Basal Levels Research Articles

Evaluation of the diagnostic value of different serum biomarkers in girls with central precocious puberty

BackgroundAlarming trends show an increase in referrals of girls with suspected early puberty. However, variations in defining precocious puberty exist. GnRH stimulation test is the gold standard diagnostic test, which is expensive and challenging to interpret. Moreover, intravenous GnRH might not be available. Therefore, we sought to evaluate the diagnostic performance of basal serum Luteinizing Hormone and Anti-Mullerian hormone in girls with central precocious puberty, as an easier alternative to GnRH stimulation test. The study was conducted on 26 Egyptian girls presenting with early puberty at age < 8 years. Several evaluations including Tanner staging and GnRH stimulation test were performed. Basal serum luteinizing hormone, follicle stimulating hormone, and anti-Mullerian hormone were analyzed, and ROC curve was performed.ResultsThe mean age of the patients at diagnosis was 5 ± 2.8 years. Most patients had breast Tanner 3 (76.9%). Bone age was advanced in 65.4% of patients. Basal luteinizing hormone level significantly differed between central precocious puberty and controls (p < 0.001), with an area under the curve of 0.840. A cut-off of 0.29 IU/L provided 84.62% sensitivity and 76.92% specificity.ConclusionsBasal luteinizing hormone out-performs follicle-stimulating hormone and anti-Mullerian hormone as a screening tool and valuable indicator of central precocious puberty, potentially reducing the necessity of GnRH stimulation test.

EROD activity in the native fish Cnesterodon decemmaculatus as a biomarker for assessing aquatic pollution by AhR agonist chemicals within the Rio de la Plata Basin

The 7-ethoxyresorufin-O-deethylase (EROD) activity was first time characterized in the neotropical fish Cnesterodon decemmaculatus as a biomarker for assessing environmental health in aquatic ecosystems of the Rio de la Plata Basin impacted by organic pollutants agonist of the aryl-hydrocarbon receptor (AhR). Both laboratory and field studies were conducted. Laboratory experiments were run using β-naphthoflavone (BNF) as an AhR agonist model. A clear concentration-response relationship was found between 1 and 100 μg/L, with a NOEC and LOEC of 1 and 10 μg/L. A fast time-dependent response was observed with a significant induction after 24 h and a plateau from 24 to 48 h up to 264 h of exposure. Differences in basal activity were found between juveniles, females, and males, but induction levels were similar. Both basal activities and induction levels were distinct in the whole body, liver, gill, muscle, brain, and embryos. Fold-change inductions in the respective tissues were: 20, 114, 3, 5, 1, and 14. Maternal transfer and early cyp1a activation were unveiled by embryonic induction. Clear differences in EROD activity were found among juveniles collected in hydrocarbon-polluted streams, beside the La Plata Petrochemical hub, and a reference stream. Similar EROD activities were observed in laboratory and feral fish, usually with values below or above 1000 pmol/min x mg protein for unexposed or exposed organisms. The study contributes with original information about EROD activity in C. decemmaculatus that encourages the use of both the response as a robust biomarker of exposure and the species as a good sentinel organism to be included in surveillant programs for assessing aquatic pollution by AhR agonist chemicals within the Rio de la Plata Basin within the One Health paradigm.

1β-Hydroxydeoxycholic Acid as an Endogenous Biomarker in Human Plasma for Assessment of CYP3A Clinical Drug-Drug Interaction Potential.

4β-Hydroxycholesterol (4β-HC) in plasma has been used as a biomarker to assess CYP3A drug-drug interaction (DDI) potential during drug development. However, due to the long half-life and narrow dynamic range of 4β-HC, its use has been limited to the identification of CYP3A inducers, but not CYP3A inhibitors. The formation of 1β-hydroxydeoxycholic acid (1β-OH DCA) from deoxycholic acid (DCA) is mediated by CYP3A, thus 1β-OH DCA can potentially serve as an alternative to 4β-HC for assessment of CYP3A DDI potential. To study this feasibility, we developed a sensitive LC-MS/MS method for the simultaneous quantitation of 1β-OH DCA and its glycine and taurine conjugates in human plasma with the LLOQ of 50 pg/mL, which enabled the quantitation of basal levels and further reduction. The method was applied to a DDI study to assess how 1β-OH DCA and its glycine and taurine conjugates would respond to CYP3A induction or inhibition. Rifampin induction resulted in an increase of 1β-OH DCA and its conjugates in plasma, with 6.8-, 7.8-, 8.3-, 10.3-fold increases of AUCLST, AUC24h, Cmax and mean concentrations for total 1β-OH DCA (total of all three forms), respectively. Importantly, inhibition with itraconazole resulted in notable reduction of these biomarkers, with 84%, 85%, 82%, 81% reductions of AUCLST, AUC24h, Cmax and mean concentrations for total 1β-OH DCA, respectively. This preliminary data demonstrates for the first time that total 1β-OH DCA in plasma has the potential to serve as a biomarker for CYP3A DDI assessment in early clinical development and may provide key advantages over 4β-HC. Significance Statement We have reported the use of total 1β-Hydroxydeoxycholic Acid (1β-OH DCA) (sum of 1β-OH DCA and its glycine and taurine conjugates) plasma concentration as a biomarker for CYP3A activity. Itraconazole inhibition led to an 81-85% decrease of total 1β-OH DCA plasma exposures, while rifampin induction led to a 6.8-10.3 fold increase of total 1β-OH DCA plasma exposures. Using 1β-OH DCA exposures in plasma also provides benefit of allowing PK and biomarker assessment using the same matrix, thus simplify collection procedures.

