lecture 14: endometrial receptivity and pregnancy

Question 1

What is shed at menses?

Answer 1

• the female endometrium

Question 2

How do the sperm fertilise the egg?

Answer 2

• many sperm make light work
• digestion of complete holes through the zona pellucida by the acrosome reaction requires release of many acrosomes – initial sperm do the hard work for the others
• once one sperm has entered, hardening of the zona pellucida via intiation of the cortical response blocks polyspermy

Question 3

What are stages of embryo development and hatching?

Answer 3

• ther fertilised oocyte is called a zygote
• the zygote undergoes continuous division (2-cell, 4-cell, 80cell) but does not grow
• once the cell number is greater than 16 the structure is called a morula
• by day 5 the structure is known as a blastocyst – it contains a fluid filled cavity, and different cell types:
  
  • trophoblast – forms the placenta
  • inner cell mass (embryoblast) - forms the baby
• the zona pellucida starts to break down
• caused by lytic factors from the uterine cavity and the blastocyst itself
• the embryo hatches from the zona pellucida and is now ready to implant into the uterus

Question 4

What is implantation into the uterus?

Answer 4

• takes place ~6-7 days after fertilisation
• requires an appropriately receptive endometrium
• endometrium secretes proteins, lipids and other metabolites which can act as ‘nutrients’ for the blastocyst during implantation and later placentation
• endometrium produces ‘pro-implantation’ factors to enter into a dialogue and let the blastocyst know it is ready
• this process is absolutely critical for full pregnancy - many women lose the embryo at this point and never realise they are pregnant
• luminal epithelium lining the endometrium

Question 5

What are the stages of blastocyst implantation?

Answer 5

• transport, orientation, hatching
• apposition
  
  • not at full contact with the maternal endometrium at this point
  • just floating above and thinking about implanting
  • can enter into intense dialogue at this point
  • maternal endometirum can alter expression of adhesion proteins at this point
  • changed adhesion
• attachment
• invasion
  
  • day after attachment
• there is also decidualisation of the endometrial lining
• presence of leukocytes, macrophages, natural killer cells
• trophoblast cells invading maternal endometrium and

Question 6

What is needed between embryo and endometrium?

Answer 6

• synchrony of development
• when we have low embryonic quality (developed too far or not enough) then implantation is impossible
• endometrium also has to be adequately prepared
• their levels of preparedness need to be matched - both ready at the same time
• implantation is possible when egg is somewhat developed and endometrium kind of prepared
• but might not be proper or whatever

Question 7

What is endometrial receptivity?

Answer 7

• gradually achieved over ~20 days of a 28 day menstrual cycle under the influence of oestrogen (follicular/proliferative phase) and progesterone (after ovulation, luteal/secretory phase)
• endometrium is receptive for around 4 days – known as the ‘window of implantation’
• spans days 20-24/LH+6-10
• associated with secretory transformation of endometrial cells and progressive decidual transformation of stromal cells

Question 8

What are endometrial epithelial changes for receptivity and implantation?

Answer 8

• changes in endometrial epithelial cells
• under influence of oestrogen and progesterone glandular epithelial cells become secretory
• luminal epithelial cells alter adhesion molecules and integrity/tightness of junctions between cells
• epithelial surface becomes less polarised
• secreted factors enter into a communication with the blastocyst
• top picture: proliferative phase (endometrium not ready to receive the embryo)
  
  • glandular epithelial cells are very small and compact, not secretory
  • very small blood vessels
  • stroma: tissue glue that holds it together is very tightly packed
• secretory phase
  
  • endometrium ready to accept blastocyst
  • presence of secretions within the uterine gland epithelial cells
  • stromal cells become more oedematous
  • starting to undergo decidual transformation under the influence of oestrogen and progesterone
  • presence of highly enlarged blood vessels
  • important to increase the vascularity of the tissue at this point and also allow influx of leukocytes

Question 9

How can we be sure endometrial factors are required for implantation?

Answer 9

• uterine gland knockout sheep
  
  • neonatal sheep administered progesterone
  • no uterine glands
  • retarted conceptus development
  • no implantation
  • no loss of adhesion molecules
  • due to lack of secretory products
• knockout mice
  
  • leukaemia inhibitory factor knockout
  • implantation failure due to uterine defect
  • blastocysts recovered from knockout implanted into wild type mother
  • therefore LIF, an endometrial factor, was absolutely essential for implantation
• examination of factors with human uterine cavity
  
  • presence of specific proteins can predict implantation of an embryo in an IVF cycle
  • absence of certain factors in infertile women
  • endometrial lavage

Question 10

What happens to endometrial secreted factors?

