Assisted Reproductive Technologies

Assisted Reproductive Technologies: 

Assisted Reproductive Technologies Dr Ravikanth G.O. KVG Medical College Sullia D.K .

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

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In 1944 a lab technician named Miriam Menkin became the first person to witness the creation of life outside the human body .

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Robert edwards Patrick step toe Nobel prize in2010

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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.

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Dr Kumar, a reproductive biologist, was known for having created India’s first test-tube baby. His collaboration with gynaecologist Indira Hinduja had resulted in the widely publicized birth of Harsha Chawda in Mumbai on 6 August 1986.

MAJOR ASSISTED REPRODUCTIVE TECHNOLOGIES (ART): 

MAJOR ASSISTED REPRODUCTIVE TECHNOLOGIES (ART) In vitro fertilization and embryo transfer (IVF-ET) Direct intra-peritoneal insemination (DIPI) Gamete intra-fallopian transfer (GIFT) Zygote intra-fallopian transfer (ZIFT) Intracytoplasmic sperm injection (ICSI)

Causes of infertility: 

Causes of infertility

Indications of ART : 

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

Tubal factor: 

Tubal factor First developed indication Surgery/IVF DISTAL TUBAL OBSTRUCTION SURGERY –only in young women mild disease Results with surgery in severe 10-35% Ectopic preg 5-20% Hydrosalphinginges should removed toxic /mechanical

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Proximal tubal obstruction HSG Hysteroscopic cannulation Microsurgery Bipolar Tubal Obstruction Previous Tubal Sterilisation poor prognosis sugery failure avoid surgery single additional pregnancy

Endometriosis : 

Endometriosis Mech interferes ovum capture oocyte devolopment early embryogensis reduced endometrial receptivity Poor oocyte quality Advanced disease- IVF GnRH agonast Mild disease - OI,IUI,---IVF

Male factor infertility: 

Male factor infertility

Male factor infertility: 

Male factor infertility Sperm count < 13.5 million/ml < 32% progressive motility <9 normal morphology Infertility increases with no of abn parameter 1f 2-3, 2f -5-7, 3f- 16times IUI IVF ICSI

Unexplained infertility: 

Unexplained infertility 10-30% Results Expectented Mx 1.3% IUI with Clomophene 8.3% IUI with Gonadotropins 17% IVF 28%

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Ovarian failure Diminished ovarian reserve

Prognostic Factors of ART: 

Prognostic Factors of ART Maternal age Ovarian reserve

Maternal age: 

Maternal age Young good result Previous live birth carries better result

s: 

s

u: 

u

Ovarian reserve: 

Ovarian reserve No of follicle at various age At higher age inhibin B decreases Giude but not to deny Rx Cycle Day 3 Serum FSH >10-15iu/L peak E2 level No of Oocytes Pregnancy Live Birth

Ovarian reserve: 

Ovarian reserve Cycle day 3 Serum Estradiol > 75-80pg/ml Clomiphene citrate challenge test day 3 Serum FSH & E2 Clomiphene 5-9 day day 10 serum FSH (>2SD)

Evaluation Before IVF: 

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: 

Typical ART cycle COH Monitoring with TVS and Serum E2 Prevention of premature LH surge and Ovltion Oocyte maturing with HCG Oocyte retrival Fertilization by IVF/ICSI Invitro embryo culture Luteal support Transfer of fresh embryo/ cryopresev of excess First trimister preg monitoring

Ovarian stimulation regimens: 

Ovarian stimulation regimens ideal regimen Low cancellation rates Minimal drug costs,risks and side effects Limited monitoring Maximum singleton pregnancy

Ovarian stimulation regimens: 

Ovarian stimulation regimens Natural cycle Minimum stimulation i.e.clomiphene Mild stimulation I,e . CC+ low dose Gn Aggressive stimulation high dose Gn +/- GnRHa / GnRH ant

Natural cycle: 

Natural cycle First birth form IVF Cycle cancellation are high Low pregnancy rate Indication -poor response to stimulation Advantage -Less monitoring Less costly

Clomiphene cycle: 

Clomiphene cycle 100mg on 5-8 days Yield 1-3 follicle Better than natural cycle GnRHa /HCG can be used

