Endometrial aging and uterine receptivity: endometrial receptivity analysis (ERA) outcomes in female patients of diverse age groups.

This retrospective cohort study included patients seeking fertility treatment at a single academic institution, the University of California, San Francisco (UCSF). Between 01/2019 and 05/2024, 210 patients underwent endometrial biopsy for the endometrial receptivity analysis (ERA). Five patients were excluded from the analysis: three due to insufficient biopsy material, one due to a “non-informative” result, and one due to a compromised sample during transit. Demographic information, including age, body mass index (BMI), infertility diagnosis, and pregnancy history at the time of biopsy, was obtained through chart review. Additionally, data on the number of prior implantation failures, including the total number of embryos transferred and the total number of euploid embryos transferred, were recorded for each patient. Implantation failure was defined as a negative serum human chorionic gonadotropin (hCG) following embryo transfer. This study was approved by the UCSF Institutional Review Board. Standard technique for endometrial biopsy was performed using a Pipelle catheter (CooperSurgical, Trumbull, CT). The biopsies were performed in the luteal phase under three different endometrial preparation methods: natural cycle (NC), modified natural cycle (mNC), and programmed cycle (PC). The timing of the biopsy was performed according to the instructions provided by the manufacturer of the ERA test. At our center, ERA testing is primarily performed under programmed cycle conditions. However, there was a minority of patients who underwent ERA biopsy in either a natural cycle (NC) ( n  = 10; 4.8%) or a modified natural cycle (mNC) ( n  = 25; 12.2%). The programmed cycle group received exogenous estradiol either in the form of estradiol patches (300 mcg every 3 days) or oral Estrace 6 mg daily to stimulate endometrial lining development. There was a minimum of 12 days of estrogen-only phase before the patients were brought in for a transvaginal ultrasound to evaluate the endometrial lining. After an ultrasound evaluation of the endometrium, progesterone (IM Progesterone in oil, 50 mg daily) was initiated. Endometrial biopsy in the PC group was performed on the 6th day of progesterone therapy. In both NC and mNC endometrial preparation groups, the endometrial biopsy was performed either at luteinizing hormone (LH) surge + 6 days or 7 days after administration of choriogonadotropin alfa, respectively (EMD Serono, Rockland, MA). The mNC group also received luteal phase vaginal progesterone support (Prometrium 200 mg twice daily or Endometrin 100 mg twice daily). All endometrial biopsy samples were processed and sent to Igenomix according to the manufacturer’s instructions for ERA testing. The primary outcome of this study was the proportion of non-receptive ERA results (pre- and post-receptive combined) stratified by female age group. Demographic, treatment parameters, and embryo transfer outcomes were compared across age groups using the Kruskal–Wallis test, chi-squared test, or Fisher’s exact test as appropriate. The proportions of non-receptive ERA results by age group were compared using Fisher’s exact test. A sensitivity analysis, including only patients who were biopsied for ERA during a programmed cycle, was also performed. Univariable logistic regression analyses were performed to identify potential predictors of a non-receptive ERA result. Baseline- and treatment-related characteristics that were significantly different among age groups, and factor(s) with a significance level of p  < 0.08 in the univariable analyses, were included in a multivariable logistic regression model. An additional multivariable logistic regression was performed that included BMI due to biologic plausibility that this variable could affect our outcome of interest. All statistical tests were two-sided, and significance was defined as p < 0.05. Analyses were conducted using Stata v18.0 (StataCorp, College Station, TX).

