fertilisation Flashcards
1
Q
In pigs, how many sperm reach the oviduct?
A
1 in 100,000
2
Q
What stain is used to simply observe sperm on slides
A
Toluidine blue
3
Q
How does live dead staining work?
A
-Based on plasma membrane integrity
-If the plasma membrane is compromised, the red dye mixture is able to get into the spermatozoa
4
Q
How does JC-1 staining work?
A
-Mitochondrial membrane potential
- If the mitochondria are active, ie producing ATP, then the middle piece will show as yellow
5
Q
How can flow cytometry be used when assessing sperm quality?
A
Can automatically assess the amount of sperm with specific dyes
6
Q
Male factors affecting fertility: What problems can arise in the testis?
A
-Sperm assembly
-DNA condensation
7
Q
Male factors affecting fertility: What problems can arise in the epididymis?
A
-Sperm maturation
-lipid and protein modification
-sperm storage
8
Q
Male factors affecting fertility: What problems can arise in the seminal plasma?
A
-Accessory secretions
-adsorbance to sperm surface
9
Q
Male factors affecting fertility: What problems can arise in the sperm transport?
A
-Highly complex interactions with the female tract
10
Q
Which animal exhibit cooperative sperm swimming?
A
-South American marsupials
- Mice
11
Q
Sperm selectivity: which species have intravaginal insemination and what prevents sperm entry to the uterus?
A
-Sheep
-Rodents
-Primates
-Cervical mucus and complex anatomy
12
Q
Which species has transcervical insemination?
A
Pigs
13
Q
What may prevent sperm entry to the oviduct via the UTJ?
A
-If the cell surface lacks specific proteins
-ADAM, calmegin and calreticulin
14
Q
How does sperm binding to the OEC protect sperm?
A
Binding induces HSP synthesis, which enters the oviduct and protects the sperm surface
15
Q
Which hormone secretion stimulates selective sperm chemotaxis and hyperactivation?
A
Progesterone
16
Q
What process does sperm entry into the uterus induce?
A
Post-mating inflammatory response and leukocyte invasion
17
Q
What does sperm selection at the UTJ involve?
A
-head and tail morphology
-swimming behaviour
-correct response to extracellular signals
18
Q
What modulates gene expression in oviductal epithelial cells?
A
Sperm microRNA
19
Q
What modulates sperm motility in oviductal fluid?
A
Bicarbonate
20
Q
What reduces polyspermy and causes ZP hardening?
A
Oviductal fluid
21
Q
In humans, what proportion of sperm reach the site of fertilisation?
A
1 in 30,000
22
Q
How can sperm properties be assessed?
A
- Can assess using microscopy but this is limited
- should observe them in action
-Cervical mucus penetration test
-Migration efficiency
23
Q
How can DNA contribute to poor sperm quality?
A
-Direct damage to purines/pyrimidines
-Single/double-strand breaks
-crosslinking
-chromosomal rearrangements
24
Q
How can mitochondria contribute to poor sperm quality
A
Damage to mDNA causes decreased ATP production which causes impaired motility
25
How does sperm membrane contribute to poor sperm quality?
-Lipid peroxidation of polyunsaturated fatty acids cause impaired motilty
26
Why isn't mitochondrial DNA passed on paternally?
-The entire sperm enters an oocyte
- the mitochondrial is tagged by ubiquitin which causes it to be degraded
27
how many couples with infertility involve male factors?
50%
28
What are ROS?
Free radicals with unpaired electrons
29
What events are ROS important for?
-Capacitation
-acrosome reaction
-binding
-traversing ZP
30
What damage can oxidative stress cause?
-Poor motility
-morphological abnormalities
-DNA damage
-cell death
31
What is considered a normal concentration of sperm in semen analysis?
more than 20x10^6
32
What is considered normal motility in semen analysis?
motility above 50% progressive
33
How reliable is semen concentration determined by semen analysis?
dependent on the technician who does it
34
What percentage of normal sperm morphology is considered adequate?
3-4%
35
What does azoospermia mean?
No sperm at all
36
What does globozoospermia mean?
Sperm with large heads and no acrosome
37
Which partner is more likely to have antibodies against sperm?
female
38
Why does DNA fragmentation cause problems?
Single/double-strand break which causes problems with embryonic development
39
How can sperm DNA be repaied?
By the oocyte
40
Name 4 factors associated with higher amounts of DNA fragmentation.
-heavy smoking
-moderate smoking
-obesity
-heavy drinking
41
What happens to DNA fragmentation with incubation?
It increases
42
What test is used to assess sperm DNA damage?
The halo sperm test
43
What is the affect of cysteine on DNA fragmentation?
The more cysteine an animal has in its DNA the more stable it is and less likely to fragment
44
What are the 3 steps of cryopreservation?
-Cooling
-Freezing
-Thawing
45
How are semen samples frozen?
-Cryoprotectant is added a few hours before freezing
-sperm samples are cooled slowly to about 5 degrees c to avoid cold shock
-samples are left to equilibrate for a a few hours
-Samples are frozen according to optimal protocols
46
What is the most commonly used cryoprotectant?
Glycerol
47
How do cryoprotectants change cell osmolarity?
1) they raise solution osmolarity to high concentrations
2) water leaves cells causing them to shrink
3) Cells resume original volume and re-equilibrate
48
What are the 3 stages of freezing sperm?
- supercooling
-ice crystallisation
-cooling is resumed
49
How are sperm damaged during freezing?
prolonged exposure to the salts in the solutions
50
what are the 2 effects of freezing sperm?
-reduces the number of surviving sperm
-reduces the competency of survivors
51
How is the optimal cooling rate for sperm determined?
Its a compromise between the cell damage caused by solution effects and intracellular ice
52
What are the requirements of successful cryopreservation?
1) Majority of spermatozoa should retain an intact plasma membrane
2) cell functions in the live population shouldn't be impaired
3) All organelles should be intact and functional;
4) Sperm DNA should be intact and able to support development
53
What affect does cryopreservation have on sperm lifespan?
Massively reduces it, they have capacitation like changes
54
What is the effect of cryopreservation on net fertility?
It's reduced
55
What percentage of spermatozoa survive cryopreservation?
60%
56
How does cooling affect sperm membranes
causes damage
57
What does cold shock do to sperm
Causes a form of premature capacitation
58
Name 3 common additives to sperm cyropreservation
- Egg yolk
-skimmed milk
soybean lecithin
59
How can ice crystal formation be avoided when preserving sperm?
Cooling it quickly through vitrification, which usually requires high concentrations of cryoprotectant, sperm cannot survive the initial dilution, however vitrification can be done without cryoprotectant
60
what chemical is used for vitrification with human sperm?
Liquid nitrogen, sperm is dropped directly into it and closed in straws
61
How is fertility measured?
By coohort:
-completed fertility
-parity progression ratio
By time period
- crude birth rate
-generala fertility rate
-age specific fertility rate
-total fertility rate
62
63
what does the 2017 sperm-decline meta analysis predict?
