Importance of Assisted Reproductive Biotechnology in Humans & Animals

Atul Kaushal, M.Tech Ist semAnimal Biotechnology Center

National Dairy Research InstituteKarnal

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.

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.

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.

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.

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

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.

Assisted Reproductive Technology (ART)

• Insemination Sperm collection/sperm donation

• In vitro fertilization Egg retrieval/egg donation

• Surrogacy Selection of surrogate mother

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.

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.

In Vitro Fertilization (IVF)

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.

In Vitro Fertilization (IVF)• Treating infertility through assisted reproductive

technology Ovulation induction Monitoring of hormone levels and follicle scans with ultrasound Ovaries Zygote Uterus

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

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.

Retrieved Egg

Human Metaphase-I oocyte with extruded polar body

Human Metaphase-II oocyte

Human Metaphase-II oocyte with prominent spindles

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.

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.

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.

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.

Intracytoplasmic sperm injection (ICSI)

A revolutionary technique that demands precision and expertise

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.

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.

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

Gamete/embryo transfer

• 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.

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.

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.

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.

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.

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.

The End

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