Investigation of the PIWI interacting RNA expression differences in normoresponder and diminished ovarian reserve follicular fluid

Study question Does ferroptosis, through its regulation of Hippo signaling pathway, contribute to follicular atresia, thereby leading to diminished ovarian reserve (DOR) and associated infertility? Summary answer Ferroptosis in cumulus cells may induce follicular atresia and causing DOR through Hippo signaling pathway. What is known already Ferroptosis is a novel form of regulated cell death associated with oxidative stress and lipid peroxidation. Its activation has been implicated in various pathologies, including ovarian cancer, endometriosis, polycystic ovary syndrome and primary ovarian insufficiency. The Hippo signaling pathway regulates cellular growth and apoptosis, and recent evidence suggests a potential interaction between ferroptosis and this pathway in ovarian cells. Oocytes are dependent on cumulus cells for their energy supply, with the energy necessary for oocyte maturation, fertilization and early embryonic development. However, the relationship between ferroptosis and Hippo signaling in the context of DOR has not been fully elucidated. Study design, size, duration This cross-sectional study was conducted in an IVF clinic between July 2023 and December 2024. 81 women aged between 18 and 39 years undergoing IVF treatment were included, divided into two groups: 46 patients diagnosed with DOR with a serum AMH level of < 1.1 ng/ml and/or ≤3 oocytes retrieved in prior IVF cycles. 35 with normal ovarian reserve (NOR) defined by serum AMH levels between 1.1 and 3.5 ng/ml,and 4-15 oocytes retrieved during egg collection. Participants/materials, setting, methods Cumulus-oocyte complexes were retrieved after ovarian stimulation with recombinant FSH and GnRH antagonist protocol and ovulation trigger with recombinant HCG. Cumulus cells were collected during egg retrieval, mechanically using a 31G insulin syringe. Ferroptosis-related (GPX4, EMP1) and Hippo pathway-related genes (MST, YAP, LATS) were analyzed by qRT-PCR. Gene expression levels were compared between DOR and NOR groups by using delta-CT values. Additionally, iron and reactive oxygen species levels in follicular fluid were measured. Main results and the role of chance Significantly higher expression levels of EMP1 and GPX4 were observed in the DOR group compared to the NOR group (p = 0.024 and p = 0.015, respectively), while no significant difference was found in the expression of other genes. The increase in both EMP1 and GPX4 in the DOR group suggest the activation of ferroptosis pathway in cumulus cells, which, in turn, may reflect a compensatory activation of the antioxidant system. No difference was observed in follicular fluid iron levels between the DOR and NOR groups; however, reactive oxygen species (ROS) levels were significantly elevated in the DOR group (p = 0.008). The increase in ROS levels may serve as an indirect indicator of the ferroptosis activation triggered by oxidative stress. Although these results support a relationship between ferroptosis and DOR, the influence of confounding factors such as age and ovarian stimulation protocols cannot be entirely excluded. Limitations, reasons for caution The study’s limitations include its cross-sectional design and the small sample size. The findings, while promising, may not be fully generalizable due to the limited number of cases. Additionally, the complexity of cellular interactions and the lack of longitudinal follow-up limits the ability to establish causality. Wider implications of the findings These findings highlight the potential role of the relationship between ferroptosis and the Hippo signaling pathway in the pathophysiology of DOR. Understanding these mechanisms could open new avenues for therapeutic strategies aimed at preserving ovarian reserve and improving fertility outcomes in women with DOR. Trial registration number No

Elevated Early Follicular Phase Estradiol Does Not Predict Unsuccessful Pregnancy in Infertile Women with Polycystic Ovarian Syndrome (PCOS)

