in vitro fertilization and embryo transfer "ivf"; overview on the story from a to z ……

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Ahmed Mowafy 2015

1931 Aldous Huxley published a science fiction novel called “Brave New World”. In this novel , Huxley realistically described the technique of IVF near we know now. in 1937, The New England Journal of Medicine describes this as “note worthy !!“.

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1958 Carl Gemzell obtained the first pregnancies following treatment with human pituitary gonadotrophin (hPG) and human menopausal gonadotrophin (hMG),

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1958 – 1959 Min Chang succeeded in growing rabbit embryos derived from oocytes fertilized in vitro, and in 1959 achieved a live birth by transfer of an in-vitro-fertilized oocyte.

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1961 Palmer from France described the first retrieval of oocytes by laparoscopy.

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1973 The first IVF pregnancy was reported by the Monash research team of Professors Carl Wood and John Leeton in Melbourne, Australia. Unfortunately, this resulted in early miscarriage

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1978 Louise Brown the first ever IVF baby occurred in Oldham, England on July 25, 1978. This birth was the result of the collaborative work of Patrick Steptoe and Robert Edwards

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Louise Brown Louise Joy Brown, the first test-tube baby 1978

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1983 • First pregnancy after oocyte donation

• The first vaginal egg retrieval using an abdominal ultrasound

1984 • First surrogacy embryo transfer baby born in

California, USA

• First report on pregnancy following translaparoscopic gamete intrafallopian transfer “GIFT”

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1985 Human pregnancy by in vitro fertilization (IVF) using sperm aspirated from the epididymis

1986 First report on pregnancy after translaparoscopic zygote intrafallopian transfer “ZIFT”

1989 First report on biopsy of human preimplantation embryos and sexing by DNA amplification

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1992 • The first pregnancy after intracytoplasmic sperm

injection “ICSI”

• Successful in-vitro fertilization and embryo transfer after treatment with recombinant human FSH

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Ahmed Mowafy 2015

(Centres for Disease Control and Prevention, 2013. National Summary and Fertility Clinic Reports. Atlanta, GA, 2013.) A H M E D M O W A F Y 2 0 1 5

Congenital absent or secondary to surgery which cannot be corrected by surgery and when surgery fails.

In patients with polycystic ovarian disease (PCOS) and other ovulatory problems.

Patients with endometriosis, often have tubal involvement and ovarian cysts (endometrioma).

Mullerian anomalies that requires a surrogate mother (prohibited in Moslem countries).

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due to poor quality semen parameters; Azoospermia, oligozoospermia, asthenozoospermia, anti-sperm antibody etc.

When woman's ovarian function is diminished with age. In many cases, this reduced function can be overcome through the use of IVF

after failure of ovulation induction and IUI.

Hostile cervical mucus which cannot be treated by IUI.

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1.preimplantation genetic diagnosis (PGD) to rule out presence of genetic disorders, Gender selection

2.Pregenetic screening (PGS)

3.egg donation and surrogacy "; where the woman providing the egg is not the same who will carry the pregnancy to term. (Prohibited in Muslims countries)

4.Fertility preservation: oocyte, sperm and embryo cryopreservation for further use , oocyte cryopreservation can be used for women who are likely to lose their ovarian reserve due to undergoing chemotherapy

5.same-sex couples, single and unmarried parents (ethical debate)

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Ahmed Mowafy 2015

Oocytes and sperms are placed into the fallopian tube. The procedure is applicable in patients who have normal fallopian tubes.

The fertilized oocytes in the pronuclear stage (zygotes) are placed into the fallopian tube. It has the same indications as GIFT. In this case the male and female pronuclei are seen in the cytoplasm.

The embryos (zygotes after cleavage) are placed into the fallopian tube.

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The oocytes and sperms are placed into the pelvic cavity.

A hole is made in the zona pellucida and 3-6 spermatozoa are introduced into the subzonal space.

A single sperm or spermatid is injected into the cytoplasm of the oocyte under the control of the microscope.

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Ahmed Mowafy 2015

1. The questions your patient is going to ask (FAQs)

2. The NBs you should tell your patients about

3. Instructions in the consent

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1. What would be the success rate of my ICSI?

• 32.2% for women aged under 35

• 27.7% for women aged between 35–37




• 1.9% for women aged 45 and over

( Human fertilization and embryology authority HFEA 2010 )

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1. What would be the success rate of my ICSI?

( Human fertilization and embryology authority HFEA 2010 )

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2. The Cost ?!!

• Argentina 4600$

• Austria 2500 EUR

• India 3000 – 3500 $.