Physiological responses of invasive round goby (Neogobius melanostomus) to environmental stressors across a latitudinal span

AbstractEcosystems worldwide are struggling with increasingly high levels of human-induced stressors, impacting their ecological health at local, regional, and global scales. Among the factors affecting freshwater organisms, temperature stands out, especially given the recent escalation of global warming, with possible adverse effects. In this study, we examined the basal levels of oxidative stress parameters in the round goby Neogobius melanostomus, spanning its native and non-native ranges of occurrence. We aimed to assess five populations across transcontinental scale (Turkey, Croatia, Slovakia, Poland, Finland) to determine whether individuals already experiencing conditions other than their thermal optimum (such as those in Turkey and Finland) would display elevated non-enzymatic indicators (level of lipid peroxidation and reduced glutathione levels) and enzymatic indicators (activity of glutathione peroxidase and catalase activity) of oxidative stress compared to those inhabiting milder ecosystems. The results obtained did not align with our initial predictions. The native Turkish population exhibited the lowest values for both antioxidants and oxidative damage, indicating an efficient redox system in this species. All tested parameters showed variation among populations, as well as between tissues and sexes. Temperature did not emerge as significant factor in any of the parameters tested, while for glutathione peroxidase, our analysis indicated a positive relationship with enzyme activity and salinity. The lack of a continuous latitudinal pattern in the non-native populations suggests the species’ potential for adaptive shifts, indicating superior adaptive abilities, especially in human affected ecosystems, rather than strictly adhering to environmental gradients.

Differential regulation by CD47 and thrombospondin-1 of extramedullary erythropoiesis in mouse spleen

Extramedullary erythropoiesis is not expected in healthy adult mice, but erythropoietic gene expression was elevated in lineage-depleted spleen cells from Cd47−/− mice. Expression of several genes associated with early stages of erythropoiesis was elevated in mice lacking CD47 or its signaling ligand thrombospondin-1, consistent with previous evidence that this signaling pathway inhibits expression of multipotent stem cell transcription factors in spleen. In contrast, cells expressing markers of committed erythroid progenitors were more abundant in Cd47−/− spleens but significantly depleted in Thbs1−/− spleens. Single-cell transcriptome and flow cytometry analyses indicated that loss of CD47 is associated with accumulation and increased proliferation in spleen of Ter119−CD34+ progenitors and Ter119+CD34− committed erythroid progenitors with elevated mRNA expression of Kit, Ermap, and Tfrc. Induction of committed erythroid precursors is consistent with the known function of CD47 to limit the phagocytic removal of aged erythrocytes. Conversely, loss of thrombospondin-1 delays the turnover of aged red blood cells, which may account for the suppression of committed erythroid precursors in Thbs1−/− spleens relative to basal levels in wild-type mice. In addition to defining a role for CD47 to limit extramedullary erythropoiesis, these studies reveal a thrombospondin-1-dependent basal level of extramedullary erythropoiesis in adult mouse spleen.

Differential regulation by CD47 and thrombospondin-1 of extramedullary erythropoiesis in mouse spleen.

Extramedullary erythropoiesis is not expected in healthy adult mice, but erythropoietic gene expression was elevated in lineage-depleted spleen cells from Cd47-/- mice. Expression of several genes associated with early stages of erythropoiesis was elevated in mice lacking CD47 or its signaling ligand thrombospondin-1, consistent with previous evidence that this signaling pathway inhibits expression of multipotent stem cell transcription factors in spleen. In contrast, cells expressing markers of committed erythroid progenitors were more abundant in Cd47-/- spleens but significantly depleted in Thbs1-/- spleens. Single-cell transcriptome and flow cytometry analyses indicated that loss of CD47 is associated with accumulation and increased proliferation in spleen of Ter119-CD34+ progenitors and Ter119+CD34- committed erythroid progenitors with elevated mRNA expression of Kit, Ermap, and Tfrc. Induction of committed erythroid precursors is consistent with the known function of CD47 to limit the phagocytic removal of aged erythrocytes. Conversely, loss of thrombospondin-1 delays the turnover of aged red blood cells, which may account for the suppression of committed erythroid precursors in Thbs1-/- spleens relative to basal levels in wild-type mice. In addition to defining a role for CD47 to limit extramedullary erythropoiesis, these studies reveal a thrombospondin-1-dependent basal level of extramedullary erythropoiesis in adult mouse spleen.

Enhancing Therapeutic Approaches in Glioblastoma with Pro-Oxidant Treatments and Synergistic Combinations: In Vitro Experience of Doxorubicin and Photodynamic Therapy

Glioblastoma (GBM) is an aggressive brain cancer characterized by significant molecular and cellular heterogeneity, which complicates treatment efforts. Current standard therapies, including surgical resection, radiation, and temozolomide (TMZ) chemotherapy, often fail to achieve long-term remission due to tumor recurrence and resistance. A pro-oxidant environment is involved in glioma progression, with oxidative stress contributing to the genetic instability that leads to gliomagenesis. Evaluating pro-oxidant therapies in brain tumors is crucial due to their potential to selectively target and eradicate cancer cells by exploiting the elevated oxidative stress levels inherent in these malignant cells, thereby offering a novel and effective strategy for overcoming resistance to conventional therapies. This study investigates the therapeutic potential of doxorubicin (DOX) and photodynamic therapy (PDT) with Me-ALA, focusing on their effects on redox homeostasis. Basal ROS levels and antioxidant gene expression (NFE2L2, CAT, GSR) were quantitatively assessed across GBM cell lines, revealing significant variability probably linked to genetic differences. DOX and PDT treatments, both individually and in combination, were analyzed for their efficacy in inducing oxidative stress and cytotoxicity. An in silico analysis further explored the relationship between gene mutations and oxidative stress in GBM patients, providing insights into the molecular mechanisms underlying treatment responses. Our findings suggest that pro-oxidant therapies, such as DOX and PDT in combination, could selectively target GBM cells, highlighting a promising avenue for improving therapeutic outcomes in GBM.

