76. Assisted reproductive technology. In vitro fertilization and embryo transfer. Gamete transfer , ICSI and other modifications. Flashcards
what is assisted reproductive technology ?
IVF:
controlled ovarian hyperstimulation - having atleast 4 follicle
follicle puncture
oocyte retrieval
followed by in vitro fertilisation
embryo culture
embryo transfer in the uterus
luteal support with progesterone
gamete intra- fallopian
also cryopreservation of human gametes and embryo’s
and intrauterine insemination
what are the different protocols in suppression of hypothalamicpitatory axis is and why do we do this ?
long protocol
short flare protocol - 1st day of menstruation scan is done to see the thickening of the endometrium and to rule out prescience of cyst in ovaries
if everything is normal the Gnrh analogue can be given
1 or 2 days later - daily application of hormone fsh
when atleast 3 follicles have grown to size of 18 mm
follicular maturation triggered by hcg
antagonist protocol
1st day of menstruation scan is done to check the thickening of the endometrium
and rule out presence of cysts in the ovaries
subcutaneous FSH started if everything is satisfactory
gnrh antagonist after 6 days of gonadotropin stimulation - prevents premature ovulation
3 follicles have grown to the size of 18 mm
final follicular maturation triggered with with hormone hcg
progesterone given vaginally
2-5 days later we can do embryo transfer
how does the standard long protocol work ?
gnrh is started in the mid luteal (day 21) phase or early follicular phase of previous cycle
causing an initial flare effect of LH
the pitutory release of LH is suppressed through negative feedback
preventing premature ovulation of the follicle during stimulation
(1st day )around time of menstruation we check for thickening of the endometrium and rule out cysts in ovaries
and if everything is normal FSH is initiated
serum level of estradiol is measured less than 40 pg/ml and no follicle seen more than 10mm confirming the pituitary down regulation
Controlled ovarian hyperstmulation with gonadotropins such as FSH can then start with step up and step down depending on level of estradiol and follicle development
given atleast when 3 follicles of atleast 18mm with US
around the 12th day final induction of final maturation and triggering oocyte release by HCG in replacement of lh
Oocyte is retrieved 36 hours after the hCG is given.
followed by daily supplement of hormone progesterone given vaginally
2-5 days after oocyte recovery embryo should be transferred
2 weeks later a pregnancy test is performed
how can we see the follicular growth ?
transvaginal monitoring is performed daily or every other day
what are the criteria for Hcg triggering ?
presence of atleast 3 follicles of atleast 18mm
estradiol level lower than 0.7 mol/l
what are other medications for ovulatory induction not for controlled ovary stimulation ? and what is the difference n terms of follicle ?
antiestrogen -inhibition of negative feedback with estrogen resulting in increase FSH
- clomiphene citrate
- letrozole
- tamoxifen
controlled - multifoliclar
not controlled - unifollicular
when is puncture and aspiration of the oocyte made ?
36 hr after the application of 5000E or 10000E hCG ultrasound guided needle piercing
the eggs are then aspirated from the follicles
10-30 eggs are retrieved
after the eggs are aspirated? what happens
eggs are prepared and stripped from surrounding cells
examined for maturity and quality
mature eggs are then paced in an IVF culture medium and transferred to an incubator to wait fertilisation
when should the sperm be collected for fertilisation ?
60-90 minutes prior to fertilisation
after the sperm is collected what is done to the sperm ?
Sperm washing involves removing any mucus and non-motile sperm in the semen to improve the chances of fertilization and to extract certain disease-carrying material in the semen.
A culture medium is carefully placed on top of the semen. The medium is a hospitable environment for the sperm, and healthy sperm will swim up into it
or density gradient centrifugation
the capacitated sperms placed into the culture media containing the oocyte with 4-6 hours of retrieval
if the partner cant give sperm sample what is done ?
percutaneous epididymal sperm aspiration
what are other fertilisation methods done ?
ICSI - intracytoplapmic sperm injection
when ICSI is done the results of fertilisation is checked after ?
