Blastocyst culture in Assisted Reproductive Technology

Index

1. Introduction to blastocyst culture in Assisted Reproductive Technology

2. In- VITRO Fertilizatiion

3. Materials and Method of treatment in IVF

4. Blastocyst culture in IVF : Later on development of Blastocyst

5. Advantages and disadvantages of Blastocyst culture: Success rates in Blastocyst culture

6. Cryopreservation

7. Ethics and issues

8. Future aspects

9. References

Chapter 1:

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Introduction to blastocyst culture in ART

IVF is a major treatment in infertility when other methods of assisted reproductive

technology fails. The vitro fertilisation (IVF) process involves hormonally controlling the

ovulatory process, removing ova (eggs) from the woman's ovaries and letting sperm fertilise

them in a fluid medium in vitro, The fertilised egg i.e zygote is then transferred to the

patient's uterus with the intent to establish a successful pregnancy.

Whereas in blastocyst culture the entire process of IVF is same just in case of implanting

embryo in uterus , it is now possible to grow embryos in the laboratory to the blastocyst stage

of development, which occurs on day five after fertilization when the embryo has 50-200

cells. Typically, the strongest, healthiest embryos make it to blastocyst stage as they have

survived key growth and division processes and have a better chance of implanting once

transferred. The selection of potentially more viable embryos allows the embryologist to

transfer fewer embryos, often one or two, lowering the risk of high order multiples while

maintaining high pregnancy rates.

BLASTOCYST is compact viable cellular structure formed by the merger of numerous

blastomeres after the stage of morula post compaction is termed as blastula. It is the ultimate

live stage which could be cultured in the laboratory after which it has only one objective and

that is to subsequently get itself implanted in the receptive endometrium ultimately resulting

in a pregnancy. It could be termed as the end part of lab culture in an IVF laboratory and as it

enjoys this privilege it albeit has the maximum implantation rate, thus making it the most

wanted “developing stage” in any Assisted Reproduction Unit. Being at the ultimate end of

procedures it deserves immense care and caution in its culturing which makes “Blastocyst

Culture” very challenging. A beautifully extended blastocyst with well compacted inner cell

mass, thin evenly lined trophectodermal cells and a well swollen blastocoel cavity is not all

that easily available especially from oocytes of women undergoing stimulation protocols – a

mode of treatment for infertility. Rapid strides in instrumentation, culture techniques,

hormone therapy and various other application support protocols all help achieve ideal

blastocyst culture, much to the joy of unfortunate childless couples.

Chapter 2.

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In- Vitro Fertilization:

The term in vitro, from the Latin root meaning in glass, is used, because early biological

experiments involving cultivation of tissues outside the living organism from which they

came, were carried out in glass containers such as beakers, test tubes, or petri dishes. Today,

the term in vitro is used to refer to any biological procedure that is performed outside the

organism it would normally be occurring in, to distinguish it from an in vivo procedure,

where the tissue remains inside the living organism within which it is normally found. A

colloquial term for babies conceived as the result of IVF, test tube babies.

The first IVF baby in the world was born in July of 1978 in England. More than 250,000

babies have been born since then as a result of using the in vitro fertilization technique. IVF

offers infertile couples a chance to have a child who is biologically related to them...

Who all are treated with IVF?

• IVF can be used as an effective treatment for infertility of all causes except for

women with infertility caused by an anatomic problem with the uterus , such as severe

intrauterineadhesions.

Couples who have failed to conceive after at least one year of trying, who also have

one or more of the following:

1. Blocked fallopian tubes or pelvic adhesions with distorted pelvic anatomy.

2. Women that have had tubectomy and are considering tubal anastomosis as well as

men that are considering vasectomy reversal surgery might also consider IVF. 

3. Severe male factor (low sperm count or low motility)

4. Failed 4-6 cycles of ovarian stimulation with intrauterine insemination

5. Advanced female age - over 38 years

6. Reduced ovarian reserve, which means lower quantity (and sometimes quality) of

eggs. A day 3 AMH, Inhibin B, FSH and Estradiol measurements are often done as

screening tests for egg quantity (and quality).