• Kenya 4000$

• Malaysia 5000$

• UAE 6000$

• USA 7000 $

• Israel 5000 $

• UK 6000$

8000 – 12000 L.E A H M E D M O W A F Y 2 0 1 5

2. The Cost ?!!

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3. For how long should I stay in bed after embryo transfer?

Actually : no role.

The recommended time is

20-30 minutes of bed rest after ET.

(Marieke J et al, Ultrasonographic Evidence that

Bedrest after Embryo Transfer Is Useless Gynecol Obstet

Invest 2009;68:122-126)

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4. Does ICSI increase the liability of congenital defects?

• No ↑ the risk of CM.

( K. Paul Katayama et al, 2011 Meta-analysis genetics

Journal of Assisted Reproduction and genetics..)

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Alarm the patient of warning symptoms :

• rapid weight gain

• dyspnea

• palpitation

• vomiting

= Risk of OHSS

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For PCOS patients:

• ↑ Risk of OHSS.

• ↑ Duration of stimulation phase.

• The value of weight loss.

• Better chance for embryo vitrification

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For poor responders:

• ↓ success rate.

• Cancellation:

• Poor response.

• Poor quality oocytes.

• Failure of fertilization

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Remind your patient with:

• The time of B-HCG.

• The schedule of follow up visits after +ve pregnancy test.

• The higher possibility of multiple pregnancy.

• The possible need for fetal reduction

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1. Detailed history and clinical examination

2. Ovarian reserve testing

3. Uterine cavity exam

4. (mock) embryo transfer

5. Semen analysis

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• Duration of infertility

• Age of the patients

• Assessment of cause of infertility

• Body mass index “BMI”

• Previous investigations

• Previous surgical operations

• Previous diagnostic procedures (laparoscopy, hysteroscopy …etc)

• Previous trials of ICSI

• Special habbits (smoking, alcolism, caffeine intake ..etc)

• Basic trans-vaginal ultrasound

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a. Passive tests

• Anti Mullerian Hormone (AMH)

• Follicle Stimulating Hormone (FSH) - early follicular phase

• estradiol level- early follicular phase

• Basal FSH/ LH ratios

• FSH and estradiol

• Inhibin B level- early follicular phase

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a. Passive tests

• Antral Follicular Count (AFC)

• Ovarain Volume

• Ovarian blood flow

• Ovarian biopsy

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b. Dynamic tests

• Chlomiphene citrate challenge test (CCCT)

• Exogenous FSH Ovarian Response Test (EFORT)

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AMH Based COH

• < 0.5 --------------> don’t stimulate.

• 0.5 – 1 ------------ > don’t use long protocol.

• > 1 -------------- > use for long protocol.

• AMH (>4 ) ----> care for OHSS.

• Serum frozen immediately to avoid low levels.

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• 3D trans-vaginal ultrasonography

• Office hysteroscopy

• Saline infusion sonohysterography (SIS)

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• To assess the cervix and to make sure there are no abnormalities such as tumors, cysts or anything else that could possibly halt embryo transfer

• To determine the depth of your uterine cavity and the technique most likely to successfully place the embryos into your uterus.

• To determine what type and size of catheter he should use during the actual embryo transfer process.

• Insertion of a catheter, “Wallace catheter”, into the cervix and uterus as in actual embryo transfer

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• As a part of your initial fertility evaluation

• Semen is tested before IVF. If semen abnormalities are identified, consultation with andrologist.

• To determine the method of sperm collection

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• Infectious disease screening including HIV,HBV

• Prolactin , T3 ,T4 , TSH , DHEAS

• Genetic studies

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Ahmed Mowafy 2015

I. GnRH agonist drugs

Short Acting : • Triptorelin acetate (Decapeptyl) • Leuprolide acetate (Lucrin) • Buserelin acetate (Superfact)

Long Acting : • Goserelin acetate( Zoladex)

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I. GnRH agonist drugs

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II.GnRH antagonist drugs

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I. Urinary gonadotrophins

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II.Recombinant gonadotrophins

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Preparation Trade name Route price company

Progesterone Cyclogest 200 mg

Cyclogest 400 mg

Vaginal pessaries

Vaginal pessaries

90 LE

125 LE

Activis UK

Prontogest 200 mg

Prontogest 400 mg

Prontogest

Vaginal pessaries

Vaginal pessaries

ampoules

72 LE

102 LE

82.5 LE

Marcyrl co

Crinone 8% Vaginal gel 236 LE Merck

sharp co

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1. COCS 2. Anti oxidants 3. Folic Acid 4. Low dose Aspirin 5. Metformin 6. Vasodilators ; sildenafil , L-Arginine 7. Growth hormone 8. DHEA

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COCS

In the cycle preceding the IVF cycle

Regulation of the cycle if irregular.

prevent ovarian cysts, which sometimes develop during GnRH analog therapy.

