Implanatation/endometrium during the menstrual cycle

Question 1

Define implantation

Answer 1

The embedding of the blastocyst in the endometrial stroma.
The process by which an embryo attaches to the uterine wall and penetrates first the epithelium and then the circulatory system of the mother to form the placenta.
Occurs 1-3 days after the morula enters the uterine cavity (between day 5-7 post fertilisation).
Secretory endometrium essential
Progesterone essential
Decidualisation essential

Question 2

Decidualisation

Answer 2

Decidualisation essential (endometrial fibroblasts transform into secretory decidual cells, filled with glycoprotein and lipids)
Decidualisation confers immune tolerance, regulates trophoblast invasion, nourishes and protects the developing fetus.

Question 3

Timing, location, chance of implantation per cycle and stages

Answer 3

5-10days after the LH surge (WOI)
Upper, posterior wall midsaggital plan,
40%
Apposition
Adhesion
Invasion

Question 4

Key parts of apposition

Answer 4

Embryo hatches from ZP.
EPF (early pregnancy factor) released - helps with immune tolerance and cell proliferation and growth.
hCG secreted from day 7 post fertilisation - help with endometrial receptivity and stimulates ongoing progesterone production from corpus luteum.
Pinopodes develop on surface epithelial cells in endometrium.
Microvilli of trophoblast attach to pinopodes
Key chemicals - cytokines - VEGF, IL-1, G-CSF, platelet activating factor

Question 5

key part of adhesion

Answer 5

Adherence process involves adhesion molecules on the endometrium and blastocyst - integrins, selectins and trophinins.
Bond is strengthened through release of chemokines and cytokines.
Key ones - CSF-1 (stimulates macrophages), LIF (leukaemia inhibiting factor) and IL-1.
Increased capillary permeability thorugh VEGF also essential
Several progesterone receptor regulated genes are essential = WNT4 HOXa10

Question 6

invasion

Answer 6

Trophoblast invade thourgh the uterine epithelium by intruding between epithelial cells.
Almost no necorsis
Most of the trophoblastic surface is non-adhesive which adds invasion.
Occurs thorugh proteinase degradation of the extracellular matrix
Prostaglandin E2 major regulator.
VEGF and angiopoietins essential for vascular remodelling.
Insulin-like growth factor II stimluates invasion.
Increased macrophages, uNK cells,

Question 7

key steps and key modulators of implantation

Answer 7

Apposition:
1. Pinopodes + microvilli
2. hCG and early pregnancy factor
Adhesion:
1. selectins + trophonin + integrins
2. Cytokine mediators: G-CSF, IL1, LIF, VEGF, platelet activating factor
Invasion:
1. Differentiate into syncytiotrophoblast and cytotrophoblast
2. No necrosis/apotosis, invade through degradation of ECM by proteinases and metalloproteinases
3. Important: uNK cells, prostaglandin, IGF-1, increased macrophages.

Question 8

Immune process of implantation

Answer 8

• IL 4, 5, 10 –> shift to Th2 immunity which promotes tolerance
• TGF-B, IL-10 –>increase in Treg –> suppress activation of immune system
• Uterine NK cells increase –> produce cytokines which stimulate growth of trophoblast (LIF, G-CSF)
• Trophoblasts do not express MHC 1a antigens (HLA-A, B) –>hence not recognised as foreign
• Trophoblasts express HLA-C antigens –>uNK cells express inhibitory receptors which bind to HLC-C molecules on trophoblast so semi-allogenic fetus not attacked

Question 9

Endometrial receptivity

Answer 9

Self-limited period during which the endometrium transiently transforms to receive a blastocyst and support implantation
Mediated by immune cells, cytokines, growth factors, chemokines, adhesion molecule etc.
WOI has been postulated to open 5 days after endogenous or exogenous progesterone action and close 2 days later
Requirements
Receptive endometrium
Functionally normal blastocyst
Cross-communication
Molecular diagnosis of uterine receptivity
Objective evaluation

Transcriptomics enables identification of a specific gene expression profile of a tissue under concrete conditions or treatments

WOI plays critical role in establishing bidirectional communication
Cell-to-cell communication mechanisms occur involving the delivery of endometrial miRNAs from the maternal endometrium to preimplantation embryo
Proteomics, secretomics and lipidomics of endometrial receptivity
Proteomic analyses reflect what is happening in the tissue at a cellular level
Global study of the proteomics of the endometrium will reveal the molecular changes that occur in the endometrium

Question 10

ERA

Answer 10

ERA = a customized microarray covering 238 genes that are differentially expressed between receptive and non-receptive endometria.
Once WOI located, a personalised embryo transfer can be performed within a manufactured FET.
Synchronization depends on timing of progesterone exposure

Systematic review and meta-analysis -
Arian 2022 Fert Ster - NO evidence of improvement in LBR with use of ERA including in a subgroup of those with 2 or more previous failed transfers.

