CHAPTER – I

INTRODUCTION

[1]
Natural Fertilization Process

Fertilization is an initial in the process of

development of a new organism . The fusion of male and female
gametes is defined as Fertilization . Fertilization that occurs within
the body of female organisms is called as Natural Fertilization.

During the natural fertilization process of a woman of

childbearing age , a number of follicles develop in the ovary every
month, due to hormonal activity of the pituitary glands and ovaries.
The oocytes (eggs) are contained within these follicles. Usually ,
each month, a single follicle of all , reaches maturation . the mature
oocyte is released into the oviduct (fallopian duct) in preparation
for fertilization . this process is called ovulation .

If no pregnancy develops , the oocyte will

disintegrate and the uterine mucosa (endometrium), which had
been preparing for pregnancy , will shed in the form of menstrual
bleeding . In order for fertilization to take place and an embryo to
form , a spermatozoon ( sperm cell) must fertilize the oocyte.

The natural fertilization process takes place in the

oviduct, when a single spermatozoon penetrates the envelop of the
oocyte and activates a mechanism preventing other spermatozoa
form penetrating .This ensures normal fertilization. The fertilized
oocyte divides many times , forming many cells that form the
developing embryo . At the same time , the embryo moves down
the oviduct in the direction of the uterus and about a week after
fertilization , implants in the uterus and continues to develop .

[2]
 When a woman unable to become pregnant ,
another artificial medical techniques are used i.e. IN VITRO
FERTILIZATION (IVF).

IVF is form of Assisted Reproductive Technology

(ART) .It is also called as TEST TUBE BABY . In Vitro Fertilization is a
procedure in which eggs (ova) form a woman’s ovary are removed .
They are fertilized with sperm in a laboratory procedure , and then
the fertilized egg (embryo) is returned to the woman’s uterus .
Many women struggling with infertility choose in vitro fertilization
as an assisted conception method . It is used for infertility
treatment and gestational surrogacy .

[3]
 In Vitro Fertilization (IVF) is the most common form
of assisted reproductive technology and is used in the management
of patients with difficulty undergoing conception. This activity
revolves around the etiology , evolution, and role of the
interproffessional team in management strategies for IVF.

In Vitro Fertilization (IVF) is a complex series of

procedures used to help with fertility or prevent genetic problems
and assist with the conception of a child.

During IVF mature eggs are collected (retrived) from

ovaries and fertilized by sperm in a lab . Then the fertilized egg (
embryo) or egg (embryos) are transferred to a uterus. One full cycle

[4]
of IVF takes about 3 weeks. Sometimes these step split into
different parts and process can take longer.

IVF is the most effective form of assisted

reproductive technology. The procedure can be done using a
couple's own eggs sperms or IVF may involve eggs , sperm or
embryos from a known or anonymous donor. In some cases , a
gestational carrier someone who has an embryo implanted in the
uterus might be used.

Your chances of having a healthy baby using IVF

depend on many factors such as your age and the cause of
infertility. In addition IVF can be time consuming , expensive and
invasive. If more than one embryo is transferred to the uterus ,
IVF can result in a pregnancy with more than one fetus (multiple
pregnancy).

You should contact the doctor and discuss

everything with him properly for best results.

Techniques that involve manipulation of oocytes

outside the body are termed assisted reproductive technology
(ART) with In Vitro Fertilization (IVF) as the most common form.
The term in vitro means outside a living organism as oocytes
mature in vivo in the ovary and embryos develop into pregnancy in
the uterus , but the oocytes are fertilized in a petridish . Robert
Edwards , ph.D. and Patrick steptoe , MD , reported the first live
birth from IVF in July 1978 in England. This achievement later earn
Dr. Edwards the Nobel prize in medicine in 2010.

Since this major break through in the treatment of

infertility the field of reproductive endocrinology , infertility (REI)
has progressed rapidly , and IVF now accounts for 1.6% and 4.5% of

[5]
all live births in the US and Europe , respectively. Initially developed
as a way to bypass irreparable tubal disease , IVF is now widely
applied for the treatment of infertility due to a variety of causes ,
including endometriosis , male factor , and unexplained infertility.
Women who can't use their own oocyte due to primary ovarian
insufficiency (POI) or age related decline in oocyte number can now
become successfully pregnant utilising donor oocyte IVF.

Anatomy And Physiology

Recognition of female pelvic anatomy is paramount
in properly understanding and completing IVF. In order to achieve
optimal results in IVF , certain types of anatomy appear to be
associated with success rates of oocyte retrival and embryo
transfer. The uterus is a mullerian structure of the female pelvis
that provides an environment to harbor a growing pregnancy. The
uterus is composed of three layers , the serosa , the myometrium
and the endometrium. The endometrium or the lining of the uterus
is a structure of glandular tissue that undergoes predictable
changes during the menstrual cycle in response to level of
circulating hormones.

Approximately 10% - 15% of couples will have

difficulties getting pregnant. Infertility is defined as no conception
after 12 months of unprotected sexual intercourse in woman less
than 35 or 6 months in women of 35 years old . If the female
partner has oligomenorrhea or amenorrhea , endometriosis , tubal
disease or there is known male issue , an earlier ovulation is
indicated. The work up for couples experiencing infertility includes
an assessment of ovulatory function , ovarian reserve , uterine
cavity , tubal latency and a semen analysis. If there is evidence of

[6]
endometriosis , tubal occlusions , or adrenal adhesions then a
diagnostic laproscopy should be considered.

Indications

Approximately 25% to 35% of infertile women are

found to have tuboperitoneal disease , with pelvic inflammatory
disease (PID) being the most common cause of tubal damage. PID is
usually a result of Chlamydia trachomatis infection.

Bacterial infection can result in tubal occlusion or

peritubular adhesions that render in vivo fertilization unlikely. IVF
by passes the tubal damage by transferring embryos directly into
the uterus.

Endometriosis , a chronic inflammatory disease

defined by the presence of endometrial tissue outside of the
uterine cavity , is significantly more prevalent in woman with
infertility compared to those without.