A total of 210 patients underwent endometrial sampling for ERA testing during the study period. Five patients were excluded from the analysis due to inconclusive testing. Of the remaining 205 ERA results, 166 (81.0%) were classified as “receptive,” while 39 (19.0%) were non-receptive (pre- or post-receptive). Among the non-receptive results, 6 (2.9%) were “post-receptive” and 33 (16.1%) were “pre-receptive.” Demographic and treatment parameters by age group category are shown in Table  1 . The median age in the youngest group (age < 35) was 33 (interquartile range; IQR 32–34); in the 35–37 group, median (IQR) age was 36 (35–37); in the 38–40 group, median (IQR) age was 39 (38–40); and in the oldest group (age ≥ 41), the median age (IQR) was 43 (42–44) ( p  < 0.01). The median BMI was not significantly different among the age groups: in the youngest group, median BMI was 23.1 (IQR 21.8–25.5), age 35–37 median BMI was 22.5 (IQR 21.3–26.5), age 38–40 median BMI was 23.2 (IQR 21.8–26.6), and the median BMI in the age ≥ 41 group was 24.7 (IQR 22.0–27.5) ( p  = 0.20). The distributions of nulliparity status, number of previous miscarriages, and primary infertility diagnosis were not statistically different across age groups (all p  > 0.05). Additionally, the number of previous implantation failures was similar across all age groups: in the < 35 age group, median was 1 (IQR 1–2), age 35–37 median was 2 (IQR 1–2), age 38–40 median was 1 (IQR 1–2), and for the ≥ 41 age group, the median was 1 (IQR 0–2) ( p  = 0.75). The number of previous failed euploid embryo(s) transferred approached but did not meet statistical significance. The youngest group (age < 35) had a median of 0 previous failed euploid embryo transfers (IQR 0–2). For the next age group (35–37), the median was 1 (IQR 0–1); similarly, for the group aged 38–40, median was 1 (IQR 0–2); and for the oldest group (age 41 and above), the median was 0 (IQR 0–1) ( p  = 0.054). Table 1 Patient characteristics by age category Demographics  < 35 ( N  = 35) 35–37 ( N  = 58) 38–40 ( N  = 53) 41 and above ( N  = 59) p -value Age a 33 (32, 34) 36 (35, 37) 39 (38, 40) 43 (42, 44)  < 0.01 BMI a 23.1 (21.8, 25.5) 22.5 (21.3, 26.5) 23.2 (21.8, 26.6) 24.7 (22.0, 27.5) 0.20 Nulliparous b   Yes 30 (85.7%) 52 (89.7%) 40 (75.5%) 46 (78.0%) 0.19   No 5 (14.3%) 6 (10.3%) 13 (24.5%) 13 (22.0%) Number of previous miscarriages a 0 (0, 1) 0 (0, 2) 0 (0, 1) 1 (0, 1) 0.20 Primary infertility diagnosis b   Unexplained 20 (57.1%) 37 (63.8%) 29 (54.7%) 37 (63.8%) 0.64   Polycystic ovary syndrome 3 (8.6%) 2 (3.5%) 3 (5.7%) 3 (5.2%)   Male factor 3 (8.6%) 5 (8.6%) 6 (11.3%) 8 (13.8%)   Tubal factor 1 (2.9%) 3 (5.2%) 1 (1.9%) 1 (1.7%) Endometriosis 0 (0%) 2 (3.5%) 0 (0%) 0 (0%) Uterine 1 (2.9%) 3 (5.2%) 2 (3.8%) 1 (1.7%) Recurrent pregnancy loss 1 (2.9%) 3 (5.2%) 1 (1.9%) 4 (6.8%) Other* 6 (17.1%) 3 (5.2%) 11 (20.8%) 5 (8.6%) Number of implantation failures a 1 (1, 2) 2 (1, 2) 1 (1,2) 1 (0, 2) 0.75 No. of failed euploid embryos transferred a 0 (0, 2) 1 (0, 1) 1 (0, 2) 0 (0, 1) 0.054 a Data presented as median (25th, 75th percentile) b Data presented as n (%) * Other includes diminished ovarian reserve, advanced reproductive age of female, primary ovarian insufficiency, history of gonadotoxic chemotherapy, need for donor gamete(s), and inability to have intercourse Patient characteristics by age category a Data presented as median (25th, 75th percentile) b Data presented as n (%) * Other includes diminished ovarian reserve, advanced reproductive age of female, primary ovarian insufficiency, history of gonadotoxic chemotherapy, need for donor gamete(s), and inability to have intercourse Endometrial receptivity analysis (ERA) results by age category are shown in Table  2 . In the age < 35 group ( n  = 35), the majority of ERA results were receptive ( n  = 28; 80%). Of the non-receptive results in this age group ( n  = 7; 20.0%), 6 were pre-receptive (17.1%) and one was post-receptive (2.9%). In the age 35–37 group ( n  = 58), 48 biopsies were receptive (82.8%). In this age group, 10 biopsies (17.2%) were non-receptive: 7 were pre-receptive (12.0%) and 3 were post-receptive (5.2%). The age 38–40 group ( n  = 53) had 44 receptive (83.0%) and 9 non-receptive ERA (17.0%) results, of which 7 were pre-receptive (13.2%) and 2 were post-receptive (3.8%). The age 41 and above group ( n  = 59) consisted of 46 receptive results (78.0%) and 13 non-receptive (22.0%) results, all of which were pre-receptive. There was no significant difference in the proportions of