That by 2045 we will have a median sperm count of 0
64
Describe a cohort fertility measure
-Number of children born to a cohort of women (women born or marries within a specific timer period) over their lifetime
65
Describe a period fertility rate
-Number of children born within a specific time period, usually expressed per 1000 women alive in the middle of the period
66
What is completed ferility?
The number of children a woman has had over her lifetime
67
why is completed fertility useful?
Reports what happened within the survey population
68
What are the issues with completed fertility?
-accuracy
-survivor bias
-comprehensiveness
-retrospectiveness
69
What is the parity progression ratio?
The proportion of women (or couples)
with at least n children (parity n)
who go on to have at least one more child
(parity n+1)
e.g. P01 the proportion of childless couples who
go on to have at least 1 birth
P12 the proportion of couples who have had
1 child who go on to have a second birth etc
70
What is the advantage or parity progression ratio?
Predictive of likely future patterns
71
What are the disadvantages of parity progression ratio
Couple bias (P01) Single mothers?
Unhelpful re never-married childlessness
72
What is crude birth rate?
The ratio of live births in (say) a calendar year to the average population in
that period (often the mid year population).
Expressed as: births per 1000 population
(Births/Population) x 1000
73
What are the advantages of crude birth rate?
Easy to calculate and understand – annual
births/population
74
What are the disadvantages of a crude birth rate?
Requires universal vital registration, and accurate
censuses (ie not Nigeria)
Denominator affected by:
Changing Mortality
Migration
75
What is general fertility rate?
General Fertility Rate: GFR
(Births in period/N of Women aged
15-49 at mid period) *1000
76
What are the advantages of general fertility rate?
Sensitive to short-term change
(eg COVID; economic
downturns)
Accounts for migration
77
What are the disadvantages of general fertility rate?
-Requires universal vital
registration, and accurate
censuses (national-level; or
HDSS (Health & Demographic
Surveillance System)
-Doesn’t reveal change in fertility
over reproductive lifecourse
78
What is age specific fertility rate?
Age Specific Fertility
Rate
(Births to women
aged x /N of Women
in age group x at mid
period) *1000
79
What are the advantages of age specific fertility rate?
Reveals how fertility has
changed over the
lifecourse
Doesn’t require universal
vital registration, and
accurate censuses
[sample surveys are an
effective proxy]
80
What are the disadvantages of age specific fertility rate?
Retrospective, so relies
on memory [but usually
only over short period]
81
Describe the spacing pattern of fertility
Women space their children over their reproductive lifetime
82
Describe the stopping pattern of fertility
Women have children ealy and then stop over time
83
Describe the starting pattern of fertility
women have children a bit later in life and then gradually stop
84
Describe a synthetic cohort measure in terms of measuring fertility
Number of children who
would be born per
woman if she lived to the end of
her childbearing years
and bore children at each
age in accordance with
prevailing age-specific
fertility rates
85
How is synthetic cohort fertility measured?
Women Children per
aged 1000 women
15-19 127
20-24 232
25-29 250
30-34 207
35-39 129
40-44 57
45-49 16
15-49
1018 total
Women spend 5 years in each age group so multiply
total by 5
1018*5 = 5090 children per 1000 women
Express for 1 woman who survives to her 50th
birthday:
5090/1000= 5.09
86
What are the advantages of synthetic cohort fertility measure?
Easily comparable, and
comprehensible (a proxy for
births per woman)
87
What are the disadvantages of cohort fertility measure?
A synthesis – doesn’t actually
represent the fertility of any
specific woman [or cohort]
88
What is capacitation?
The final maturational stage of spermatozoa that takes
place in the female genital tract, before spermatozoa gain
the ability to fertilize oocyte.
-Involves a change in the plasma membrane and rapid progressive motility
89
How are sperm stored in the female reproductive tract?
Oviductal cells provide nutrients which nurture them and support them
90
How long can sperm survive in the human reproductive tract?
5 days
91
How long can sperm survive in the chicken reproductive tract?
21-30 days
92
How long can sperm survive in the reproductive tracts of snakes?
up to 7 years
93
How long can sperm survive in the reproductive tract of stick insects?
77 days
94
At capacitation, which factors are changed when the sperm interacts with the oviductal cells, and through which mechanisms?
-PH and Calcium ions
-Receptor-ligand signal transduction
95
Which cells may alter gene and protein expression in oviductal cells?
-Oocytes
-Sperm
96
What types of signals modulate peri-conception?
-Long and short range
97
Describe short range signals in peri-conception
Local responses to gametes and embryo
98
Describe long range signals in periconception
*Environmental factors
*Nutrition
*Hormonal status
99
Why it is important to study maternal communication with gametes and
embryos (Peri-conception Environment)?
-Promoting embryo production
-Improving implantation and maintenance of pregnancy
-Improving offspring health (DOHAD)
100
Which ZP protein do spermatozoa initially bind to?
ZP3, this initiates the acrosome reaction
101
Which ZP protein do acrosome-reacted sperm interact with?
ZP2
102
Which ZP protein's function is currently unknown?
ZP1
103
Why is contraception using the ZP untannable?
It destroys the ovaries
104
What types of cells are spermatozoa and oocytes in terms of genetics?
Haploid
105
Describe cortical granule-reaction
The sperm binds with the ZP proteins
enzymes are released, the plasma membrane of the oocyte changes and blocks more sperm from entering, preventing polyspermia
106
Which ZP protein is involved in cortical granule reaction?
ZP2 changes to ZP2F
107
When does calcium oscillation begin?
10 mins after fertilisation
108
What is the purpose of calcium oscillation?
-Activates the egg and genome
-pronuclei of the oocyte and sperm develop
-Synergy forms the zygote
-Division of the zygote begins
109
When do the trophoblast and inner cell layer form?
Blastocyst
110
Why is there no net growth during the pre-implantation period?
The number of individual blastomeres increases, however overall embryo size is consistent
111
How common is infertility in the UK?
1 in 6 couples
112
What causes 70% of failures in ART?
Implantation failure
113
When does blastocyst hatching happen?
day 5/6 post conception
114
when does apposition happen in implantation
day 6/7 post conception
115
When does adhesion happen in implantation?
day 7/8 post conception
116
When does invasion happen in implantation
day 8/9 post conception
117
When is the window of implantation?
days 19-21 of the cycle
118
How long does it take an embryo to become a blastocyst?
5-6 days
119
Which structure in the embryo-mother communication makes TGFB?
Epithelium
120
What is the effect of TGFB on the implanting blastocyst?
-Increases invasiveness
-promotes adhesion of trophoblast
-promotes pre and post implantation embryo development
121
What is the effect of TGFB on the endometrium?
Promotes proliferation
decidualisation
promotes implantation
remodulation of the endometrium
122
What are the roles of oestrogen and progesterone in implantation?
-facilitate blastocyst attachment
-Regulates IL6 secretion
-IGF1 production in the endometrial stroma
-Oestrogen leads to IL-11 production, which promotes decidualisation
123
What role does activin A have in the endometrial stroma in implantation?
promotes decidualisation
124
What role does HB-EGF have in the endometrial stroma in implantation?
