Mechanisms of endometrial aging: lessons from natural conceptions and assisted reproductive technology cycles

Abstract

Until recently, the study of age-related decline in fertility has focused primarily on the ovary; depletion of the finite pool of oocytes and increases in meiotic errors leading to oocyte aneuploidy are well-established mechanisms by which fertility declines with advancing age. Comparatively little is known about the impact of age on endometrial function. The endometrium is a complex tissue comprised of many cell types, including epithelial, stromal, vascular, immune and stem cells. The capacity of this tissue for rapid, cyclic regeneration is unique to this tissue, undergoing repeated cycles of growth and shedding (in the absence of an embryo) in response to ovarian hormones. Furthermore, the endometrium has been shown to be capable of supporting pregnancies beyond the established boundaries of the reproductive lifespan. Despite its longevity, molecular studies have established age-related changes in individual cell populations within the endometrium. Human clinical studies have attempted to isolate the effect of aging on the endometrium by analyzing pregnancies conceived with euploid, high quality embryos. In this review, we explore the existing literature on endometrial aging and its impact on pregnancy outcomes. We begin with an overview of the principles of endometrial physiology and function. We then explore the mechanisms behind endometrial aging in its individual cellular compartments. Finally, we highlight lessons about endometrial aging gleaned from rodent and human clinical studies and propose opportunities for future study to better understand the contribution of the endometrium to age-related decline in fertility.

Full text

Mechanisms of endometrial
aging: lessons from natural
conceptions and assisted
reproductive technology cycles
Anat Chemerinski*, Jessica Garcia de Paredes, Kristin Blackledge,
Nataki C. Douglas and Sara S. Morelli
Department of Obstetrics, Gynecology and Reproductive Health, Rutgers New Jersey Medical School,
Newark, NJ, United States
Until recently, the study of age-related decline in fertility has focused primarily on
the ovary; depletion of the finite pool of oocytes and increases in meiotic errors
leading to oocyte aneuploidy are well-established mechanisms by which fertility
declines with advancing age. Comparatively little is known about the impact of
age on endometrial function. The endometrium is a complex tissue comprised of
many cell types, including epithelial, stromal, vascular, immune and stem cells.
The capacity of this tissue for rapid, cyclic regeneration is unique to this tissue,
undergoing repeated cycles of growth and shedding (in the absence of an
embryo) in response to ovarian hormones. Furthermore, the endometrium has
been shown to be capable of supporting pregnancies beyond the established
boundaries of the reproductive lifespan. Despite its longevity, molecular studies
have established age-related changes in individual cell populations within the
endometrium. Human clinical studies have attempted to isolate the effect of
aging on the endometrium by analyzing pregnancies conceived with euploid,
high quality embryos. In this review, we explore the existing literature on
endometrial aging and its impact on pregnancy outcomes. We begin with an
overview of the principles of endometrial physiology and function. We then
explore the mechanisms behind endometrial aging in its individual cellular
compartments. Finally, we highlight lessons about endometrial aging gleaned
from rodent and human clinical studies and propose opportunities for future
study to better understand the contribution of the endometrium to age-related
decline in fertility.
KEYWORDS
endometrium, aging, senescence, pregnancy, assisted reproductive technology,
endometrial microbiome, endometrial receptivity
1 Introduction
The average maternal age at first birth has been steadily rising, reflecting both a decline
in births to teenage and young adult mothers as well as an increase in first births in women
in the fourth and fifth decades of life (Osterman et al., 2023). While the age at peak
fecundability has been estimated to occur at 29–30 years for parous women (women who
have had a prior delivery) and 27–28 years for nulliparous women (women who have never
had a delivery) (Rothman et al., 2013), the birth rate for women aged 30–34 years has
surpassed that of women aged 25–29 years since 2015 (Osterman et al., 2023). It is well
OPEN ACCESS
EDITED BY
Lin Zhao,
Duke University, United States
REVIEWED BY
Yiwen Qin,
Cornell University, United States
Rujuan Zuo,
Oslo University Hospital, Norway
*CORRESPONDENCE
Anat Chemerinski,
ac2059@njms.rutgers.edu
RECEIVED 03 November 2023
ACCEPTED 09 February 2024
PUBLISHED 28 February 2024
CITATION
Chemerinski A, Garcia de Paredes J,
Blackledge K, Douglas NC and Morelli SS (2024),
Mechanisms of endometrial aging: lessons from
natural conceptions and assisted reproductive
technology cycles.
Front. Physiol. 15:1332946.
doi: 10.3389/fphys.2024.1332946
COPYRIGHT
© 2024 Chemerinski, Garcia de Paredes,
Blackledge, Douglas and Morelli. This is an
open-access article distributed under the terms
of the Creative Commons Attribution License
(CC BY). The use, distribution or reproduction in
other forums is permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original publication in this
journal is cited, in accordance with accepted
academic practice. No use, distribution or
reproduction is permitted which does not
comply with these terms.
Frontiers in Physiology frontiersin.org01
TYPE Review
PUBLISHED 28 February 2024
DOI 10.3389/fphys.2024.1332946

established that the ovarian factor—the decline in oocyte quantity
and quality, which occurs because of an increase in meiotic errors in
this finite pool—becomes a more important consideration with age
(Crawford and Steiner, 2015). Though the dogma was called into
question in 2004 (Horan and Williams, 2017) when oocyte stem cells
were isolated from mouse ovaries (Johnson et al., 2004), most
current evidence points to the existence in human ovaries of a
more or less finite, non-renewable, pool of oocytes which is
gradually depleted until menopause. Therefore, given the trend
towards delaying childbearing and the impact of age on ovarian
reserve and fecundability, it is not surprising that the focus of
scientific inquiry with respect to fertility and aging has been
the ovary.
For many years, the data from assisted reproductive technology
(ART) cycles aligned with the ovary-centric paradigm, and the age-
related decline in fertility was ascribed mostly or entirely to ovarian
aging (Navot et al., 1991;Navot et al., 1994). As a result, many
technologies were developed with the aim of circumventing ovarian
aging. The hurdle of ovarian aging can now be overcome via oocyte
or ovarian tissue cryopreservation at a younger age, or by the use of
non-autologous oocytes, though the former requires financial means
and advanced planning while the latter adds financial burden and is
not accepted by all patients as an alternative to the use of autologous
oocytes. The use of preimplantation genetic testing for aneuploidy
(PGT-A) may decrease the chance of transferring aneuploid
embryos, the rates of which predictably increase with advancing age
in women undergoing in vitro fertilization (IVF) (Franasiak et al., 2014).
However, none of these technologies considers the potential impact of
the age of the uterus, and more specifically the impact of aging of the
endometrium (uterine mucosal lining) on pregnancy outcomes.
More recently, a reexamination of the assumptions about
endometrial aging has brought the topic to the forefront. A
2022 analysis of endometrial gene expression by age revealed
altered expression of genes related to cilia motility and epithelial
cell proliferation in women over 35 years (Devesa-Peiro et al., 2022),
identifying mechanisms that may contribute to endometrial aging. A
2023 systematic review and meta-analysis of >11,000 euploid embryo
transfers found a higher ongoing pregnancy rate or live birth rate in
women <35 years compared with their oldercounterparts (Vitagliano
et al., 2023), suggesting the role of a non-ovarian factor in dictating
pregnancy outcomes in older women. In an attempt to address the
contribution of the endometrium to pregnancy success, the last
decade has seen a rise in endometrial receptivity testing.
Endometrial receptivity refers to the limited time during which the
human endometrium, after undergoing morphologic and functional
changes under the influence of ovarian hormones, permits embryo
attachment (Achache and Revel, 2006). Endometrial receptivity
testing is therefore based on the premise that the window of
implantation has a distinct transcriptomic signature that should be
targeted in order to personalize the timing of an embryo transfer
(Ruiz-Alonso et al., 2013). This technology allowed for the
characterization of the endometrium as pre-receptive, receptive, or
post-receptive; it was initially considered to be a panacea in terms of
correcting endometrial factor infertility, including the potential to
correct for an adverse impact of age on endometrial receptivity.
However, the utility of this testing has been the subject of heated
debate (Quaas and Paulson, 2021;Ruiz-Alonso et al., 2021)andhas
not yet been assessed for applicability to women of older reproductive
age (>37 years). Therefore, it is now time to reconsider the impact of
age on endometrial function.
Herein we review the existing literature on endometrial aging
and its impact on pregnancy outcomes. We begin by reviewing the
cycle of endometrial growth, differentiation, shedding and
regeneration as an overview of the principles of endometrial
physiology and function. We then explore mechanisms that
contribute to endometrial aging, examining each individual
cellular compartment. Finally, we highlight lessons about
endometrial aging that can be gleaned from studies of natural
conceptions and those achieved using ART, as well as the
relationship between endometrial aging and disorders of
pregnancy that originate from placental dysfunction.
2 Endometrial physiology and function
2.1 Endometrial structure and function
The endometrium is a dynamic tissue, undergoing morphologic
and functional changes in response to ovarian-derived steroid
hormones (Figure 1). These changes prepare the endometrium
for embryo implantation, and in the absence of successful
implantation and initiation of a pregnancy, the human
endometrium sheds during menses and regenerates in the
subsequent cycle. The endometrium is composed of two layers:
the functionalis (upper two-thirds) layer, which is shed during
menstruation, and the basalis (lower one-third) layer which is
adjacent to the myometrium and does not shed. The functionalis
layer is comprised of a columnar surface (luminal) epithelium which
invaginates to form the lining of the endometrial glands, as well as
stromal, vascular and immune cells. The endometrium is highly
responsive to cyclic changes in the ovarian steroid hormones
estradiol and progesterone, which largely exert their effects via
their cognate nuclear receptors and downstream regulation of
gene transcription within multiple cellular compartments of the
endometrium (Jain et al., 2022).
Although both isoforms of the nuclear estrogen receptor (ER),
ER-alpha and ER-beta, are expressed in all parenchymal
endometrial cell types (including glandular epithelial and
stromal) across all phases of the menstrual cycle, ER-alpha is the
predominant form uniformly (Matsuzaki et al., 1999). ER-alpha is
encoded by the ESR1 gene on chromosome 6 and ER-beta is encoded
by the ESR2 gene on chromosome 10 (Jain et al., 2022). Progesterone
receptors (PR) are encoded by a single gene on chromosome 11 and
occur in two isoforms: PR-A and PR-B. PR-A is the predominant
form within the endometrium. Expression of endometrial PR-A is
upregulated by estrogens via ER-alpha, and thus rising estradiol
levels during the proliferative phase of the menstrual cycle are
required for the endometrium to respond to progesterone in the
secretory phase (Yu et al., 2022).
2.2 Endometrial growth during the
proliferative phase
Following menses, the proliferative phase of the menstrual cycle
is characterized by endometrial growth and re-epithelialization
Frontiers in Physiology frontiersin.org02
Chemerinski et al. 10.3389/fphys.2024.1332946

