: The blastulation-rate per day4 viable embryo was similar in the control and study group (n=915/1873,49% versus 365/732, 50%). Blastocyst quality was also similar (excellent: 47% versus 50%, good:20% versus 15%, average:12% versus 14%, poor: 21% versus 21%). The euploidy-rates were 36% (n=330/915) and 37% (n=136/365). No difference was reported in clinical-pregnancy- (n=232/378,61% versus n=83/137,61%), miscarriage- (n=34/220,15% versus n=10/76,13%) and sustained-implantation-rates (n=186/378,49% versus n=66/137,48%). Media refresh in 10 µl for ≥40hr from day4 did not impact blastocyst development (multivariate-OR 0.99,95%CI:0.83-1.2, p=0.97) and sustained-implantation (multivariate-OR 0.96,95%CI:0.64-1.42, p=0.83).Conclusions: This study supports single embryo culture in a reduced volume of media from day4. This strategy is critical to minimize the risk for external contamination and to increase the concentration of embryonic-molecules (cell-free-DNA, mtDNA, miRNAs, metabolites) for non-invasive embryo-selection purposes. Background and Aim: Omic analyses on spent-culture-media are emerging as non-invasive schemes for embryo selection. Media refresh and reduced culture volume are critical to improve their specificity and sensitivity. However, limited evidence exists to support embryo culture in reduced volumes and some authors claimed this practice might influence developmental and reproductive potential altering the concentration of autocrine factors and/or of potentially-toxic metabolites. It is therefore critical to outline suitable protocols to preserve embryonic competence while improving non-invasive embryo-selection strategies. Methods: Between March2018 and December2019, 1357 oocyte-pick-ups for PGT-A were conducted. All couples could choose to participate to a pilot non-invasive PGT-A study (ClinicalTrials.gov:NCT03520933) whose design involved culture media refresh the morning of day4 from 30 µl single drops of continuous-single-culture-media (CSCM) to 10 µl for ≥40hr until blastocyst full-expansion or developmental arrest. If not consenting to the study, no refresh was performed, and embryo culture was concluded in 30 µl of CSCM. ICSI, blastocyst-culture under mineral-oil (5%O2/6%CO2), grading according to Capalbo et al (2014), trophectoderm-biopsy, NGS-based analysis and vitrified-warmed euploid single-embryo-transfer (SET) were performed. Overall, 389 and 151 cycles met the inclusion criteria (maternal age <44yr, own gametes, ejaculated sperm, no severe-oligoasthenoteratozoospermia) in the control and study group. Only viable embryos in day4 were included: 1873 and 732 in the control and study group. The primary outcome was the blastulation-rate per day4 viable embryos. Secondary outcomes were blastocyst quality, euploidy, clinical-pregnancy-, miscarriage- and sustained-implantation-rate per euploid single-embryo-transfer. All transfers performed up to August 2021, and issuing from these cycles, were included (N=378 and 137, respectively). Fisher's exact tests and logistic-regression-analyses adjusted for confounders were conducted. Results: The blastulation-rate per day4 viable embryo was similar in the control and study group (n=915/1873,49% versus 365/732, 50%). Blastocyst quality was also similar (excellent: 47% versus 50%, good:20% versus 15%, average:12% versus 14%, poor: 21% versus 21%). The euploidy-rates were 36% (n=330/915) and 37% (n=136/365). No difference was reported in clinical-pregnancy- (n=232/378,61% versus n=83/137,61%), miscarriage- (n=34/220,15% versus n=10/76,13%) and sustained-implantation-rates (n=186/378,49% versus n=66/137,48%). Media refresh in 10 µl for ≥40hr from day4 did not impact blastocyst development (multivariate-OR 0.99,95%CI:0.83-1.2, p=0.97) and sustained-implantation (multivariate-OR 0.96,95%CI:0.64-1.42, p=0.83). Conclusions: This study supports single embryo culture in a reduced volume of media from day4. This strategy is critical to minimize the risk for external contamination and to increase the concentration of embryonic-molecules (cell-free-DNA, mtDNA, miRNAs, metabolites) for non-invasive embryo-selection purposes.