Nucleus Accumbens Corticotropin-Releasing Hormone Neurons Projecting to the Bed Nucleus of the Stria Terminalis Promote Wakefulness and Positive Affective State.

The nucleus accumbens (NAc) plays an important role in various emotional and motivational behaviors that rely on heightened wakefulness. However, the neural mechanisms underlying the relationship between arousal and emotion regulation in NAc remain unclear. Here, we investigated the roles of a specific subset of inhibitory corticotropin-releasing hormone neurons in the NAc (NAcCRH) in regulating arousal and emotional behaviors in mice. We found an increased activity of NAcCRH neurons during wakefulness and rewarding stimulation. Activation of NAcCRH neurons converts NREM or REM sleep to wakefulness, while inhibition of these neurons attenuates wakefulness. Remarkably, activation of NAcCRH neurons induces a place preference response (PPR) and decreased basal anxiety level, whereas their inactivation induces a place aversion response and anxious state. NAcCRH neurons are identified as the major NAc projection neurons to the bed nucleus of the stria terminalis (BNST). Furthermore, activation of the NAcCRH-BNST pathway similarly induced wakefulness and positive emotional behaviors. Taken together, we identified a basal forebrain CRH pathway that promotes the arousal associated with positive affective states.

Enhancing Therapeutic Approaches in Glioblastoma with Pro-Oxidant Treatments and Synergistic Combinations: In Vitro Experience of Doxorubicin and Photodynamic Therapy.

Glioblastoma (GBM) is an aggressive brain cancer characterized by significant molecular and cellular heterogeneity, which complicates treatment efforts. Current standard therapies, including surgical resection, radiation, and temozolomide (TMZ) chemotherapy, often fail to achieve long-term remission due to tumor recurrence and resistance. A pro-oxidant environment is involved in glioma progression, with oxidative stress contributing to the genetic instability that leads to gliomagenesis. Evaluating pro-oxidant therapies in brain tumors is crucial due to their potential to selectively target and eradicate cancer cells by exploiting the elevated oxidative stress levels inherent in these malignant cells, thereby offering a novel and effective strategy for overcoming resistance to conventional therapies. This study investigates the therapeutic potential of doxorubicin (DOX) and photodynamic therapy (PDT) with Me-ALA, focusing on their effects on redox homeostasis. Basal ROS levels and antioxidant gene expression (NFE2L2, CAT, GSR) were quantitatively assessed across GBM cell lines, revealing significant variability probably linked to genetic differences. DOX and PDT treatments, both individually and in combination, were analyzed for their efficacy in inducing oxidative stress and cytotoxicity. An in silico analysis further explored the relationship between gene mutations and oxidative stress in GBM patients, providing insights into the molecular mechanisms underlying treatment responses. Our findings suggest that pro-oxidant therapies, such as DOX and PDT in combination, could selectively target GBM cells, highlighting a promising avenue for improving therapeutic outcomes in GBM.

HIF-1 Transcriptionally Regulates Basal Expression of STING to Maintain Cellular Innate Immunity.

Stimulator of IFN genes (STING) is a critical component of the innate immune system, playing an essential role in defending against DNA virus infections. However, the mechanisms governing basal STING regulation remain poorly understood. In this study, we demonstrate that the basal level of STING is critically maintained by hypoxia-inducible factor 1 (HIF-1)α through transcription. Under normal conditions, HIF-1α binds constitutively to the promoter region of STING, actively promoting its transcription. Knocking down HIF-1α results in a decrease in STING expression in multiple cell lines and zebrafish, which in turn reduces cellular responses to synthetic dsDNAs, including cell signaling and IFN production. Moreover, this decrease in STING levels leads to an increase in cellular susceptibility to DNA viruses HSV-1 and pseudorabies virus. These findings unveil a (to our knowledge) novel role of HIF-1α in maintaining basal STING levels and provide valuable insights into STING-mediated antiviral activities and associated diseases.

Effects of preduodenal ileal surgical transposition on enteroendocrine intestinal cells in wistar rats: Histomorphological and serum changes.

In our study, we focused on the role of the distal ileum as a main endocrine actor in relation to the pancreas. We investigated the effects of intestinally released hormones on the pancreas in terms of type 2 diabetes mellitus (T2DM) improvement, as a main effect of bariatric surgeries. To specifically study the importance of the ileum, we used an experimental surgical model performed in healthy Wistar rats. After preduodenal transposition of the ileum, we analyzed the histology and enterohormonal cells of the intestine. We measured the plasma level of several hormones and effectors in this enteropancreatic axis. We used a surgical control (Sham) group and a surgical group, where ileum preduodenal transposition (PDIT) was performed. We measured basal glycemia and serum levels of several incretins, including GLP-1, PYY, and GIP, and we performed a glucose overdose test. After two test periods, the basal glycemia and glucose overdose results were not different between groups, however, the PDIT group had significantly increased expression of GLP-1, with increased cellular release in the ileum and duodenum compared with the Sham group. Both plasma GIP levels and GIP tissue expression were decreased in the PDIT group compared with the sham group. There were no differences in PPY hormone levels. The ileum crypts and villi of the PDIT group showed improvement in histological parameters. We concluded that model animals had an altered transposed ileum related to the enterohormonal adaptation of the ileum. Our results indicated that the ileum is important in the hormonal control of the enteropancreatic axis.