non-receptive ERA results by age group ( p  = 0.52). Additionally, a sensitivity analysis was performed, including only patients who were biopsied for ERA during a programmed cycle. There was still no significant difference in the proportion of non-receptive ERA results by age group ( p  = 0.25). These results can be found in Supplemental Table 3 . Table 2 Proportion of non-receptive ERA by age category  < 35 (N = 35) 35–37 ( N  = 58) 38–40 ( N  = 53) 41 and above ( N  = 59) p -value Non-receptive ERA 7 (20.0%) 10 (17.2%) 9 (17.0%) 13 (22.0%) 0.52 Pre-receptive 6 (17.1%) 7 (12.0%) 7 (13.2%) 13 (22.0%) Post-receptive 1 (2.9%) 3 (5.2%) 2 (3.8%) 0 (0%) Receptive ERA 28 (80%) 48 (82.8%) 44 (83.0%) 46 (78.0%) Proportion of non-receptive ERA by age category Univariable logistic regression was first performed to evaluate potential associations between individual patient demographics or treatment-related factors and a non-receptive ERA result. As shown in Table  3 , no significant associations were found between older age groups and non-receptive ERA. Compared to the reference group (age < 35), those aged 35–37 had a non-significant increased odds of a non-receptive ERA (odds ratio; OR 1.20 [95% CI 0.41–3.51], p  = 0.74). Similarly, age 38–40 had non-significant, mildly increased odds of a non-receptive ERA (OR 1.22 [95% CI 0.41–3.66], p  = 0.72). The oldest group, age 41 and above, had a nonsignificant decrease in odds of a non-receptive ERA (OR 0.88 [95% CI 0.32–2.48], p  = 0.82). BMI, primary infertility diagnosis, number of previous miscarriages, number of implantation failures, and number of failed euploid or total embryos transferred preceding the endometrial biopsy were not significantly associated with higher odds of non-receptive ERA in our cohort (all p  > 0.05). Given the trend toward a difference in the number of prior failed euploid embryo transfers across age groups ( p  = 0.054) and its potential association with ERA outcomes, this variable was included in a multivariable logistic regression model. After adjusting for this factor, there remained no statistically significant difference in non-receptive ERA results by age category. Compared to the reference group (age < 35), the adjusted OR (95% CI) for a non-receptive ERA was aOR 1.21, (95% CI 0.41–3.55) for age 35–37, aOR 1.23 (95% CI 0.41–3.69) for age 38–40, and aOR 0.84 (95% CI 0.30–2.38) for age 41 and above (all p  > 0.05). An additional multivariable logistic regression model was conducted, including both the number of prior failed euploid embryo transfers and BMI, as there is biologic plausibility that BMI could affect the hormonal milieu of the uterus. There remained no significant difference in the outcome of non-receptive ERA. Compared to the reference group (age  0.05). Table 3 Logistic regression analyses of possible predictors of non-receptive ERA Variable Unadjusted OR—univariable logistic regression Adjusted*OR—multivariable logistic regression Adjusted** OR—multivariable logistic regression OR (95% CI) p -value OR (95% CI) p -value OR (95% CI) p -value Age category (compared to age < 35) Age 35–37 1.20 (0.41–3.51) 0.74 1.21 (0.41–3.55) 0.73 1.23 (0.42–3.63) 0.70 Age 38–40 1.22 (0.41–3.66) 0.72 1.23 (0.41–3.69) 0.71 1.30 (0.43–3.93) 0.64 Age 41 and over 0.88 (0.32–2.48) 0.82 0.84 (0.30–2.38) 0.75 0.98 (0.34–2.85) 0.97 BMI 0.96 (0.90–1.03) 0.24 0.96 (0.90–1.03) 0.22 Primary infertility diagnosis 0.97 (0.86–1.09) 0.57 No. previous miscarriages 1.06 (0.75–1.48) 0.75 No. implantation failures 1.07 (0.79–1.44) 0.67 No. failed euploid embryo(s) transferred 0.87 (0.61–1.23) 0.43 0.85 (0.60–1.21) 0.36 No. total failed embryo(s) transferred 0.98 (0.81–1.20) 0.86 * Adjusted for the number of failed euploid embryo transfers ** Adjusted for the number of failed euploid embryo transfers and BMI Logistic regression analyses of possible predictors of non-receptive ERA * Adjusted for the number of failed euploid embryo transfers ** Adjusted for the number of failed euploid embryo transfers and BMI Pregnancy outcomes in those who completed a subsequent FET after ERA testing are shown in Supplemental Table  1 . There were no significant differences in implantation, clinical pregnancy, or live birth rates in the subsequent FET cycle in women < 35 as compared to older age groups. When the data was limited to only patients who had a non-receptive ERA result, there was again no difference in pregnancy outcomes between the reference group (age < 35) and older age groups with ERA-guided timing (Supplemental Table  2 ).

