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IN VITRO FERTILISATION
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The specifically prepared sperms
and the incubated eggs are combined in a test
tube or petri dish in prescribed number ratio
and left for few hours for them to unite.
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After 18 hours they are examined
under stereo zoom microscope for fertilization
which is obvious if two pronucleus are seen clearly
. Each pronucleus denote the decondensation of
male and female nucleus (DNA). Once they are identified
fertilization is confirmed. This day could be
accounted as Day 1
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| Pronuclear stage embryo |
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| Two cell stage
( 24 hours) |
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The culture dishes are kept
in a modular chamber which holds the gaseous
environment very strictly and these are kept
in turn in a CO2 incubator. Our IVF lab utilizes
TRIGAS (O2, CO2, N2 mixture) incubator instead
of CO2 alone , since the presence of 5 % O2
improves the culture Conditions and integrity
of embryos.
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After 48 hours ,properly growing
embryos shows 4 cells inside,and after 72 hours
they show 8 cell divisions. Only then they are
considered as good embryos. Once the cell division
seems to be insufficient and the embryos have
odd number of cells and unequal cells they are
considered to be "blocked" ones and
they have meager chances to make blastocysts
at the end of Day 5 . Depending on the number
of 8 cell stage embryos available on day 3,
the type of embryo transfer and the day of embryo
transfer are decided. The success rates are
also destined according to the number of 8 cell
staged, grade I embryos available in the culture
system for that particular couple.
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| Four cell stage embryo
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| Eight cell
stage embryo |
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STIMULATION OF OVARIES
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Normally a women after attaining
menarche will produce one egg per month around
14 th day after menstrual period . For our In
Vitro Culture program, one egg may not suffice
and the whole procedure and success depend on
the number and quality of the eggs obtained. So
, we hyper stimulate the ovaries in a safe manner
using a method called controlled ovarian hyperstimulation
by "Down Regulation" under long protocol.
After downregulating the system we give Hormone
injections of International Quality e.g. Rec.
FSH, Pure FSH, HMG , etc which helps the wife
to produce more number of better quality eggs.
The dose and number depends on the age , weight
and the Basal hormonal status of the women. We
teach the spouse to give injections to his wife
since most of the medicine comes in self injectable
syringes . Once the follicles harbouring the eggs
reach sufficient size "egg pick up"
is planned and the whole procedure is explained
to the couple. The nature of anesthesia could
be the couple's choice, provided it doesn't alter
the egg quality. Usually we give IV analgesia
for shorter time .
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EGG PICK UP
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Usually planned in the day working
hours, under IV sedation which will not harm the
eggs. An Ultrasound machine is used to pick up
the eggs through vaginal vault using a specific
instrument called TRANSVAGINAL PROBE with It`s
guide through which the egg pick up needle is
introduced and the follicle is punctured. The
needle system is a complex one having two ports,
one for aspirating the follicular fluid and the
other one for flushing the empty follicle with
specific medias. This needle system is connected
to a low power suction motor which aspirate the
fluid very gently without harming the eggs. The
aspirated fluid which contains the eggs are collected
in a Polystyrene test tube and sent to the adjoining
IVF lab. The aspirate is examined by an embryologist
under stereozoom microscope to identify the eggs
and keep them separately in an ideal nutritious
culture media in incubator till they are inseminated.
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This identification, separation
and , handling needs an ideal gas mixture and
warm(37*c) environment under a sterile zone called
Integrated Laminar Flow Work station . The whole
procedure takes maximum 30 minutes and the amount
of bleeding or pain will be very minimum.
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SPERMS AND PREPARATION
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Sperm collection and processing
is to be done with great care to avoid infection
. Before this IVF program is designed the husband
must have a trial preparation of sperms to see
the integrity and biological nature of the sperms
and it`s survival hours. Now after egg pick up
the husband is asked to give the semen sample
within 2 hours. If he think it might be a problem
in these tension hours , he must have done it
during earlier days ,which could be cryopreserved.
Same applies to the husbands working abroad ,
or undergoing surgery / radio, chemotherapy .
The husband could be given non toxic condom if
he is unable to produce the sample by masturbation.
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EMBRYO CULTURE
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The culture dishes are kept in
a modular chamber which holds the gaseous environment
very strictly and these are kept in turn in a
CO2 incubator. Our IVF lab utilizes TRIGAS (O2,
CO2, N2 mixture) incubator instead of CO2 alone
, since the presence of 5 % O2 improves the culture
status and integrity of embryos .
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After 48 hours the cell division
shows 4 cells inside the embryos and after 72
hours they show 8 cell divisions. Depending on
the number of 8 cell stage the viability of the
culture system is assessed. The success rates
are also decided according to this.
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EMBRYO TRANSFER
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Depending on the no; of available
grade I day 3 embryos, either DAY 3 ET or
Day 3 & Day 5 embryo transfer which is called
SEQUENTIAL ET is done .
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Day 3 Transfer : After 72 hrs
of culture day 3 Embryos will have 8 inner cells.
