Abstract
STUDY QUESTION
Is there a relationship between serum and endometrial progesterone (P4) levels, including P4 and metabolites (oestrone, oestradiol and 17α-hydroxyprogesterone), and ...endometrial receptivity?
SUMMARY ANSWER
Serum P4 levels were not correlated with endometrial P4, nor associated with endometrial receptivity as determined by the ERA® test; however, endometrial P4 and 17α-hydroxyprogesterone levels were positively correlated and related to endometrial receptivity by ERA.
WHAT IS KNOWN ALREADY
Acquisition of endometrial receptivity is governed by P4, which induces secretory transformation. A close relationship between serum P4 and pregnancy outcome is reported for hormone replacement therapy (HRT) cycles. However, the relationship between serum and uterine P4 levels has not been described, and it is unknown whether uterine receptivity depends more on serum or uterine P4 levels.
STUDY DESIGN, SIZE, DURATION
A prospective cohort study was performed during March 2018–2019 in 85 IVF patients undergoing an evaluation-only HRT cycle with oestradiol valerate (6 mg/day) and micronised vaginal progesterone (400 mg/12 h).
PARTICIPANTS/MATERIALS, SETTING, METHODS
Patients were under 50 years of age, had undergone at least one failed IVF cycle, had no uterine pathology, and had adequate endometrial thickness (> 6.5 mm). The study was conducted at IVI Valencia and IVI Foundation. An endometrial biopsy and a blood sample were collected after 5 days of P4 vaginal treatment. Measures included serum P4 levels, ERA®-based evaluation of endometrial receptivity, and endometrial P4 levels along with metabolites (oestrone, oestradiol and 17α-hydroxyprogesterone) measured by ultra-performance liquid chromatography–tandem mass spectrometry.
MAIN RESULTS AND THE ROLE OF CHANCE
Seventy-nine women were included (mean age: 39.9 ± 4.6, BMI: 24.2 ± 3.9 kg/m2, endometrial thickness: 8.2 ± 1.4 mm). The percentage of endometria indicated as receptive by ERA® was 40.5%. When comparing receptive versus non-receptive groups, no differences were observed in baseline characteristics nor in steroid hormones levels in serum or endometrium. No association between serum P4 and endometrial steroid levels or ERA result was found (P < 0.05). When the population was stratified according to metabolite concentration levels, endometrial P4 and 17α-hydroxyprogesterone were significantly associated with endometrial receptivity (P < 0.05). A higher proportion of receptive endometria by ERA was observed when endometrial P4 levels were higher than 40.07 µg/ml (relative maximum) and a lower proportion of receptive endometria was associated with endometrial 17α-hydroxyprogesterone lower than 0.35 ng/ml (first quartile). A positive correlation R2 = 0.67, P < 0.001 was observed between endometrial P4 and 17α-hydroxyprogesterone levels.
LIMITATIONS, REASONS FOR CAUTION
This study did not analyse pregnancy outcomes. Further, the findings can only be extrapolated to HRT cycles with micronised vaginal progesterone for luteal phase support.
WIDER IMPLICATIONS OF THE FINDINGS
Our findings suggest that the combined benefits of different routes of progesterone administration for luteal phase support could be leveraged to ensure an adequate concentration of progesterone both in the uterus and in the bloodstream. Further studies will confirm whether this method can optimise both endometrial receptivity and live birth rate. Additionally, targeted treatment to increase P4 endometrial levels may normalise the timing of the window of implantation without needing to modify the progesterone administration day.
STUDY FUNDING/COMPETING INTEREST(S)
This research was supported by the IVI-RMA Valencia (1706-VLC-051-EL) and Consellería d’Educació, Investigació, Cultura, i esport Generalitat Valenciana (Valencian Government, Spain, GV/2018//151). Almudena Devesa-Peiro (FPU/15/01398) and Cristina Rodriguez-Varela (FPU18/01657) were supported by the FPU program fellowship from the Ministry of Science, Innovation and Universities (Spanish Government). P.D.-G. is co-inventor on the ERA patent, with non-economic benefits. The other authors have no competing interests.
