Embryology III
PERIMPLANTATION
DEVELOPMENT
autumn 2024
Zuzana Holubcová
Department of Histology and Embryology
zholub@med.muni.cz
Preparing uterine
tissue for
implantation
Uterine tissue remodeling
Mutter et al 2023
~28 days
~ 400 cycles per reproductive life
- day 1 = 1st day of menstruation bleeding
❖Menstrual cycle
= cyclical endometrial tissue turnover and rejuvenation
Scar-free
repair
RegenerationShedding
Endometrial remodelling
❖Menstrual cycle
- coordinated with the ovarian cycle
and estrogen (E2) and progesteron (P4)
secretion
Cyclic endometrial changes
Salamonsen et al 2021
Functionalis
- hormone sentitive
- comprises luminal
epithelium and
vertical glands
- sheds during
menstruation
Basalis
- unresponsive to
hormones
- comprises
horzontal glands
network, stromal
cells, vasculature
and stem cells
- intact during cycle
vertical
Cyclic endometrial changes
- tissue shedding, bleeding, and rapid scar-free repair (~48 hours )
of zona functionalis of endometrium
- highly regulated inflammatory response to P4 withdrawal
- absence of anti-luteolytic signal hCG
Menstrual phase
↓P4
Inflammation
Vasoconstriction
ECM degradation
Hypoxia
activation of MMPs
e.g. cathepsin, elastase
Menstrual phase
→ secretion of
▪ cytokines
▪ chemokines
▪ prostaglandin synthesizing enzymes
❖INFLAMMATORY RESPONSE
massive influx of
immune cells
ECM degradation
and tissue breakdown
Evans and Salamonsen et al 2012
local
vasoconstriction
hypoxia
Functionalis
Basalis
Myometrium
- caused by local vasonstriction
Maybin et al 2021
Critchley et al 2006
- women with heavy
menstrual bleeding have
decreased HIF-1 during
menstruation
- genetic and
pharmacological
reduction of
endometrial HIF-1 in
mice causes prolonged
menstrual bleeding
- hypoxia-induced stabilization of HIF-1a
physiologically drives endometrial repair
after shedding
Hilary Critchley
Menstrual phase
❖HYPOXIA
Salamonsen 2021 (modified)
Menstrual phase
❖MENSTRUAL BLEEDING
Stem cells/progenitors
- menstrual fluid environment + preservation of basalis → no scarring
- the restoration of an intact epithelial layer
- re-epithelialization of denuded areas occurs simultaneously with tissue breakdown and
is completed within ~48 hours of initiation of shedding
- no scarring due to preservation of basalis
Martinez Aguilar et al 2020
Menstrual phase
❖ENDOMETRIAL REPAIR
1) resurfacing of
luminal epithelium
2) angiogenesis in
sub-epithelial
stroma
3) repair of damaged
transverse arteries
Menstrual phase
Luminal epithelium resurfacing
← migration of epithelial cell
progenitors from exposed horizontal
endometrial glands in basalis
←stomal cell transformation to luminal
epithelium = MESENCHYMAL-TO-EPITHELIAL
TRANSITION (MET)
❖ENDOMETRIAL REPAIR
Menstruation Proliferative phase
Cousins et al 2022
- post-menopausal period (starts ~ day 4, last ~ 10 days)
- increase of endometrial linning thickness (from ~ 0.5 mm to ~ 7-8 mm)
- rapid regrow and regeneration of functional layer due to massive cellular proliferation
- activation of growth factor signaling pathways, high vascular perfusion, and transient tissue edema
- positional proliferation, cell specification, and angiogenesis
- can occur only once the epithelial surface is covered
Proliferative phase
Salamonsen et al 2021
- E2-dependent
- E2-receptor present in epithelial
and stromal part
+ role of androgens
(initiate gland reformation)
- E2 induces cell proliferation
and expression of P4 receptor
→ endometrium
thickening and priming
for the structural changes
that will undergo during
the secretory phase
Proliferative phase
Proliferative phase
- rare clonogenic population with high proliferative potential
- capable of self-renewal and differentiation to one or more lineages of specialized tissue cells
- reside predominantly in basalis endometrial layer (epithelial + stromal compartment)
▪ Epithelial progenitors → glandular epithelium
▪ Endometrial mesenchymal stem cells (eMSCs) → stromal and endothelial cells
▪ Side population (SP) cells → epithelial, stromal, and endothelial cells
❖UTERINE STEM CELLS
- essential for endometrial, stromal
and vascular regeneration
following menstruation and
parturition
- distinct populations giving rise to
different endometrial cell types,
multizonal differentiation hierarchy
- repopulate functionalis and
