ASSISTED REPRODUCTIVE TECHNIQUES

PROF. M.C. BANSALM.B.B.S , M.S. , M.I.C.O.G, F.I.C.O.G.Founder Principal& Controller;Jhalawar Medical College and Hospital , Jhalawar.Ex. Principal & Controller;Mahatma Gandhi Medical college And Hospital, Sitapura, Jaipur.

All technique involving direct manipulation of oocyte/sperm outside the body

History of ART

• 1978- first successful birth using In Vitro Fertilization

• 1984- first successful birth using Gamete Intra Fallopian Transfer

• 1986-first successful birth using Zygote Intra Fallopian Transfer

Robert edwardsPatrick step toeNobel prize in2010

• The world’s second and India’s first IVF baby, Kanupriya, alias Durga, was born 67 days later on October 3, 1978, through the efforts of Dr. Subhas Mukherjee and his two colleagues in Kolkata.

Abdulkareem Sultan Al-Olama 6

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 7

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.

Causes of infertility

Indications of ART

• Tubal factor infertility• Endometriosis• Male factor infertility• Unexplained infertility• Ovarian failure and diminished ovarian reserve• Pelvic malignancy• Mullerian anomaly• Genetic risk

MAJOR ASSISTED REPRODUCTIVE TECHNOLOGIES (ART)

• In vitro fertilization and embryo transfer (IVF-

ET)• Direct intra-uterine insemination • Gamete intra-fallopian transfer (GIFT)• Zygote intra-fallopian transfer (ZIFT)• Intracytoplasmic sperm injection (ICSI)

Artificial Insemination

• Sperm is collected and placed into a woman’s vagina, cervical canal or in the uterus.

• Sperm can come from partner or an anonymous donor.

•Insemination is when sperm is collected and processed. The sperm is then placed into a woman’s vagina, cervical canal or directly into the uterus.

•Insemination may be used if the mucus around cervix is not compatible with partner’s sperm, or may have problems with immune system. This can cause sperm to be killed before egg is fertilized

•Artificial Insemination is when the sperm used comes from partner.

DONATED SPERM(INTRAUTERINE INSEMINATION)

• Doctor puts donated sperm in woman

• Seminal fluid washed from sperm

• Donor selection possible

Natural cycle

• First birth form IVF is from oocyte collected from natural cycle

• Cycle cancellation are high (25%-75%)• Low pregnancy rate• Indication -poor response to stimulation• Advantage -Less monitoring Less costly

STIMULATION PROTOCOLS

• Protocol A (suppression and stimulation)• Protocol B(flare up)• Protocol C(cc+ HMG)• Protocol D

PROTOCOL A

• Suppression /down regulation• GnRH analogue Inj suprefact(Buserelin

acetate) 0.5 cc s/c BD(i.e 20 units of insulin syringe)=0.5 mg from Day 22 of previous cycle (i.e day 7 of post ovulation)

• If the cycle is anovulatory/totally irregular then,E2+progesterone along with buserelin

On D1 do E2 and LH estimationa. If E2 is <26 and LH<4 down regulation has been

doneb. If either of the values are more than that ,continue

the same dose of down regulation for another 2 days.if only marginally high OD x 2 days

If marginally low no need to repeatStart the stimulation along with 4 units of insulin

syringe i.e 0.1 cc OD Suprefact

The initial dose of exogenous gonandotrophins used to stimulate ovarian follicular development depends on individual need

• Typical starting dose of HMG (1ampule= 75IU) 3-4 amp I/M daily

Suprefact (0.1 cc) I/M or S/CAge 35 yrs,wt 50 kg,FSH-10,previous h/o surgery for

endometriosis/PCOD then increase the dose by 1 amp for each cause but not>than 6 amp daily

After 3rd day i.e from 4th day onward,start USG for follicular study.We should have a cohort 4-5 follicles on each side and size of >7-8 mm each and about E2>100

• Continue with the same dose .If <2 follicles are leading then stop suprefact and HMG both

• If size <7 mm increase the dose by 1 amp of HMG• Growth should be 2mm/day.If <2 mm/day,then

increase the dose by 1 amp step by step.When the leading follicle is 18-20 mm give HCG 10000 iu IM

• CALL after 36 hrs for ovum pick up(confirm availability of embryologist and other preparations)