TRIAL REGISTRATION NUMBER
NCT03456375.
More than 10% of women diagnosed with breast cancer during reproductive age carry hereditary germline pathogenic variants in high-penetrance BRCA genes or in others genes involved in DNA repair ...mechanisms such as PALB2, BRIP or ATM. Anticancer treatments may have an additional negative impact on the ovarian reserve and subsequently on the fertility of young patients carrying such mutations. Recently, the combination of carboplatin and paclitaxel is being recommended to these BRCA-mutated patients as neoadjuvant therapy. However, the impact on the ovary is unknown. Here, we investigated their effect of on the ovarian reserve using mice carriers of BRCA1-interacting protein C-terminal helicase-1 (BRIP1) mutation that plays an important role in BRCA1-dependent DNA repair. Results revealed that the administration of carboplatin or paclitaxel did not affect the ovarian reserve although increased DNA double-strand breaks were observed with carboplatin alone. Co-administration of carboplatin and paclitaxel resulted in a significant reduction of the ovarian reserve leading to a lower IVF performance, and an activation of the PI3K-Pten pathway, irrespective of the genetic background. This study suggests that co-administration of carboplatin and paclitaxel induces cumulative ovarian damage and infertility but a heterozygote genetic predisposition for DNA damage related to BRCA1 gene function does not increase this risk.
Abstract
STUDY QUESTION
Can severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA be detected in the reproductive tract of asymptomatic patients undergoing ART?
SUMMARY ANSWER
SARS-CoV-2 ...mRNA is not detectable in semen, follicular fluid, vaginal secretions or residual medulla from ovarian tissue cryopreservation procedures in asymptomatic patients who undergo ART, irrespective of the results of a triage questionnaire and a nasopharyngeal SARS-CoV-2 RNA detection test.
WHAT IS KNOWN ALREADY
The SARS-CoV-2 pandemic had a huge impact on the activities of fertility clinics. Although some studies reported the presence of SARS-CoV-2 mRNA in the reproductive system during or after acute COVID-19 symptomatic infections, uncertainties remain regarding the presence of viral mRNA in the reproductive material and follicular fluid of asymptomatic patients undergoing ART.
STUDY DESIGN, SIZE, DURATION
An observational cohort trial of residual material samples including semen, follicular fluid, vaginal secretions and ovarian medulla was conducted during the second pandemic wave in Brussels from September 2020 to April 2021.
PARTICIPANTS/MATERIALS, SETTING, METHODS
All patients who underwent ART (IUI, IVF/ICSI, oocyte and ovarian tissue cryopreservation) responded to a triage questionnaire at the beginning and end of the cycle and underwent nasopharyngeal swab collection for SARS-CoV-2 RNA detection by RT-PCR before the procedure according to standard recommendations. For semen analysis, only the questionnaire was requested the day before the sample collection. The ART cycles of patients with positive nasopharyngeal SARS-CoV-2 RNA detection tests and/or questionnaires were cancelled except for those that could not be postponed. After providing informed consent, swabs on residual materials were collected the day of the oocyte, ovarian tissue or semen collection and were processed for RT-qPCR.
MAIN RESULTS AND THE ROLE OF CHANCE
A total of 394 samples from 291 patients were analysed. Amongst them, 20 samples were obtained from patients with a positive questionnaire but negative nasopharyngeal SARS-CoV-2 test and 20 others were from patients with a positive nasopharyngeal SARS-CoV-2 test. The remaining samples were collected from patients with a negative or unknown nasopharyngeal SARS-CoV-2 test and/or a negative or unknown triage questionnaire. Viral RNA for SARS-CoV-2 was undetectable in all of the samples.
LIMITATIONS, REASONS FOR CAUTION
Considering the cancellation policy, only a limited number of samples from patients with positive triage questionnaires or nasopharyngeal SARS-CoV-2 tests were included in the analysis.
WIDER IMPLICATIONS OF THE FINDINGS
The study suggested that there was no risk of reproductive tract contamination by SARS-CoV-2 in asymptomatic patients, irrespective of the results from a triage questionnaire or nasopharyngeal SARS-CoV-2 test. The results suggested that no additional measures to prevent staff or cross-patient contamination need to be implemented in the IVF and andrology laboratories.
