Embryo Transfer Technology

8/3/2019 Embryo Transfer Technology

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EMBRYO TRANSFER

TECHNOLOGY


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Click to edit Master subtitle style

Embryo Transfer

Refers to a step in the process of assistedreproduction.The development of Embryo TransferTechnology (ETT) has emerged as a very

powerful tool after artificial insemination forgenetic improvement programme.

It is a complex process and involves multiplesteps resulting in the insemination and

fertilization of oocytes (eggs) in the laboratory.

Used in connection with In vitro fertilization(IVF)),

It may be used in humans or in animals, in


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Refers to the technology by which fertilized

ova are collected from the reproductive tractof genetically superior female (donor) andtransferre

d to that of another female(recipient) which is genetically inferior. ETT now offers new opportunity to

improve the dairy industry.


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HistoricalPerspective:

In 1890 Waiter Heape, student at Cambridge

University performed the first embryotransfer experiment.

He removed surgically two embryo (4-cellstage) from Angora rabbit and transferred it ina Belgian female hare.

Afterwards the events occurring indifferent year are as follows:

1933:- J. S. Nicholson successfullytransferred embryo in rat. 1951:- El willett usefully transferred

embryo in cattle.


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1960:- Kvasnickni, a Russian scientist producedfirst successful embryo transplanted offspringin swine.

1964:- Mutter and co-workers transferred abovine embryo non surgically and getting aviable offspring.

1979:- Steptoe and Edwards got success ingetting a human baby girl through ETT.

1983:- Drost got calf born by ETT.

1991:- Mishra and coworker successfullyproduced calf from frozen thawed buffaloembryo.


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What is embryo transfer?

Embryo transfer takes place after eggshave been collected and fertilised in thelaboratory.

Depending on your situation betweenone and three of the best qualityembryos are selected and then

transferred to the womans womb. An embryo must successfully attach

itself to the wall of the womb for

pregnancy to begin.


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Embryo Transfer - Is It Necessary?

It has enabled many infertile couples toexperience the joy of parenthood.

This is specifically used in the artificialinsemination for both humans and

animals alike.The process is called In Vitro

Fertilization (IVF); this involves

inserting multiple embryos in the uterusof the female with the purpose oftriggering a successful pregnancy.


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Why is embryo transfer necessary?

There are many, many reasons why this processmay be applied. In humans, this is a boon forinfertile couples. This is specifically helpful when the sperm count is low and pregnancy cannot

occur due to lack of contact between the eggand the sperm.

the conditions of the woman's reproductive

organs are such that they prevent fertilizationthough the eggs are okay.


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Ovulation


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The Process Of

Ovulation


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Video Illustrating the detailed process ofOvulation


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Superovulation

Designed to stimulate the ovaries to produce

several eggs (oocytes) rather than the usualsingle egg as in a natural cycle. During the normal reproductive cycle, two

hormones - follicle-stimulating hormone (FSH)

and luteinizing hormone (LH) - are required toinitiate and complete the process of eggmaturation.


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Medications are administered to womeneither orally or by injection to increaselevels of both LH and FSH.

The goal ofovulation induction (OI) is togrow and ovulate an egg in a women whonormally does not ovulate, while the goalofsuperovulation (SO) is to produce more

than one egg to improve fertility in a

women who already ovulates.

C l d f tilit di ti


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Commonly used fertility medicationsClomiphene Citrate tablet brands are taken by mouthusually at a dose of 50 mg daily for 5 days between days 5-9 of yourmenstrual cycle. In patients with shorter cycles the medication is

sometimes started earlier on day 3 of cycle.

Serophene

ClomidInjectable Gonadotropin pens are given to help a woman

produce and release healthy eggs when ovulation is a problem or totimulate the ovaries to produce multiple eggs during In Vitro

Fertilization (IVF)..

Follistim

GonalF


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HCG trigger an HCG medication given to "trigger"ovulation when ultrasound shows that the largest(dominant) follicle is between 16-20 mm. The medication is

sometimes utilized to maintain adequate progesteroneproduction from the ovary after ovulation.

Progesterone preparations utilized to maintain

optimal progesterone levels after ovulation and throughthe first 12 weeks of pregnancy. The medications can begiven in the form of vaginal tablets, vaginal suppositoriesor injections.

ometrin tablets

Crinonecream

Progesterone injection


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GnRH medications

GnRH agonists are used are given to suppress pituitary

function during IVF.In the treatment of endometriosis or uterine fibroids.During IVF it important to prevent your own trigger toovulation.