The mechanism of how endometriosis causes

infertility are not yet fully understood , but research has shown
that pelvic adhesions , chronic intraperitoneal inflammation ,
disturbed folliculigenesis and decreased embryo implantation have
all been described in women with endometriosis. Laproscopic
surgery has been found to increase the role of pregnancy from 4.7%
to 30.7% indicating the importance of restoring normal pelvic
anatomy for a spontaneous pregnancy. Unfortunately , women
with endometriosis have a significantly lower success rate with
compared to other causes of infertility , with more advanced
disease correlated to inferior outcomes.

[7]
 Poor semen quality is sole cause of infertility in 20%
of couples and contributes to fertility issue in another 20% .
Decreased sperm count , motility or morphology (shape of the
sperm) can be successfully treated medically or surgically in
approximately 50% of men. Intrauterine insemination can also
increase pregnancy rates in couples where the male partner has
low number of motile sperm . If such treatments fail , IVF with or
without intra cytoplasmic sperm injection (ICSI) can be used. Sperm
extracted from testicle or epidydamis in case of obstructive
azoospermia or testicular hypofunction can only be used in an IVF
cycle with ICSI as the sperm have not undergone the final in vivo
maturation process , allowing it to fertilize an oocyte.

Woman who are unable to produce their own

oocytes due to POI or dimished ovarian reserve are able to become
pregnant with donor oocytes or donor embryos. IVF can't overcome
the impact of age on oocyte function and fertilizability.

Why it's done ?

In Vitro Fertilization ( IVF) is a treatment for
infertility or genetic problems. If IVF is performed to treat inf
ertility , you and your partner might be able to try less- invasive
treatment options before attempting IVF , including fertility drugs
to increase production of eggs or intrauterine insemination - a
procedure in which sperm are placed directly in the uterus near the
time of ovulation.

Sometimes , IVF is offered as a primary treatment for

infertility in women over age 40. IVF can also be done if you have

[8]
certain health conditions. For example , IVF may be an option if you
or your partner has :-

i. Fallopian tube damage or blockage:- If ovulation is

infrequent or absent , fewer eggs are available for
fertilization.
ii. Ovulation disorders:- If ovulation is infrequent or absent ,
fever eggs are available for fertilization.
iii. Endometriosis:- Endometriosis occurs when tissue similar to
the lining of the uterus implants and grows outside of the
uterus often affecting the function of the ovaries , uterus and
fallopian tube.
iv. Uterine Fibroids:- Fibroids are benign tumors in the uterus.
They are common in women in their 30s and 40s . Fibroids can
interfere with implantation of the fertilized egg.
v. Previous tubal sterilization or removal:-
Tubal ligation is a type of sterilization in which the fallopian
tube are cut or blocked to permanently prevent pregnancy. If
you wish to conceive after tubal ligation , IVF may be an
alternate to tubal ligation reversal surgery.
vi. Impaired sperm production or function:- Below average
sperm concentration , weak movement of sperm ( poor mobility)
or abnormality in sperm size and shape can make it difficult
for sperm to fertilize an egg. If semen abnormalities are
found, a visit to an infertility specialist might be needed to
see if there are correctable problems or underlying health
concerns.
vii. Unexplained infertility:- Unexplained infertility means no
cause of infertility has been found despite evaluation for
common causes.

[9]
viii. A genetic disorder:- If you or your partner is at risk of
passing on a genetic disorder to your child , you may be
candidates for preimplantation genetic testing - a procedure
that involves IVF . After the eggs are harvested and fertilized ,
they are screened for certain genetic problems , although not
only genetic problems can be found. Embryos that don't
contain identified problems can be transferred to the uterus .
ix. Fertility preservation for cancer or health conditions:-
If you are about to start cancer treatment - such as radiation
or chemotherapy - that could harm your fertility, IVF for
fertility preservation may be an option. Women can have eggs
harvested from their ovaries and frozen in an unfertilized
state for later use or the eggs can be fertilized and frozen as
embryos for future use.

[10]
 1890 - The very first in vitro manipulation of eggs/embryos was
performed by WALTER HEAPE , when he transferred in vivo fertilized
eggs form one female rabbit to another .

 1959 - M.C. CHANG , successfully conducted IVF in rabbits .

 February 15, 1969 - The journal Nature published a paper authored by

R.G. EDWARDS , B.D. BAVISTER and P.C. STEPTOE : “ Early stages of
fertilization in vitro of human oocytes matured in vitro ” .

 July 25, 1978 – The first IVF pregnancy in human was successfully
conducted by ROBERT EDWARD (PHYSIOLOGIST) and PATRICK STEPOE (
GYANECOLOGIST) and LOUISE JOY BROWN was born and she was the
WORLDS FIRST TEST TUBE BABY .

 October 25 , 1978 – DR. SUBHASH MUKHOPADHYAY (PHYSICIAN) was

form Kolkata , India, who created the WORLD’S SECOND and INDIA’S
FIRST TEST TUBE BABY , KANHUPRIYA AGARWAL (DURGA) who was
born 67 days after the first IVF and baby in United Kingdom .

 2012 – It was estimated that 5 million children had been born

worldwide using IVF and other assisted reproductive techniques .

[11]
[12]
[13]
OBJECTIVES

 Female Infertility ( Approximately 40% of cases )

a. Fallopian tube blockage/damage

b. Ovulation disorder
c. Endometriosis Uterine Fibroids
d. Previous tubal sterilization / removal

 Woman’s Age

 Male Infertility (Approximately 40% of cases)

 Unexplained Infertility ( Approximately 20% of cases)

 Genetic Disorders

[14]
[15]
MATERIALS
PHYSICAL ARRANGEMENTS
The embryo laboratory should be located in an area
immediately adjacent to the operating room where the follicular aspirations
and embryo transfers are to be performed. During the course of a follicular
aspiration, it is vital that the clinician be able to communicate directly with
the laboratory scientist(s) and discuss the source of the aspirate, i.e., the size
and appearance of the follicles, the character of the aspirate, whether or not
the aspirate contains granulose cells , apportion of the cumulus mass, and/or
the oocyte itself .

