Aging promotes accumulation of senescent and multiciliated cells in human endometrial epithelium.

STUDY QUESTION: What changes occur in the endometrium during aging, and do they impact fertility? SUMMARY ANSWER: Both the transcriptome and cellular composition of endometrial samples from women of advanced maternal age (AMA) are significantly different from that of samples from young women, suggesting specific changes in epithelial cells that may affect endometrial receptivity. WHAT IS KNOWN ALREADY: Aging is associated with the accumulation of senescent cells in aging tissues. Reproductive aging is mostly attributed to the decline in ovarian reserve and oocyte quality, whereas the endometrium is a unique complex tissue that is monthly renewed under hormonal regulation. Several clinical studies have reported lower implantation and pregnancy rates in oocyte recipients of AMA during IVF. Molecular studies have indicated the presence of specific mutations within the epithelial cells of AMA endometrium, along with altered gene expression of bulk endometrial tissue. STUDY DESIGN SIZE DURATION: Endometrial transcriptome profiling was performed for 44 women undergoing HRT during the assessment of endometrial receptivity before IVF. Patients younger than 28 years were considered as the young maternal age (YMA) group (age 23-27 years) and women older than 45 years were considered as the AMA group (age 47-50 years). Endometrial biopsies were obtained on Day 5 of progesterone treatment and RNA was extracted. All endometrial samples were evaluated as being receptive based on the expression of 68 common endometrial receptivity markers. Endometrial samples from another 24 women classified into four age groups (YMA, intermediate age group 1 (IMA1, age 29-35), intermediate age group 2 (IMA2, age 36-44), and AMA) were obtained in the mid-secretory stage of a natural cycle (NC) and used for validation studies across the reproductive lifespan. PARTICIPANTS/MATERIALS SETTING METHODS: A total of 24 HRT samples (12 YMA and 12 AMA) were subject to RNA sequencing (RNA-seq) and differential gene expression analysis, 20 samples (10 YMA and 10 AMA) were used for qPCR validation, and 24 NC samples (6 YMA, 6 IMA1, 6 IMA2 and 6AMA) were used for RNA-seq validation of AMA genes across the woman's reproductive lifespan. Immunohistochemistry (IHC) was used to confirm some expression changes at the protein level. Computational deconvolution using six endometrial cell type-specific transcriptomic profiles was conducted to compare the cellular composition between the groups. MAIN RESULTS AND THE ROLE OF CHANCE: Comparisons between YMA and AMA samples identified a lower proportion of receptive endometria in the AMA group (P = 0.007). Gene expression profiling identified 491 differentially expressed age-sensitive genes (P adj < 0.05) that revealed the effects of age on endometrial epithelial growth and receptivity, likely contributing to decreased reproductive performance. Our results indicate that changes in the expression of the cellular senescence marker p16INK4a and genes associated with metabolism, inflammation, and hormone response are involved in endometrial aging. Importantly, we demonstrate that the proportion of multi-ciliated cells, as discovered based on RNA-seq data deconvolution and tissue IHC results, is affected by endometrial aging, and propose a putative onset of age-related changes. Furthermore, we propose that aging has an impact on the transcriptomic profile of endometrial tissue in the context of endometrial receptivity. LARGE SCALE DATA: The raw sequencing data reported in this article are deposited at the Gene Expression Omnibus under accession code GSE236128. LIMITATIONS REASONS FOR CAUTION: This retrospective study identified changes in the endometrium of patients undergoing hormonal replacement and validated these changes using samples obtained during a NC. However, future studies must clarify the importance of these findings on the clinical outcomes of assisted reproduction. WIDER IMPLICATIONS OF THE FINDINGS: The findings reported in this study have important implications for devising future strategies aimed at improving fertility management in women of advanced reproductive age. STUDY FUNDING/COMPETING INTERESTS: This research was funded by the Estonian Research Council (grant no. PRG1076), Horizon 2020 innovation grant (ERIN, grant no. EU952516), Enterprise Estonia (grant no. EU48695), MSCA-RISE-2020 project TRENDO (grant no. 101008193), EU 874867 project HUTER, the Horizon Europe NESTOR grant (grant no. 101120075) of the European Commission, the EVA specialty program (grant no. KP111513) of the Maastricht University Medical Center (MUMC+), MICIU/AEI/10.13039/501100011033 and FEDER, EU projects Endo-Map (grant no. PID2021-12728OB-100), ROSY (grant no. CNS2022-135999), and the National Science Fund of Bulgaria (grant no. KII-06 H31/2). The authors declare no competing interests.

The endometrial transcriptome of infertile women with and without implantation failure.

