Predicting risk of endometrial failure: a biomarker signature that identifies a novel disruption independent of endometrial timing in patients undergoing hormonal replacement cycles.

OBJECTIVE: To propose a new gene expression signature that identifies endometrial disruptions independent of endometrial luteal phase timing and predicts if patients are at risk of endometrial failure. DESIGN: Multicentric, prospective study. SETTING: Reproductive medicine research department in a public hospital affiliated with private fertility clinics and a reproductive genetics laboratory. PATIENTS: Caucasian women (n = 281; 39.4 ± 4.8 years old with a body mass index of 22.9 ± 3.5 kg/m2) undergoing hormone replacement therapy between July 2018 and July 2021. Endometrial samples from 217 patients met RNA quality criteria for signature discovery and analysis. INTERVENTION(S): Endometrial biopsies collected in the mid-secretory phase. MAIN OUTCOME MEASURE(S): Endometrial luteal phase timing-corrected expression of 404 genes and reproductive outcomes of the first single embryo transfer (SET) after biopsy collection to identify prognostic biomarkers of endometrial failure. RESULTS: Removal of endometrial timing variation from gene expression data allowed patients to be stratified into poor (n = 137) or good (n = 49) endometrial prognosis groups on the basis of their clinical and transcriptomic profiles. Significant differences were found between endometrial prognosis groups in terms of reproductive rates: pregnancy (44.6% vs. 79.6%), live birth (25.6% vs. 77.6%), clinical miscarriage (22.2% vs. 2.6%), and biochemical miscarriage (20.4% vs. 0%). The relative risk of endometrial failure for patients predicted as a poor endometrial prognosis was 3.3 times higher than those with a good prognosis. The differences in gene expression between both profiles were proposed as a biomarker, coined the endometrial failure risk (EFR) signature. Poor prognosis profiles were characterized by 59 upregulated and 63 downregulated genes mainly involved in regulation (17.0%), metabolism (8.4%), immune response, and inflammation (7.8%). This EFR signature had a median accuracy of 0.92 (min = 0.88, max = 0.94), median sensitivity of 0.96 (min = 0.91, max = 0.98), and median specificity of 0.84 (min = 0.77, max = 0.88), positioning itself as a promising biomarker for endometrial evaluation. CONCLUSION(S): The EFR signature revealed a novel endometrial disruption, independent of endometrial luteal phase timing, present in 73.7% of patients. This EFR signature stratified patients into 2 significantly distinct and clinically relevant prognosis profiles providing opportunities for personalized therapy. Nevertheless, further validations are needed before implementing this gene signature as an artificial intelligence (AI)-based tool to reduce the risk of patients experiencing endometrial failure.

The adenomyosis/endometriosis IVF patient - call for clinical focus.

Endometriosis and adenomyosis are distinct clinical conditions that carry the same pathophysiological features. In recent years the clinical focus on assisted reproductive technology patients with either condition (E/A) has increased, in the recognition that this subgroup of patients might need special attention to obtain reproductive success. Endometriosis and adenomyosis are characterized by a disruption of progesterone and oestrogen signalling pathways, resulting in local oestrogen dominance and progesterone resistance at the receptor level. Recent scientific evidence suggests that the endometrial progesterone receptor resistance encountered in E/A patients can be overcome by a freeze-all policy, followed by down-regulating circulating oestradiol concentrations prior to frozen embryo transfer (FET), in combination with an increase in exogenous luteal phase progesterone supplementation in hormonal replacement therapy (HRT) FET cycles. Specifically, for adenomyosis patients who do not respond to gonadotrophin-releasing hormone agonist down-regulation in terms of a decrease in circulating oestradiol concentrations, a small case series has suggested that the addition of an aromatase inhibitor for 21 days prior to HRT-FET is a valid option. Endometriosis and adenomyosis are hormonally active diseases, which need to be treated by controlling local hyperoestrogenism and progesterone resistance. Based on physiology and recent preliminary clinical data, the authors of this opinion paper wish to stimulate discussion and spark interest in research in E/A patients.

Transcriptome analysis of adenomyosis eutopic endometrium reveals molecular mechanisms involved in adenomyosis-related implantation failure and pregnancy disorders.

BACKGROUND: Women with adenomyosis are characterized by having defective decidualization, impaired endometrial receptivity and/or embryo-maternal communication, and implantation failure. However, the molecular mechanisms underlying adenomyosis-related infertility remain unknown, mainly because of the restricted accessibility and the difficult preservation of endometrial tissue in vitro. We have recently shown that adenomyosis patient-derived endometrial organoids, maintain disease-specific features while differentiated into mid-secretory and gestational endometrial phase, overcoming these research barriers and providing a robust platform to study adenomyosis pathogenesis and the associated molecular dysregulation related to implantation and pregnancy disorders. For this reason, we aim to characterize the dysregulated mechanisms in the mid-secretory and gestational endometrium of patients with adenomyosis by RNA-sequencing. METHODS: Endometrial organoids were derived from endometrial biopsies collected in the proliferative phase of women with adenomyosis (ADENO) or healthy oocyte donors (CONTROL) (n = 15/group) and differentiated into mid-secretory (-SECorg) and gestational (-GESTorg) phases in vitro. Following RNA-sequencing, the significantly differentially expressed genes (DEGs) (FDR < 0.05) were identified and selected for subsequent functional enrichment analysis and QIAGEN Ingenuity Pathway Analysis (IPA). Statistical differences in gene expression were evaluated with the Student's t-test or Wilcoxon test. RESULTS: We identified 1,430 DEGs in ADENO-SECorg and 1,999 DEGs in ADENO-GESTorg. In ADENO-SECorg, upregulated genes included OLFM1, FXYD5, and RUNX2, which are involved in impaired endometrial receptivity and implantation failure, while downregulated genes included RRM2, SOSTDC1, and CHAC2 implicated in recurrent implantation failure. In ADENO-GESTorg, upregulated CXCL14 and CYP24A1 and downregulated PGR were related to pregnancy loss. IPA predicted a significant inhibition of ID1 signaling, histamine degradation, and activation of HMGB1 and Senescence pathways, which are related to implantation failure. Alternatively, IPA predicted an inhibition of D-myo-inositol biosynthesis and VEGF signaling, and upregulation of Rho pathway, which are related to pregnancy loss and preeclampsia. CONCLUSIONS: Identifying dysregulated molecular mechanisms in mid-secretory and gestational endometrium of adenomyosis women contributes to the understanding of adenomyosis-related implantation failure and/or pregnancy disorders revealing potential therapeutic targets. Following experimental validation of our transcriptomic and in silico findings, our differentiated adenomyosis patient-derived organoids have the potential to provide a reliable platform for drug discovery, development, and personalized drug screening for affected patients.