generate proangiogenic and
paracrine factors promoting
angiogenesis and
immunosuppression
- dysregulated function leads to
cancer
Proliferative phase
❖UTERINE STEM CELLS
Chen et al. 2022
Proliferative phase
❖LYMPHOID AGGREGATES
- reside in the basal endometrial layer
- clumps of several hundreds of immune cells
- core of B-cells surrounded by a circle of T-cells
and a halo of macrophages
- established in each cycle by the recruitment
of circulating immune cells
- regulate spatial responsiveness of
endometrial tissue to ovarian hormones
Shen et al 2021
Proliferative phase
Muter et al 2023
- cellular specification and tissue patterning in
endometrial tissue
• INF secreted by activated T-cells
residing in lymphoid aggregates is a
potent inhibitor of estrogen and P4
signaling and cellular proliferation
• WNT signaling (WNT7A) expressed
predominantly in luminal epithelium
promotes ciliogenesis in response to
estrogen
Endometrialgrowth
fast
slow
Cytokine and morphogen gradient:
→ cyclic tissue remodeling restricted to
superficial layer
→ spatial patterning for P4 action
in the secretory phase
- elevated replication stress and senescence
markers in epithelial and stromal cells are
associated with pathologically thin endometrium,
E2 resistance and implantation failure
Features:
- ↓ endometrial cell proliferation
- ↑ adhesive capacity of epithelial cells
- ↑ glandular secretion
- differentiation of stromal cells
- development of spiral arteries
- stromal edema
- stem cell recruitment
- influx of immune cells
Salamonsen et al 2021
Secretory phase
- post-ovulation stage (~ day 14-day 28)
- Ovulation → rapid drop of estrogen production (↓E2)
→ secretion of progesteron by corpus luteum (↑P4), peaks in mid secretory phase (+7-8 days)
IMPLANTATION
WINDOW
- profound mophological and functional tranformation of endometrial stromal cells
- P4-dependent process acting on E2-primed cells
- spontaneous in humans
- regulates tropholast invasion during implantation → essential for establishing pregnancy
- decidua (lat. „deciduus“) = maternal uterine tissue, shed off during parturition and in non-conceptous cycle
Secretory phase
❖DECIDUALIZATION (= DECIDUAL REACTION)
Deryabin et al 2020
decidual
markers
Secretory phase
❖DECIDUALIZATION
Jan Brosens
Essential role of cAMP signalling
G-protein-coupled receptors
- cAMP analogs, activators of adenylate cyclase
(AC) and PDE inhibitors are potent inductors of
decidualization in vitro
- pharmaceutical modulation of cAMP signallng
pathway can affect implantation efficiency
P4 activation of its nuclear receptor (PGR)
is critical for maintaining decidualization
process but insufficient for initiation of
differentiation process
Trigger mechanism:
Yoshie et al 2015
- morphological change:
Secretory phase
❖DECIDUALIZATION
Gellersen and Brosens 2003
- accumulation of glycogen, lipids and
glycoproteins
- the presence of giant mitochondria, prominent
rER and GA, and dilated sER cisternae
- tendency to polyploidisation
- connection by gap junctions
- ↓ tissue roughness and stiffness
Cornillie, et al 1985
fibroblastic-phenotype
Spindle-shaped cells
large rounded cells with large nucleii
and abundant cytoplasm
*
* deciadualization starts around spiral arteries
- decidualized cells express a variety of cytokines, chemokines,
growth factors, and angiogenic factors
- altered expression of steroid hormone receptors
- metabolic changes
(e.g. P4-induced downregulation of DIO2 critical for ↓T4-to-T3 conversion,
silencing of stress- activated signaling and increasing ROS scavenging activity)
- upregulation of ion channels and protein transporters
(→ increased absorption of uterine fluid facilitating embryo-endometrial
interaction during implantation, increased vascular permeability )
- secretion and remodeling of extracellular matrix
(→ deposition of hyaluronan → water perfusion → stoma edema and
reduced stiffness)
- release of proinflamatory regulators
(particularly in decidual-like senescent cells damaged by replication stress in
the proliferation phase)
- accumulation of uterine NK cells (uNK)
Secretory phase
❖DECIDUALIZATION
- functional change:
IL-6, LIF
Secretory phase
❖DECIDUALIZATION
Glycogen
• Uterine Natural Killer cells (uNK)
- subset of immune cells abundant in
secretory endometrium and decidua