largely in response to rising estradiol secreted by the dominant
ovarian follicle (Figure 1, left panel). By cycle days 5–6 (where cycle
day 1 is the first day of menses), the entire cavity is re-epithelialized
and stromal growth begins. In the early proliferative phase,
endometrial glands are narrow, tubular, and are lined by low
columnar epithelial cells (Noyes et al., 1975). The blood supply
to the endometrium originates from the uterine arteries, which
subsequently branch into the arcuate arteries, radial arteries, and
spiral arterioles which are steroid hormone-responsive vessels
(Farrer-Brown et al., 1970). Throughout the proliferative phase,
there is widespread cellular proliferation in all of the principal
cellular compartments of the endometrium including epithelial,
stromal and vascular cells; proliferation peaks on cycle days
8–10 and coincides with the highest concentration of estrogen
receptors in the endometrium (Bergeron et al., 1988). During the
proliferative phase, the endometrium grows, on average, from
4–5mm–11 mm in thickness, and produces the trilaminar
appearance on transvaginal ultrasound (Bakos et al., 1993).
Endometrial growth is also promoted by an increase in insulin-
like growth factors (IGF) and epidermal growth factors (EGF), both
of which are stimulated by rising estradiol levels during the
proliferative phase (Hofmann et al., 1991;Giudice et al., 1993).
Other hormones influencing endometrial growth are
glucocorticoids, specifically cortisol, and androgens. Androgen
receptors are predominantly expressed in the endometrial stroma
during the proliferative phase with reduced expression during the
secretory phase, and androgens have an anti-proliferative effect on
endometrial epithelial and stromal cells (Gibson et al., 2016).
Glucocorticoid receptors are expressed in human endothelial
cells, and their activation in the proliferative phase reduces
angiogenesis (Logie et al., 2010). Finally, Brighton et al. suggested
that an organized process of senescence in, and ultimately removal
of, a subgroup of endometrial stromal cells contributes to the overall
remodeling and regrowth of the endometrium during the
proliferative phase (Brighton et al., 2017).
Cellular senescence is a phenomenon described in proliferative
tissue including the endometrium and can occur under physiologic
conditions when a cell reaches its final telomere length or under the
influence of oxidative stress and other stressors such as DNA damage,
inflammation, and epigenetic modifications (Deryabin et al., 2020). It
is especially important to distinguish the global process of aging as a
clinical entity from the cellular process of senescence: cellular
senescence is present throughout the lifespan and is not an
inherently pathologic process. Senescent cells are present in
healthy tissue, arise from replicative exhaustion, and are cleared by
the immune system. In the endometrium, stromal cells differentiate
during the process of decidualization into either mature decidual cells
or senescent cells. These senescent cells remain metabolically active,
secreting pro-inflammatory cytokines, chemokines, matrix
metalloproteases and growth factors, a senescence-associated
secretory phenotype (SASP) that mediates communication with the
surrounding microenvironment (Coppe et al., 2008). Via secretion of
SASP, senescent cells can be recognized and quickly cleared by
immune cells, thus creating an appropriately regulated
inflammatory environment for embryo implantation (Deryabin
et al., 2020).
While serving a physiologic function, senescence may also be
associated with pathological conditions such as neurodegeneration
and atherosclerosis, cancer and immune system dysregulation. Many
of these conditions are “age-related”in that damage is accrued over
time. And indeed, during natural aging, immune clearance of
senescent cells in the endometrium may be impaired. These
senescent cells are not able to decidualize properly and their
secretory phenotype can create a pro-inflammatory
microenvironment that may interfere with endometrial receptivity
(van Deursen, 2014;Deryabin et al., 2020). To this end, the
elimination of accumulated senescent cells is currently being
investigated as a strategy to alleviate various age-related diseases
and tissue malfunction (Baker et al., 2011;Zhu et al., 2015)and
may have future applications to the endometrium.
FIGURE 1
Ovarian and endometrial events in the human menstrual cycle. In the proliferative phase, estradiol secreted from the developing follicle leads to
growth and development of the endometrium (left). Magnified inset shows that multiple cell types comprise the endometrium: epithelial, stromal, stem,
immune and endothelial. Following ovulation, in the secretory phase, the endometrium undergoes various transformative events, including stromal cell
decidualization, in preparation for possible embryo implantation (right). Images created with Biorender.com.
Frontiers in Physiology frontiersin.org03
Chemerinski et al. 10.3389/fphys.2024.1332946