PROTOCOL B

• FLARE UP• This is suitable for aged patients:where

ovarian functions are poor• D1 & D2 Inj buserelin 0.5cc s/c• D3 onwards in morning inj buserelin 0.5 cc s/c

and in evening inj HMG 3 amp• D5 onwards start follicular study other steps

as before

PROTOCOL C

• Clomiphene citrate 150-250 mg from D1-D5• TVS ON D5/6• HMG started on D5,follicle 5 mm in each ovary• Next all steps as protocol A

Protocol D

• CC from D2-D6• Follicular study USG from D9/D10• If endometrium is very thin(<5mm) (tab lynoral

0.05 mg x 5 days/progynova 2 mg x 5 days) may be continued throughout the cycle

• When the follicle size >18-20 mm then give inj HCG IM

• If 1 follicle-inj HCG 5000 IU• If >2 follicle –inj HCG 10000 IU

Prognostic Factors of ART

• Maternal age • Ovarian reserve

Maternal age

• Young good result• Previous live birth carries better result

Ovarian reserve

• No of follicle decreases with age• At higher age inhibin B decreases due to shrinkage of

follicular pool n FSH progressively rises• Ovarian reserve tests even when grossly abnormal should be

used to Guide rather than to deny Rx• Tests are Cycle Day 3 Serum FSH <10-15iu/L peak E2 level ie <75-80pg/ml No of Oocytes Pregnancy Live Birth

Ovarian reserve

• Cycle day 3 Serum Estradiol > 75-80pg/ml• Clomiphene citrate challenge testProvocative test. It includes day 3 Serum FSH & E2 Clomiphene 5-9 day day 10 serum FSH (>2SD then abnormal)

Evaluation Before IVF

• Ovarian reserve• Male factor• Infectious disease chlamydial ,HIV,HBV,HCV,• Mock embryo transfer• Evaluation of uterus HSG,Hysteroscopy,Sonohysterography.

Typical ART cycle

• COH• Monitoring with TVS and Serum E2• Prevention of premature LH surge and Ovulation• Oocyte maturing with HCG• Oocyte retrival• Fertilization by IVF/ICSI• Invitro embryo culture• Luteal support• Transfer of fresh embryo/cryopreserved • First trimister preg monitoring

In Vitro Fertilization (IVF)

Oocyte retrival

• 36 h after hCG• Laproscopically• TVS• I.V. sedation propofol/Midazolam/Fentanyl.• Prophylactic antibiotic• No antiseptic ,clean with NS• 5-7 MHz, TVS 16G needle,vaccum100-200mmhg,the

follicle wall rapidly collapse but donot obstruct the needle lumen

• All folicle > 10mm aspirated• Empty follicle syndrome

• Complications haemorrhage pelvic infection rupture of a cyst laceration of sacral vein lumbosacral osteomyelitis

Oocyte maturation

• 20-30% of retrieved are immature• hCH triggers resumption of meosis

Oocyte maturity

• Expansion of cumulus• Radiance of corona• Size and cohesivness of granulosa cells• Shape and color of oocyte• first polar body• Germinal vesicle

Mature oocyte

• Cumulus cells are expanded Luetinised• corona cells –sunburst appearance

Metaphase 1 oocyte

• No polar body• Dense cumulus cells• Germinal vesicle and nucleous faded

Immature oocyte

Fertilization • Semen is collected by mastrubation • Sperm preparation swim up density gradient • Incubation in high protien media for 0.5-4hr• Each oocyte incubated with 50-100 thousand motile sperm in 5% CO2 in air, 98%humidity,37°C for 12-18hr

• Conventional ivf – 50-60% fertilization• Achieves second meiotic division• Extrude second polar body• Look for polyploidy.it can be observed in 5-

10% embryos

Intracytoplasmic sperm injectionICSI

• Zona drilling (micropipette and acidified tyrode solution)

• Partial zona dissection• Subzonal insertion This all requires sperm to interact with oolemma and did not prevent polyspermic fertilization

Intracytoplasmic sperm injection

• Intracytoplasmic sperm injection (ICSI, pronounced "eeksee") is an in vitro fertilization procedure in which a single sperm is injected directly into an egg.