Sequential Rx with CC+Gn: 

Sequential Rx with CC+Gn CC100mg for 5 days Low dose exogenous Gn Pregnancy rates are good OHSS is less

Ex Gn with GnRHa down regulation: 

Ex Gn with GnRHa down regulation Prevent premature LH surge thus cycle cancel Egg yield and pregnancy rates are higher Coordinate the cycle for group of women Widely preferred for forms of ART Dis adv of GnRHa – Blunt the respone of Gn costly

GnRHa treament Long protocol : 

GnRHa treament Long protocol Midluteal of previous cycle high yield flare effect will not affect recruitment low LH will decrease the androgen luprolide 1mg/d s.c.for 10 days/ till mensus 0.5mg/d s.c.untill HCG nafarlen intranasal spray 400mcg then 200mcg

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Poor responder half the dose discontinue after 5 days of Gn /completely Good response E2 <40pg/ml No ovarian follicle >10-15mm at Gn

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Gn 225-300 iu uFSH,rFSH,uHMG . Step up/step down Step down protocol is most preferred All Gn including HCG can s.c. Long acting rFSH t1/2 3 times longer

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Day 3-5 of Gn serum E2 Later every 1-3 day serum E2 and ovarian scan Most need 7-12 day of Rx Goal > 2 follicle >17-18mm fewmm >14-16 (200pg/ml) Endometrium 8-9mm good(poor 6-7mm) hCG 5000-10,000iu rhCG 250 micro gram

Mx of high responders: 

Mx of high responders Multiple follicle Ovarian enlargement E2 >3000pg/ml Cycle cancellation OCP+GnRHa low dose Gn Coasting- GnRHa ctd 1-3day hCG after E2 level moderate Proceeding with oocyte rtrival & fertilisation but freezing all embryo Delay transfer by 5 days to observe for OHSS

Mx of poor responders: 

Mx of poor responders <3 follicle >16mm Single dominant follicle Serum E2 <500pg/ml Long protocol with higher dose of Gn (mx 450iu) Decrease the dose of GnRHa / discontineue it after Gn Use GnRHant Sequentianl Rx CC+Gn

Sequential GnRHa+Gn short/flare protocol: 

Sequential GnRHa+Gn short/flare protocol

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Leuprolide 1mg on D2-4 Reduced to 0.5mg Gn D3 150-450iu Monitor and hCG is same Preffered in poor responder to long protocol Pregnancy is low Decreased flexibilty Significant increase in progestirone affect late CL rescue

Ultrashort protocol: 

Ultrashort protocol GnRHa for 3 days Discontineu Gn premature LH surge inferior results

OCP-microdose GnRHa: 

OCP- microdose GnRHa 14-21 days OCP Microdose leuprolide 40mcg bid form D3 High dose Gn form D3 of leuprolide does not increase Progestirone /androgens Preferred in poor responders for long protocol

Simulteneous Gn+GnRHant: 

Simulteneous Gn+GnRHant Advantage of GnRH ant duration of Rx is shorter no supressive effect ovavry no flare response so no cysts in PCOD prevent premature LH surge agonist can be used for LH surge Disadvantage Strict compliance is necessary Supress endogeneous Gn completely Low pregnancy rate compared to long protocol

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Ganirelix , Cetrorelix 0.25mg 5-6 days of Gn /lead follicle13-14mm Single large dose cetrorelix 3mg 6-7 day of Gn

Oocyte retrival: 

Oocyte retrival 36 h after hCG Laparoscopically TVS I.V. seadtion propofol /Midazolam/Fentanyl. Prophylactic antibiotic doxy 100mg/ cefoxitin 2g No antiseptic clean with NS 5-7 MHz, TVS 16G needle,vaccum100-200mmhg

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All folicle > 10mm aspirated Empty follicle syndrome Complications haemorrhage pelvic infection rupture of a cyst laceration of sacral vein lumbosacral osteomyelitis

Oocyte maturation: 

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

Oocyte maturity: 

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: 

Mature oocyte Cumulus cells are expanded Luetinised corona cells –sunburst appearance

Metaphase 1 oocyte: 

Metaphase 1 oocyte No polar body Dense cumulus cells Germinal vesicle and nucleous faded Immature oocyte