2.3 Endometrial changes and remodeling
during the secretory phase
Whereas during the proliferative phase the endometrium is
mostly responsive to estradiol alone, after ovulation, in the
secretory phase (Figure 1, right panel), the endometrium
responds to both estradiol and progesterone secreted by the
ovarian corpus luteum formed post-ovulation. During the
secretory phase, the total endometrial thickness remains relatively
fixed, despite continued elevated estradiol levels (Bakos et al., 1993).
This is at least partly attributable to the action of progesterone, which
limits growth of the endometrium via decreased mitosis and DNA
synthesis (Moyer and Felix, 1998). Furthermore, progesterone
stimulates the activity of 17beta-hydroxysteroid dehydrogenase and
sulfotransferase, which converts estradiol to estrone sulfate, the form
of estrogen that is more rapidly cleared from cells (Gurpide et al.,
1976;Falany and Falany, 1996). Finally, progesterone antagonizes
estrogen-mediated stimulation of oncogenes, which would otherwise
promote further endometrial growth (Kirkland et al., 1992).
Progesterone receptor expression in endometrial cells is stimulated
by increased circulating estradiol during the proliferative phase via
ER-alpha. In a negative feedback loop, further ER-alpha receptor
expression in endometrial cells is inhibited by progesterone (West and
Brenner, 1985). While PR-A levels start to decline, PR-B expression
levels remain constant during the secretory phase and PR-B plays a
role in the control of glandular secretion. In a tightly controlled
feedback loop, progesterone then limits expression of ER-alpha via
PR-A (McDonnell and Goldman, 1994). This feedback loop is
essential for creating the specific microenvironment that allows for
endometrial response to estrogen in the proliferative phase, while
simultaneously preventing an over-response, leading to endometrial
hyperplasia.
As the endometrium prepares for the possibility of embryo
implantation it undergoes a process of remodeling that includes
functional and morphological changes. Approximately 7-8 days
after ovulation, circulating levels of estradiol and progesterone
promote an increase in circulating prostaglandins and VEGF.
These factors influence capillary permeability, thereby resulting in
stromal edema, a hallmark of this phase of the cycle (Plaisier, 2011).
Rising progesterone triggers a marked increase in multiple leukocyte
populations, which ultimately comprise up to 40% of all endometrial
cells (King, 2000). These leukocytes serve multiple roles, including
immunoprotection as the endometrium prepares for implantation,
regulation of trophoblast invasion, and in the absence of pregnancy,
breakdown of endometrial tissue in the menstrual phase via secretion
of matrix metalloproteinases (Mihm et al., 2011). Towards the end of
the secretory phase, prior to the onset of menses, the endometrium
has differentiated into three zones: the deepest layer remains the
unchanged basalis layer, the middle layer is the stratum spongiosum,
or loose edematous stroma containing tightly coiled spiral vessels and
dilated glands, and the most superficial layer is the more stromally
dense stratum compactum (Heremans et al., 2021). A hallmark of
endometrial remodeling in the secretory phase is the transition of
endometrial stromal fibroblasts into epithelioid-like decidualized
stromal cells, characterized by increasing amounts of glycogen and
lipids, and an expanded cytoplasm (Owusu-Akyaw et al., 2019).
Decidualized stromal cells secrete both prolactin and insulin-like
growth factor binding protein (IGFBP-1), which stimulate
trophoblast growth and invasion, along with other actions that
together produce the morphologic changes that aid embryo
implantation (Owusu-Akyaw et al., 2019). The degree to which
this process is completed optimally results in endometrial
receptivity, which refers to the preparation of the endometrium for
embryo implantation during the specific4–6day“window of
implantation”in the secretory phase (Lessey and Young, 2019).
2.4 Endometrial shedding
In the absence of pregnancy, demise of the corpus luteum leads
to withdrawal of estradiol and progesterone, initiating the process by
which two-thirds of the endometrium is shed during menstruation.
Immediately after hormone withdrawal, the tissue height
(particularly within the upper zone of the functionalis layer or
the stratum compactum) decreases. The reduction in blood flow
within spiral arterioles triggers vasoconstriction which causes local
ischemia. Menstruation occurs due to resulting endometrial
ischemia as well as enzymatic degradation from the release of
lysosomes containing lytic enzymes, which promote apoptosis in
both glandular epithelial and stromal cells (Rosenwaks and Seegar-
Jones, 1980;Ferenczy, 2003;Jain et al., 2022).
2.5 Endometrial regeneration
The process of endometrial regeneration originates, at least in part,
from epithelial and stromal stem/progenitor cell populations residing in
the basalis layer (Salamonsen et al., 2021). Re-epithelialization of the
exposed surface of the endometrium is accomplished by endometrial
epithelial stem cells located in glands within the basalis layer (Cousins
et al., 2021). Circulating estradiol, secreted by a new dominant ovarian
follicle, begins to increase following menses, prompting the glandular
proliferation necessary for regenerating the functionalis layer (Gargett
et al., 2016). By days 2–3 of the cycle, DNA synthesis occurs in the areas
of the basalis that have now been exposed due to menses. Garry et al.
observed that menstrual shedding occurs in a piecemeal manner, with
areas of shedding and healing endometriumexistingsimultaneously
(Garry et al., 2009). The regeneration process is supported by stromal
fibroblasts which direct regrowth of the endometrium via autocrine and
paracrine mechanisms; endometrial stromal cells contribute to the
regeneration not only of the stromal compartment but also the
epithelial compartments via mesenchymal-epithelial transition
(Owusu-Akyaw et al., 2019). The repair processes occurs rapidly;
new epithelium covers more than two-thirds of the endometrial
cavity by cycle day 4. The repair process is also, at least in part,
likely a response to injury and inflammation caused by high levels of
circulating cytokines and proteolytic enzymes (Salamonsen et al., 2021).
3 The aging of individual cell types in
the human endometrium
3.1 Histopathologic changes
There are few reports that describe how the clinical
characteristics of the uterus and endometrium change with age.
Frontiers in Physiology frontiersin.org04
Chemerinski et al. 10.3389/fphys.2024.1332946

Uterine size and volume are known to decrease following
menopause (Merz et al., 1996). In a recent study of 146 Chinese
women, Li et al. (Li et al., 2020) found that the mean uterine volume
decreased from 44.9 ± 17.8 cm
3
prior to menopause, to 16.3 ±
10.6 cm
3
after menopause; mean endometrial thickness decreased
from 4.8 ± 2.1 to 3.3 ± 0.5 mm. In a sub-analysis of patients that did
not have fibroids, uterine volume was noted to decrease even prior to
the final menstrual period (Li et al., 2020). Histologically, the aging
endometrium has been characterized as inactive in the decade after
menopause, to senescent or atrophic thereafter (Ferenczy and
Bergeron, 1991). This distinction is used to differentiate between
a tissue without mitotic activity but whose cells contain estrogen
receptors and are able to respond to hormonal stimulation
(inactive), and cells that are atrophic and no longer express
estrogen receptors (severely atrophic) (Ferenczy and Bergeron,
1991). However, in order to better understand the cellular
changes occurring in the endometrium over the reproductive and
post-reproductive lifespan, the individual cellular compartments
(stromal, epithelial, vascular, immune and stem cells), and their
role with respect to embryo implantation, must be examined
(summarized in Table 1).
3.2 Stromal cells
Endometrial stromal fibroblasts, mesenchymal in origin,
proliferate during the proliferative phase of the menstrual cycle
and undergo decidualization following exposure to progesterone
after ovulation. Stromal cells depart the cell cycle to differentiate into
decidualized cells, a process mediated by transcription factors
including C/EBPs, FOXO1, CREB and STAT5 (Deryabin et al.,
2020). Decidualized endometrial stromal cells secrete a variety of
peptide hormones and growth factors, including prolactin, relaxin,
renin, insulin-like growth factors (IGFs) and insulin-like growth
factor-binding proteins (IGFBPs) (Fritz and Speroff, 2011).
Senescence, the point at which mitotic activity ceases in normal
cells, can be measured by specific markers including p21 and
senescence-associated β-galactosidase (SA-β-Gal). Cellular
senescence is a physiologic process that can also occur after
exposure to non-physiologic stressors (Deryabin et al., 2020).
Senescence of endometrial fibroblasts has been associated with
poor reproductive outcomes. Tomari et al. examined the impact of
stromal cell senescence on endometrial receptivity, using
endometrial biopsies taken at the time of oocyte retrieval, 2 days
TABLE 1 Age-related changes in individual cellular compartments.
Cell type Cell function Changes with age
Stromal cells
Undergo decidualization following exposure to progesterone; decidualized
stromal cells secrete peptide hormones and growth factors that stimulate
trophoblast growth and invasion
- Increased cellular senescence associated with poorer pregnancy outcomes
(Tomari et al., 2020)
- Differential gene regulation, particularly PR and ESR1/2 (Erikson et al.,
2017)
- Decreased proliferation (Berdiaki et al., 2022)
- Decreased expression of decidualization markers (Berdiaki et al., 2022)
Luminal epithelial
cells
Represents the first point of contact with the implanting embryo
- Increased p16 and p21 expression (Lv et al., 2022)
- Increased expression of profibrotic PTGS2 (Lv et al., 2022)
- Decreased expression of cell cycle related gene PCNA (Lv et al., 2022)
- Upregulation of NF-kappa B signaling pathway and extracellular matrix
receptor interaction associated with cellular senescence (Lv et al., 2022)
- Lower secretion of bactericidal substances (e.g., SLPI) in post-menopausal
women (Fahey and Wira, 2002)
Glandular
epithelial cells
Secretory products signal to embryo and provide fetus with nutrition during
organogenesis
Increased somatic mutation burden (Moore et al., 2020)
Endothelial cells Express steroid hormone receptors, undergo remodeling in response to
ovarian steroid hormones
- Diminished vasoreactivity in post-menopausal uterine arteries
(Nicholson et al., 2017)
- Increased myometrial artery calcifications (Hessler et al., 2015)
Immune cells Protect against infection, while creating an environment that is not hostile to
allogenic sperm and permissive of embryo implantation
- Higher cytolytic potential in CD3
+
T cells in post-menopausal subjects
(White et al., 1997)
- Increased cytotoxic activity of CD8
+
T cells after menopause
(Rodriguez-Garcia et al., 2020)
- Higher frequency of Th17, CD8
+
T cells, and CD8
+
tissue-resident
memory T cells after menopause (Rodriguez-Garcia et al., 2014;
Rodriguez-Garcia et al., 2018)
Stem cells Contribute to endometrial regeneration and serve immunomodulatory
functions in endometrium
Reduced expression of SHH and increased expression of SERPINB2 (Cho
et al., 2019)
SLPI, secretory leukocyte protease inhibitor; SHH, sonic hedgehog.
Frontiers in Physiology frontiersin.org05
Chemerinski et al. 10.3389/fphys.2024.1332946