Impact of adenomyosis on in vitro fertilization outcomes in women undergoing donor oocyte transfers: a prospective observational study.

OBJECTIVE: To prospectively examine the association between adenomyosis type, location, and severity with reproductive outcomes in patients undergoing single embryo transfer (SET) with embryos derived from donor oocytes. DESIGN: A prospective observational cohort study. SETTING: University-affiliated in vitro fertilization center. PATIENTS: Patients with infertility with (n = 114) and without (n = 114) adenomyosis who received their first donor oocyte transfer between January 2019 and January 2023 were included in this study. INTERVENTIONS: Adenomyosis was confirmed with the presence of at least one direct feature visualized by 2- or 3-dimensional transvaginal ultrasound and classified according to type (diffuse or focal), localization (inner or outer myometrium and/or junctional zone [JZ]), and uterine extension (mild, moderate, or severe). After an artificial or natural endometrial preparation cycle, patients underwent SET in the blastocyst stage. MAIN OUTCOME MEASURES: The primary outcome was the implantation rate. The secondary outcomes were the clinical pregnancy, live birth, and miscarriage rates after SET. RESULTS: The presence of adenomyosis did not significantly affect the implantation, clinical pregnancy, or live birth rates. However, women with adenomyosis had a significantly higher miscarriage rate than those without adenomyosis (35.4% vs. 18.1%, respectively). The multivariate analysis assessed possible risk factors for each clinical outcome considered in the study and showed that adenomyosis affected the risk of miscarriage. Specifically, transvaginal sonography detection of adenomyosis in the JZ was associated with over threefold higher relative risk of miscarriage (relative risk [RR], 3.28; 95% confidence interval [CI], 1.38-7.78). Conversely, adenomyosis features detected exclusively in the outer myometrium were associated with a higher ongoing pregnancy rate (RR, 0.30; 95% CI, 0.13-0.72). Diffuse adenomyosis in the JZ and severe adenomyosis increased the relative risk of miscarriage two-fold (RR, 2.29; 95% CI, 1.22-4.30 and RR, 2.20; 95% CI, 1.19-4.04, respectively). CONCLUSIONS: This study demonstrated that although adenomyosis did not significantly reduce the odds of implantation, the direct signs of adenomyosis in the JZ and disease severity are significant risk factors for miscarriage in patients receiving donor oocyte transfers. This study highlights the importance of thorough ultrasound examination and detailed adenomyosis classification in the assessment and management of patients with infertility.

Serum progesterone is lower in ovarian stimulation with highly purified HMG compared to recombinant FSH owing to a different regulation of follicular steroidogenesis: a randomized controlled trial.