Study question Are TINAGL1 levels in follicular fluid associated with ovarian reserve in patients undergoing ovarian stimulation for in-vitro fertilization (IVF)? Summary answer TINAGL1 concentration was lower in the follicular fluid (FF) of patients with DOR vs patients without infertility or polycystic ovarian syndrome (PCOS). What is known already TINAGL1 is an extracellular matrix protein involved in cell adhesion, proliferation, migration, and differentiation. TINAGL1 has previously been shown to be expressed in the mouse ovary and to be a serum biomarker that varies throughout the mouse estrous cycle, peaking in the estrous phase. Furthermore, TINAGL1 knockout mice have been shown to be infertile. TINAGL1 expression in the human ovary has not been previously evaluated. Study design, size, duration Between 2019 and 2023, a prospective study of 136 patients undergoing ovarian stimulation and oocyte retrieval (OR) was conducted at a single academic fertility center to assess TINAGL1 levels in luteinized FF and granulosa cells. Study groups were determined by infertility diagnosis: DOR n = 24, polycystic ovary syndrome (PCOS) n = 27, and control n = 85 (undergoing fertility preservation or had a partner with male factor only). Participants/materials, setting, methods TINAGL1 protein levels were quantified in the FF of patients that underwent OR via commercial enzyme-linked immunosorbent assay (ELISA). Granulosa cells were recovered from FF after OR, underwent lysis, RNA extraction, cDNA amplification and TINAGL1 mRNA expression was quantified via quantitative real-time polymerase chain reaction (qPCR). Demographics and infertility diagnoses were collected from the medical record. TINAGL1 levels were compared between the three groups. Statistical analysis was performed with parametric and non-parametric tests as appropriate. Main results and the role of chance Patients with DOR were younger when compared to the control and the PCOS group [Mean(SD) 32.7 (3.8) vs 34.45(3.9) vs 37.7 (3.0) years, p < 0.001] and had lower AMH levels [1.19 (0.8) vs 3.96 (3.03) vs 8.67 (4.56) ng/ml, p < 0.001]. TINAGL1 protein concentration in FF was measured by ELISA. Mean (SD) TINAGL1 levels were highest in PCOS, and lowest in the DOR group. Mean TINAGL1 levels were higher in the FF of the PCOS group when compared to the control group [4.73(7.15) vs 4.45 (4.57) ng/ml, p = 0.83] but this difference did not reach statistical significance. However, mean TINAGL1 FF levels were significantly lower in the DOR group when compared to the control group [2.54 (1.6) vs 4.48 (4.57) ng/ml, p = 0.04]. TINAGL1 was also found to be expressed in the granulosa cells with qPCR. TINAGL1 expression was upregulated by (+89%) in the PCOS group vs the control group (p = 0.0095). Importantly, there was a trend for decreased (-52%) TINAGL1 expression in DOR when compared to the control group (p = 0.67). The latter result should be interpreted with caution since only a subset of samples had sufficient granulosa cells for RNA extraction (DOR=4, control=18, PCOS=5). Limitations, reasons for caution This is a single institution study with a small number of patients in the DOR group (n = 24). Wider implications of the findings This is the first study to show that TINAGL1 is expressed in human granulosa cells. Decreased TINAGL1 may be a biomarker associated with DOR. Future studies should assess serum TINAGL1 levels at baseline and during ovarian stimulation and its contribution to the pathophysiology of DOR. Trial registration number Not applicable