-promotes glandular secretion
-endometrial cellular proliferation
-decidualisation
125
What role does COX 2 have in implantation in the endometrial epithelium
leads to prostaglandins which:
-increases vascular permeability
-promotes implantation
-promotes adhesiveness to the uterine lining
126
When do oestrogen levels peak in the cycle?
Just before ovulation
127
when do progesterone levels peak during the cycle?
after ovulation
128
What are the 2 types of oestrogen receptors?
alpha and beta
129
What are the 2 types of oestrogen receptors?
A and B
130
How do ER mediate the biological affects of oestrogen?
interacting with the site-specific DNA and other proteins
131
How do PRs enact the effects of progesterone
activating PRs to act in a genomic fashion to regulate transcriptional responses of implantation related genes
132
What does autocrine signalling mean?
A cell secreted factors which affect that same cell
133
What are the effects of human chorionic gonadotropin?
-Autocrine effects on trophoblast
-Paracrine effects on maternal ovary and endometrium
134
What is the role of interleukin-1a and B in implantation?
-First response of the blastocyst to the receptive endometrium, inducing a second wave of cytokines
135
What is the role of IGFBP-1?
IGFBP-1-modulate the mitogenic and metabolic
effects of insulin-like growth factors IGF1 and IGF2
136
Where can IGF1R and IGF2R be found?
localized to the predecidual stromal cells in late
secretory-phase endometrium and to decidual cells
during pregnancy
137
What does IGFBP-1 interact with?
The IGFBP-1 interacts with the IGF2 synthesized by
trophoblasts
138
What inhibits IGFBP-1
IGF2 and IL-1β are inhibitory to IGFBP-1
139
Describe mucin 1
A highly glycosylated polymorphic mucin-like protein-
“barrier to implantation
140
Describe the expression of mucin 1
expressed at a high level in the mid-secretory phase
and being more abundant in fertile then infertile
women.
141
What is the role of mucin 1 and how has this been demonstrated in vitro?
establishment of stromal decidualization and its down-regulation locally at the region of implantation sites
In vitro- MUC l staining had disappeared from
epithelial cells beneath the attached embryo and that staining was unaffected in cells not at the implantation
site
142
What type of molecules are interleukins?
Cytokines
143
Desribe osteopontin in a receptive endometrium?
Upregulated
144
Where is osteopontin expressed?
epithelial, immune and vascular cells
145
Which hormone impacts osteopontin and when in the cycle does this happen?
upregulated by progesterone in the mid secretory, receptive phase of the menstrual cycle.
146
What does osteopontin bind to ?
binds to cell surface receptors and signalling through these adhesion proteins, and co receptors includingIGF1R, EGFR and FGFR
147
Define extracellular vesicle
Definition: a membrane-enclosed nano-sized
vesicle discharged by a cells that carry DNA,
RNA, and proteins between different cells
148
What are the 3 classes of EVs?
EVs are generally subdivided into three
classes: apoptotic bodies, microvesicles, and
exosomes
149
What is the size of small EV's/ exosomes
40-100 nanometers
150
What is the size of large EV's/microparticles
100-1000 nanometers
151
What is the size of apoptotic bodies?
bigger than 1 micrometer
152
Which cells do EV's derive from in the tumour microenvironment?
immune and inflammatory cells,
stromal fibroblasts, and endothelial cells forming the
blood vessels
153
How do Ev's contribute to oncogenesis?
carrying several surface markers and signalling
molecules, oncogenic proteins and nucleic acids that can be transferred horizontally to the stromal target cells and condition the tumour microenvironment for an improved tumour growth, invasion, and metastasis
154
What functions do EV's play in the cardiovascular system?
Cardiovascular function
* biological roles in maintaining normal cardiac
structure and function under physiological
conditions.
* EVs contribute to the development of cardiovascular
diseases
155
Describe EV's on the pre-implantation stage
EVs from uterine fluid during the pre-implantation stage which contained a higher abundance of proteins
involved in cell apoptosis
156
Describe EV's in the implantation stage
EVs derived from the implantation stage had
a higher abundance of proteins involved in cell
adhesion
157
Give an example of the role of EVs in implantation
Endometrial EVs modify trophectodermal cells for embryo
adhesion and invasion
* EVs act in a paracrine manner via communicating
between the endometrium and embryo
158
In the Dutch Famine Birth Cohort Study, what was the effect of famine in between conception and the 1st trimester?
A higher risk of coronary heart disease
159
In the Dutch Famine Birth Cohort Study, what was the effect of famine in between the 1st trimester and the 2nd trimester?
Increased prevalence of obstructive airways disease
160
In the Dutch Famine Birth Cohort Study, what was the effect of famine in between the 2nd and 3rd trimesters?
Higher bp and mental diseases
161
What diseases is someone more disposed to if they are born with a small body weight?
-Adult cardiovascular disease
-Type 2 diabetes
-Osteoporosis
-Schizophrenia
-Depression
162
What diseases is someone more disposed to if they are born with a large body weight?
cancer
163
What are the effects of maternal low protein diet from fertilisation to blastocyst stage?
Causes offspring to exhibit cardiovascular disease:
* Increased blood pressure throughout adult life
* Smaller heart mass (females)
* Increased lung ACE activity (enzyme that increase blood vessel contraction)
* Reduced capacity to dilate arterial vessels
164
Describe the epigenetic landscape theory
-processes by which the genotype gives rise to the phenotype
-stably maintained mitotically (and potentially meiotically) heritable patterns of
gene expression that occur without changes in the DNA sequence
165
Are epigenetic modifications reversible?
Yes
166
Give 3 methods of epigenetic modification
-Histone acetylation
-Histone methylation
-cpG DNA methylation
167
Give 3 factors which may modify someones epigenetic profile
-Healthy food
-Healthy lifestyle
-medications
168
Describe genome-wide reprogramming of methylation
-Fetal germ cells, low
-mature gametes, high methylation
-zygote, high methylation
-embryo-blastocyst, low
-developing fetus, increasing
169
List 3 things that Maternal-embryonic communication regulates
* Features of blastocyst morphogenesis
* Coordination of implantation
* Maternal immunotolerance
170
What is developmental plasticity?
Developmental plasticity – ‘selecting’ the
right phenotype to fit the anticipated future
environment
171
Which diseases see an increased incidence in children born from IVF?
Beckwith-Wiedemann and Angelman syndrome
172
173
How often does cleavage occurs pre-implantation?
10-12 hours
174
At what stage in embryo formation is an embryo ready to implant?
Blastocyst
175
When does compaction happen?
When an embryo has 16-32 cells (morula)
176
Which part of the embryo forms the placenta?
trophectoderm
177
Describe the maternal to zygotic transition
Maternal to zygotic transition (MZT) is the period during which zygotic genes are activated and maternal transcripts are cleared.
MZT
178
How are maternal RNAs decayed in yhr MZT?
-maternal-factor-mediated
zygotic-factor-mediated pathways
179
How are epigenetics modified (5)
-methylation
-acetylation
-ubiquitination
-phosphorylation
-histone modification
180
What is compaction dependent on?
calcium ions
181
How does the trophectoderm form?