INTRODUCTION: Implantation failure after transferring morphologically "good-quality" embryos in in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) may be explained by impaired endometrial receptivity. Analyzing the endometrial transcriptome analysis may reveal the underlying processes and could help in guiding prognosis and using targeted interventions for infertility. This exploratory study investigated whether the endometrial transcriptome profile was associated with short-term or long-term implantation outcomes (ie success or failure). MATERIAL AND METHODS: Mid-luteal phase endometrial biopsies of 107 infertile women with one full failed IVF/ICSI cycle, obtained within an endometrial scratching trial, were subjected to RNA-sequencing and differentially expressed genes analysis with covariate adjustment (age, body mass index, luteinizing hormone [LH]-day). Endometrial transcriptomes were compared between implantation failure and success groups in the short term (after the second fresh IVF/ICSI cycle) and long term (including all fresh and frozen cycles within 12 months). The short-term analysis included 85/107 women (33 ongoing pregnancy vs 52 no pregnancy), excluding 22/107 women. The long-term analysis included 46/107 women (23 'fertile' group, ie infertile women with a live birth after ≤3 embryos transferred vs 23 recurrent implantation failure group, ie no live birth after ≥3 good quality embryos transferred), excluding 61/107 women not fitting these categories. As both analyses drew from the same pool of 107 samples, there was some sample overlap. Additionally, cell type enrichment scores and endometrial receptivity were analyzed, and an endometrial development pseudo-timeline was constructed to estimate transcriptomic deviations from the optimum receptivity day (LH + 7), denoted as ΔWOI (window of implantation). RESULTS: There were no significantly differentially expressed genes between implantation failure and success groups in either the short-term or long-term analyses. Principal component analysis initially showed two clusters in the long-term analysis, unrelated to clinical phenotype and no longer distinct following covariate adjustment. Cell type enrichment scores did not differ significantly between groups in both analyses. However, endometrial receptivity analysis demonstrated a potentially significant displacement of the WOI in the non-pregnant group compared with the ongoing pregnant group in the short-term analysis. CONCLUSIONS: No distinct endometrial transcriptome profile was associated with either implantation failure or success in infertile women. However, there may be differences in the extent to which the WOI is displaced.

An integrative analysis of endometrial steroid metabolism and transcriptome in relation to endometrial receptivity in in vitro fertilization patients.

OBJECTIVE: To study the relationship between the steroid concentration in the endometrium, in serum, and the gene expression level of steroid-metabolizing enzymes in the context of endometrial receptivity in in vitro fertilization (IVF) patients. DESIGN: Case-control study of 40 IVF patients recruited in the SCRaTCH study (NTR5342), a randomized controlled trial investigating pregnancy outcome after "endometrial scratching." Endometrial biopsies and serum were obtained from patients with a first failed IVF cycle randomized to the endometrial scratch in the midluteal phase of the natural cycle before the next fresh embryo transfer during the second IVF cycle. SETTING: University hopsital. PATIENTS: Twenty women with clinical pregnancy were compared with 20 women who did not conceive after fresh embryo transfer. Cases and controls were matched for primary vs. secondary infertility, embryo quality, and age. INTERVENTION: None. MAIN OUTCOME MEASURE(S): Steroid concentrations in endometrial tissue homogenates and serum were measured with liquid chromatography-mass spectrometry. The endometrial transcriptome was profiled by RNA-sequencing, followed by principal component analysis and differential expression analysis. False discovery rate-adjusted and log-fold change >|0.5| were selected as the threshold for differentially expressed genes. RESULT(S): Estrogen levels were comparable in both serum (n = 16) and endometrium (n = 40). Androgens and 17-hydroxyprogesterone were higher in serum than that in endometrium. Although steroid levels did not vary between pregnant and nonpregnant groups, subgroup analysis of primary women with infertility showed a significantly lower estrone concentration and estrone:androstenedione ratio in serum of the pregnant group (n = 5) compared with the nonpregnant group (n = 2). Expression of 34 out of 46 genes encoding the enzymes controlling the local steroid metabolism was detected, and estrogen receptor ß gene was differentially expressed between pregnant and nonpregnant women. When only the primary infertile group was considered, 28 genes were differentially expressed between pregnant and nonpregnant women, including HSD11B2, that catalyzes the conversion of cortisol into cortisone. CONCLUSION(S): Steroidomic and transcriptomic analyses show that steroid concentrations are regulated by the local metabolism in the endometrium. Although no differences were found in endometrial steroid concentration in the pregnant and nonpregnant IVF patients, primary women with infertility showed deviations in steroid levels and gene expression, indicating that a more homogeneous patient group is required to uncover the exact role of steroid metabolism in endometrial receptivity. CLINICAL TRIAL REGISTRATION NUMBER: The study was registered in the Dutch trial registry (www.trialregister.nl), registration number NL5193/NTR5342, available at https://trialsearch.who.int/Trial2.aspx?TrialID=NTR6687. The date of registration is July 31, 2015. The first enrollment is on January 1, 2016.