ObjectiveTo investigate the changes in bile acid (BA) metabolites within the follicular fluid (FF) of patients with diminished ovarian reserve (DOR) and to identify novel diagnostic markers that could facilitate early detection and intervention in DOR patients.DesignA total of 182 patients undergoing assisted reproductive technology (ART) were enrolled and categorized into the normal ovarian reserve (NOR) group (n = 91) or the DOR group (n = 91) to measure BA levels in FF. To identify the changes in granulosa cells (GCs), we collected GCs from an additional 7 groups of patients for transcriptome sequencing.SettingReproductive medicine center within a hospital and university research laboratory.PopulationA total of 182 patients undergoing assisted reproductive technology were enrolled and categorized into the NOR group (n = 91) or the DOR group (n = 91).MethodsIn this study, BA metabolites in FF of DOR and NOR patients were analyzed in detail by targeted metabolomics, and the correlation between BA levels in FF and clinical indicators was discussed. Then, we constructed a diagnostic model for DOR using the random forest algorithm based on five different BAs. Additionally, we performed a functional enrichment analysis on differentially expressed genes (DEGs) in GCs from both DOR and NOR patients.Main outcome measuresBA levels in FF and their correlation with clinical indicators; the areas under the curve (AUCs) of the random forest diagnostic model for DOR; and the DEGs and corresponding functional enrichment results of GC RNA analysis.Result (s)The levels of lithocholic acid, chenodeoxycholic acid, ursodeoxycholic acid, deoxycholic acid and cholic acid in FF of DOR group were lower than those of NOR group. And significant reductions in total, primary, secondary, and unconjugated BA levels were observed in the DOR group. The above five BAs levels were closely related to indicators of ovarian reserve. The AUC of the diagnostic model based on the above five BAs was 0.964. Based on transcriptome sequencing data from two groups of GCs, a total of 482 up-regulated and 654 down-regulated DEGs were identified. Gene ontology analysis revealed that the metabolic and biosynthetic processes of fatty acids, steroids, and cholesterol were enriched in these DEGs, whereas Kyoto Encyclopedia of Genes and Genomes analysis indicated enrichment of fatty acid and ovarian steroidogenesis.Conclusion(s)The levels of multiple BA metabolites in FF are significantly lower than those in patients with DOR and are closely related to the evaluation of ovarian reserve function.

BackgroundInsulin-like peptide (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. Our study investigates if circulating and follicular fluid INSL3 levels serve as biomarkers for ovarian reserve and IVF success in women with unexplained infertility (UI) and diminished ovarian reserve (DOR).MethodsThis prospective study included 75 women (25 with DOR, 24 with UI) undergoing IVF and 26 controls with normal ovarian reserve. Serum and follicular fluid INSL3 levels were measured, and their association with ovarian reserve markers, pregnancy rates, and live birth rates (LBR) was analyzed.ResultsCirculating (p = 0.001) and follicular fluid (p < 0.001) INSL3 levels, AMH levels (p < 0.001) and AFC (p < 0.001) were significantly lower and basal E2 level (p < 0.001) were significantly higher in DOR group compared to the UI and control groups. Circulating INSL3 positively correlated with serum anti-Müllerian hormone (AMH) and antral follicle count (AFC), and negatively correlated with follicle-stimulating hormone (FSH). Positive pregnancy rates and LBR were significantly lower in the DOR group. Basal FSH was identified as a significant predictor of LBR.ConclusionsThe current study presents that although the serum and follicular fluid INSL3 levels are significantly lower in women with DOR, the narrow margin between the DOR and control groups indicates that INSL3measurement may be insufficient on its own to be of diagnostic value for DOR. Further research with larger sample sizes is needed to validate these findings and explore the role of INSL3 in ovarian aging and infertility treatment.

BackgroundPrevious studies have discussed the pregnancy outcomes of diminished ovarian reserve (DOR) patients. However, data on embryonic development potential, neonatal outcomes, and maternal complications of DOR patients still remained unknown. This is the first study to investigate the risk of DOR on pregnancy and perinatal outcomes among women < 38 years.MethodsRetrospective cohort study was conducted. Patients (< 38 years of age) undergoing their first oocyte retrieval cycle were included. Patients were divided into DOR group and non-DOR group. Pregnancy outcomes of fresh cycle and cumulative live birth rate and perinatal outcomes after one oocyte retrieved cycle were compared between DOR and non-DOR group.Result(s)From January 2016 to September 2020, there were 8,179 patients involved: 443 patients in the DOR group and 7,736 patients in the non-DOR group. The incidences of live birth and clinical pregnancy did not differ significantly between patients with or without DOR after fresh cycle transfer, but the cumulative live birth rate was significantly lower in DOR group. Among women who had singleton live births, after binary logistic regression, the rates of maternal complications and neonatal outcomes were comparable in the two groups.Conclusion(s)DOR patients (< 38 years of age) showed similar pregnancy outcomes in the first fresh embryo transfer cycle but a lower chance of live birth after a whole oocyte retrieval cycle to non-DOR patients and DOR is not associated with adverse perinatal outcomes.