• This procedure is used to overcome male infertility problems, although it may also be used where eggs cannot easily be penetrated by sperm, and occasionally as a method of in vitro fertilization, especially that associated with sperm donation.

Procedure

• Single sperm is immobilized• Drawn in to pipette• Oocyte is stabilized• Polarbody is 6/12 o’ clock position• Oocyte is entered 3 o’clock• Pipette pierce zona and oolemma• 50-70% fertilization

Indication of ICSI

• Male factor• Oligospermia <5%• Asthenospermia <5%• Teratospermia <4%• PGD• Poor IVF/failed IVF

Embryo culture

• 4-7% CO2 conc.• Incubation volume 10-50microL• Embryo group size 1-4• Protein supplement High serum albumin Recombinant albumin Synthetic serum substitute

Co-culture system

• Efforts to create optimal culture led to development of co-culture system

• Human tubal fluid • Maternal serum/protien substitute• Autologus endometrial cells

Risk of infection FDA has not approvedUsed in failed IVF

• Many of the large programs have attempted blastocyst culture, but returned to day 3 transfers because they had trouble getting the embryos to grow the blastocyst stage. Growing the embryos to the blastocyst stage requires great attention to detail - a luxury not afforded in a big program performing dozens of procedures a day.

• Programs performing less than 200 cycles per year have had the best luck with blastocyst culture and transfer with reported (but unverified) pregnancy rates in the 50-70% range for younger patients and egg donor cycles.

Extended (Blastocyst) culture

• First human birth• Now many use cleavage stage embryo(2-3day)With knowledge of physiologic requirement,there is development of sequential media that varies in composition with stage of embryo developmentPrecompaction embryo(morula) need pyruvate and non essential a aPost compaction embryo(blastocyst) glucose and essential AA

Advantage of blastocyst culture

• True viability assesment is better• Excludes embryos that have limited

Devolopmental potential• Synchronize the stage of development• Reduce the abnormal endometrial milieu• Reduce risk of expulsion• Allows PGD• Few embryo can be transformed

Disadvantage

• Doesn’t improve the quality of embryo• Lesser quality embryo may fail to grow• Multiple pregnancy• Need extended culture

Preimplantation Genetic Diagnosis

• It offers couples who carry serious genetic disordes the opportunity to have healthy child

• Aneuploidy• Structural abnomalites (translocations,invertions)• Inherited gene disorder Cystic fibrosis Thalassemia Haemophelia Duchnemuscular dystrophy

• Equipment and procedure is same as ICSI• 1-2 cells are extracted• Polar bodies first polar body second polar body paternal chromosome are not assesed• The most common aproach to PGD is Cleavage

stage embryo biopsy typically performed on day 3 of fertization before embryo starts to compact

PREIMPLANTATION GENETIC DIAGNOSIS (PGD)

Embryo transfer

Can transfer zygote to blastocystDay 3 cleavage embryo 6-8cells equal size no cytoplasmic fragmentation

Day 5 blastocyst

• Blastocoel cavity less than half the volume of the embryo

Many cells, tightly packed

Transfer technique

• Cervical mucus plug aspirated slowly• No blood on catheter tip• Catheter tip examined microscopically after transfer• Soft catheter/ stiff catheter• Volume of media < 50 micro L• Catheter tip does not touch fundus and transfer occus at

level of 0.5 cm below fundus.whenever possible ,mucus ,blood ,ut.contractions should be avoided.

• A preliminary trial transfer is done to identify women who may benefit from cervical dilatation before treatment begins

Fertilization• After the fertilization,

embryos are transferred into uterus, anywhere from one to six days later,

• but usually this happens between 2 to 3 days after the egg retrieval.

• During this time, the fertilized egg separates to become a 2-4 cell embryo.

STEPS

Assisted hatching• In vivo zona dissolves on zona- endomet interface• In vitro embryo make opening in zona and escapes ,leaving behind an empty

zona• Methods Zona drilling with acidic tyrode’s solution partial zona dissection with micro glass needle

Laser photo ablation Enzymatic Hatching Use of Piezo -micromanipulator Indication failed IVF poor prognostic factorDisadv.-Hatching may cause embryo demage and the risk of monozygotic the risk of monozygotic twinning inceases

Embryo cryopresevation• SUCCESS WITH FROZEN EMBRYO CYCLE significantly increases the cumulative pregnancy

rate/retrival of oocyte• OHSS is avoidedPrincipleIt has two distinct stages –freezing and thawing

Object of freezing is to avoid crystallization of intercellular water cell water gradually replaces cryoprotectent dimethyl sulfoxide propanediol glycerol

embryos are sealed in vials and cooled -30°C to -110◦C and then stored in liquid nitrogen After thawing, process is reversed, gradually passing the embryo through decreasing conc. of cryoprotectant.