Answer 10

• taken up by placental cells and support development
• endometrium is still 5-6 mm thick at 6 weeks of gestation, with highly active glands that discharge into the placenta
• endometrial secretions produced by the glands are taken up by the trophoblast cells of the placenta
• NB: no significant placental blood flow until around week 12 of gestation – nutrition must come from another source

Question 11

What are the endometrial stromal changes for receptibity and implantation?

Answer 11

• progesterone dependent but also driven by certain cytokines (IL-11, activin)
• changed phenotype – take on many features of epithelial cells
• secrete many new proteins
  
  • including chemokines, growth factors etc
• produce high levels of inhibitors of proteases (e.g. TIMPs, cystatin)
• decidualisation occurs into the mid-late secretory phase of a normal menstrual cycle
• in pregnancy these cells turn into the decidua of pregnancy

Question 12

How do we know a decidualising endometrium is important for implantation?

Answer 12

• decidualising stromal cells act as sensors of embryo ‘health’
• culture of decidualising stromal cells with ‘bad’ embryos inhibited production of growth factors and cytokines
• decidualising stromal cells from fertile women will not migrate towards a bad embryo
• decidualising stromal cells from women with recurrent pregnancy loss WILL migrate
  
  • cannot discriminate between a good and bad embryo
  • reason for recurrent pregnancy loss – allow bad embryos to implant
  • embryos cannot survive long because they are genetically abnormal, just long enough for a positive pregnancy test (hCG)

Question 13

What are endometrial leukocyte (white blood cell) changes receptivity and implantation?

Answer 13

• leukocyte composition changes dramatically from non-receptive (proliferative) to receptive (mid-secretory) phase
• largest changes seen in uterine natural killer and macrophage numbers
• numbers change even more dramatically in first trimester of pregnancy
  
  • uNK cells = 70% of total leukocytes
  • macrophages = 20% of total leukocytes

Question 14

What are the natural killer cells in the endometrium?

Answer 14

• phenotypically and functionally very different to natural killer cells in the circulation (peripheral NK cells)
• the presence of cell surface marker CD56 (CD56^hi) and the absence of CD16 (CD19^-) uterine NK cells (uNK)
• they represent 70% of leukocytes at the implantation site
• they may be regulated by signals from the maternal decidua
• during pregnancy they can respond to foetal signals
• they produce a range of soluble products, including angiogenic cytokines (such as angiopoeitin-2 and vascular endothelial growth factor C)
• they are found in close proximity to uterine spiral arteries (blood vessels) and facilitate remodelling during pregnancy
• may help in tolerance of foetal allograft
• mice deficient in uterine natural killer cells have placental defeects

Question 15

What do signals produced by uterine NK cells influence?

Answer 15

• trophoblast function
• uterine/decidual natural killer cells isolated from first trimester of pregnancy endometrium/decidua
• measured factors produced by the natural killer cells
• invasive placental cells (trophoblast) isolated from first trimester of pregnancy samples
• stimulated with natural kill cell factors
• migration (mimicking placental invasion of the endometrium) examined
• IL-8, IP-10, VEGF, PLGF (last two important for remodelling blood vessels)

Question 16

What mediates tolerance of the foetus?

Answer 16

• interaction between ligands on trophoblasts and receptors on NK cells
• HLA-C2 present on trophoblast
• receptor, KIRDS1 present on NK cell
• foetal HLA-maternal KIR in this case appears to allow allo-recognition of the ‘foreign’ object
• allows tolerance of the foetus
• close association between uNK cells and trophoblast invading into endometrium/decidua

Question 17

What does successful implantation require?

Answer 17

• hatched blastocyst
• receptive endometrium
• maternal progesterone
• synchrony of development of endometrium and blastocyst
• signals
  
  • between blastocyst and endometrium
  • between different endometrial cells

Question 18

How can the blastocyst signal to facilitate its own implantation?

Answer 18

• hCG is produced by the blastocyst from the 8 cell stage
• traditionally thought to be a luteotrophic factor only (maintenance of CL – more later)
• hCG stimulation of endometrium/endometrial cells elevates pro-implantation factors
• in baboons treated with hCG we see elevated levels of LIF in their uterus, also elevation of other factors involved in implantation such as VEGF, PROK1
• all of these factors are involved in the maternal side of implantation and help this process be achieved

Question 19

What is blastocyst apposition?

Answer 19

• blastocyst positioned close to, but not touching, the maternal endometrium
• likely ‘homing signals’ secreted by the endometrium play a role in this process
• in rodents, pinopodes (greek for drinking foot) seem to ‘pull’ the blastocyst into close contact with the maternal endometrium
• mechanism in humans less clear
• this is the stage at which changes in epithelial cell polarity have to occur otherwise the 2 polarised epithelial (trophectoderm and maternal luminal epithelium) will repel each other
• just before adhesion the trophoblast differentiates into two different cell masses: the outer syncytiotrophoblast (ST) and the inner cytotrophoblast (CT)

Question 20

What is blastocyst adhesion?