Improved response to gonadotropins

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Metformin

Increases the sensitivity of body tissue to insulin effects

decreases high levels of testosterone.

May reduce 1st trimester pregnancy loss in women with PCOS.

reduces the incidence of gestational DM.

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Anti-Oxidants

Prevent oxidative damage to embryos

May improves fertilization and implantation rate

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Vasodilators ; sildenafil , L-arginine

increase blood flow in uterus and stimulate the development of endometrium may improve endometrial receptivity

DHEA , Growth hormone

May have a role in poor responders

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Ahmed Mowafy 2015

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Ahmed Mowafy 2015

1. Pre-stimulation preparation

2. Down regulation of pituitary hormones

3. Ovarian stimulation

4. Monitoring follicle development with ultrasound and serum hormone levels

5. Final oocytes maturation and hCG administration

6. Transvaginal ultrasound-guided oocyte retrieval

7. Fertilization in vitro

8. Embryo transfer

9. Luteal phase support

10.Pregnancy test and early pregnancy follow-up

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• Stop (smoking – alcohol)

• Reduce caffeine intake limiting caffeine intake to 200 mg per day. (One cup of coffee contains 100 mg caffeine)

• Increase Fluid Intake

A daily intake of 8 glasses of water daily is advised.

• Well balanced diet

• Reduction of body weigh (in obese patients) A H M E D M O W A F Y 2 0 1 5

• Folic acid

• multivitamins ( vitamin B12 – vitamin E )

• Anti-oxidants

• Low dose aspirin 75-81 mg

• Others : DHEA , sildenafil

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• COCs: in the cycle prior to the ART cycle

ensures that GnRH analog therapy will start at the proper time if you have irregular cycles.

prevent ovarian cysts, which sometimes develop during GnRH analog therapy

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• To avoid premature ovulation (premature LH surge) which indicates cancellation of the cycle.

• Prevents spontaneous ovulation and allows complete control of the cycle by exogenous hormones only.

• Drains the pituitary from its hormones leading to initial flare up effect followed by prolonged suppression

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International Society for Mild Approaches in Assisted Reproduction (ISMAAR), 2007

1. Natural cycle Unstimulated,

spontaneous cycle

Single

oocyte No medication

2. Modified natural

cycle

Semi-natural,

controlled natural cycle

IVF

Single

oocyte

hCG only

GnRHan and FSH/HMG

add-back

3. Mild

Soft, minimal

stimulation, ‘friendly’

IVF

2-7

oocytes

Low dose FSH/HMG,

oral compounds and

GnRHan

4. Conventional Standard, routine, COS > 8

oocytes

GnRHa or antagonist

conventional

FSH/HMG dose

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• Dose adjusted based on US and E2 monitoring

• Usually requires 10-12 days of stimulation A H M E D M O W A F Y 2 0 1 5

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HCG induces final follicular maturation.

When:

At least 3 follicles >18 mm .

E2: 150 pg/ml per >15mm follicles. .

Endometrium: Thickness >8mm, Triple line

IM injection of HCG 10,000 IU administered 34-36 h before the scheduled time of oocyte retrieval

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Do not trigger ovulation with the intention of fresh ET in women who have:

E2>3500 pm/l

or >20 follicles on US (NICE, 2013)

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The egg retrieval is performed at a time usually between 34 and 36 hours after hCG injection.

Egg retrieval is usually accomplished by transvaginal ultrasound aspiration.

It is done under short general anesthesia, and is 20 to 30 minutes procedure.

In some circumstances, one or both ovaries may not be accessible by transvaginal ultrasound. Laparoscopy may then be used .

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The eggs are aspirated (removed) from the follicles through the needle connected to a suction device.

Usually around 10-15 oocytes are aspirated

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After the eggs are retrieved, they are examined in the laboratory for maturity and quality.

Mature eggs are placed in an IVF culture medium and transferred to an incubator to await fertilization by the sperms

Shortly before or after the oocyte collection the male partner will be asked to give a sperm sample.

Intra-cytoplasmic sperm injection (ICSI) is performed.

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Fertilization check is performed the next day approximately 18 hours after sperm injection or insemination of the eggs.