-Cell-cell adhesion increases
-outer cells become polarised and form the trophectoderm
182
How can you tell if an embryo is compacted?
The individual blastomeres become indistinct
183
Which transmembrane cell-cell adhesion molecule drives compaction?
E-cadherin
184
Describe the expression and location of E-cadherin.
E-cadherin is expressed from early cleavage but relocated to regions of cell-cell contact at the time of compaction, probably
through activation of Pk-C
185
How does E-cadherin connect blastomeres?
Connect each other to each others cytoskeleton
186
Describe the blastocyst formation model
Blastocyst formation is dependent upon the polarized distribution of the Na/K- ATPase confined to the basolateral membrane domains of the trophectoderm
This establishes a trans-trophectoderm ion gradient that facilitates movement of
water across the epithelium facilitated by the presence of both apical and basolateral
AQPs
These events combined with the establishment of a trophectoderm tight junctional seal to prevent the loss of fluid out of the embryo through paracellular routes results in the expansion of the embryo and the formation of the blastocyst
187
What animal is 2-cell arrest common in?
- mice
188
Give 3 signs of 2-cell arrest
-Cell shrinkage and lysis, possible autophagic cell death
- presence of midline cellular fragmentation
- complete embryo fragmentation
189
What animal does abnormal embryo development happen in?
Humans
190
Give 3 examples of abnormal development that may be seen in human embryos
- Fragmentation without loss of blastomeres
-embryo arrest
-fragmentation with loss of blastomeres
191
Describe preimplantation human development up to day 6
day1- fertilised pronucleate eggs
day 2- 2 to 4-cell
day 3- 8-16 cells
day 4- compaction and morula formation
day 5- cavitation
day6- blastocyst formation and expansion
192
Describe the metabolism in pro-nucleate and cleavage stage emrbyos
Before compaction, the embryo has a metabolism based on low levels of oxidation of pyruvate, lactate, and specific
amino acids
193
Describe the role of cumulus cells in pro-nucleate and cleavage stage emrbyos
-The ovulated oocyte is surrounded by, and is directly connected to, cumulus cells which actively produce pyruvate and lactate from glucose.
* This creates a high concentration of pyruvate and lactate and a low concentration of glucose around the fertilized oocyte
194
Describe the ratio of ATP:ADp in pro-nucleate and cleavage stage emrbyos
The early embryo is characterized by a high ATP:ADP level, which in turn allosterically inhibits phosphofructokinase (PFK), thereby limiting the flux of glucose through the glycolytic pathway before
compaction
195
Briefly describe embryo metabolism from cleavage to blastocyst
-Energy metabolism via pyruvate is dependent on oxidative phosphorylation and this happens in the earlier stages of embryo development, however glycolysis becomes more used in the later stages
-TCA cycle happens in the mitochondria and happens in the earlier stages, however, the embryo then becomes dependent on glycoloysis
196
Describe production of ATP from from cleavage to blastocyst
-Overall ATP production increases
- ATP production from oxidation decreases, whilst ATP production from glycolysis increases to a greater extent
197
What causes cumulus cell expansion?
LH surge
198
What allows the oocyte to detach from the follicle wall?
LH surge
199
When does an embryo require loads of energy and oxygen?
After compaction, the embryo exhibits greatly increased oxygen consumption (and an increased capacity to use glucose as an energy source.
200
Why may oxygen and energy requirements increase so much after compaction?
-The increase in oxygen consumption possibly reflects the considerable energy required for the formation and maintenance of the blastocele,
-the increase in glucose utilization reflects an increased demand for biosynthetic precursors
201
What is the effect of compaction on the ATP:ADP ratio
Consequently, there is a reduction in the ATP:ADP ratio, and a concomitant increase in AMP, which has a positive allosteric effect on PFK, thereby facilitating a higher flux of glucose through glycolysis
202
what products is glucose needed for in energy metabolism?
Glucose provides the pentose moieties for nucleic acid synthesis and is required for phospholipids and non-essential amino acids biosynthesis
203
Which glucose transporter genes are expressed consistently from oocyte to blastocyst?
-Glut1,3,8 and SGLT-1
204
From oocyte to blastocyst when is glut4 present?
blastoocyst
205
From oocyte to blastocyst when is glut5 present?
8-cell to blastocyst
206
Name 4 things that embryos take in from culture medium
-glucose
-pyruvate
-amino acids
-oxygen
207
Name 4 things that embryos put into culture medium
-Lactate
-ammonium
-amino acids
-human leukocyte antigen-G
208
How is glucose intake related to embryo viability?
higher glucose=higher viability
209
which gender of embryo has a higher glucose uptake and why?
-female
-4% more DNA on the X chromosome
210
How is viability affected by glycolytic activity?
-too low unviable
-too high unviable
211
When does oxygen consumption peak in embryo formation?
at the blastocyst stage due to compaction
212
Out of the oviduct and uterus, which has higher oxygen levels?
The oviduct
213
What are the levels of oxygen in the oviduct?
2-8%
214
How does oxygen affect glucose uptake?
-hpoxia=higher uptake of glucose
215
What is the role L-glutamine and L-alanine in foetal growth?
-Increases foetal growth and viability
216
What is the role methionine in foetal growth?
Enhance emrbyo survival and exert antioxidant function
217
What is the role of L-arginine in foetal growth?
Increase embryo survival and exert antioxidant function
218
What is the role of N-carbamyl-glutamate in foetal growth?
Increases embryo survival and suppresses embryo losses
219
What is the role of taurine in foetal growth?
Possess antioxidant and anti-inflammatory properties
220
What is the role of leucine in foetal growth?
Positive impact on embryo implantation and embryo survival
221
What is the role of Proline in foetal growth?
Improve reproductive performance
222
What is the role of glutamine in foetal growth?
Improves pre-implantation embryo development
223
What is glycine used for?
Amino acids fill several niches in embryo physiology, such as the use of glycine as buffer of intracellular pH (pHi)
224
Name 2 amino acids which are used as energy sources by the early embryo?
-glutamine
-aspartate
225
Why can amino acids be used a marker for the selection of embryos for transfer?
-Amino acids may be consumed by the embryo orsecreted either intact or metabolized.
*Therefore, amino acid turnover can be a suitable marker for the selection of embryos for transfer
226
How is embryo arrest linked to amino acids?
-high amino acid depletion were more likely to arrest
-Those with a gradual utilisation were more likely to develop
227
How does oxygen saturations at the cleavage stage relate to amino acid turnover?
Embryos cultured in high oxygen at the cleavage stage have a high amino acid turnover compared to those cultured in a low o2 environnment
228
How does oxygen saturations at the blastocyst stage relate to amino acid turnover?
Embryos cultured in high oxygen at the blastocyst stage had a lower amino acid turnover compared to those cultured in low o2
229
How does amino acid turnover relate to DNA damage?
High amino acid turnover = high DNA damage
230
Name 4 invasive methods for assessing embryo metabolism
-Taking polar bodies at the pronuclei stage
-taking a blastomere at the cleavage stage
-taking some trophectoderm at the blastocyst stage
-taking blastocoel fluid through blastocentesis
231
Name 4 non-invasive methods to assess embryo quality
-Time-lapse system
-spent culture media
-on-chip analysis
-live cell imaging system
232
What are the ideal qualities of metabolomic analysis?