18-22h after insemination
which zygotes are cultivated for embryo transfer ?
zygotes with two pronuclei and 2 polar bodies
what determines the number of transferred embryo ?
usually 2 embryo of best quality in blastocyst phase are transferred
when unsuccessful preceding of IVF or patient over 40 years of age - more than 2 embryos transferred
what are some of the regulations during embryo transfer ?
full urinary bladder
removal of cervical mucus and treatment of posterior vaginal fornix and cervix with culture media
avoid cervical tenaculum
usage of soft aseptic catheter under US
trans abdominal controlled ET
slow insertion of the embryo 15mm from fundus
after 1 minute in the fundus the catheter is slowly removed
check catheter for emitting embryo
how do we maintain the luteal phase after embryo transfer for the embryo to grow in optimal condition ?
intravaginal progesterone applications
starting from day of follicle puncture and lasting 14-17 days until pregnancy test is done
when positive result of pregnancy test - until 12-13 gestational weeks
prior to ICSI what should be done to the oocytes ?
granulose cells are cleaned with the hypo of hyaluronidase enzyme with pasteur pipette
explain the mechanism of ICSI ?
in a small petri dish sperms exposed to PVP to reduce motility
after that mineral oil to prevent them from dying
the tail is pressed bottom of the petridsih followed by aspiration
orientation and fixtation of the oocyte holding pipette so polar body is at 6 or 12 o clock
and micropunture of zona pellucida at 3 o clock position through injecting pipette
the sperm is injected pass the zona pellucida and oocyte cell membrane
what determines the success of ICSI ?
availability os sperm and their normal morphology
oocyte is good quality
adequate equipment
artificial insemination occurs in two ways what are they ?
partner’s sperm
and donor’ sperm - which is frozen
what are the indication for artificial insemination in partners sperm ?
hypospadias of the urethera
impotence
oligospermia with normal sperm motility
vaginismus
what is an indication of done sperm artificial insemination ?
azoospermia
rh incompatibility
heridatory diseases
what are the there techniques artificial insemination can occur ?
intrvaginal - low freq because of vaginal fluid / can be done at home
targeting to flood the vagina with semen
intracervical - immunological conflict due to antibodies in cervical
mucus
commonly used in home - wit semen
intrauterine - washed sperm
unwashed semen is used, it may elicit uterine cramping, expelling the semen and causing pain, due to content of prostaglandins.
for artificial insemination to be successful what is needed ?
fallopian tubes open and empty
normal ovulation or you need to have ovulation stimulation
menstrual cycle is closely observed, often using ovulation kits, ultrasounds or blood tests
sperm washing - where the sperm is separated fro the semen
Resting on the table for fifteen minutes after an IUI
what is cryopreservation used for ?
to preserve embryo to use in future IVF if present pregnancy fails or wanting a later pregnancy
what is the mechanism of cryopreservation ?
cyroprotectnt penetrate the cels and cause intracellular dehydration and prevent the formation of ice crystals
in cryopreservation what should be seen for successful embryo transfer ?
embryo should process intact zona pellucida
half of the blastomeres should be intact
other than embryo what else can be cryopreserved ?
testicular tissue
sperm
ovarian tissue
what are the complication of ART ?
after the transfer of three or more embryo into the uterine multiple pregnancy can occur
during follicle puncture - intraabdominal bleeding might occur due to injury of blood vessels
ovarian torsion and necrosis or rupture of theca lutein cyst in OHSS
due to the presence of multiple luteinising cyst nd vascular hyperpermability
stoping the use of hCG and using gnRH reduces OHSS
hemorrhagic shock can occur by using hcG induces vascular permeability leading to ascitis and hypovolumic shock
other mild cases - hyperestrogenmia , activation of the
infectious complication if antispectc equipments used
what is the ideal patient selection for assisted reproductive technology ?
less than 35 age
prescence of ovarian reserve
normal seminogram
couple must be screened negative for HIV
presence of hydrpsalpinges
fibroid uterus especially submucus
smoking
how can we test and ovarian reserve ?
FSH LEVELS in the first 2-3 days of menstrual cycle
estradiol level of the 2-3 days
antimullarian hormone - hormone produced individual follicles
antral follicle count through vaginal ultrasound
must be screened for HIV and hepatitis
normal uterine activity evaluated by hysteroscopy and sonohysteroscopy
what are the indication for IVF ?
tubal disease
endometriosis
unexplained fertility
failed ovulation induction
when is cryopreservation indicated ?
chemotherapy
what is gamete intrafallopian tube transfer ?
sperm and unfertilised oocytes are transferred into fallopian tubes
fertilisation is then achieved in vivo
what is important when having gamete transfer ?
normal fallopian tubes
what is the procedure of gamete transfer ?
super ovulation as done through IVF collect two occytes and a lot of motile spermfor each fallopian tube
all this placed in a plastic tube container - then passed through laparoscopically and inserted 4cm into the distal end of fallopian tube
what are some other way of assisted reproductive technology ?
zygote intrafallopian tube transfer
micromanipulation
gestational surrogacy
embryo or oocyte donation