Based on these characteristics and observation, the operating

surgeon can decide whether to proceed with the aspiration of the next
follicle ( if an oocyte has been successfully recovered ) or to irrigate the
previously aspirated follicle in an attempt to recover an oocyte that was not
recovered with the initial aspirate . If the initial aspirate contained clear fluid
and few or no granulose cells, the surgeon can assume that an abnormal
follicle (probably degenerating or atretic ) has been aspirated and need not
make further attempts at irrigating and reaspiration that follicle .

Part of the current quality control procedures for human IVF –

ET require the availability of mouse embryos fertilized in vivo at the 2-cell
stage that can be grown to the hatched blastocyst stage in the same culture
media and under the same culture conditions as the human oocytes and
embryos . Because few, if any , operating room committees would permit
small animal experimentation immediately adjacent to the operating room,
there must be other suitable laboratory facilities available for the recovery of
these 2-cell embryos .

In addition, there must be suitable facilities for preparation of

the tissue culture media as well as for the preparation of the sperm for the
insemination . depending upon space constraints, these facilities may either
be combined with the embryo laboratory adjacent to the operating room or
be combined with the animal facilities . In addition, there should be facilities
for washing the glass wares and other materials needed for the procedure .

[16]
MAJOR EQUIPMENTS
LAMINAR FLOW HOOD (Fig.i)
All manipulations of human oocytes and embryos as well as
the final preparation of the culture media must be accomplished under a
horizontal laminar flow hood (e.g.:- Baker EG3252 or EG6252, Baker
Company, Sanford, ME) .

The horizontal laminar flow hood surrounds the items in the

hood with filtered air, thus chances of microbiological contaminations . These
hoods should be equipped with germicidal (ultraviolet) lamps, which should
be turned on when the hood is not being used. All the working surfaces
should be cleaned with 70% alcohol .

MICROSCOPE (Fig.ii)
Appropriate microscopes are critical to all phases of the IVF
system. A standard laboratory compound microscope (such as the Nikon
Labophot, Nikon, Garden City, NK) is necessary for sperm count and motility
assessments .

A stereoscopic (dissecting) microscope is critical for all phases

of oocyte identification, oocyte handling, and loading the ET catheter. We
have found

[17]
the best stereomicroscope system to be a combination of the Wild M8
optical body (Wild, Rockleigh, NJ) and the Nikon diascopic base and
illuminator . This combination microscope with attached video camera within
the laminar floe hood is illustrated . In addition, a high quality inverted
microscope (such as the Nikon Diaphot) equipped for differential
interference-contrast and phase- contrast optics with photo micrographic
capabilities is highly desirable for high resolution evaluation of embryo
quality, as well as permanent documentation .

INCUBATOR (Fig.iii)
Two different gaseous atmospheres have been used for
successful human IVF: 5% CO2 ,5% O2 , 90% N2 , 5% CO2 in air . Although there
has not as yet been a demonstration that either environment is more
advantageous for human IVF , we use the three gas mixture and would
recommend the acquisition of an incubator system capable of providing
either atmosphere .

We currently use a Forma model 3315 or 3187 (Forma

Scientific, Marietta, OH) which has a gas processor that mixes nitrogen, room
air and carbon dioxide for the desired percentage mix . A double chamber
incubator provides adequate space to keep eggs separate from different
patients and also provides backup in the event of equipment malfunction .
We have used several Queue incubators (Queue Systems, Parkersburg, WV )
but have found them to be unreliable and would not recommend their use .

An alternate arrangement for the establishment of a triple-

gas mixture would be the use of a 5% CO2 in air incubator (such as Forma
3158 or 3326 ) in which is placed a small chamber supplied with a premixed
gas of the desired composition at a constant flow rate .

The disadvantage of this system is the limited space available

in the inner chamber, particularly when embryo cultures are performed in
organ culture dishes . Depending upon the incubator chosen, tanks of
compressed nitrogen and/ or carbon dioxide with appropriate regulators are
attached to the incubator .If the incubator is designed to use ambient air, it
must also be supplied with compressed air, either form a central source or
from a freestanding air pump .

[18]
Fig-i : LAMINAR FLOW HOOD

Fig-ii : MICROSCOPE

Fig-iii : INCUBATOR

[19]
OTHER EQUIPMENTS

1. MEDIA PREPARATION :-

 An Analytic Balance
 An Osmometer
 A pH meter

2. SEMEN COLLECTION &

PREPARATION

 Semen container Jar

 A centrifuge
 A Hemocytometer Set
 Sperm Rinse

3. CULTURE MEDIA

OTHERS

OSMOMETER Ph METER

[20]
ANALYTIC BALANCE SEMEN CONTAINER JAR

CENTRIFUGE HEMOCYTOMETER SET

[21]
COVERSLIP BD SYRINGE WITHOUT NEEDLE HEATING BLOCK

DISTILLED WATER VIBRATOR

CATHETER CONICAL TUBE

[22]
 NEEDLE CULTURE MEDIA

SPERM RINSE SLIDES

BD SYRINGE PIPETTE

ROUND BOTTOM TUBE

[23]
[24]
METHODS
Starting IVF can be a very exciting time – it is another step
closer to becoming parents . Naturally , you will feel hopeful about a
successful outcome but you also need to prepare yourself for around two
months of medications, numerous procedures and testing . The success rate
of modern fertility treatments is high, but for the majority of couples,
multiple treatment cycle may be necessary . The basic stages involved in
IVF procedure are detailed below . The whole process up to the embryo
transfer stage will usually take six to eight weeks .