-Non invasive
-reproducible
-objective
-rapid
-easy to use routinely
-cost effective
-ethically sound
-independent of other parameters
233
What are the benefits of high pressure liquid chromatography- MS?
can generate information on multiple compounds within a sample of culture media
234
What are the negatives of high pressure liquid chromatography- MS?
-Expensive equipment
* Expensive to run
* Requires trained personnel
* Longer analysis time
235
Describe time-lapse microscopy
Time-lapse microscopy (TLM) captures
images over time provides a combination of
morphological, dynamic and quantitative
information about developmental events
236
What information can time-laspe microscopy provide?
-Time to formation of 2-cell
embryos (first cleavage)
* Regularity in duration of
cell cycles
* Morphology of embryos,
fragmentation
237
What is a pro of time-lapse microscopy?
Does not need to open
incubators to monitor
embryo development,
reduces perturbation of
embryo environment
238
Give 3 signs that an embryo is of poor quality
-uneven cleavage
-multinucleated blastomeres
-high fragmentation
-high number of apoptotic cells
239
What is fertilisation?
Human fertilisation is the union/fusion of haploid gametes of a human egg and sperm to form the diploid zygote.
240
Where does fertilisation happen?
ampulla
241
Describe what happens to the spermatozoa in fertilisation
Spermatozoa
- Sperm Binding --- zona pelucida
- Acrosome Reaction --- enzymes to digest the zona pellucida
- Membrane Fusion - between sperm and egg, allows sperm nuclei passage into egg cytoplasm
242
Describe what happens to the oocyte during fertilisation
Membrane Depolarization
Cortical Reaction
Meiosis 2 - completion of 2nd meiotic division
243
Define cell cleavage
The zygotes of many species undergo rapid cell cycles with no significant overall growth, producing a cluster of cells the same size as the original zygote
244
Describe blastulation
blastula precedes the formation of the gastrula (a hollow cup-shaped structure having three layers of cells) in which the germ layers of the embryo form
245
Why is the zona peluza important?
-sperm interactions
-protects the embryo
-stops embryo attaching to the fallopian tube
246
What are the 3 stages of implantation?
-apposition
-attachment
-invasion
247
What is required for implantation to take place?
-Hatched blastocyst
-Receptive endometrium
-communication between the mother and embryo
248
When does the endometrium become receptive?
mid-secretory phase (days 19-23), window of implantation
249
Where is the inner cell mass positioned during apposition?
very close to the endometrium
250
Which cells underly the ZP?
Trophoblast
251
What is the effect of oestrogen on the endothelium?
proliferation and differentiation
252
when does progesterone peak in the cycle?
day 17/18-22/23
-secretory phase
253
Describe the features of an unreceptive endometrium
-Long apical microvilli
-High surface negative charge
-Thick mucin layer
254
What are the features of a receptive endometrium?
-Shortening of the microvilli
-Loss of surface negative charge
-Thinning of mucin coat
-Formation of pinopodes
255
What is a reliable marker for human endometrial receptivity?
The presence of pinopodes
256
What covers the EEC ?
Glycocalyx, heavily glycosylated mucin
257
What is the effect of glycocalyx on the endometrium?
-protects epithelial surface from infection
-provides lubrication
258
What is a potential negative of glycocalyx?
may act to deter the blastocyst implantation
259
What happens to glycocalyx in the presence of a blastocyst?
-Selectively cleared
-Exposure of blastocyst to epithelial apical epitopes and further interaction with the trophoblast
260
What does glycocalyx removal allow for?
-Trophoblast pinopodes direct contact and the beginning of the attachment phase
261
What are pinopodes?
Cytoplasmic projections that arise from the apical surface of the EEC
262
Describe the effects of hormones n pinopodes
-progesterone dependent
-inhibited by the presence of oestrogen
263
How do the epithelial cells of the endometrium change during the receptive phase?
The cells become less polar and become flatter
264
What is decidualisation?
differentiation of elongated, fibroblast‐like mesenchymal cells in the uterine stroma to rounded, epithelioid‐like cells during the menstrual cycle and pregnancy
265
What happens during decidualisation?
Transformation of endometrial stromal fibroblasts into specialized secretory decidual cells.
Decidualizing stromal cells acquire the unique ability to regulate trophoblast invasion, to resist inflammatory and oxidative insults, and to dampen local maternal immune responses.
266
When does decidualisation occur in humans?
mid luteal phase, independent of pregnancy
267
Which hormone drives decidualisation and what changes happen
Decidualization is happening at mid-luteal phase of menstrual cycle and is progesterone (from CL) driven that makes changes at both transcriptome and proteomic level.
268
When endometrial basal cells undergo decidualisation, how does their shape change?
From stromal to decidual (round)
269
Why are decidualised cells important?
they have the unique ability to regulate the trophoblast invasion
270
What produces HCG and what is its affect?
-The embryo
-increased receptivity
271
Describe the molecular messages seen in attachment
- switch off, Mucin1
-Come hitcher, LIF, adhesion molecules( integrins, selectins, E-cadherins)
272
Describe the molecular messages of invasion
-MMPS and TIMPs
-PGs
-COX-2
-IL-11
-VEGF
273
How does the embryo invade the epithelium?
It releases endolytic enzymes which dissolve it
274
Describe the endometrial factors involved in implantation failure
thin endometrium, altered expression of adhesive molecules and immunological factors
275
Describe the embryonic factors of implantation failure
genetic abnormalities of the male or female, sperm defects, embryonic aneuploidy or zona hardening
276
How long is the embryogenic phase?
14 days
277
How long is the embryonic phase?
6 weeks
278
How long is the foetal phase?
220 days
279
What is the baby called in the embryogenic phase?
The conceptus
280
When is HCG produced?
-Made in the trophoblast of the implanting blastocyst as early as 6-7 days after fertilisation
281
What does the ectoderm give rise to?
-Skin cells
-Neurons of brain
-melanocytes
282
What does the mesoderm give rise to?
-Cardiomyocytes
-skeletal myocytes
-tubule of kidneys
-Erythrocytes
-Smooth muscle cells in the gut
283
What does the endoderm give rise to?
Alveolar cells
Thyroid cells
pancreatic cells
284
What can be used to induce abortion before 7 weeks?
progesterone antagonists
285
Surgically, what may result in pregnancy loss?
Removal of the corpus luteum
286
When is progesterone production taken over by the placenta?
12 weeks gestation
287
Which structures make HCG?
Trophoblast cells
placenta
288
Where in GnRH released from, where does it act and what does it do?
Released from the hypothalamus, travels to anterior pituitary via portal vessels, acts on gonadotrophs to release the gonadotrophins FSH and LH
289
What does FSH do in men and women?
Causes follicles to grow and produce oestrogen in women
sperm production in men
290
What does LH do in men and women?
