Assisted Reproductive Technology (ART)

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Assisted Reproductive Technology (ART)

• Prepared by Dr. Abdelsalam Talafha
• Collage of veterinary medicine, JUST
• American Board Certified, Comparative Veterinary
  Obstetrics and Gynecology

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ART

• Infertility
• Inability to conceive after 1 year of unprotected
  and regular sexual intercourse
• Primary infertility
• Couples have never had children
• Secondary infertility
• Couples initiated conception in the past and then
  had difficulty

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ART

• USA women infertility rate
• Ages 20-24 4.1
• Ages 25-29 5.5
• Ages 30-34 9.4
• Ages 35-39 19.7
• 80 of infertility cases can be diagnosed
• 85 of cases can be successfully treated

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ART

• Female infertility
• Disorders of ovulation 27
• Fallopian tube disorders 22
• Pelvic adhesions 12
• Endometriosis 5 to 15
• Hyperprolactinemia 7

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Male Infertility
100
Total
2104
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Male Infertility
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ARTAssisted Conception
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Assisted Conception

• Techniques designed to assist couples in their
  effort to procreate
• Reason
• Identification and correction of female, male or
  a combination of both infertility causes

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Assisted Conception

• Objective
• To bring sperm and oocyte close to each other to
  promote chances of fertilization and, ultimately,
  achieve a pregnancy

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Assisted Conception

• Required procedures
• Superovulation
• Sperm preparation
• Assisted fertilization

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Superovulation

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

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Superovulation

• Drugs used
• Human menopausal gonadotropin
• Taken from urine of postmenopausal women
• Follicle stimulating hormone (FSH) and
  luteinizing hormone (LH) activity
• Recombinant FSH
• Recombinant LH

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Superovulation- protocol

• Gonadotropin for 9-11 days
• Monitoring follicular development by transvaginal
  ultrasound
• Follicles 16 -18 mm in diameter
• 10,000 IU hCG
• Oocytes maturation
• Ovulation

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Sperm Preparation

• Select PMNS (Progressively motile normal sperm)
• Remove seminal plasma, WBC, and bacteria
• Sperm capacitation
• Coating of sperm with seminal plasma proteins
• Allow sperm to become fertile
• In vivo or in test tube

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Intrauterine Insemination

• Sperm sample deposited in uterus just before
  release of an oocyte (s) in a natural or
  stimulated cycle using a Soft catheter
• Give hCG at injection or up to 24 hrs later
• Sperm volume 0.2-0.3 ml
• Pregnancy rates
• Around 15 per cycle

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Gamete Intrafallopian Transfer

• Laparoscopic technique in which oocyte and sperm
  placed in fallopian tube, allowing in vivo
  fertilization
• Procedure
• Superovulation
• US guided transvaginal oocyte retrieval
• 0.1-0.2 mil sperm with 2-3 oocytes

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In vitro fertilization - IVF

• Taking oocytes from woman
• Fertilizing them in lab with her partner's sperm
• Transferring resulting embryos back to her uterus
  3 or 5 days later

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IVF

• Procedure
• Superovulation
• Insemination
• Embryo transfer
• Luteal support

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IVF - Superovulation

• Gonadotropin stimulation
• Monitoring follicular development
• Ultra Sound guided transvaginal oocyte retrieval
• Oocyte fertilization with sperm

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IVF - Insemination

• Containers used
• Test tubes, Petri dishes, multi-well dishes
• Each oocyte inseminated with 0.5-1.0 mil PMNS
• Fertilization detected 12-20 hrs later by
  presence of
• 2 pronuclei in oocyte cytoplasm
• 2 polar bodies in perivitelline space

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IVF - Insemination

• Syngamy (combination of maternal and paternal
  pronuclei 24 hrs after insemination)
• Further cleavages occur at 24 hr intervals

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IVF - Embryo transfer

• Embryos transferred to uterus on 2nd or 3rd day
  after in vitro insemination
• 4-8 cells embryos
• 2-3 embryos transferred in 20 µl of culture fluid
• Transabdominal US to see fluid placed in uterus
• Cryopreserve excess embryos

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IVF - Luteal support

• Progesterone (P4) necessary for pregnancy
  maintenance
• Premature luteolysis in some superovulatory
  regimens
• P4 supplementation until menses occur or woman
  has positive pregnancy test

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Intracytoplasmic Sperm Injection - ICSI

• Injection of single sperm into single oocyte in
  order to get fertilization
• Procedure
• Superovulation
• US guided transvaginal oocyte retrieval
• IVF
• Oocytes injected with sperm using special
  microscopes, needles and micromanipulation
  equipment

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ICSI - Indications

• Low sperm concentration, motility, abnormal
  morphology
• Antisperm antibodies
• Fertilization failure after conventional IVF
• Ejaculatory disorders
• Absence of vas deferens or obstruction of
  ejaculatory ducts

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Assisted Hatching

• Indications
• Couples having IVF with
• Female partner's age over 37
• Poor quality embryos
• Excessive fragmentation
• Slow rates of cell division

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Assisted Hatching Procedure

• Embryo held with a specialized holding pipette
• A needle used to expel an acidic solution against
  ZP
• A small hole made in ZP
• Embryo washed and put back in culture in
  incubator
• ET shortly after hatching procedure
• Hope for the best

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Further Advances And Uses Of Assisted Conception
Technology

• Cryopreservation of
• Sperm
• Embryo
• Oocyte
• Ovarian tissue
• Growth of human follicles and oocytes in vitro
• In vitro maturation and transplantation of human
  spermatozoa

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Preimplantation Genetic Diagnosis (PGD)

• Identify genetic conditions in embryo before ET
• Hemophilia
• Cystic ?brosis
• Aneuploidy

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PGD

• Hemophilia
• Hereditary bleeding disorder
• Absence of a blood protein essential for clotting
• Types A lack of factor VIII
• Type B lack of factor IX

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PGD

• Cystic Fbrosis
• Genetic disease
• Defective gene causes body to produce abnormally
  thick, sticky mucus that obstruct
• Lungs
• Pancreas

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PGD

• Aneuploidy
• Having less than or more than normal diploid
  number of chromosomes
• Monosomy
• Trisomy
• Triploidy

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PGD

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

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Three day old embryos
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PGD

• Genetic analysis
• Polymerase chain reaction (PCR)
• Multicolor ?uorescence in situ hybridization
  (FISH)

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Polymerase Chain Reaction (PCR)

• In vitro technique for generating large amounts
  of a specific fragment of DNA from a small amount
  of template
• DNA amplification allows for
• Identification of point mutations
• DNA diagnostics

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FISH

• Detects
• Number of chromosomes
• Sexing embryos
• Sex chromosome aneuploidy
• Whole-chromosome paints for detection of
  rearrangements and identi?cation of marker
  chromosomes
• Analyze polar bodies

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FISH

• Fluorescent probes that bind to speci?c
  chromosomes are labeled with biotin for detection
  by speci?c ?uorochrome-conjugated antibodies
  under a ?uorescent microscope

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Interphase nucleus
X
Y
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Triploid
Normal Diploid
Tetraploid
Missing chromosomes
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PGD
Monosomy
Double trisomy
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PGD
Monosomy
Double trisomy
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ART

• Infertility
• Female partner 35
• Male partner 35
• Both partners 20
• Unknown cause 10
• Infertility more common with increasing age