new technologies in assisted reproduction Flashcards
what is the definition of infertility?
failing to get pregnant after 2 years of unprotected sex.
causes for infertility?
male - 30% female - 30% combined - 10% unexplained - 25% other - 5%
how many couples does infertility affect?
1 in 6 couples
what is the aim of assisted reproductive technology?
to bring the sperm and oocyte close together to increase chances of fertilisation and achieve pregnancy
what of the types of assisted reproductive technology?
IVF intra cytoplasmic sperm injection (ICSI) preimplantation genetic diagnosis mitochondrial donation gene editing
what is causing infertility in animals?
pollution
deforestation
rising global temperature
1/3 of male fish in UK rivers are feminised due to pollution events.
reasons people may want assisted reproductive technology?
infertility - female and male
absence of a partner (lesbian, gay transgender parent, single woman, death of spouse)
genetic engineering (eliminate hereditary disorders, sex selection, promote beneficial trades, generate transplant tissue, produce engineered species)
what are the steps in IVF?
super ovulation semen collection insemination embryo culture embryo transfer luteal support.
how is super ovulation achieved?
stimulation of ovaries to produce multiple follicles - manipulation of the hypothalamic pituitary gonadal hormone axis
- suppression of normal cycle using GnRH agonist
- high dose FSH and LH increase follicle number
what are the risks with super ovulation?
there are mild and severe effects
if drug regime is not ideal for the individual
- ovarian hyper stimulation syndrome - acute inflammatory condition that can be fatal
- IVF patients more likely to develop ovarian cancer - thought not to be from treatment but from original pathology.
what is the process of oocyte collection?
monitor follicular development by transvaginal ultrasound.
when there is 2+ follicles 16-18mm diameter
10000 IU hCG (LH like action) induces oocyte maturation
oocytes aspirated by ultrasound guided catheter
tell me about the process of sperm preparation and selection?
preparation - removal of seminal plasma, WBC and bacteria.
selection - sperm viability (motility - percale swim up, plasma membrane integrity, acrosome integrity), genetic characteristics (sex sorting)
what are the different ways of collecting sperm?
natural
electro-ejaculation (if men are unable to produce samples) - insert a probe into rectum next to the prostate gland - you get lots of seminal plasma but very few sperm as the testis are not being stimulated.
in animals you can use an artificial vagina
boar mate - a chemical which is the same as the pig male pheromone - they will mate with the woman because they think there is competition. It can also be used to see if woman are in oestrous
how can sperm viability be tested?
- basic sperm assessment: motility, morphology
- fluorescent staining assess viability
- plasma membrane integrity - DNA specific probe SYBR14 stains sperm with an intact plasma membrane green, DNA specific probe propidium iodine stains sperm with damaged membranes red.
- acrosome integrity - FITC PNA (peanut agglutin) stains outer acrosomal membrane green.
- assess using flow cytometry ( evaluates thousand sperm in a short time, measurements in real-time allowing continuous assessment in response to an experimental treatment)
how does flow cytometry work?
- sperm stained with fluorescent probes
- each fluorescent probe emits light at a specific wavelength when excited
- The flow Cytometer consists of: (a laser beam directed onto a stream fluid containing fluorescent labelled sperm, fluorescent detectors aimed at the point where the fluid passes through the laser)
- passage through the laser excites the fluorescent particles in the sperm causing them to emit light
- fluorescent detectors assess the number of sperm passing through the laser beam emitting light of a specific wavelength.
- Determine the proportion of membrane intact sperm
how can sex be sorted?
- it is a specialised type of flow cytometry
- sorts sperm into sub-populations based on fluorescent labelling
- one cell at a time
- FACS machine assigns electrical charge to sperm fluorescing at a specified wavelength
- sperm sorted by electrical charge
(routinely done in animals but can’t be done in humans) - common in pigs as testosterone makes pigs taste bad so farmers want females.
what is robotic sperm?
it is sperm controlled by magnets - a robotic addition to sperm
it has a corkscrew shaped motor
was created in germany 2016
been tested in bovine sperm and oocytes
drives sperm with abnormal motility
could be used to deliver drugs? - put drug in sperm head and drive it towards cancer.
when does cleavage occur in IVF?
embryo cleavage is 24 hours after IVF
what happened when bovine embryos were exposed to human maturation media prior to blastocyst stage?
large offspring syndrome
often fatal for mother and calf at birth
organ defects in surviving calf
when are the embryos from IVF transferred to the uterus?
