The document discusses the importance of assisted reproductive biotechnology in humans and animals. It notes that about 1 in 6 couples is infertile in the US and look to clinics in India and Asia for help. It also discusses efforts to save endangered species like the Pashmina goat and wild buffalo through cloning techniques. The document then goes on to discuss various causes of male and female infertility and techniques used in assisted reproduction like IVF, ICSI, and embryo transfer.

1. Importance of Assisted
Reproductive Biotechnology in
Humans & Animals
Atul Kaushal, M.Tech Ist sem
Animal Biotechnology Center
National Dairy Research Institute
Karnal

2. Infertility, Conservation & Innovation
 A human couple which does not use any contraceptive, but still can
not conceive within 2~3 years are considered infertile.
 About one out of every 6 couples is infertile in the US, and they look
towards Indian & other Asian infertility clinics.
 More relevantly, the efforts of National Dairy Research Institute in
coordination with SKUAST, Jammu are significant in saving the
Pashmina goat by producing Noori by ART.
 A female calf named ‘Deepasha’, which was a clone of a wild buffalo
named ‘Asha’, that has been kept in semi-captivity at Chhattisgarh,
was born on December 12, 2014 by normal parturition. Wild buffalo is
an endangered species.
 A male cloned calf named ‘Rajat’ and a female cloned calf named
‘Lalima’ were produced through hand-guided cloning technique in
NDRI, Karnal.
 The first produced buffalo clone in the world at NDRI campus is
named ‘Garima’ and was done so for selective breeding.

3. Male Infertility
• Infertility due to male factor accounts for at least
40% of infertility cases. For this reason, it is very
important that men also be investigated for fertility
problems. While blockages and problems producing
healthy sperm are often the main reasons for male
infertility, there are a variety of issues that are
diagnosed.
• Once a diagnosis has been made by a fertility
specialist, a proper course of treatment can be
recommended. Depending on the cause of male
infertility, possible forms of treatment may include
surgery, drugs, or assisted reproductive technologies,
like ICSI – IntraCytoplasmic Sperm Injection.

4. Decreasing human male/female fertility
rates
• Recent studies have identified several factors causing
a decline in male fertility.
• The anti-impotence drug Viagra is suspected to
damage sperm acrosomes, and lowering the taker’s
ability to conceive (40% less).
• Another study suggests that pollution from chemicals
such as dioxin can lower a man's sperm count.
Similar pollutants are known to harm female fertility.
• Endocrine-disrupting chemicals are also thought to
be affecting fertility rates.

5. Menevit, the 'first ever drug for male
infertility',
• Menevit, developed in Australia, contains
antioxidants which work by acting on free
radicals that fragment sperm, the main cause
of infertility.
• In a preliminary study of 60 infertile men, the
rate of pregnancy was increased significantly,
but larger clinical trials are required before the
drug can be merited.

6. Female infertility
• Ultrasound is likely to be done first, as this can alert
the specialist to any possible problems. There are
also a number of blood tests that can be performed.
In women, the most common ones include:
• Day 3 FSH testing
• Luteinizing hormone (LH) testing
• Progesterone testing
• Estradiol Level testing
• Hysterosalpingogram
• For women who are suspected of having low ovarian
reserves, a Day 3 Inhibin and a Clomiphene
Challenge test may be performed.
• Antibody against husband’s sperm

7. Treating Infertility: Assisted Reproductive
Technology (ART)
• A large percentage of couples face difficulties in
getting pregnant, many have found success with
Assisted Reproductive Technology (ART).
• ART involves a number of different procedures to
help address fertility problems and increase the
likelihood of pregnancy. Only a reproductive
biotechnologist can correctly advise a couple that are
aspiring to be parents through ART.

8. Assisted Reproductive Technology (ART)
• Insemination
 Sperm collection/sperm donation
• In vitro fertilization
 Egg retrieval/egg donation
• Surrogacy
 Selection of surrogate mother

9. Who needs IVF?
• Couple has infertility problem.
– Men with low sperm count, abnormal sperm, no mature
sperm, antibody against sperms etc.
– Women with oviduct obstruction, endometriosis,
endocrine imbalance, reproductive organ infection,
antibody against partner’s sperms etc.
• The couple both carry recessive genetic disorder
on the same genetic locus, or male with X-/Y-linked
genetic disorder or female with X-linked genetic
disorder and desire a healthier progeny.

10. Who needs IVF?
• Humans aspiring to be parents/Endangered
species.
– Males with low sperm count, abnormal sperm, no
mature sperm, antibody against sperms etc.
– Females with oviduct obstruction, endometriosis,
endocrine imbalance, reproductive organ infection,
antibody against partner’s sperms etc.
• The couple both carry recessive genetic disorder on the
same genetic locus, or male with X-/Y-linked genetic
disorder or female with X-linked genetic disorder and desire
a healthier progeny.

11. In Vitro Fertilization (IVF)

12. In Vitro Fertilization (IVF)
• IVF is the uniting of egg and sperm in vitro (in
a test tube inside the laboratory).
• Subsequently the embryos are transferred
into the uterus through the cervix and
pregnancy is allowed to begin.

13. In Vitro Fertilization (IVF)
• Treating infertility through assisted reproductive
technology
 Ovulation induction
 Monitoring of hormone levels and follicle scans with ultrasound
 Ovaries
 Zygote
 Uterus

14. Procedures for IVF
• Day 0 Egg retrieval, Sperm collection, and
preparation before insemination in vitro
• Day 1 Check eggs for fertilization (the presence of
two pronuclei or PN's)
• Day 2 Embryos at the 4-cell or more stage of
development
• Day 3 Embryos at the 8-cell or more stage of
development
• Day 4 Embryos at the compacted morula (16-32 cell)
stage
• Day 5 Embryos at the blastocyst stage of
development

15. Inducing Superovulation
• Injections of Lupron, Metrodin, Pergonal or
Humegon allow the ovaries to develop many follicles
as seen in this ultrasound immediately prior to egg
retrieval.