Programmed RNA editing with an evolved bacterial adenosine deaminase.

Programmed RNA editing presents an attractive therapeutic strategy for genetic disease. In this study, we developed bacterial deaminase-enabled recoding of RNA (DECOR), which employs an evolved Escherichia coli transfer RNA adenosine deaminase, TadA8e, to deposit adenosine-to-inosine editing to CRISPR-specified sites in the human transcriptome. DECOR functions in a variety of cell types, including human lung fibroblasts, and delivers on-target activity similar to ADAR-overexpressing RNA-editing platforms with 88% lower off-target effects. High-fidelity DECOR further reduces off-target effects to basal level. We demonstrate the clinical potential of DECOR by targeting Van der Woude syndrome-causing interferon regulatory factor 6 (IRF6) insufficiency. DECOR-mediated RNA editing removes a pathogenic upstream open reading frame (uORF) from the 5' untranslated region of IRF6 and rescues primary ORF expression from 12.3% to 36.5%, relative to healthy transcripts. DECOR expands the current portfolio of effector proteins and opens new territory in programmed RNA editing.

Serum androgen dynamics in young women aged 18-40 treated with chemotherapy for breast cancer: an observational, multicentric, prospective study in France

Although the deleterious impact of chemotherapy regimen used to treat women of reproductive age with breast cancer on ovarian reserve has been extensively studied, hardly anything has been reported on the effect of these protocols on theca cell function and ovarian androgen secretion. The aim of this prospective multicentric cohort study was to describe serum levels of total testosterone and androstenedione during chemotherapy and 24-month follow-up in 250 patients <40 years treated for breast cancer. Mean basal levels of androstenedione and total testosterone at diagnosis were 1.68 ng/mL and 0.20 ng/mL respectively. No correlation with age was found. Serum levels of androstenedione and total testosterone rapidly decreased after chemotherapy completion, before slowly increasing and almost returning to basal levels in all patients during 2-year follow-up. In conclusion our study demonstrates a chemotherapy-induced alteration of ovarian thecal function, resulting in a significant decrease in serum androgen levels. This alteration of theca cell function adds to the well-known alteration of granulosa cell function, resulting in a global, but partly transient, ovarian failure in young women treated for breast cancer. These data bring new insight into ovarian physiology and emphasize the need for pre and post-treatment ovarian follow-up. Trial registration: ClinicalTrial.gov identifier NCT01114464

Study of the Relationship between Diabetic Retinopathy, Basal Insulin Therapy (Glargine or NPH) and Insulin-Like Growth Factor 1 Serum Level

Abstract Background Diabetic retinopathy (DR) is the major cause of preventable blindness worldwide. DR is a microvascular complication of diabetes mellitus that is more prevalent in patients with type 1 diabetes. Type 1 diabetes mellitus is an autoimmune disease that leads to insulin deficiency so patients require long term therapy with basal insulin for optimum glycemic control. Insulin- like growth factor 1 (IGF-1) is an important growth factor that is involved in cell proliferation and angiogenesis. There is a lot of evidence that IGF-1 is tightly related to DR development. Insulin Glargine is a long-acting insulin analogue which is peak-free and less hypoglycemic as compared to the intermediate-acting NPH insulin. Glargine has an increased affinity to IGF-1 receptor and this raised concern about its role in development of DR. The relationship between diabetic retinopathy, basal insulin therapy and IGF-1 serum level is still in debate. Aim we aimed to compare the incidence and severity of diabetic retinopathy in type 1 diabetic patients on basal insulin therapy (Glargine or NPH) and its relation to serum IGF-1 level. Methods The current study included 88 subjects with type 1 diabetes divided into 44 on basal insulin therapy with Glargine and actrapid ( Group A) and 44 on NPH and actrapid ( Group B). Hemoglobin A1c, Total cholesterol, Triglycerides, High density lipoproteins, Low density lipoproteins and serum IGF-1 levels were assessed. Full clinical examination including (weight, height, BMI), vital signs (pulse, blood pressure) was done and Fundus examination by Optomed Aurora® IQ handheld fundus camera. Results On comparing the 2 studied groups, There was no statistical significant difference as regarding fundus examination findings between studied groups (P value = 0.429). There was no statistically significant relation between diabetic retinopathy and serum IGF-1 (P value = .080 with Mean ± SD of serum IGF-1 level in patients with Normal fundus 14.8 ± 11 vs. Patients with Diabetic retinopathy 21.3 ± 16.9). There was statistically significant association between diabetic retinopathy and duration of diabetes (P value&amp;lt; 0.05 with Mean ± SD of diabetes duration in patients with Diabetic retinopathy 17.7 ± 6.9 vs. Patients with Normal fundus 14.6 ± 5.6). There was a statistical significant difference as regarding age (P value&amp;lt; 0.05 with Mean ± SD of age in Group B regimen 28.4 ± 6.7 vs. Group A 25.5 ± 4.9). Conclusion Serum IGF-1 levels showed no statistically significant difference in patients either on Glargine or NPH and also in patients either with normal fundus or with diabetic retinopathy. The incidence and severity of diabetic retinopathy in patients with type 1 diabetes mellitus is directly proportionate to duration of disease. No noted difference concerning diabetic retinopathy changes between patients on basal insulin therapy either Glargine or NPH.