IVF involves 6 stages :-

STAGE 1 : Ovarian Stimulation and Monitoring

STAGE 2 : Egg (oocyte) retrieval [Egg pick up]
STAGE 3 : Collection of Sperm
STAGE 4 : Culture Media
STAGE 5 : Co-incubation (Fertilization)
STAGE 6 : Embryo Culture / Embryo Development
STAGE 7 : Embryo Selection
STAGE 8 : Pre Implantation Genetic Screening (PGS)
STAGE 9 : Embryo Transfer
STAGE 10 : Luteal Phase
STAGE 11 : Cryopreservation
STAGE 12 : Surrogacy

[25]
STAGE 1 : OVARIAN STIMULATION AND MONITORING
At the beginning of the menstrual cycle, the hypothalamus (
the part of the brainthat controls many bodily functions) releases a hormone
called Gonadotrophin Releasing Hormone ( GnRH) . GnRH in turn causes the
pituitary gland to release a hormone called Follicle Stimulating Hormone
(FSH) to prepare one egg for release . When the egg mature , the pituitary
gland produces another hormone called Luteinizing Hormone (LH) . This
promotes the follicle to release the one egg into the fallopian tube in the
process known as Ovulation . Follicles are the fluid-filled sacs in which eggs
grow to maturity . With IVF, having a greater number of mature eggs
available for fertilization increases the chances of pregnancy .

Certain medications are used to prevent an early release of

eggs, while other medications are used to stimulate the ovaries to develop
more ovarian follicles . By having several mature eggs available for
attempted fertilization and transfer – usually between five and ten – it is
hoped that at least one will result in pregnancy .

FERTILITY MEDICATIONS
GONADOTROPHINS : Gonadotrophins (also spelt as
‘gonadotropins’ ) act directly on the ovary , promoting follicular
development. Gonadotrophins are available as a synthetic form of the
naturally occurring hormone Follicle Stimulating Hormone (FSH), You may
hear these medications commonly referred to as ‘FSH’ rather than
gonadotrophin . You may also be given a mixture of FSH and LH ; a non-
synthetic hormone showing both FSH and LH activity.

These medications are taken by a self-administered injection

under the skin (subcutaneous), usually via an easy to use pen – like device .
The injections are given under the skin of the tummy or thigh . The length of
treatment varies for each patient . Your doctor will advise the length of your
treatment and your dose . For further information about these medications,
including side effects , plese speak within your healthcare team or read the
Consumer Media Information (CMI) available from the TGA website in
Australia (www.tga.gov.au) or Medsafe website in NZ (medsafe. govt.nz) .

[26]
HUMAN CHORIONIC GONADOTROPHIN (HcG): Given by injection one
to two days after the last dose of HcG causes the final maturation and release
of an egg . You will probably do this injection yourself at home at a
specified time .

LUTEINISING HORMONE (LH) : This medication is similar to the LH found

naturally in humans . LH supplementation is recommended for the treatment
of women who have been shown to produce very low levels of some of the
hormones involved in the natural reproductive cycle . It is used together with
a gonadotrophin to bring about the development of follicles .

GONADOTROPHIN RELEASING HORMONE (GnRH) AGONIST : Daily

administration of a GnRH agonist (or analogue) will first stimulate the
pituitary gland at the base of the brain to make extraFSH and LH, but then
causes these hormones to drop right down . As a result, after around two
weeks daily administration, your normal menstrual cycle , hormones and
ovulation are all shut down . This helps control premature ovulation (egg
release) and can also relieve pain of hormonally controlled conditions such
as endometriosis and fibroids .

When used in combination with injected gonadotrophins, its allows for more
reliable timing of the eggs being available for IVF . It includes the medications
NAFARELIN ACETATE and LEUPRORELIN ACETATE . Nafarelin acetate is given
by nasal spray morning and night and leuprorelin acetate is gaven by a daily
subcutaneous (under the skin) injection .

GnRH ANTAGONISTS : Another class of injectible medication, GnRH

antagonists – CETRORELIX ACETATE and GANIRELIX ACETATE– working by
dropping the levels of FSH and LH without first causing an increase in these
levels (as do the GnRH agonists) . This means they can be given for a shorter
period of time . As with GnRH agonists, using this medication allows the
continued stimulation of follicle growth whilst minimizing the risk of
premature egg release prior to egg collection .

[27]
PROTOCOLS
A ‘protocol’ is a plan or schedule of how your IVF cycle will be
done. It will usually include the medications you will be taking, instructions
on how to take them, when you will need to have ultrasounds and blood
tests and the procedures you need to follow throughout the cycle . There are
a few standard protocols used and your doctor will choose the one that is
right for you . The most common ones are :-

LONG DOWN REGULATION :

During ovulation, the release of the egg is triggered by a
sudden surge of the hormone LH at mid-cycle . During an IVF cycle, we do not
want an LH surge to trigger an early release of these eggs. A GnRH agonist is
used to temporarily turn off your own LH and FSH secretion in what is known
as ‘PITUITARY SUPPRESSION’ or DOWN REGULATION’ .

It involves the use of a nasal spray containing nafarelin acetate

or a daily injection of leuprorelin acetate for at least 10 days . FSH is then
given and both drugs are continued until the timing of egg pick up is
determined .

ANTAGONIST TREATMENT CYCLE :

The length of GnRH antagonist administration is generally
shorter then the agonist because it is usually given shortly after
commencement of FSH injections, compared to GnRH agonist which has to
start prior to FSH injections. With this protocol, a daily injection of either
cetrorelix acetate or ganirelix acetate is used to suppress LH surge,
commencing five or six days after starting the FSH injections or up to the day
that sufficient follicles of adequate size are present (as measured by
ultrasound ) . In both cases, daily injections of FSH and GnRH antagonists are
continued .

OVARIAN STIMULATION
 Ovarian Stimulation is used to producer multiple mature follicles, rather
than the single egg normally developed each month .
 Produces many good follicles to be fertilized .

[28]
 Multiple eggs are stimulated because some eggs will not fertilize or
develop normal after fertilization .
 Regular monitoring by ultrasound scan is done .
 Generally, 8 to 14 days of stimulation is required .

