76. Assisted reproductive technology. In vitro fertilization and embryo transfer. Gamete transfer , ICSI and other modifications.

Question 1

what is assisted reproductive technology ?

Answer 1

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

Question 2

what are the different protocols in suppression of hypothalamicpitatory axis is and why do we do this ?

Answer 2

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

Question 3

how does the standard long protocol work ?

Answer 3

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

Question 4

how can we see the follicular growth ?

Answer 4

transvaginal monitoring is performed daily or every other day

Question 5

what are the criteria for Hcg triggering ?

Answer 5

presence of atleast 3 follicles of atleast 18mm

estradiol level lower than 0.7 mol/l

Question 6

what are other medications for ovulatory induction not for controlled ovary stimulation ? and what is the difference n terms of follicle ?

Answer 6

antiestrogen -inhibition of negative feedback with estrogen resulting in increase FSH

• clomiphene citrate
• letrozole
• tamoxifen

controlled - multifoliclar
not controlled - unifollicular

Question 7

when is puncture and aspiration of the oocyte made ?

Answer 7

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

Question 8

after the eggs are aspirated? what happens

Answer 8

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

Question 9

when should the sperm be collected for fertilisation ?

Answer 9

60-90 minutes prior to fertilisation

Question 10

after the sperm is collected what is done to the sperm ?

Answer 10

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

Question 11

if the partner cant give sperm sample what is done ?

Answer 11

percutaneous epididymal sperm aspiration

Question 12

what are other fertilisation methods done ?

Answer 12

ICSI - intracytoplapmic sperm injection

Question 13

when ICSI is done the results of fertilisation is checked after ?

Answer 13

18-22h after insemination

Question 14

which zygotes are cultivated for embryo transfer ?

Answer 14

zygotes with two pronuclei and 2 polar bodies

Question 15

what determines the number of transferred embryo ?

Answer 15

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

Question 16

what are some of the regulations during embryo transfer ?

Answer 16

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

Question 17

how do we maintain the luteal phase after embryo transfer for the embryo to grow in optimal condition ?

Answer 17

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

Question 18

prior to ICSI what should be done to the oocytes ?

Answer 18

granulose cells are cleaned with the hypo of hyaluronidase enzyme with pasteur pipette

Question 19

explain the mechanism of ICSI ?

Answer 19

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

Question 20

what determines the success of ICSI ?

Answer 20

availability os sperm and their normal morphology

oocyte is good quality

adequate equipment

Question 21

artificial insemination occurs in two ways what are they ?

Answer 21

partner’s sperm

and donor’ sperm - which is frozen

Question 22

what are the indication for artificial insemination in partners sperm ?

Answer 22

hypospadias of the urethera

impotence

oligospermia with normal sperm motility

vaginismus

Question 23

what is an indication of done sperm artificial insemination ?

Answer 23

azoospermia
rh incompatibility
heridatory diseases

Question 24

what are the there techniques artificial insemination can occur ?

Answer 24

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.

Question 25

for artificial insemination to be successful what is needed ?

Answer 25

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

Question 26

what is cryopreservation used for ?

Answer 26

to preserve embryo to use in future IVF if present pregnancy fails or wanting a later pregnancy

Question 27

what is the mechanism of cryopreservation ?

Answer 27

cyroprotectnt penetrate the cels and cause intracellular dehydration and prevent the formation of ice crystals

Question 28

in cryopreservation what should be seen for successful embryo transfer ?

Answer 28

embryo should process intact zona pellucida

half of the blastomeres should be intact

Question 29

other than embryo what else can be cryopreserved ?

Answer 29

testicular tissue
sperm
ovarian tissue

Question 30

what are the complication of ART ?

Answer 30

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

Question 31

what is the ideal patient selection for assisted reproductive technology ?

Answer 31

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

Question 32

how can we test and ovarian reserve ?

Answer 32

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

Question 33

what are the indication for IVF ?

Answer 33

tubal disease
endometriosis
unexplained fertility
failed ovulation induction

Question 34

when is cryopreservation indicated ?

Answer 34

chemotherapy

Question 35

what is gamete intrafallopian tube transfer ?

Answer 35

sperm and unfertilised oocytes are transferred into fallopian tubes

fertilisation is then achieved in vivo

Question 36

what is important when having gamete transfer ?

Answer 36

normal fallopian tubes

Question 37

what is the procedure of gamete transfer ?

Answer 37

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

Question 38

what are some other way of assisted reproductive technology ?

Answer 38

zygote intrafallopian tube transfer
micromanipulation
gestational surrogacy
embryo or oocyte donation