Camellia Seed Cake Extract Supports Hair Growth by Abrogating the Effect of Dihydrotestosterone in Cultured Human Dermal Papilla Cells.

Autocrine and paracrine factors play key roles in the process of Androgenetic alopecia (AGA), which are secreted by balding dermal papilla cells (DPCs) after dihydrotestosterone (DHT) induction. Camellia seed cake is an oriental oil extraction byproduct, and its extract has been traditionally used to wash hair in China. This study elucidated the hair growth-promoting effects of Camellia seed cake extract (CSCE) in DHT-treated cultured DPCs and its underlying mechanisms. The effect of CSCE on cell viability and release of inflammatory factors IL-6 and IL-1α was performed on human dermal papilla cells (DPCs) incubated with DHT. Relative expression of bax, bcl-2, p53, androgen receptor (AR) and 5α- reductase type II (SRD5A2) was determined by PCR. Senescence-associated was examined by β-galactosidase (SA-β-Gal) assays. CSCE restored DHT-induced cell damage in a dose-dependent manner, and effectively reduced the production of IL-6 and IL-1α in DHT-treated DPCs. CSCE exhibited an anti-apoptotic effect, which increased the expression of bcl-2, and decreased the expressions of bax and p53 in DHT-incubated DPCs. CSCE also showed an anti-androgenic effect reversing the increase in AR and SRD5A2 expressions in DPCs driven by DHT incubation. In addition, CSCE inhibited the β-galactosidase enzyme activity and slowed down the cell senescence of DPCs which is crucial for AGA progression. In this study, we found that CSCE may have the potential to prevent and alleviate AGA by abrogating the effect of DHT in cultured DPCs.

Inflammation and Oxidative Stress Induce NGF Secretion by Pulmonary Arterial Cells through a TGF-β1-Dependent Mechanism.

Expression of the nerve growth factor NGF is increased in pulmonary hypertension (PH). We have here studied whether oxidative stress and inflammation, two pathological conditions associated with transforming growth factor-β1 (TGF-β1) in PH, may trigger NGF secretion by pulmonary arterial (PA) cells. Effects of hydrogen peroxide (H2O2) and interleukin-1β (IL-1β) were investigated ex vivo on rat pulmonary arteries, as well as in vitro on human PA smooth muscle (hPASMC) or endothelial cells (hPAEC). TβRI expression was assessed by Western blotting. NGF PA secretion was assessed by ELISA after TGF-β1 blockade (anti-TGF-β1 siRNA, TGF-β1 blocking antibodies, TβRI kinase, p38 or Smad3 inhibitors). TβRI PA expression was evidenced by Western blotting both ex vivo and in vitro. H2O2 or IL-1β significantly increased NGF secretion by hPASMC and hPAEC, and this effect was significantly reduced when blocking TGF-β1 expression, binding to TβRI, TβRI activity, or signaling pathways. In conclusion, oxidative stress and inflammation may trigger TGF-β1 secretion by hPASMC and hPAEC. TGF-β1 may then act as an autocrine factor on these cells, increasing NGF secretion via TβRI activation. Since NGF and TGF-β1 are relevant growth factors involved in PA remodeling, such mechanisms may therefore be relevant to PH pathophysiology.

Effect of leptin on the growth and expression of STAT3 in yak mammary epithelial cells.