There is biologic plausibility that the WOI environment may change with age. However, the ERA test does not appear to capture this difference. The proportion of non-receptive ERA was not significantly higher in older reproductive age groups, even in those over the age of 41. Alternatively, our results may also support that the WOI is not significantly impacted by age when assessed by transcriptomic levels of canonical receptivity markers. Based on our results, we suggest caution in using the ERA solely on the basis of increased maternal age, as our findings indicate that the ERA may not reliably detect age-related differences in the window of implantation. Rigorous, prospective clinical and basic science studies are needed to further understand endometrial aging and its potential clinical impact in fertility treatment.

The field of assisted reproductive technology has made strides in addressing infertility associated with ovarian aging using in vitro fertilization, pre-implantation genetic testing for aneuploidy, and oocyte donation. However, critical determinants of success from the uterine perspective and the endometrium—the soil for implantation—have remained largely elusive and controversial despite significant efforts. In particular, there is a growing focus on endometrial aging as a potential target for improving pregnancy rates and healthy live births, especially as the age of motherhood in modern society continues to rise [ 36 ]. Given the potential impact of chronological aging on the cellular machinery associated with endometrial remodeling, we investigated whether advanced female reproductive age is associated with aberrant expression of canonical receptivity markers on the ERA, many of which relate to decidualization and progesterone effects. Contrary to what we hypothesized, our study did not find increased odds of non-receptive ERA results associated with increased female age. Animal studies have demonstrated an inverse relationship between age and estrogen and progesterone receptor density in the endometrium, suggesting that endometrial tissue may become less hormone-sensitive with increasing age [ 12 , 13 ]. This observation provides biologic plausibility for impaired uterine receptivity with aging, as implantation depends on hormone-mediated endometrial remodeling. For example, thinner endometrial lining has been observed in older females [ 37 ], and mean serum estradiol levels also decline with advancing age [ 38 , 39 ]. Several observational studies also reported declining pregnancy and live birth rates in oocyte recipients as recipient age increases [ 34 , 40 , 41 ], although there is no consensus on the threshold age in humans at which uterine receptivity steadily declines [ 30 – 32 ]. The endometrial receptivity analysis (ERA) test was developed to assess the transcriptomic profile of 238 genes that largely represent canonical receptivity markers that were extrapolated from menstrual cycle phase and logically reflect downstream effects of progesterone. Importantly, recent high-quality data raise substantial concerns about the lack of benefit with ERA. A large randomized controlled trial with single euploid frozen embryo transfers (FETs) demonstrated no significant improvement in pregnancy or live birth rates when embryo transfers occurred at the timing recommended by the ERA compared to standard timing [ 42 ]. Similarly, a meta-analysis by Zolfaroli et al. concluded that ERA-guided transfer does not confer significant advantages [ 43 ]. These findings suggest that, while ERA may provide biological insights at the transcriptomic level, its clinical value in improving embryo transfer outcomes appears limited based on current evidence. Of note, there have been multiple recent publications implicating endometrial aging as a factor affecting implantation success and receptivity; hence, it is worthwhile to assess the utility of the ERA in this specific context [ 34 , 40 , 41 , 44 ]. Our study conclusions suggest that the ERA also does not adequately capture potential age-related differences in endometrial receptivity. One possible explanation is that mRNA patterns of a gene panel may not be sensitive enough to detect or report subtle age-related changes in specific genes. It is also possible that the age-related impact occurs at the post-transcriptional stages and instead affects protein levels that may