techniques of art
TRANSCRIPT
ART
Satin Patel, MDDecember 2, 2009
Objectives
• History of Assisted Reproductive Technologies• Understand ovarian stimulation and methods of
monitoring follicular development• Understand oocyte retrieval methods• Compare cleavage stage vs blastocyst transfer• Guidelines for # of embryos transferred• Review techniques of embryo transfer• Review complications of ART and their prevention
and treatment
History of ART
• Early 1970’s– First attempt at GIFT by Patrick Steptoe & Roberts
Edwards, United Kingdom• Used HMG/hCG simulation and laparoscopy for egg
retrieval.• Transferred 1 to 2 eggs and millions of sperm into
ampulla
History of ART1978 2003
World's first test-tube baby Louise Brown has a child of her own
Louise Brown, the first test-tube baby in the world, has given birth to a child of her own. The boy named Cameron was conceived naturally and without IVF. He was born weighing 5lb 6oz at St Michael's Hospital in Bristol just before Christmas and Louise describes him as "tiny but perfect" in an interview with The Mail on Sunday.
History of ART
• 1980s– 15% per cycle pregnancy rate with natural cycle IVF
• 1984– Two pregnancies following transfer of frozen-
thawed embryos (Zeilmaker)
• 1985– Transvaginal US-guided oocyte retrieval (Wikland)
History of ART
• 1990– PGD introduced, initially to screen for X-linked
disorders (Handyside)
• 1992– Advent of ICSI (Belgium)
Abdulkareem Sultan Al-Olama 7
Definition of Infertility & ART
• Infertility is defined classically as the inability to conceive after 1 year of unprotected intercourse. This definition is based on the cumulative probability of pregnancy:
Abdulkareem Sultan Al-Olama 8
Definition of Infertility & ART Cont’d
Month Monthly Probability Cumulative Probability
1 0.2 0.20
2 0.2 0.36
3 0.2 0.49
4 0.2 0.59
5 0.2 0.67
6 0.2 0.74
7 0.2 0.79
8 0.2 0.83
9 0.2 0.86
10 0.2 0.89
11 0.2 0.91
12 0.2 0.93
Abdulkareem Sultan Al-Olama 9
Definition of Infertility & ART Cont’d
• ART refers to all techniques involving direct retrieval of oocytes from the ovary
• ART procedures include IVF, GIFT, ZIFT, and ICSI.
• The simplest ART procedure, IVF has been around for over 20 years and is perhaps the most commonly recognized ART of all procedures.
AGING AND INFERTILITY
Abdulkareem Sultan Al-Olama 11
Common causes of infertility Cont’d
• Female factors– Cervical factor– Ovulatory factor (PCOs)– Uterine factor (endometriosis)– Pelvic factor
12
IVF
1. Hyper ovulation2. Egg Retrieval3. Artificial
Insemination 4. Embryo Transfer
ART
Types of ART (2006)
IVF - Indications
• Tubal Factor• Severe male factor• Diminished ovarian reserve• Endometriosis• Advancing age• Third party reproduction• Unexplained infertility
IVF – Current Methods
• Precycle Work-up• Ovarian Stimulation• Egg Retrieval• Embryo Transfer• PGD• Laboratory Handling
Precycle Workup
• Assess Ovarian Reserve– Day 3 FSH; AFC; CCT; AMH
• Assess Uterine Cavity– Sonohysterogram; HSG; Office Hysteroscopy
• Semen Analysis• Male and Female ID labs• Genetic screening
Ovarian Stimulation
• Natural cycle IVF– Very frequent monitoring for spontaneous surge– Retrieval can occur anytime, day or night– Low pregnancy rates (~15%)– Low efficiency and practicality
Ovarian Stimulation
• Gonadotropin COH– Starting dose• Age• Ovarian reserve testing• Diagnosis • BMI• Response in prior cycles
IVF –Monitoring
Dose adjusted based on US and E2 monitoring Usually requires 10-12 days of stimulation
Ovarian Stimulation
• GnRH Agonists (Lupron)– Increase pregnancy rates, decrease cycle
cancellations due to poor response or premature LH surges and allow batching
• GnRH Antagonists (Cetrotide, Antagon, Ganirelex)– Lack the “flare effect” of agonists– May be used to suppress the ovary prior to cycle start– May used to prevent premature LH surge (~14mm)
Ovarian Stimulation
• OCP Pretreatment– Prevents cyst formation by GnRH agonists,
decreases cycle cancellation, increases oocyte number and fertilization rates
• hCG– Purified Human (5,000 – 10,000 U) or Recombinant
hCG (250 mcg) is given when 3 follicles are > 18 mm in average diameter
– Resumption of meiosis and oocyte maturation and GC luteinization
Ovarian Stimulation - Protocols
Transvaginal Egg Retrieval
Assisted Hatching
Assisted Hatching
• Various protocols have been described– Partial zona dissection– Acid Tyrode’s assisted hatching– Laser-assisted hatching– Zona pellucida thinning
Laser vs Mechanical AH
Assisted Hatching
Assisted Hatching
• AH does not appear to improve the pregnancy rate or implantation rate in 1st IVF attempts.