-Causes ovulation in women
-causes testosterone production
291
List things may which disrupt the HPO axis
-Stress
-low BMI
-excess exercise
-pituitary adenoma
-sheehan syndrome
-premature ovarian insufficiency
292
Describe a primordial follicle
Primordial follicle is the primary oocyte (arrested in 1st meiosis prophase) surrounded by flattened granulosa cells and a basement membrane
293
Which cells secretes oestrogen and has FSH receptors?
Granulosa cells
294
What is the functional unit of the ovary?
Primordial follicles
295
Why is only one egg ovulated per month?
-Antral follicles undergo atresia unless
“rescued” by high enough level of FSH -
requires good luck and timing
-a follicle starts maturing everyday, so only1 is timed correctly with the FSH surge
296
Which cells produce anti-mullerian hormone?
Granulosa cells
297
How can we measure the follicle count in women undergoing IVF?
By using AMH as a surrogate marker for the amount of follicles
298
When are hormone at the basal level?
Onset of menstruation
299
What causes a gradual rise in FSH?
Less negative feedback from oestrogen and progesterone
300
How many follicles per cycle are usually dominant?
1
301
Which cells produce LH receptors?
Granulosa cells in the dominant follicle
302
What causes the LH surge observed just before ovulation?
The effect of oestrogen on the pituitary switching from being negative to being positive
303
What does the LH surge lead to? (4 things)
* completion of first meiotic division (and then arrests at metaphase in meiosis 2)
* Rapid increase in size of follicle
* Granulosa cells lose FSH and oestrogen receptors and produce progesterone - progesterone receptors develop which allow positive feedback (important
later)
* Ovulation!
304
What happens to the granulosa cells after ovulation?
They hypertrophy and form lipid-rich lutein cells
305
How long will the corpus luteum live without pregnancy?
14 days
306
When is peak progesterone levels?
day 7 after ovulation (so usually day 21)
307
Why do developing follicles undergo atresia after ovulation?
Cos of suppression of FSH and LH from progesterone
308
How does the combined oral contraceptive work?
* Constant level of oestrogen and
progesterone
* Negative feedback on FSH and
LH
* Prevents development of follicles
and LH surge
* INHIBIT OVULATION
309
When is an appropriate time to check someone's baseline FSH and LH?
day 2-5 of their cycle
310
What may elevated levels of LH and FSH suggest?
That the person is in peri-menopause
311
When should someone's peak progesterone be checked?
7 days before their next period
312
What causes menstruation?
-Fall in progesterone leads to
* Spasm of spiral arterioles
* Ischaemic necrosis of endometrium
* Uterine contraction (fundus to cervix)
313
Which days are the proliferative phase?
days 5-14
314
How does oestrogen act during the proliferative phase?
Oestrogen causes proliferation of endometrium and
stroma
* Thickened endometrium
* Increased metabolic activity
* Increase number of progesterone receptors
* Oestrogen encourages myometrial contraction from
cervix to fundus (? aids sperm transport)
315
When is the secretory phase?
days 14-28
316
describe the uterus in the secretory phase
-Progesterone causes secretory changes in
endometrium
* Glands producing glycoprotein, amino
acids and glucose
* Spiral arterioles develop
* Myometrial activity is suppressed
317
How is PCOS characterised?
-Polycystic ovaries (multiple follicles develop)
* Hyperandrogenism (phenotypic or biochemical)
* Oligo/amenorrhoea
318
What causes polycystic ovaries and oligo/amenorrhoea?
High levels of free androgen disrupt HPO axis, multiple follicles develop but ovulation
occurs infrequently if at all (hence polycystic ovaries and oligomenorrhoea)
319
Why are heavy periods and an increased risk of endometrial cancer observed in PCOS?
Without ovulation there is no corpus luteum and without progesterone there is no
menstruation - endometrium keeps proliferating under influence of oestrogen
* Leads to infrequent, heavy periods and increases risk of endometrial cancer
320
How is subfertility treated in PCOS?
Treatment - fertility is ovarian induction with clomiphene, metformin, letrozole. IVF if this fails
321
What is done to decrease the risk of endometrial cancer in women with PCOS?
To reduce risk of endometrial cancer need to ensure regular period (at least 4x yearly) or
endometrial suppression normally achieved using progesterone contraception
322
How is a high BMI associated with PCOS?
-Obesity increases amount of aromatase which converts androgens to oestrogens
* Reduces amount of sex hormone binding globulin (therefore increasing free androgen)
* Improves insulin sensitivity - insulin resistance is implicated in reduced SHBG and increased production of androgens
323
What is lonng term PCOS associated with?
Long term PCOS increases risk of T2DM, endometrial Ca, dyslipidaemia and cardiovascular disease
324
How are women with recurrent miscarriage treated?
Progesterone supplementation to improve ongoing pregnancy rates
325
What is menopause?
Menopause is where the ovaries lose the capacity to ovulate
326
What causes symptoms associated with menopause?
-Falling oestrogen levels
327
What symptoms are associated with menopause?
-Vasomotor instability (hot flushes, poor sleep, migraines)
* Urogenital atrophy (vaginal dryness, pain during sex, recurrent UTI, prolapse)
* Altered mood and libido
* Osteoporosis
328
When is it normal to experience the menopause?
After 45
329
At what age can someone have premature ovarian insufficiency?
Under 40
330
What hormone can be used to screen for premature ovarian insufficiency?
FSH for women under 45
331
Describe HRT treatment and the considerations that need to be had before commencing someone on HRT
-HRT is primarily oestrogen replacement, effective to treat vasomotor symptoms, prevent osteoporosis, and has some protective effects on the cardiovascular system
* This is especially important in women experiencing early menopause
* If the woman has a uterus OR has had endometriosis they need a progesterone with their oestrogen replacement to prevent endometrial hyperplasia and endometrial cancer
* Considerations - oestrogen increases VTE risk, progesterone increases breast cancer risk
although absolute risk remains small
332
How many patients present for IVF due to unexplained infertility?
1/3
333
What is an acceptable number of normal sperm ina sample?
4%
334
Name 4 factors which may mean a male presents for IVF treatment
-Oligospermia
* Asthenospermia
* Teratospermia
* Azoospermia (OA / NOA)
335
How can sperm be collected in cases of obstructive azoospermia?
PESA : Percutaneous Epididymal Sperm Aspiration
TESE: Testicular Sperm Extraction
336
How is sperm extracted in cases of autonomic dysfunction?
Electroejaculation probe
337
When sperm look for the oocyte, what are they aiming for?
hyaluronic acid
338
What is the name of the model which explains how only one oocyte is ovulated per cycle?
Goujon model
339
How can the pituitsary be respressed and why is this necessary?
- agonists or antagonists, can cause ovulation which would prevent harvesting
340
Which method of pituitary repression causes an initial LH spike?
agonists
341
Which type of patient would be at risk of ovarian hyperstimulation?
A lady with a good ovarian reserve who is young
342
What does the dosage of drugs given in controlled ovarian hyperstimulation depend on?
Age
* Body weight
* Ovarian reserve (baseline FSH, LH, AMH)
* Antral Follicle Count
343
How is controlled ovarian hyperstimulation monitored?