BackgroundFemales with diminished ovarian reserve (DOR) have significantly lower cumulative live birth rates (CLBRs) than females with normal ovarian reserve. A subset of young infertile patients, whose ovarian reserve is declining but has not yet met the POSEIDON criteria for DOR, has not received the attention it merited. These individuals have not been identified in a timely manner prior to the initiation of assisted reproductive technology (ART), leading to suboptimal clinical pregnancy outcomes. We categorized this overlooked cohort as the “high-risk DOR” group.ObjectiveThe primary aim of this study was to identify high-risk DOR patients through anti-Mullerian hormone (AMH) and antral follicle counts (AFCs).MethodsA total of 10037 young women (≤ 35 years old) who underwent their first initial oocyte aspiration cycle at a single reproductive medicine center were included and further classified into three groups, based on the thresholds for AMH and AFC established through receiver operating characteristic (ROC) analysis and in alignment with the POSEIDON criteria. Two ROC analyses were performed to identify the cutoff values of AMH and AFC to obtain one viable embryo (one top-quality embryo or one viable blastocyst). The cutoffs of ROC were measured by sensitivity and specificity. The primary outcome was the cumulative live birth rate (CLBR) per oocyte aspiration cycle. The secondary outcomes included the number of oocytes retrieved and the number of viable embryos formed. Pearson’s chi-square tests were conducted to compare the clinical outcomes among the three groups. Furthermore, univariate logistic regression analyses were performed to investigate the associations between ovarian reserve and clinical outcomes. All of the above comparisons between the high-risk DOR and NOR were further confirmed by propensity score matching (PSM) (1:1 nearest-neighbor matching, with a caliper width of 0.02).ResultsAccording to the ROC analyses and POSEIDON criteria, the present study identified a population of high-risk DOR patients (1.20 ng/mL < AMH values < 2.50 ng/mL, with 6 ≤ AFC ≤ 10; n = 682), and their outcomes were further compared to those of DOR patients (positive control, AMH values ≤ 1.2 ng/mL, and/or AFC ≤ 5; n = 1153) and of NOR patients (negative control, 2.5 ng/mL ≤ AMH values ≤ 5.5 ng/mL, and 11 ≤ AFC ≤ 20; n = 2649). Patients in the high-risk DOR group had significantly lower CLBRs than those in the NOR group (p < 0.001) but higher CLBRs than those in the DOR group (p < 0.001). Logistic regression further demonstrated that high-risk DOR was associated with a lower likelihood of cumulative live birth chance (OR 0.401, 95% CI: 0.332–0.486, p < 0.001) than NOR was, with a greater likelihood of cumulative live birth chance (OR 1.911, 95% CI:1.558–2.344, p < 0.001) than DOR was. To investigate the effects of embryo development stage, the outcomes of D3 embryos and blastocysts were analyzed separately. Significant differences in pregnancy outcomes were detected only in D3 embryo ET cycles among the three groups (high-risk DOR vs. NOR, all p < 0.05; DOR vs. NOR, all p < 0.05). DOR/high-risk DOR did not influence the pregnancy loss rates or pregnancy outcomes (clinical pregnancy rates and ongoing pregnancy rates) per positive HCG cycle (all p > 0.05). After PSM, the differences in ovarian response and pregnancy outcomes between the high-risk DOR and NOR groups were consistent with the results before PSM.Conclusion(s)Our study revealed that the CLBR of the high-risk DOR patients was significantly lower than that of females with normal ovarian reserve and greater than that of females with DOR. The values of AMH ranging from 1.2 to 2.5 and AFC ranging from 6 to 10 appeared to constitute meaningful thresholds in females with mildly reduced ovarian reserve.

