1. Programming and Assisted Reproductive Technologies Modules 18 and 19 AnS 536 Spring 2014

2. Fetal Programming  Hypothesis  The developing fetus responds to nutritional and oxygen shortages by diverting resources from other organs to the brain  Potential adverse affects may occur later in life  Adaptations include:  Vascular response  Metabolic response  Endocrine response

3. Fetal Programming  Exogenous maternal malnutrition during pregnancy  May cause lifelong, persisting adaptation to the fetus  Low birth weight  ↑ Cardiovascular risk  Non-insulin dependent diabetes  Critical periods of vulnerability to suboptimal conditions during development  Vulnerable periods occur at different times for various tissues  Greatest risk: rapidly dividing cells

4. Fetal Programming  ‘Fetal origins’ hypothesis  Poor in utero environment induced by maternal dietary or placental insufficiency may program susceptibility later in fetal development and life  ‘Thrifty phenotype hypothesis’  If in utero nutrition is poor, then predictive adaptive responses are made by the fetus to maximize uptake and conservation of any nutrients available, resulting in a conservative metabolism  Problems occur when postnatal diet is adequate and plentiful and exceeds the range of predicted adaptive response

5. Fetal Programming  Prevalent in developed and developing countries  Dutch famine (limited intake of 1680-3360 kJ)  During late gestation was associated with increased adult obesity and glucose intolerance  During early gestation resulted in hypertension  Disadvantageous populations in USA, South Africa, the Caribbean, India, and Australia  Shown cardiovascular risk to be greater in populations suffering from poor in utero nutrition

6. Fetal Programming  Permanent affects of programming  Modifies susceptibility to disease  Structural changes to organs  Might pass across generations  Different effects on males and females  Placental effects  Fetus will attempt to compensate for womb deficiencies

7. Metabolic Syndrome  Cluster of abnormalities occurring together, increase your risk of heart disease, stroke, and diabetes  Largely attributed to altered dietary and lifestyle factors favoring central obesity  Characterized by a group of metabolic risk factors in a person  Abdominal obesity  Atherogenic dyslipidemia  Elevated blood pressure  Insulin resistance  Proinflammatory states  Prothrombotic states

8. Metabolic Syndrome  Abdominal obesity  Strongly associated with metabolic syndrome  Atherogenic dyslipidemia  ↑ triglycerides, ↓ concentrations of HDL cholesterol, ↑ remnant lipoproteins, ↑ apolipoprotein B, small LDL particles nad small HDL particles

9. Metabolic Syndrome  Elevated blood pressure  Strongly associated with obesity  Commonly occurs in insulin-resistant individuals  Insulin resistance  Commonly associated with metabolic syndrome  Usually leads to glucose intolerance  diabetic- level hyperglycemia  Independent risk factor for cardiovascular disease

10. Metabolic Syndrome  Proinflammatory states  ↑ levels of C-reactive protein  Excess adipose tissue release inflammatory cytokines  Multiple mechanisms contribute to inflammatory state  Prothrombotic states  ↑ Plasma plasminogen activator inhibitor-1  ↑ fibrinogen  Rises in response to a high cytokine state  Acute phase reactant  Proinglammatory and prothrombotic states are interconnected

12. Metabolic Syndrome  Underlying risk factors for this condition:  Abdominal obesity  Insulin resistance  Physical inactivity  Aging  Hormonal imbalance  Genetic predisposition  Fetal environment

13. Non-genomic Intergenerational Effects  Significant evidence that programmed phenomena can be disturbed in later generations  Offspring exposed to a poor uterine environment  Prenatal programming by nutrition or exercise (animal models)  Postnatal programming by nutrition or handling (animal models)  Effects:  Birth weight  Glucose tolerance  Hypothalamic-pituitary axis in subsequent generations

14. Non-genomic Intergenerational Effects  Effects on birth weight  Black and white hooded rats (Steward, 1975)  Continued poor maternal nutrition produced amplified effects on birth weight through a number of generations  Accidental introduction of less-palatable food in control animals resulted in a period of self-imposed calorie restriction  Evidence that poor nutrition in one generation can produce effects on birth weight in subsequent generations

15. Non-genomic Intergenerational Effects  Effects on birth weight, cont…  First generation pups (Pinto and Shetty, 1995)  Exercise during pregnancy resulted in low birth weigh first generation pups  First generation offspring were sedentary during pregnancy and second generation offspring were also found to be growth retarded  Suggesting adverse intergenerational influence of maternal exercise stress on fetal growth

16. Non-genomic Intergenerational Effects  Metabolic parameters and blood pressure  Female rabbits with surgically induced hypertension were mated with normotensive males  Female offspring had increased blood pressure as adults when compared to the offspring of sham-operated females  Blood pressure in male offspring was unaffected