of pregnancy
- tissue-specific characteristics
- derived from circulating NK cells
- differentiation in response to local
clues
- affect uterine spiral remodeling
and immunological tolerance
- alteration in uNK number/function
causes infertility, miscarriage,
or pregnancy complications
Muter et al 2023
Secretory phase
❖DECIDUALIZATION
• Uterine Natural Killer cells (uNK)
- switch from pro-inflammatory phenotype to immunomodulatory, cytokine-producing,
and angiogenic phenotype
KIRs+ (killer cells
immunoglunoglobulin-like
receptors)
CD56bright CD16−
CD39+ CD9+ CD49a+ CD57−
Ki67 nuclear proliferation marker
- maintain endometrial homeostasis by selectively eliminating senescent decidual cells
- the activity of uNKs is affected the quality of implanting embryo
Farag and Caligiuri 2006
Menstruation versus pregnancy
↓P4 ↑P4
Endometrium → decidua bed of pregnancyEndometrium breaks down → menstruation
Senescent decidual cells
- damaged by replication stress
during the proliferation phase
- fail to differentiate into
decidual cells during the
secretory phase
- insensitive to progesterone
- produce complex secretome
- P4 sensitive decidual cells engage uNK to
eliminate senescent cells
Muter et al 2021
- paracrine induction of senescence in neighbouring
cells → sterile inflammation and ECM breakdown
Menstruation versus pregnancy
Menstruation versus pregnancy
Endometrial receptivity
- uterine lining preparation for an embryo
implantation
- WINDOW OF IMPLANTATIN (WOI)
= limited time interval (3-6 days, ~day 20-24 of the cycle )
during the mid-secretory phase, when the
endometrium is ready to receive an embryo
- optimal timing for IVF embryo transfer
Endometrial receptivity
✓COMPACTION OF ENDOMETRIUM
- post-ovulatory decrease in endometrium thickness
(during luteal phase increases density of endometrium but not its
volume)
- detectable by ultrasound
- indicative of P4 responsiveness and degree of
decidualization
✓PINOPODES
- surface protrusions facilitating embryo implantation
- appear on apical side of luminal epithelial cells in the
mid secretory phase
(~day 20-21, but ~5 day inter-individual timing variation!)
- their development is associated with level of P4,
expression of implantation promoting factors
(L-selectin ligand, LIF, V3 integrin)
Luddi et al 2020
Muter et al 2023
- gene expression profiling assays
- NGS of 248 genes related to endometrial
receptivity
identification of
patient´s WOI for
personalized
embryo transfer
- inter-patients differences in WOI transcriptomic
signature
- inconsistency between individual patient´s cycles
(E2 and inflammation)
- invasiveness of biopsies (mini inavasive procedure)
- NGS results available with a delay
- cost burden
✓ ENDOMETRIAL RECEPTIVITY ARRAY (ASSAY/ANALYSIS) – „ERA“
Endometrial receptivity
Uterine microbiome
- uterine cavity is not sterile
- inter-/intra-individual species diversity
- dysbiosis associated with adverse
reproductive outcomes
- ideal composition?
- benefits of species diversity?
Molina et al . 2020
Inverseti et al . 2023
Uterine microbiome
Zhu et al . 2022
Uterine microbiome
❖ Uterine microbiome diagnostic tests
Cyclic changes of junctional zone
- specialized layer of circular smooth muscle that
surrounds the endometrium (inner myometrium)
- visible of high-resolution ultrasound and magnetic
resonance imaging
- undegoes homone-dependent contraction and
remodelling during the menstrual cycle
➢ trans-differentiation stromal fibroblast to myocytes
➢ cervico-fundal contractions facilitate → sperm transport during
the fertile window; fundo-cervical contractions → flow of effluent
during menstruation stratum basalisjunctional zoneouter myometrium
Triple line measurement
Role of uterine cyclic remodeling
Brosenset al 2009
- brief exposures of any
organ to low levels of
stress confers resistance
to stress levels that
otherwise cause tissue
damage
- repeated menstruation
cycles might
precondition the
uterus for future
pregnancy
❖ Inflammatory ‛memory’
Menstrual disturbances
➢Menopausal amenorrhea
➢Thin endometrium
➢Abnormal uterine bleeding
➢Intrauterine adhesions
➢Asherman syndrom
➢Endometriosis
- thin (<4 mm) endometrium consisting of
stratum basalis only
- glands are sparse and have low secretory
activity, could be dilated and produce cysts