prior to embryo transfer into the uterus (Tomari et al., 2020).
Patients with implantation failure (i.e., who did not achieve
pregnancy after embryo transfer) were classified as having a non-
receptive endometrium. β-galactosidase activity, a marker of cellular
senescence, was found to be increased in human endometrial
stromal cells (HESCs) isolated from non-receptive endometrium
as compared to HESCs isolated from receptive endometrium. Cell
cycle arrest is a hallmark of senescent cells (Ogrodnik et al., 2019);
whereas HESCs isolated from women who achieved pregnancy were
more frequently in S phase (actively replicating), HESCs isolated
from subjects with implantation failure were more frequently in G0/
G1 phase, suggesting senescent cells in cell cycle arrest. Cellular
senescence-related genes CDKN1A and CDKN2A were also more
highly expressed in ESCs isolated from the endometrium of subjects
with implantation failure (Tomari et al., 2020). The results of this
study, demonstrating an inverse association between HESC
senescence and embryo implantation, suggest that implantation
failure may be attributed, at least in part, to aging of HESCs.
In addition to assessing markers of stromal cell senescence,
other methods have been used to show the impact of age on
endometrial stromal cells. In a transcriptomic analysis of pre-
menopausal and peri-menopausal stromal fibroblasts, over
1,000 genes were found to be differentially expressed between
thesetwoagegroups(Erikson et al., 2017). Ingenuity pathway
analysis demonstrated the pathways of interest to be related to
cell-cycle processes (“organization of cytoskeleton”,“formation
of filaments”“formation of actin stress fibers”), as well as to
fibroblasts (“proliferation of fibroblasts”,“migration of fibroblast
cell lines”), and identified differential regulation of additional
pathways common with senescent or aging cells. Quantitative
real-time PCR validation of specific genes revealed
downregulation of both the progesterone receptor (PR)and
estrogen receptor beta (ESR2), and upregulation of estrogen
receptor alpha (ESR1) in the younger cohort (Erikson et al.,
2017). This study also examined the transcriptomic profile of
endometrial mesenchymal stem cells (eMSCs) in these two age
groups and performed a four-way comparison (pre- and peri-
menopausal stromal fibroblasts, and pre- and peri-menopausal
eMSCs). They found that the gene expression profile of
perimenopausal stromal fibroblasts more closely resembled
that of the eMSC groups, suggesting that the perimenopausal
stromal fibroblasts are less differentiated than premenopausal
stromal fibroblasts (Erikson et al., 2017).
In vitro studies have demonstrated that hypoxia-induced
endometrial stromal cell senescence leads to impaired
decidualization as evidenced by incomplete morphological
transformation and reduced expression of decidualization
markers IGFBP1 and PRL (Deryabin and Borodkina, 2022).
Kusama et al. found that in vitro decidualization of HESCs,
confirmed by upregulated expression of PRL,IGFBP1 and
FOXO1, resulted in an increase in expression of senescence
markers p21 and p53, suggesting that decidualization may
promote cellular senescence (Kusama et al., 2021). Treatment of
decidualized HESCs with Dasatinib and Quercetin, which have been
extensively studied in combination as a senolytic treatment due to
their selective clearance of senescent cells (Xu et al., 2018;Islam et al.,
2023), increased expression of decidualization markers such as PRL
and decreased the number of senescent cells (Kusama et al., 2021).
In a study of endometrial stromal cells from women aged
25–46 years, endometrial biopsies were taken in the proliferative
phase and proliferation was assessed using a fluorometric assay.
Women aged 25–35 years had significantly higher levels of stromal
cell proliferation compared with women aged 36–46 years. Women
in the younger age group had significantly higher expression of
BMP2 and STAT3, two important regulators of stromal cell
proliferation and differentiation. Following in vitro
decidualization, endometrial stromal cells from younger women
also had significantly higher expression of PRL and IGFBP-1, two
key markers of decidualization (Berdiaki et al., 2022). Collectively
these data implicate aberrations in stromal cell functions during the
process of endometrial aging, including changes in cell cycle
regulation, decreased proliferative capacity and impaired
decidualization, as well as alterations in the transcriptomic
profiles of stromal fibroblasts to resemble stem cells more closely.
These age-related changes may be at least partially mediated by the
altered expression of steroid hormone receptors PR and
ESR1 and ESR2.
3.3 Epithelial cells
The epithelial cells of the endometrium can be subdivided into
those lining the endometrial glands (glandular epithelium) and
those lining the lumen (luminal epithelium). The luminal
epithelial cells are the first point of contact with the implanting
embryo; the secretory products of glandular epithelial cells signal to
the embryo and provide the fetus with nutrition during
organogenesis (Burton et al., 2002). Epithelial cells, too, have
been investigated with respect to their role in endometrial aging.
A recent study linked accelerated cellular senescence in epithelial
cells with inadequate endometrial development, a clinical condition
known as “thin endometrium”that is associated with decreased
clinical pregnancy and live birth rates. While an association between
thin endometrium and age has not yet been established, this study
points to a possible link between the two entities. The authors
explored important changes in the luminal epithelium of subjects
with thin (<7 mm) compared with normal (8–14 mm)
endometrium, using single cell RNA sequencing on endometrial
biopsy samples taken in the late proliferative phase of a natural
menstrual cycle in women who presented for evaluation to a fertility
clinic (Lv et al., 2022). The authors found that despite having similar
cell types, thin endometria had decreased numbers of proliferating
stromal cells relative to normal endometria, as well as decreased
luminal epithelium with relatively increased glandular epithelium.
They additionally found increased immunostaining for p16 and p21,
markers of cellular senescence, as well as changes in gene expression
in thin endometria, such as increased expression of the profibrotic
enzyme PTGS2 (COX-2) and decreased expression of cell cycle-
related gene PCNA; these aberrations are suggestive of excessive
collagen deposition. Finally, they report changes in signaling
pathways, such as upregulation of NF-kappa B signaling and
extracellular matrix receptor interaction pathways in thin
endometria. Taken together, these findings suggest an
endometrial phenotype characterized by accelerated cellular
senescence and increased fibrosis in the epithelial cells of thin
endometria (Lv et al., 2022).
Frontiers in Physiology frontiersin.org06
Chemerinski et al. 10.3389/fphys.2024.1332946

In addition to their role as the first point of contact with an
implanting embryo, luminal epithelial cells play a crucial role in
protecting the endometrium against pathogens via tight junctions to
regulate passage of molecules across the barrier. The permeability of
this layer is mediated, at least in part, by fluctuations in estradiol
(Wira et al., 2015). Luminal epithelial cells also exhibit immune
properties which change with aging; luminal epithelial cells secrete
substances such as secretory leukocyte protease inhibitor (SLPI), a
protein with bactericidal properties which has been shown to be
higher concentrations in premenopausal as compared to
postmenopausal. In this study, pre-menopausal endometrial
epithelial cells were found to have greater ability to kill
Staphylococcus aureus and Escherichia coli in culture than
endometrial cells from post-menopausal women and this
correlated directly with SLPI levels (Fahey and Wira, 2002).
The glandular epithelium has been implicated in endometrial
aging as well. A study of the somatic burden of endometrial glands of
women aged 19–81 years found that approximately 29 base
substitutions were added per gland per year, without a
correlation to parity (Moore et al., 2020). Together, these studies
suggest that epithelial cell senescence may be the consequence of
alterations in proliferation, changes in the composition of the
microenvironment, and increasing mutational burden.
3.4 Vascular cells
Each layer of the endometrium is supplied by a network of
vessels originating from the uterine arteries which course through
the myometrium: the basal arteries supply the basalis layer, spiral
arterioles feed the functionalis layer, and the subepithelial capillary
plexus supports the subepithelial zone (Massri et al., 2023). Human
endometrial vasculature undergoes cyclic remodeling under the
influence of estrogen in the proliferative phase and progesterone
in the secretory phase. Endometrial endothelial cells express
estrogen and progesterone receptors and undergo remodeling in
response to ovarian steroid hormones, as well as in response to
angiogenic factors such as VEGF and FGF2 secreted by epithelial
and stromal cells (Massri et al., 2023).
Mechanisms governing aging of the endometrial vasculature
can only be inferred at best, based on studies demonstrating
adverse effects of age on uterine artery function. The
vasoreactivity of the uterine arteries was examined in
hysterectomy samples of pre- and post-menopausal subjects,
and menopause was found to be associated with diminished
vessel constriction and relaxation, as well as an impairment in
the vasodilatory response with exposure to 17β-estradiol
(Nicholson et al., 2017). In a separate study, myometrial
artery calcifications were evaluated in women who had
undergone hysterectomy. No calcifications were observed in
women aged 45–49 years, and the rate of myometrial artery
calcification increased with increasing age with 50% of women
aged 70–81 years demonstrating calcifications (Hessler et al.,
2015). Although these studies examine uterine artery aging, it is
plausible that the processes reported in these larger vessels are
occurring in the smaller endometrial vessels derived from them,
and even that aging in larger vessels may impact the vessels that
branch from them.
3.5 Immune cells
The immune cells in the human endometrium have the dual role
of protecting against ascending infections, while creating an
environment that is not hostile to allogenic sperm and is
permissive of embryo implantation (Wira et al., 2015).
Endometrial immune cell populations must be responsive to sex
steroids (Shen et al., 2021), and indeed, multiple studies have
confirmed the suppressive effects of estradiol on endometrial
T cells (Nilsson and Carlsten, 1994;Rijhsinghani et al., 1996;
White et al., 1997;Rodriguez-Garcia et al., 2020;Shen et al.,
2021). T cells, key mediators of the adaptive immune system, are
present in the endometrium and their populations change with age.
In an early study of T cell function in the endometrium in pre- and
post-menopausal subjects, CD3
+
T cells were found to have higher
cytolytic potential in the proliferative phase compared with the
secretory phase of pre-menopausal subjects, and highest cytolytic
potential was found in post-menopausal subjects, suggesting that the
cytolytic activity is suppressed in preparation for embryo
implantation in a hormone-mediated manner and that this
function is lost after menopause (White et al., 1997). More recent
studies have found that pre-treatment of CD8
+
T cells with estradiol
at physiologic concentrations, followed by co-culture with allogenic
target cells, reduced the number of dead target cells relative to co-
culture of untreated T cells and target cells suggesting an estradiol-
mediated reduction in T cell cytotoxicity (Shen et al., 2021).
Other studies have found increased cytotoxic activity of CD8
+
T cells (Rodriguez-Garcia et al., 2020) and changes in T cell
populations following menopause: the frequencies of Th17 and
CD8
+
T cells were found to be increased in menopausal
endometrium (Rodriguez-Garcia et al., 2014;Rodriguez-Garcia
et al., 2018) and CD8
+
tissue-resident memory T cells, identified
as CD103
+
, have been found in greater abundance in
postmenopausal endometrial samples (Rodriguez-Garcia et al.,
2018). Collectively, these studies point to an estrogen-dependent
suppression of endometrial T cells which becomes less robust with
the age-related decline in estrogen concentrations.
3.6 Stem cells
The lower basalis layer serves as the source for cyclic
regeneration of the endometrium (Salamonsen et al., 2021).
Given that the regenerated tissue is composed of various cell
types, the origin, location and characteristics of endometrial stem
cells are of great interest (Gargett et al., 2016). Epithelial stem/
progenitor cells and mesenchymal stem cells (eMSC) have been
isolated from human and rodent endometrium (Bozorgmehr et al.,
2020;Cousins et al., 2021) and have been shown to have classic
properties of stem cells, including clonogenicity and in the case of
eMSC, multilineage differentiation potential (Gargett et al., 2009).
Stem cells play an important role in the maintenance, repair or
regeneration of nearly all tissues in the human body. These cells
reside in various organs in a quiescent state and are called upon to
differentiate in response to growth or injury (Jung and Brack, 2014).
However, stem cells are susceptible to age-related damage that
impairs their regenerative capacity (Liu et al., 2022). Certain
tissues, such as the intestinal mucosa, undergo frequent
Frontiers in Physiology frontiersin.org07
Chemerinski et al. 10.3389/fphys.2024.1332946