STUDY FUNDING/COMPETING INTEREST(S)
This study was funded by the Université Libre de Bruxelles and by a grant from Ferring. A.D. and I.D. received a grant from Ferring for the study. The authors have no other conflict of interest to declare related to this study.
TRIAL REGISTRATION NUMBER
N/A.
► The laccase–phenol treatment could result in the grafting of these phenols onto the fibres. ► These enzyme treatments may permit to give novel or improve properties to flax fibres. ► Bioactive ...papers have been created: treated papers present high antimicrobial activity. ► The antimicrobial agents grafted are immobilized in the flax fibres.
Unbleached flax fibres for paper production were treated with laccase from Pycnoporus cinnabarinus and low molecular weight phenols (syringaldehyde – SA, acetosyringone – AS and p-coumaric acid – PCA) to evaluate the potential of this treatment to biomodify high cellulose content fibres. After the enzymatic treatment with the phenols, an increase in kappa number was found, probably due to a covalent binding of the phenoxy radicals on fibres. Grafting was more evident in pulps treated with PCA (an increase of 4 kappa number points with respect to the laccase control was achieved). Paper handsheets from treated pulps showed antimicrobial activity against the bacteria tested: Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumoniae. An important reduction on microbial count was obtained after incubation of liquid cultures of the bacteria with grafted handsheets. AS and PCA grafted fibres showed a high antibacterial activity on K. pneumoniae, getting a nearly total growth inhibition. AS fibres also caused a high reduction in bacterial population of P. aeruginosa (97% reduction). Optical properties of handsheets from treated pulps were also determined, showing a brightness decrease and increase in coloration, evaluated by CIE L*a*b* system, caused by the laccase induced grafting of the phenols. The results suggest that these low molecular weight phenols, covalently bound to the flax fibres by the laccase treatment, can act as antimicrobial agents and produce handsheets with antimicrobial activity.
Abstract
STUDY QUESTION
What are the key considerations for developing an enhanced transcriptomic method for secretory endometrial tissue dating?
SUMMARY ANSWER
Multiple gene expression signature ...combinations can serve as biomarkers for endometrial dating, but their predictive performance is variable and depends on the number and identity of the genes included in the prediction model, the dataset characteristics and the technology employed for measuring gene expression.
WHAT IS KNOWN ALREADY
Among the new generation of transcriptomic endometrial dating (TED) tools developed in the last decade, there exists variation in the technology used for measuring gene expression, the gene makeup and the prediction model design. A detailed study, comparing prediction performance across signatures for understanding signature behaviour and discrepancies in gene content between them, is lacking.
STUDY DESIGN, SIZE, DURATION
A multicentre prospective study was performed between July 2018 and October 2020 at five different centres from the same group of clinics (Spain). This study recruited 281 patients and finally included in the gene expression analysis 225 Caucasian patients who underwent IVF treatment. After preprocessing and batch effect filtering, gene expression measurements from 217 patients were combined with artificial intelligence algorithms (support vector machine, random forest and k-nearest neighbours) allowing evaluation of different prediction models. In addition, secretory-phase endometrial transcriptomes from gene expression omnibus (GEO) datasets were analysed for 137 women, to study the endometrial dating capacity of genes independently and grouped by signatures. This provided data on the consistency of prediction across different gene expression technologies and datasets.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Endometrial biopsies were analysed using a targeted TruSeq (Illumina) custom RNA expression panel called the endometrial dating panel (ED panel). This panel included 301 genes previously considered relevant for endometrial dating as well as new genes selected for their anticipated value in detecting the secretory phase. Final samples (n = 217) were divided into a training set for signature discovery and an independent testing set for evaluation of predictive performance of the new signature. In addition, secretory-phase endometrial transcriptomes from GEO were analysed for 137 women to study endometrial dating capacity of genes independently and grouped by signatures. Predictive performance among these signatures was compared according to signature gene set size.
