Embryo Flashcards
What are the sections of the fallopian tube?
isthmus
ampulla
infundibulum
fimbriae
Where do sperm mature?
epididymis
Where is seminal fluid made? What does it do?
70% from seminal vesicles
30% from prostate
transport medium, antioxidant + metabolic support to sperm
What are the granulose cells that surround the oocyte called?
granulosa cells
When is development arrested in the egg?
metaphase II of meiosis
What factors in the seminal fluid help the sperm reach the upper part of the FRT?
initial propulsion > ejaculation
coagulation of vaginal/testicular fluid by coagulase to make sperm latch onto vagina > then uncoagulated by fibrinolysis to release sperm
relaxin to aid motility of sperm
PGs relax smooth muscle in uterus > retropulsion to suck and squeeze sperm up
= largely transported by own movement
What factors in the FRT help the sperm reach the upper part of the FRT?
ciliated surface of cervical OS
muscle contraction
post-coital change in oviductal transcriptome profile > changes in secretion of proteins specific to sperm (+oocyte)
low levels of prog = thinner mucus
Where are sperm stored before the egg is ready? How are they kept alive?
in the isthmus
bind temporarily to epithelial cells > signal transduction > Ca/pH/gene/protein changes in the cell occurs to improve the FRT environment for the sperm > keep sperm viable
= delays capacitation + motility depressed
Why does the FRT need to act to keep sperm alive when being stored?
sperm have no mechanism to repair themselves = short lifespan
depend on FRT for survival
can survive for hours in cervical crypts nourished by mucoid secretions
What types of taxis occur in the oviduct to guide the sperm to the oocyte?
- thermotaxis - sperm move towards warmer 39 degree ampulla to meet oocyte (sensors = TRPM8 + opsins)
- chemotaxis - cumulus cells release P4 = chemoattractant
- rheotaxis - face towards oncoming current > rotation of flagella when CatSper is activated
How does the oviduct move the oocyte towards the sperm? Which cells regulate this?
ovulated oocyte + cumulus cells picked up by fimbriae
passed to ampulla > adheres to epithelium
propulsive contractions + cilia beat move it towards isthmus
oviduct motility regulated by pacemaker activity in interstitial cells of Cajal
What sperm parameters are needed for high fertility?
sufficient number of competent spermatozoa for a sufficient duration
chances of conception won’t increase even with increasing sperm once they reach a certain level
Male infertility is associated with how many infertile couples?
50%
40-80% a/w ROS
How can DNA damage in sperm be tested?
chromatin structure assay
TUNEL assay
DNA oxidation
How can poor motility or morphology in sperm be treated?
ICSI (intra cytoplasmic sperm injection)
- put sperm directly onto oocyte membrane or be inserted into the egg
What is cryopreservation of sperm?
Preservation by cooling to very low temperatures to stop any processes that would cause functional or material damage
What are the 3 steps of cryopreservation of sperm?
- cooling
- freezing
- thawing
When can damage of sperm in cryopreservation occur?
contamination > disease
sterility of liquid nitrogen + tanks
microorganisms also preserved during freezing
What is the zona pellucida?
layer of glycoproteins covering the oocyte
Which ZP proteins make up the ZP?
ZP1, 2 + 3
Which ZP proteins are involved in fertilisation and how?
intact sperm bind to ZP3 > trigger acrosome reaction > interacts with ZP2 > penetrates ZP > fertilises egg
What part does ZP1 play in fertilisation?
only structural
Which ZP protein is triggered after fertilisation?
ZP2 to change to ZP2f > anymore sperm trying to bind are blocked = prevents polyspermy
What is the preconception environment modulated by?
short range signals:
- local responses to gametes/embryo
- changes to FRT environment
long range signals:
- environmental factors
- nutrition
- hormonal status
What is capacitation?
final stage of maturation of the sperm
before this they are unable to pass through corona radiata and undergo fertilisation
Where does capacitation occur?
in the FRT
interact with uterus epithelial cells to facilitate it
What happens to the sperm during capacitation?
- hyper activated motility > stronger and wider amplitude beats
- stripping of glycoprotein coat/seminal plasma proteins from head > membrane fluidity changed > primed for acrosomal reactions
What reaction occurs after capacitation?
acrosome reaction
What happens in the acrosome reaction?
- sperm binds with ZP 2/3/4 > membranes fuse
- Ca2+ runs into the sperm
- trypsin and acrosin released
- shedding of cap to expose acrosome
- sperm digests through ZP
What steps occur after the acrosome reaction for fertilisation to occur?
- growth of acrosomal processes > sperm in the perivitelline space > microvilli on oocyte membrane envelop sperm head
- fusion of plasma membranes > JUNO on oocyte + IZUMO on the sperm > sperm nucleus released into oocyte
fusion triggers Ca2+ release within oocyte > resumption of oocyte meiosis
- cortical reaction (prevents polyspermy)
Define pregnancy
upon fertilisation, pregnancy initiated when the conceptus successful signalled presence to mother, continues with development of 1+ offspring (embryo/foetus) in a women’s uterus
What processes occur in the oocyte during fertilisation?
membrane depolarisation
cortical reactions
completion of 2nd meiotic division
How is polyspermy prevented?
- fusion with oocyte membrane > openings in oocyte membrane > Na+ enters oocyte > depolarisation of oocyte membrane
increase in IC Ca2+ in oocyte (caused by phospholipase zeta on sperm) causes:
- cortical reaction > cortical granules released from egg > cross-link ZP proteins > makes it harder
- enzymes in cortical granules e.g. ovastacin > cleave ZP2 > sperm can’t bind
- egg sheds JUNO receptors
Which receptors on sperm surface bind to integral like molecules on the oolemma?
ADAM 1/2/3
How is the oviduct involved in embryo development?
- early embryo relies on oxidative metabolism (from pyruvate + lactate via TCA cycle) > oviductal fluid provides energy source
- at blastocyst stage, glycolytic metabolism occurs in now mature mitochondria > oviduct can supply glycogen for energy during cleavage
- amylase produced by oviductal epithelial cells > converts glycogen to glucose (glycogenolysis)
- oviductal epithelial cells provide embryotrophic factors e.g. RGF, FGF, IGF, TGT
How is the oviduct involved in embryo protection against stress + immune responses?
- oviductal antioxidants reduce oxidative stress in embryo
- epithelial cells product heat shock protein family (HSP25 and 70) > handle heat stress
- oviductal fluid contains catalase, superoxide dismutases, glutathione peroxidase > reduce stress of embryos from ROS
- protects embryo from own immune system through E2/ESR1 signalling by inhibiting production of AMPs + excess protease activity
What processes occur in the oviduct to transport the embryo?
tubal muscle contraction (by endothelins + E2)
increased fluid secretion + flow (prolactin)
motility of ciliated epithelial cells (E2)
What is the main energy source in early and late stage embryos?
early = pyruvate late = glucose
When does the energy source switch in a developing embryo?
at compaction
switches from krebs/oxidative phosphorylation > glycolysis
Why is pyruvate the main source of energy in an early stage embryo? How does it provide energy to embryo?
pyruvate produced by cumulus cells > into oocyte > Krebs > ATP for embryo
high ATP:ADP > high ATP inhibits PFK > no glycolysis = pyruvate is the main source
as oocytes mature > cumulus cells mature around it + expand > utilise glucose for glycolysis > produce pyruvate for Krebs > ATP
Why is glucose the main source of energy in a late stage embryo? How does it provide energy to embryo?
utilise ATP > atp reduces > ADP:ATP increases > stimulates PFK expression > glycolysis
produces NADH
What role does NADH have in embryo development?
produced in glycolysis
> DNA synthesis
aspartate > AA goes into embryo > converted to malate with NADH > important in development
What levels of glycolysis are needed for high viability embryos?
low levels
high = increased apoptosis
What are glucose receptors used for in the developing embryo?
nucleic acid synthesis, phospholipids, non-essential AA synthesis
= glucose uptake can predict viability
Which glucose receptors are used during embryo development?