17. Non-genomic Intergenerational Effects  Postnatal effects  Second generational alterations on glucose homeostasis has been seen when overfeeding takes place in the neonatal period  In rodents, naturally occurring variations in maternal behavior is associated with different hypothalamic- pituitary-adrenal stress responsiveness in offspring  Postnatal environmental manipulations to the hypothalamic-pituitary-adrenal axis stress response may produce intergenerational effects

18. Assisted Reproductive Technologies (ART)  Artificial insemination (AI)  Sexed semen and embryo sexing  Embryo transfer (ET)  In vitro fertilization (IVF)  Intracytoplasmic sperm injection (ICSI)  Gamete intrafallopian transfer (GIFT)  Zygote intrafallopian transfer (ZIFT)  Donor egg, sperm or embryo  Cloning (SCNT)

19. Artificial Insemination (AI)  Used commonly in livestock  Method of banking semen (genetics) without keeping a sire on site (cryopreservation)  Challenges  Difficulty passing AI gun through cervix  Potentially reduced pregnancy rates  Breeding when animal is in estrus  Damage to reproductive tract  Reduced fertility with cryopreserved sperm

20. Artificial Insemination (AI)  Management approaches  Goal is to increase conception rates  Implementing appropriate methods of heat detection  Skilled technician in AI  Time of year, time of day, or temperature on day of breeding can affect conception rates

21. Sexed semen and embryo sexing  Biological mechanism  Sexed semen uses flow cytometry to sort genetically male and female sperm  Female (XY) sperm have 4% more DNA than male sperm  Embryo sexing entails obtaining a biopsy of the inner cellular mass (ICM) of the embryo to determine male or female status  Used in livestock industry (dairy cattle)  Ethical considerations in humans

22. Sexed semen and embryo sexing  Challenges  Sexed semen  Reduced fertility  Higher concentration of sperm needed to ensure pregnancy  Embryo sexing  Reduced viability of embryo  Multiple pregnancies can occur

23. Sexed semen and embryo sexing Management approaches  Sexed semen is preferred method, less risk to developing embryo  Less invasive  Sexed semen is used in combination with IVF technologies or AI  Embryo sexing require embryo transfer technique

24. In vitro fertilization (IVF)  Commonly used practice in humans and livestock (cattle)  Biological mechanisms  Dam is administered a series of reproductive hormone (GnRH) to stimulate the development of Graafian follicles, a.k.a., superovulation  Oocytes are collected via aspiration and are incubated in an artificial lab environment, mimicking the environment of the uterus  Sperm is introduced to the oocytes and fertilization occurs  Embryos are developed to the blastocyst stage prior to transfer to the mother or dam

25. In vitro fertilization (IVF)  Challenges  Patients or recipients using IVF technology usually face moderate to severe infertility problems  Poor quality ovum or sperm  Uterine rejection  May be used as a ‘last ditch effort’ for pregnancy  Incidence of multiple births are high  Ectopic pregnancies

26. In vitro fertilization (IVF)  Management approaches  Age of the patient  Inversely related to the probability of multiple pregnancies and overall pregnancy success  Implantation rate  Attributed to many factors including quality of embryo  Selecting embryos with the greatest potential for survival  Matching synchrony of uterus to embryo stage of development  Number of embryos transferred  Directly related to risk of multiple pregnancies  Most controllable of the variables

27. Embryo transfer (ET)  Biological mechanisms (in livestock)  A donor animal is super ovulated, bred by a sire (AI or live cover)  Fertilization occurs in vivo and embryos are collected prior to the implantation stage  Collected embryos can be then be transferred to a recipient (surrogate) animal with the same estrus synchrony as the donor or can be cryopreserved for a later implantation date

28. Embryo transfer (ET)  Challenges  Reduced rate of pregnancy as compared to natural conception  Fresh embryos have better conception rate as compared to cryopreserved embryos  Synchronizing recipient animals with the donor animal  Retained embryos in donor animal resulting in pregnancy

29. Embryo transfer (ET)  Management approaches  Optimizing synchrony for maximum pregnancy rates  Selecting appropriate recipients for breed and birth weight of offspring  Use of prostaglandin in donor animals to eliminate pregnancy due to retained embryo

30. Intracytoplasmic sperm injection (ICSI)  Biological Mechanism  A single sperm is injected into unfertilized oocyte and is transferred to a recipient  Treatment for male factor infertility  Challenges  Potentially abnormal sperm can fertilize ova  Long term health affects, including genetic abnormalities  Lower birth weight  Abnormalities on the Y chromosome  Greater potential for developmental delays