17. Retrieved Egg

18. Human Metaphase-I oocyte with extruded polar body

19. Human Metaphase-II oocyte

20. Human Metaphase-II oocyte with prominent spindles

22. Fertilization of retrieved egg
• Retrieval of maturing eggs via laparoscopy.
• Each egg is incubated with > 3.5 x 106 sperms. The
fertilized egg contains two pronuclei. Each contains
half the chromosomal material from each one of the
genetic parents.

23. Embryo at 4-cell stage
• A four cell pre-embryo. Each of the cells is called
a blastomere. The embryo is surrounded by a
protein matrix "shell" called the zona pellucida.

24. Transferring of 8 cell-stage embryo
• On the third day after egg retrieval eight cell pre-
embryos can be transferred to the uterus. On
average, 15 to 25% of embryos will implant after
being transferred.

25. Hatching in vivo prior to implantation
• Prior to implantation, the pre-embryo, now at the
blastocyst stage, must hatch out of the zona
pellucida prior to implantation.

26. Intracytoplasmic sperm
injection (ICSI)
A revolutionary technique that
demands precision and expertise

27. IntraCytoplasmic Sperm Injection (ICSI)
• Intracytoplasmic sperm injection (ICSI,
pronounced "eeksee") is an in vitro fertilization
procedure in which a single sperm is injected
directly into an egg.
• This procedure is used to overcome male
infertility problems, although it may also be
used where eggs cannot easily be penetrated by
sperm, and occasionally as a method of in vitro
fertilization, especially that associated with
sperm donation.

28. How is ICSI performed?
• Using micromanipulation technology, ICSI allows
fertility specialists to fertilize an egg using just one
sperm. While it is preferred to use sperm from a semen
sample, specialists can retrieve sperm from the testicles
if it is necessary. Once sperm has been collected, the
specialist will draw a single sperm into a needle and
inject it directly into an egg that has been collected from
the female partner through the usual retrieval methods.
• This process bypasses the conventional IVF methods of
fertilization, thereby ensuring that fertilization has
taken place. The fertilized eggs are then left to culture
for a few days before being transferred back to the
woman’s uterus. ICSI is always used alongside IVF.

29. Who needs ICSI?
• Men who have very poor semen quality or
azoospermia should be offered ICSI.
– Low sperm count
– Poor sperm motility
– Abnormally high amount of morphologically atypical
sperm
– Experienced fertilization failure for unknown reasons in a
previous IVF cycle
– Lack of any sperm in ejaculate due to CAVD, failed
vasectomy reversal, failure to produce sperm, or an
obstruction in the epididymus due to past inflammation
– Retrograde ejaculation
– Immunological factors

30. Gamete/embryo transfer

31. • Many of the large programs have attempted
blastocyst culture, but returned to day 3
transfers because they had trouble getting the
embryos to grow the blastocyst stage. Growing
the embryos to the blastocyst stage requires
great attention to detail - a luxury not afforded
in a big program performing dozens of
procedures a day.
• Programs performing less than 200 cycles per
year have had the best luck with blastocyst
culture and transfer with reported (but
unverified) pregnancy rates in the 50-70%
range for younger patients and egg donor
cycles.

32. Gamete intrafallopian transfer
• In gamete intrafallopian transfer (GIFT), eggs
are removed from the woman ovary, and
placed in one of the fallopian tubes, along
with the man's sperm. This allows the
fertilization to take place inside the woman’s
body. Therefore, this variation is actually an in
vivo fertilization, and not an in vitro
fertilization.

33. Zygote Intrafallopian transfer
• Zygote IntraFallopian Transfer (ZIFT) is an
infertility treatment where a blockage in the
fallopian tubes are the cause.
• Egg cells are removed from a woman's ovaries,
and in vitro fertilized.
• The resulting zygote is placed into the
fallopian tube by the use of laparoscopy.

34. IVD: IVFed eggs developed in vivo
• The treatment, named in vivo development, or IVD,
has been termed a more natural alternative to IVF
and there are hopes that it will lead to fewer
embryos with genetic abnormalities being produced.
• In normal IVF eggs are fertilized with sperm and
allowed to grow for a few days in a laboratory dish
containing chemicals and nutrients, before being
transferred into the woman's womb for implantation.

35. The creation of 'female' sperm and
'male' eggs
• If this technological breakthrough ever comes about,
it is suggested that such artificial gametes could offer
the possibility of genetic reproduction to people in
same sex relationships.
• In fact artificial gametes offer people to have
genetically related offspring regardless of age, gender,
relationship status or sexuality. Women could use the
technique to produce eggs even after having gone
though the menopause.
• Individuals who cannot find reproductive partners
could even use artificial gamete technology to create
complementary gametes from their own bodies to
fertilize their 'natural' sperm or eggs.

36. Applications of ART
• Endangered species can be protected. Work is
already being carried out by institutions like
NDRI with animals like Noori, the first ART-
born Pashmina goat by combined efforts with
SKUAST, Jammu.
• ART, allows in vitro fertilized (IVFed) embryos
for preimplantation genetic screening (PGS)
evaluation. The is not used to look for a
specific disease but a technique to identify
embryos at risk.

37. The End
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