* Scan
* Serum Estrogen
* Progesterone
344
How is ovulation triggered in controlled ovarian hyperstimulation?
LH
345
What is the criteria for egg collection in controlled ovarian hyperstimulation?
* 3 or more follicles > 17mm diam
* 10,000 iu hCG
* Trans vaginal Oocyte Recovery 36 hours later (after LH)
* GA or Sedation
* Risks 1:2000
346
How is embryo transfer done?
- Abdominal USs guided
-catheter
-maximum of 2 embryos
-1-2cm from fundus
347
How long is lutel support given for and what hormone is used?
2 weeks
progesterone
348
Why do we transfer blastocycts and not earlier embryos?
50% of day 3 embryos won't form blastocysts
-improve chance of pregnancy (embryonic activation around day 2/3)
-single embryo transfer
349
When can an embryo be transfered?
-when the lady is in her receptive endometrium period so day 19-21, so 5 days after ovulation
-If not menstruating regularly can build up the endometrium using progesterone
350
What are the possible complications of IVF?
-Multiple Pregnancy
* Ectopic Pregnancy
* Miscarriage
* Disappointment
* Infection – rare
* OHSS - rare
351
according to the older system for grading cleavage stage embryos, what is a grade 4 embryo?
Cells are of equal or unequal size;
fragmentation is moderate to heavy
352
according to the older system for grading cleavage stage embryos, what is a grade 3 embryo?
Cells are of unequal size; no fragmentation
to moderate fragmentation
353
according to the older system for grading cleavage stage embryos, what is a grade 2.5 embryo?
Cells are mostly of equal size; moderate
fragmentation
354
according to the older system for grading cleavage stage embryos, what is a grade 2 embryo?
Cells are of equal size; minor fragmentation
only
355
according to the older system for grading cleavage stage embryos, what is a grade 1 embryo?
Cells are of equal size; no fragmentation
seen
356
How are blastocysts graded?
-Are graded 1 - 4 according to degree of
expansion (and 5-6 degree of hatching)
* Blastocysts of grades 3 or higher are further
graded A-C for Inner Cell Mass (ICM) and
Trophectoderm
A - Tightly packed, many cells
B - Loosely packed, several
C - Very few cells
357
Describe embryo-endometrium communication day 5
Free floating blastocyst in utero
358
Describe embryo-endometrium communication day 6
Blastocyst hatching – Start of Window
359
Describe embryo-endometrium communication day 7
Blastocyst apposition to endometrium at
the beginning of the implantation window
360
Describe embryo-endometrium communication day 8
Blastocyst adhesion occurring when LIF is
maximally expressed by the endometrium
and blastocyst expresses the LIF receptor
361
Describe embryo-endometrium communication day 9
Blastocyst Invasion
362
Describe embryo-endometrium communication day 10
Implantation complete– End of Window
363
What male factors can contribute towards poor embryo quality?
Unexplained
– Iatrogenic (surgery, radiation etc)
– Chromosomal / Genetic
– Congenital / varicocoele
364
What factors can contribute towards a reduced ovarian reserve and poor embryo quality?
Age
– Iatrogenic
– Chromosomal / Genetic
– Congenital
365
What factors can lead to a poor endometrium which can lead to implantation failure?
* Unexplained
* Poor endometrial development
* Infections
* Polyps
* Submucous fibroids
* Congenital (Uterine Septa)
366
What uterine factors may lead to implantation failure?
* Fibroids
* Adenomyosis
* Mullerian dysgenesis
367
What tubal factor may lead to implantation failure?
* Hydrosalpinges
368
What endocrine factors may lead to implantation failure?
* Hypothyroid
* Hyperthyroid
* Diabetes Mellitus
* Polycystic Ovary Syndrome
* Premature ovarian failure
369
What immune disorders may lead to implantation failure?
* Autoimmune thyroid disorders
* Auto-immune gonadal diseases
* Abnormal endometrial cytokines– Elevated NK cells in endometrium
370
How is implantation failure defined
-Failure to achieve a clinical pregnancy
* following the transfer of at least four embryos
* at least 3 transfer cycles
, fresh or frozen
* in which embryos were good quality
* in women aged <40 years
371
What are the outcomes of IVF?
* Ongoing clinical pregnancy
* Cycle cancellation
* Failed fertilisation
* Failed implantation
* Miscarriage
* Ectopic pregnancy
* Implantation failure (repeated)
372
What investigations may be done in someone with repeated failure of implantation?
-Ovarian reserve tests: FSH, AMH,
antral follicle count
* Parental karyotype (?aneuploidies)
* Hereditable/acquired thrombophilia
* Pelvic imaging (TVS/TAS)
* Hysterosalpingography
* Hysteroscopy
373
What tests may be done to investigate the ovarian reserve?
FSH
AMH
antral follicle count
374
Which tests may be done in the interest of research in someone with repeated implantation failure
* Sperm DNA
fragmentation
* Natural Killer Cells
375
What general measures may be taken to improve embryo quality?
* Encourage couples to try early
* Good diet
* Reduce / Cigarette smoking
* Reduce alcohol
* Weight control
376
What specific measures may be taken to improve embryo quality?
* Ovarian stimulation protocol
* Gamete donation (for parental aneuploidies)
* Insemination by ICSI
* Careful embryo selection - embryoscope
* Blastocyst transfer
* Assisted hatching
* Improved embryo transfer techniques
* Sperm DNA fragmentation tests ???
377
What is antiphospholipid syndrome?
* A systemic autoimmune disease characterized by
vascular thrombosis and/or pregnancy morbidity
in the persistent presence of antiphospholipid
antibodies (aPL).
378
How is antiphospholipid syndrome evaluated?
aPL are currently evaluated by three tests – Abs against beta-2 glycoprotein I (anti-β2GPI abs) – Abs against cardiolipin (aCL), – Lupus anticoagulant (LA).
379
How does antiphospholipid syndrome cause defective placentation?
by interacting with both sides of the placenta.
* At the decidual level, aPL is proinflammatory
with neutrophil infiltration, secretion of
cytokines and complement activation
* At trophoblast level, aPL down-regulate hCG, integrins and cadherins resulting in reduced trophoblast proliferation and growth
380
What can treatment of antiphospholipid improve?
Reccurent miscarriages, not implantation failure
381
What is thyroid autoimmunity?
Described as the presence of autoantibodies
against thyroid peroxidase (TPO) and/or
thyroglobulin (TG)
382
What is thyroid autoimmunity associated with?
Associated with repeated implantation failure
(RIF)
383
What is the treatment for thryoid autoimmunity
Treatment is Thyroxine supplements
384
What are natural killer cells?
Natural killer (NK) cells are subpopulation of
lymphocyte in peripheral blood
385
Describe the role are uterine natural killer cells
Currently unknown, clinicians may test for them, and provide treatments however there is no evidence that they help
386
What are the causes of fluid within the endometrium?
* Ovarian stimulation (uterine or cervical)
* High dose Estrogen
* Low Estrogen
* Tubal - Hydrosalpinges
* Endometrial
– Polyp or fibroid
– Asherman’s synechia
– Cervical stenosis
– Chronic infection
* Others
387
How would a poorly developed endometrium appear on an US?