Simon 2020. RBMO. - ITT showed no difference in LBR between PET, FET and fresh ET. PP showed ss improvement in PET arm but this has received wide criticism.

Question 11

Placental functions

Answer 11

Question 12

parts of the blastocyst

Answer 12

Question 13

placental hormones

Answer 13

Promtoes maternal
plasma volume expansion
inflammation
thrombosis
Insulin resistence
weight gain
Immune tolerance
altered liver/kidney/lung CVS

Fetus
Fetal growth

Question 14

Immunology of placental implantation

Answer 14

Most hypomethylated tissue in the body
Trophectoderm very biosynthetically active (very high mitochondrial number).
From 6-10weeks hypoxic environment and then turns into a normal O2 environment from 13-23 weeks.

Question 15

Morphology of endometrial layers

Answer 15

1. Upper two thirds – functionalis layer (prepared for blastocyst implantation, and is the site of proliferation, secretion, and degeneration – i.e. changes during menstrual cycle, responsive to hormones)
   a. Simple columnar epithelium
   b. Stratum compactum
   c. Stratum spongiosum
2. Lower one third – basalis layer (provides regenerative endometrium following menstrual loss of the functionalis, no change during menstrual cycle, unresponsive to hormones).

Question 16

Five endometrial phases of a normal menstrual cycle

Answer 16

1. Menstrual endometrium
2. Proliferative phase
3. Secretory phase
4. Preparation of implantation
5. Endometrial breakdown

Question 17

Menstrual endometrium

Answer 17

Thin, dense tissue composed of Basalis layer and small amount of residual stratum spongiosum.
Stratum spongiosum – disarray, breakage of glands, fragmentation of vessels and stroma, necrosis, white cell infiltration.
It is a transitional state so at the same time as the above occurs so too does the beginnings of endometrial regeneration originating in epithelial stem cells within the glands of the basalis layer.
DNA synthesis occurs within the basalis layer by day 2-3 of the menstrual cycle.
New surface epithelium emanates from the flanks of stumps of glands in the basalis layer.
Rapid reepithelialisation occurs supported by fibroblasts and stem cells.
By the end of menstruation, the entire uterine cavity is re-epithelised and stromal growth begins.

Question 18

Proliferative phase

Answer 18

Driven by oestrogen secretion associated with ovarian follicle growth.
All the components – glands, stroma and epithelial cells, endothelial cells (what line blood vessels) show proliferation, marked by increased mitosis and increased nuclear DNA and cytoplasmic RNA synthesis. Peaks day 8-10 (reflects rising E2 and maximum E2 receptors within endometrium).
Endometrium grows from 0.5mm (menstrual endometrium) to 3.5mm-6mm in height of each singular layer. On USS this is seen as the trilaminar appearance where the endometrium originating from both sides of the uterus is 3.5mm-6mm thick and the junction of the two.

Question 19

Secretory phase -

Answer 19

Separated into early and late
Occurs after ovulation - mediated by oestrogen and progesterone
Glycogen accumulation, glands become tortuous and sacculated. Large stromal cells, stromal oedema.
Progesterone receptor (A and B) expression in endometrial cells - occurs through exposure to oestrogen and ERalpha.
PR-A - stromal, critical in decidualisation.
PR-B - glandular, glandular secretions
Glandular secretions develop in late secretory phase, allows sectreion of glycoproteins and peptides into the endometrial cavity.
Peak secretory level Day 7 after LH surger - coinciding with blastocyst implantation.

Question 20

Implantation phase

Answer 20

Stromal oedema
Secondary to P4 and E2 mediated increase in prostaglandins and VEGF production that causes an increase in vascular permeability.
Leukocyte infiltration - predominantly uNK cells and macrophages.
uNK have an immunoprotective role in implantation and placentation.
Stromal cells transform into decidual cells under the influence of P4 and mediated by autocrine and paracrine factors.
The decidua becomes an important structural and biochemical tissues of pregnancy, the decidual cells control the invasive nature of the trophoblast and the products of the decidua (prolactin, relaxin, renin, insulin-like growth factors (IGFs), and insulin-like growth factor– binding proteins (IGFBPs)) play important roles in fetal and maternal tissues.
These cells are key to both menstruation and endometrial haemostasis (implantation and placentation). The withdrawal of oestrogen and progesterone (which occurs when implantation is not achieved) lead to endometrial breakdown and menstruation.

Question 21

Endometrial breakdown

Answer 21

Stratum compactum formed in upper part of functionalis layer by day 25. In the absence of fertilisation and implantation and the lack of hcG from the trophoblast, the fixed life span of the corpus luteum is complete and E2 and P4 levels wane.
This withdrawal of sex steroids causes – vasomotor reactions, apoptosis, tissue loss, and finally menstruation.
Enzymatic autodigestion of the functional layer of the endometrium and capillary plexus leads to menstruation.
Lysosomes, MMPs, TNG-Alpha all important.