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7. Reduced egg quantity and quality is usually treated with either IVF, or with IVF using

egg donation from another younger woman.

8. Severe endometriosis.

Factors to Consider:

Age: Women younger than 35 years who do not have problems with their partners'

sperm may try IVF.

Multiple births: Generally, in women who use IVF to establish a live birth, about 63%

are single babies, 32% are twins, and 5% are triplets or more.

Cost: One cycle of IVF costs an average of $12,400.

Reduced surgery: If a woman has IVF, she may not have to undergo surgery on her

fallopian tubes. It is estimated that the IVF technique has reduced such surgeries by

half.

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Chapter 3.

Materials and Method of treatment in IVF:

Materials Used:

Injectable Gonadotrophins

Aspiration needle

Inverted microscope

Centrifuge

CO2 incubator

Plastic catheter

Fertilization media

Sequential media of Blastocyst culture

Methods:

The IVF treatment for infertility has several stages: ovulation induction, egg harvesting,

insemination and fertilization, and embryo transfer or blastocyst transfer. In some rare cases,

you can also select to undergo preimplantation genetic diagnosis (PGD) to check for

chromosomal abnormalities.

Ovarian stimulation or ovulation induction:

Treatment cycles are typically started on the third day of menstruation and consist of a

regimen of fertility medications to stimulate the development of multiple follicles of the

ovaries. In most patients injectable gonadotropins (usually FSH analogues) are used under

close monitoring. Such monitoring frequently checks the estradiol level and, by means of

gynecologic ultrasonography, follicular growth. Typically approximately 10 days of

injections will be necessary. Spontanenous ovulation during the cycle is prevented by the use

of GnRH agonists or GnRH antagonists, which block the natural surge of luteinising hormone

(LH).

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In order to maximize success rates with IVF we need to get a good number of eggs from the

woman. We prefer to get about 8 - 10 high-quality eggs at the egg retrieval procedure.

IVF success rates are strongly correlated with the number of eggs retrieved.

The eggs develop in structures in the ovaries called follicles. Each follicle contains one egg

and can be seen on ultrasound.

Egg collection

When follicular maturation is judged to be adequate, human chorionic gonadotropin (β-hCG)

is given. This agent, which acts as an analogue of luteinising hormone, would cause ovulation

about 36 hours after injection, but a retrieval procedure takes place just prior to that, in order

to recover the egg cells from the ovary the following procedure takes place:

• A needle is passed through the top of the vagina under ultrasound guidance to get to

the ovary and follicles.

• Woman is given anaesthesia so that she will not have significant pain during the

procedure.

• The actual aspiration procedure takes about 20-30 minutes in average..

The follicles are fluid-filled structures in the ovary that are surrounded by cells from the

ovary called granulosa cells.

The egg is microscopic and cannot be seen on ultrasound.

Eggs are surrounded by a mass of ovarian cells called cumulus cells

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Oocyte with surrounding granulosa cells

Egg identification

The oocyte cumulus complex is removed from the follicle when the fluid is aspirated through

the needle. The fluid and the oocyte-cumulus complex as well as some granulosa cells are

collected in tubes and passed to the Embryology lab.

The contents of the tubes are poured into flat dishes so that eggs can be searched under

stereo-microscope. The oocyte-cumulus complexes are what can be then identified.

“Naked” Egg

Fertilisation

The semen is prepared for fertilisation by removing inactive cells and seminal fluid. If semen

is being provided by a sperm donor, it will usually have been prepared for treatment before

being frozen and quarantined, and it will be thawed ready for use. The sperm and the egg are

incubated together (at a ratio of about 75,000:1) in a CO2 incubator in the culture media for

about 18 hours. By that time fertilisation should have taken place and the fertilised egg would

show two pronuclei. In situations where the sperm count is low, a single sperm is injected

directly into the egg using intracytoplasmic sperm injection (ICSI).

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The fertilised egg is stored in Ham's F-10 medium supplemented with fetal cord serum,for

48-72 houre to grow around the6 to 8-cell stage.