GLUT 1, 3, 8, SGLT1 > present up to blastocyst stage
GLUT 5 = from 8-cell stage onwards, GLUT 4 = blastocyst stage only > needed when glucose demand increases
When is O2 demand in the developing embryo the highest?
sudden increased demand at blastocyst stage for compaction, expansion etc
decreases straight after
Why is more O2 needed later in embryo development?
early cleavage > access to more of the O2 in oviduct due to higher SA
later on > cells more compact > access to O2 lower > need higher supply to survive
When is O2 toxic to the developing embryo?
high O2 (levels in the air) = toxic > oxidative stress > influences cell devision
more proliferation can occur at lower levels (e.g. 5%)
How AAs used in the developing embryo?
protein synthesis
cell function
buffer for pH control
energy source
How does AA depletion change as the embryo progresses?
increases
How can AA depletion be used as a marker of embryo viability?
high metabolic activity early on > used up lots of AAs > stressed by condition > embryo becomes arrested at blastocyst stage > won’t develop
pattern of depletion = good marker
How do RNA levels change in the developing embryo?
reduces as embryo cleaves
utilises RNA from 4-8 cell stage onwards
starts to synthesis own mRNA as maternal source degrades
How does circulating IGI-I + insulin affect the follicle?
synergises with FSH + LH > stimulates oestradiol
= promotes follicle/oocyte growth, oestrous behaviour, ovulation
acts on secretory epithelia in the tract > enhance embryo growth/reduce loss
How does circulating IGI-I + insulin affect the CL?
synergises with LH >
stimulates progesterone
acts contract to promote embryo growth and development
What are signs of a successful preimplantation embryo? What is an abnormal embryo?
uniform cells, appropriately sized ICM + trophoblasts
abnormal:
fragmentation > fragmented blastoma bits are toxic are in vitro > can kill other blastomas > have to be removed
arrest > develops to certain stage
Describe the 4 key methods of measuring embryo quality?
- count no of cells with differential staining of ICM, TE + apoptotic cells
- time-lapse microscopy imaging (embryoscope)
- metabolic analysis
- NMR or MS coupled with separation methods e.g. HPLC-MS
What is important to look for when counting cells to evaluate embryo quality?
ICM:TE important > little/no ICM = no chance of pregnancy
lots of apoptotic cells = poor quality
How does time-lapse microscopy work in evaluating embryo quality?
captures images every 10 mins over several days > produces film
e.g. time to first cleavage, regularity in duration of cell cycles, morphology, fragmentation
+/- of counting cells to evaluate embryo quality?
- invasive method > only used in research
+ provides lots of detail
+/- of time-lapse microscopy work in evaluating embryo quality?
+ can leave for days, non-invasive, don’t need to open incubator = reduces perturbation to environment, can predict likelihood of pregnancy
What parameters can be looked at in metabolomic analysis when evaluating embryo quality?
uptake of glucose, AA depletion, pyruvate, lactate,
O2, lipid
production of H20, Co2, lactate, NH4+, enzymes, hormones
+/- of metabolomic analysis in evaluating embryo quality?
+ non-invasive, reproducible, easy, can predict embryo survival, no ethics, fast, independent of other parameters
What parameters can be looked at in NMR/MS when evaluating embryo quality? +/-?
peaks related to specific substances e.g. AAs
+ shows levels of lots of different substances in different embryos/times
- expensive, requires trained personnel, long analysis time
Summarise what factors in the embryo environment affect embryo development?
glucose, energy substrates, AAs, GFs, steroid hormones, cytokines, metabolic regulators
What factors in the in vitro culture affect embryo development?
protein supplements
media composition
What factors in the in vivo environment affect embryo development?
diet
body composition
What are the potential short term responses to changes in embryo/in vitro/in vivo environment?
= developmental plasticity
epigenetic modifications altered IC signalling metabolic stress gene expression changes apoptosis cell proliferation disturbed
What are the potential long term responses to changes in embryo/in vitro/in vivo environment?
reduced implantation capacity unbalanced foetal/placental allocations altered maternal nutrient provision abnormal foetal growth altered setting of neuroendocrine axis abnormal birthweight/postnatal growth CVD + metabolic syndromes
Outline the steps from fertilisation to implantation
- fertilisation
- syngamy
- cleavage
- compaction
- blastulation
- expansion
- hatching
- secretion of hCG
- apposition
- adhesion
- invasion
- implantation
Where does fertilisation occur in humans?
ampulla-isthmus junction
What is syngamy?
cytoplasmic contents of sperm cell membrane passes into the oocyte cytoplasm after fusion
= completes fusion
What happens in the cleavage stage?
ooplasm divides in 2 = 2-cell stage (conceptus)
> 4-cell > 8-cell > 16-cell
at 16+ = morula
asynchronous process
How many days does it take to form the morula?
3-4 days
each cleavage occurs every 10-12 hours
What happens in compaction?
- cells flatten and maximise IC contents > cell demarcations disappear and they are tightly connected
- cell-cell adhesion + attrition increases
- polarisation of outer cells which become the trophectoderm
What 2 molecules drive compaction?
Ca2+ dependant > if blocked, compaction doesn’t happen
e-cadherin (transmembrane cell-cell adhesion molecule) > relocated to regions of cell-cell contact by activation of Pk-C
What happens if e-cadherin gene is deleted in the early embryo?
trophectoderm cells don’t form > morula cells start to compact but then dissociate at blastocyst stage
same happens in a-catenin knockouts
= cell-cell junctions very important
Give some examples of adhesion molecules that drive compaction
e-cadherin
a-catenin
occludins
How to cell-cell junctions aid embryo development?
at earlier stages, embryo = permeable
proteins polarised on surface > tight junctions help seal embryo > nothing but water can come in + out
number of junctional proteins increase with number of cells in embryo
When does the blastocyst reach the endometrium?
day 5 after fertilisation
What is blastulation?
formation of blastocyst from the morula
What are the names of the cells formed up to the gastrula?
morula > blastocoel (liquid in the blastocysts, indicator of complete development of the blastocyst) > blastula > gastrula
What is a gastrula? What does it form?
hollow cup shaped structure with 3 layers of cells
differentiates to form the germ layers of the embryo
> endoderm, mesoderm + ectoderm
How does expansion to the blastocyst occur?