Frozen Embryos

• Embryos may be taken from an individual and stored for later use.

• Once ready to use, they can be thawed and then placed into the uterus.

• This allows a higher chance of pregnancy.

• All stage of Embryo can be frozen• For indefinite time• Embryo survival 50-90%• Better for zygote than clevage stage and blastocyst• Overall success 15-20%• Thawing can be done sequentially until the no. of transfer embryos is reached or the larger no of embryo is thawed to select with best morphology

• Embryo can be refrozen• Embryo can be transferred natural cycle artificial cycle GnRHa down regulation E2 (micronized E2 4-6mg/d, transdemal 0.1-0.2mg after mensus)

(inhibit rise in FSH) estimate serum P <1ng/mL time of transfer to synchronize stage of embryo

Results of IVF

• Measured by % of pregnancy,live birth• 18 % of pregnancy- miscarriage 15% induced abortion 0.9% still birth 0.6% ectopic pregnacy 0.7%Overall 28.4% pregnancy/retrieval 16% clinical pregnancy/ET in frozen embryo

ART outcome over yearsAge of patients < 35y 35-37y 38-40y 41-42

ART out come in 1996(live birth/ ET)

33.6% 29% 21.6% 11.5%

ART out come in 2001(live birth/ET)

41.1% 35% 25.4% 14.5%

• Results are better with <35 year previous live birth previous success IVF• poor result diminished ovarian reserve uterine factor multiple factor

Multiple pregnancy

35% of ART are multiple pregnancy 30.7% are twins 4.3% are triplets 3% in general population

Risks of IVF

• Ectopic pregnancy• OHSS Earlier worries of possible link b/n Ovarian Ca and OI drugs has declined but still linger

Ectopic pregnancy

• Two time more• Risks tubal factor infertility ET placed high in tube larger volume of media difficult transfer high hormones• Heterotropic pregnancy 1in 10.000pregnancy

Risks of OHSS

• High exogenous Gn• High /rapidly increase in E2• Higher /repeated dose of hCG• Multiple pregnancy

Prevention of OHSS

• Elevated/rapidly rising E2 ,then coasting• Use low dose hCG(5000iu)• Use GnRHant in PCOD• Use GnRHa for LH surge• If symptoms of OHSS oocyte retrival and freezing • Prophylactic i.v. albumin infusion 20-50G

Offspring from IVF

• Prematurity• LBW• Delayed neurological devolopment• Congenital bith diffects- two time higher NTD Alimentary atresia Omphalocele Hypospadias

ICSI

Genetic/epigenitic abnormalities Sex chromosomal abnomalities1. angelman syndrome MR Delayed motor development poor balance abnormal movement absent speech

2.Beckwithian syndrome Macrosomia Macroglossia Midline abdominal wall defect predisposition to embryonal Ca

Oocyte donaton

• First reported in 1983• Achieved by IVF • Recipients partners sperm• Transferred to synchronized uterus

Indication

• Ovarian failure• Genetically transmitted disease• Diminished ovarian reserve• Inaccessible ovaries

Evaluation of recipients

• Similar in IVF• Psychological counseling• Turner syndrome • Marfans syndrome cardiac disease aortic root dissection

Controlled endometrial devolopment

Endogenous hormones are suppressed-GnRHaFollicular phase 7days-3 weeks by E2Window of endometrial receptivity 3day (max 5d) controlled by duration of Progesterone can be administerd intramuscularly from the day of retrieval/ 4days before transfer in a dose of 20-50mg/d to achieve serum conc. of 20ng/mLTVS endometrium >6-7mm

Donor screening

• 21-34 y• History and examination rule/out STD,Genetic disease preconception testing Blood group Rhtype rubella and varicella HIV 1&2,HBV,HCV,Gonrrhea ChlamydiaPschologic evaluation