MAIN RESULTS AND THE ROLE OF CHANCE
Testing of the ED panel allowed development of a model based on a new signature of 73 genes, which we termed ‘TED’ and delivers an enhanced tool for the consistent dating of the secretory phase progression, especially during the mid-secretory endometrium (3–8 days after progesterone (P) administration (P + 3–P + 8) in a hormone replacement therapy cycle). This new model showed the best predictive capacity in an independent test set for staging the endometrial tissue in the secretory phase, especially in the expected window of implantation (average of 114.5 ± 7.2 h of progesterone administered; range in our patient population of 82–172 h). Published sets of genes, in current use for endometrial dating and the new TED genes, were evaluated in parallel in whole-transcriptome datasets and in the ED panel dataset. TED signature performance was consistently excellent for all datasets assessed, frequently outperforming previously published sets of genes with a smaller number of genes for dating the endometrium in the secretory phase. Thus, this optimized set exhibited prediction consistency across datasets.
LARGE SCALE DATA
The data used in this study is partially available at GEO database. GEO identifiers GSE4888, GSE29981, GSE58144, GSE98386.
LIMITATIONS, REASONS FOR CAUTION
Although dating the endometrial biopsy is crucial for investigating endometrial progression and the receptivity process, further studies are needed to confirm whether or not endometrial dating methods in general are clinically useful and to guide the specific use of TED in the clinical setting.
WIDER IMPLICATIONS OF THE FINDINGS
Multiple gene signature combinations provide adequate endometrial dating, but their predictive performance depends on the identity of the genes included, the gene expression platform, the algorithms used and dataset characteristics. TED is a next-generation endometrial assessment tool based on gene expression for accurate endometrial progression dating especially during the mid-secretory.
STUDY FUNDING/COMPETING INTEREST(S)
Research funded by IVI Foundation (1810-FIVI-066-PD). P.D.-G. visiting scientist fellowship at Oxford University (BEFPI/2010/032) and Josefa Maria Sanchez-Reyes’ predoctoral fellowship (ACIF/2018/072) were supported by a program from the Generalitat Valenciana funded by the Spanish government. A.D.-P. is supported by the FPU/15/01398 predoctoral fellowship from the Ministry of Science, Innovation and Universities (Spanish Government). D.W. received support from the NIHR Oxford Biomedical Research Centre. The authors do not have any competing interests to declare.
Abstract
Study question
Does first-line chemotherapy prior to ovarian tissue cryopreservation and diseases impact on the signaling pathways governing follicle activation and survival in pre-pubertal ...and adult patients?
Summary answer
Chemotherapy increases follicle apoptosis rate without affecting early follicular activation. Moreover, damages varied according to the time of last exposure and the diseases.
What is known already
Chemotherapeutic agents can impair the ovarian stockpile inducing a decrease of the reproductive potential of female cancer survivors. Among fertility preservation methods, cortical tissue cryopreservation is offered in patients who already started their treatment or cannot postpone it, and it is currently the only available procedure for pre-pubertal patients. Few studies did not report negative impact of prior first-line chemotherapy on ovarian tissue cryopreservation outcomes, the consequences of the treatment on the follicle activation in human remain to be further investigated. Identifying the signaling modifications induced by chemotherapy exposure is essential to address this question.
Study design, size, duration
Fragments of cryopreserved ovarian tissue from young adult (12-29 years old, n = 8) and pre-pubertal (3-10 years old, n = 7) cancer patients donated for research were thawed and cultured for 24h. Analyses of the follicle and stroma survival, as well as the PI3K/AKT/mTOR and Hippo signaling pathways were conducted at thawing and after culture. The different patients were grouped according to their pubertal stage and those who received chemotherapy before ovarian banking were compared to non-exposed patients.
Participants/materials, setting, methods
To assess the distribution and morphology of the follicles, the structure of the stroma, and the apoptosis, histological investigations were performed through follicular counting, Sirius Red staining, immunostaining, and TUNEL staining. The PI3K/AKT/mTOR and Hippo signaling pathways were investigated to explore the follicular activation among the different groups by gene expression and protein analyses on isolated follicles and cortex, respectively.