ATPase on basolateral side of trophoblast cell > takes Na+ into blastocyst > creates gradient > water follows through aquaporins on apical + basolateral side > blastocyst expands
- depends on polarised distribution of Na+/K+/ATPase
- causes ZP to thin
What day does hatching occur?
day 6 = start of implantation window
What happens during hatching?
blastocyst hatches out of the ZP
When does the blastocyst start to produce hCG? Which cells produce it?
day 6-7
trophoblast cells
What is the role of hCG release on day 6-7?
prevents decline of CL > ensures synthesis of progesterone until placenta is formed
What happens if prog levels get too low at the blastocyst stage?
vessels spasm > rupture > endometrial cells turn necrotic > endometrial lining is shed > embryo is shed
prog levels must be maintained!
How much net growth is there during the cleavage stage? What about once the blastocyst stage is reached?
embryos develop through cell division > embryos don’t increase in size > blastoma cells become smaller and smaller as they cleave
at blastocyst stage, embryo size increases + ZP becomes thinner
What is the maternal to zygotic transition? When does it happen?
when development comes under the exclusive control of the zygotic genome rather than the maternal (egg) genome - zygotic genes are activated + maternal transcripts are cleared
occurs between 2-cell + morula stage
How is early embryo development regulated?
can’t surpass 3 or 4 cell cycles unsupported
provided by GFs + cytokines from oviduct/uterus/maternal circulation/embryo
e.g. IGF-I/II, TGF-a/B, FGF-4, ILs
= must be provided in vitro
What are the 4 key molecular players in blastocyst formation?
Oct4
Cdx2
Nanog
Gata 6
What is the expression of oct4 in the blastocyst?
uniform expression
only in ICM
repressed in trophoblast cells by cdx2
What is the expression of cdx2 in the blastocyst?
stochastic expression
only in trophectoderm by 16 cell stage
What is the expression of nanog in the blastocyst?
stochastic expression, then salt + pepper expression in ICM repressed in trophectoderm by cdx2
What is the expression of gata6 in the blastocyst?
stochastic expression
salt + pepper expression in ICM induced by Grbt2
What is the basic structure of a blastocyst?
surrounded by ZP > later degenerates to be replaced by underlying trophoblastic cells
trophoblast cells around outside - provides nutrients to embryo > later develops into placenta
fluid filled cavity = blastocoel
inner cell mass - source of embryonic stem cells
What are the 3 essentials for implantation to occur?
- Window of implantation (cycle day 19-23/day 6 post fertilisation i.e. mid secretory phase) > structural and functional changes to become a receptive endometrium
- good quality healthy embryo at blastocyst stage
- communication between mother + baby at time of implantation
What are the stages leading up to implantation once the blastocyst has arrived at the endometrium?
zygote signals presence to mother = orientation
> apposition > attachment > invasion
What is apposition?
lining up of trophoblast cells so the ICM is adjacent to the endometrium
> causes further decidualisation of stroma
can still be dislodged at this stage and needs further adhesion
What factors are required for apposition to occur?
pinopods on endometrium
steroid hormones, gonadotrophin, cytokines, ECVs
Which factors are switched on/off during the attachment stage?
switch off mucin 1 = blocks attachment
switch on:
- LIF = promotes luminal epithelial receptivity to attachment
- adhesion molecules e.g. fibronectins, integrins, selectin, e-cadherins
- HB-EGF = binds to conceptus > sheds zona to assist in attachment
How does the arrangement of cells change in the blastocyst during invasion?
trophectoderm further differentiates >
cytotrophoblast proliferates into endometrium > cell membranes disintegrate > nuclei + cytoplasm released into an are > form finger like processes = syncytiotrophoblast > invade into the maternal blood vessels = gain supply of O2 + nutrients > becomes the placenta
cytotrophoblast cells continue to proliferate into the syncytiotrophoblast
> totally embedded
What factors are involved in the invasion stage of implantation?
MMPs digest stromal compartments > TIMPs control this = switch on
VEG-2 stimulate angiogenesis
Proinflammatory endometrial reaction following attachment due to PGs controlled by COX2
Blastocyst secretes cytokines > stimulates LIF expression in endometrium
What is the histology of a receptive endometrium?
Shortening of microvilli
Loss of negative surface charge
Thinning of the mucin coat
Formation of pinopodes
What is decidualization?
significant changes to cells of endometrium in preparation/during pregnancy
What cells changes happen during decidualization?
differentiation of elongated fibroblast-like mesenchymal cells > rounded epithelioid cells
in the uterine stroma
during the menstrual cycle + pregnancy
When does decidualization occur?
mid-luteal phase of menstrual cycle, independent of pregnancy
What hormone is decidualization driven by?
progesterone from the CL > makes changes at transcriptome/proteomic level
What are decidualized stromal cells able to do?
- regulate trophoblast invasion
- resist inflammatory and oxidative insults
- dampen local maternal immune responses
What is the phase between conceptus and embryo called and how long is it?
embryogenic phase
14 days
hCG synthesised in the trophoblast from 6-7 days after fertilisation
What is the phase between embryo and foetus called and how long is it? What happens?
embryonic
6 weeks
cell + tissue types differentiate > basic body plan laid down > tiny foetus formed
What is the phase between foetus and baby called and how long is it?
Foetal phase
220 days - 2nd + 3rd trimester to term
What cells does the endoderm later form?
epithelial lining of lungs, digestive tract, urethra, bladder, reproductive system
alveolar lung cells
thyroid cells
liver + pancreas
What cells does the mesoderm later form?
cardiac muscle cells skeletal muscle cells tubule cells of kidney RBCs notochord smooth muscle cells in gut
What cells does the ectoderm later form?
skin cells of epidermis
neurons
pigment cells - cornea and lens of eye
Where are oestrogen + progesterone receptors expressed in the endometrium?
in epithelium + stromal cells
What are the actions of oestrogen during implantation?
Increases during luteal phase but not required to open implantation window
required to develop receptive endometrium
= stimulates proliferation + differentiation of uterine epithelial cells
When is the endometrium receptive?
mid-secretory phase (days 7 to 10 after ovulation)
What are the actions of progesterone during implantation?
prostagenic domination required for uterus + implanting blastocyst to engage effectively
PRs in uterine glandular/luminal epithelium down-regulated > prog stimulates glandular production but inhibits glandular secretion
stimulates proliferation + differentiation of stromal cells
How do PR antagonists affect pregnancy?
induce abortion before 7 weeks
= prog essential for implantation
What occurs if the CL is removed?
loss of pregnancy
adequate prog production by CL = critical to maintenance for pregnancy until placenta takes over function at 12 wks
What is hCG synthesised by?
trophoblast cells (later by the placenta)
What is the role of prog in the maintenance of pregnancy?
causes proliferation of uterine lining, vascularisation of endometrium, promotes myometrial quiescence, increases maternal ventilation, promotes glucose deposition in fat stores
What are the autocrine and paracrine affects of hCG?
autocrine: promotes differentiation + migration of extra villous trophoblasts
paracrine: on maternal ovary + endometrium
When can hCG be detected?
found in blood immediately
found in urine a few days after missed period
How is IL-1a/B involved in implantation? When and where are they found?
expressed throughout menstrual cycle, increases in mid-luteal phase
on surface epithelium
increase secretion of cytokines > cause molecular changes > up-regulation of adhesion molecules
How are IGFs involved in implantation?
localised to predecidual stromal cells in late-secretory phase + decidual cells in pregnancy
IGFBP-1 modulates mitogenic + metabolic effect of IGF1/2 = key importance in growth, apposition, development
IGF2 + IL-1B inhibit IGFBP-1 > inhibits decidualization
What molecule acts as a barrier to implantation in the uterus?
mucin 1 = anti-invasion
highly glycosylated polymorphic mucin-like protein
must be inhibited at site of implantation for implantation to occur
When is mucin 1 highly expressed?
mid-secretory phase
more abundant in fertile women
What role does osteopontin have in implantation? When is it upregulated?
upregulated in mid-secretory receptive endometrium by prog
binds to cell surface receptors + signals through adhesion proteins/co-receptors
What are ECVs?
phospholipid membrane enclosed nano sized vesicle discharged by cells that carry DNA, RNA + proteins between different cells
= facilitate paracrine communication between mother + embryo
What are the 3 types of ECVs?