Leptin (LEP) is an autocrine and paracrine factor produced by the fat pad and acinar epithelial cells of the breast. This study aimed to investigate the effects of LEP on yak mammary epithelial cells (YMECs) and the expression of STAT3. In addition, we evaluated the possible effects of prolactin (PRL) on the function of LEP. The YMECs were treated with 0, 50, 100, 200, 400, and 800 ng/mL LEP for 48 h in the absence of PRL and the presence of 500 ng/mL PRL. The growth activity of YMECs was measured using the cell counting kit-8 assay. The changes in the lactation signaling pathway-related factor STAT3 were detected at the mRNA, protein, and protein phosphorylation levels using the reverse transcriptase-quantitative polymerase chain reaction and Western blotting. To explore whether LEP affects the activation of STAT3 through JAK2/JAK3 in YMECs, the JAK2/3 signaling pathway inhibitor AG490 was used at a fixed concentration of LEP. Each concentration of LEP significantly promoted the expression of STAT3 mRNA (p < 0.05) in YMECs in the presence of PRL. In the absence of PRL, all concentrations of LEP were found to inhibit the expression of the STAT3 protein (p < 0.05). The expression of the STAT3 protein in YMECs was found to first increase followed by a decrease with an increase in the concentration of LEP. In addition, the phosphorylation level of STAT3 increased in all groups, except the 100 ng/mL concentration group. The STAT3 phosphorylation trend and protein expression were different, such that the level of protein phosphorylation was higher than that of the STAT3 protein (p < 0.05). The addition of AG490 reduced the expression of the STAT3 mRNA, STAT3 protein, and STAT3 phosphorylation in the LEP and LEP + PRL groups. Altogether, the results indicated that different concentrations of LEP exerted varying effects on the growth of YMECs and the expression of STAT3, and the activity of STAT3 was primarily activated by JAK2. The addition of LEP can effectively inhibit the downregulation of the JAK2/STAT3 signal pathway by AG490, mitigate its inhibitory effect on the proliferation of YMECs, and reduce apoptosis. We believe that these findings will provide a theoretical and experimental basis for future research in this field.

Cellular context alters EGF-induced ERK dynamics and reveals potential crosstalk with GDF-15.

Cellular signaling dynamics are sensitive to differences in ligand identity, levels, and temporal patterns. These signaling patterns are also impacted by the larger context that the cell experiences (i.e., stimuli such as media formulation or substrate stiffness that are constant in an experiment exploring a particular variable but may differ between independent experiments which explore that variable) although the reason for different dynamics is not always obvious. Here, we compared extracellular-regulated kinase (ERK) signaling in response to epidermal growth factor treatment of human mammary epithelial cells cultures in either well culture or a microfluidic device. Using a single-cell ERK kinase translocation reporter, we observed extended ERK activation in well culture and only transient activity in microfluidic culture. The activity in microfluidic culture resembled that of the control condition, suggesting that shear stress led to the early activity and a loss of autocrine factors dampened extended signaling. Through experimental analysis we identified growth differentiation factor-15 as a candidate factor that led to extended ERK activation through a protein kinase C-α/β dependent pathway. Our results demonstrate that context impacts ERK dynamics and that comparison of distinct contexts can be used to elucidate new aspects of the cell signaling network.

Immune responses in diabetic nephropathy: Pathogenic mechanisms and therapeutic target

Diabetic nephropathy (DN) is a chronic, inflammatory disease affecting millions of diabetic patients worldwide. DN is associated with proteinuria and progressive slowing of glomerular filtration, which often leads to end-stage kidney diseases. Due to the complexity of this metabolic disorder and lack of clarity about its pathogenesis, it is often more difficult to diagnose and treat than other kidney diseases. Recent studies have highlighted that the immune system can inadvertently contribute to DN pathogenesis. Cells involved in innate and adaptive immune responses can target the kidney due to increased expression of immune-related localization factors. Immune cells then activate a pro-inflammatory response involving the release of autocrine and paracrine factors, which further amplify inflammation and damage the kidney. Consequently, strategies to treat DN by targeting the immune responses are currently under study. In light of the steady rise in DN incidence, this timely review summarizes the latest findings about the role of the immune system in the pathogenesis of DN and discusses promising preclinical and clinical therapies.

CCL2 overexpression is associated with paclitaxel resistance in ovarian cancer cells via autocrine signaling and macrophage recruitment