They can be given beginning a week before your

menses .They can also be given together with the gonadotropin

injections beginning on day 2-3 of cycle (micro-dose orshort protocol).

GnRH antagonists such as Ganirelix or Cetrotide aregiven daily, beginning on day 6 of gonadotropininjections.

Cetrotide Lupr

on

h b h i k f d i


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What about the risks of gonadotropintherapy?

As with any medical treatment, there aresome risks specific to fertility medications: Multiple pregnancy- (mostly twins) occur in

approximately 20% of ovulation induction cycles utilizing

gonadotropin injections. Poor response - treatment is discontinued during

IVF when there is no response or when less than 3 eggsdevelop. When this happens, a different stimulation

protocol utilizing higher gonadotropin dose is usuallyadvised. Cycle cancellation Gonadotropin injections are

discontinued and ovulation is prevented when too manyeggs develop or when the estrogen level is very high, as

this may increase the risk of multiple pregnancies and
http://www.bocafertility.com/IVFhttp://www.bocafertility.com/IVF


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Ovarian Hyperstimulation Syndrome(OHSS) - is a potentially life threateningcomplication resulting from overstimulation ofthe ovaries.

The condition is associated with ovarianenlargement,

torsion (twisting) of the ovaries, weight gain, accumulation of abdominal fluid, decrease of blood volume and low blood

pressure,


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All these symptoms lead to hospitalization and aggressivetreatment. OHSS may occur even with mild stimulation.Fortunately, the severe form of Ovarian Hyperstimulation

Syndrome is uncommon, occurring in less than 1% ofpatients.

R t i i th O t ( t i l)


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Retrieving the Oocytes (egg retrieval)

Collection of eggs is performed under transvaginal

ultrasound guidance. To accomplish this, a needle is inserted (under IV

sedation) through the vaginal wall into the ovaries usingultrasound to locate each follicle.

The follicular fluid is drawn up into a test tube to obtain

the eggs. Although patients are given pain medications

intravenously and are carefully monitored by ananesthesiology staff,

Some women may experience some discomfort duringthe procedure. Generally, the oocyte (egg) retrieval takes 20-30

minutes. Patients are usually discharged home within hours after

the retrieval.


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Risks of oocyte (egg) retrieval may include, but arenot limited to, the following:

Potential reactions from the drugs and procedures usedin the administration of anesthesia.

Risks associated with the passage of the needle throughthe vagina into the ovaries (including infection, bleedingetc..)


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Collecting and Preparing the Sperm:- A semen sample will be obtained from the partner by

masturbation on the day of the oocyte (egg) retrieval. This is usually obtained while the retrieval is being

performed. After the specimen is produced, the sperm will be

prepared for inseminating the collected eggs in thelaboratory.

Men who feel that they may have difficulty producing asemen specimen have the opportunity to have theirspecimens frozen by the laboratory ahead of time for usein this situation.

Testicular biopsy can also be performed as a method toextract sperm for IVF.

I i ti f E /F tili ti P


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Insemination of Eggs /Fertilization Process

Following egg retrieval, the follicular fluid is

immediately transferred to the laboratory foridentification of eggs, evaluation, andpreparation for insemination.

In the process of collecting the follicular fluid, it

is possible that a large number of eggs may beretrieved. It is strongly recommended that all of these

eggs be inseminated to maximize the number

of embryos available for subsequent transfer.The prepared sperm is added to each egg and

they will be allowed to incubate overnight undercontrolled laboratory conditions.

The next day, each egg is evaluated for


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However, it is possible that no eggs arefertilized. If this happens, the laboratory staff

will re-inseminate the eggs or performintracytoplasmic sperm injection (ICSI) inhopes of obtaining embryos for transfer

If fertilization still does not occur, the eggs will

be discarded and the remainder of theprocedure will be cancelled.

In the case of severe male factor, the couplemay be asked to consider the option of using

anonymous donor sperm (obtained through alicensed sperm bank for use as a "backup" orsecondary sperm source) if it is not possible toobtain sufficient sperm from the partner at the

time of fertilization.

A fertilized egg is called an embryo


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A fertilized egg is called an embryo. An embryo goes through stages of development until it

is ready for transfer to the uterus.

For the eggs that have fertilized


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For the eggs that have fertilized They will be allowed to develop for two or more

additional days under controlled laboratory conditionsbefore they are placed inside the woman's uterus.

