Programming and
Assisted Reproductive
Technologies
Modules 18 and 19
AnS 536
Spring 2014
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
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
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
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
Fetal Programming
 Permanent affects of programming
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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
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
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Abdominal obesity
Atherogenic dyslipidemia
Elevated blood pressure
Insulin resistance
Proinflammatory states
Prothrombotic states
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
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  diabeticlevel hyperglycemia
 Independent risk factor for cardiovascular disease
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
Metabolic Syndrome
 Underlying risk factors for this condition:
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Abdominal obesity
Insulin resistance
Physical inactivity
Aging
Hormonal imbalance
Genetic predisposition
Fetal environment
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
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
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
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
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 hypothalamicpituitary-adrenal stress responsiveness in offspring
 Postnatal environmental manipulations to the
hypothalamic-pituitary-adrenal axis stress response
may produce intergenerational effects
Assisted Reproductive
Technologies (ART)
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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)
Artificial Insemination (AI)
 Used commonly in livestock
 Method of banking semen (genetics) without
keeping a sire on site (cryopreservation)
 Challenges
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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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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Social implications
Lack of genetic history
Predisposition to risk of disease
Children may never know their parents
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
Cloning (SCNT)
 Challenges
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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
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
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
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)
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?
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
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
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)
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