P-214 Direct unequal cleavage-1 (DUC1) in IVF/ICSI cycles: relationship with patient characteristics and insemination method

Abstract Study question Does the incidence of DUC1 in IVF/ICSI cycles vary with the maternal profile and insemination method? Summary answer The incidence of DUC1 cycles is higher in patients with a more favourable prognosis. DUC1 embryos are more frequent in ICSI than in IVF cycles. What is known already The emergence of Time-Lapse Microscopy (TLM) has enabled a dynamic and thorough evaluation of in vitro embryo development, unveiling the occurrence of morphological phenomena unobserved with static observation, such as direct unequal cleavage (DUC), which is associated with a drastically reduced competence to implant. The incidence of DUC-1 (DUC immediately after syngamy) has been reported to vary from 8.3% to 26%, but the reasons underlying its occurrence and variability are unknown. There is evidence that the maternal profile and insemination method can impact embryo developmental competence, but whether they impact the incidence of DUC is also unknown. Study design, size, duration The study includes 2920 first IVF/ICSI cycles (2018 to 2022) providing 12492 embryos. Cycles presenting male infertility were excluded from the analysis. The percentages of DUC1 embryos in relation to total embryos and of cycles providing at least one DUC1 embryo in relation to total cycles were compared between AMA (advanced maternal age; ≥35) and pre-AMA (&amp;lt;35) cycles. Patient/cycle characteristics were compared between cycles with and without DUC1 occurrence in AMA and pre-AMA cycles, separately. Participants/materials, setting, methods Ovarian stimulation was induced with rFSH or with a combination of FSH and LH using classic antagonist or short agonist protocols. Oocytes were inseminated with either IVF or ICSI standard methodology. Embryo culture was performed in an integrated embryo culture Time-lapse System (EmbryoScope; Vitrolife). Differences between percentages were assessed by the Fisher’s exact test, while differences between continuous variables were assessed with the Wilcoxon sum rank test. Main results and the role of chance The percentage of DUC1 embryos in relation to total embryos did not vary between pre-AMA (5.4%) and AMA (5.2%; p &amp;gt; 0.05) cycles. Alternatively, the incidence of cycles providing at least one DUC1 embryo was higher in pre-AMA (21.7%) as compared with AMA patients (16.0%; p &amp;lt; 0.0001). In both pre-AMA and AMA cycles, DUC1 occurrence was associated with higher oocyte yield (13.5±7.7 vs. 10.6±6.7 and 11.0±6.3 vs. 7.5±5.5, respectively; p &amp;lt; 0.0001), higher AMH serum levels (4.4±4.7 vs. 3.4±3.2 and 3.0±3.6 vs. 2.1±2.1ng/mL, respectively; p &amp;lt; 0.004) and lower basal FSH serum levels (7.0±2.1 vs. 7.6±2.9 and 7.8±3.3 vs. 8.5±3.4mU/mL, respectively; p &amp;lt; 0.0001). Overall, the percentage of DUC1 embryos was higher in ICSI (5.6%) as compared to IVF (4.4%, p &amp;lt; 0.01) cycles. However, when the data was stratified by maternal age, DUC1 incidence was significantly higher in ICSI vs. IVF in AMA (5.6 vs 4.0%; p = 0.002) but not in pre-AMA (5.7 vs 5.0%; p = 0.19). All 21 DUC1 embryos transferred during the study failed to implant. Limitations, reasons for caution Our study is limited by its retrospective nature. The present conclusions must be confirmed in other patient populations with different race/genetics and habits. Wider implications of the findings The occurrence of cycles providing DUC1 embryos is paradoxically associated with a more favourable patient prognosis, and the percentage of DUC1 embryos is higher in ICSI as compared to IVF cycles in AMA patients. Our findings represent new valuable clues for the clarification of DUC1 underlying causes. Trial registration number NA

P-615 Can the use of progestin in egg donation cycles influence morphokinetic parameters of embryos when compared with GnRH antagonist during ovarian stimulation? A case-control study

Abstract Study question Are there differences in clinical and morphokinetic parameters of embryos obtained from fresh egg donation cycles using GnRH antagonist versus PPOS on same donor? Summary answer Lower number of oocytes retrieved and faster early morphokinetics parameters were observed in PPOS protocol. What is known already Controlled ovarian hyperstimulation (COH) is a crucial step in assisted reproduction treatments and the ideal protocol should be chosen according to the patient’s profile, based on literature results, intending to achieve high effectiveness. The use of PPOS in ovarian stimulation protocols is getting attention because of its safety, efficacy and proved to be patient friendly and an economical alternative to avoid premature LH surge. In the last years, morphokinetics parameters started to be considered in embryo classification and selection, as they correlate to embryo implantation potential. Here, morphokinetics parameters were compared from two different COH in donated oocyte cycles. Study design, size, duration Case-control study including cycles from 10 oocyte donors and 20 recipient patients, whose embryos formed with fresh oocytes were cultured in a time-lapse system (Embryoscope® Plus), in a single private IVF center between Jun/2020 and Oct/2022. Each donor were submitted to two cycles: first stimulation with standard GnRH antagonist (ant) short protocol (group A) and in a posterior cycle: a second stimulation with progesterone (PPOS protocol with dydrogesterone, group B). Participants/materials, setting, methods All donors used menotropin for COH (doses adjusted according to patient’s profile). Group A started GnRh ant with follicle above 14 mm. On group B, dydrogesterone 10 mg was started on the first day of COH and maintained until the day of ovulation trigger, which was performed with GnRH agonist. Clinical and morphokinetics parameters were compared. Paired t-test, t-test or Mann-Whitney were used properly, values of p &amp;lt; 0.05 were considered significant. Main results and the role of chance A total of 70 blastocysts were analyzed, 39 from group A and 31 from group B. The following morphokinetics parameters were annotated: time of PN fading (tPNf), time to two cells (t2), three cells (t3), four cells (t4), five cells (t5), eight cells (t8), time to morula (tM), time to blastulation (tB) and KIDScoreTM. Statistically significant differences were observed for tPNf: group a) 24,01 ± 3,28h versus b) 22,59 ± 2,48h, p = 0,0484, t2: a) 26,30 ± 3,24h versus b) 24,76 ± 2,27h, p = 0,0211; t3: a) 37,04 ± 4,44h versus b) 35,53 ± 2,93, p = 0,0347 and t4: a) 38,38 ± 5,15h versus b) 36,05 ± 3,32h, p = 0,0152. Clinical characteristics of the donors were also analyzed: age, body mass index (BMI), total gonadotropin dose, antral follicular count (AFC), follicle stimulating hormone (FSH), basal hormone levels, follicle count, oocyte pick-up (OPU), altered oocytes, number of germinal vesicles, metaphase I (MI) and metaphase II (MII) oocytes. Differences were observed only between two clinical aspects: patients’ age: a) 24,2 ± 3,6 versus b) 25,0 ± 3,3, p = 0,0031 and OPU: a) 23,3 ± 9,1 versus b) 18,6 ± 8,3, p = 0,004, however both variables does not seem to affect embryo formation potential. Limitations, reasons for caution The results should be interpreted with caution due to the limitations of evaluate a small dataset of donors, resulting in low number of embryos in each group of protocols for analysis. Wider implications of the findings In this case-control study, a lower number of oocytes retrieved and faster early morphokinetics times were observed in PPOS compared to antagonist protocol. The implications of these differences still need further data analyses to compare if these findings can change clinical outcomes. Trial registration number Not applicable.