STUDY QUESTION: Does ovarian stimulation with highly purified (hp)-HMG protect from elevated progesterone in the follicular phase compared to recombinant FSH (r-FSH) cycles through a different regulation of follicular steroidogenesis? SUMMARY ANSWER: hp-HMG enhanced the Δ4 pathway from pregnenolone to androstenodione leading to lower serum progesterone at the end of the cycle, while r-FSH promoted the conversion of pregnenolone to progesterone causing higher follicular phase progesterone levels. WHAT IS KNOWN ALREADY: Elevated progesterone in the follicular phase has been related to lower clinical outcome in fresh IVF cycles. Progesterone levels are positively correlated to ovarian response, and some studies have shown that when r-FSH alone is used for ovarian stimulation serum progesterone levels on the day of triggering are higher than when hp-HMG is given. Whether this is caused by a lower ovarian response in hp-HMG cycles or to a difference in follicular steroidogenesis in the two ovarian stimulation regimens has not been well characterized. STUDY DESIGN, SIZE, DURATION: A randomized controlled trial including 112 oocyte donors undergoing ovarian stimulation with GnRH antagonists and 225 IU/day of r-FSH (n = 56) or hp-HMG (n = 56) was carried out in a university-affiliated private infertility clinic. Subjects were recruited between October 2016 and June 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: The women were aged 18-35 years with a regular menstrual cycle (25-35 days) and normal ovarian reserve (serum anti-Müllerian hormone (AMH) = 10-30 pMol/l) undergoing ovarian stimulation for oocyte donation. FSH, LH, estradiol (E2), estrone, progesterone, pregnenolone, 17-OH-progesterone, androstenodione, dehidroepiandrostenodione, and testosterone were determined on stimulation Days 1, 4, 6, and 8 and on day of triggering in serum and in follicular fluid. Samples were frozen at -20°C until assay. Total exposures across the follicular phase were compared by polynomic extrapolation. MAIN RESULTS AND THE ROLE OF CHANCE: Subjects in both groups were comparable in terms of age, BMI, and AMH levels. Ovarian response was also similar: 17.5 ± 7.9 (mean ± SD) versus 16.5 ± 7.5 oocytes with r-FSH and hp-HMG, respectively (P = 0.49). Serum progesterone (ng/ml) on day of trigger was 0.46 ± 0.27 in the hp-HMG group versus 0.68 ± 0.50 in the r-FSH group (P = 0.010). Differences for progesterone were also significant on stimulation days 6 and 8. The pregnenolone: progesterone ratio was significantly increased in the r-FSH group from stimulation day 8 to the day of trigger (P = 0.019). Serum androstenodione (ng/ml) on day of trigger was 3.0 ± 1.4 in the hp-HMG group versus 2.4 ± 1.1 in the r-FSH group (P = 0.015). Differences in adrostenodione were also significant on stimulation Day 8. The pregnenolone:androstenodione ratio was significantly higher in the hp-HMG group (P = 0.012) on Days 6 and 8 and trigger. There were no other significant differences between groups. Follicular fluid E2, FSH, LH, dehidroepioandrostenodione, androstenodione, and testosterone were significantly higher in the hp-HMG than r-FSH group. No differences were observed for progesterone, estrone, 17-OH-progesterone, and pregnenolone in follicular fluid. LIMITATIONS, REASONS FOR CAUTION: All women included in the study were young, not infertile, and had a normal BMI and a good ovarian reserve. The findings might be different in other patient subpopulations. Hormone analyses with immunoassays are subject to intra-assay variations that may influence the results. WIDER IMPLICATIONS OF THE FINDINGS: Stimulation with hp-HMG may prevent progesterone elevation at the end of the follicular phase because of a different follicular steroidogenesis pathway, regardless of ovarian response. This should be considered, particularly in patients at risk of having high progesterone levels at the end of the follicular phase when a fresh embryo transfer is planned. STUDY FUNDING/COMPETING INTEREST(S): Roche Diagnostics provided unrestricted funding for all serum and follicular fluid hormone determinations. J.L.R., M.M., and A.P. have nothing to declare. E.B. has received consulting fees from Ferring, Merck, Gedeon Richter, and Roche and has participated in a research cooperation with Gedeon-Richter. In addition, the author has participated in speakers' bureau and received fees from Ferring, Gedeon Richter, Merck, and Roche. P.A. has received consulting fees from MSD and has participated in speakers' bureau and received fees from Ferring. P.A. also declares travel/meeting support from MSD. E.L. has received consulting fees from Ferring and MSD. In addition, the author has participated in a research cooperation with Gedeon-Richter. Also, the author has participated in speakers' bureau and received fees from Ferring and IBSA, as well as travel/meeting support from IBSA and Gedeon Richter. E.B., P.A., and E.L. also own stocks in IVIRMA Valencia. TRIAL REGISTRATION NUMBER: NCT: NCT02738580. TRIAL REGISTER DATE: 19 February 2016. DATE OF FIRST PATIENT'S ENROLMENT: 03 October 2016.

Systemic changes induced by autologous stem cell ovarian transplant in plasma proteome of women with impaired ovarian reserves.

Patients with poor ovarian response (POR) and premature ovarian insufficiency (POI) are challenging to treat, with oocyte donation remaining as the only feasible option to achieve pregnancy in some cases. The Autologous stem cell ovarian transplantation (ASCOT) technique allows follicle development, enabling pregnancies and births of healthy babies in these patients. Previous results suggest that growth factors and cytokines secreted by stem cells are partially responsible for their regenerative properties. Indeed, ASCOT beneficial effects associate with the presence of different bone marrow derived stem cell- secreted factors in plasma. Therefore, the aim of this study was to assess whether ASCOT induce any modifications in the plasma proteomic profile of patients with impaired ovarian reserves. Discriminant analysis highlighted clear distinctions between the plasma proteome before (PRE), during stem cell mobilization and collection (APHERESIS) and three months after ASCOT (POST) in patients with POR and POI. Both the stem cell mobilization and ASCOT technique induced statistically significant modifications in the plasma composition, reversing some age-related protein expression changes. In the POR group, functional analysis revealed an enrichment in processes related to the complement cascade, immune system, and platelet degranulation, while in the POI group, enriched processes were also associated with responses to oxygen-containing compounds and growth hormones, and blood vessel maturation. In conclusion, our findings highlight the potential proteins and biological processes that may promote the follicle activation and growth observed after ASCOT. Identifying plasma proteins that regenerate aged or damaged ovaries could lead to more effective, targeted and/or preventive therapies for patients.

Hybrid Endometrial-Derived Hydrogels: Human Organoid Culture Models and In Vivo Perspectives.

The endometrium plays a vital role in fertility, providing a receptive environment for embryo implantation and development. Understanding the endometrial physiology is essential for developing new strategies to improve reproductive healthcare. Human endometrial organoids (hEOs) are emerging as powerful models for translational research and personalized medicine. However, most hEOs are cultured in a 3D microenvironment that significantly differs from the human endometrium, limiting their applicability in bioengineering. This study presents a hybrid endometrial-derived hydrogel that combines the rigidity of PuraMatrix (PM) with the natural scaffold components and interactions of a porcine decellularized endometrial extracellular matrix (EndoECM) hydrogel. This hydrogel provides outstanding support for hEO culture, enhances hEO differentiation efficiency due to its biochemical similarity with the native tissue, exhibits superior in vivo stability, and demonstrates xenogeneic biocompatibility in mice over a 2-week period. Taken together, these attributes position this hybrid endometrial-derived hydrogel as a promising biomaterial for regenerative treatments in reproductive medicine.