regeneration similar to the endometrium, and these stem cells, too,
are susceptible to aging. Intestinal stem cells which are found at the
base of the intestinal crypts, bear some similarities to endometrial
epithelial stem/progenitor cells that reside in the basalis layer of the
endometrium and contribute to the repair and re-epithelialization
that takes places following menses (Cousins et al., 2021).
Endometrial epithelial stem/progenitor cells and intestinal stem
cells share certain properties—namely that they are tissue
resident, localized to the deeper layers (basalis layer of the
endometrium and intestinal crypts, respectively), and permit the
tissue to undergo constant renewal (Baulies et al., 2020;Bozorgmehr
et al., 2020). Thus, though studies describing the impact of aging on
endometrial stem cells are limited at present, findings in the intestine
may provide relevant insights. Age-related changes in intestinal stem
cells are reflected in the decreased number of crypts, increased crypt
length and width, and fewer numbers of proliferating stem cells
noted with aging (Liu et al., 2022). In murine studies, intestinal stem
cells in aged mice exhibited functional impairment, with decreased
migration capacity from crypt to villus (Nalapareddy et al., 2017).
In the endometrium, several studies have explored the molecular
changes that stem cells incur with age, in order to better understand
the implications for reproductive health. One such study (Cho et al.,
2019) examined the sonic hedgehog (SHH) protein, which functions
principally as a morphogen during embryonic development but has
also been found to play a role in age-related conditions such as
neurodegenerative disease and atherosclerosis (Queiroz et al., 2012;
Cho et al., 2019). In this study, endometrial tissues from aging mice
were found to express significantly lower levels of SHH mRNA and
protein compared to young mice. Treatment of human endometrial
mesenchymal stem cells with SHH alleviated senescence-induced
dysfunctions, improving self-renewal, migratory, and multilineage
differentiation capacities. These effects, mediated by downregulation
of SERPINB2, provided new insights into the mechanisms
regulating endometrial stem cell aging, and identified
SERPINB2 as a potential biomarker and therapeutic target in
aging endometrial stem cells (Cho et al., 2019).
Endometrial stem cell dysfunction has been implicated in
adverse pregnancy outcomes such as recurrent pregnancy loss
(RPL), a clinical entity that is more common with advancing
female reproductive age (Magnus et al., 2019). W5C5 is a marker
of perivascular endometrial mesenchymal stem cells (eMSCs);
W5C5-eMSCs can self-renew, demonstrate multilineage
differentiation, and can reconstitute mesodermal tissue (Masuda
et al., 2012). In a study of secretory phase endometrial biopsies,
subjects with RPL were found to have reduced clonogenicity in both
W5C5+ (perivascular) and W5C5-(non-perivascular) stromal cells
(the latter representing a heterogenous group of non-stem cells such
as stromal, vascular or immune cell types) relative to controls (Lucas
et al., 2016). In a study of secretory phase endometrial biopsies,
subjects with RPL were found to have reduced clonogenicity in both
W5C5+ (perivascular) and W5C5- (non-perivascular) stromal cells
relative to controls (Lucas et al., 2016). Further, the number of
clonogenic cells demonstrated a negative correlation with the
number of miscarriages (Lucas et al., 2016). Subjects with RPL
were also found to have hypomethylation of the HMGB2 gene, a
marker of reproductive senescence in stromal cells, associated with
reduced potential for proliferation and diminished endometrial
plasticity (Lucas et al., 2016). These studies suggest that
increasing miscarriage risk in older women may not be due to
embryo aneuploidy alone, and imply a role for aging eMSC and
other endometrial cell types; but proof of these concepts requires
additional study.
In addition to tissue regeneration, mesenchymal stem cells also
serve immunomodulatory functions; MSCs have been shown to
inhibit the functions of the innate and adaptive immune systems by
impairing dendritic cell function, diminishing natural killer cell
cytotoxicity, and preventing T cell differentiation (Sotiropoulou
et al., 2006;Willis et al., 2018;He et al., 2019;Lee et al., 2021).
In the endometrium, MSCs exert paracrine effects on their
neighboring stromal cells, as evidenced by in vitro studies in
which endometrial stromal cells co-cultured with MSCs
demonstrate increased proliferation, migration and invasion
relative to controls (Zhao Q. et al., 2023). In the context of
endometrial aging, it is interesting to note that MSCs with
clonogenic activity have even been isolated from inactive post-
menopausal endometrium and appear to have similar self-
renewal capacity to MSCs isolated from premenopausal
endometrium (Schwab et al., 2005;Ulrich et al., 2014). However,
whether MSCs isolated from postmenopausal endometrium retain
their paracrine/immunomodulatory effects on neighboring
endometrial cells, and/or capacity to regenerate tissue in vivo, has
not been proven. Given the crucial role of these stem cells in
regenerating the endometrium and maintaining its principal
functions, and given their presence throughout the lifespan, the
question of their role in regulating endometrial aging is particularly
interesting and should be examined in future studies.
4 Insights from rodent studies
Because of similarities between rodents and humans, studies
in rodent models have contributed greatly to our understanding
of human pathophysiology. While human reproductive anatomy
and physiology overlaps substantially with the rodent, certain key
morphologic and functional differences between the two species
should be noted. These include differences in anatomy, lack of
menstruation in rodents, cycle length, timing of ovulation and
trigger for and extent of decidualization. These important
distinctions are summarized in Figure 2. Despite these
differences, rodent studies remain crucial to the study of
human disease as they contribute in vivo mechanistic data (for
example, knockout models) that cannot be obtained from
human studies.
To determine whether aging impacts the response of the
endometrium to estradiol, Bader et al. (Bader et al., 2012)
employed aromatase knockout mice (ArKO) as a model of
estrogen deficiency. Markers of estrogen response in the uterus,
including lactoferrin (Ltf), estrogen receptor alpha (Esr1), and
estrogen receptor beta (Esr2), were measured. Exposure of young
adult (3 months) and aged adult female mice (12 months) to
estradiol resulted in an age-dependent response with significantly
higher expression of Esr1 in young adult mice. While Ltf expression
was found to increase in both age groups of mice after estradiol
exposure, the effect was ten times more pronounced in young adult
animals. Together, these changes suggest an age-dependent
reduction in endometrial responsiveness to estrogen.
Frontiers in Physiology frontiersin.org08
Chemerinski et al. 10.3389/fphys.2024.1332946

