Assisted Reproductive Technology (ART)Assisted Reproductive Technology (ART)

Prepared by Dr. Abdelsalam TalafhaPrepared by Dr. Abdelsalam TalafhaCollage of veterinary medicine, JUSTCollage of veterinary medicine, JUST

American Board Certified, Comparative Veterinary American Board Certified, Comparative Veterinary Obstetrics and GynecologyObstetrics and Gynecology

ARTART

Infertility Infertility Inability to conceive after 1 year of unprotected Inability to conceive after 1 year of unprotected

and regular sexual intercourse and regular sexual intercourse Primary infertilityPrimary infertility

Couples have never had childrenCouples have never had children Secondary infertilitySecondary infertility

Couples initiated conception in the past and then Couples initiated conception in the past and then had difficulty had difficulty

ARTART

USA women infertility rateUSA women infertility rateAges 20-24: 4.1%Ages 20-24: 4.1%Ages 25-29: 5.5%Ages 25-29: 5.5%Ages 30-34: 9.4%Ages 30-34: 9.4%Ages 35-39: 19.7%Ages 35-39: 19.7%

80% of infertility cases can be diagnosed80% of infertility cases can be diagnosed 85% of cases can be successfully treated85% of cases can be successfully treated

ARTART

Female infertilityFemale infertilityDisorders of ovulation: 27%Disorders of ovulation: 27%Fallopian tube disorders: 22%Fallopian tube disorders: 22%Pelvic adhesions: 12%Pelvic adhesions: 12%Endometriosis: 5% to 15% Endometriosis: 5% to 15% Hyperprolactinemia: 7%Hyperprolactinemia: 7%

Male Infertility

Total 2104 100

Male Infertility

ARTARTAssisted ConceptionAssisted Conception

Assisted ConceptionAssisted Conception

Techniques designed to assist couples in their Techniques designed to assist couples in their effort to procreateeffort to procreate

ReasonReason Identification and correction of female, male Identification and correction of female, male

or a combination of both infertility causesor a combination of both infertility causes

Assisted ConceptionAssisted Conception

ObjectiveObjectiveTo bring sperm and oocyte close to each To bring sperm and oocyte close to each

other to promote chances of fertilization and, other to promote chances of fertilization and, ultimately, achieve a pregnancy ultimately, achieve a pregnancy

Assisted ConceptionAssisted Conception

Required proceduresRequired proceduresSuperovulation Superovulation Sperm preparation Sperm preparation Assisted fertilizationAssisted fertilization

SuperovulationSuperovulation

Hormonal manipulation to enhance ovulation Hormonal manipulation to enhance ovulation and release multiple oocytes during ovulatory and release multiple oocytes during ovulatory cycle cycle

SuperovulationSuperovulation

Drugs usedDrugs used:: Human menopausal gonadotropinHuman menopausal gonadotropin

Taken from urine of postmenopausal womenTaken from urine of postmenopausal womenFollicle stimulating hormone (FSH) and Follicle stimulating hormone (FSH) and

luteinizing hormone (LH) activity luteinizing hormone (LH) activity Recombinant FSH Recombinant FSH Recombinant LHRecombinant LH

Superovulation- protocolSuperovulation- protocol

Gonadotropin for 9-11 daysGonadotropin for 9-11 days Monitoring follicular development by transvaginal Monitoring follicular development by transvaginal

ultrasoundultrasound Follicles 16 -18 mm in diameterFollicles 16 -18 mm in diameter

10,000 IU hCG 10,000 IU hCG Oocytes maturation Oocytes maturation OvulationOvulation

Sperm PreparationSperm Preparation

Select PMNS (Progressively motile normal sperm) Select PMNS (Progressively motile normal sperm) Remove seminal plasma, WBC, and bacteria Remove seminal plasma, WBC, and bacteria Sperm capacitationSperm capacitation

Coating of sperm with seminal plasma proteins Coating of sperm with seminal plasma proteins Allow sperm to become fertileAllow sperm to become fertile In vivo or in test tubeIn vivo or in test tube

Intrauterine InseminationIntrauterine Insemination

Sperm sample deposited in uterus just before Sperm sample deposited in uterus just before release of an oocyte (s) in a natural or stimulated release of an oocyte (s) in a natural or stimulated cycle using a Soft catheter cycle using a Soft catheter

