B8.065 Assisted Reproductive Technology Flashcards
what is ART
in vitro handling of both human oocytes & sperm OR embryos for purpose of establishing a pregnancy
types of ART
IVF embryo transfer gamete and embryo cryopreservation oocyte and embryo donation gestational carriers
what is NOT ART
intrauterine insemination
structure and function of FSH
heterodimeric glycoprotein (a chain similar to other anterior pituitary hormones, B chain makes it unique)
3-4 hour half life
stimulates granulosa cells > critical regulator of follicular development
regulation of FSH levels
suppressed by rising E levels from developing follicle
when is FSH the highest during the cycle
cycle day 3
level reflective of maternal age and ovarian reserve (ie, it will be higher if you have less eggs bc there is less negative feedback)
how is FSH used in ART
recombinant injection used to stimulate oocyte development
function of E2 in ART
most important product of granulosa cell from developing follicle
given to patients to thicken endometrial lining (but can slow follicular growth)
when does E2 peak during the cycle
late follicular phase
peak level around 200
structure and secretion of LH
heterodimeric glycoprotein (similar to HCG) 20 min half life secreted in a pulsatile manner -follicular q 90 min -luteal q 2-3 hr
function of LH in cycle
stimulates mature granulosa cells
stimulates lutenized cells of corpus luteum to produce progesterone
use of LH in IVF
dont give LH bc it rapidly degrades, give hCG instead (longer half life and less expensive)
given to mature and/or release oocyte with fertility treatment (for egg retrieval)
what happens when the oocyte responds to the LH surge
resumption of oocyte meiosis
completion of metaphase I
polar body extrusion
metaphase II arrest
response of follicle to LH surge
breakdown of follicular wall
cumulus-oocyte complex ovulated
lutenization of remaining granulosa and theca cells
when does ovulation occur with respect to the LH surge
34-36 hours after
function of the corpus luteum
secretes P4 and lipid droplets accumulate in cells
function of P4
prepared endometrium for implantation
maintains fetal-placental unit until placenta takes over (8-10 wks gestation)
P4 in ART
given to patients in luteal phase of fertility treatment to support possible pregnancy
steps of fertilization (start w ovulation)
- oocyte remains in ampulla of fallopian tube for 3 days (waiting for sperm)
- sperm reaches zona pellucida
- acrosomal reaction
- sperm head binds to sperm receptor
- fusion of sperm and egg plasma membranes
- cortical reaction
- zona pellucida hardening
- oocyte nucleus completes maturation to yield female pronucleus and second polar body
what is the acrosomal reaction
leads to breakdown of zona pellucida
acrosome reacts with perivitelline space
what is the cortical reaction
microvilli on oocyte surface surround sperm head, oocyte releases cortical granules
THIS LEADS TO zona pelludica hardening which prevents polyspermia
prezygote
fertilized pronuclear oocyte with male and female pronuclei
zygote
fertilized one cell stage without nuclei before cell cleavage
preembryo
conceptus during cleavage stages and until 14d post-fertilization (when primitive streak develops)
morula
preembry stage from 16 cell to blastocyst formation
72-96 hours post insemination
blastocyst
postmorula preembryonic stage in which fluid filled blastocele develops
embryo
conceptus from primitive streak until major organ development at the end of the 8th post ovulatory week
when does hatching of the blastocyst occur
day 6-7
when does implantation occur
day 8-9
embryo development within the fallopian tube
transport area of egg/sperm
fertilization occurs in ampulla
location of all embryo development until blastocyst stage
outcome of blastocyst components
inner cell mass > fetus
outer cell mass > placenta and fetal membranes
hatches from zona pellucida around day 7 and implants in uterus
process of implantation
- requires prior conditioning by P4 (similar success rates with various protocols, just important that it is given)
- P4 causes stromal cell swelling and accumulation of glycogen, lipids, and protein
- hCG from blastocyst stimulates CL to secrete P4 - blastocyst approaches uterine wall (adplantation)
- blastocyst attaches to uterine wall (adhesion)
- invades trophoblast and embeds
when is ART needed
bilateral tubal disease
severe male factor (total motile sperm < 5 mil)
genetic disorder
failed other treatment modalities
what is IVF?