To explore the effect of steroid hormones on uterine
expression of estrogen-responsive genes Ltf and C3,and
progesterone-responsive genes Ihh, Lif, Areg, Hoxa10 and
Hand2,Lietal.(Li et al., 2017) studied ovariectomized mice
between 2- and 12-months-old supplemented with subcutaneous
estradiol or progesterone. They found no difference between
young and aged mice in expression of Ltf and C3 after
treatment with estradiol; however, Ltf expression was lower in
aged mice following progesterone treatment. Expression of
progesterone-induced implantation-related genes Lif and
Hand2 as well as Indian hedgehog (Ihh), ageneinvolvedin
mediating the communication between the endometrial
epithelium and stroma required for embryo implantation, was
reduced in aged mice. The effects of age were also assessed by the
detection of telomerase activity with real-time PCR after
exposure to steroid hormones. Telomerase is an enzyme that
prevents shortening of telomeres, and reduced expression may
suggest diminished protection from telomere shortening and
cellular senescence. In this study, 12-month-old mice had
significantly lower levels of telomerase expression compared to
2-month-old mice after exposure to progesterone. Overall, this
study suggests that the age-related decline in fertility may be
associated with an alteration in endometrial responsiveness to
progesterone, as well as decreased telomerase activity.
Cellular senescence may also contribute to aging of the rodent
endometrium. To explore age-related changes in genes and
pathways related to cellular senescence, Kim et al. (Kim and You,
2022) studied mRNA and microRNA expression from uterine tissue
of young (3 months) and old (11 months) mice in the metestrus
phase of the estrous cycle. More than 500 differentially expressed
genes were identified; pathway analysis revealed changes in cellular
senescence-associated pathways including arachidonic acid and
glutathione metabolism. Seven microRNAs were identified as
interacting with differentially expressed genes in relevant
pathways such as arachidonic acid and glutathione metabolism,
suggesting a role in regulating endometrial aging. Furthermore,
administration of the traditional herbal medicine Samul-tang to
the aged mice reduced expression of one of these microRNAs (miR-
223–3p), identifying potential therapeutic targets for regulating
cellular senescence in the endometrium. Taken together, rodent
studies have provided some insight into the age-related changes in
the endometrium—such as increase senescence and diminished
hormone responsiveness—which may partially explain some of
the mechanisms behind the observed age-related decline in fertility.
FIGURE 2
Mouse and Human Comparative Reproductive Anatomy and Physiology. Female mouse uteri contain two horns, while human uteri generally have a
single cavity. The mouse estrous cycle is composed of proestrus, estrus, metestrus and diestrus stages, with ovulation marking the transition from
proestrus to estrus. The human menstrual cycle is divided into the proliferative and secretory phases, with ovulation marking the transition between the
two. In the mouse, the trigger for stromal cell decidualization is embryo implantation, after which stromal cells undergo mesenchymal to epithelial
transition and adopt the decidualized phenotype. In the human, the trigger for decidualization is ovulation. Images created with Biorender.com.
†
Reprinted from “Vascular changes in the cycling and early pregnant uterus”by Massri N, Loia R, Sones JL, Arora R, Douglas NC, 2023, JCI Insight.
2023 June 8; 8 (11):e163422. Copyright [2023] by the American Society for Clinical Investigation. Reprinted with permission.
Frontiers in Physiology frontiersin.org09
Chemerinski et al. 10.3389/fphys.2024.1332946

5 Insights from human clinical studies
Along with rodent studies, clinical studies have been designed to
address the question of endometrial aging as well (summarized in
Figure 3 and Table 2). In women, the decline in fertility typically
begins around age 30 and becomes clinically significant between the
ages of 35 and 40 years, after which there is an even greater decrease
(Menken et al., 1986). In addition to the well-established ovarian
contribution to reproductive aging, the endometrium may play an
important role; indeed, this tissue may be responsible for as many as
two-thirds of known implantation failures and may be involved in
the IVF-associated clinical entity of recurrent implantation failure,
defined as women who have had three failed transfers of good-
quality embryos (Bashiri et al., 2018;Craciunas et al., 2019).
However, until recently, the role of the endometrium in
reproductive aging had been underestimated and understudied.
Early studies emphasized ovarian aging as the predominant
factor in predicting IVF outcomes in women of advanced
FIGURE 3
Cellular Compartments of the Endometrium Undergo Age-Related Changes Which May Contribute to Adverse Pregnancy Outcomes. (A) The
endometrium is composed of two layers—the functionalis (upper two-thirds), and the basalis (lower one-third)—and contains various cellular
compartments including epithelial, stromal, vascular and immune. (B) The cellular compartments of the endometrium undergo age-related changes
including impaired decidualization, increased fibrosis, vascular calcification, alterations in the microbiome and immune cell populations, increased
oxidative stress, and cellular senescence. (C) These changes may contribute to the early and late pregnancy complications observed with age. Images
created with Biorender.com.
Frontiers in Physiology frontiersin.org10
Chemerinski et al. 10.3389/fphys.2024.1332946

reproductive age. Navot et al. (Navot et al., 1991) compared IVF
outcomes in infertile women aged ≥40 years who underwent IVF
using autologous oocytes or oocytes from younger donors and
unsurprisingly found higher implantation, pregnancy and
delivery rates in recipients of oocyte donation compared to
women undergoing IVF with autologous oocytes, underscoring
the importance of oocyte quality. Since this study was not able to
isolate the contribution of the endometrium, a subsequent
prospective study by the same group was undertaken (Navot
et al., 1994). In this second study, donor oocyte-derived embryos
from young women with a mean age 30.2 years were transferred in
younger (<40 years) and older (≥40 years) subjects. The clinical
pregnancy rate did not differ between the younger and older subjects
(21.6% vs. 23.5%), with similar pregnancy loss rates as well (9.1% vs.
16.7%). These early studies were limited, however, by relatively small
sample sizes and the use of procedures less applicable in modern
practice such as the transfer of a high number of cleavage stage
embryos (mean 4.1 embryos per recipient). In more recent years, an
expanding body of literature supports a more important role for
endometrial aging in the age-related decline in fertility. A
2012 prospective study by Gupta et al. (Gupta et al., 2012)
assessed 270 patients undergoing the transfer of high-quality,
untested embryos from egg donors aged 21–31 years. Ongoing
pregnancy rates were significantly lower in donor egg recipients
aged 40–44 years compared with recipients younger than 40 years
(24.6% vs. 38.4%, respectively), and an even greater decrease was
observed over the age of 45 years. Although lack of extended culture
limited optimal assessment of embryo quality (embryos were
transferred at day 2 or 3), these results suggested the possibility
of diminished endometrial function with aging.
5.1 Pregnancies conceived through assisted
reproductive technology (ART)/euploid
embryo transfers
Despite continued advancement in ART technologies, including the
advent of preimplantation genetic testing for aneuploidy (PGT-A) to
select euploid embryos for transfer, live birth rates have not increased
substantially in the last decade (Osterman et al., 2023). The relatively
low live birth rate of 45%–65% (Cimadomo et al., 2023) following
transfer of a euploid embryo indicates that factors other than ploidy are
required for successful embryo implantation and pregnancy
continuation, including the importance of the endometrium in
supporting a healthy implantation.
The “embryo factor”refers to the impact of embryo ploidy on
pregnancy rates. Pregnancies conceived from autologous oocytes in
women of advanced reproductive age are more likely to be aneuploid
(Franasiak et al., 2014) and are at increased risk of miscarriage (Magnus
et al., 2019) and pregnancy complications (Pinheiro et al., 2019); it is
thus more difficult to separate ovarian and endometrial aging. Though
previous studies attempted to minimize the embryo factor by evaluating
embryos derived from young oocyte donors, recent studies
incorporating extended embryo culture and embryo biopsy for
PGT-A followed by transfer of euploid embryos have provided the
ability to further reduce the influence of the “embryo factor”and better
isolate the endometrial factor. A systematic review and meta-analysis by
Vitagliano et al. (Vitagliano et al., 2023) investigated the effect of
maternal age on ART success ratesafterthetransferofeuploid
embryos derived from autologous oocytes. An analysis of 11,335 ET
cycles indicated that the live birth rate (LBR) declined incrementally
from the youngest age group (<35 years, LBR 54.8%) to the oldest (>42,
LBR 46.2%), further supporting the hypothesis that key changes in
endometrial function contribute to decreasing IVF success rates in
women aged 35 years or older. Although strengths of this study include
the large sample size and the use of narrow age categories, limitations
include confounders such as age-related changes in embryos
independent of ploidy and the lack of information about uterine
pathologies which may be more common with age and which may
interfere with embryo implantation.
A systematic review of 18 studies by Zhao et al. investigated the
impact of advanced maternal age (AMA), defined as 40 years or
older, on endometrial receptivity (Zhao J. et al., 2023). The main
objective was to assess the impact of advancing age on implantation,
clinical pregnancy, miscarriage and live birth rates in infertile
women undergoing ART after oocyte donation. The results
TABLE 2 Age-related changes in the endometrium: human studies.
Biological
process
Changes with age Study limitations References
Endometrial
receptivity
Differential gene expression Pregnancy outcomes not assessed Devesa-Peiro et al.
(2022)
DNA methylation Increased DNA methylation in the endometrium Clinical outcomes not assessed Olesen et al. (2018)
Endometrial
microbiota
Increased rates of endometrial dysbiosis with advancing age Pregnancy outcomes not assessed Fujii and Oguchi
(2023)
Oxidative stress Oxidative stress may increase with age, estrogen may have
protective effects
Few studies, not a well-established effect Kaltsas et al. (2023)
Pregnancy after IVF Lower live birth rates in older women undergoing transfer of
euploid embryos derived from autologous oocytes
Lower LBRs may be attributable to unidentified age-
related changes in the embryo other than ploidy
Vitagliano et al.
(2023)
Lower live birth rate and higher miscarriage rate in older women
undergoing transfer of embryos derived from donor oocytes
Variable definitions of advanced age of embryo
recipients
Zhao et al. (2023b)
Lower embryo implantation rates in women with evidence of
premature endometrial aging
Premature aging of the endometrium is not a well-
established entity
Shapiro et al. (2016)
IVF, in vitro fertilization; LBR, live birth rate.
Frontiers in Physiology frontiersin.org11
Chemerinski et al. 10.3389/fphys.2024.1332946