Tumor cells can secret various cytokines and chemokines, which affect the tumor cells themselves and the neighboring cells. Here, we observed that human ovarian cancer (OC) cells developed resistance to paclitaxel treatment following culture with the conditioned medium (CM) derived from paclitaxel-resistant OC (OCTR) cells. A cytokine array revealed that both OCTR cells secreted large amounts of CC chemokine ligand 2 (CCL2). CCL2 and its receptor, CCR2, were overexpressed in OCTR cells. CCL2 expression was associated with worse progression-free survival in patients with ovarian cancer. The inhibition of the CCL2/CCR2 axis suppressed the chemoresistance induced by OCTR-CM. The enhanced expression and production of CCL2 in OC cells were mediated via the NF-κB pathway, and stimulated the activation of the PI3K/Akt pathway, which resulted in the development of paclitaxel resistance in OC cells. Additionally, the OCTR cells significantly increased the migration of macrophages, which was also associated with the overproduction of CCL2 in chemoresistant cancer cells. The macrophages stimulated by OCTR cells expressed high levels of markers of M2 phenotype, and their CM significantly decreased the paclitaxel responsiveness of OC cells. The administration of a CCR2 inhibitor to a murine model significantly improved the paclitaxel sensitivity. These data suggested that apart from inducing chemoresistance in OC cells by acting as an autocrine factor, CCL2 also functions as a chemokine that induces the chemotaxis of macrophages, which may contribute to chemoresistance. Therefore, targeting the CCL2/CCR2 signaling axis may improve the therapeutic response of patients with ovarian cancer to paclitaxel.

The importance of kidney calcium handling in the homeostasis of extracellular fluid calcium.

Extracellular fluid calcium concentration must be maintained within a narrow range in order to sustain many biological functions, encompassing muscle contraction, blood coagulation, and bone and tooth mineralization. Blood calcium value is critically dependent on the ability of the renal tubule to reabsorb the adequate amount of filtered calcium. Tubular calcium reabsorption is carried out by various and complex mechanisms in 3 distinct segments: the proximal tubule, the cortical thick ascending limb of the loop of Henle, and the late distal convoluted/connecting tubule. In addition, calcium reabsorption is tightly controlled by many endocrine, paracrine, and autocrine factors, as well as by non-hormonal factors, in order to adapt the tubular handling of calcium to the metabolic requirements. The present review summarizes the current knowledge of the mechanisms and factors involved in calcium handling by the kidney and, ultimately, in extracellular calcium homeostasis. The review also highlights some of our gaps in understanding that need to be addressed in the future.

Value of autotaxin in predicting refractory Mycoplasma pneumoniae pneumonia in children and its correlation with inflammatory cytokines

To study the value of autotaxin (an autocrine motility factor) level in serum and bronchoalveolar lavage fluid (BALF) in predicting refractory Mycoplasma pneumoniae pneumonia (RMPP) in children and its correlation with interleukin-6 (IL-6), interleukin-8 (IL-8), and C-reactive protein (CRP). A retrospective analysis was performed on 238 children with Mycoplasma pneumoniae pneumonia who were admitted from January 2019 to December 2021. According to disease severity, they were divided into two groups: RMPP (n=82) and general Mycoplasma pneumoniae pneumonia (GMPP; n=156). The two groups were compared in terms of the levels of autotaxin, IL-6, IL-8, and CRP in serum and BALF to study the value of autotaxin level in serum and BALF in predicting RMPP in children, as well as the correlation of autotaxin level with IL-6, IL-8, and CRP in children with RMPP. Compared with the GMPP group, the RMPP group had significantly higher levels of autotaxin, IL-6, IL-8, and CRP in serum and BALF (P<0.05). For the children with RMPP, the levels of autotaxin, IL-6, IL-8, and CRP in serum and BALF in the acute stage were significantly higher than those in the convalescent stage (P<0.05). The receiver operating characteristic (ROC) curve showed that the level of autotaxin in serum and BALF had a good value in predicting RMPP in children, with an area under the curve of 0.874 (95%CI: 0.816-0.935) and 0.862 (95%CI: 0.802-0.924), respectively. The correlation analysis showed that the level of autotaxin in serum and BALF was positively correlated with IL-6, IL-8, and CRP levels (P<0.001). The level of autotaxin in serum and BALF increases and is correlated with the degree of disease recovery and inflammatory cytokines in children with RMPP. Autotaxin can be used as a predictive indicator for RMPP in children.

Construction and Verification of a Fibroblast-Related Prognostic Signature Model for Colon Cancer.