in vitro fertilization
eggs surgically removed from ovary
sperm mixed with egg outside of body
fertilized egg (embryo) placed into uterus
-<5% of all fertility treatments in the US involve IVF
describe controlled ovarian hyperstimulation
- daily gonadotropin injection (mainly FSH) for 10 days to mimic follicular phase
- GnRH agonists given to prevent ovulation
- lead follicles 18-22 mm in diameter indicate possible MII oocyte
- hCG given for oocyte maturation
- retrieval 34-36 hours after hCG
how is oocyte retrieval performed
transvaginal ultrasound guided
performed under conscious sedation
no betadine for vaginal prep (just normal saline + abx afterward to prevent PID)
needle attached to pump to suction out the follicular fluid and cumulus-oocyte complex
fertilization in ART
ICSI or conventional
- activated oocyte releases 2nd polar body (1N DNA)
- male and female pronuclei form and approach each other
- 2 pronuclei in 6-10 hours suggests fertilization
- 1 or >2 pronuclei suggests abnormal fertilization
what is ICSI and when is it indicated
intracytoplasmic sperm injection
-injection of sperm into mature oocyte (at metaphase II)
indicated with severe male factor infertility (TMS < 5 mil)
steps of ICSI
- semen processing: centrifugation to remove seminal fluid
- oocyte preparation: gonadotropin stimulation, hCG injection, retrieval 34-36 hrs after hCG
- microinjection setting: inverted microscope
- sperm selection: put sperm in a dense solution to stop movement
- oocyte maturity: inject at MII
- oocyte penetration
- evaulation of fertilization
when do you use conventional insemination for IVF
no significant male infertility
no significant concern for failed fertilization
steps of conventional insemination
- fresh semen sample collected by masturbation
- select highly motile sperm with normal morphology
- incubated in protein rich media to achieve capacitation
- 50-100,000 motile sperm incubated with each oocyte for 12-20 hours
embryo culture for IVF
replicate conditions in fallopian tube
- temperature
- electrolyte, protein, carbohydrate concentrations
- pH
- osmolarity
- exposure to light
what is sequential media
mimics native environment
- D1-2 nonessential amino acids and pyruvate
- D3+ essential amino acids and glucose
when do you do an embryo transfer
@ the blastocyst stage (day 5-6)
- higher implantation rate
- lower twin rate
- ability to assess “true” viability
how are embryo transfers done
US guided
placement 2 cm from fundus
technique associated w success rates (don’t bang around in there)
embryo selected by blastomere number/uniformity and degree of fragmentation
factors that negatively impact IVF success rate
advanced maternal age cigarette smoking obesity communicating hydrosalpinges increasing duration of infertility low ovarian reserve
trouble with advanced maternal age
cohort of growing follicles diminished
chromosomal segregation errors in female meiosis associated with embryo aneuploidy
-most errors in meiosis 1
-defects in spindle assembly
discuss tubal infertility
30% of female factor infertility
salpingitis primary etiology
diagnosis of tubal infertility
hysterosalpingogram gold standard
how to work with tubal inferility issues
IVF higher success rate than tubal surgery
salpingectomy before embryo transfer recommended if communicating hydrosalpinx
-50% increase in success rate
-removes reflux of cytotoxic fluid from hydrosalpinx to uterine cavity
preimplantation genetic testing for monogenic disorders
direct testing of IVF embryos, typically for single, inherited genetic diseases (CF)
preimplantation genetic testing for aneuploidy
direct testing of IVF embryos for large-scale chromosome abnormalities (trisomy, deletions, duplications)
may improve success rates for women with advanced for maternal age and/or women with multiple number of high quality blastocysts
techniques for preimplantation genetic testing
PCR
- single gene defects
- primers are developed to surround mutation
- affected individuals saliva tested to ensure mutation can be detected using designed primers