POSSIBLE SIDE EFFECTS OF OVARIAN STIMULATION

 Discomfort, bruising or swelling at injection site

 Rash
 Allergic Sensitivity
 Headache
 Mood Swings
 Chance of Hyperstimulation Syndrome (OHSS)

OVARIAN HYPERSTIMULATION SYNDROME

Ovarian Hyperstimulation Syndrome (OHSS) is a potentially
life-threatening medical condition which may occur when your ovaries have
been overly stimulated by various fertility medications. The ovaries may
increase in size and produce large amount of fluid.

It is characterized by pain and bloating in your abdomen and if

severe can cause problems with breathing or urination . Contact a member of
your healthcare team immediately if you believe you have any of these
symptoms.

MONITORING OVULATION
Throughout this first stage, your response to FSH will be
carefully monitored for Ovarian Hyper stimulation Syndrome (OHSS) – see
above – and to gain a clearer picture of what is happening to the follicles so
the right timing and dose can be determined .

ULTRASOUND
Your clinic ( doctor, nurse or sonographer ) will often use one
or more ultrasound scans to obtain an actual image of the ovaries and to
regularly monitor follicle growth in the ovary beginning on or before day

[29]
eight of the cycle . As follicles mature , they grow larger . Through ultrasound,
your doctor can observe the effects of treatment on follicle growth and size,
and decide when to give HcG to assist with egg release . Ultrasound may be
preformed abdominally or more commonly vaginally, using a slender probe a
little thicker than a tampon .The sound waves can not be felt and procedure
is minimally invasive and usually painless .

BLOOD TESTS
Testing the blood every few days for estrogen levels can
monitor the response to treatment with FSH . Developing follicles secrete
increasing Amounts of the estrogen hormone , in particular oestradiol (E2) –
one of the main types of estrogen. Together with ultrasound, this can help
determine the best timing for giving the Hcg injection to stimulate ovulation

[30]
 OVARIAN STIMULATION

OHSS

[31]
ULTRASOUND

BLOOD TEST

[32]
 ANTAGONIST TREATMENT CYCLE

STAGE 2 : EGG (OOCYTE) RETRIEVAL

Eggretrieval also known as ‘egg pick up’ is arranged just prior

to expected ovulation . Egg retrieval is usually performed 36 to48 hours after
the administration of the ovulation inducing drugs HcG or LH . Your doctor
will try to retrieve as many mature eggs as possible, although all the eggs
(oocytes) may not be used in the 4 current IVF cycle . Egg retrieval is
performed under mild sedation, local anesthesia or , in some cases, general
anesthesia, most commonly by ultrasound guided fine needle (aspiration) .

The mature follicle are identified using ultrasound, and then a

needle is passed through the vaginal wall into the follicles and the fluid
withdrawn form the mature follicle with genital suction . The fluid is
immediately examined under a microscope to see if an egg has been
retrieved . The process is repeated for each mature follicle in both ovaries .
All retrieved Sometimes laparoscopy is used where a doctor uses a tube with
a tiny camera on the end of it to guide it to the ovarian follicles .

An aspiration system then uses light suction to retrieve the

egg form the follicle . Lapartoscopy, which requires general anesthesia, is
usually only used when the ovaries are inaccessible via the transvaginal
approach (e.g. when large fibroids are present).

[33]
NUMBER OF EGGS IN THE FOLLICLES
Not every follicle contains an egg, or some may contain
mature eggs which may not be capable of being fertilized . So don’t be
surprised if the number of eggs retrieved is less than the number of follicles
you have been watching develop on ultrasound . The average number of eggs
retrieved is between eight and nine and retrieval process lasts approximately
20-30 minutes .

FOLLICULAR ASPIRATION AND EGG RETRIEVAL

[34]
THE FEMALE OVARY AND DEVELOPMENT OF FOLLICLE

RISKS ASSOCIATED WITH EGG RETRIEVAL

[35]
DRAWBACKS
 More clomiphene leads to abortion
 HcG increases the continuity
 HcG has side effects
 Administration of HcG done at about 1.9cm size follicle has to be
confirmed by ultrasound imaging

Difficulties
 Ovulation estimated on basis of temperature chart
 Change of cervical mucosa score
 Oestrogen level in blood / urine
 Level of LH in blood / urine
 Determination of follicular size

STAGE 3 : COLLECTION OF SPERM

About two hours before egg pick up, a semen sample is

collected form the male partner . Two to three days abstinence form
intercourse/masturbation is preferred prior to the sample collected day . The
sperm sample is usually Produced by masturbation at the clinic . The sperm is
processed to select the strongest, most active sperm . This is called ‘SPARM
WASHING’ .

If sperm are not present in the ejaculate, sperm collection may

be attempted surgically (see ‘surgical sperm extraction’ information next
page ) .

If undergoing IVF, the sperm are then placed with the eggs in
an incubator set to the same temperature as a woman’s body The next day,
the eggs are examined under a microscope to determine whether
fertilization has occurred and you will be phoned about how many of your
eggs have been fertilized . The resulting embryos will be either transferred to
the uterus two to five days later, or frozen for later transfer .

If undergoing ICSI, the eggs are prepared for injection and

their maturity confirmed . In what is a delicate laboratory procedure, a single

[36]
sperm is placed directly into the cytoplasm (the centre) of the egg – hence
the name intra cytoplasmic sperm injection . Fertilization can then be
identified in a similar fashion to IVF after about 20 to 24 hours .

SURGICAL SPERM EXTRACTION

When a man does not have any sperm in his ejaculated semen
because of blockage, failed sterilization reversal, or other reason, sperm can
be surgically extracted from the epididymis or testicular tissue, where they
are stored . Fertilization is then attempted by placing the sperm and the egg
together either by IVF or ICSI .

SPERM PREPARATION TECHNIQUES

1. SEMEN COLLECTION

The semen consists of a suspension of spermatozoa stored in

the epididymis that at the moment of the ejaculation, is mixed with the
secretions of the accessory glands . These glands are mainly the prostate and
the seminal vesicles, while the bulbourethral glands and the epididymis
represent only the minor contribute on of the ejaculate . Two main fractions
are present in the seminal fluid; the first one is prostatic, rich in
spermatozoa. The last fraction of the semen consists of vesicular fraction,
less rich in spermatozoa (Bjorndahl & Kvist , 2003 ).