Depending upon the couple's wishes, some fertilizedeggs/ embryos may be frozen and stored for future use.

Embryos may be transferred to the patient on day 1(zygote stage) of development,

on day two (two-cells to four-cells stage), on day three (six-cells to eight-cells stage), on day four (morula), or on days five to seven (different blastocyststages)

Growth of an embryo from the 2 cell stage


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Growth of an embryo from the 2-cell stageto Blastocyst.

In these pictures actual photographs of an


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In these pictures actual photographs of anembryo is shown under magnification:


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Three to five days following the Egg Retrievalthe patient will have the Embryo Transfer (ET).

During this time the fertilized eggs (embryos)have been allowed to grow and divide in theincubator.

The patient would have also been started on

Progesterone injections and/or suppositories theday of HCG injection to prepare the uterinelining for implantation.

Transferring Embryos to the Uterus


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Transferring Embryos to the Uterus The embryo transfer typically takes place

under sterile conditions, even though you will

not be placed under anesthesia.Steps of Embryo transfer Include:-

1. With the assistance of the embryologist, the

embryos are loaded into a special catheter.

1. A speculum is placed into the vagina to allowvisualization of the cervix, which will then be

cleaned.

1. Under ultrasound guidance, the catheter isplaced through the cervix and into the uterus.
http://pcos.about.com/od/normalmenstrualcycle/f/vagina.htmhttp://pcos.about.com/od/glossary/g/cervix.htmhttp://pcos.about.com/od/glossary/g/cervix.htmhttp://pcos.about.com/od/normalmenstrualcycle/f/vagina.htm


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4. When the catheter has been placedappropriately, the embryos are gently inserted

into the uterus where they will hopefullyimplant.

5. Typically, two to four embryos are be

transferred in one treatment cycle.

Th P f b t f


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The Process of embryo transferDiagrammatic View


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Video Illustrating Embry TransferProcess


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Catheter(images)

S l


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Speculum:-A speculum is a medical tool for investigatingbody cavities, with a form dependent on the body cavity for which itis designed. Like an endoscope, a speculum allows entry into a bodycavity; endoscopes, however, tend to have optics while a speculum is

intended for direct vision.


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After the embryos are transferred to thewomb, the woman will continue progesteronesupplementation that begins on the evening of

your egg retrieval procedure. Progesterone can be taken as a combination of

oral troches and rectal/vaginal suppositories orby injections.

Administration of these medications after eggcollection has been shown to create a more

favorable uterine environment for

the embryos, which therefore increasespregnancy rates.


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Side effects ofprogesterone may include thefollowing:

1. Vaginal dryness;2. Bloating,3. Breast tenderness;4. Depression,5. Mood swings;6. Delay of menses

Additional aspects of successful


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Additional aspects of successfulimplantation include:-

Bed rest following embryo transfer, The absence of hydrosalpinx (blocked, fluid-filled fallopian tubes) that could lead towashing out of the implanted embryos by

intermittent leakage of the hydrosalpingealfluid. If uterine contractions occur after transfer, the

embryos could be expelled down through the

cervix, or up into the fallopian tubes, instead ofimplanting in the uterus.

Additionally, the presence ofblood on theoutside of the transfer catheter tip correlates

with lower rates of successful implantation.

Risk of Embryo Transfer!
http://science.jrank.org/pages/965/Blood.htmlhttp://science.jrank.org/pages/965/Blood.html


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Risk of Embryo Transfer!

Embryo transfer can cause: Mild cramping, During the embryo transfer the embryo(s) may

be displaced through the cervix (causing loss ofembryos) or

Into the fallopian tubes (causing possible tubalpregnancy).

There is a small risk of bleeding or infection asa result of the transfer procedure.

Interaction between the embryo and the


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Interaction between the embryo and theendometriumVideo Showing the implantation process

T h i f F i


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Technique of FreezingEmbryo

Cryopreservation. Crucial procedure for success of embryo

transfer in livestock species.

Here embryo is dehydrated with the help of acryoprotectant like glycerol. After the equilibration is achieved embryos are

cooled very slowly from 25C to - 38C. The

embryo at -38C are plunged into liquidnitrogen (-196C) and stored for posterity andused whenever needed.


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Cryopreservation has the followingadvantages:-

It relieves for having simultaneoussynchronization of

oestrus in donor and recipient animals. Easy and safe transport of valuable germplasm

throughout the globe. Conservation of superior genetic material. Protection of valuable strains of experimentalanimals against possible loss through disease,

accident or genetic drift. Possibility of shortening the generation intervalfor progeny testing programme.