Main results and the role of chance
The deleterious impact of chemotherapy exposure prior to tissue banking was observed specifically on quiescent follicles by TUNEL staining at thawing and after culture in both adult and pre-pubertal patients. After 24h of culture, tissues previously exposed to chemotherapy displayed a higher rate of follicles containing DNA damages than the non-exposed fragments in adults (40.9% versus 19.1%), and pre-pubertal (48.3% versus 17.6%) patients. The atresia rates and DNA damages appeared to be lower into the tissue of patients that received their last cure 4-5 months before the cryopreservation among the treated adult and pre-pubertal groups. DNA reparation mechanisms were assessed through gH2Ax staining and revealed a correlation with the diseases as a higher phosphorylation rate of H2Ax protein was observed in tissues from acute leukemia patients compare to the others at thawing (p = 0.009) and after 24h (p < 0.001). Although protein analyses on Kit Ligand, key regulator of the follicle activation, showed an increase of expression into the groups treated by chemotherapy, genes and protein involved in the PI3K/AKT/mTOR and Hippo pathways showed similar expression levels in the chemo-treated groups compared to the non-exposed groups, irrespective of the age.
Limitations, reasons for caution
The study of follicle survival was limited to histological analyses, while follicular activation evaluations were limited to the evaluation of two main signaling, PI3K/AKT/mTOR and Hippo. Moreover, considering the heterogeneity of the human model, and the limited number of patients included, the results should be interpreted with caution.
Wider implications of the findings
Our results highlight the deleterious impact of previous chemotherapeutic treatment on follicle survival without affecting follicular activation. Besides sustaining the “burn out effect” theory, our results suggested that the time of last exposure and the pathology of the patient could impact the outcomes of the cryopreserved ovarian tissue.
Trial registration number
not applicable
The archaeological record shows that large pre-Inca agricultural systems supported settlements for centuries around the ravines and oases of northern Chile's hyperarid Atacama Desert. This raises ...questions about how such productivity was achieved and sustained, and its social implications. Using isotopic data of well-preserved ancient plant remains from Atacama sites, we show a dramatic increase in crop nitrogen isotope values (δ
N) from around AD 1000. Maize was most affected, with δ
N values as high as +30‰, and human bone collagen following a similar trend; moreover, their carbon isotope values (δ
C) indicate a considerable increase in the consumption of maize at the same time. We attribute the shift to extremely high δ
N values-the highest in the world for archaeological plants-to the use of seabird guano to fertilize crops. Guano-'white gold' as it came to be called-thus sustained agricultural intensification, supporting a substantial population in an otherwise extreme environment.
To investigate the specific and combined effects of personal concentrations of some per- and polyfluoroalkyl substances (PFAS), other persistent organic pollutants (POPs), and chemical elements ...–measured in individuals’ blood several years before the pandemic– on the development of SARS-CoV-2 infection and COVID-19 disease in the general population.
We conducted a prospective cohort study in 240 individuals from the general population of Barcelona. PFAS, other POPs, and chemical elements were measured in plasma, serum, and whole blood samples, respectively, collected in 2016–2017. PFAS were analyzed by liquid chromatography-triple quadrupole mass spectrometry. SARS-CoV-2 infection was detected by rRT-PCR in nasopharyngeal swabs and/or antibody serology in blood samples collected in 2020–2021.
No individual PFAS nor their mixtures were significantly associated with SARS-CoV-2 seropositivity or COVID-19 disease. Previously identified mixtures of POPs and elements (Porta et al., 2023) remained significantly associated with seropositivity and COVID-19 when adjusted for PFAS (all OR > 4 or p < 0.05). Nine chemicals comprised mixtures associated with COVID-19: thallium, ruthenium, lead, benzobfluoranthene, DDD, other DDT-related compounds, manganese, tantalum, and aluminium. And nine chemicals comprised the mixtures more consistently associated with SARS-CoV-2 seropositivity: thallium, ruthenium, lead, benzobfluoranthene, DDD, gold, and (protectively) selenium, indium, and iron.