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Embryo transfer may be performed on day 2, 3 or 5 post fertilization.

One or more embryos suspended in a drop of culture medium are drawn into a transfer catheter, a long, thin sterile tube with a syringe on one end.

The physician gently guides the tip of the transfer catheter through the cervix and places the fluid containing the embryos into the uterine cavity under guidance of trans-abdominal ultrasound on a full bladder

.

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Precautions and instructions for successful embryo transfer:

1. Avoiding the use of a tenaculum:

2. Removal of the cervical mucus plug:

3. Ultrasound-guided transfer: improves the placement of the catheter tip with respect to the endometrial surface.

4. Be sure that is no uterine bleeding

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Precautions and instructions for successful embryo transfer:

5. Do not touch the fundus: Since embryo transfer is often a blind shot, contact between the catheter and the uterine walls may result in trauma.

6. Keeping catheter stationary for at least one minute

7. Giving antiprostaglandins to prevent contractions: The insertion of a cannula into the uterus may cause contractions, and can hinder implantation.

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SART/ASRM Guidelines for the Number of Embryos Transferred (2008)

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SART/ASRM Guidelines elective single embryo transfer (2012)

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Day 5 (blstocyst) embryo transfer

1. Synchronizing embryo replacement with the endometrium

2. Enhance embryo selection

3. Higher implantation rate

4. Decrease the number of embryos transferred ; Decrease multiple gestations

5. Longer time in culture provides opportunity for Preimplantation Genetic Diagnosis (PGD)

6. Lower incidence of ectopic pregnancy

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Day 5 (blstocyst) embryo transfer

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SART/ASRM Guidelines Blastocyst culture and transfer in clinical-assisted reproduction (2013)

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Luteal phase supplementation , after controlled ovarian stimulation for IVF, has been a routine practice in IVF/ ET because stimulated IVF cycles are associated with a defective luteal phase in almost all patients . Pabuccu R, Akar ME. Curr Opin Obstet Gynecol. 2005;17:277–81.

Why ?

due to removal of large quantities of granulosa cells during the oocyte retrieval

prolonged pituitary recovery that followed the GnRH agonist downregulation

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When ?

A window for progesterone start time between the night after oocyte retrieval and day 5. (A systematic review and meta analysis. ASRM 2014)

Till 5 weeks after conception (7th week of gestation)

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How ?

Progesterone

Oral Route: Significantly associated with lower imlantation and pregnancy rate , higher miscarriage rate or both. ( fert. Sterility 1999, Human reproduction 1999)

Intra-muscular route

Vaginal route

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How ?

Progesterone

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How ?

Progesterone

IM Versus vaginal Route:

Intramuscular Progesterone in oil (50 mg/day) generates circulating Progesterone concentrations at or above the physiological range.

Vaginally administered Progesterone yields lower serum levels, but nonetheless achieves endometrial tissue concentrations up to 30-fold greater than those achieved with IM Progesterone

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How ?

Others

Estrogen and progesterone (COCs)

Controversy

GnRH agonist

hCG

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How ? ASRM Guidelines for progesterone supplementation in LP (2008)

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B-hCG is done after 14 days of embryo transfer

The theoretical LMP can be considered as:

IVF and ICSI: Reduction of 14 days (2 weeks) to the day of egg collection.

Replacement of frozen-thawed embryo (3 days): Reduction of 17 days to the day of embryo replacement.

Replacement of frozen-thawed embryo (blastocyst): Reduction of 19 days to the day of embryo replacement

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Ahmed Mowafy 2015

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Ahmed Mowafy 2015

International Society for Mild Approaches in Assisted Reproduction (ISMAAR), 2007

1. Natural cycle Unstimulated,

spontaneous cycle

Single

oocyte No medication

2. Modified natural

cycle

Semi-natural,

controlled natural cycle

IVF

Single

oocyte

hCG only

GnRHan and FSH/HMG

add-back

3. Mild

Soft, minimal

stimulation, ‘friendly’

IVF

2-7

oocytes

Low dose FSH/HMG,

oral compounds and

GnRHan

4. Conventional Standard, routine, COS > 8

oocytes

GnRHa or antagonist

conventional

FSH/HMG dose

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>> According to ovarian reserve

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I.GnRH agonist protocols

Types :

1.Long GnRH agonist protocol

2.Short GnRH agonist protocol

3.Ultra short GnRH agonist protocol

Effects :

1. Flare effect: Within 12 h and lasting 24-48 h : 5 fold increase of FSH 10 fold rise in LH & 4 fold elevation in E2.

2. down-regulation of pituitary hormones with suppression of the pituitary Gnt synthesis & liberation >> Arrest of follicular development

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Types :


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Types :


GnRH agonist From middle of the luteal phase (D19-21)

Gonadotrophins while GnRH agonist therapy is maintained.