- apoptotic bodies - released from apoptotic cells
- microvesicles - outward budding of membrane in normal cells
- exosomes - from multivesicular compartments by inversion of plasma membrane > compartment fuses with membrane > releases it
What roles do ECVs have outside of fertility?
modulators of tumour environment
monitor/diagnose conditions
carry drugs/tx
vaccines
What role do ECVs play in implantation?
- endometrial receptivity
- highest no of ECVs in uterine fluid in luteal phase
- contain lots of apoptotic proteins before implantation, more cell adhesion proteins from implantation - modulation of receptive endometrium
- ECVs released from embryo regulated by hCG/IL-1B > signal to endometrium > quality control to see if embryo is developmentally incompetent > if competent, send signals to stop endometrium supporting embryo - modify trophectoderm cells for adhesion/invasion
- from endometrial epithelial cells > communicate between endometrium + embryo > improve implantation - implantation cytokines
- on surge of prog/PR > embryo signals to ECVs > expression of cytokines essential for implantation
What is the most limiting factor in ART procedures?
implantation failure = 70% of ART failures
What are the endometrial causes of IF?
thin endometrium - can’t support foetus
altered expression of adhesive molecules/immunological factors
What are the embryonic causes of IF?
genetic abnormalities sperm defects embryonic aneuploiidy zona hardening = most have txs for
What is the difference in causes between IF and recurrent miscarriage?
IF = unreceptive endometrium RM = endometrium overreactive > allows poor quality embryos to implant/can't maintain embryo
What is tolerance in pregnancy?
Allows the development of an immunologically distinct organism by reducing the negative impact of the pathogen on the individual without actual fighting the pathogen
What is the role of NF-kappaB in implantation?
Activate IL-6/8 > stimulate migration + invasion
Regulates MMPs > destruction of maternal spiral arteries after invasion of the trophoblasts
What is the role of NF-kappaB in pregnancy?
inhibited
linked to development of Th1 response > when down regulated > suppressed Th1 response + promotes Th2 response > viviparity of foetus
Which process is responsible for acute allograft rejection? How does this shift in pregnancy?
Th1 cytokine immune response
shifts to Th2-immune response > to allow foetus to develop without rejection from mother’s immune system
How are embryos graded?
1st number = number of cells
2nd number = degree of idealised blastomeres (4 = highest) i.e. what are they meant to look like at this size
3rd number = degree of fragmentation
higher number = better quality
How are blastocysts graded?
day 5-8 embryo where some differentiation has occurred
grade 1-4 = degree of expansion
grade 5-6 = degree of hatching
grades 3+ = further graded A-D for ICM and trophectoderm (a = tightly packed, many cells > d= degenerating cells)
Define implantation rate
Number of embryo implanted out of a number transferred over a period of time
Define failed implantation
failure to reach a stage in which there is ultrasound evidence of intrauterine pregnancy (intra-uterine gestational sac)
Define recurrent IF
Failure to achieve a clinical pregnancy following the transfer of at least 4 good quality embryos, in at least 3 transfer cycles (fresh/frozen) in a women aged <40yrs
What is the difference between repeated and recurrent IF?
repeated - due to age + uterus/ovaries/tubes/embryo factors
recurrent - only due to uterus/ovaries/tubes as it excludes poor quality embryos + age >40 according to definition
What are the 3 possible outcomes of IVF?
- Ongoing clinical pregnancy - success
- Failed IVF - cycle cancellation, failed fertilisation/implantation, biochemical pregnancy, clinical miscarriage, ectopic pregnancy
- Recurrent IF
What are the embryonic causes of RIF?
Sperm – unexplained, iatrogenic (surgery, radiation etc), chromosomal, genetic, congenital, varicocoele
Reduced ovarian reserve – age, iatrogenic, chromosomal, genetic, congenital
how many cumulus cells should be removed?
What are the lifestyle causes of RIF?
age
Diet and supplements – folate and vitamin D
BMI <18/>30
Smoking > increases FSH dose needed, reduces egg yield, embryo quality + implantation
Stress - lack of pregnancy causing the stress?
What are the lab factor causes of RIF?
STIMULATION PROTOCOLS, serum progesterone at HCG trigger
Handling eggs - collection, denudings eggs insemination, ICSI
Cheap culture media, culture dishes and embryo transfer catheters
aldehydes/noxious volatile substances
Exposure of embryos to light
Incubator conditions - pH, thermal environment
Embryo transfer technique
What are the endometrial causes of RIF?
Unexplained
Infections
Poor endometrial development
Submucous fibroids – benign tumours of myometrium
Congenital (uterine septa)
Adenomyosis – tissue lining endometrium infiltrates into wall of womb
Polyps – soft tissue growth on lining of womb
Uterine synechia (Asherman’s syndrome) – ashesions of the womb lining
What are the uterine + tubal causes of RIF?
uterine:
Fibroids
Adenomyosis
mullerian dysgenesis
tubal:
hydrosalpinges
What are the endocrine causes of RIF?
Poorly controlled thyroid diseases
DM
PCOS
POF
What are the immune causes of RIF?
AI diseases: thyroid, gonadal
abnormal endometrial cytokines eg elevated NK cells
Antiphospholipid syndrome
What ix should be done for RIF?
FSH, AMH, Antral follicle count – look at ovarian reserve, AI ovarian disorders
Testosterone, Sex Hormone Binding Globulin and Free androgen index – PCOS
TSH and thyroid perioxidase antibody - thyroid disorder, AI
Lupus anticoagulant, antiphospholipid Ab
HbA1C – DM
Pelvic ultrasound and transvaginal US, hysterosalpingography - uterine, endometrial, tubal or ovarian factor
Second line:
Hysteroscopy/laparoscopy - to confirm uterine, endometrial, tubal or ovarian factor
Parental karyotyping - only if strongly indicated
What are the tests of doubtful value in RIF ix?
sperm DNA fragmentation test endometrial sampling for natural killer (uNK) cells
blood for pNK cells
What are the general measures that can be done to improve embryo quality?
Try early Good diet Supplements – folate and Vit D. Fertility care for men Stop smoking Reduce alcohol Weight control
What are the specific measures that can be done to improve embryo quality?