P-664 Insulin-like peptide 3 as a potential biomarker in predicting IVF success and ovarian reserve in women with unexplained infertility and diminishing ovarian reserve

Abstract Study question Could insulin-like peptide 3 (INSL3) predict ovarian reserve and in vitro fertilization (IVF) success in women with unexplained infertility (UI) and diminishing ovarian reserve (DOR)? Summary answer In women with DOR, reduced intrafollicular and serum INSL3 levels correspond with low AMH and high FSH levels, similar to established ovarian reserve markers. What is known already INSL3, belonging to the insulin-like peptide family, is produced by theca interna cells within antral follicles and the corpora lutea. It is hypothesized that INSL3 is integral to the initial development and function of antral follicles, specifically through its regulatory effect on androgen biosynthesis in the thecal cells of these follicles. Moreover, INSL3 is implicated in the modulation of the ovarian microenvironment, which is essential for facilitating the maturation of oocytes. Study design, size, duration This research was carried out at the IVF centers of two tertiary university hospitals, assessing 49 infertile women with either UI (n = 24) or DOR (n = 25) during IVF treatment. A control group (n = 26) comprised women with normal ovarian reserve undergoing IVF due to male or tubal factors. Outcome parameters are ovarian reserve markers (serum FSH, estradiol, AMH, INSL3 concentration and antral follicle count [AFC]), positive pregnancy rate and live birth rate (LBR) (gestational age ≥28 weeks). Participants/materials, setting, methods Women aged between 20-40 with a body mass index (BMI)&amp;lt;30 kg/m² were included. Serum FSH, estradiol, AMH, INSL3 levels and AFC were evaluated on menstrual cycle days 2 or 3, and INSL3 in follicular fluid from dominant follicles (&amp;gt;14 mm) were evaluated during oocyte retrieval. Exclusions included endocrine abnormalities (e.g., polycystic ovary syndrome, hyperprolactinemia, hypo and hyperthyroidism) cycle cancellation due to lack of a viable embryo, uncorrected Mullerian anomalies, recurrent miscarriage history and hydrosalpinx presence. Main results and the role of chance The mean age in the study groups was 28.41±3.5 (UI), 32.40±4.21 (DOR), and 29.65±3.91 (control). In the DOR group, female age (p = 0.001), BMI (p = 0.049), duration of infertility (p = 0.006), previous IVF attempts (p &amp;lt; 0.001), basal FSH (p &amp;lt; 0.001), and basal estradiol (p &amp;lt; 0.001) were higher, while AMH (p &amp;lt; 0.001), AFC (p &amp;lt; 0.001), and follicular fluid INSL3 (p &amp;lt; 0.001) were lower. Serum INSL3 in DOR was lower but not statistically significant. No correlation was found between serum INLS, follicular fluid INSL3, ovarian reserve markers, positive pregnancy rates, miscarriage, and LBR. DOR group had longer stimulation durations (p = 0.017), but lower max estradiol (p &amp;lt; 0.001), fewer oocytes (p &amp;lt; 0.001), mature follicles (p &amp;lt; 0.001), and PN (p &amp;lt; 0.001). Pregnancy rates were 54.1% (UI), 24% (DOR), and 53.84% (control); LBRs were 54.1% (UI), 20% (DOR), and 42.3% (control), with significant differences in pregnancy tests (p = 0.04) and LBR (p = 0.03), both lower in DOR. Univariate analysis identified duration of infertility (p = 0.019) and basal FSH (p = 0.009) as significant LBR predictors. Multivariable analysis confirmed basal FSH (p = 0.033) as significant, with increased duration of infertility and basal FSH level reducing LBR probability. Limitations, reasons for caution The limited sample size of our study potentially undermines the robustness of circulating INSL3 as a predictive biomarker for DOR, evidenced by the non-significant decrease in serum INSL3 levels in the DOR group. Moreover, the lack of INSL3 level assessment during oocyte retrieval further constrains our findings. Wider implications of the findings Our research explored INSL3 as a viable biomarker for predicting ovarian reserve and IVF success in UI and DOR cases. Reduced INSL3 levels in circulation and follicular fluid in DOR patients could be beneficial in clinical practice. Trial registration number not applicable