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Embryo

Sexing Embryo sexing enable us to have desired sexaccording to our requirement. Example - If we want animals for meat purposes

with faster growth and lean carcasses then certainlywe would like to have more males, but if we wantanimal for milk purposes than we will like to havemore female for dairy purpose. Now these desired

traits can be obtained by sexing embryo.

There are two approaches for the control ofsex of offspring:1. regulation of sex of embryo2. identification of sex of embryo.


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Regulation of Sex of Embryo are Based on:

Sperm separation

Nuclear transplantation

Parthenogenesis and

Removal of few cell from an embryo at an earlystage of development and to make a preparation from

these cells in which the chromosomes can beexamined. Such removal of cells do not restrict itsfurther developmental capacity.


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Sperm separation

Sperm sorting is an advanced technique thatsorts sperm "in vitro" by flow cytometry. Thisshines a laser at the sperm to distinguish Xand Y chromosomes, and can automaticallyseparate the sperm out into different samples.

The technology is already in commercial usefor animal farming.It is currently being trialed

on humans in the US under the trademarkMicroSort; it claims a 90% success rate but isstill considered experimental by the FDA


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Parthenogenesis

Parthenogenesis is a form of asexual reproductionfound in females, where growth and developmentof embryos occur without fertilization by a male.In plants, parthenogenesis means development of an

embryo from an unfertilized egg cell, and is acomponent process of apomixis.

The word "parthenogenesis" comes fromthe Greek , parthenos, meaning "virgin", and

, genesis, meaning "birth".The term issometimes used inaccurately to describe reproductionmodes in hermaphroditic species which can reproduceby themselves because they contain reproductiveorgans of both sexes in a single individual's body.


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Identification of Sex of Embryo:- This can be doneby:

Karyotyping-can be done by using quinacirinemustard.

Identification of sex chromatin.

Assay of sex linked enzymes, like glucose-6-phosphate dehydrogenase.

Identification of DNA probe or Y -chromosome and

Serological determination of H-Y antigen which is amale specific antigen in mammals. These includeattempts to develop monoclonal antibodies against H-

Y antigen and to detect sex by means of ELISA.

Micro Manipulation of


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Micro-Manipulation ofEmbryoThe technique is based on the developmental and

regulatory capacity of individual cells or groups of cellsfrom embryo at early cleavage stage.

By micromanipulation of embryo we can have

Monozygotic twins in sheep, cattle, pigs and horses.This have been derived by separating the blastomeres

from either two, four or eight-celled embryo into theequal groups.

A large number of identical animals from a singleembryo by the simple technique of separatingblastomeres. (Single blastomere from 8-celled embryoor group of two blastomere from I6-celled embryoseldom produce a viable foetus).


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A set of identical quadruplet lambs derived from single8-celled embryo.

Chimeric animals by this technique. Chimera is acomposite animal living cells from more than one cellline. The main objective is to combine the best genetictraits of each donor in the chimeric animaL Sheep-goat

chimera has been successfully produced.

Many exciting possibilities for manipulation of animalsof selected genotype.

v


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vTransfer Technology:

1. It permits exploitation of superior femalegenotype giving more offspring from thesame genetic donor than would arise under

normal breeding condition.2. Twin production, introduction of new gene

into cloned herds, manipulation of embryo

and transgenic animals have been possibleby EIT.

3. Infertility, especially of the older animalsis a major handicap in animal breedingro ramme. Such animals of su erior


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6. It increases the reproductive capacity

of a superior heifer or a cow, since semenfrom one male can be used for a largenumber of females. A single superiorfemale can be made to give a largenumber of ovules through superovulation.

7. Old superior cow that are unable tomaintain pregnancy can still donate

ovules for embryo transfer.

8. We may not need to import a superiorquality cow, instead we may have

imported frozen embryo which can grow


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9. In an animal (cow and buffalo) in vitro

production of embryo has considerablepotential value in for disseminating geneticimprovement and shortening thegeneration interval as compared to

progeny testing.

10. ETT can be used to resolve severalreproductive enigmas like embryo-utero

relationship, endocrine requirement formaintenance of pregnancy, the relativeinfluence of genetic and environment onfetal growth and the biology of zoneellucida and blastomere.


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Embryo Transfer Technology

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