MICROMANIPULATION

Submitted By…Satyendra Das

M.Sc 3rd Sem, Roll No. 17

Dept. of Life Science and Bioinformatics

Submitted to…Dr. Shubhadeep RoychoudhuryDept. of Life Science and Bioinformatics

INTRODUCTION

Micromanipulation refers to the procedures which are performed using microscopic instruments to manipulate oocytes (eggs), sperm, and embryos.

These microscopic techniques are designed to increase the chances of a successful in vitro fertilization (IVF) cycle

Preimplantation genetic diagnosis(PGD) ,ICSI, and assist hatching are offered to patients who can benefit from these techniques.

Why Micromanipulation ? During fertilization inside the human body, normally every few sperm

arrive at the egg. With IVF, many thousands of sperm reach the vicinity of the egg, greatly improving the chances of successful fertilization.

During conventional in vitro fertilization (IVF), eggs from the woman are collected and placed in a petri dish or test tube containing many sperm cells .

The gametes are then left in an incubator overnight for fertilization to occur. When this method fails, or when only a few sperm cells are available, then assisting fertilization using micromanipulation needs to be applied.

Widely used techniques for Micromanipulation

1. Intra cytoplasmic sperm injection (ICSI)

2. Preimplantation genetic diagnosis (PGD)

3. Assisted hatching

PREIMPLANTATION GENETIC DIAGNOSIS (PGD)

What is it?

Genetic analysis of a single cell from an eight-cell embryo done in conjunction with in vitro fertilization (IVF) to improve the chances of a “normal” pregnancy.

Why consider PGD in addition to IVF?

1. recurrent miscarriages

2. one child already affected with a genetic disease

3. family history of inherited disease

4. maternal age older than 38

5. prior failure with IVF

6. family “balancing” for sex

The Methods of Preimplantation Genetic Diagnosis

1. A single cell from the 6-8-cell embryo is removed using a fine glass needle by puncturing the the zona pellucida and aspirate the cell.

- In skilled hands, this generally does not harm the developing embryo. - Each cell is called a blastomere.

Blastomere removal for PGD testing

2. Prepare a metaphase spread of chromosomes to assess karyotype (number and integrity of each chromosome)

3. Two types of assessment techniques are common:a. chromosome “painting” (or FISH) using fluorescent probes specific

for each chromosome. These allow number and size of each chromosome to be checked.

• useful for identifying aneuploidies (incorrect chromosome numbers) and translocations

• limited number of chromosomes can be checked simultaneously; some abnormalities undetectable

Chromosome 1 in a normal cell, “painted” with red fluorophore

Fluorescence in situ hybridization (FISH)

Screening aneuploids with multiple probes

Aneuploidy is the most frequent cause of spontaneous abortions.

(b) Polymerase chain reaction (PCR) - amplification of DNA specific to a gene of interest (family history guides choice of genes)

Examples of genetic disorders detectable via PCR-based tests: Tay Sachs (autosomal recessive; ~98% accuracy) Cystic fibrosis (autosomal recessive; ~85% for common allele mutation) Huntington’s disease (autosomal dominant) Thalassemias (autosomal recessive blood disorder) Duchenne muscular dystrophy (X-linked recessive) Spinal muscular atrophy

As more genetic tests are developed as diagnostic tools, more will be used for predictive purposes in PDG.

Assisted hatching is an IVF technique in which the zona is treated prior to embryo transfer in order to weaken the wall of the embryo and thus improve the likelihood of successful hatching and embryo implantation.

Just prior to embryo transfer, the developing embryo must "hatch” out of its outer shell (zona pellucida).

Some embryos seem to have a thicker shell that may decrease their ability to implant

In such cases Advance technique of IVF with assisted hatching is performed.

3. Assisted Embryo Hatching

Techniques used in assisted hathing

1. Mechanical hatching

2. Chemical hatching

3. laser hatching

Mechanical hatching• In this the embryo cover is slit open mechanically with

the help of a thin long drawn out glass needle.

• This is done with the help of a machine called micromanipulator

Procedure:

• The zona pellucida is pierced with a very thin glass micro needle through both sides, the needle tip being controlled in the perivitelline space by eye.

• Then the suction of the holding pipette is stopped and the holding pipette is rubbed against the trapped area of the zona until this area has been completely abraded.

Mechanical technique: partial zona dissection

Creating a hole in the zona pellucida of an embryo by using acidic tyrode’s solution .

Procedure:

The fine micropipette containing acidic tyrode’s solution is brought very close to the zona pellucida and the acidic solution expelled gently over a small area (~20-30mm) until the zona is dissolved through to the inside.

After the procedure the embryos are thoroughly washed in fresh

medium and cultured until the time of transfer.

Chemical hatching

• This technique involves the creation of a precise gap in the zona pellucida of selected embryos using a 1.48 micron infrared diode laser.

Contact laser / Non contact laser

• Contact laser: laser radiation has to be delivered directly to the zona called contact laser

• Non contact laser: laser gamete manipulation has described in the non-contact mode. Using various wavelengths.

It allows direct delivery of laser beam through the objectives.

1.48 micron diode laser is advantageous over all other techniques as at 1.48 wave length , radiation is non mutagenic

The radiation is administered in the non contact mode

without embryo micromanipulation.

Procedure:

The embryo is held with gentle suction using a glass pipette

The red “pilot light “ provides a visual target for the laser.The white circle marks the safety region based on temperature.

The laser is usually fired 3 times to produce a small hole in the zone

Assisted hatching done by using laser light

This specifically designed laser system includes the laser, which serves as the energy source to create an opening in the zona pellucida.

A computer which allows the operator to precisely control the laser energy output, laser pulse duration and gap size.

Takes less time and does not expose the embryos to potentially adverse chemicals.

This procedure appears to be quick, precise and is chemical free.

Importance of Laser Assisted Hatching

CONCLUSIONMicromanipulation simply describes a set of tools and techniques. However, the stools and techniques have played a key role in advancing knowledge in reproductive biology and expanding the repertoire of clinical methodology and options. They have essentially solved the dilemma of male factor infertility, they allow for the diagnosis and circumvention of inherited genetic conditions and they hold great promise for further advancement in the future.

REFERENCES1. Malter H.E., “Micromanipulation in assisted Reproductive Technology”, (2016)32, pp 339-347.

2. IVF FIORID Reproductive Associates, Reproductive biomed contene. Micromanpulation: pp-339-347

3. Karl Kraus (1874-1936), Austrian Scientist,Pro domo et Mundo, Ch 7 (1912).

4. Cohen J., Malter H.E., Beth T., “Gamete and Embryo micromanipulation of infertility Treatment.” 1992, pp 86-87, 350.

5. Cohen J., Elsmer C., Kort H., Malter K., Massey J., Mayer M.P., Weimer K., “Impairedment of hatching process following IVF in Human”. 1990, pp. 7-13.

6. Malter H.E., Cohen J., “Blastocyst formation and hatch in vitro following zona drilling of mouse and human embryo gametic.” pp. 24, 67-80.

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