techniques of art

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