[37]
 During ejaculation, it is very important to collect the entire
volume of the sample if the first fraction (rich in spermatozoa ) is lost, the
assessment of the semen features will be more difficult. In case of the AI, the
semen sample will not contain the best portion of the spermatozoa . For
these reason, the first step throughout the sperm preparation, is the correct
sperm collection .

The semen collection is strongly recommended after an

abstinence period of 2-3 days (Jurema et al .2005 ; Marshburn et al.,2010 ) to
maximize the conception rate . A sterile container (non-toxic for the
spermatozoa) will be used and the collection of the semen will occur in a
private room very close to the laboratory . All of these elements are
mandatory for the therapeutic use . After the collection, the name of the
couple should be clearly written on the container .

2. CHOICE OF THE TECHNIQUE

The techniques for the select one of the most efficient

spermatozoa are very important for clinical practice . The choice of the best
technique for semen preparation, before the AI, strictly depends on the
quality of the sample (Canale at al.,1994) .So, if we have a sample with
normal count, motility and morphology of sperms we choose a sperm
washing or a swim up method .

By contrast with a suboptimal quantity sample we usually

prefer a density gradient centrifugation . With the first methods, we obtain
good quality sperms: while the density gradient centrifugation is usually
preferred for the greater number of T-Mobile spermatozoa selected form
poor characteristics samples ( low number, motility and morphology
samples). Each technique can be changed or improved with simple changes,
in order optimize the recovery of the sperms .

The efficiency of the sperm selection is expressed as the

concentration of spermatozoa with normal motility (that is progressively
motile spermatozoa, according to the definition of the World Health
Organization Manual of 2010) (WHO Manual, 2010) . Glass-wool columns are
reported to be as effective as density gradient for the separation of
spermatozoa also with intact acrosome form semen with sub optimal

[38]
characteristics (Rhemrev et al., 1989; Sterzik et al., 1998) , but this technique
is less used .

The swim up method and the density gradient centrifugation

procedure different levels of contamination in the sample in the final
preparation . In fact, the swim up technique produces an higher level of non-
sperm components (e.g. debris, bacteria) and the diffusion of other
substances (e.g. the prostatic zinc) form the semen into the overlaying
medium respect of the density gradient centrifugation (Bjorndahl et al.,2005)
. Some differences also exist in the presence and the product on of the
Reactive Oxygen Species

(ROS) and the sperm DNA damage, associated with high level of ROS, after
the application of the two main techniques (Irvine et al.,2000 ; Zini et al.,
1993.2009) .

The final volume of the preparation depends on the technique

performed . If the Intra Uterine Insemination (IUI0 is preformed, 0.3-0.4
milliliters(ml) of spermatozoa resuspended in sterile medium is required . If
the case of the Follopian tube Sperm Perfusion (FSP), the volume of the
suspension must be 4 ml, because it must perfuse the uterus and the both
tubes . Because of its simplicity the first technique is the most used, even if
some authors, comparing the IUI versus FSP, demonstrate the superiority of
the FSP technique about the pregnancy rate in stimulated cycles (Fanchin,
1995) .

3. THE SPERM COUNT

“Before and after the treatment of the seminal fluid, the

following parameters must be evaluated” in line with WHO Manual 2010.

 Volume (ml)
 Concentration (millions/ml)
 Motility (Progressive motility)
 Morphology (% normal sperms)

In addition, it is very important to establish the concentration

of spermatozoa with progressive motility in the final preparation. The

[39]
concentration of the progressive spermatozoa is calculated by multiplying
the percentage(%) of the progressive sperm(PS) for the concentration of the
sperms in the final preparation

[PS] =%PS X[S]f

E1
The total number of the progressive spermatozoa (TPS) is
calculated by multiplying the concentration of the progressive sperms for the
final volume of the suspension .

TPS =[PS] X Vf
E2
The total number of the progressive sperms in the preparation
before the AI may be defined as a threshold value in predicting outcome in AI
.This threshold is not absolute and may vary from study to study , even if
some authors have identified this value 10 million sperms (Miller et al.,2002 ;
Van Voorhis et al.,2001).

4. SPERM WASHING

For the best quality samples (number and motility of sperms)

the sperm washing is often performed (Boomsma et al., 2004) for the AI . The
procedure simply consists in the washing of the semen with a sterile medium
added with human albumin . After the fluidification of the sample , the entire
volume is divided in fractions of not more than 2ml into centrifuge tubes .
The sterile medium of the equal volume (e.g. for the volume of the sample of
2 ml the medium added is 2 ml ) is added in each tube and gently mix with a
sterile pipette .

After that , the samples are centrifuged at 300g (the rpm must
be calculated for the centrifuge in each laboratory) for 10min and than the
supernatant is very carefully removed with a sterile pipette . The pellet is
resuspended in 1ml of the medium, gently mixed and centrifuged again and
the final pellet is resuspended in sterile medium for the AI . It is very
important to determine the count and the motility of the final preparation

[40]
before the insemination . In spite of the simplicity and velocity of the
method, it must be reminded that the repeated centrifugations without the
separation of the good sperms from leukocytes and dead sperms can
produce many oxidative species and the damage of the sperms function
(Aitken &Clarkson, 1988) .

5. SWIM UP METHOD

The swim up is the most common technique used in IVF

laboratories and is preferred if the semen sample has a normal number of
good sperms (normozoospermia) . By this technique, the sperms are selected
on their motility and the capability to swim out the seminal plasma.