Oocyte is injected during ICSI

8-cell embryo for transfer

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Selection

Laboratories have developed grading methods to judge oocyte and embryo quality. Embryos

are graded by the embryologist based on the number of cells, evenness of growth and degree

of fragmentation. Typically, embryos that have reached the 6-8 cell stage are transferred three

days after retrieval.

In blastocyst culture:

The embryos are placed into an extended culture system with a transfer done at the

blastocyst stage, especially if many good-quality day-3 embryos are available. Blastocyst

stage transfers have been shown to result in higher pregnancy rates. The inner cell mass is

formed after this stage. It contains EMBRYONIC STEM CELLS which can be extracted and

preserved for future use.

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Blastocyst for transfer

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Embryo transfer

The number to be transferred depends on the number available, the age of the woman and

other health and diagnostic factors. A maximum of two embryos are transferred except in

unusual circumstances. The embryos judged to be the "best" are transferred to the patient's

uterus through a thin, plastic catheter, which goes through her vagina and cervix. Several

embryos may be passed into the uterus to improve chances of implantation and pregnancy.

The woman should then remain in a resting position for the next hour or so.

PREGNANCY

The woman is given certain hormones for the next 2 weeks. About 14 days after the transfer,

a blood test for hormone B- hCG is carried out ,If implantation works (the egg or eggs attach

to the uterine wall and grow), the pregnancy test result is positive.

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Chapter 4

Blastocyst culture in IVF

Definition of a blastocyst An embryo that has developed for five to seven days after fertilization and has 2

distinct cell types and a central cavity filled with fluid (blastocoel cavity)

The cells in a blastocyst have just started to differentiate

The surface cells that surround the cavity (just under the outer shell) are called the

trophectoderm and will later develop into the placenta

A more centrally located group of cells - the inner cell mass, will become the fetus.

In the past, it was difficult to get high quality blastocysts with in vitro culture systems

- unless "feeder" cells were utilized - called coculture.

However, since 1998 more advanced culture media have been commercially available

that (if used properly) can yield high blastocyst formation rates.

Now blastocyst embryo transfer is a viable IVF treatment option for many couples

When is blastocyst transferred in IVF:

The embryos are placed into an "sequential media"which attempts to reproduce the natural

environment of the maternal reproductive tract. The nutrients are designed to meet the

requirements of the rapidly developing embryo and have led to the development of

blastocysts with better viability and higher implantation rates Blastocyst stage transfers have

been shown to result in higher pregnancy rates.

One of media used is serum-free media G 1.2 and G 2.2 media.

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Blastocyst development The zygote continues to divide, creating an inner group of cells which will become

embryo with an outer shell which will become the membranes that nourish and

protect it. This stage is called a blastocyst. It is formed on day 5 as fluid builds within

the compacted morula

A healthy blastocyst often begins hatching from its outer shell, called the zona

pellucida between day 5 to day 7 after fertilization

Within 24 hours after hatching, embryo implantation after IVF begins as the embryo

invades into the uterine lining. At this point in the mother's menstrual cycle, the lining

of the uterus has grown and is ready to support a baby. The blastocyst sticks tightly to

the lining, where it receives nourishment via the mother's bloodstream.

The blastocyst releases HCG hormone (the pregnancy test hormone) which leaks into

the mother's blood as the embryo implants.

The cells of the embryo now multiply and begin to take on specific functions. This

process is called differentiation. It leads to the various cell types that make up a

human being (such as blood cells, kidney cells, and nerve cells).

There is rapid growth, and the baby's main external features begin to take form. It is

during this critical period (most of the first trimester) that the growing baby is most

susceptible to damage.

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Chapter 5

ADVANTAGES AND DIS-ADVANTAGES OF BLASTOCYST CULTURE:

Reduce risks for multiple pregnancies Transferring blastocysts following IVF provides potential benefit - reducing

possibility for multiple pregnancy

Many 2 or 3-day-old embryos do not have the capacity to become high quality

blastocysts and make a viable pregnancy. However, on day two or three of culture we

don't have methods to determine which embryos will be viable long-term, and which

will soon arrest their development.