Sperm DNA fragmentation tests Ovarian stimulation protocol Gamete donation for parental aneuploidies ICSI/IMSI careful embryo selection - embryoscope blastocyst transfer assisted hatching improved embryo transfer techniques
What is antiphospholipid syndrome?
systemic autoimmune disease characterized by vascular thrombosis and/or pregnancy morbidity in the persistent presence of antiphospholipid antibodies (aPL)
What are the 3 tests for antiphospholipid syndrome?
abs against beta-2 glycoprotein I
abs against cardiolipin
lupus anticoagulant
How does aPL affect pregnancy?
causes defective placentation by interacting with both sides of the placenta
at decidual level > aPL is proinflammatory with neutrophil infiltration, secretion of cytokines + complement activation
at trophoblast level, aPL down-regulates hCG, integrins + cadherins > reduced trophoblast proliferation and growth
= linked to recurrent miscarriages not usually IF
What abs are present in thyroid AI disease? How is it treated?
abs against TPO +/- TG
a/w RIF
tx = thyroxine
What are natural killer cells? Are they present in pregnancy?
lymphocyte in peripheral blood
similar cells which are poor killers (uNK) populate uterine lining at implantation > role in regulating placentation + trophoblast cell invasion
can test for it but no agreed normal range of normality
What are the causes of persistent endometrial fluid?
Ovarian stimulation (uterine or cervical) High/low oestrogen Tubal > hydrosalpinges Endometrial – Polyp or fibroid – Asherman’s synechia – Cervical stenosis – Chronic infection
What are the causes of a poorly developed endometrium?
Poor ovarian reserve or POI Iatrogenic - medical, radiation Endometrial – infection, ashermans Uterine fibroids Adenomyosis
What types of cells do the trophectoderm split into?
syncytiotrophoblast and cytotrophoblast
What 2 cell types does the ICM form as part of the bilaminar disc?
epiblast (amniotic cavity above)
hypoblast (primitive yolk sac below)
What changes occur in weeks 1-3 of the developing embryo?
- Trophoblast splits into syncytiotrophoblast and cytotrophoblast. ICM forms bilaminar disc
- Synctiotrophpblast becomes conintinuous with endometrium + invades maternal blood vessels = placenta formed
- gastrulation occurs > differentiation of epiblast into 3 layers
Innate immunity: specificity memory non-reactivity to self cellular/molecular barriers blood proteins cells
specificity: no (conserved region of related microbes produced by damaged cells)
memory: no
non-reactivity to self: yes
cellular/molecular barriers: skin, mucus, AMPs
blood proteins: complement
cells: macrophages, neutrophils, NK cells
Adaptive immunity: specificity memory non-reactivity to self cellular/molecular barriers blood proteins cells
specificity: for microbial/non-microbial antigens
memory: yes
non-reactivity to self: yes
cellular/molecular barriers: lymphocytes, abs
blood proteins: abs
cells: lymphocytes
What are possible defects in the immune system?
chronic inflammation, AI disease, immunodeficiency
What features must an immune system have?
discrimination: distinguish self from non-self and harmless vs harmful non-self
flexibility: not all changes in environment need immune reactions
managing infection
memory of previous infection
What are the features of innate immunity?
1st line of defence
- Physical + chemical barriers (skin and mucus)
- Immediate action (anti-microbial peptides) - directly kill microbes/indirectly by modulating host defence
- Non-specific - pattern recognition receptors, generalised response to bacteria/virus
- Leukocytes of innate immunity (neutrophils, macrophages, NK cells and dendritic cells)
What are the key types of pattern recognition receptors (PRRs)?
membrane bound PRRs e.g. TLRs, c-type lectin (recognise carbs in cell membrane of bacteria)
cytoplasmic PRRs e.g. NOD-like (recognise DNA/RNA of viruses = inflammatory response), RIG-I-like (recognise viruses)
What do PRRs recognise?
PAMPs (conserved between species to recognise PAMPs)
What are PAMPs?
pathogen associated molecular patterns
synthesised by microorganisms and damage associated molecular patterns (DAMPs)
What are DAMPs?
a/w components of host cells that are released during cell damage/death
What are toll like receptors?
type 1 transmembrane proteins
always membrane bound on cell/endosome
What role do TLRs play in the immune response?
stimulated by specific ligand > cell signalling with other molecules > translocation of NF-kappa-B or AP1 (TFs) > increase expression of genes specific to original ligands > stimulate adaptive immune response
recognise PAMP > recruit specific set of adaptor molecules > initiate downstream signalling > secretion of inflammatory cytokines, type I IFN, chemokine, antimicrobial peptides
recruitment of neutrophils, activation of macrophages, induction of IFN stimulated genes > direct killing
maturations of DCs > induction of adaptive immunity
What are TLRs made up of?
an ectodomain containing leucine-rich repeats > mediate recognition of PAMPs
a transmembrane region
cytosolic TIR domains > activate downstream signalling pathways
Which TLRs are within cells and recognise DNA/RNA of viruses?
3, 7, 8, 9
Which TLRs recognise bacteria + fungi pathogen particles like flagella, toxins?
1, 4, 5, 6
What happens if TLRs are activated during implantation e.g. in infection?
-ve effect
immune system has to handle something else > can’t concentrate on implantation
What is the end product of the TLR signalling pathway?
AMPs
How do TLRs respond to commensal bacteria in the vagina?
stimulates TLRs > basal expression of AMP = not harmful
more bacteria > stimulates TLRs > express more AMP > break mucous barrier of FRT > invade cells > stimulate innate response etc
Which parts of the FRT are sterile?
upper tract
lower = colonised by bacteria > exposed to non-self entities, not all harmful e.g. sperm, embryo
Which TLR is not expressed in the vagina?
TLR4
recognises bacteria > vagina exposed to outer environment/consenal bacteria > constantly activated
When are TLRs most highly expressed?
2nd half of secretory phase when implantation occurs
increased AMP expression > favours embryo implantation
What are the components of the innate immune system in the FRT?
epithelial cells = physical barrier + produce AMPs e.g. defensins
expression of TLRs in all epithelial cells in tract
What is the innate immune system in the FRT regulated by?
ovarian hormones
oestrogen > increases susceptibility to infection + allowance of sperm in tract (oestrogenic contraceptives increase infection susceptibility)
progesterone > increases immune surveillance, prepare tract for implantation, increase expression of TLRs
What challenges does the FRT face?
menstruation fertilisation Implantation and pregnancy Defence against microorganisms Parturition i.e. much more active than MRT
When is immunity highest during menstruation?
after prog surge
arrival of neutrophils + macrophages to uterine decidua > surround degraded tissues to remove them
production of enzymes (MMP, TIMP) to degrade connective tissues of endometrium
clearance of tissue fragments
= happens after action of prog stops
What are the 5 cardinal signs of inflammation?
heat, redness, swelling, pain, loss of function
What are the stages of pregnancy which require the strongest inflammatory response?
implantation, placentation + 1st and early 2nd trimester of pregnancy
= resemble open wound
How much inflammation occurs in 2nd trimester?
development + growth = much less inflammation
How much inflammation occurs in 3rd trimester?
renewed inflammation
influx of immune cells to myometrium > promote uterine contraction, expulsion of baby + rejection of placenta
What are the 3 ways an individual protects themselves from infectious disease?
avoidance - decreasing chance of exposure
resistance - fighting infection
tolerance - reducing negative impact of pathogen without fighting it = what is needed from maternal immune system
What does tolerance of microorganisms by the maternal immune system allow for?
creation of commensal flora of bacteria in digestive tract/FRT
acceptance of embryo during implantation
How do immune cells behave in the 1st trimester on the invading embryo?