Deciphering reproductive aging in women using a NOD/SCID mouse model for distinct physiological ovarian phenotypes.

Female fertility is negatively correlated with age, with noticeable declines in oocyte quantity and quality until menopause. To understand this physiological process and evaluate human approaches for treating age-related infertility, preclinical studies in appropriate animal models are needed. Thus, we aimed to characterize an immunodeficient physiological aging mouse model displaying ovarian characteristics of different stages during women's reproductive life. NOD/SCID mice of different ages (8-, 28-, and 36-40-week-old) were employed to mimic ovarian phenotypes of young, Advanced Maternal Age (AMA), and old women (~18-20-, ~36-38-, and >45-years-old, respectively). Mice were stimulated, mated, and sacrificed to recover oocytes and embryos. Then, ovarian reserve, follicular growth, ovarian stroma, mitochondrial dysfunction, and proteomic profiles were assessed. Age-matched C57BL/6 mice were employed to cross-validate the reproductive outcomes. The quantity and quality of oocytes were decreased in AMA and Old mice. These age-related effects associated spindle and chromosome abnormalities, along with decreased developmental competence to blastocyst stage. Old mice had less follicles, impaired follicle activation and growth, an ovarian stroma inconducive to growth, and increased mitochondrial dysfunctions. Proteomic analysis corroborated these histological findings. Based on that, NOD/SCID mice can be used to model different ovarian aging phenotypes and potentially test human anti-aging treatments.

The effect of vitrification on blastocyst mitochondrial DNA dynamics and gene expression profiles.

PURPOSE: Does vitrification/warming affect the mitochondrial DNA (mtDNA) content and the gene expression profile of blastocysts? METHODS: Prospective cohort study in which 89 blastocysts were obtained from 50 patients between July 2017 and August 2018. mtDNA was measured in a total of 71 aneuploid blastocysts by means of real-time polymerase chain reaction (RT-PCR). Transcriptomic analysis was performed by RNA sequencing (RNA-seq) in an additional 8 aneuploid blastocysts cultured for 0 h after warming, and 10 aneuploid blastocysts cultured for 4-5 h after warming. RESULTS: A significant decrease in mtDNA content just during the first hour after the warming process in blastocysts was found (P < 0.05). However, mtDNA content experimented a significantly increased along the later culture hours achieving the original mtDNA levels before vitrification after 4-5 h of culture (P < 0.05). Gene expression analysis and functional enrichment analysis revealed that such recovery was accompanied by upregulation of pathways associated with embryo developmental capacity and uterine embryo development. Interestingly, the significant increase in mtDNA content observed in blastocysts just after warming also coincided with the differential expression of several cellular stress response-related pathways, such as apoptosis, DNA damage, humoral immune responses, and cancer. CONCLUSION: To our knowledge, this is the first study demonstrating in humans, a modulation in blastocysts mtDNA content in response to vitrification and warming. These results will be useful in understanding which pathways and mechanisms may be activated in human blastocysts following vitrification and warming before a transfer.

Long-term effect of chemotherapy after ovarian decortication on the ovarian function in women surviving cancer.

PURPOSE: Ovarian decortication may affect ovarian function. We investigated the status of ovarian reserve after ovarian decortication plus chemotherapy at a stage of presumed stabilized recovery in women surviving cancer. METHODS: We searched our database for cancer survivors subjected to ovarian decortication and chemotherapy at least 3 years previously. Ovarian function was explored for levels of anti-Müllerian hormone (AMH), follicle-stimulating hormone (FSH), and estradiol (E2), and menstrual pattern. RESULTS: Forty women (mean age 29.6 (SD, 6.1) years) were assessed at a mean of 4.7 (1.5) years after surgery. The predecortication levels of AMH and FSH changed at post-treatment from 2.2 (1.4) to 0.5 (1.3) ng/mL for AMH (p < 0.001) and from 4.7 (2.1) to 16.7 (21. 6) IU/L for FSH (p < 0.001). Amenorrhea consistent with primary ovarian insufficiency (POI) was diagnosed in 11 women, and normal ovarian reserve (AMH ≥ 1.0 ng/mL) was found in 4 of the 21 women who recovered regular cycles. Logistic regression confirmed AMH as an independent predictor of diminished ovarian reserve (OR = 0.24, 95% CI: 0.04-0.63, p = 0.025) and POI (OR = 0.11, 95% CI: 0.01-0.52, p = 0.027), and age was predictive of POI (OR = 1.36, 95% CI: 1.08-1.96, p = 0.035) and of irregular menstrual cycle (OR = 1.20, 95% CI: 1.03-1.46, p = 0.034). CONCLUSION: Ovarian decortication plus chemotherapy had a deleterious effect when assessed at a stage of stabilized ovarian recovery, but whether ovarian decortication had a specific impact cannot be revealed from our data.

Reproductive outcomes of oocyte donation in patients with uterine Müllerian anomalies.