P-588 Serum Luteinizing Hormone levels before progesterone supplementation are significantly associated with live birth rate in single euploid frozen embryo transfers performed in hormonal replacement cycles

Abstract Study question Do luteinizing hormone (LH) levels measured prior to initiating progesterone supplementation influence live birth rate (LBR) in artificial frozen-thawed single euploid blastocyst transfer cycles? Summary answer Serum LH levels, measured prior to progesterone initiation in artificial frozen-thawed single euploid blastocyst transfer cycles, are significantly associated with LBR. What is known already In the absence of a dominant follicle, estrogen supplementation in artificial endometrial preparation for frozen embryo transfer (FRET) can lead to a rise in endogenous LH levels, as observed in a natural cycle. Since the LH rise is a key feature in natural conception and the embryo, the endometrium, and the myometrium display receptors for LH, the rise observed during FRET preparation may have an impact on the clinical outcomes of the treatment. Previous studies found contradictory results hence none of them excluded embryo aneuploidy by implementation of preimplantation genetic testing for aneuploidies and the transfer of euploid embryos. Study design, size, duration Retrospective analysis of 639 frozen-thawed single euploid blastocyst transfer cycles performed in a tertiary referral IVF center from January 2018 to August 2023. Cycles with hormonal pre-treatment or pituitary suppression were excluded. Participants/materials, setting, methods Single euploid FRET cycles were included. Oral estradiol administration commenced on cycle-day 2/3. Estradiol, progesterone, and LH were measured at the start of endometrial preparation and up to 3 days before progesterone initiation, and the slope of LH was calculated. Patients were stratified in groups according to their LH values before progesterone administration, and the impact of LH on LBR and implantation rate was assessed. Logistic regression analysis was performed to adjust for potential confounders. Main results and the role of chance In total 639 cycles were included. Patients’ characteristics (mean+/-SD) were: age 33.2±5.9 years; BMI 27.5±4.9 kg/m2. The median duration of estrogen administration was 15 days (range: 11-27 days). The overall implantation rate and LBR were 65.9% (421/639) and 46.9% (300/639), respectively. Patients were stratified into six groups according to their LH levels prior to progesterone supplementation: &amp;lt;10th percentile (≤6mIU/ml, n = 57), p10th-p25th (&amp;gt;6 to ≤ 8.5mIU/ml, n = 81), p25th-p50th (&amp;gt;8.5 to ≤ 12.4mIU/ml, n = 176), p50th-p75th (&amp;gt;12.4 to ≤ 18.1mIU/ml, n = 180), p75th-p90th (&amp;gt;18.1 to ≤ 25.0mIU/ml, n = 91) and &amp;gt;p90th (&amp;gt;25.0mIU/ml, n = 54). A significant trend for higher LBR with increasing serum LH levels was observed (Cochran-Armitage Test, P = 0.041) but not for implantation rate (P = 0.158). Those with higher basal serum estradiol levels before estrogen supplementation had a steeper LH increase from basal to progesterone-supplementation day (0.40 mIU/mL higher per 10pmol/L increase in basal estradiol, P = 0.027). Regression analysis identified as independent predictors of LBR BMI (OR 0.96, 95%CI: 0.93-0.99) and embryo quality (CC vs. AA p &amp;lt; 0.001, CB vs. AA p = 0.027, BC vs. AA p = 0.013). Patients with steeper LH increases from estrogen to progesterone-supplementation day had higher LBR in both univariable (OR:1.27, 95%CI: 1.00–1.63, P = 0.052) and multivariable analyses (OR:1.26, 95%CI: 0.97–1.64, P = 0.085) but the difference didn’t reach statistical significance. Limitations, reasons for caution The retrospective design of the study is a limitation. Results should be interpreted with caution due to the small number of patients in the lowest and highest LH percentile groups. Wider implications of the findings The results of this study suggest that serum LH levels measured before progesterone supplementation may have an impact on the clinical outcome of artificial frozen-thawed blastocyst transfer cycles. Further studies with a higher sample size are necessary to confirm or refute our findings. Trial registration number not applicable