DC, NK cells, macrophages, dendritic cells > infiltrate decidua > accumulate around invading embryo
absence of these cells > -ve effects on placental development, implantation + decidualisation
Is the innate immune response in favour or against implantation?
presence of immune cells = not response to foreign foetus > facilitates and protects pregnancy
immune system is active and carefully controlled in pregnancy NOT suppressed
How does maternal immune system identify the embryo from a harmful non-self entity?
in apposition stage, embryo signals presence to mother > educates maternal immune system to distinguish embryo from bacteria/viruses
What are epigenetics in pregnancy?
Periconception environment affects health of offspring and have genetic influences on gene expression
Same gene can produce 2 different phenotypes
What is the difference between genetics and epigenetics?
hardware = genetics, DNA sequence, irreversible software = epigenetics, modifications on DNA sequence, reversible
What are some examples of epigenetic modification?
methylation > works on DNA, acts to repress gene expression
acetylation > works on histones to increase transcription
What is developmental plasticity?
selecting the right phenotype to fit the anticipated future environment
When can epigenetic profiles be changed?
Different profiles are affected at different times – gametes, zygote, embryo, foetus, birth, lifestyle
by food, exercise, medications, sperm/oocyte etc
What was the main finding of the dutch famine birth cohort study?
in utero exposure to famine > higher incidence of CHD, COPD, high BP, mental health
depending on when famine occurred in the pregnancy
What was the main finding of the barker early/foetal origins/thrifty phenotype study?
poor maternal nutrition > low infancy weight > increased death by IHD later in life
What is the key idea from the waddington epigenetic landscape theory?
landscapes are influenced by environment
we are like balls rolling down a hill > depending on the environment we will go in different directions
genes/dna sequence is fixed
epigenetic modifications are not > environment influences base on which proteins are coded
Define cellular differentiation
processes by which the genotype gives rise to the phenotype
What was the main finding of the DOHaD study?
small body size at term increases risk of CVD, T2DM, OP, schizophrenia + depression
large body size at term increases risk of cancer
How does IVF culture + a lack of early embryo-maternal communication affect epigenetic modification?
don’t know exactly what occurs in the mothers body > cannot replicate this exactly in an in vitro culture
embryos epigenetic profile will be set in this culture > don’t know long term consequences of this
ivf children not old enough to show this yet - oldest is 44
increase incidence of imprinting disorders e.g. Beckwith-Wiedemann, Angelman syndrome
animals likely to have different regulation but e.g. cows > large offspring syndrome
What does maternal-embryonic communication regulate that IVF babies may miss out on?
Features of blastocyst morphogenesis Co-ordination of implantation Maternal immuno-tolerance Developmental plasticity - "selecting" the right phenotype to fit the anticipated future environment Implications - DOHaD, ART
Define infertility
a couple are subfertile If conception has not occurred after a year of regular unprotected intercourse (2-3 times a week)
How many couples are affected by infertility?
15%
50% male, 50% female cause
1/3rd unexplained
How often does pregnancy occur from IVF?
<30%
What is primary vs secondary IVF failure?
primary = the female has never conceived
secondary = she has previously conceived even if the pregnancy ended in miscarriage or termination
What is the preconception advice given for IVF patients?
Intercourse 2-3x per week Folic acid – 0.4mg Smear Stop smoking Manage pre-existing medical conditions Alcohol cessation Weight management
What are the conditions for a couple to be approved for IVF?
Under 42 BMI under 30 FSH < 8.9 No previous children for either person No vasectomy reversal
What problems can occur with the sperm that cause infertility?
Oligospermia - number of sperm is too low
Asthenospermia - motiltiy of sperm reduced, less capacity to reach egg in time
Teratospermia - morphology more abnormal, difficulty fertilising
Azoospermia - no sperm in ejaculate (obstructive - in testis, non-obstructive - rate of production is so low = no sperm to spill into ejaculate)
When do sperm need to be manually extracted? What are the main methods?
if sperm is coming into ejaculate > can use them however abnormal they are as there will be some normal ones to inject into the egg
TESE + PESA
How is sperm recovered from men with obstructive azoospermia?
surgical sperm recovery from testis/epididymis
How are sperm recovered from men with retrograde ejaculation?
ejaculate goes into urine > collect sperm from urine
What is PESA?
percutaneous epididymal sperm aspiration
fine needle goes into epididymis (ready sperm stored here)
sperm usually in a good state
fairly non-invasive + can be frozen so man doesn’t have to have another procedure
What is TESE?
testicular sperm extraction
men with no sperm in epididymis > retrieve from testicular tubules under microscope
How can eggs be retrieved in an anovulatory woman?
need good number AND quality of eggs as not all eggs can provide an embryo
controlled ovarian hyperstimulation to induce ovulation to generate enough follicles for IVF/ICSI > use FSH, LH + GnRH
What are some causes of an ovulation?
PCOS Hypothalamic hypogonadism Hyperprolactinaemia Pituitary damage Premature ovarian failure Hypo or hyperthyroidism Androgen secreting tumours
What is the Goujon model?
chosen egg growing 2-3 months ago
factors during that time can affect the quality of it e.g. illness
What is the FSH threshold concept when deciding a COH protocol?
dose + time given = very important
very few follicles sensitive to low levels of FSH, increasing dose > mobilises + recruits more follicles
FSH rises briefly at beginning of month in natural cycle > enough to grow and recruit one follicle
In IVF > recruit 5-10 eggs so give FSH at a higher than natural dose and for a bit longer
Why must an LH surge be avoided during a COH protocol?
LH = prepares follicle for maturation + ovulation
if LH surge > lose all the eggs so have to interfere
What medications can be used to prevent a premature LH surge in a COH protocol?
GnRH agonists (injections/nasal drop) > initial stimulation then suppression of LH if maintained daily, start week before needed to avoid LH surge later
GnRH antagonists > immediately downregulates pit so no LH/FSH is released
= can use lower doses for half the time and don’t need to start 10 days early > more commonly used
What are the stages of a COH before egg retrieval?
- choose protocol
- decide dose - depends on age, body weight, ovarian reserve, antral follicle count
- follicle monitoring
- trigger ovulation > simulate LH surge
What parameters in the blood can be used to indicate ovarian reserve?
baseline FSH + LH = day 2-3
AMH = no variation within cycle
How is AFC estimated?
on US
How are follicles monitored after COH protocol?
US - diameter/number of follicles
serum oestrogen - direct product of follicles > indicates how much they’ve matured
consider measuring LH + progesterone
What are the criteria for egg collection after COH?