31. Intracytoplasmic sperm injection (ICSI) Management approaches  Men and women should have genetic screening for potential chromosomal abnormalities prior to fertility treatment  Men lacking a vas deferens can carry mutations increasing the risk of offspring with cystic fibrosis

32. Gamete Intrafallopian transfer (GIFT)  Biological mechanisms  An unfertilized oocyte and sperm are combined outside of the uterus and are surgically transferred to the site of normal fertilization in the fallopian tube via laparoscopic technique  Fertilization occurs in vivo  Implantation occurs naturally

33. Gamete Intrafallopian transfer (GIFT)  Challenges  Surgical intervention causes trauma and scarring  More invasive technique  Multiple pregnancies  Management approaches  Other techniques are more widely used (IVF) due to higher success rates

34. Zygote intrafallopian transfer (ZIFT)  Biological mechanisms  Similar to GIFT however, oocyte and sperm are combined outside of the uterus and are not transferred until an embryo is produced  Management approaches  Other techniques are more widely used (IVF) due to higher success rates

35. Donor ova, sperm or embryo  Donor oocyte, sperm or embryos can be used to generate offspring if poor quality ova or sperm exist or if there is a lack of a female or male counterpart  Challenges  Social implications  Lack of genetic history  Predisposition to risk of disease  Children may never know their parents

36. Cloning (SCNT)  Producing genetically identical copies of a biological entity  Different types of methods:  Reproductive  Natural identical twinning  Somatic cell nuclear transfer (SCNT)  Non-reproductive  Recombinant DNA Technology  Therapeutic cloning

37. Cloning (SCNT)  Challenges  Low conception rates  Increased birth weights  Increased incidence of genetic abnormalities  Decreased neonatal survival  Increased placentation abnormalities  Decreased life span of animal??  Increased dystocia and prolonged gestation  Decreased genetic variation

38. Cloning (SCNT)  Biological mechanisms  Low conception rates  Research is being done to explore this reality  Current methods of cloning are very artificial and vastly differ from normal in vivo embryo development  Methods to promote a more similar environment to what the embryo experiences in vivo  Increased birth weights  Possible link to media used in incubating cloned embryos  Fetal calf serum (FCS) promotes excessive growth of embryo

39. Cloning (SCNT)  Biological mechanisms, cont…  Increased incidence of genetic abnormalities  Possible link to problems in cell reprogramming with SCNT  Electric charge fuses cells to promote cell proliferation  Decreased neonatal survival  Offspring can be less vigorous initially after birth  Anemia, enlarged organs, metabolic disturbances, problems thermoregulating, hypoxia can all contribute

40. Cloning (SCNT)  Biological mechanisms, cont…  Increased placentation abnormalities  Mechanisms unknown  Hydrops amnion is a condition that is seen during gestation in cattle and sheep  Less frequent attachment sites but increased size of codyledons as compared to normal pregnancies in cattle  Intrauterine Growth Restriction (IUGR)  Decreased life span of animal ??  “Dolly” the sheep only lived to 6 years of age  Controversial studies that cloning affects life span of offspring  Decreased telomere length has been associated with a decreased life span  Age of animal being cloned may affect life span of offspring (increased age shortens telomere length)

41. Cloning (SCNT)  Biological mechanisms, cont…  Increased dystocia and prolonged gestation  Recipient animals carrying cloned animals fail to recognize the onset of parturition near term or the cloned fetus fails to induce parturition  Increased birth weights contribute to dystocia  Decreased genetic variation  Selection of cloned animal can potentially promote a genetically inferior or superior animal  Breeding pool can be narrowed  Long term effects?

42. Cloning (SCNT)  Management approaches  Low conception rates  Matching synchrony of recipient animal with stage of embryo  Increased birth weights  Selecting larger framed, multi-parous recipient animals  Awareness of breed of embryo and potential birth weight  Caesarian section deliveries

43. Cloning (SCNT)  Management approaches, cont…  Increased incidence of genetic abnormalities  Humane euthanasia or abortion in severe cases  Preventing the perpetuation of genetically inferior animals through selection  Decreased neonatal survival  Intensive care and monitoring of animal first week of life  Ensuring colostrum uptake  Temperature regulation

44. Cloning (SCNT)  Management approaches, cont…  Increased placentation abnormalities  Close monitoring of recipient animals for hydrops amnion  Abort early in gestation if necessary  Pregnancy palpations/ultrasound to determine fetal well being  Decreased life span of animal ??  Age of animal being cloned may affect life span of offspring (increased age shortens telomere length)

45. Cloning (SCNT)  Management approaches, cont…  Increased dystocia and prolonged gestation  Know expected parturition dates  Induce parturition if necessary  Caesarian sections  Decreased genetic variation  Criteria for animal selection  Promoting healthy animals – not just based on phenotype