Traditionally, cancer-associated fibroblasts (CAFs), an essential component of tumor microenvironment, were exert a crucial part in colon cancer progression. In this study, single-cell RNA-sequencing (scRNA-seq) data from 23 and bulk RNA-seq data from 452 colon cancer patients were extracted from the GEO database and TCGA-COAD and GEO databases, respectively. From single-cell analysis, 825 differentially expressed genes (DEGs) in CAFs were identified between each pair of six newly defined CAFs, named enCAF, adCAF, vaCAF, meCAF, erCAF, and cyCAF. Cell communication analysis with the iTALK package showed communication relationship between CAFs, including cell autocrine, cytokine, and growth factor subtypes, such as receptor-ligand pairs of TNFSF14-LTBR, IL6-F3, and IL6-IL6ST. Herein, we demonstrated the presence and prognostic value of adCAF and erCAF in colon cancer based on CIBERSORTx, combining single-cell marker genes and transcriptomics data. The prognostic significance of the enCAF and erCAF has been indirectly proved by both the correlation analysis with macrophages and CAFs, and the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) experiment based on 20 paired tumor samples. A prognostic model was constructed with 10 DEGs using the LASSO Cox regression method. The model was validated using two testing datasets, indicate a significant survival accuracy (p < 0.0025). Correlation analyses between clinical information, such as age, gender, tumor stage and tumor features (tumor purity and immune score), and risk scores revealed our CAF-related model’s robustness and excellent performance. Cell infiltration analysis by xCell revealed that the interaction between CAFs and multiple non-specific immune cells such as macrophages and the dendritic cell was a vital factor affecting immune score and prognosis. Finally, we analyzed how common anti-cancer drugs, including camptothecin, docetaxel and bortezomib, and immunotherapy, such as anti-PD-1 treatment, could be different in low-risk and high-risk patients inferred from our CAF-related model. In conclusion, the study utilized refined colon cancer fibroblast subsets and established the prognostic effects from the interaction with nonspecific immune cell.

O-264 Testosterone potentiates the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the maturation of spermatogonial cells in vitro from prepubertal mice

Abstract Study question Can granulocyte-macrophage colony-stimulating factor (GM-CSF) and testosterone induce spermatogonial cells to develop spermatogenesis in vitro using methylcellulose culture system (MCS)? Summary answer GM-CSF and testosterone induced the development of premeiotic and meiotic cells in vitro. Testosterone also induced and potentiate the development of post-meiotic cells by GM-CSF. What is known already Spermatogenesis is a complicated process of sperm generation. During this process spermatogonial cells proliferate and differentiate to meiotic, postmeiotic stages that continue in spermiogenesis to generate mature sperm. This process is under regulation of autocrine and paracrine factors provided by developed germ cells and somatic cells such as Sertoli, peritubular and Leydig cells. In addition, endocrine factors such as FSH, LH and testosterone are crucial for complete spermatogenesis. Different in vitro culture systems were used to induce the development of complete spermatogenesis in vitro, however this was not yet achieved. Study design, size, duration Sexually immature mice (7-day-old) were used to isolated cells from the seminiferous tubules. These isolated ells were cultured in methylcellulose culture system (MCS) as a 3-dimensinal in vitro system. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and testosterone were added in the beginning of the cultures and after two weeks. The cultures were determined after 4 weeks. Participants/materials, setting, methods Cells were enzymatically isolated from the seminiferous tubules and cultured (2x105/well/0.5ml) in MCS that contained StemPro-34 medium, KSR, rEGF, rGDNF, rLIF, and r-bFGF. The cultures were grown in the presence/absence of GM-CSF (0.1 or 1ng/ml) and/or testosterone (10-7M) and incubated for four weeks in a CO2 incubator at 37°C. The developed cells and colonies/spheroids were examined microscopically, and quantified the developing cells of the different stages of spermatogenesis by immunofluorescence staining (IF) and/or qPCR analyses Main results and the role of chance Our results show the presence of GM-CSF and its receptor (GM-CSFR) in different testicular cells. We demonstrate the development of colonies/spheroids which contain premeiotic, meiotic and post-meiotic cells in MCS, as examined by IF and qPCR analyses. The percentage of pre-meiotic and miotic stage in the cultures with GM-CSF were significantly higher than those in the control group (p &amp;lt; 0.001), and the percentage of cells of the post-meiotic stage were significantly higher in the cultures with GM-CSF and testosterone together compared to each one separately (p &amp;lt; 0.001). In conclusion, testicular cells express GM-CSF/GM-CSFR, and GM-CSF affects only the development of premeiotic and meiotic cells in vitro. In addition, testosterone not only increased the development of cells from the premeiotic, meiotic and post-meiotic stages, but it also potentiated the effect of GM-CSF on the development of post-meiotic cells in vitro. Limitations, reasons for caution no limitation Wider implications of the findings These findings providing evidence that GM-CSF and testosterone could be used to improve the maturation of spermatogonial cells in vitro and that they might play a role in the maturation process of spermatogonial cells in vivo. Trial registration number not applicable