3+ follicles >17mm
What is the egg collection process?
give hCG/LH/GnRHa to induce maturation
transvaginal oocyte recovery under sedation 34-36 hrs after injection
TV US > needle goes into ovary connected to a tube and pumping mechanism > collect oocytes
Give an overview of the steps in IVF
ovarian stimulation + monitoring (2wks) > egg collection > insemination/ICSI > fertilisation check = day 1 > embryo culture (2-3 days) > embryo transfer > luteal support > pregnancy test
How does embryo transfer occur? How many are transferred?
catheter inserted into uterus under USS > insert outer protective catheter for sterile tunnel > load embryo into inner catheter > thread in inner catheter and advance high into uterus > push culture media containing embryo into uterus > withdraw catheter
prog causes secretory phase > becomes receptive
max 2 embryos transferred
+/- cryopreservation
What support is given during the luteal phase in IVF patients?
supplement with extra prog for 6 weeks to prepare endometrium for implantation
+ oestrogen/hCG if deficient
Why is support needed during the luteal phase in IVF patients
CL would pump out oestrogen/progesterone to support the embryo
on egg collection, GCs which form the CL are drained when sucking up the oocyte > compromises ability of CL to produce enough oestrogen/progesterone
What is the risk of giving hCG supplementation to IVF patients?
can cause false +ve pregnancy tests
When is a pregnancy confirmed on US?
intrauterine pregnancy shows gestation sac with embryo + yolk sac inside
Define pregnancy rate
measures +ve pregnancy tests
Define clinical pregnancy
can identify foetus on US
Define take home rate
live baby rate - excludes pregnancy loss e.g. miscarriage, ectopic
Define fertilisation rate
number of eggs that have been fertilised
Define cleavage rate
after fertilisation, the number of eggs which start dividing
What is the main negative SE of IVF?
multiple pregnancy > riskier for mum and babies
getting better at success with less embryo transfers > reducing multiple pregnancies
What are the possible complications of IVF?
multiple pregnancy ectopic pregnancy miscarriage disappointment infection = rare ovarian hyperstimulation syndrome (OHSS) = now rare increased risk of cancer
What can an ectopic pregnancy be confused with?
pseudogestation sac - fluid in the uterus
ectopic = risk of haemorrhage if it ruptures so must be treated asap
What is OHSS triggered by? Why does it happen?
triggered by hCG
causes by dose and time FSh is given + body’s response to it
usually avoidable if protocol chosen carefully
can happen early after egg retrieval or later after pregnancy
How does OHSS present?
bloating, nausea, abdominal/pubic tenderness, vomiting, diarrhoea, sudden weight increase
very large follicles on US
What is the possible dangerous complication of OHSS?
hypercoaguability of the blood > DVT, embolism
ascitic fluid may need to be drained
Best tx for OHSS?
prevention
What future research in IVF is needed?
Embryo culture systems
Time - lapse morphokinetics (how we observe the embryos)
Metabolomics proteomics (what the embryo produces)
Epigenetics
PGD/PGS - genetic testing
Embryo-endometrial dialogue
What blood results are indicative of ovulation having occurred?
high prog in mid luteal phase (day 21)
What are the treatment options for anovulatory women to induce ovulation?
PCOS: clomifene - oestrogen receptor blocker > causes release of gonadotrophins
letrozole: oral aromatase inhibitor > induces ovulation
laparoscopic ovarian diathermy
gonadotrophins if these fail - daily SC injections of FSH and LH
What are the possible consequences of using SC gonadotrophin to induce ovulation?
can cause more than one follicle to develop
= needs to be monitored on US
once of adequate size > stimulation can occur by hCG/LH
Examples of ART
IVF, ICSI, PGD, mitochondrial donation, gene editing
When might ART be used?
infertility
absence of partner e.g. homosexual couples
genetic engineering
What is the sperm collection + preparation process in IVF?
collected through natural or electro ejacualtion
sperm prepared > seminal plasma removed
selection based on variability/genetic characteristics undergo motility/morphology/plasma membrane + acrosome integrity tests using flow cytometry
When are oocytes fertilised in vitro and then embryos transferred?
fertilised > cleavage occurs 24hrs later > mature 2-5 days after fertilisation
transferred at 4-8 cell stage (2-5 days)
extra embryos are cyropreserved
Why is luteal support required in IVF?
multiple follicles are made during IVF > multiple corpus luteum secrete progesterone > sharp increase in prog which isn’t physiologically normal > sharp decline of prog production after luteal phase
GnRH causes short luteal phase
What is given for luteal support during IVF?
progesterone
can use hCG to provide combined E and P secretion but more likely to cause OHSS
How long is luteal support given for after IVF?
up to 12 weeks gestation
What happen during ICSI?
sperm injected directly into oocytes
= higher success rates
When might ICSI be required?
if sperm unable to fertilise due to morphology/motility/damaged acrosome/immature
What is PGD?
pre-implantation genetic diagnosis
= screen embryos for genetic disorders eg Huntington’s, CF, sickle cell
What is the procedure used during PGD?
routine IVF
zona drilling at 8 cell stage (2-3 days) > remove 1-2 cells > cells screened > healthy embryos transferred/frozen > affected embryos allowed to perish
What happens during mitochondrial donation?
nucleus from pt transplanted into oocyte with healthy mitochondria
followed by IVF/ICSI and ET
What technology can be used for gene editing?
CRISPR-Cas9 - identifies, removes and replaces faulty genes
How does CRISPR-Cas9 work? What are its problems?
create a small piece of RNA with a short “guide” sequence that attaches to a specific target sequence in a cell’s DNA > this guide RNA also attaches to the Cas9 enzyme > when introduced into cells, the guide RNA recognizes the intended DNA sequence > Cas9 enzyme cuts the DNA at the targeted location > synthetic DNA chains replace to introduced desired trait
lots of ethical concerns - rewriting germ lines
gene editing for organ transplant > creating a donor baby
What is a reproductive technology?
intervention used to control reproduction
emphasis on improving/limiting fertility to prevent inbreeding
What is the extinction vortex? What can prevent them?
small population leads to inbreeding, disease, inbreeding depression, smaller population > extinction
genetic biobanks/assisted reproduction can help slow/block this
What reproductive technologies using embryos are used in animals?
IVF
- in vivo derived > embryos flushed form tract from super ovulated and naturally mated cow
- in vitro > oocytes extracted from ovaries
- embryo splitting and transfer > AI followed by embryo flushing, split to produce more embryos, implanted into foster uterus
What reproductive technologies using sperm are used in animals?
semen extraction - semen taken from desirable bulls
- highly profitable, cost effective
- can be sex sorted > put into flow cytometer, charge detected, XX and XY are different
AI - semen inserted into uterus laparoscopically/long catheter vaginally during oestrus of female (controlled using intravaginal prog sponges)
What is the basic process of cloning in animals? What can go wrong?
genetically unimportant + genetically valuable > recover oocytes from GU + remove chromosome and polar body + create cell line from GV > inject cell into enucleated oocyte of GU > reconstructed oocyte developed into embryo > transferred to recipient embryo > offspring born possibly with phenotypic abnormalities
matured cloned individual with abnormalities naturally mated > 2nd generation offspring with normal phenotype
What contraception options are there for animals?
suppression of GnRH with vaccine
= stops pituitary LH and FSH
for population control eg in rats
What is a stem cell?
primitive cell that is capable of self-renewal, making a range of cell types and can convert to different cell types by differentiation
allows them to build embryos and tissues and repair tissues (regneration)
What is a primitive stem cell?
only makes 1 type of cell
What is a totipotent stem cell?
can form all cell types found in an embryo plus extraembryonic/placental cells ie a fertilised oocyted
What are the only type of totipotent cells?
embyronic cells within first couple of cell divisions after fertilisation
What are pluripotent stem cells?
can form all cell types but not placenta
ie embryonic stem cells
What are multipotent stem cells?
can form limited number of cell types in a particular lineage
Name the embryonic sources of stem cells?