better reflect the functional state of the endometrium. It is also worth noting that the distribution of ERA results in our cohort differed from that reported in several other studies [ 45 , 46 ]. For example, Barrentexea et al. and others reported an approximately 50–60% “receptive” rate, whereas in our cohort, nearly 80% of ERA tests were “receptive” [ 27 , 32 , 46 ]. Several factors may account for this discrepancy. First, a large majority of our patients (83%) underwent programmed cycles with intramuscular progesterone in oil (50 mg daily), whereas the studies cited above relied exclusively on vaginal progesterone or mixed vaginal/intramuscular combination. Exogenous replacement of steroid hormones may compensate for a reduction in estradiol/progesterone production and/or receptor concentrations or responsiveness with aging. Additionally, serum and endometrial progesterone concentrations differ substantially between vaginal and intramuscular administration routes, potentially altering luteal phase molecular expression and physiology [ 27 , 47 , 48 ]. All ERA samples were processed and analyzed by Igenomix in a blinded fashion such that our institution had no influence on assay execution or result classification. Our findings do not support the notion that older age groups would exhibit a significantly increased proportion of non-receptive ERA results. Advanced reproductive age itself should therefore not be an indication to undergo ERA testing. Our study was strengthened by its large sample size, diverse age distribution, and relatively uniform treatment protocols within a single center. We did not exclude patients based on infertility diagnosis, which helps augment the generalizability of our results. The ERA test and determination of “endometrial receptivity” were analyzed and reported by a third-party company (Igenomix) that was blinded to the details of a patient’s medical history. Potential confounders for ERA non-receptivity were assessed and adjusted for as appropriate in our analysis. We also acknowledge the limitations of our study. As a retrospective cohort study, we attempted to reduce bias by making a detailed assessment of demographic and treatment-related parameters that could affect our outcome of interest. Despite our methodology, it is possible that we omitted a variable that exerts an effect on ERA results, given the limited knowledge on proven clinical factors that impact endometrial receptivity on a transcriptomic level. Despite our relatively large cohort size, it is still possible that the sample size did not provide enough power to detect small but significant differences. However, as above, the translational value of the ERA is also being contested [ 42 , 49 ]. It is possible that clinically relevant endometrial aging is not detectable using this tool. In Supplemental Tables 1  and 2 , we evaluated clinical outcomes of the subsequent FET following ERA. Across age groups, and acknowledging the limitations of our sample size, we observed no differences in implantation, clinical pregnancy, or live birth rates, regardless of whether transfer timing was adjusted based on ERA results. Given the relatively small number of pregnancies included, larger cohort studies are needed to validate these findings. Additionally, because our clinical protocol involves adjusting transfer timing when the ERA recommends a change, we do not have comparable data for patients who underwent transfers at standard timing despite a non-receptive result. Our results highlight the need for additional scientific investigations into the cellular and molecular changes that occur in the aging endometrium, which may be better identified in extreme ranges of reproductive age [ 28 ]. Prospective clinical data on IVF outcomes, adjusting for female age, embryo quality, endometrial factors, and treatment history/indication, can further substantiate the phenomenon of endometrial aging. Modern techniques utilizing single-cell sequencing may also provide a more granular view of endometrial changes with chronological aging, particularly in cell types beyond the epithelial and stromal populations [ 28 , 39 ]. These efforts will help further understand which molecular changes associated with endometrial aging are clinically relevant, and inform therapeutic development on how to best overcome these alterations in the endometrial environment.