P-422 A retrospective cohort study on the usefulness of Endometrial Receptivity Analysis (ERA) prior to a gestational carriers’ (GC) first euploid frozen embryo transfer (FET)

Abstract Study question Does determination of endometrial receptivity by ERA improve the outcome of FET for GC prior to their first FET using a euploid embryo? Summary answer GC with ERA prior to FET had 92.3% implantation and 84.6% live birth rate (LBR) compared to 81.5% implantation rate and 72.2% LBR without ERA. What is known already The endometrial window of implantation is approximately 24 hours wide with a complex interaction of autocrine, paracrine and endocrine factors. Successful embryo implantation involves a 3-step process of apposition, adhesion and invasion of an embryo into a receptive endometrium. The ERA was developed using the expression profile of 248 genes using Next Generation Sequencing to determine if the endometrium is receptive, early receptive, late receptive, pre-receptive or post-receptive to objectively determine the optimal timing of progesterone exposure prior to embryo transfer. Some studies have shown statistically significant improvements in live birth rates while other studies have shown no difference. Study design, size, duration A retrospective cohort study compared the implantation and live birth rates of 26 GC who chose to have a personalized embryo transfer using their ERA profile vs. 54 GC who used our standard FET protocol without an ERA prior to their first euploid FET. All GC having their first FET from January 2018 – December 2020 were included. Participants/materials, setting, methods A single private practice fertility clinic performed 80 euploid FET cycles on first-time GC from January 2018 – December 2020. All intended parents were offered the opportunity to complete an ERA for their GC prior to their first FET cycle. Implantation and live birth rates were calculated for all FET cycles using a GC for the first time with associated p – values. Main results and the role of chance GC were 22 – 38 years old with a mean age of 30 and a history of 2.4 previous spontaneous births prior to their first FET. GC who used a standard FET protocol with 5 days of progesterone (P + 5) exposure before FET had an implantation rate of 81.5% and a live birth rate of 72.2%, compared to an implantation rate of 92.3% and a live birth rate of 84.6% for GC who used a personalized embryo transfer protocol based on their ERA profile (p = 0.17). 19.2% of GC had a receptive endometrial profile using P + 5, 26.9% of GC were pre-receptive needing P + 5.5 and 53.8% needed P + 6. No GC were found to have a post-receptive endometrial profile (P + 4 or P + 4.5). Our retrospective cohort study was not powered to find a statistically significant difference, though our data trended towards an improvement in both implantation and live birth rates by over 10% if a GC were to complete an ERA prior to her first FET. Limitations, reasons for caution Implantation and live birth rates for the clinic during the study period were 75% and 68% respectively using euploid embryos. Our high baseline implantation and live birth rates, coupled with low numbers of patients included in this study, may make these results less generalizable to the IVF population at large. Wider implications of the findings It is controversial to recommend an ERA to a good prognosis patient such as a GC without a prior unsuccessful euploid FET. These results should prompt further study to confirm our findings with a larger prospective randomized controlled trial of gestational carriers using ERA profiles prior to their first FET. Trial registration number N/A

P-424 Endometrial receptivity as determined by Endometrial Receptivity Analysis (ERA) can change after a successful live birth from a frozen embryo transfer (FET)