showed no significant difference in implantation or clinical
pregnancy rates in women with AMA compared to their younger
counterparts. A significantly higher miscarriage rate and a lower live
birth rate were found in older compared to younger women. Thus,
the findings of this study overall suggest a negative impact of age on
endometrial function; the results should be interpreted with caution
given the heterogeneity in the included studies, the retrospective
design of the studies, use of different definitions of AMA, and
different endometrium preparation protocols.
5.2 Impact of aging on endometrial
receptivity and function
Endometrial receptivity refers to the ability of the endometrium to
“accept”the embryo for implantation within a specifictimeframeofthe
secretory phase, and to provide an optimal environment for pregnancy
development (Bui et al., 2022). A recent systematic review and meta-
analysis of studies examining pregnancy outcomes from embryos
derived from non-autologous oocytes demonstrated a higher
miscarriage rate and lower live birth rate in women with AMA,
though the analysis included studies with variable definitions of
AMA and a wide range of cutoffs (Zhao J. et al., 2023). A negative
correlation has also been noted between advancing age and expression
of the implantation marker HOXA10 in endometrial explants, which is
implicated in endometrial-blastocyst communication (Fogle et al.,
2010). More recently, Devesa-Peiro et al. (Devesa-Peiro et al., 2022)
evaluated endometrial gene expression in women <35 compared to
those ≥35 years and identified 5,788 differentially expressed genes
between the two groups. Pathway analysis revealed alterations in
cilia motility and ciliogenesis pathways, pointing to these cellular
changesintheluminalepitheliumassomeofthecrucial
dysregulated functions associated with advancing maternal age.
Anotherfactorrequiredforsuccessful embryo implantation is
synchronicity of embryonic development relative to the timing of
the relatively narrow window of implantation during the secretory
phase. Shapiro et al. (Shapiro et al., 2016) investigated whether
indicators of endometrial-embryo asynchrony, such as prematurely
elevated serum progesterone levels and delayed embryo development
were increased in women of advanced reproductive age, defined in this
study as 35 years of age and older. Day 5 blastocyst transfer and low
serum progesterone levels (i.e., progesterone level lower than 1.5 ng/mL
during ovarian stimulation) were definedasmarkersforasynchronous
endometrium. Notably, patients with advanced reproductive age had a
significantly lower proportion of synchronous transfers than younger
women (31.9% vs. 50.0%). While these results may suggest a more
frequently displaced window of implantation in the endometrium of
older women undergoing IVF with fresh embryo transfer, this study
does not separate the contribution of the embryo (which is more likely
to be aneuploid in older subjects) from the contribution of the
endometrium even though synchrony is a composite of the two.
5.3 Recurrent implantation failure and
recurrent pregnancy loss
Clinical entities such as recurrent pregnancy loss (RPL) and
recurrent implantation failure (RIF) may be at least partially
attributed to an underlying endometrial factor. As such, elucidating
the mechanisms behind these pathologies and understanding the role of
aging—if any—on their natural history is a topic of interest requiring
further investigation. Evaluation of DNA methylation levels, a method
known as Horvath’s epigenetic clock, has previously been shown to
closely correlate with chronologic age in other human tissues (Horvath,
2013). Olesen et al. (Olesen et al., 2018) used CpG methylation sites to
estimate the biological age of the endometrium. Nine patients aged
18–38 years underwent endometrial biopsy in two consecutive
menstrual cycles in the mid-secretory phase (7 days after the LH
surge). The authors found a significant correlation between
chronologic age and biologic age of the endometrium. While this
study was limited by a small sample size and lacked pregnancy
outcomes data, it provided novel and compelling evidence that the
endometrium ages with time.
It has been suggested that a discrepancy between the biological and
chronological age of the endometrium (i.e., premature aging of the
endometrium) in young women may account for some cases of
endometrial factor infertility. A 2022 RNAseq analysis by Chen
et al. (Chen et al., 2022) explores this question. In this study, the
transcriptomes of 245 women <35 years with history of RIF were
evaluated. Twenty-nine women >40 years served as a reference
group. All subjects underwent endometrial biopsy in the mid-
secretory phase (7 days after the LH surge in a natural menstrual
cycle). In the younger cohort, two clusters of subjects were identified
based on differential gene expression: cluster 1 was defined by
upregulation of pathways involved in immune function while cluster
2wasdefined by upregulation of metabolic pathways. The
immunologically active cluster had higher expression of genes
associated with endometrial receptivity. In contrast, subjects in the
metabolically active cluster shared more genes with the reference group
(women >40) and had lower implantation rates than those in the
immunologically active cluster. Overall, this study suggests that
premature aging of the endometrium could contribute to the clinical
entity of recurrent implantation failure in younger women with RIF.
However, premature aging is a relatively under-studied area of
endometrial aging and additional studies are needed to elucidate the
causes and characteristics of the prematurely aging endometrium.
5.4 Endometrial microbiota, endometrial
receptivity
Though long held to be a sterile environment, mounting evidence
now supports the existence of a physiologic endometrial microbe
population which is impacted by hormonal changes and represents
another element of the embryo-endometrial interface (Toson et al.,
2022). Multiple studies support a connection between the female
reproductive tract microbiome and infertility (Rowe et al., 2020;
Cela et al., 2022;Elkafas et al., 2022), but the mechanisms by which
changes in the endometrial microbiome contribute to female infertility
remain unknown. Further, although the bacterial composition of the
endometrium is known to be influenced by age (Fujii and Oguchi,
2023), the role of probiotics and the endometrial microbiota have not
been well-studied in the context of age-related decline in fertility.
Lactobacillus species are the most common bacteria extending
throughout the female reproductive tract, with fewer bacteria in the
endometrium compared to the vagina (Chen et al., 2017). A recent
Frontiers in Physiology frontiersin.org12
Chemerinski et al. 10.3389/fphys.2024.1332946