Give hCG at injection or up to 24 hrs laterGive hCG at injection or up to 24 hrs later Sperm volume: 0.2-0.3 mlSperm volume: 0.2-0.3 ml Pregnancy rates Pregnancy rates

Around 15% per cycle Around 15% per cycle

Gamete Intrafallopian TransferGamete Intrafallopian Transfer

Laparoscopic technique in which oocyte and Laparoscopic technique in which oocyte and sperm placed in fallopian tube, allowing in vivo sperm placed in fallopian tube, allowing in vivo fertilization fertilization

ProcedureProcedureSuperovulationSuperovulationUS guided transvaginal oocyte retrievalUS guided transvaginal oocyte retrieval

0.1-0.2 mil sperm with 2-3 oocytes0.1-0.2 mil sperm with 2-3 oocytes

In vitro fertilization - IVFIn vitro fertilization - IVF

Taking oocytes from womanTaking oocytes from woman Fertilizing them in lab with her partner's sperm Fertilizing them in lab with her partner's sperm Transferring resulting embryos back to her Transferring resulting embryos back to her

uterus 3 or 5 days lateruterus 3 or 5 days later

IVFIVF

ProcedureProcedureSuperovulationSuperovulation InseminationInseminationEmbryo transfer Embryo transfer Luteal support Luteal support

IVF - SuperovulationIVF - Superovulation

Gonadotropin stimulation Gonadotropin stimulation Monitoring follicular developmentMonitoring follicular development Ultra Sound guided transvaginal oocyte retrievalUltra Sound guided transvaginal oocyte retrieval Oocyte fertilization with spermOocyte fertilization with sperm

IVF - InseminationIVF - Insemination

Containers used Containers used Test tubes, Petri dishes, multi-well dishesTest tubes, Petri dishes, multi-well dishes

Each oocyte inseminated with 0.5-1.0 mil Each oocyte inseminated with 0.5-1.0 mil PMNS PMNS

Fertilization detected 12-20 hrs later by Fertilization detected 12-20 hrs later by presence of presence of 2 pronuclei in oocyte cytoplasm 2 pronuclei in oocyte cytoplasm 2 polar bodies in perivitelline space2 polar bodies in perivitelline space

IVF - InseminationIVF - Insemination

Syngamy (combination of maternal and paternal Syngamy (combination of maternal and paternal pronuclei 24 hrs after insemination)pronuclei 24 hrs after insemination)

Further cleavages occur at 24 hr intervalsFurther cleavages occur at 24 hr intervals

IVF - Embryo transferIVF - Embryo transfer

Embryos transferred to uterus on 2Embryos transferred to uterus on 2ndnd or 3 or 3rdrd day day after in vitro inseminationafter in vitro insemination

4-8 cells embryos 4-8 cells embryos 2-3 embryos transferred in 20 µl of culture fluid2-3 embryos transferred in 20 µl of culture fluid Transabdominal US to see fluid placed in uterus Transabdominal US to see fluid placed in uterus Cryopreserve excess embryosCryopreserve excess embryos

IVF - Luteal supportIVF - Luteal support

Progesterone (P4) necessary for pregnancy Progesterone (P4) necessary for pregnancy maintenance maintenance

Premature luteolysis in some superovulatory Premature luteolysis in some superovulatory regimens regimens

P4 supplementation until menses occur or woman P4 supplementation until menses occur or woman has positive pregnancy testhas positive pregnancy test

Intracytoplasmic Sperm Injection - ICSIIntracytoplasmic Sperm Injection - ICSI

Injection of single sperm into single oocyte in order Injection of single sperm into single oocyte in order to get fertilization to get fertilization

ProcedureProcedureSuperovulation Superovulation US guided transvaginal oocyte retrieval US guided transvaginal oocyte retrieval IVF IVF

Oocytes injected with sperm using special Oocytes injected with sperm using special microscopes, needles and micromanipulation microscopes, needles and micromanipulation equipmentequipment

ICSI - IndicationsICSI - Indications

Low sperm concentration, motility, abnormal Low sperm concentration, motility, abnormal morphology morphology

Antisperm antibodiesAntisperm antibodies Fertilization failure after conventional IVF Fertilization failure after conventional IVF Ejaculatory disordersEjaculatory disorders Absence of vas deferens or obstruction of Absence of vas deferens or obstruction of

ejaculatory ductsejaculatory ducts

Assisted HatchingAssisted Hatching

IndicationsIndications Couples having IVF with Couples having IVF with

Female partner's age over 37Female partner's age over 37Poor quality embryos Poor quality embryos