P-332 Decoding autophagy dynamics in adenomyosis: insights of a murine model

Abstract Study question This study aimed to highlight the role of autophagy in the pathogenesis of adenomyosis using a murine model of medically induced adenomyosis. Summary answer Autophagy seems to play a role in the early stages of the disease and is likely not the primary mechanism driving the progression of adenomyosis. What is known already Autophagy is an intracellular mechanism involved in the recycling of aging organelles and non-functional proteins. Initially described as a phenomenon activated under stress or fasting conditions (non-selective autophagy), it is now accepted that a basal level of autophagy is essential for several cellular functions to maintain cellular homeostasis (selective autophagy). Autophagy is essential for cell survival and tissue growth, and an alteration of the autophagic pathway has been described in endometrial pathologies such as endometrial hyperplasia and carcinoma, infertility, and endometriosis. The contribution of autophagy to adenomyosis is still unclear due to conflicting findings in the existing literature. Study design, size, duration This experimental study was conducted from October 2022 until January 2024. A total of 170 mice pups were treated (90 treated with tamoxifen – 80 with vehicle only). Participants/materials, setting, methods Female neonatal CD1 mice received oral doses of 2.7µmol/kg tamoxifen from days 2 to 5 after birth. Control mice received only the vehicle. After synchronizing their estrus cycle, the mice were euthanized at 1 month or 3 months old. The diagnostic and grading of adenomyosis were performed by histological examination (hematoxylin-eosin and immuno-fluorescence). RT-PCR and Western Blot were used to study the modulation of autophagy pathways between the uterus of the two groups. Main results and the role of chance Histological analysis of 3-month-old tamoxifen-treated group showed that 97% of the mice developed the disease. The grading method was defined by assessing the depth of lesions. After sensitivity enhancement through immunofluorescence staining (alpha-SMA, EpCAM and DAPI), grade 3 adenomyosis was diagnosed in up to 91% of our samples (vs 84% when analyzed by H&amp;E only). Neonatal treatment of CD1 mice with tamoxifen reliably produces severe adenomyotic lesions. Immunofluorescence enhances sensitivity in histological examinations of myometrial lesions, combining alpha-SMA staining for smooth muscle detection with EpCAM for identifying epithelial cells. RTqPCR analysis of lc3b, akt, bcl2, bclxl, ulk2, ulk1, cxcr4, tnfs10, dapk1, igf1, atg9, pik3c3, and bid showed an up-regulation of autophagy in one-month-old adenomyosis mice. This was also indicated by a decrease in akt and bcl2, which are inhibitors of the pathway, along with an increase in lc3b, a marker for phagophore formation. However, the conflicting decrease in ulk2, a component of the induction complex, raises questions. Importantly, these changes in autophagy are not observed in 3-month-old mice, suggesting that autophagy may play a role in the early stages of disease development. There were no statistical differences in Protein expression of pakt/akt, lc3b, and bcl2 between the groups. Limitations, reasons for caution The analysis used protein or mRNA extracted from the whole uterus. Variations in results may arise if the endometrial and myometrial components are analyzed separately. Techniques like laser capture microdissection could prove valuable in this context. Continued research focusing on investigating the role of apoptosis in the study is essential. Wider implications of the findings Given the frequent association of adenomyosis with endometriosis and/or uterine fibroids, using an experimental model offers the advantage of eliminating bias from associated comorbidities. This approach presents a unique opportunity to unravel the molecular basis of adenomyosis, while enabling a translational analysis when juxtaposed with clinical studies. Trial registration number not applicable

P-426 Comprehensive study of the endometrial early/mid secretory phase during natural cycles reveals conserved molecular dynamics involved in the window of implantation

Abstract Study question Is there a common transcriptomic signature in the endometrial early/mid secretory phase of healthy women in natural cycles? Summary answer We observed a conserved transcriptomic regulation of the endometrium around the window of implantation (WOI), consistent across donors, and not influenced by the biopsy procedure. What is known already Extensive research has been conducted on the gene expression changes that characterize the WOI, aiming to identify signals that indicate its onset. However, much of this work has utilized whole-transcriptome techniques and has involved IVF patients, who require luteal phase support. The few studies performed in natural cycles often relied on restricted methods, such as qPCR that examines only a few targets simultaneously. As such, an extensive transcriptomics study in volunteer donors in true natural cycles is lacking. This may provide deeper insights into the physiological changes during the WOI in young women driven by the endogenous effects of progesterone. Study design, size, duration 34 healthy donors were enrolled. Each donor provided one endometrial sample, on a day between LH + 2 and LH + 9 during their natural cycle. A minimum of two and a maximum of five donors were allocated to each day. Ovulation was detected via ecographic inspection and hormonal determination of LH/E2/P4. LH + 0 was established when the LH peak was detected (&amp;gt;2.8 times basal levels), together with a subsequenc decrease in E2 (-30%) and increase in P4 (&amp;gt;1.6ng/ml) Participants/materials, setting, methods When the biopsy was of sufficient size, we separated it in two parts and extracted RNA separately to assess concordance between different areas of the same biopsy. RNAseq libraries were prepared with NEBNextUltraII Directional RNA LibraryPrep and sequenced on a NovaSeq6000. Fastq files were aligned with STAR to GRCh38. Principal component analyses (PCA) were created with plotPCA, gene expression time course analysis was performed with DEseq2, and pseudotime analysis was performed using the slingshot package. Main results and the role of chance Of the 34 donors, we obtained good quality RNAseq data (minimum 20 million reads) from 41 samples, with 27 donors having one sample sequenced, while 2 independent samples from the same biopsy were obtained from 7 volunteers. Analysing same-biopsy concordance via PCA revealed a great degree of similarity between all the pairs, which clustered close to each other. Samples from different donors collected on the same day also showed a good clustering, apart from a single day LH + 4 sample. Pseudotime analysis revealed a distinct trajectory in transcriptomic profiles changing progressively from day LH + 2 to LH + 9. This analysis clearly delineated three phases: LH + 2 to LH + 5, LH + 6 to LH + 7, and LH + 8 to LH + 9. Accordingly, we categorized these into pre-receptive, receptive and post receptive groups, and conducted a time course experiment. We identified a total of 3,311 genes with varying expression across these three timepoints, primarily associated with progesterone signalling, metabolism and cell-to-cell communication. Based on their expression patterns, we isolated a subset of 538 genes which increased their expression during the receptive phase only. These genes could represent novel markers for a more in-depth characterization of the WOI. Limitations, reasons for caution The samples analysed are in bulk, and the genes identified could be expressed either in the stroma or in the epithelial compartment. These findings are not applicable to artificial cycles, as the absence of the corpus luteum and varying levels of exogenous progesterone administered may change the transcriptomic signatures. Wider implications of the findings Our study revealed good concordance of whole transcriptome profiles of donor’s endometria according to cycle progression. This may lead to the identification of novel markers involved in the regulation of the WOI that may not have been found in previous studies performed on artificial cycles. Trial registration number not applicable