Assisted Reproductive Technologies

1. Assisted Reproductive Technologies

2. Outline • History of Assisted Reproductive Technologies • Artificial insemination • Embryo transfer • In-vitro fertilization • Cloning • Problems that arise • Non-implantation • Ectopic pregnancies • Large Offspring Syndrome

3. History of Assisted Reproductive Technologies

4. Artificial Insemination • First known method of reproductive technology • Leeuwenhoek (1678) was the first person to see sperm cells • He called them animalcules • Spallanzani (1784) was the first known person to artificially inseminate • Practice involved dogs • It wasn’t until the 20th century that A.I. became a practical procedure. • In Russia (Ivanow) used in dogs, foxes, rabbits, poultry, and horses.

5. Embryo Transfer • First successful embryo transfer was in rabbits in 1890 • First bovine embryo was recovered in 1930 • Hartman, Lewis, Miller and Swett of the Carnegie Laboratory of Embryology in Baltimore, MD • First successful embryo transfer was in 1950 • First calf born at University of Wisconsin • Non-surgical methods expanded the commercial use of E.T. and by 2002 over 25,000 E.T. calves were registered.

6. In-Vitro Fertilization • Research and experiments began in 1878 and continued until the first successful reports of IVF were recorded around 1953. • The discovery of sperm capacitation in 1951 was the key component to successful IVF. • Hamster gametes were the first mediums of using spermatozoa capacitated in-vitro (Yanagimachi and Chang, 1963, 1964) • Chang (1959) showed the rabbit eggs fertilized in-vitro could develop into normal functioning young. • Crucial to the acceptance of human IVF as a clinical treatment for infertility

7. Cloning • First successful clone was in the late 1800’s by Hans Dreisch • Performed on sea urchins • In 1951 a team of scientists cloned a frog embryo. • They took the nucleus out of a fertilized embryo and placed it in an unfertilized embryo. • First cloned mammal was a sheep. • Took differentiated cells from the mammary gland to culture and create another sheep.

8. Problems that Arise

9. Implantation Failure • Pregnancy rates after one cycle of IVF or ET are ~60%

10. Assumed etiologies for repeated implantation failure (RIF) • Decreased endometrial receptivity Uterine cavity abnormalities Thin endometrium Altered expression of adhesive molecules Immunological factors Thrombophilias • Defective embryonic development Genetic abnormalities (male/female/gametes/embryos) Zona hardening Suboptimal culture conditions • Multifactorial effectors Endometriosis Hydrosalpinges Suboptimal ovarian stimulation

11. Decreased Endometrial Receptivity • Altered expression of adhesive molecules • Many cases were found to be caused by local dysregulation of cytokines. • Absence of a specific integrin–αVβ3 • High levels of aromatase p450 mRNA • Decrease in pinopode expression • High matrix metalloproteinases

12. Defective Embryonic Development • Zonapellucidahardening • Hardens naturally after fertilization • Increases thickness during • In-vitro culturing • In-vivo ageing • Zona thickness prevents hatching

13. Multifactorial Effectors • Hydrosalpinges • Fluid is slightly alkaline • Contains cytokines, prostaglandins, other inflammatory compounds • Reflux of hydrosalpinx fluid • Causes diminishing embryonic endometrial apposition

14. Ectopic Pregnancies • The implantation of a fertilized ovum outside the endometrial cavity

15. Ectopic Pregnancies • Most commonly caused by damaged fallopian tubes • ~97% occur in fallopian tubes • Occurs in 1-2% of all pregnancies • Due to the nature of IVF tubal implantation is a common problem. • In a study of 3000 clinical pregnancies 4.5% had ectopic pregnancies due to in-vitro fertilization.

16. Ectopic Pregnancies

17. Large Offspring Syndrome • Only observed in bovine and ovine • IVP or cloned embryos • Series of pathologic changes have extended gestation and increased birth weight • Causes breathing difficulties, reluctance to suckle, and sudden perinatal death

18. Large Offspring Syndrome • In one study (Garry et al., 1996) large calves born were hypothermic, some hypoglycemic and some showed signs of metabolic acidosis and hypoxia • Further tests showed • Fourfold increase of plasma concentrations of insulin at birth • Plasma glucagon concentrations increased with birth weight • Found no relationship of maternal malfunction with these symptoms.

19. Large Offspring Syndrome • No exact known causes of LOS • Extrauterine environments and manipulations during in-vitro or cloning are associated with embryos resulting in LOS • During the in-vitro culture phase the embryos can be perturb enough to cause drastic changes • A study showed that mature and fertilized eggs recovered from sheep donors then cultured in-vitro for 6 days showed an increase in mean birth weight at day 125 of gestation (Sinclair et al., 1998)

20. Large Offspring Syndrome • Asynchrony in gestational age between embryo and recipient female • Placing embryos in an advanced uterine environment may initiate the accelerated growth • Receives more secretory signals

21. Large Offspring Syndrome • Types of culture embryos are mediated in have effects on growth • Lazzariet al.,(2002) showed that embryos cultured in high concentrations of serum or BSA showed increased growth and birth weights • Showed increase in number of cells in Day 7 blastocysts • Increase size in Day 12 blastocysts • Increased abundance of specific gene transcripts.

22. FIG. 2.Expression patterns of various developmentally important gene transcripts in bovine blastocysts of different origin. Vertical striped bars: in vivo; open bars: ligated sheep oviduct; shaded bars: SOF-BSA; solid bars: SOF-serum. Bars with different superscripts differ significantly (a:b, P ≤ 0.05)