- stroma is less cellular, and contains more
collagen fibers
- no apparent mitotic activity (senescence)
- physiological postmenopausal amenorrhea
- stem/progenitor cells are in a dormant state
- quiescent stem/progenitor cell can be
reactivated by exogenous E2 during hormonal
replacement therapy, but their clonic
efficiency is lower than in premenopausal
endometrium
postmenopausal endometrium
normal endometrium
(late proliferative phase)
Menopausal amenorrhea
= fall in estrogen production due to ovarian reserve
exhausition → atrophic endometrium
Thin endometrium
Triple line ultrasound measurement
Thin endometrium
Possible Causes
- Low estrogen levels
- Luteal defects
- Advanced age
- Fibroids
- Genetic factors
- Intrauterine Adhesions
- Poor blood flow
- Pelvic surgeries/inflammation
- Iatrogenic effect
Symptoms
- Irregular menstrual cycle
- Painful or inadequate menses
- Fertility issues
Treatment
- Hormonal therapy
- Uterine surgeries and interventions
(e.g. endometrial scratching)
- Growth factor (PRP) therapy
- Sildenafil (off-label)
(fibroids)
- altered tissue oxygenation and HIF-regulation are suspected to underlie AUB conditions
Martinez Aguilar et al 2020
Abnormal uterine bleeding (AUB)
- damage of basalis layer and loss of stem/progenitor cells
Intrauterine adhesions (IUA)
→ failure of adequate repair and regeneration
Cen et al 2022
Intrauterine adhesions (IUA)
MSC-secreted exosomes
- anti-inflammatory
- anti-apoptic
- proangiogenic
- immuno-modulatory
→↑ epithelisation and wound healing
→↑collagen maturity
→↓ scarring
- wound healing therapeutical potential
❖ STEM CELLS/PROGENITORS CELLS THERAPY
- non-invasive harvest from
the menstrual fluid
- short-lived
- risk of off-target migration and tumor
growth
Cen et al 2022
Asherman syndrom
- etiology unknown, risk factors
include uterine surgery,
pregnancies, trauma, pelvic
infections, genital tuberculosis,
and obesity
- excessive intrauterine adhesions,
scarring and synechiae
- causes dysmenorhea, irregular cycles,
miscarriages, and placental anomalies
- regenerative potential of
stem celll demonstrated
in clinical trials
Carlos Simon
Endometriosis
Endometriosis lesions origin
✓ retrograde menstruation
(shedding fragments of endometrium
containing uterine stem cells into the
Fallopian tube and pelvic cavity)
✓ ectopic adhesion and survival
of uterine stem cells
(enhanced by genetic background,
eMES/ePSC population composition and
proliferation profile, and/or local
environment)
✓ persistence and invasion of
small superficial lessions
(dependent on proliferation, penetration,
migration, proinflamatory and angiogenic
capacity of deposited endometriotic cells)
Masuda et al 2021
Dysmenorrhea and AUB
Dyspareunia
Painful defection and urination
Pelvic and back pain
Infertility
= presence of cycling endometrial
tissue outside of the uterine cavity
Chronic
inflammation
Caroline Gargett
- occurs in ~5% of newborn girls
(typically post-term)
- results from P4 withdrawal from
neonatal circulation upon birth
- cervix blocked → premenarchial
retrograde uterine bleeding
- visible bleeding from the vagina
indicates intense tissue shedding
with a higher risk of retrograde
menstruation
- possible predisposition for early
onset of endometriosis in
adulthood
Endometriosis
Gargett al 2014
❖ Neonatal „menstrual-like“ bleeding
➢ ENDOMETRIAL ORGANOIDS
- hollow spherical structures consisting of multiple cell types, including epithelial,
stromal, glandular, and vascular cells
- spontaneous self-organization in a defined serum-free medium („assembloids“ )
- responsiveness to E2 and P4
- lack of complex organization of endometrial tissue
Research of endometrial physiology
❖ 2D in vitro models
← primary endometrial cells from biopsies
← endometrial cells isolated from menstrual fluid
➢ endometrial cancer cell lines
- Ishikawa, ECC-1, KLE, RL95-2 And Hec50co
- genetically abnormal, single-cell type
➢ biopsy material
- heterogenous character
- different cycle stage
- individual genetic background
Disease modeling
Drug screening
Embryo-endometrial cross talk research
❖ 3D in vitro models
Research of endometrial physiology
Cousin et al 2022
Opportunity to model complexity of
uterine tissue regeneration
Matrix-based systems
for co-culture of
multile cell types