The PFAS studied were not associated with SARS-CoV-2 seropositivity or COVID-19. The results confirm the associations between personal blood concentrations of some POPs and chemical elements and the risk of COVID-19 and SARS-CoV-2 infection in what remains the only prospective and population-based cohort study on the topic. Mixtures of POPs and chemical elements may contribute to explain the heterogeneity in the risks of SARS-CoV-2 infection and COVID-19 in the general population.
•With a clear time sequence, this is the only prospective cohort study on the topic.•We analyzed 120 chemicals, including the 8 PFAS most usually detected.•Such PFAS were not associated with SARS-CoV-2 seropositivity or COVID-19.•Adjusting for PFAS, mixtures of POPs and elements remained associated with COVID-19.•Some POPs and elements may contribute to explain the heterogeneity in the two outcomes.
Abstract
Study question
Does carboplatin and paclitaxel regimen have a toxic cumulative impact on the ovarian reserve of young breast cancer patients with germline Breast Cancer (BRCA) mutation?
...Summary answer
Based on follicle classification and survival through apoptosis and DNA repair mechanism recruitment analyses, gonadotoxic but no cumulative effect was observed in BRCA-mutated ovarian tissue.
What is known already
Ovarian function recovery and fertility issues remain major concerns for young patients diagnosed with cancer. Breast cancer is the first cancer diagnosed among women under 40 years and around 10% of them are BRCA germline mutation carriers. Beside their predisposition to breast and ovarian cancers, BRCA carriers might be more sensitive to chemotherapy-induced gonadotoxicity due to the role of BRCA in DNA repair mechanism. Current therapies for BRCA-mutated breast cancer patients often include carboplatin and paclitaxel, drugs that are both considered as moderately gonadotoxic. However, the effect of combined therapy in BRCA-mutated ovarian tissue is unknown.
Study design, size, duration
Ovarian tissue cryopreserved for fertility preservation before chemotherapy and donated for research by breast cancer patients with or without BRCA1/2 mutation (< 35 years at diagnosis) were used in this study. Two models were investigated: 3 days in vitro culture with carboplatin (10 µg/mL) and/or paclitaxel (1 µM) and ovarian tissue xenograft into mice followed by 3 weeks injections of carboplatin (50mg/kg/week) and paclitaxel (10mg/kg every 3 days). Control conditions were performed for each model.
Participants/materials, setting, methods
Ovarian tissue from 3 BRCA-mutated and 3 non-mutated patients were thawed and exposed in vitro and in vivo to chemotherapy. Follicle density (number of follicles/mm²) as well as ratios of quiescent/activated and healthy/atretic follicles were evaluated following haematoxylin-eosin staining. Apoptosis and DNA repair mechanisms were analysed using TUNEL/GDF9 co-staining and phosphorylated H2A histone family member X (γH2AX)/ZP-3 co-staining, respectively. Genes expression level will be evaluated on isolated follicles to assess the activation process.
Main results and the role of chance
First, the impact of in vitro culture and in vivo xenotransplantation was evaluated by follicle counting and classification. A decrease of follicular density (5 to 95% irrespective of patient, model, nor condition) and an increase of activated and atretic follicles was observed after both in vitro and in vivo experiments in all conditions. However, a slight increase of quiescent follicle pool was observed in the in vitro control condition, with no difference between mutated and non-mutated fragments. Then, immunofluorescence staining was performed to evaluate follicle damage. For quantification, 30 to 150 follicles per patient were counted within each condition and classified as positive if at least one cell was stained. Apoptosis was assessed with TUNEL/GDF9 co-staining. In both models, the ratio of positive follicles was higher in fragments treated with chemotherapy (40-60%) compared to control conditions (15-20%). Recruitment of DNA repair complex was analysed using γH2AX/ZP3 co-staining. In both models, an increase in positive follicles ratio was observed in treated fragments (15-40%) compared to control conditions (5-10%). Interestingly, the positive follicles ratio was lower in BRCA-mutated (15-18%) than in non-mutated fragments (25-40%) after 3-days in vitro culture, irrespective of the treatment used.