Three of them are transfered to the uterus ( Choosing
the best day 3 embryo willbe ideal to augment
the success rate). Rest of the 8 celled embryos
will be left for Blastocyst culture till day 5
/ day 6.
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Day 5 / 6 Transfer : Blastocyst
are examined under inverted microscope for identifying
the viable ones, by comparing the INNER CELL MASS
and outer cell TROPHECTODERM Ratio. Best one is
chosen for ET, using very soft embryo Transfer
catheter.
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Transfer Techniques are unique
for each center which plays major role in deciding
the success rates. To improve the implantation
we use "Embryo Glue" , while transfering
them into uterus. Embryo glue is nothing but HYALARUNON
, an essential molecule which is found naturally
in the reproductive tract of a woman to aid in
further growth and nidation .
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ICSI - INTRACYTOPLASMIC
SPERM INJECTION
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This is a procedure in which
a sperm is injected in to the cytoplasm of one
egg using micro injection needle with the help
of micromanipulators under inverted microscopes
with X 400 magnification . The main advantage
of the Method is that, it needs only very few
sperms equivalent with the number of eggs obtained.
This stands as the first line of treatment in
extreme male infertility like severe Oligospermia,
asthenospermia etc. Surgically extracted few sperms
are ideally used for this procedure in males who
are otherwise Azoospermic( nil sperms in the ejaculate).
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PROCEDURE
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Sperms are immobilized using
PVP ( latest being LASER immobilization ) and
they are aspirated singly in the microinjection
needle and the egg is held using a holding pipette
. Then the loaded sperm is injected into the cytoplasm
of the egg. All these techniques need great deal
of skill and hitech equipments . By doing this
several barriers of fertilization are overcome
and fertilization occur in almost all eggs injected.
The overall success rates is around 33 % in day
3 transfer and 41 % success in day 5/6 transfers
in our center.
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Before undertaking this treatment
males are supposed to subject themselves for blood
karyotyping test which must be normal as a pre
requisite for ICSI.
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SURGICAL SPERM EXTRACTION : This
is done when there is no sperms in the ejaculate
. Very few live sperms or spermatids ( younger
sperms) could also help in doing ICSI and getting
babies. Methods explained as below are done according
to the need of the male partner.
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1.MESA : Micro Epididymal Sperm Aspiration
, using microscopic equipments and microsurgical
procedures , few sperms could be extracted
by this. When IVF is planned later in a
convenient time, these sperms could be preserved
by cryopreservation.
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| 2.
PESA : Percutaneous Sperm Aspiration , is
done in place of MESA in few centers. |
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: Testicular Sperm Extraction method will
extract sperms from the testes directly. Even
if few Spermatids ( younger sperm cells) are
extracted this will serve the purpose of fertilization
in ICSI procedure. |
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Whatever may be the mode of sperm extracted for
ICSI, the sperm injected eggs are fertilized for
90% of the time. Failure in the fertilization ,
thereby loosing the costly eggs are conquered by
this method , which definitely promote the success
rates because of more no; of embryos . The success
rates comparatively less than routine IVF , because
of male pronucleus problem during fertilization
and further growth.
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ASSISTED HATCHING METHODS
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The outer covering of embryo
is called Zona pellucida which is a cover protecting
the integrity of the blastomeres inside. They
should be dissolved or breached before the embryo
/ blastocyst get attached with endometrial lining
of uterus for proper nidation. When they are resistant
for natural enzymes in the endometrial cavity
which help in dissolving this , or when the women
concerned is aged whose eggs may have harder thicker
zona , we may have to dissolve the zona partially
before transferring them in to uterus. It is believed
to help nidation in time. The following are the
methods used:
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1. Chemical - using Acid Tyrode
solution
2. Enzymatic using - Protease
3. Laser assisted hatching
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Out of these we follow the latest
one called "LASER ASSISTED HATCHING"
system, which augments the success rates significantly
by improving the implantation rates. This technique
helps the blastocyst to hatch in time, by which
the implantation potential is improved. This procedure
will help the eggs of elderly women whose eggs
will have thicker ZONA PELLUCIDA. Cryopreserved
Embryos and Blastocyst also have thicker ZONA,
which will be helped out by this procedure.
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CRYOPRESERVATION
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It means preserving the gametes -
eggs , sperms , embryos & blastocysts in minus
centegrades without spoiling the inner cell integrity
for future use. There are two standard methods called
slow freezing technique and rapid freezing technique,otherwise
known as VITRIFICATION - the latest one. Vitrification
helps to form rapid ice crystallization of cytoplasm
which preserves cell integrity well. THAWING is
a procedure which brings the tissues back to its
original room temperature for the purpose of further
culture or embryo transfer.
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LIVE 3D/4D SCAN SYSTEM
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Recently we have introduced latest
MEDISON live 3D/4D colour Doppler scan system
which gives all details of the fetus from 8th
week of gestation onwards. Absolutely defects
are studied in detail before deciding the management.
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