• It does appear to be beneficial in patients with prior implantation failures.
• It is unclear whether AH helps patients in FET cycles, of advanced age or with thick ZPs.
• There is insufficient evidence to routinely recommend AH in patients undergoing ART.
ICSI
ICSI
ICSI
Indications for ICSI
• Moderate to severe male factor• Epididymal or testicular sperm• History of failed fertilization with IVF• Antisperm antibodies• Low egg number
ICSI Concerns
• Damage to the oocyte (meiotic spindle)
• Override natural safeguards that serve to prevent fertilization by abnormal sperm– Transmission of paternal genetic abnormalities
• Sex chromosomal abnormalities• Y chromosome microdeletions
• Karyotyping and Y chromosome deletion analysis should be offered to all men with severe male factor infertility who are undergoing ICSI.
ICSI Use
Embryos (Day 1 and Day 3)
Day 1: male and female pronuclei
Day 3: 8 cell embryo
2-cell 4-cell 8-cells
Embryo Development
hatched blastocyst
Blastocyst Transfer
Blastocyst (Day 5)
ICM – Inner cell Mass
TE - trophectoderm
Embryo TransferAbdominal USN guided
Day 3 or Day 5 Transfer
DAY 3 DAY 5
Day of Embryo Transfer
Blastocyst Transfer (Day5)
• Advantages:– Synchronizing embryo replacement with the
endometrium
hatched blastocyst
Blastocyst Transfer
Blastocyst Transfer (Day 5)
• Advantages:– Synchronizing embryo replacement with the
endometrium
– Enhance embryo selection• Higher implantation rate• Decrease the # of embryos transferred
• Decrease multiple gestations
• Longer time in culture provides opportunity for Preimplantation Genetic Diagnosis (PGD)
Blastocyst Transfer
• Disadvantages– More demanding of embryology lab personnel
and equipment– Requires modified cryopreservation and thawing
protocols– More cycles are cancelled due to lack of embryos
to transfer (Less embryos to freeze)– Increase rate of twinning
Day of Transfer Success Rates
Techniques of Embryo Transfer
USN-guided Embryo Transfer
Randomized-controlled trial (2002) – Vizcaya, Spain• abdominal US guidance (n = 255 women) • clinical touch embryo transfer (n = 260)
Clinical pregnancy rates (87% Day 3 transfers):• 26.3% (67/255) in the US-guided transfer group• 18.1% (47/260) in the clinical touch transfer group (P < 0.05)
Implantation rate – 11.1% vs. 7.5%
“Easy” transfers – 97% vs. 81%
Cochrane 2007
Cochrane 2007
Human Reproduction 2007
Anesthesia and IVF
• Adequate pain control is important for patient safety and comfort
• Wide variation in anesthetic technique amongst centers internationally.
• In the US, 95% of centers use conscious sedation
Anesthesia and IVF
• Studies have demonstrated a higher pregnancy rate with conscious sedation (28.2%) than under general anesthesia (16.3%)
• Important factors to consider when choosing an anesthetic agent• Whether the substance enter the follicular fluid• The toxicity of the anesthetic agent
Anesthesia and IVF
• Propofol is widely used during egg retrieval procedures
• The safety of Propofol has been extensively described in the ART literature– Follicular fluid concentrations do not appear to
increase with time of retrieval (length of sedation)– No difference in fertilization, cleavage and embryo
cell number with Propofol
Anesthesia and IVF
• Midazolam (Versed)– Most commonly used benzodiazepine for
conscious sedation– Minimal amounts are found in follicular fluid– No adverse effect on fertilization
Anesthesia and IVF
• Patients are commonly given Versed 1 mg; Fentanyl 50 µg; Propofol 1.5-2 mg/kg.
• Spontaneous ventilation via face mask O2
Cryopreservation (Freezing)
• Embryos– Can be frozen at any state from 2PN zygotes to
blastocysts– Slow freezing vs vitrification– Freezing at the blast stage may enhance
identification of the best oocytes for subsequent transfer
– ~80-85% of frozen blasts survive thawing and rexpand
Cryopreservation (Freezing)
• Sperm– Cryopreserved husband or donor sperm may be
used for ICSI or IVF and couples may freeze husband’s sperm as a back-up in case of difficulty collecting the day of retrieval
– In cases where epididymal or testicular extraction is performed, donor sperm-back up in recommended
• Oocytes– Currently experimental
Age and ART
Effect of Maternal Age
Ovarian physiology
PREPUBERTAL MENOPAUSAL
follicular pool
atretic follicles
atretic folliclesfollicula
r pool
atretic follicles
ovulated folliclesgrowing follicles
ovulated follicles
FERTILE ADULT
6-7 w
eeks
20 w
eeks
at b
irth
puberty
men
opau
se
Ooc
ytes
(M
illio
ns)
0
1
2
3
4
5
6
7
8
Numberofoocytes
A woman goes into puberty with about 400,000 eggs.During the reproductive years, usually only a single egg matures each cycle
Ovarian Physiology
Who is at risk for decreased ovarian reserve?