Embryo endometrial synchronization

• Progesterone therapy started on day the donor undergo retrieval

• Day 2 embryo – third day of Progesterone therapy

• Day 3 Embryo –fourth day• Day 5 Embryo –sixth day

Luteal support

• 5-7week • 10 week for added support

Results of oocyte donation

• Age is important• 35 y• 47% live birth with avg 2.9 ET

Gestational surrogacy

Indication Absence of uterus Irreparable uterus Congenital Ascherman Syndrome life threatening medical disorderPerson related/nonrelated parous healthy

Gamete intrafallopian transfer

• In gamete intrafallopian transfer (GIFT), eggs are removed from the woman, and placed in one of the fallopian tubes, along with the man's sperm. This allows fertilization to take place inside the woman's body. Therefore, this variation is actually an in vivo fertilization, and not an in vitro fertilization.

Zygote intrafallopian transfer

• Zygote intrafallopian transfer (ZIFT) is an infertility treatment where a blockage in the fallopian tubes are the cause. Egg cells are removed from a woman's ovaries, and in vitro fertilized. The resulting zygote is placed into the fallopian tube by the use of laparoscopy.

Gamete Intra-Fallopian Transfer(GIFT)

• A mixture of a woman’s eggs and sperm are placed into the fallopian tube during a laparoscopy.

• Once inserted, fertilization is allowed to occur.

GIFT,ZIFT

• GIFT oocyte and sperm are transferred• Zygote in ZIFT • Embryo transfer is done by laparoscopy 4 cm inside the fimbriaGIFT 27%ZIFT 27.9%Indication difficult IVF ReligiousEctopic pregnancy

Surrogacy

• Two types:– Egg donor surrogacy– Gestational surrogacy

• Surrogate may be relative, friend, or paid stranger

Applications of ART

• Endanger species• Assisted reproductive technology, allows in

vitro fertilized (IVF ET) embryos for preimplantation genetic screening (PGS) evaluation. The is not used to look for a specific disease but a technique to identify embryos at risk.

Ovarian tissue cryopresevation

Indication pt on chemo/radiotherapy Orthotropic transplantationHeterotropic transplantation

Under trial

Oocyte cryopreservation

• No surgery• Few Ca patient have time for stimulation• Vitrifaction high concentration of cryopresevent is used glass like state thawed oocyte –pregnancy is similar to fresh

Sperm retrieval technique

• Sperm retrival is done in cases of -• Ejaculatory failure• Obstructive/Nonobstructive azoospermia• Retrograde ejaculation

Testicular sperm aspiration (TESA)

Testicular sperm aspiration (TESA) is a procedure performed for men who are having sperm retrieved for in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI). It is done with local anesthesia in the operating room or office and is coordinated with their female partner’s egg retrieval. A needle is inserted in the testicle and tissue/sperm are aspirated. TESA is performed for men with obstructive azoospermia (s/p vasectomy). Often TESA doesn’t provide enough tissue/sperm and an open testis biopsy is needed.

Testicular sperm extraction (TESE)/Testis biopsy/Testis mapping

TESE/testis biopsy/testis mapping are procedures performed for men who have testis failure. The procedure is performed to see if there are sperm present as well as for pathologic diagnosis to evaluate for malignancy. It is either done as a scheduled procedure or is coordinated with their female partner’s egg retrieval. TESE is usually performed in the operating room with sedation, but can be performed in the office with local anesthesia alone.. Patients usually cryopreserve sperm during this procedure for future IVF/ICSI. This diagnostic biopsy is usually performed to evaluate for an obstructive etiology – microdissection TESE has replaced this as the optimal form of retrieval for testis failure patients.

Microepididymal Sperm Aspiration (MESA)

MESA is a procedure performed for men who have vasal or epididymal obstruction (s/p vasectomy, cystic fibrosis). It is either done as a scheduled procedure or is coordinated with their female partner’s egg retrieval. MESA is performed in the operating room with general anesthesia under the operating microscope. Patients usually cryopreserve sperm during this procedure for future IVF/ICSI.

Microdissection TESE (microdissection testicular sperm extraction)

Microscopic TESE is a procedure performed for men who have testis failure. Microdissection TESE is performed in the operating room with general anesthesia under the operating microscope. Patients cryopreserve sperm during this procedure for future IVF/ICSI.

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