Thin
388
What are causes of a poorly developed endometrium?
* Poor ovarian reserve or POF
* Iatrogenic (medical, radiation
* Endometrial– Asherman’s synechia– Chronic infection
* Uterine fibroids
* Adenomyosis
* Others
389
What is level 1+ of evidence when managing implantation failure?
high quality meta-analyses of RCTs or RCT
with a low risk of bias
390
What is level 1-of evidence when managing implantation failure?
meta analyses or RCTs or RCT with a high
risk of bias
391
What is level 2 of evidence when managing implantation failure?
systematic review of case-control or well
conducted case control cohort studies
or cohort studies
392
What is level 3 of evidence when managing implantation failure?
case reports or case series
393
What is level 4 of evidence when managing implantation failure?
expert opinion
394
What is a stem cell?
A stem cell is a (relatively) primitive cell that is capable of:
Self-renewal - making a copy of oneself
Make a range of cell types (Potency).
Convert to a different cell types (Differentiation).
395
What can stem cells do?
Build embryos and tissues (development)
Repair tissues (regeneration)
396
What are iPS stem cells?
Induced pluripotency
397
What does it mean if a cell is pluripotent?
It can form any type of cell
398
How are human embryonic stem cells derived?
-Blastocyst
- Anti-trophectoderm anti-body to remove the trophectoderm
- Complement, to induce complement-mediated killing
- ICM cells then re-plated onto feeder cells (originally mouse fibroblasts)
399
How can human pluripotent stem cells be characterised?
-Flow cytometry/ in situ staining
-genetic
-epigenetic
- Gene expression: through microarrays/RNA-seq/ QPCR
-Differentiation: in-situ-staining, teratoma, clonogenic assays
400
In the context of stem cells, which is more important, molecular markers or functional tests?
Functional tests
401
What is the gold standard for functional tests in stem cells?
-Chimera or teratoma formation
402
What des it mean if a human embryonic stem cell is "normal"?
Death is favoured over self renewal and differentiation
403
What does it mean if a human embryonic stem cell is adapted?
It favours renewal over death or differentiation
404
What does it mean if a human stem cell is nullipotent?
Favours self-renewal over death, it cannot differentiate
405
What plays a role in human embryonic stem cells?
Selection pressure, affects self-renewal, differentiation and death
406
How can selection pressures be relieved in human embryonic stem cells?
-genomic alterations
-gross chromosomal changes
smaller changes
-point mutations
407
What are the advantages and disadvantages of using embryoid body to test pluripotency?
Pro: 3D structure
Con: not reproducible, not directed
408
What are the advantages and disadvantages of using spin embryoid body to test pluripotency?
Pros: 3D structures, differentiation is reproducible
Cons: not directed, needs different size EB's tp cover all lineages
409
What are the advantages and disadvantages of using spin embryoid body + growth factors to test pluripotency?
Pros: differentiation is reproducible and directed, 3D structures
Cons: Needs different size EB's to cover all lineages,many different methods and markers
410
What are the advantages and disadvantages of using monolayer growth factors to test pluripotency?
Pros: differentiation is reproducible and directed
Cons; No 3D structure, many different methods and markers
411
What are the advantages and disadvantages of using teratoma to test pluripotency?
Pros: 3D structure, extensive differentiation
Cons:Not reproducible, not directed, very expensive, difficult to quantitate
412
Why is differentiation normally irreversible?
-chromatin and methylation changes the prevent the re-expression of genes (epigenetic changes)
413
What method of non-integrating delivery of reprogramming factors is favoured?
Transient transfection of mRNA
414
How can you identify different colonies?
-Morphology
-Protein expression
-gene expression
-differentiation
-epigenetics
415
Name 5 animals which are bread by ART
Pigs
sheep equines
deer
chickens
turkeys
honeybees
416
Describe in vivo embryo technologies in livestock
superovulated cows, natural mating, embryos flushed from the track
417
Describe in vitro derived embryo technologies in livestock
Ovaries obtained from abattoirs, oocytes extracted, matures and fertilised in vitro
418
Are embryo technologies used in wild species?
Rarely
419
What is oestrus?
Period before ovulation where females want to mate
420
Which drug is used to synchronise oestrus?
PGF2 alpha
421
What can be used to prevent luteal regression?
Controlled internal drug release device or intravaginal progesterone
422
Describe the 2 principles of oestrus synchronisation for AI
1) shortening the lifespan of the corpus luteum vua PGFalpha
2) prolonging luteal phase progesterone/progestagen
423
Name 3 methods of semen collection
1) Artificial vagina
2) electroejaculation
3)massage of ampulla
424
How may semen samples be used to inseminate animals?
1) Through the cervix and into the uterus (cattle and pigs)
2) Directly into the vagina or partly through the cervix
3) Insemination into the uterine horns by laparoscopy (sheep and deer)
4) Non-surgical use of a very long (2m) catheter in pigs
425
What type of semen is used for cattle AI?
Frozen
426
What types of animals is it not possible to cryopreserve sperm in?
Marsupials
427
What counts as successful sperm cryopreservation?
-majority of sperm intact plasma membrane
-Cell functions in the live population shouldn't be impaired
-all organelles should be intact and functional
-Sperm DNA should be intact and able to support develppment
428
What are the problems with frozen semen?
-Typically 50% of sperm don't retain their plasma membranes
-They usually have a shortened lifespan
429
How does sex sorting semen affect conception and pregnancy rates?
It lowers pregnancy and conception rates
430
What are the applications for cloning technology in livestock?
-Multiplying outstanding F1 crossbred animals
-Rapid production of a small number of genetically superior animals for breeding
431
What are the problems with cloned animals?
-Higher perinatal and postnatal mortality than normal animals
-13% of clconed bovine embryos resulted in full-term calves, compared to 45% in vitro produced emrbyos
-only 64% of cloned calves survive 3 months
432
Describe mitochondrial heteroplasmy
The recipient oocyte has maternal mitochondria and the cell from the nuclear donor has mitochondria in it, so the resulting embryo has mixed mitochondria
433
433
What can be used to track the oestrus cycles of animals?
Faecal or urinary hormones (progestagen)
434
What can be a non-invasive method of monitoring age, sex and pregnancy status of animals?
Faecal/ urinary DNA and hormones
435
What are the benefits of using non-invasive techniques to assess populations?
-Sampling doesn't disturb or distress animals
-large, field-based and sequential data sets
-may provide a continuous view of reproductive fitness in wild populations
436
Describe the methods of mammalian cloning in endangered species
-Nucelei from one species are fused with enucleated eggs from another species to produce viable embryos
-embryos are transferred to females of a closely related species
437
What are the problems with mammalian cloning?
-Mitochondrial heterogeneity possibly creating mitochondrial diseases
-low success rate
-Animals produced have a poor survival
438
What are the ethical concerns for de-extinction?
1) Animal welfare:
-Poor health and social behaviour
-Concern for numerous egg donors and embryo recipients
2) Housing of potentially infirm animals in zoos
3) Natural habitats may no longer exist
439