Excessive fragmentationExcessive fragmentationSlow rates of cell divisionSlow rates of cell division

Assisted Hatching – ProcedureAssisted Hatching – Procedure

Embryo held with a specialized holding Embryo held with a specialized holding pipettepipette

A needle used to expel an acidic solution A needle used to expel an acidic solution against ZP against ZP

A small hole made in ZP A small hole made in ZP Embryo washed and put back in culture in Embryo washed and put back in culture in

incubatorincubatorET shortly after hatching procedureET shortly after hatching procedure

Hope for the bestHope for the best

Further Advances And Uses Of Assisted Further Advances And Uses Of Assisted Conception TechnologyConception Technology

Cryopreservation ofCryopreservation ofSperm Sperm EmbryoEmbryoOocyteOocyteOvarian tissue Ovarian tissue

Growth of human follicles and oocytes in vitro Growth of human follicles and oocytes in vitro In vitro maturation and transplantation of human In vitro maturation and transplantation of human

spermatozoaspermatozoa

Preimplantation Genetic Diagnosis (PGD)Preimplantation Genetic Diagnosis (PGD)

Identify genetic conditions in embryo before ET Identify genetic conditions in embryo before ET HemophiliaHemophiliaCystic fibrosisCystic fibrosisAneuploidyAneuploidy

PGDPGD

HemophiliaHemophiliaHereditary bleeding disorderHereditary bleeding disorderAbsence of a blood protein essential for Absence of a blood protein essential for

clottingclottingTypes A: lack of factor VIIITypes A: lack of factor VIIIType B: lack of factor IXType B: lack of factor IX

PGDPGD

Cystic FbrosisCystic FbrosisGenetic diseaseGenetic diseaseDefective gene causes body to produce Defective gene causes body to produce

abnormally thick, sticky mucus that obstruct abnormally thick, sticky mucus that obstruct LungsLungsPancreasPancreas

PGDPGD

AneuploidyAneuploidyHaving less than or more than normal diploid Having less than or more than normal diploid

number of chromosomesnumber of chromosomesMonosomyMonosomyTrisomy Trisomy TriploidyTriploidy

PGDPGD

Performed with IVF Performed with IVF 8-cell stage (3 days old) embryo biopsy 8-cell stage (3 days old) embryo biopsy Obtain 1-2 blastomeres for molecular analysisObtain 1-2 blastomeres for molecular analysis

Three day old embryos

PGDPGD

Genetic analysisGenetic analysisPolymerase chain reaction (PCR) Polymerase chain reaction (PCR) Multicolor fluorescence in situ hybridization Multicolor fluorescence in situ hybridization

(FISH)(FISH)

Polymerase Chain Reaction (PCR)Polymerase Chain Reaction (PCR)

In vitro technique for generating large amounts of a In vitro technique for generating large amounts of a specific fragment of DNA from a small amount of specific fragment of DNA from a small amount of templatetemplate

DNA amplification allows for DNA amplification allows for Identification of point mutationsIdentification of point mutationsDNA diagnosticsDNA diagnostics

FISHFISH

Detects Detects Number of chromosomes Number of chromosomes Sexing embryos Sexing embryos Sex chromosome aneuploidy Sex chromosome aneuploidy Whole-chromosome paints for detection of Whole-chromosome paints for detection of

rearrangements and identification of marker rearrangements and identification of marker chromosomeschromosomes

Analyze polar bodies Analyze polar bodies

FISHFISH

Fluorescent probes that bind to specific Fluorescent probes that bind to specific chromosomes are labeled with biotin for chromosomes are labeled with biotin for detection by specific fluorochrome-conjugated detection by specific fluorochrome-conjugated antibodies under a fluorescent microscopeantibodies under a fluorescent microscope

X

Y

Interphase nucleus

Normal DiploidTriploid

Tetraploid Missing chromosomes

Monosomy Double trisomy

PGDPGD

Monosomy Double trisomy

PGDPGD

ARTART

Infertility Infertility Female partner: 35% Female partner: 35% Male partner: 35% Male partner: 35% Both partners: 20%Both partners: 20%Unknown cause: 10%Unknown cause: 10%

Infertility more common with increasing ageInfertility more common with increasing age