To explore the changes and correlations of anti-Müllerian hormone (AMH) and stem-cell factors (SCF) in different ovarian reserve patients during controlled ovarian hyperstimulation (COH) and the effects on COH outcomes. Serum at six different timepoints during GnRH-antagonist protocol and follicular fluid (FF) on oocyte retrieval day of 52 patients with polycystic ovary syndrome (PCOS), 61 patients with normal ovarian reserve (NOR) and 42 patients with diminished ovarian reserve (DOR) were collected. AMH and SCF were assessed using enzyme-linked immunosorbent assay. During COH, AMH in the PCOS group was the highest, but SCF did the opposite, and serum AMH gradually decreased, while SCF inversely increased. In the PCOS group, SCF on the first and fourth days of gonadotropin (Gn) administration was negative with Gn dosage (r = - 0.362, P < 0.05; r = - 0.344, P < 0.05). In the NOR group, the basal AMH was also negative with Gn dosage (r = - 0.297, P < 0.05) and positive with COH outcomes (number of retrieved oocytes, MII oocytes, and 2PN fertilization) as well as serum SCF after Gn administration. In the DOR group, both AMH and SCF were significantly associated with COH outcomes. Serum AMH in the DOR group after Gn administration and FF AMH showed a negative correlation with SCF. Serum AMH decreased, while SCF increased during COH. AMH and SCF are effective for Gn time and dosage adjustment and predicting COH outcomes for NOR and DOR patients. In addition, serum AMH in DOR patients after Gn administration and FF AMH has a negative effect on SCF.

We aimed to evaluate intracytoplasmic sperm injection (ICSI) outcomes in young patients with diminished ovarian reserve (DOR) plus severe male factor (SMF) compared with age-matched controls with DOR. A total of 189 infertile women under 35 years with DOR undergoing ICSI procedures were included retrospectively. Participants whose partners' sperm analysis was normal considered as the DOR group (n=154) and whose partners' had SMF considered as the DOR + SMF group (n=35). The two groups were compared regarding cycle characteristics and pregnancy outcomes. Demographic features except infertility duration were similar between two groups. The duration of infertility was significantly longer in the DOR + SMF group compared to the DOR group (p=0.02). Ovarian stimulation characteristics, oocyte retrieval parameters, fertilization rate, quality of embryos, embryo cancellation rate, and development up to blastocyst stage were found similar between two groups. Implantation, clinical pregnancy, abortion, and live birth rate, multiple pregnancy rate per cycle were distributed homogenously between the DOR and DOR + SMF groups. Regarding perinatal and neonatal outcomes of groups, fetal height and weight were significantly lower in DOR + SMF group than in DOR group (p=0.001 and 0.01, respectively). Gestational week at delivery was lower in the DOR + SMF group compared to the DOR group (p < 0.0001). Fetal anthropometric measures were lower regarding to preterm delivery in the DOR + SMF group than the DOR group. Large sample-sized studies should be performed to explain the decreased gestational week at the time of delivery in the DOR + SMF group.

BackgroundFollicular microenvironment has been proposed as an important factor for oocyte grown and maturation. We sought to evaluate the oxidative stress and inflammatory levels in follicular fluid (FF) and association with embryo quality in patients with diminished ovarian reserve (DOR).MethodsThe current research included 46 DOR cases and 56 normal ovarian reserve (NOR) cases. Twelve representative oxidative stress markers and eight representative inflammatory factors were measured in the FF.ResultsOxidative stress markers total GSH (T-GSH) was decreased in the FF from women with DOR compared with that in NOR group (P = 0.041). More modest differences were observed for reduced GSH (rGSH) and rGSH/GSSG. Women with DOR compared to controls had higher level of TNF-α (P = 0.000) and lower level of IL-18 (P = 0.013). Correlation analysis revealed that GSSG was negatively correlated with normal fertilization rate in NOR group (r = -0.358, P = 0.008), and reduced GSH was negatively correlated with normal fertilization rate in DOR group (r = -0.299, P = 0.049). Moreover, as the regression analysis data showed, the GSSG level was significantly associated with embryo quality indicator.ConclusionsThe FF in DOR patients was accompanied by increased oxidative stress and inflammatory levels. Follicular development of women with DOR might be influenced by unusual IL-18 and TNF-α levels in FF. And oxidative stress marker GSSG in NOR group was a negative predictor for embryo quality.