OBJECTIVE: To evaluate live-birth rates per embryo transfer in patients with uterine Müllerian anomalies (UMAs). Secondary objectives were to compare reproductive outcomes between the normal uterus group, the different UMA types, and UMA subgroups with and without required surgery. DESIGN: This retrospective study compared two cohorts, one with UMAs and other with normal uteri of our oocyte donation program at 12 Instituto Valenciano De Infertilidad/Reproductive Medicine Associates University affiliated clinics from January 2000 to 2020. The oocyte donation reduces confounding because of differences in embryo quality. The primary outcome was the live-birth rate per embryo transfer. Secondary outcomes included the rates of implantation, clinical pregnancy, miscarriage, and ongoing pregnancy. We calculated odds ratios with 95% confidence intervals. PATIENTS: Infertile women undergoing oocyte donation with UMAs. INTERVENTION: None. MAIN OUTCOME MEASURES: The rates of implantation, clinical pregnancy, miscarriage, ongoing pregnancy, and live birth. RESULTS: We analyzed 58,337 cycles of oocyte donation: 57,869 patients had no uterine malformation, and 468 women had UMAs. Compared with patients with normal uteri, patients with UMAs had lower rates of live births (36.67% [32.84-40.65] vs. 38.1% [95% confidence intervals {CI}: 37.82-38.42]) and ongoing pregnancy (39.74% [35.93-43.66] vs. 41.5% [41.24-41.83]). The miscarriage rate was higher in patients with UMAs (19.5% [16.55-22.85] vs. 16.6% [16.47-16.92]). Specifically, patients with a unicornuate uterus (n=29) had lower rates of implantation (24.07% [13.49-37.64] vs. 42.85% [95% CI: 42.6-43.09]), pregnancy (41.86% [27.01-57.87] vs. 59.51% [59.22-59.81]), ongoing pregnancy (16.67% [6.97-31.36] vs. 41.54% [41.24-41.83]), and live births (16.67% [6.97-31.36] vs. 38.12% [37.83-38.42]). In addition, patients with a partial septate uterus (n=91) had a higher miscarriage rate (26.50% [18.44-34.89] vs. 16.7% [16.47-16.92]). Compared with the normal uterus group, the live-birth rates were lower in the UMA without surgery group (33.09% [27.59-38.96] vs. 38.12% [37.83-38.42]). CONCLUSION: Among patients who received embryos derived from donated oocytes, live birth and ongoing pregnancy rates were lower in patients with UMAs compared with patients with normal uteri. A higher miscarriage rate was found in patients with UMAs. Patients with a unicornuate uterus had worse reproductive outcomes. Our results show that the uterus is less competent in patients with UMAs. TRIAL REGISTRATION: This study was registered at clinicaltrial.gov (NCT04571671).

Human blastocysts uptake extracellular vesicles secreted by endometrial cells containing miRNAs related to implantation.

STUDY QUESTION: Are the extracellular vesicles (EVs) secreted by the maternal endometrium uptaken by human embryos and is their miRNA cargo involved in implantation and embryo development? SUMMARY ANSWER: Data suggest that EVs secreted by human endometrial epithelial cells are internalized by human blastocysts, and transport miRNAs to modulate biological processes related to implantation events and early embryo development. WHAT IS KNOWN ALREADY: Successful implantation is dependent on coordination between maternal endometrium and embryo, and EVs role in the required cell-to-cell crosstalk has recently been established. In this regard, our group previously showed that protein cargo of EVs secreted by primary human endometrial epithelial cells (pHEECs) is implicated in biological processes related to endometrial receptivity, embryo implantation, and early embryo development. However, little is known about the regulation of these biological processes through EVs secreted by the endometrium at a transcriptomic level. STUDY DESIGN, SIZE, DURATION: A prospective descriptive study was performed. Endometrial biopsies were collected from healthy oocyte donors with confirmed fertility on the day of oocyte retrieval, 36 h after the LH surge. pHEECs were isolated from endometrial biopsies (n = 8 in each pool) and cultured in vitro. Subsequently, conditioned medium was collected and EVs were isolated and characterized. Uptake of EVs by human blastocysts and miRNA cargo of these EVs (n = 3 pools) was analyzed. PARTICIPANTS/MATERIALS, SETTING, METHODS: EVs were isolated from the conditioned culture media using ultracentrifugation, and characterization was performed using western blotting, nanoparticle tracking analysis, and transmission electron microscopy. EVs were fluorescently labeled with Bodipy-TR ceramide, and their uptake by human blastocysts was analyzed using confocal microscopy. Analysis of the miRNA cargo of EVs was performed using miRNA sequencing, target genes of the most expressed miRNA were annotated, and functional enrichment analysis was performed. MAIN RESULTS AND THE ROLE OF CHANCE: EVs measured 100-300 nm in diameter, a concentration of 1.78 × 1011 ± 4.12 × 1010 (SD) particles/ml and expressed intraluminal protein markers Heat shock protein 70 (HSP70) and Tumor Susceptibility Gene 101 (TSG101), in addition to CD9 and CD81 transmembrane proteins. Human blastocysts efficiently internalized fluorescent EVs within 1-2 h, and more pronounced internalization was observed in the hatched pole of the embryos. miRNA-seq analysis featured 149 annotated miRNAs, of which 37 were deemed most relevant. The latter had 6592 reported gene targets, that in turn, have functional implications in several processes related to embryo development, oxygen metabolism, cell cycle, cell differentiation, apoptosis, metabolism, cellular organization, and gene expression. Among the relevant miRNAs contained in these EVs, we highlight hsa-miR-92a-3p, hsa-let-7b-5p, hsa-miR-30a-5p, hsa-miR-24-3p, hsa-miR-21-5p, and hsa-let-7a-5p as master regulators of the biological processes. LIMITATIONS, REASONS FOR CAUTION: This is an in vitro study in which conditions of endometrial cell culture could not mimic the intrauterine environment. WIDER IMPLICATIONS OF THE FINDINGS: This study defines potential biomarkers of endometrial receptivity and embryo competence that could be useful diagnostic and therapeutic targets for implantation success, as well as open insight further investigations to elucidate the molecular mechanisms implicated in a successful implantation. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Spanish Ministry of Education through FPU awarded to M.S.-B. (FPU18/03735), the Health Institute Carlos III awarded to E.J.-B. (FI19/00110) and awarded to H.F. by the Miguel Servet Program 'Fondo Social Europeo «El FSE invierte en tu futuro¼' (CP20/00120), and Generalitat Valenciana through VALi+d Programme awarded to M.C.C.-G. (ACIF/2019/139). The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER: N/A.