XEN - extraembryonic endoderm stem cells (from yolk sac)
TS – trophoblast stem cells
hESCs – human embryonic stem cells = pluri (from early epiblast)
EpiSC – Epiblast stem cells (post-implantation from late epiblast) = pluri
What are the types of cell from sources other than embryonic?
foetal SC
adult SC - tissue specific
induced pluripotent SC
What is the procedure by which SCs are derived?
derived from blastocyst in vitro > immunosurgery to remove trophectoderm using anti-trophectoderm abs + complement to induce complement mediated killing > leaving just ICM > replated, isolated and cultured onto feeder cells
How does the derivation method of human SCs compare to mouse SCs?
derivation methods very similar to mouse ES cells
human ES cells different from mouse phenotypically but same as mouse EpiSCs
How can the stem cell fraction be determined?
surface markers eg genes
When is the fate of a SC determined?
selective pressures at decision points > genetic changes in SCs
pressure can be relieved by alterations in genome, gross chromosomal changes, smaller changes (CNVs), point mutations
What processes do SCs undergo in a normal environment?
Self renewal
Apoptosis
Differentiation
What changes might a SC make to alter the epigenome in order to survive compared to a normal environment?
normal = self renewal, death or differentiation
adapted/genetically abnormal > more self renewal than death or differentiation
- growth of SCs in culture > risk they won’t grow in optimal conditions > mutations occur and overrun culture > favour self renewal > selection advantage in culture
= occurs in cancer
> cell cultures require continuous monitoring to maintain quality
What are nullipotent cells?
nullipotent = self renewal/death, cannot differentiate (happens in human embryonal carcinoma)
How can pluripotent stem cells be differentiated? What would influence the method choice?
many ways to differentiate pluri SCs - picked by whether you need to quantitate, ease of getting germ layers, reproducibility
eg by assay embryoid body, spin EB, monolayer
What can different SCs be differentiated into?
Different cell lines have different capacities for making different llineages
resulting cells need to be functionally tested
What is in vitro differentiation directed by?
exogenous signalling ligands
What do induced pluri SCs start off as?
somatic differentiated SCs
How are somatic cells induced to become pluri SCs?
using viruses and certain genes (only 4 original factors needed) > selection > colony formation > IPS cells
What are induced pluri SCs very similar to?
ES cells
Why is the formation of induced pluri SCs irreversible?
irreversible due to chromatin + methylation changes preventing re-expression of genes
loose genetic material + epigenetic changes = turning back on genes very difficult
What kind of delivery of reprogramming factors is best?
non-integrating delivery
inserting genetic material into a cell > risk of insertional mutagenesis
What are the applications of induced pluri SCs?
grow forever!
somatic cells from patients and normal person > is differentiation affected in iPS cells + what is the affected cell phenotype?
- drug screen: what affect does it have on differentiation/can drug relieve the disease cell phenotype/effect on normal phenotype
test SEs - does drug affect certain genotypes differently to others > tailored drug options
When are induced pluri SCs harder to use in drug development/disease modelling?
some diseases harder to model when we don’t know how they work eg schizophrenia, alzheimer’s
How is pluripotency maintained in induced pluri SCs?
by signals in balance which are context dependant
governed by tgf-B + fgf signalling > added to media to keep them renewing
What are the uses of stem cells?
Can capture specific genome from a population/disease
Drug testing on normal and effected phenotypes
regenerative medicine
drug discovery
toxicology
disease models
How are stem cells used in disease therapy? Give examples.
Need to make desired cell, transplant correctly, ensure it integrates well
parkinson’s - transplantation of foetal neuronal cells
T1DM - islet transplantation
age related mascular degeneration - RPE cells
What are the risks of using stem cells in disease therapy?
lack of efficacy, side effects, cancer, immunorejection
Why can SCs be more difficult to use in therapy for some diseases more than others?
diabetes difficult - low availability of cells, young patients, difficult to monitor transplanted cells, consequences of unexpected problems are serious
not all diseases suitable - have to be able to make desired cell, transplant to correct pace and ensure it integrates
Define fertility
The number of live births actually achieved by a woman (or man, couple, or population)
Define fecundity
physiological capability of a woman (or man or a couple) to produce a live birth
Define live birth
complete expulsion or extraction from its mother of a product of conception,
irrespective of the duration of pregnancy, which after such separation, breathes or shows any other evidence of life, such as beating of the heart, pulsation of the umbilical cord or definite movement of voluntary muscles, whether or not the umbilical cord has been cut or the placenta is attached
How is epidemiology of fertility collected?
via registration systems (birth/death registers, population registers), census qs, surveys eg world fertility surveys, contraceptive prevalence surveys, reproductive health survey
Define cohort fertility rate?
number of children born to a cohort of women (women born/married within a specific time period) over their lifetime or up to a certain age
Define completed fertility
average no of children born by cohort of women by the end of their reproductive yrs
Define parity progression ratio
proportion of women with at least n children (parity = n) who go on to have at least on more (n+1) eg P01 - proportion of childless couples who then have 1, P12 - 1 child, go to have another one
can be within a certain time frame
What is the most common parity progression ratio?
P01, P12 close, P23/34 much lower usually
Define period fertility
number of children born within a specific time period, expressed as per 1000 women alive in the middle of the period
What is the retrospective cohort approach to fertility epidemiology?
Medical hx of child’s births and when union with partner occurred eg when they moved in and became at risk of becoming pregnancy
What is the prospective cohort approach to fertility epidemiology?
Number of children a woman has over a life time
Map it as she has children > takes much longer
What has happened to fertility worldwide?
dropped over time in most countries
except US
What is the average number of children worldwide?
2.432
2 or less is normal everywhere
except belt in central africa = still 4+
What problems does the drop in fertility cause?
populations are very top heavy > affecting population pyramids
What would be the problem in trying to reduce the population now?
changes in society are needed for a smooth transition
Many problems with this - is it actually what the world needs for climate change etc
Define crude birth rate
ratio of live births per yr to the average population in that period (births per 1000 population)
What is the problem with crude birth rates? Use examples.
dependant on population structure
eg nigeria - lots of children, very few old people
vs japan - very high proportion of old people, very low proportion of young people
both have high proportions of people who can’t have children but very different crude birth rates
= are CBRs comparable?
need to remove people that are not at risk of giving birth
define general fertility rate
births in period/N of women aged 15-49 at mid period)*1000
define age specific fertility rate
births to women aged x/N of women in age group x at mid period *1000
Define spacing in fertility epidemiology. Give an example where this happens.
countries with higher rates of breastfeeding due to recently being pregnant > amenorrhoeic for longer > takes longer to get pregnant again > fertility reduces eg cameroon
Define stopping in fertility epidemiology. Give an example where this happens.
set amount of children wanted > choose method of contraception that ensures they decide final family size eg india
in india, fertility drops dramatically after 2 children > most popular method of contraception is sterilisation
Define starting in fertility epidemiology. Give an example where this happens.
increasingly delaying having children out of choice/finding partner later > less fertile when they try > have less children than they hoped to achieve eg greece
Define synthetic cohort measure in fertility epidemiology?
total fertility rate calculated (age specific rates at particular point and assume it’s followed)
Period measure but expressed as a cohort measure = synthetic cohort measure
No of children who would be born per woman if she lived to the end of their childbearing yrs
What factors affect the use of contraceptives in different women?
geographical locations - cultural beliefs, number of women in union (higher risk in these women)