Criteria of suppression:

i.Hormonal: E2 <50 pg/ml ,Progesterone < 1 ng/m , LH <5 IU

ii.US: No ovarian cysts , endometrial thickness <6 mm

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Types :

2. Short GnRH agonist protocol (flare protocol)

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Types :

3. Ultra short GnRH agonist protocol

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Types :

Ultra Long GnRH agonist protocol

GnRH could be given for three months before the start of hMG.

May be used in patients with severe endometriosis before the start of the treatment

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II.GnRH antagonist protocols

Types :

1.Small daily dose (LubecK)

2.Single dose (French)

Effects :

Inhibition of LH & FSH immediately without the initial flare up effect.

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Types :


HMG or FSH: From day 2 or 3 of the cycle

Cetrorelix or Ganirelix: 0.25 mg daily SC: from stimulation day 5 or 6 (fixed protocol) or leading follicle14 mm (Flexible protocol) onwards until the day of HCG

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Types :


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Types :

2. Single dose (French)

HMG or FSH: from day 2 or 3 of the cycle

Cetrorelix: single dose, 3 mg SC, on stimulation day 7

HCG is given when the follicles are mature by U/S &/or E2

GnRha single dose can be given instead of HCG to reduce incidence of OHSS

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Types :

2. Single dose (French)

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>> According to ovarian reserve

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Ahmed Mowafy 2015

1. Semen analysis and preparation

2. Oocyte collection and denudation

3. Incubation for 30 – 120 min

4. Intracytoplasmic sperm injection

5. Incubation and embryo grading

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Normal semen parameters

WHO 2010

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Ways to Collect Semen

I.Ejaculation

II.Surgical

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I.Ejaculation

1.Masturbation, directing the semen into a clean sample cup

2.Coitus interruptus - withdrawing the penis from the partner just before ejaculating

3.Coitus - by using a special silicon condom that does not contain any spermicides

4.Assisted ejaculation – electro-ejaculation used in paralegics

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II.Surgical

1.PESA =percutaneous epididymal sperm aspiration

2.MESA =microsurgical epididymal sperm aspiration

3.TESA =testicular sperm aspiration

4.TESE =testicular sperm extraction

5.Micro TESE = microsurgical testicular sperm extraction

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Cases with retrograde ejaculation :

semen is ejaculated into the bladder. The acidity of the urine will kill sperms quickly. Alkalination of the urine is very important in order to recover live motile sperms

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Sperm preparation :

1. Simple Wash

2. Swim-Up

3. Swim-Down

4. Migration-Sedimentation

5. Density Gradient Centrifugation

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Oocyte Denudation :

1. Mechanical

2. Chemical

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Oocyte Denudation :

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Oocyte grading :

1. Empty zona “EZ”

2. Germinal vesicle “GV”

3. Metaphase I “M I”

4. Metaphase II “M II”

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Oocyte grading :

Empty zona “EZ” Germinal Vesicle “GV”

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Oocyte grading :

Metaphase I “M I” Metaphase II “M II”

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embryo grading :

One-cell embryo embryo containing four on day 1 cells on day 2

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embryo grading :

eight-cell embryo day 5 blastocyst stage on day 3

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Ahmed Mowafy 2015

The IMSI process involves the assessment of sperm, at

very high magnification, using an inverted microscope.

This digitally enhanced microscope provides a much

greater magnifying power (about 6000x magnification)

compared to the one normally used in ICSI (200x).

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• It is a micro-surgical procedure on pre-embryos before embryo transfer

• Performed by making a small hall in the zona or outer shell of pre-embryos

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• Indications include :

1.More than 3 failed IVF trials

2.Poor embryos growth and morphology (less than 6 cells on day 3)

3.Thickening of the zona (more than 17 mm)

4.Cytoplasmic fragmentation (more than 20%)

5.Advanced maternal age ( more than 37 years old)

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Used in :

1.Sex selection

2.Diagnosis of aneuploidy

3.Diagnosis of balanced translocations

4.Single gene defect

Commonly, more than 100 diseases can be detected through testing, including (Hemophilia A,

Muscular dystrophy, Tay-Sachs disease, Cystic fibrosis and Down Syndrome)

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in vitro fertilization and embryo transfer "ivf"; overview on the story from a to z ……

Health & Medicine