Cytoplasm and germinal vesicle transfer: 

Cytoplasm and germinal vesicle transfer

Fertilization : 

Fertilization Semen is collected by mastrubation Sperm preparation swim up density gradient Incubation in high protien 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

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Conventional ivf – 50-60% fertilization Achives second meotic division Extrude second polar body Look for polyploidy

Sperm retrieval technique: 

Sperm retrieval technique Ejaculatory failure Obstructive/ Nonobstructive azoospermia Retrograde ejaculation genetic evaluation for cystic fibrosis Medical methods prevent retrograde ejaculation Imiprimine 25mg bid,50mgbid Psedoephidrine 50mg Ephidrine 25-50mg qid Phenylpropranalamine 50-75mgbid

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Alkanizing the urine sodabicorbanate 650mg qid 1-2 days before restricting fluid intake Vibrostimulatory electroejaculation Epidemal sperm aspiration MESA,open , / percutaneous. Testicular sperm extraction(TESA)

Intracytoplasmic sperm injection ICSI: 

Intracytoplasmic sperm injection ICSI Zona drilling (micropipette and acidified tyrode solution) Partial zona dissection Subzonal insertion polyspermy

Procedure : 

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: 

Indication of ICSI Male factor Oligospermia <5% Asthenospermia <5% Teratospermia <4% PGD Poor IVF/failed IVF

Embryo culture: 

Embryo culture 4-7% CO2 Incubation volume 10-50microL Embryo goup size 1-4 Protien suppliment High serum albumin Recombinent albumin Synthetic serum substitute

Co-culture system: 

Co-culture system Human tubal fluid Maternal serum/ protien substitute Autologus endometrial cells Risk of infection FDA has not approved Used in failed IVF

Extended (Blastocyst) culture: 

Extended (Blastocyst) culture First human birth Now many use cleavage stage embryo(2-3day) With knowledge of physiologic requirement Precompaction embryo( marula ) need pyruvate and non essential a a Post compaction embryo glucose and essential a a

Advantage of blastocyst culture: 

Advantage of blastocyst culture True viability assesment Devolopmental potential Synchronize the stage of development Reduce the abnormal endometrial milieu Reduce risk of expulsion Allows PGD Few embryo can be transformred

Disadvantage : 

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

Preimplantation Genetic Diagnosis: 

Preimplantation Genetic Diagnosis First select female embryo in 1990 Aneuploidy Structural abnomalites ( translocations,invertions ) Inherited gene disorder Cystic fibrosis Thalassemia Haemophelia Duchnemuscular dystrophy

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Equipmqnt and procedure is same as ICSI 1-2 cells are extracted Polar bodies first polar body second polar body paternal chromosome are not assesed Clevage stage embryo

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Chromosomal analysis FISH (fluorescence insitu hybridization) Comparative genomic hybridization Detection of single gene disorder PCR Cloning cleavage stage dividing Somatic cell nuclear transfer

Embryo transfer : 

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

Day 5 blastocyst: 

Day 5 blastocyst Blastocoel cavity less than half the volume of the embryo Many cells, tightly packed

Transfer technique: 

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 0.5 cm below fundus Difficult cases Cervical dilatation USG guided Transmyometrial Expert physician

2004START/ASRM GIUDELINES for ET: 

2004START/ASRM GIUDELINES for ET <35y <2 embryo 1 in fav first ivf prv suces ivf good quality E cryp 35-37y 2 fav 3 othr 38-40y 3 fav 4 othr >40 y 5

Age independent : 

Age independent Additional embryo unfavrable prognosis Defined by age of donor One more oocyte than embryo GIFT

Assisted hatching: 

Assisted hatching In vivo zona dissolves on zona - endomet interface In vitro embryo make open in and escapes Methods acidic tyrode’s solution partial zona dissection with micro glass needle Laser photo ablation Enzymatic Hatching Piezo -micromanipulator Indication failed IVF poor prognostic factor

Embryo cryopresevation: 

Embryo cryopresevation Improves cumulative pregnancy/ retrival OHSS is avoided Principle avoid crystallization of intrcell water cell water gradually replaces cryoprotectent dimethyl sulfoxide propanediol glycerol embryos are sealed in vials and cooled -30 ° C to -110◦C thawing process is reversed