Answer 20

• physical adhesion between foetal trophoblast/trophectoderm layer and maternal luminal epithelium
• mediated via interactions between adhesion molecules
• best characterised are interactions between extracellular matrix factors and integrin adhesion molecules
  
  • fibronectin on maternal side
  • integrins

Question 21

What is blastocyst invasion and initiation of placentation?

Answer 21

• the trophoblast cells of the blastocyst penetrate through the maternal luminal epithelium
• the cytotrophoblast consists of an inner irregular layer of ovoid, single-nucleus cells. this is also wehre intensive proliferation takes place
• the syncytiotrophoblast produces enzymes and secretes factors that cause apoptosis of the endometrial epithelial cells
• the syncytiotrophoblast crosses the basal lamina and penetrates into the stroma that lies below
• with the implantation of the blastocyst in the endometrium the syncytiotrophoblast develops quickly and will entirely surround the embryo as soon as it has completely embedded itself in the endometrium

Question 22

What is the placentation process?

Answer 22

• placentation is the progressive formation of a fully functioning placenta – during the first trimester the major functions are:
  
  • production of hCG
  • invasion of maternal blood supply
• the placenta consists of a foetal component (the chorion) and a maternal component (the decidua)
• in the first trimester when placenta is still forming the foetal chorion and maternal decidua can still be easily separated
• in the first trimester (up until week 12) the trophoblast cells are still remodelling the maternal spiral arteries – no significant blood flow to the placenta
• trophoblast cells also plug the spiral arteries to prevent stress caused by pulsatile, high blood flow during the delicate time of early development
• progressively during the first trimester the spiral arteries are transformed into high capacitance (allow lots of blood flow) low resistance vessels

Question 23

What is early placentation in the first trimester?

Answer 23

• humans have haemochorial placentation
• most invasive form of placentation – trophoblasts invade into the muscular myometrium layer
• trophoblasts (endovascular) invade and remodel the maternal vessels to come into contact with maternal blood
• endovascular trophoblasts disrupt endothelial cells lining the blood vessels and can even disrupt the muscular lining
• allows non-pulsatile smooth blood flow – no damage to developing foetus

Question 24

What is the placenta and placental function?

Answer 24

• foetal component of the placenta: chorionic villi develop from trophoblast – contains foetal vasculature
• maternal component: comprises decidua: invasion and remodelling of maternal vasculature by foetal trophoblasts allows maternal blood to pool in intervillous space
• placental functions (once fully formed)
  
  • provide substrates for foetal metabolism and excretes waste
  • acts a foetal ‘lung’ (gas exchange)
  • produces hormones important for the maintenance of pregnancy
• abnormalities in placental development cause gestational complications

Question 25

What are hormones for pregnancy and maternal recognition of pregnancy?

Answer 25

• during the luteal phase of the menstrual cycle the corpus luteum produces progesterone
• maintenance of progesterone is essential for pregnancy
• in the first few weeks the syncytiotrophoblast cells of the developing placenta produce human chorionic gonadotropin (hCG) – this is the hormone measured by pregnancy tests
• hCG binds activates the same receptor as luteinising hormone (the LH-CG-receptor)
• hCG activates a luteotrophic response in the corpus luteum to maintain steroid hormone production (oestrogen and progesterone)

Question 26

What are hormone levels as pregnancy progresses?

Answer 26

• syncytiotrophoblast derived hCG maintains corpus luteum up until approximately week 9 of pregnancy
• any pertubations in progesterone until this point may be detrimental to pregnancy
• after week 9 the placenta takes over production of progesterone – luteal placental shift
• placenta produces hormones which are important for maternal adaptation to pregnancy:
  
  • oestrogen
  • progesterone
  • relaxin
  • hCG
  • human placental lactogen

Question 27

How do defective maternal-foetal communication and placentation lead to gestational complications?

Answer 27

• communication between mother and foetus essential for appropriate implantation and placentation:
  
  • endometrial secretions and implantation
  • appropriate decidualisation
  • immune cell function
• alterations in this dialogue can result in:
  
  • recurrent miscarriage (endometrium cannot differentiate between good and bad embryo)
  • spontaneous abortion (defective maternal foetal communication)
  • pre-eclampsia and intra-uterine growth restriction (IUGR)
• problems may be observed in ART cycles – progesterone supplementation usually required
• defective placentation thought to underlie pre-eclampsia and IUGR (shallow invasion of uterine arteries and defective remodelling) – stressed foetus

Question 28

What are the take home messages?

Answer 28

• endometrial lining must be ready and receptive for implantation
• implantation and placentation is a multi-stage process
• invasion and remodelling of maternal blood supply is essential to provide foetus with nutrients and gas exchange
• maternal recognition of pregnancy via hCG is essential to maintain progesterone
• inadequate maternal/foetal communication can result in gestational complications