1. Assisted Reproductive Technology (ART) Prepared by Dr. Abdelsalam Talafha Collage of veterinary medicine, JUST American Board Certified, Comparative Veterinary Obstetrics and Gynecology

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

3. ART USA women infertility rate USA women infertility rate Ages 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 diagnosed 80% of infertility cases can be diagnosed 85% of cases can be successfully treated 85% of cases can be successfully treated

4. ART Female infertility Female infertility Disorders 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%

5. Male Infertility Total 2104 100

6. Male Infertility

7. ART Assisted Conception

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

9. Assisted Conception Objective To bring sperm and oocyte close to each other to promote chances of fertilization and, ultimately, achieve a pregnancy To bring sperm and oocyte close to each other to promote chances of fertilization and, ultimately, achieve a pregnancy

11. Assisted Conception Required procedures Superovulation Superovulation Sperm preparation Sperm preparation Assisted fertilization Assisted fertilization

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

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

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

16. Sperm 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 capacitation Sperm capacitation Coating of sperm with seminal plasma proteins Coating of sperm with seminal plasma proteins Allow sperm to become fertile Allow sperm to become fertile In vivo or in test tube In vivo or in test tube

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

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

21. In vitro fertilization - IVF Taking oocytes from woman Taking 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 uterus 3 or 5 days later Transferring resulting embryos back to her uterus 3 or 5 days later

22. IVF Procedure Procedure Superovulation Superovulation Insemination Insemination Embryo transfer Embryo transfer Luteal support Luteal support

23. IVF - Superovulation Gonadotropin stimulation Gonadotropin stimulation Monitoring follicular development Monitoring follicular development Ultra Sound guided transvaginal oocyte retrieval Ultra Sound guided transvaginal oocyte retrieval Oocyte fertilization with sperm Oocyte fertilization with sperm

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

27. IVF - Insemination Syngamy (combination of maternal and paternal pronuclei 24 hrs after insemination) Syngamy (combination of maternal and paternal pronuclei 24 hrs after insemination) Further cleavages occur at 24 hr intervals Further cleavages occur at 24 hr intervals

28. IVF - Embryo transfer Embryos transferred to uterus on 2 nd or 3 rd day after in vitro insemination Embryos transferred to uterus on 2 nd or 3 rd day after in vitro insemination 4-8 cells embryos 4-8 cells embryos 2-3 embryos transferred in 20 µl of culture fluid 2-3 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 embryos Cryopreserve excess embryos

29. IVF - Luteal support Progesterone (P4) necessary for pregnancy maintenance Progesterone (P4) necessary for pregnancy maintenance Premature luteolysis in some superovulatory regimens Premature luteolysis in some superovulatory regimens P4 supplementation until menses occur or woman has positive pregnancy test P4 supplementation until menses occur or woman has positive pregnancy test

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

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

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

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

47. Further Advances And Uses Of Assisted Conception Technology Cryopreservation of Cryopreservation of Sperm Sperm Embryo Embryo Oocyte Oocyte Ovarian 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 spermatozoa In vitro maturation and transplantation of human spermatozoa

48. Preimplantation Genetic Diagnosis (PGD) Identify genetic conditions in embryo before ET Identify genetic conditions in embryo before ET Hemophilia Hemophilia Cystic fibrosis Cystic fibrosis Aneuploidy Aneuploidy

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

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

51. PGD Aneuploidy Aneuploidy Having less than or more than normal diploid number of chromosomes Having less than or more than normal diploid number of chromosomes Monosomy Monosomy Trisomy Trisomy Triploidy Triploidy

52. PGD 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 analysis Obtain 1-2 blastomeres for molecular analysis

53. Three day old embryos

59. PGD Genetic analysis Polymerase chain reaction (PCR) Polymerase chain reaction (PCR) Multicolor fluorescence in situ hybridization (FISH) Multicolor fluorescence in situ hybridization (FISH)

60. Polymerase Chain Reaction (PCR) In vitro technique for generating large amounts of a specific fragment of DNA from a small amount of template In vitro technique for generating large amounts of a specific fragment of DNA from a small amount of template DNA amplification allows for DNA amplification allows for Identification of point mutations Identification of point mutations DNA diagnostics DNA diagnostics

61. FISH Detects Number of chromosomes Number of chromosomes Sexing embryos Sexing embryos Sex chromosome aneuploidy Sex chromosome aneuploidy Whole-chromosome paints for detection of rearrangements and identification of marker chromosomes Whole-chromosome paints for detection of rearrangements and identification of marker chromosomes Analyze polar bodies Analyze polar bodies

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

63. X Y Interphase nucleus

64. Normal Diploid Triploid Tetraploid Missing chromosomes

65. MonosomyDouble trisomy PGD

66. MonosomyDouble trisomy PGD

67. ART 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 age Infertility more common with increasing age