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All stage of Embryo can be frozen For indefinite Embryo survival 50-90% Better for zygote than clevage stage and blastocyst Overall success 15-20% Thawing sequential larger no of embryo

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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

Luteal phase support: 

Luteal phase support COH with Gn – multiple corpus luteam GnRHa /ant Progestirone orally 300-800mg/d vaginally 100-600mg/d injection 25-50 mg/d hCH 1500-2500iu / 3d For 5 weeks

Results of IVF: 

Results of IVF Mesured by % of prgnancy,live birth 18 % of pregnancy- miscarriage 15% induced abortion 0.9% still birth 0.6% ectopic pregnacy 0.7% Overall 28.4% pregnancy/ retrival 16% clinical pregnancy/ET in frozen embryo

ART outcome over years: 

ART outcome over years Age 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%

PowerPoint Presentation: 

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

Multiple pregnancy: 

Multiple pregnancy 2000 35% of ART are multiple pregnancy 30.7% are twins 4.3% are triplets 3% in general population

Live birt/ET and multiple births <35 y : 

Live birt /ET and multiple births <35 y ET Live birth/ET Singleton Twins Triplet+ 1 30% 100% 2 51.7% 59% 39.6% 1.2% 3 46.7% 53.4% 38.8% 7.8% 4 42.9% 49.1% 41.3% 9.6% 5+ 43.1% 43.3% 46.8% 9.9%

Risks of IVF: 

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

Ectopic pregnancy: 

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: 

Risks of OHSS High exogenous Gn High /rapidly increase in E2 Highe r /repeated dose of hCG Multiple pregnancy

Prevention of OHSS: 

Prevention of OHSS Elevated/rapidly rising E2 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: 

Offspring from IVF Prematurity LBW Delayed neurological devolopment C ongenital bith diffects - t wo time higher NTD Alimentary atresia O mphalocele Hypospadias

ICSI: 

ICSI Genetic/ epigenitic abnormalities Sex chromosomal abnomalities angelman syndrome MR Dlayed motor devolopment poor balnce abnormal movement absent speech

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

Oocyte donaton: 

Oocyte donaton First reported in 1983 Achived by IVF Recipients partners sperm Transfared to synchronized uterus

Indication : 

Indication Ovarian failure Genetically transmitted disease Diminished ovarian resrve Inaccessible ovaries

Evaluation of recipients: 

Evaluation of recipients Similar in IVF Psychological counseling Turner syndrome Marfans syndrome cardiac disease aortic root dissection

Controlled endometrial devolopment: 

Controlled endometrial devolopment Endogenous hormones are suppressed- GnRHa Follicular phase 7days-3 weeks by E2 Window of endometrial receptivity 3day (max 5d) controlled by duration of Progestirone form the day of retreival / 4days before transfer 20-50mg/d serum 20ng/mL TVS endometrium >6-7mm

Donor screening: 

Donor screening 21-34 y History and examination r/o STD,Genitic disease preconception testing Blood group Rhtype rubella and varicella HIV 1&2,HBV,HCV,Gonrrhea Chlamydia Pschologic evaluation

Embryo enddometrial synchronization: 

Embryo enddometrial synchronization Day 2 embryo – third day of P Day 3 Embryo –fourth day Day 5 Embryo –sixth day

Luteal support: 

Luteal support 5-7week 10 week for added support

Results of oocyte donation : 

Results of oocyte donation Age is important 35 y 47% live birth with avg 2.9 ET

Gestational surrogacy: 

Gestational surrogacy Indication Absence of uterus Irreparable uterus Congenital Ascherman Syndrome life threatening medical disorder Person related/nonrelated parous healthy

GIFT,ZIFT: 

GIFT,ZIFT GIFT oocyte and sperm are transferred Zygote in ZIFT Et is done by laparoscopy 4 cm inside the fimbria GIFT 27% ZIFT 27.9% Indication difficult IVF Religious Ectopic pregnancy

Ovarian tissue cryopresevation: 

Ovarian tissue cryopresevation Indication CT RT Orthotropic transplantation Heterotropic transplantation Under trial

Oocyte cryopreservation: 

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