Women are increasingly delaying childbearing worldwide, especially in developed nations. In the USA, the average maternal age rose from 24.6 in 1970 to 27.2 in 2000, with an even greater increase in urban areas [ 1 ]. By 2017, the average maternal age at first birth in large urban counties was 29.0 [ 2 ]. This trend is driven by factors such as career and educational priorities, greater contraceptive access, relatively limited awareness of age-related fertility decline, and personal choice [ 3 ]. While the exact age at which fertility begins to rapidly decline is somewhat variable, it is well established that after age 35, ovarian reserve and oocyte quality diminish, leading to increased chromosome missegregation, lower pregnancy rates, and higher miscarriage rates [ 4 – 6 ]. Preimplantation genetic testing for aneuploidy (PGT-A) and oocyte donation have become more widely used to mitigate these effects of ovarian aging. However, live birth rates following in vitro fertilization treatment have now plateaued around 50–60% per euploid embryo transfer, with emerging research suggesting that endometrial aging may also contribute to implantation failure, miscarriage, and lower clinical pregnancy rates in women over 35 [ 7 – 9 ]. Traditionally, the uterus and endometrium were thought to age more slowly—or perhaps, less clinically significantly—than the ovaries. The effects of endometrial aging have been comparatively overlooked in the literature compared to ovarian aging. However, recent studies challenge this view, showing age-related epigenetic alterations in the endometrium [ 10 , 11 ]. In animal models, genome-wide analysis has revealed declining estrogen and progesterone receptor expression with age, potentially disrupting normal endometrial function [ 12 , 13 ]. This, in turn, could lead to perturbations in biological pathways that critically depend on steroid hormone action, such as endometrial decidualization and the establishment of the window of implantation (WOI). Human clinical studies further support this notion, demonstrating a potentially inverse relationship between maternal age and endometrial function. A prospective study in oocyte donor recipients found that pregnancy rates declined and miscarriage rates increased with recipient age, implicating uterine aging in implantation failure [ 14 ]. Similarly, another study that examined pregnancy outcomes of recipients sharing the same pool of oocytes from young donors observed that younger recipients exhibited better outcomes compared to recipients over the age of 40 [ 15 ]. Interestingly, basic science studies have also identified potential molecular signatures associated with endometrial senescence in women under age 35 following recurrent implantation failure, highlighting endometrial biological aging independent of chronological age as a possible contributor to implantation failure [ 16 ]. The WOI opens in the early to mid-luteal phase, when the endometrium is considered optimally receptive for embryo implantation. There has been a focus on classifying the gene expression pattern within the endometrium during this critical period to define a clinically relevant signature for the WOI [ 17 – 20 ]. Some have proposed that dyssynchrony between the embryo and the WOI endometrium can result in implantation failure or miscarriage if an embryo is transferred too early (pre-receptive) or too late (post-receptive) [ 21 , 22 ]. The endometrial receptivity analysis (ERA) assesses transcriptomic profiles of 238 genes identified from healthy controls to help clinicians gauge whether the endometrium displays a molecular signature that is pre-receptive, receptive, or post-receptive in relation to the WOI signature [ 20 ]. This clinical tool purports to provide personalized embryo transfer timing based on the patient’s specific receptivity profile in relation to the duration of progesterone exposure [ 23 ]. Of note, data regarding the practical applications of the ERA test remain mixed, and the appropriate indication and patient population that could benefit from the ERA remain to be determined [ 24 – 26 ]. Although the clinical utility of the ERA for the general infertility patient population appears limited [ 26 – 32 ], a recent study showed transcriptomic evidence of suboptimal endometrial receptivity as well as changes in endometrial cellular composition with advanced maternal age [ 33 ]. Additionally, reports have demonstrated an association between advancing maternal age and reduced implantation rates, even in the context of frozen embryo transfers with exclusively euploid embryos or embryos derived from donor oocytes [ 34 , 35 ]. These data raise renewed focus on the endometrium impacting implantation and may focus consideration of the ERA as a tool to evaluate implantation timing based on maternal age, underscoring the importance of further investigation in this area. Given the previously reported molecular, cellular, and epigenetic changes observed in the endometrium of women with advanced reproductive age, we hypothesize that there could be a higher prevalence of WOI displacement with increasing female age. If this hypothesis is confirmed, there may be utility in employing the ERA specifically in older female patients to guide embryo transfer timing and aid reproductive outcomes. Herein, our study aims to investigate whether increasing female chronological age is associated with a higher prevalence of non-receptive ERA secondary to endometrial aging.

Below is the link to the electronic supplementary material. Supplementary file1 (DOCX 16.9 KB) Supplementary file1 (DOCX 16.9 KB)