If the “direct swim uo” is performed, after the fluidification of

the sample, the entire volume (well mixed) is divided in fractions of 1ml into
centrifuge tubes (round bottom is preferred) . 1.3ml of culture medium is
placed over the semen with extreme attention in each tube . The tubes must
be put in the incubator, inclined at an angle around 45o and incubated 37o C
for 30- 60 min . By inclining the tube at 45o , we increase the surface between
the medium and the semen and we improve the capability of the sperms to
swim out of the semen and to reach the medium . After that, the tube must
be returned in the vertical position and 1ml of the supernatant of each tube
can be gently removed, aspirating the sperms from the upper meniscus
downwards with a sterile pipette (Henkel et el.,2003)

In alternative, culture medium can be placed each tubes and

the semen can be stratified under the medium, in order to obtain a much
cleaner surface between the semen and the medium . In addition, the
recovery of the sperms can be optimized by increasing the number of the
tubes and decreasing the volume of the semen in each tube . 2ml of medium
are added to the supernatant is removed again and the pellet is resuspended
in the sterile medium for the AI .

[41]
6. DENSITY GRADIENT CENTRIFUGATION

This is the preferred technique to select the greater number of

motile spermatozoa in cases of severe oligozoospermia , teratozoospermia or
asthenozoospermia . In this method, good quality sperms can be separated
form dead sperms, leukocytes and the other components of the seminal
plasma by a density discontinuous gradient . Cells with different density and
motility can be selected during the centrifugation by the colloidal silica
coated with saline of the gradient; the sperms with high motility and good
morphology are at the bottom of the tube, finally free from dead
spermatozoa, leukocytes, bacteria and debris .

The most applied discontinuous density gradient is a two

layers density gradient, formed by a top layer of 40% (v/v) and a lower of
layer 80% (v/v) . Density gradient media are available in commerce ready to
use or ready to make the different density layers; the top layer phase (40%) is
prepared by adding 4ml of density gradient medium to 6ml of isotonic sterile
medium (BBW ,Earle, Ham F-10 or HTF) supplemented with HAS (Human
Serum Albumin) ; the lower layer phase (80%) is prepared by adding 8ml of
density gradient medium to 2ml of isotonic sterile medium . The density
gradient is prepared by layering 1ml of 40% medium in a conical centrifuge
tube (not the round bottom tube! ). The number of the tubes depends on the
volume could be divided in not more of 1ml of semen per tube .

After the fluidfication, 1ml of the semen is layered over the

upper layer (40%) and centrifuged at 300g for 15 minutes . If the volume of
each layer is reduced (<1ml) the spermatozoa have to migrate for a less
distance between the layers and so the greater number of motile
spermatozoa can be recovered . The centrifugation time and force can be
varied depending on the quality of the sample: for example, the
centrifugation time can be increased for specimens with high viscosity . After
the centrifugation , most of the supernatant must be gently removed and the
pellet is placed into a new, clean tube; here the pellet is well resuspended in
5ml of medium to remove the density gradient medium . It is centrifuged at
200g for 10 minutes . At the end of the centrifugation, the supernatant is

[42]
removed and 5ml of new medium are added . The centrifugation is repeated
again and the final pellet is resuspended in the sterile medium for the AI

The concentration and the motility after the preparation can

be determined . It must be stressed that the sterile conditions and materials
are essential when we perform the technique for therapeutic application .

Nevertheless, the two main techniques produce different

levels of contamination in the sample and of the production of ROS . In
addition, the swim up technique produces an higher level of non-sperm
components respect of the density-gradient centrifugation . The density
gradient centrifugation recovers spermatozoa with improved motility but
lower DNA integrity instead of the swim up technique, as the literature
suggests (Zini et al.,1999, 2000) . Several studies demonstrate that sperm
DNA damage is associated with lower natural pregnancy rates (Loft et al.,
2003; Spano et al., 2000) and lower IUI pregnancy rates (Evenson et al.,
2008) .

[43]
DONOR SPERMS

Donor sperms is used when the male partner dos not produce
sperm , when the sperm are of very poor quality or if there is a high risk of
passing on genetic diseases . Donor sperms are used less frequently these
days because of the improvement in sperm extraction techniques . The
semen selected for a couple closely matches , as much as possible, the male
partner’s characteristic, e.g. eye and hair colour, height and build . There are
many factors to consider, such as whether to tell friends or family about
using donor sperm and whether the child should know about their origins as
they grow up . Access Australia and the Donor Conception support Group
have many resources on the issues relating to donor insemination .

DONOR OOCYTES

Egg donation is one treatment option for those who wish to

have a child but who are unable to use their own oocytes (eggs) . The eggs
may be sourced form an anonymous donor or donated by a close friend or
relatives . The donor undergoes ovarian stimulation to help the recipient . A
comprehensive medical and counseling process is undertaken prior to the
initiation of such treatment cycles .

[44]
 SEMEN COLLECTION

CHOICE OF THE TECHNIQUE

THE SPERM COUNT

[45]
 SPERM WASHING

SWIM UP METHOD

DENSITY GRADIENT CENTRIFUGATION

[46]
STAGE 4 : CULTURE MEDIA
Culture media is an important part of IVF .It is a complex
solution that is used to support cell growth . It mimics the composition of
oviduct & uterine fluids to closely approximately natural environment of
developing embryo . It is made up of salt solution and other components like
carbohydrates (pyruvate, lactate, glucose) , Amino acids which together are
main source of the developmental stages .
There are three different media to culture embryos at different
developmental stages .

 FERTILIZATION MEDIA : It is used to promote the fertilization of the

oocytes.
 CLEAVAGE MEDIA : It has specific nutrients to assist the embryo in
early development form the one cell stage to around the eight cell
stage .
 BLASTOCYST MEDIA : It contains a different set of nutrients that help
the embryo continue to develop from eight cell to well over 100 cells .
There are two culture media system used for the IVF process .
 ONE MEDIA SYSTEM
 SEQUENCIAL MEDIA SYSTEM

[47]
[48]
[49]
STAGE 5 : CO-INCUBATION ( FERTILIZATION )

The sperm and eggs are incubated at a ratio 75000 :1 in culture

media in order for the actual fertilization to take place . A review in 2013
came to the result that a duration of this co-incubation for about 1 to 4 hrs
results in significantly higher pregnancy rates than 16 to 24 hours . In most
cases, the egg will be fertilized during co-incubation and will shows two
pronuclei . In certain situations, such as low sperm count or motility , a single
sperm can be injected directly into egg called Intra Cytoplasmic Sperm
Injection (ICSI ) .The Fertilized egg is passed to a special growth medium and
left for 48 hrs when the egg consists of 6-8 cells .