Limitations, reasons for caution
Reasons of caution include the high inter-variations between patients as well as intra-variations in one patient regarding follicular density within ovarian tissue. Moreover, BRCA-mutated patients included (27.67 ± 2.08) are younger than control ones (33.67 ± 1.53). Ongoing experiments with additional patients should support the results already obtained.
Wider implications of the findings
Carboplatin and paclitaxel combined exposure in vitro or in vivo does not seem to have a cumulative deleterious impact on ovarian reserve. Ovarian tissue with BRCA-mutation did not appear to be more sensitive to chemotherapy exposure. Further investigations focusing on DNA repair mechanisms will be performed in both models.
Trial registration number
Not applicable
Abstract Study question Are STAI State punctuations a potential non-invasive marker of endometrial prognosis in IVF patients? Summary answer Being stressed (determined by STAI State punctuations) is ...positively and negatively correlated with endometrial cortisol and estradiol levels, respectively, and highly associated with pregnancy consecution. What is known already Despite it is well known IVF treatments are a source of stress, this factor is frequently overlooked mainly due to the difficulty of stress evaluation and the lack of accurate biomarkers, not being standardized in the clinical setting. Steroid hormones are essential for reproductive physiology and highly related to well-known stress biomarkers such as cortisol. The lack of correlation between endometrial and serum steroid levels creates the need to look for non-invasive tools reflecting the endometrial microenvironment. In this study, by molecularly and psychologically characterizing patients, we aim to unveil non-invasive potential markers of stress associated to pregnancy consecution. Study design, size, duration This prospective cohort study included a total of 74 IVF patients (<45 years old, no uterine or systemic pathologies and good quality embryos) who underwent endometrial biopsy collection in mid-secretory phase for metabolites measurement between 2019 and 2023. These patients also underwent psychological evaluation by stress related questionnaires. Participants/materials, setting, methods The endometrial concentration of eleven steroid metabolites was measured by ultra-performance liquid chromatography-tandem mass spectrometry. Patients also underwent stress and anxiety evaluation (STAI), and reproductive outcomes in the first embryo transfer after biopsy collection were followed up. Wilcoxon and Barnard’s test were applied to compare the mean and proportion of stressed/unstressed, pregnant/not pregnant patients and metabolites levels. Correlations between metabolites’ levels and the proportion of stressed patients were evaluated by Pearson’s correlation coefficient. Main results and the role of chance Patients with STAI State punctuations > 60 presented higher mean endometrial cortisol levels (8.01 ng/g (n = 23) vs 5.08 ng/g (n = 35), p-value = 0.05) and lower mean estrone levels (54.97 ng/g (n = 23) vs 56.31 ng/g (n = 35), p-value = 0.049) than those patients with STAI State punctuations < 60. Also, in patients with high endometrial estradiol levels (>2 ng/g) a higher significant proportion of unstressed patients (STAI State < 60) was observed (88.9% vs 52% of unstressed patients in > 2 ng/g and < =2 ng/g estradiol concentrations, respectively) (n = 34, p-value = 0.036). As expected, a significant positive correlation between endometrial cortisol levels and stress (measured by STAI State questionnaire) (cor = 0.977, p-value = 0.023) and a negative correlation between endometrial estradiol levels and stress (cor = -0.9945, p-value = 0.0055) was evidenced. Additionally, a higher proportion of pregnant patients was observed when endometrial estradiol concentrations were high (>1 ng/g) (78.9% vs 50% pregnant in > 1 ng/g vs < =1 ng/g estradiol respectively, p-value = 0.035) and a higher proportion of not pregnant patients was observed when endometrial cortisol concentrations were high ( > =13.9 ng/g) (80% vs 34.4% not pregnant in > =13.9 ng/g and <13.9 ng/g cortisol respectively, p-value = 0.029). Limitations, reasons for caution Although we have employed validated psychological questionnaires and stress biomarkers, a causal relationship between stress and reproductive outcomes should be further explored. Wider implications of the findings This study demonstrated a significant correlation between stress and cortisol and estradiol levels. Since both metabolites’ levels were also related to pregnancy chances, STAI State psychological evaluation could be implemented in the clinical practice as a non-invasive method to prevent endometrial failure through psychological counseling. Trial registration number Not applicable
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