Abstract Study question Endometrial receptivity as determined by Endometrial Receptivity Analysis (ERA) can change after a successful live birth from a frozen a frozen embryo transfer (FET) Summary answer 27 of 33 patients with a previous live birth from FET had an altered endometrial receptivity profile by ERA after a subsequent unsuccessful FET. What is known already The endometrial window of implantation is approximately 24 hours wide with a complex interaction of autocrine, paracrine, and endocrine factors. Successful embryo implantation involves a 3-step process of apposition, adhesion, and invasion of an embryo into a receptive endometrium. The ERA was developed using the expression profile of 248 genes using Next Generation Sequencing to determine if the endometrium is receptive, early receptive, late receptive, pre-receptive or post-receptive to objectively determine the optimal timing of progesterone exposure prior to embryo transfer. Some studies have shown statistically significant improvements in live birth rates while other studies have shown no difference. Study design, size, duration Case series of 33 patients with successful live birth following euploid FET using 5 days of progesterone (P + 5) with subsequent unsuccessful FET using the same protocol from January 2018 – December 2020. 27 of 33 patients who repeated their ERA were found to be non-receptive using P + 5. The new ERA profile results were used for their next FET. Implantation and live birth rates were calculated. Participants/materials, setting, methods A single private practice fertility clinic performed 340 ERA cycles from January 2018 – December 2020 with 276 subsequent FET cycles. An observational study of 33 patients with a previous live birth using P + 5 elected to repeat their ERA cycle after an unsuccessful FET. Implantation and live birth rates were calculated for their subsequent FET after completing the repeat ERA. Main results and the role of chance Repeat ERA result for patients with a previous live birth using P + 5 revealed that 6 patients still needed P + 5 (18.2%) for a receptive endometrial profile, 8 patients (24.2%) needed an extra 12 hours (P + 5.5), 18 patients (54.5%) needed an extra 24 hours (P + 6) and only one patient (3.0%) needed an extra 48 hours (P + 7). No patient that repeated their ERA after a previous live birth had a post-receptive profile (P + 4 or P + 4.5) and one patient had successful live births after using 3 different ERA profiles (P + 5, P + 6 and P + 7). All patients except one had repeat ERA results that were pre-receptive with only one patient changing from P + 6 to P + 5. FET cycles using P + 5 and P + 7 had a 100% implantation and pregnancy rate, while patients using P + 5.5 had the lowest implantation rate (62.5%) and live birth rate (37.5%). Patients with an ERA profile of P + 6 had an implantation rate of 94.1% and live birth rate of 88.2%. The small number of patients in this case series make a type I error possible because of the low number of patients in each ERA profile category with insufficient power to reach statistical significance. Limitations, reasons for caution Implantation rate for the clinic during the study period was 75% and live birth rate of 68% using euploid embryos. Our high baseline implantation and live birth rates, coupled with low numbers of patients in this observational study, make these results less generalizable to the IVF population at large. Wider implications of the findings It is widely accepted that ERA results are reproducible and do not change during a woman’s reproductive life. These results suggest that some women may have an altered window of receptivity after both successful and unsuccessful FET cycles. It may be reasonable to repeat ERA cycles for carefully selected patients. Trial registration number N/A

Lentiviral interferon: A novel method for gene therapy in bladder cancer

Interferon alpha (IFNα) gene therapy is emerging as a new treatment option for patients with non-muscle invasive bladder cancer (NMIBC). Adenoviral vectors expressing IFNα have shown clinical efficacy treating bacillus Calmette-Guerin (BCG)-unresponsive bladder cancer (BLCA). However, transient transgene expression and adenoviral immunogenicity may limit therapeutic activity. Lentiviral vectors can achieve stable transgene expression and are less immunogenic. In this study, we evaluated lentiviral vectors expressing murine IFNα (LV-IFNα) and demonstrate IFNα expression by transduced murine BLCA cell lines, bladder urothelium, and within the urine following intravesical instillation. Murine BLCA cell lines (MB49 and UPPL1541) were sensitive to IFN-mediated cell death after LV-IFNα, whereas BBN975 was inherently resistant. Upregulation of interleukin-6 (IL-6) predicted sensitivity to IFN-mediated cell death mediated by caspase signaling, which when inhibited abrogated IFN-mediated cell killing. Intravesical therapy with LV-IFNα/Syn3 in a syngeneic BLCA model significantly improved survival, and molecular analysis of treated tumors revealed upregulation of apoptotic and immune-cell-mediated death pathways. In particular, biomarker discovery analysis identified three clinically actionable targets, PD-L1, epidermal growth factor receptor (EGFR), and ALDHA1A, in murine tumors treated with LV-IFNα/Syn3. Our findings warrant the comparison of adenoviral and LV-IFNα and the study of novel combination strategies with IFNα gene therapy for the BLCA treatment.