By culturing embryos to day 5 we will find that some of them have not become

blastocysts - allowing us an opportunity to choose the most competent embryos for

transfer.

Also sometimes all the embryos become ongoing pregnancies and the result is high-

order multiple gestations (triplets or greater). In such pregnancies, there are

considerable medical risks as well as financial and emotional considerations. So the

couple is faced with the agonizing decision of whether to opt for selective reduction

(the removal of one or more embryos) or to continue with a risky pregnancy.

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Although everyone agrees that every possible safeguard should be in place to avoid

such unfortunate situations, the distressing reality is that multiple pregnancies

sometimes do occur.

Transfer of fewer embryos can be done and still high pregnancy success rates are

obtained- with very little risk for having high order (triplets or higher) multiple

pregnancies.

Success rates and other advantages:

The research on pregnancy rates of day three and day five were done by many scientist across

the globe. The research paper of the practice committe of yht American society of

reproductive medicine and the practice committe of Society for assisted reproductive

technology states the implantation rate for embryos transferred at the blastocyst stage was

significantly higher than that for embryos transferred on day 3 (21.2% vs. 6%). The

pregnancy rates after transfer on day 3 or day 5 were equivalent,21.7% and 12.5%

respectively. The other advanteges are:

o Higher implantation rate

o A decrease in number of embryos transerred.

o Opportunities to select more viable embryos for transfer

o Better temporal synchronization of the embryo and endometrium at the time of

embryo transfer.

Also pre-implantation genetic diagnosis (PGD) can be done as the embryo is longer time in

culture, its done by taking single cell from the blastocyst culture which can be used to study

any genetic abnormality of the child and if that is the case then that blastocyst will not be

implanted in uterus which will save emotional and financial stress of parents.

Embroyonic stem cells can be obtained which can be further differentiated into any kind of

specialized cell types and can be used by the person any point of time in life .

Disadvantages are :

 • Blastocyst culture can be done only if adequate number of embryos is available

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In some cases very few embryos are obtained such embryos, which can give pregnancy but

do not develop till blastocyst in outside the body culture, may be lost. We have to carefully

watch embryo development daily to avoid this loss.

• Additional time and cost:

Many IVF centres charge extra money for blastocyst culture and pre- implantation diagnosis.

Also additional time and sitting are required for day 5 implant and growth of embryo till

blastocyst stage.

• Highly skilled team and excellent culture media and equipments.

Very specific conditions such as temperature, moisture and excellent environment is reruired.

Also quality media which is specific for blastocyst culture is used which has less glucose and

more of proteins and peptides required for growth .However, if the culture environment is

suboptimal, delayed embryo development and even embryonic arrest will occur in some

cases.

Therefore, if the culture system and laboratory quality control are inconsistent - good results

will not be OBTAINED

Chapter 6:

Cryopreservation:

CRYOPRESERVATION OF HUMAN EMBRYOS:

when there happens to be a surplus of embryos following selection for fresh transfer (usually

between one to four embryos are transferred to the uterus), then embryos of sufficient quality

may be considered for cryostorage. While embryos can be frozen at any preimplantation

stage between one-cell (one day old) to the blastocyst stage (5-6 days old), in an attempt to

minimize the freezing of excessive numbers of "spare" embryos and to help pre-select the

most potentially viable embryos, we generally choose to cryopreserve only at the blastocyst

stage. In certain cases where all embryos need to be frozen without a fresh transfer (e.g.,

when a woman may be at risk from ovarian hyperstimulation that might be complicated by

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pregnancy), generally all embryos are frozen the day after egg collection at the one-cell

stage.