study by Fujii et al. (Fujii and Oguchi, 2023) demonstrated changes
in the endometrial microbiota with advancing age. In this study,
authors analyzed 185 endometrial samples in the mid-secretory
phase from infertile patients ranging in age from 25 to 47 years who
had a history of at least one prior unsuccessful embryo transfer.
Transcriptomic analysis of the endometrium demonstrated that
increasing age was associated with a decrease in Lactobacillus-
dominant microbiota and the presence of dysbiotic (or disrupted)
endometrial microbiota. They also found that patient age and ultra-
low biomass were independently associated with risk of pre-
receptive endometrium, referring to an endometrium whose
transcriptomic profile indicates the window of implantation has
not yet been reached, and would thus not be conducive to the
implantation of a blastocyst. These findings suggested that the
quantity of Lactobacillus and its interaction with the host cells in
the endometrium play a role in the development of endometrial
receptivity. This study did not assess pregnancy outcomes, limiting
applicability of the findings.
Other studies have examined the impact of alterations in the
vaginal, cervical, and endometrial microbiota on ART outcomes
(Cela et al., 2022;Moreno et al., 2022;Fujii and Oguchi, 2023).
Women with a lower percentage of vaginal Lactobacillus have been
found to be less likely to have a successful embryo transfer
(Moreno et al., 2022), and a Lactobacillus-dominant
microbiota—defined as ≥80% of all identified species—is
associated with increased implantation, pregnancy, and live
birth rates (Moreno et al., 2022). In addition, persistent
inflammation of the endometrial mucosa has been shown to
impair receptivity through alterations in endometrial
decidualization (Di Pietro et al., 2013). In a recent multicenter
prospective study (Moreno et al., 2022) a dysregulation in the
balance between pathogenic bacteria and non-pathogenic
Lactobacillus was found to be associated with increased
implantation failure. In this study of 342 patients undergoing
IVF, the endometrial microbiota was profiled in the
endometrial fluid and through endometrial biopsies.
Lactobacillus was the major genus in both samples and its
presence was negatively correlated with the occurrence of
pathogenic bacteria. Results showed that patients with a
dysbiotic endometrium had increased risk of poor IVF
outcomes. Taken together, the findings that increasing age is
associated with higher risk of dysbiotic endometrium and that
dysbiosis negatively impacts ART outcomes, suggests that adverse
effects of an aged endometrium on reproduction may, at least in
part, be attributed to microbiome changes. This link, however,
remains unproven.
5.5 The possible link between oxidative
stress and endometrial aging
Oxidative stress (OS) arises from an overabundance of reactive
oxygen species (ROS) that overcomes a biological system’s ability to
counter them via the production of antioxidant defenses. ROS and
antioxidants have been identified in the female reproductive tract,
and their populations can be affected by age (Agarwal et al., 2005;
Agarwal et al., 2012;Kaltsas et al., 2023). On a molecular level, the
negative effects of OS may be the result of lipid damage, inhibition of
protein synthesis and depletion of ATP, with reproductive
consequences including the destruction of oocytes, altered
endocrine function, and endometrial damage, all of which have
adverse implications for pregnancy implantation and continuation
(Agarwal et al., 2003). If estrogen provides a protective effect against
reactive oxygen species (Kaltsas et al., 2023), estrogen may have
beneficial effects in terms of antagonizing the impact of oxidative
stress. However, this remains theoretical and mechanistic studies
are needed. Additional studies exploring the impact of ROS on the
aging endometrium are required in order to consider applying
estrogen therapy for the treatment of age-related increase in
oxidative stress.
5.6 Pregnancy complications associated
with advanced maternal age
Rodent studies have identified mechanisms by which aging
impairs endometrial decidualization.Earlystudiesintherat
compared the decidual response of young and aged
ovariectomized rats and found a diminished deciduogenic
response in aged rats (Ohta, 1987). Age-related pregnancy
complications may originate from endometrial dysfunction
leading to abnormal placentation. Indeed, there is an overall
increased incidence of spontaneous miscarriage, preeclampsia,
gestational diabetes, intrauterine growth restriction, preterm
delivery, and stillbirth among women of older reproductive
age (Wu et al., 2023). While the exact mechanisms have not
yet been elucidated, age-related aberrations in endometrial
stromal cell decidualization may play a fundamental role.
Decidualization is a requirement for healthy embryo
implantation and placentation (Wu et al., 2023). Dysregulated
endometrial decidualization is theorized to have the adverse
“ripple effect”of recurrent implantation failure and disorders
of pregnancy, including preeclampsia and recurrent miscarriages
(Agarwal et al., 2012;Garrido-Gomez et al., 2017;Wu et al.,
2023). Many of these complications may be considered problems
of endometrial aging as abnormal decidualization, placentation
and subsequent pregnancy complications exist on a continuum
(Conrad, 2020). Thus, further elucidating mechanisms
underlying the adverse effects of age on stromal cell
decidualization and endometrial remodeling may impact
prevention of age-related complications of pregnancy (Wu
et al., 2023).
6 Discussion
Female reproductive aging, and attempts to circumvent the
process, have been a major focus of reproductive research for
decades. While it has been well established that ovarian aging is the
most significant contributor to the age-related decline in female
fertility, the endometrium has been comparatively overlooked.
Although the endometrium is a self-regenerating tissue,
evidence points to senescence over time in the individual
cellular compartments. Additionally, as it is a hormone-
responsive tissue, it is susceptible to the changing hormonal
milieu that is part and parcel of reproductive aging. As
Frontiers in Physiology frontiersin.org13
Chemerinski et al. 10.3389/fphys.2024.1332946

highlighted in this review, the individual cell types in the
endometrium (stromal, epithelial, vascular, immune and stem)
are subject to the forces of aging and senescence. However, there is
a paucity of studies to determine mechanisms of endometrial aging
and its clinical sequelae; many of the studies are retrospective in
nature, report small sample sizes, have not been validated in other
studies, or have not been replicated in humans. Furthermore, the
clinical and translational studies examined in this review employ
variable definitions of advanced age—ranging from 35 to >40 years
of age—while other explore changes related to the menopausal
transition which typically occurs at age 50. In light of this, firm
criteria cannot be established to define advanced age with respect
to the endometrium. However, collectively, the studies reviewed
herein highlight age-related molecular and cellular changes that
support the concept that endometrial aging may play a significant
role in overall reproductive aging.
There exist several possible avenues for future investigation.
Beginning with the ovary, in addition to steroid hormones such as
estradiol and progesterone, the ovary is a rich source of peptide
hormones inhibin A, inhibin B, activin and others, which regulate
the pituitary-ovarian feedback loop (Muttukrishna et al., 2000). While
ovarian aging has been examined mostly in terms of the diminishing
pool of available oocytes, the reduced follicular mass also produces
fewer peptide hormones which may have direct implications for the
endometrium. Activin receptors have been described in the
endometrium (Mabuchi et al., 2010), and all of these ovarian
peptide hormones have been found to decrease with age
(Muttukrishna et al., 2000). Following ovulation, the corpus luteum
secretes estradiol, progesterone and inhibin A, and the age-related
decline in luteal function may also affect the quality of the secretory
endometrium (Broekmans et al., 2009). Although this does not
represent an active area of research, these studies highlight a gap in
the current literature and an opportunity to examine the effects of the
aging ovaries on the endometrium more closely. Finally, studies in other
tissues or organ systems may ultimately help to inform our
understanding of endometrial cellular compartments and their aging
process. For example, although age-related changes in endometrial
vasculature have not been well studied, evidence from other
regenerative tissues provides insight into the process of endothelial
cell aging that may inform our understanding of endometrial vascular
aging. In the gastrointestinal tract, for example, rodent studies
demonstrate an age-associated reduction in blood flow to the gastric
mucosa (Tarnawski et al., 2007) and endothelial cells obtained from the
gastrointestinal tract of aging rats demonstrate decreased angiogenesis
(Ahluwalia et al., 2014). Studies such as these may suggest productive
areas of investigation with respect to endometrial aging.
Aging of endometrial stem cells, and/or harnessing endometrial
stem cells as potential therapeutics for the aging endometrium, are
other potential areas of research. Although studies are currently
investigating the use of stem cells to promote endometrial
regeneration in women with intrauterine adhesions or tissue injury
(Gargett et al., 2012;Strug and Aghajanova, 2021), the lessons about
endometrial damage, repair and regeneration may have applications
in the study of endometrial aging. Various sources of stem cells exist
for this purpose. Menstrual blood stem/progenitor cells can be
collected in menstrual cups and cultured, representing an easily
accessible source of stem cells (Bozorgmehr et al., 2020). The
findings of age-related changes in endometrial stem cells, and the
possibility that these same cells can be harnessed to repair age-related
change, should lead to many fruitful opportunities for investigation.
In addition to exploring the diagnostic and therapeutic potential of
stem cells, elucidating the requirements for endometrial receptivity
may yield answers relevant to aging. In addition, the concept of
endometrial “sensitivity”, which was historically proposed to describe
the capacity of the endometrium to respond to steroid hormones,
prostaglandins, and vasoactive mediators (Psychoyos, 1984), may be
susceptible to age-related changes. The impact of endometrial aging
on sensitivity and receptivity, however, is as yet unclear.
Finally, several new and emerging interventions may have the
potential to contribute to the study and treatment of endometrial
aging. As prior studies have suggested a link between thin
endometrium and premature epithelial cell senescence (Lv et al.,
2022), treatment options that currently target thin endometria may
in the future be applied to mitigating the effects of endometrial
aging. Platelet-rich plasma has been investigated as a treatment for
thin endometrium and appears to be effective in subjects of older
reproductive age (Gangaraju et al., 2023) though studies such as this
one have very small sample sizes (fewer than 10 subjects) and firm
conclusions cannot be drawn.
The studies reviewed herein present the scope of the endometrial
aging question, from endometrial physiology and regeneration to an
exploration of the age-related changes occurring in the individual
cell types, concluding with the implications of endometrial aging
that can be inferred from clinical studies. While early studies in this
field examined the process of aging through an ovary-centric lens,
this review highlights the evidence that the biological and
chronological age of the endometrium may play a role in
pregnancy success. This review highlights important mechanisms
that may underlie the adverse impact of an aging endometrium on
fertility; however, we do not as yet have definitive molecular or
cellular markers to define aged endometrium. Future studies should
continue to explore these important facets of the reproductive aging
paradigm in order to better understand their origins and more
effectively develop therapeutic options.
Author contributions
AC: Conceptualization, Writing–original draft, Writing–review
and editing. JG: Writing–original draft. KB: Writing–original draft.
ND: Supervision, Writing–review and editing. SM:
Conceptualization, Supervision, Writing–review and editing.
Funding
The author(s) declare financial support was received for the
research, authorship, and/or publication of this article. NIH
R01AI148695 to ND.
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Frontiers in