new technologies in assisted reproduction

Question 1

what is the definition of infertility?

Answer 1

failing to get pregnant after 2 years of unprotected sex.

Question 2

causes for infertility?

Answer 2

male - 30%
female - 30%
combined - 10%
unexplained - 25%
other - 5%

Question 3

how many couples does infertility affect?

Answer 3

1 in 6 couples

Question 4

what is the aim of assisted reproductive technology?

Answer 4

to bring the sperm and oocyte close together to increase chances of fertilisation and achieve pregnancy

Question 5

what of the types of assisted reproductive technology?

Answer 5

IVF
intra cytoplasmic sperm injection (ICSI)
preimplantation genetic diagnosis
mitochondrial donation 
gene editing

Question 6

what is causing infertility in animals?

Answer 6

pollution
deforestation
rising global temperature
1/3 of male fish in UK rivers are feminised due to pollution events.

Question 7

reasons people may want assisted reproductive technology?

Answer 7

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)

Question 8

what are the steps in IVF?

Answer 8

super ovulation 
semen collection 
insemination 
embryo culture 
embryo transfer
luteal support.

Question 9

how is super ovulation achieved?

Answer 9

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

Question 10

what are the risks with super ovulation?

Answer 10

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.

Question 11

what is the process of oocyte collection?

Answer 11

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

Question 12

tell me about the process of sperm preparation and selection?

Answer 12

preparation - removal of seminal plasma, WBC and bacteria.
selection - sperm viability (motility - percale swim up, plasma membrane integrity, acrosome integrity), genetic characteristics (sex sorting)

Question 13

what are the different ways of collecting sperm?

Answer 13

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

Question 14

how can sperm viability be tested?

Answer 14

• 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)

Question 15

how does flow cytometry work?

Answer 15

• 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

Question 16

how can sex be sorted?

Answer 16

• 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.

Question 17

what is robotic sperm?

Answer 17

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.

Question 18

when does cleavage occur in IVF?

Answer 18

embryo cleavage is 24 hours after IVF

Question 19

what happened when bovine embryos were exposed to human maturation media prior to blastocyst stage?

Answer 19

large offspring syndrome
often fatal for mother and calf at birth
organ defects in surviving calf

Question 20

when are the embryos from IVF transferred to the uterus?

Answer 20

2-5 days after IVF (4-8) cell stage

Question 21

when are 2 embryos transferred?

Answer 21

in the UK if the woman are over 38 or if they have other risks.

Question 22

how is the embryo transferred?

Answer 22

using a transfer catheter, with a trans-abdominal ultrasound to guid it

Question 23

what can be done with excess embryos?

Answer 23

cryopreservation

need permission from both partners to use embryos

Question 24

what are the success rate of IVF?

Answer 24

approximately 25%

Question 25

what are some factors which affect IVF success?

Answer 25

age
sperm quality
response to super ovulation
IVF lab

Question 26

what are some risk factors of IVF?

Answer 26

multiple pregnancies
greater risk of genetic diseases
genetic imprinting (epigenetic) may affect multiple generations

Question 27

what is ICSI?

Answer 27

intra cytoplasmic sperm injection
modified IVF
sperm injected directly into oocyte

Question 28

when was the first ICSI baby?

Answer 28

1992

Question 29

why is ICSI used?

Answer 29

if sperm are unable to fertilise oocyte

• abnormal morphology
• abnormal motility
• damaged acrosome

Question 30

problems with sperm choosing in ICSI?

Answer 30

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

Question 31

risk factors with ICSI?

Answer 31

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

Question 32

what is pre-implantation genetic diagnosis?

Answer 32

you can screen embryos for genetic diseases such as huntington’s, cystic fibrosis, sickle cell anaemia.

Question 33

how is pre-implantation genetic diagnosis carried out?

Answer 33

• 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

Question 34

why is mitochondrial donation done?
what is mitochondrial disease?
who is it inherited from?

Answer 34

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.

Question 35

how is mitochondrial donation carried out?

Answer 35

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

Question 36

other than mitochondrial donation when have there been 3 parents?

Answer 36

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.

Question 37

when was the first baby born by mitochondrial donation?

Answer 37

6th april 2016

Question 38

what is gene editing in human embryos?

Answer 38

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

Question 39

when did the HFEA approve gene editing in human embryos?

Answer 39

1st feb 2016

Question 40

what are the fertility benefits of the gene editing?

Answer 40

understand gene involved in human embryo development

improve embryo development after IVF

Question 41

In what kind of cells does gene editing have a limited use?

Answer 41

differentiated cells

• specific cells must be targeted
• each individual must be treated

Question 42

implications for embryo editing?

Answer 42

• 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

Question 43

how could gene editing be used for organ transplant from PIGS?

Answer 43

• 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.