Techniques of controlled-rate freezing are utilized that slowly cool embryos in cryoprotectant

fluid ("anti-freeze" solution) from body temperature down to -196°C, at which temperature

they are stored in containers of liquid nitrogen called dewars. The embryos are actually

contained within special indelibly labeled plastic vials, or straws, that are sealed prior to

freezing. Once frozen, they are placed inside labeled tubes attached to aluminum canes and

stored in numbered canisters within the liquid nitrogen dewar. Site and label designations are

stored in three separate file systems to avoid confusion and misidentification of

cryopreserved embryos. When it comes time to thaw the embryos, all available identifiers of

the stored specimen must match and be confirmed before thawing commences. The embryos

are thawed out at room temperature, which takes about one to two minutes. However, the

most critical element of the thaw procedure is not the timing but the careful dilution of the

cryoprotectant fluid to return the embryo to its favored culture medium. This permits resumed

growth and development in vitro. Once this is done, the embryo is assessed for cryodamage

to determine if it is suitable for transfer. Experience has shown that if the embryo survives

50% or more intact, it is worthwhile to replace it. Embryos can accommodate such levels of

cellular damage and still establish healthy pregnancies. All thawed embryos routinely

undergo assisted hatching prior to transfer. The zona pellucida, which surrounds the embryo,

has been shown to suffer a certain amount of hardening during cryopreservation. This can be

overcome by artificially making an opening in the outer embryo shell.

Varying strategies may be applied according to how many and which embryos are thawed

prior to transfer. It should be noted that not every couple undergoing IVF will need to worry

about embryo freezing/thawing, since not every couple will have sufficiently large number of

"surplus" or non-transferred embryos available for freezing. Indeed, most couples have only

one or two embryos frozen, so that all are thawed and any surviving are replaced. In the event

that there are more than two or three embryos frozen, thawing is usually undertaken until two

to three healthy appearing embryos are recovered. During a medication-prepared

frozen/thawed embryo transfer cycle as a patient, you will follow a treatment schedule using

Synarel or Lupron, estrogen (pills, lozenge or patch) and progesterone (lozenge and/or

suppository) in order to achieve appropriate endometrium (uterine wall lining) for embryo

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transfer. Following embryo transfer, estrogen and progesterone will be administered daily

until the 7th to 8th week of pregnancy or until a negative pregnancy test.

Chapter 6:

Ethics and issues

The Ethics Committee of the American Society of Reproductive Medicine (ASRM) has

published guidelines for ethical consideration of human embryo:

Reduction of the risk of triplets or quadruplets embryos exceeding an optimal number

for transfer to an individual patient

Possibly increasing pregnancy rates by replacing thawed embryos during spontaneous

ovulatory cycles or cycles in which the estrogen and progesterone hormone levels do

not exceed that which occurs naturally.

Possibly decreasing the number of stimulated ovary drug treatment cycles needed for

the attainment of pregnancy.

The IVF process requires sperm, eggs and a uterus. To achieve a pregnancy any of

these requirements can be provided by a third person: third party reproduction. This

has created additional ethical and legal concerns.

In a few cases, laboratory mix-ups (misidentified gametes, transfer of wrong embryos) have

occurred leading to legal action against the IVF provider and complex paternity suits. An

example is the case of a woman in California who received the embryo of another couple and

was notified of this mistake after the birth of her son.

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Chapter 7:

Future aspect

The use of blastocyst culture in in-vitro fertilization can be used for very advance study of

foetus development,further improvement in method of treatment in IVF.

Genetic Testing And Blastocysts

Blastocyst transfer gives the ability to perform biopsies on a more highly-developed embryo

in order to test for genetic diseases.

In the future, immunofluorescent testing techniques will allow practitioners to remove a few

cells from the blastocyst, stain them, and examine them under the microscope to detect any

genetic anomalies. While that type of testing is not currently available on a day-to-day basis,

we believe it will be considered routine within the next two to five years.

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Embryonic stem cells:

Also embryonic stem cells can be obtained which can be stored and used in future for studies

or for any kind of development.

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Chapter 8

References:

• http://en.wikipedia.org/wiki/IVF

• www.ivf.com/ivffaq.html

• www.ias.ac.in/currsci/dec102005/186

• www.ias.ac.in/currsci/jan252004/254.pdf

• www.emedicinehealth.com/in_vitro_fertilization/article_em.htm

www.sumanasinc.com/webcontent/animations/content/invitrofertilization

.html

• pubmed central

• journal of the American Society of Reproductive Medicine (ASRM)

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