2-5 days after IVF (4-8) cell stage
when are 2 embryos transferred?
in the UK if the woman are over 38 or if they have other risks.
how is the embryo transferred?
using a transfer catheter, with a trans-abdominal ultrasound to guid it
what can be done with excess embryos?
cryopreservation
need permission from both partners to use embryos
what are the success rate of IVF?
approximately 25%
what are some factors which affect IVF success?
age
sperm quality
response to super ovulation
IVF lab
what are some risk factors of IVF?
multiple pregnancies
greater risk of genetic diseases
genetic imprinting (epigenetic) may affect multiple generations
what is ICSI?
intra cytoplasmic sperm injection
modified IVF
sperm injected directly into oocyte
when was the first ICSI baby?
1992
why is ICSI used?
if sperm are unable to fertilise oocyte
- abnormal morphology
- abnormal motility
- damaged acrosome
problems with sperm choosing in ICSI?
no natural selection embryologist is not objective lack of info on what makes the perfect sperm used with abnormal/non viable sperm morphology reflects genetic viability
risk factors with ICSI?
how does the embryologist know what is the best sperm
we do not know the full effects of ICSI on the offspring
are we producing genetically viable embryos
what is pre-implantation genetic diagnosis?
you can screen embryos for genetic diseases such as huntington’s, cystic fibrosis, sickle cell anaemia.
how is pre-implantation genetic diagnosis carried out?
- routine IVF treatment
- In vitro embroiled maturation 2-3 days (8 cell stage)
all cells in the embryo are genetically identical and undifferentiated (blastomeres) - zona drilling (making a very small whole in the zona pellucida
- remove 1-2 blastomeres (it has no detrimental effect on the embryo)
- cells screened for markers of genetic conditions
- embryo transfer of unaffected embryos
- affected embryos allowed to perish
why is mitochondrial donation done?
what is mitochondrial disease?
who is it inherited from?
healthy children can now be born to a mother who carrie faulty mitochondrial DNA
all mitochondrial DNA come from mothers
Defects in mitochondrial DNA affect how the cells use energy
fatal mitochondrial disease affect brain, muscles, heart, liver and sight.
how is mitochondrial donation carried out?
essentially 3 parents
- remove nucleus from an oocyte harvested from a patient with defective mitochondrial DNA.
- remove and discard nucleus from a donors oocyte with mitochondria
insert the patients nucleus into the donor oocyte with healthy mitochondria
- fertilise the oocyte with sperm (IVF/ICSI)
- implant embryo into patient
other than mitochondrial donation when have there been 3 parents?
in 1990 - poor quality oocytes were injected with cytoplasm from healthy donors to improve IVF outcomes ( cytoplasm contains donor mitochondria)
some children were born with genetic disorders and so this procedure was banned.
when was the first baby born by mitochondrial donation?
6th april 2016
what is gene editing in human embryos?
it is a form of genetic engineering (CRISPR-Cas9 technology)
allows identification, removal and replacement of faulty genes.
human embryos can be edited to remove faults
when did the HFEA approve gene editing in human embryos?
1st feb 2016
what are the fertility benefits of the gene editing?
understand gene involved in human embryo development
improve embryo development after IVF
In what kind of cells does gene editing have a limited use?
differentiated cells
- specific cells must be targeted
- each individual must be treated
implications for embryo editing?
- CRISPR-Cas9 can edit human german line
- edited information can be inherited by the patients children/grandchildren (correct a genetic flaw, enhance a desired trait.)
- prevent the spread of disease (target specific disease after infection (eliminate HIV in human cells)
- engineer carrier species (render the mosquito unable to spread malaria )
- implications of rewriting the germ line are serious and impossible to predict
how could gene editing be used for organ transplant from PIGS?
- pig organs similar size to human
- however unable to transplant pig organs as genome contains virus (porcine endogenous retrovirus PERV, capable of infecting human cells )
- CRISPR-Cas9 used remove 62 PERV genes from kidneys and lung cells.
- do this in pig embryos to eliminate pervs in all differentiated cell types
- currently carrying out porcine transplant trial in primates.
- ethical/religious problems.