A Randomized Controlled Intervention Trial with Danazol to Improve Telomeric and Fertility Parameters in Women with Diminished Ovarian Reserve: A Pilot Study.

Background: Most women who are treated at in vitro fertilization (IVF) clinics have trouble conceiving due to ovarian failure (OF), which seems to be associated to short telomeres and reduced or absent telomerase activity in their granulosa cells. Indeed, telomere pathways are involved in organ dysfunction. However, sexual steroids can stimulate the expression of the telomerase gene and have been successfully used to prevent telomere attrition. Thus, a strategy to improve IVF outcomes in women with OF could be telomerase reactivation using sexual steroids. Methods: We conducted a double-blind, placebo-controlled study. Patients with diminished ovarian reserve were randomized to Danazol or placebo for 3 months. We included patients with normal ovarian reserve in the study as untreated controls. Patients and controls underwent several ovarian stimulations (OSs). Telomere and IVF parameters were assessed. Results: We found that the mean telomere length in blood and the percentage of short and long telomeres were similar throughout the 3 months of treatment with Danazol. Remarkably, while the number of cells with one telomeric repeat-containing RNA (TERRA) focus decreased (p = 0.04) after the first month of Danazol treatment, the number of cells with 2 to 4 TERRA foci increased (p = 0.02). Regarding fertility, no differences were found in the antral follicle count. Interestingly, in OS performed after the trial, all Danazol-treated patients had a better MII oocyte rate compared to OS performed before the pilot study.EudraCT number: 2018-004400-19. Conclusions: Danazol treatment seemed to affect telomere maintenance, since both the number of TERRA foci and the ratio of MII oocytes changed. However, further research is needed to confirm these results.

Advances of xenogeneic ovarian extracellular matrix hydrogels for in vitro follicle development and oocyte maturation.

Research aimed at preserving female fertility is increasingly using bioengineering techniques to develop new platforms capable of supporting ovarian cell function in vitro and in vivo. Natural hydrogels (alginate, collagen, and fibrin) have been the most exploited approaches; however they are biologically inert and/or biochemically simple. Thus, establishing a suitable biomimetic hydrogel from decellularized ovarian cortex (OC) extracellular matrix (OvaECM) could provide a complex native biomaterial for follicle development and oocyte maturation. The objectives of this work were (i) to establish an optimal protocol to decellularize and solubilize bovine OC, (ii) to characterize the histological, molecular, ultrastructural, and proteomic properties of the resulting tissue and hydrogel, and (iii) to assess its biocompatibility and adequacy for murine in vitro follicle growth (IVFG). Sodium dodecyl sulfate was identified as the best detergent to develop bovine OvaECM hydrogels. Hydrogels added into standard media or used as plate coatings were employed for IVFG and oocyte maturation. Follicle growth, survival, hormone production, and oocyte maturation and developmental competence were evaluated. OvaECM hydrogel-supplemented media best supported follicle survival, expansion, and hormone production, while the coatings provided more mature and competent oocytes. Overall, the findings support the xenogeneic use of OvaECM hydrogels for future human female reproductive bioengineering.

Preimplantation genetic testing for aneuploidy is not related to adverse obstetric and neonatal outcomes in singleton pregnancies.

STUDY QUESTION: What is the potential impact of preimplantation genetic testing for aneuploidy (PGT-A) on obstetric and neonatal outcomes? SUMMARY ANSWER: PGT-A is not associated with increased rates of adverse maternal and neonatal outcomes in singleton pregnancies following IVF/ICSI cycles. WHAT IS KNOWN ALREADY: PGT-A pregnancies may be associated with increased risks of lower birthweight, preterm delivery, and hypertensive disorders compared with natural pregnancies. In a recent meta-analysis, the overall obstetric and neonatal outcomes of PGT-A pregnancies were favorable compared with those of IVF/ICSI pregnancies, although PGT-A pregnancies were associated with a higher risk of hypertensive disorders. STUDY DESIGN, SIZE, DURATION: A multicenter retrospective cohort study was performed in University-affiliated infertility centers. Single live births following IVF/ICSI between October 2016 and January 2021 were included in the study. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 7146 live births after single embryo transfers with (n = 3296) or without (n = 3850) PGT-A were included. The primary outcome was pre-eclampsia and secondary outcomes included gestational diabetes, low birthweight and very low birthweight, cesarean section delivery, emergency cesarean section, as well as preterm birth, birthweight, congenital abnormalities, neonatal sex, Apgar score at 5 min, and neonatal intensive care unit admission. In a subgroup analysis, were included only blastocysts screened with next-generation sequencing (NGS). MAIN RESULTS AND THE ROLE OF CHANCE: Univariate analysis showed that pre-eclampsia, cesarean section incidence, and low Apgar score were higher in women undergoing PGT-A. However, after performing multivariate logistic and linear regression models accounting for many possible confounders, pregnancies that had been conceived after embryo biopsy showed no increase in adverse obstetric and neonatal outcomes. The subgroup analysis including patients with blastocysts screened by NGS showed a decreased risk of preterm birth in the group undergoing PGT-A. LIMITATIONS, REASONS FOR CAUTION: Caution should be used when interpreting the data because of its limitations, mainly related to its retrospective design. Although this is a large multicenter study, data acquisition included self-reporting questionnaires, and the deliveries occurred in different institutions with distinct protocols. WIDER IMPLICATIONS OF THE FINDINGS: The current study does not show any major adverse clinical outcomes after PGT-A. Efforts should be made to promote good quality research on embryo biopsy in terms of neonatal and obstetric outcomes, as well as its long-term consequences. STUDY FUNDING/COMPETING INTEREST(S): No specific funding was obtained for this study. The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