The follicular fluid metabolome in infertile individuals between polycystic ovary syndrome and diminished ovarian reserve

Background/Objectives: Diminished ovarian reserve (DOR) poses significant challenges in the reproductive field, resulting in fewer mature and more low-quality eggs. Methods: We studied r-IVM in addition to standard in vitro fertilization (IVF) and compared the embryological outcomes between both DOR and NOR women. Results: We recruited 90 women (45 NOR; 45 DOR) with a younger age seen in NOR (35.2 vs. 36.5 years old) women. Otherwise, DOR women had lower levels of AMH and AFC, thus fewer retrieved follicles and collected oocytes. Most of the group presented with primary subfertility, with 55.6% in the NOR group diagnosed with polycystic ovary syndrome (PCOS), while 37.8% in the DOR group presented with aging and cancer survivorship issues. Most women in the NOR group used hCG as a trigger (82.2%), while 17.8% of the DOR group opted for a decapeptide. A total of 719 oocytes were retrieved, with 72.3% of eggs being mature in the NOR group compared to 64.9% in the DOR group. Following r-IVM, 47.69% of NOR eggs were matured compared to 60% in DOR eggs. The fertilization rates (FRs) following r-IVM were higher in the DOR group (66.7% vs. 37.8%). Overall, higher numbers and quality of D3 embryos were seen in the DOR group. Our analysis revealed that the trigger type, hCG, was the only significant factor linked to successful oocyte maturation rates. Conclusions: Our study suggests that r-IVM may enhance outcomes for women with DOR, including better egg maturity, FR, and embryo quality than NOR women.

Downregulation of glucose-energy metabolism via AMPK signaling pathway in granulosa cells of diminished ovarian reserve patients

Connecting the dots between oocyte quantity and quality in diminished ovarian reserve

BackgroundOvarian reserve reflects the quality and quantity of available oocytes and has become an indispensable measure for the better understanding of reproductive potential. Proteomic approaches are especially helpful in discerning differential protein expression patterns associated with normal and diseased states and, thus, proteomic analyses are increasingly used to identify clinically useful biomarkers. The aim of this study was to investigate proteins secreted in the urine of patients with different ovarian reserve by proteomic techniques to identify potential markers for assessing ovarian reserve.MethodsUrine samples were obtained from patients with polycystic ovary syndrome (PCOS) and diminished ovarian reserve (DOR), and from normal control (NC)participants. We used isobaric tags for relative and absolute quantification (iTRAQ) technology combined with mass spectrometry analysis to identify candidate urinary proteins in the three groups. The selected proteins were confirmed using western blot analysis and enzyme-linked immunosorbent assay (ELISA). Diagnostic performance of the selected proteins was assessed using receiver operating characteristic analysis.ResultsWhen Compared with NC samples, 285 differentially expressed proteins (DEPs) were identified in the DOR samples and 372 in the PCOS samples. By analyzing the intersection of the two groups of DEPs, we found 26 proteins with different expression trends in the DOR and PCOS groups. Vitamin D-binding protein (VDBP) was the key protein for the protein-protein interaction network. ELISA quantification of urinary VDBP revealed the highest levels in the PCOS group, followed by the NC group and the lowest levels in the DOR group (115.90 ± 26.02, 81.86 ± 23.92 and 52.84 ± 21.37 ng/ml, respectively; P < 0.05). As a diagnostic marker, VDBP had a sensitivity of 67.4% and a specificity of 91.8% for DOR, and a sensitivity of 93.8% and a specificity of 77.6% for PCOS.ConclusionsUrinary VDBP is closely associated with ovarian reserve and can be considered as a novel noninvasive biomarker of ovarian reserve. However, studies including large sample sizes are needed to validate these results.