Delayed Maturation of Oligodendrocyte Progenitors by Microgravity: Implications for Multiple Sclerosis and Space Flight.

In previous studies, we examined the effects of space microgravity on human neural stem cells. To date, there are no studies on a different type of cell that is critical for myelination and electrical signals transmission, oligodendrocyte progenitors (OLPs). The purpose of the present study was to examine the behavior of space-flown OLPs (SPC-OLPs) as they were adapting to Earth’s gravity. We found that SPC-OLPs survived, and most of them proliferated normally. Nonetheless, some of them displayed incomplete cytokinesis. Both morphological and ontogenetic analyses showed that they remained healthy and expressed the immature OLP markers Sox2, PDGFR-α, and transferrin (Tf) after space flight, which confirmed that SPC-OLPs displayed a more immature phenotype than their ground control (GC) counterparts. In contrast, GC OLPs expressed markers that usually appear later (GPDH, O4, and ferritin), indicating a delay in SPC-OLPs’ development. These cells remained immature even after treatment with culture media designed to support oligodendrocyte (OL) maturation. The most remarkable and surprising finding was that the iron carrier glycoprotein Tf, previously described as an early marker for OLPs, was expressed ectopically in the nucleus of all SPC-OLPs. In contrast, their GC counterparts expressed it exclusively in the cytoplasm, as previously described. In addition, analysis of the secretome demonstrated that SPC-OLPs contained 3.5 times more Tf than that of GC cells, indicating that Tf is gravitationally regulated, opening two main fields of study to understand the upregulation of the Tf gene and secretion of the protein that keep OLPs at a progenitor stage rather than moving forward to more mature phenotypes. Alternatively, because Tf is an autocrine and paracrine factor in the central nervous system (CNS), in the absence of neurons, it accumulated in the secretome collected after space flight. We conclude that microgravity is becoming a novel platform to study why in some myelin disorders OLPs are present but do not mature.

Multiomics analysis of male infertility

Infertility affects 8-12% of couples globally, and the male factor is a primary cause in ~50% of couples. Male infertility is a multifactorial reproductive disorder, which can be caused by paracrine and autocrine factors, hormones, genes, and epigenetic changes. Recent studies in rodents and most notably in humans using multiomics approach have yielded important insights into understanding the biology of spermatogenesis. Nonetheless, the etiology and pathogenesis of male infertility are still largely unknown. In this review, we summarized and critically evaluated findings based on the use of advanced technologies to compare normal and obstructive azoospermic versus nonobstructive azoospermic men, including whole-genome bisulfite sequencing, single-cell RNA-seq, whole-exome sequencing, and transposase-accessible chromatin using sequencing. It is obvious that the multiomics approach is the method of choice for basic research and clinical studies including clinical diagnosis of male infertility.

Shedding Light on the Complex Regulation of FGF23.

Early research has suggested a rather straightforward relation between phosphate exposure, increased serum FGF23 (Fibroblast Growth Factor 23) concentrations and clinical endpoints. Unsurprisingly, however, subsequent studies have revealed a much more complex interplay between autocrine and paracrine factors locally in bone like PHEX and DMP1, concentrations of minerals in particular calcium and phosphate, calciprotein particles, and endocrine systems like parathyroid hormone PTH and the vitamin D system. In addition to these physiological regulators, an expanding list of disease states are shown to influence FGF23 levels, usually increasing it, and as such increase the burden of disease. While some of these physiological or pathological factors, like inflammatory cytokines, may partially confound the association of FGF23 and clinical endpoints, others are in the same causal path, are targetable and hence hold the promise of future treatment options to alleviate FGF23-driven toxicity, for instance in chronic kidney disease, the FGF23-associated disease with the highest prevalence by far. These factors will be reviewed here and their relative importance described, thereby possibly opening potential means for future therapeutic strategies.