The international transportation of frozen embryos does not affect IVF outcomes.

PURPOSE: The regulated transportation of cryopreserved human embryos resulting from assisted reproduction treatments offers opportunities for patients undergoing embryo transfer in other regions/countries. However, the principal concern for fertility clinics is maintaining unaltered embryo quality to ensure satisfactory clinical outcomes. The aim of the study was to evaluate the efficacy of the transportation process comparing the survival rate and competence of transported embryos to embryos produced and transferred on-site, in frozen embryo transfer cycles. METHODS: This retrospective study assessed the outcomes of 621 blastocysts thawed at IVI Roma (Italy) between March 2021 and March 2022. Autologous or donated oocytes fertilized in vitro, cultured to the blastocyst stage, and cryopreserved in IVI Roma clinic (Group A, n = 450), were compared to embryos generated in IVI Spain clinics and transported to IVI Roma (Group B, n = 171). RESULTS: Groups A and B respectively showed no significant difference in embryo survival rates after thawing (N = 440/450, 97.8% vs. N = 168/171, 98.2%, p = 0.71), pregnancy rates (N = 221/440, 50.23% vs. N = 77/168, 45.83%, p = 0.33), clinical pregnancy rates (N = 200/440, 45.45% vs. N = 62/168, 36.90%, p = 0.06), and miscarriage rates (N = 42/221, 19,00% vs. 21/77, 28.57%, p = 0.13), even after stratification for the source of the oocyte. Logistic binomial regression considering donor oocytes, preimplantation genetic testing, and patients' age, did not show any significant results on embryo survival and IVF outcomes. CONCLUSION: The regulated transport of cryopreserved blastocysts did not affect embryo survival rate or IVF outcomes. Our data support the safety of embryo cryopreservation and medical transportation services, allowing clinics and patients to transport embryos with no significant risk to embryo competence.

Improving success rates by applying interventions in clinical practice and measuring their impact: A multicenter retrospective analysis of more than 240,000 cycles.

BACKGROUND: Systematic monitoring of key performance indicators (KPI) is an important component of quality management within the IVF laboratory and, as success of assisted reproduction depends on many variables, it is important to examine how each variable can be optimized to achieve the best possible outcome for patients. OBJECTIVE: To analyze how the design of a QMS impacts homogenization, safety, and efficacy in multiple fertility centers. Study Design Multicenter, retrospective cohort study with 188,251 patients who underwent 246,988 assisted reproductive treatments at 14 private centers belonging to IVI-RMA clinics between January 2005 and December 2019. Data were stratified by year, clinic, and patient group (standard patient cycles with no PGT-A, standard patients with PGT-A, and oocyte donation patients). Unadjusted and adjusted logistic regression models with other known predictors were made to analyze the impact and the interactions of policies. Main outcomes were determined per clinic and summarized per year as the median of the rates of the clinics; each clinic had the same weight independent of the number of cycles. RESULTS: Up to 188,251 patients were treated, for a total of 246,988 IVF cycles and 356,433 procedures. The introduction of standard operating procedures, trophectoderm biopsies, and blastocyst-stage transfers, coupled with an increased proportion of PGT-A cycles, led to improved outcomes while maximizing the number of single embryo transfers, driving a significant decrease in the number of multiple pregnancies while improving live birth rates. In terms of the live-birth rate per transfer, the interventions with greater impact over time in logistic regression analysis were 24-chromosome analysis and the introduction of benchtop incubators (odds ratio 1.92 [95% confidence interval 1.81 to 2.05]; p < 0.001). The odd ratios of the policies remained significant and very similar in the unadjusted and adjusted models. CONCLUSIONS: The greatest impact on live-birth rate per cycle was obtained with a cumulative effect of all policies, especially in egg donation patients. In patients without PGT-A changing embryo culture conditions and blastocyst stage transfer had the greatest impact; in patients with PGT-A, trophectoderm biopsy. Standardizing procedures was essential in reducing variability among clinics and implementing changes.

Impaired telomere pathway and fertility in Senescence-Accelerated Mice Prone 8 females with reproductive senescence.