• Women over 35 years of age
• Previous oophorectomy
• Heavy smokers
• History of extensive ovarian surgery
• Unexplained infertility
• Previous chemotherapy or radiation
• Uterine artery embolization
Ovarian Reserve Testing
• CCCT (Clomiphene Citrate Challenge Test)
A dynamic test of ovarian reserve
Clomid is given at a dose of 100 mg/day on cycle days 5 – 9 with measurement of FSH and E2 levels on Day 3 and repeat FSH on Day 10
Clomid Challenge Test
serum FSH
serumFSH
100mg CC
3 5 9 10cycle day
Abnormal: FSH >10 mIU/ml before or after CC, D3 or D10, or E2 > 70 on D3
FSH 10-12 borderline, increase FSH doseFSH 12-14 ↓ PRFSH >= 14 – very few clinical pregnancies
Ovarian Reserve TestingAntral Follicle Count (AFC)
Ultrasound during early follicular phase
6-10 antral follicles per ovary is normal
<6 total predicts poor response – ↑ FSH dose, reduced pregnancy rates
AFC may be a better predictor of response than FSH
Antral Follicle Count (AFC)
Normal – 6 to 10 antral follicles
Abnormal (increased)20-30 antral follicles in a PCOS ovary
AMH
• Recent studies suggest that AMH is a superior marker for diminished ovarian reserve as compared to d3 FSH and Antral Follicle count.
• AMH levels correlate well with the total developing follicular cohort as well as response to gonadotropin stimulation.
0
10
20
30
40
50
60
70
80
90
<35 36-39 >40
Abnormal OvarianReserve
Normal Ovarian Reserve
Age in Years
Pre
gnan
cy L
oss
Rate
s (%
)
Scott et al 1999
Pregnancy Loss Rates by Age and Ovarian Reserve
Effect of maternal age on pregnancy loss rates after early documentation of fetal cardiac activity by TVUS
Age SAb rate
< 35 2.1%
36-39 16.1%
> 40 20%
Smith & Buyalos 1996
Pregnancy Loss Rates after + FHM by Maternal Age
Complications of IVF
• Short-term complications– Intraperitoneal bleeding– Pelvic infections– Adnexal torsion– Ectopic pregnancy – OHSS
Complications of IVF
• Long-term Complications– Multiple pregnancy– Perinatal outcome• Lower birth weights
– Genetic abnormalities– Congenital malformations– Epigenetic changes– Ovarian cancer risk ?
Multiple Pregnancies
Complications of IVF-Multiple Pregnancies
Type of Pregnancy Ave GA at time of Delivery Ave Birth Weight
Singleton 40 weeks 7 lbs
Twins 35 weeks 5.5 lbs
Triplets 33 weeks 4 lbs
Quadruplets 29 weeks 3 lbs
Number of Embryos Transferred during ART Cycles (2006)
SART/ASRM Guidelines for the Number of Embryos Transferred (2008)
AGE # of Embryos Transferred Conditions
< 35 yr Consider 1 Favorable prognosis
No more than 2 (cleavage stage/blast) All others
35-37 yr No more than 2 (cleavage stage) Favorable prognosis
No more than 2 (blasts)No more than 3 (cleavage stage)
All others
38-40 yr No more than 3 (cleavage stage)No more than 2 (blasts)
Favorable prognosis
No more than 4 (cleavage stage)No more than 3 (blasts)
All others
> 40 yr No more than 5 (cleavage stage)No more than 3 (blasts)
PGD
Engineering 124; Spring 2003 83
Commonly, more than 100 diseases can be detected through testing, including…
• Hemophilia A• Muscular dystrophy• Tay-Sachs disease• Cystic fibrosis• Down Syndrome
Removal of one cell for testing
PGD is not a new technology, but is due to the application of old techniques to the new knowledge gained from the Human Genome Project.
Engineering 124; Spring 2003 84
Viable and Desirable?
“This information is helping parents choose which embryos they want--and which to reject as unhealthy, or merely undesirable.” (Zitner 2002)
Engineering 124; Spring 2003 85
Undesirable Embryos
Disease Free Embryos• Frozen in storage • Donated to infertile couples• Donated to stem cell research/usage
Disease Carrying Embryos
• Donated to research• Discarded
THANK YOU