Ovarian aging is the main cause of infertility and telomere attrition is common to both aging and fertility disorders. Senescence-Accelerated Mouse Prone 8 (SAMP8) model has shortened lifespan and premature infertility, reflecting signs of reproductive senescence described in middle-aged women. Thus, our objective was to study SAMP8 female fertility and the telomere pathway at the point of reproductive senescence. The lifespan of SAMP8 and control mice was monitored. Telomere length (TL) was measured by in situ hybridization in blood and ovary. Telomerase activity (TA) was analyzed by telomere-repeat amplification protocol, and telomerase expression, by real-time quantitative PCR in ovaries from 7-month-old SAMP8 and controls. Ovarian follicles at different stages of maturation were evaluated by immunohistochemistry. Reproductive outcomes were analyzed after ovarian stimulation. Unpaired t-test or Mann-Whitney test were used to calculate p-values, depending on the variable distribution. Long-rank test was used to compare survival curves and Fisher's exact test was used in contingency tables. Median lifespan of SAMP8 females was reduced compared to SAMP8 males (p = 0.0138) and control females (p < 0.0001). In blood, 7-month-old SAMP8 females presented lower mean TL compared to age-matched controls (p = 0.041). Accordingly, the accumulation of short telomeres was higher in 7-month-old SAMP8 females (p = 0.0202). Ovarian TA was lower in 7-month-old SAMP8 females compared to controls. Similarly, telomerase expression was lower in the ovaries of 7-month-old SAMP8 females (p = 0.04). Globally, mean TL in ovaries and granulosa cells (GCs) were similar. However, the percentage of long telomeres in ovaries (p = 0.004) and GCs (p = 0.004) from 7-month-old SAMP8 females was lower compared to controls. In early-antral and antral follicles, mean TL of SAMP8 GCs was lower than in age-matched controls (p = 0.0156 for early-antral and p = 0.0037 for antral follicles). Middle-aged SAMP8 showed similar numbers of follicles than controls, although recovered oocytes after ovarian stimulation were lower (p = 0.0068). Fertilization rate in oocytes from SAMP8 was not impaired, but SAMP8 mice produced significantly more morphologically abnormal embryos than controls (27.03% in SAMP8 vs. 1.22% in controls; p < 0.001). Our findings suggest telomere dysfunction in SAMP8 females, at the time of reproductive senescence.

Ovarian rescue in women with premature ovarian insufficiency: facts and fiction.

The ovary has a comparatively short functional lifespan compared with other organs, and genetic and pathological injuries can further shorten its functional life. Thus, preserving ovarian function should be considered in the context of women with threats to ovarian reserve, such as ageing, premature ovarian insufficiency (POI) and diminished ovarian reserve (DOR). Indeed, one-third of women with POI retain resting follicles that can be reactivated to produce competent oocytes, as proved by the in-vitro activation of dormant follicles. This paper discusses mechanisms and clinical data relating to new therapeutic strategies using ovarian fragmentation, stem cells or platelet-rich plasma to regain ovarian function in women of older age (>38 years) or with POI or DOR. Follicle reactivation techniques show promising experimental outcomes and have been successful in some cases, when POI is established or DOR diagnosed; however, there is scarce clinical evidence to warrant their widespread clinical use. Beyond these contexts, also discussed is how new insights into the biological mechanisms governing follicular dynamics and oocyte competence may play a role in reversing ovarian damage, as no technique modifies oocyte quality. Additional studies should focus on increasing follicle number and quality. Finally, there is a small but important subgroup of women lacking residual follicles and requiring oocyte generation from stem cells.

H3K4me3 mediates uterine leiomyoma pathogenesis via neuronal processes, synapsis components, proliferation, and Wnt/ß-catenin and TGF-ß pathways.

BACKGROUND: Uterine leiomyomas (UL) are the most common benign tumor in women of reproductive age. Their pathology remains unclear, which hampers the development of safe and effective treatments. Raising evidence suggests epigenetics as a main mechanism involved in tumor development. Histone modification is a key component in the epigenetic regulation of gene expression. Specifically, the histone mark H3K4me3, which promotes gene expression, is altered in many tumors. In this study, we aimed to identify if the histone modification H3K4me3 regulates the expression of genes involved in uterine leiomyoma pathogenesis. METHODS: Prospective study integrating RNA-seq (n = 48) and H3K4me3 CHIP-seq (n = 19) data of uterine leiomyomas versus their adjacent myometrium. Differentially expressed genes (FDR < 0.01, log2FC > 1 or < - 1) were selected following DESeq2, edgeR, and limma analysis. Their differential methylation and functional enrichment (FDR < 0.05) were respectively analyzed with limma and ShinyGO. RESULTS: CHIP-seq data showed a global suppression of H3K4me3 in uterine leiomyomas versus their adjacent myometrial tissue (p-value< 2.2e-16). Integrating CHIP-seq and RNA-seq data highlighted that transcription of 696/922 uterine leiomyoma-related differentially expressed genes (DEG) (FDR < 0.01, log2FC > 1 or < - 1) was epigenetically mediated by H3K4me3. Further, 50 genes were differentially trimethylated (FDR < 0.05), including 33 hypertrimethylated/upregulated, and 17 hypotrimethylated/downregulated genes. Functional enrichment analysis of the latter showed dysregulation of neuron-related processes and synapsis-related cellular components in uterine leiomyomas, and a literature review study of these DEG found additional implications with tumorigenesis (i.e. aberrant proliferation, invasion, and dysregulation of Wnt/ß-catenin, and TGF-ß pathways). Finally, SATB2, DCX, SHOX2, ST8SIA2, CAPN6, and NPTX2 proto-oncogenes were identified among the hypertrimethylated/upregulated DEG, while KRT19, ABCA8, and HOXB4 tumor suppressor genes were identified among hypotrimethylated/downregulated DEG. CONCLUSIONS: H3K4me3 instabilities alter the expression of oncogenes and tumor suppressor genes, inducing aberrant proliferation, and dysregulated Wnt/ß-catenin, and TGF-ß pathways, that ultimately promote uterine leiomyoma progression. The reversal of these histone modifications may be a promising new therapeutic alternative for uterine leiomyoma patients.