In gamete intrafallopian transfer , eggs are removed from the

woman and placed in one of the fallopian tubes, along with the man’s sperm
. This allows fertilization to take place inside the woman’s body . Therefore,
this allows fertilization to take place inside the woman’s body . Therefore,
this variation is actually an in vivo fertilization, not in vitro .

[50]
STAGE 6 : EMBRYO CULTURE / EMBRYO DEVELOPMENT

‘Embryo culture’ is the term used to describe the process

immediately following egg pick up . It is during the culture process that your
eggs and your partner’s sperm will be combined in order to process a
fertilized egg (known as a zygote ) . Your doctor will discuss how long they
will watch embryo development in the laboratory and how each embryo will
be ‘graded , e.g. from ‘A’ to ‘F’ depending on their quality. It is common for
transfer to be done between day two (2-4 cell stage) and day five (Blastocyst
stage – around 100 cells) of development . This allows assessment of embryo
cleavage (the way an embryo divides ) and ensure the embryo is still
developing so that only embryos capable of resulting in a pregnancy are
transferred .

Some fertility specialists prefer doing blastocyst transfers

because it is easier to choose a healthy embryo for transfer at this stage .The
latest statistics from the Australian Institute of Health and Welfare show that
there is an increasing trend towards blastocyst transfer over the traditional
‘3-day transfer’ . In 2012, blastocyst embryo transfers accounted for 59.8% of
embryo transfer cycles significantly higher than the percentage of cycles
transferring blastocysts in 2007 (33.7%) .

STAGES OF DEVELOPMENT

1. ZYGOTE

A single sperm penetrates the mother’s egg cell and the

resulting cell is called a ZYGOTE. The zygote contains all of the genetic
information (DNA) necessary to become a child . Half of the genetic
information come from the mother’s egg and half from the father’s sperm
.The zygote spends the next few days travelling down the fallopian tube and
divides to form a ball of cells . The term CLEAVAGE is used to describe the cell
division .The zygote is large compared to any other cell and undergoes
cleavage without any overall increase in size . This means that with each
successive subdivision, the ratio of nuclear to cytoplasmic material increases
. Initially the dividing cell, called blastomeres , are differentiated and

[51]
aggregated into a sphere enclosed within the membrane of glycoproteins of
the ovum .

MORULA

The blastomeres in the early cleavage stages tend to assume a

spherical shape . Their mutual pressure flatten the surfaces of the
blastomeres in contact but free surfaces remain spherical . At this 16 celled
stages, the whole embryo acquires a characteristics mulberry shape and so ,
it is called MORULA .The morula is no larger than the zygote , but keeps
producing smaller and smaller cells through cleavage . At this stage the cells
start to bind firmly together in a process called Compaction, and cleavage
continues as cellular differentiation.

2. BLASTULA

As cleavage proceeds, at about fourth and fifth cleavage

stages, a small space develops between the blastomeres of morula . The
space is initially represented as narrow crevices between the blastomeres of
the morula which gradually increases as cleavage proceeds With more rapid
multiplication of micromeres in the animal half, the space, now called as
BLASTOCOEL shifts more and more towards the animal pole . The
surrounding blastomeres secrete an albuminous fluid, termed as blastocoels
jelly into the blastocoels . The blastocoel gradually increases in size as more
and more blastocoel jelly is secreted into it . This stage of the embryo is
called BLASTULA . It consists of approximately 100 cells . The inner group of
cells will become the embryo, while the outer group of cells will become the
membrane that nourish and protect it .

3. EMBRYO

The blastocyst reaches the uterus around day five, and

implants into the uterine wall on about day six . The cells of the embryo now
multiply and begin to take on specific functions resulting in the various cell
types that make up a human being (e.g. blood cells, kidney cells and nerve
cells ).

[52]
ASSISTED HATCHING
Prior to implanting in the uterus, the embryo must emerge
from its covering in a process called HATCHING . In some women, the
membrane seems to harden, interfering with the hatching process . In such
cases, thinning the embryo membrane with a dilute acidic solution or laser
prior to embryo transfer may assist hatching . This procedure is performed on
embryos in the laboratory when needed . Older women or those who have
not achieved pregnancy after several IVF cycles are often helped by this
treatment . Assisted hatching may also be done in some cases following
cryopreservation and embryo thawinhg .

[53]
STAGE 7 : EMBRYO SELECTION

In the majority of human IVF cycles multiple embryos are

created after ovarian hyperstimulation . The viability of these embryos, and
as a consequence the chance for an embryo to successfully implant, is subject
to biological variation . To achieve the best possible live birth rates after IVF
while minimizing the risk for multiple pregnancy, one or two embryos that
are considered to have the best chance of implanting are selected for
transfer . Subsequently, supernumerary embryos with a good chance of
implanting are selected for cryopreservation and possible transfer in the
future while remaining embryos are discarded .

The best available method for embryo selection is

morphological evaluation . On the basis of multiple morphological
characteristics at one or several stages of preimplantation development,
embryos are selected for transfer (Ebner et al. ,2003; Gerris,2005) . However,
with embryo selection based on morphological evaluation implantation rates
in general do not exceed 35% , although varying results have been reported
(Centers for Disease Control and Prevention et al.,2010) . This has resulted in
a strong drive for finding alternative selection methods.

[54]
STAGE 8 : PREIMPLANTATION GENETIC SCREENING(PGS)
Preimplantation Genetic Screening (PGS) is a technique that
can be used during IVF to test embryos for a variety of genetic
disorders.Testing is done in the laboratory before the embryo is transferred
to the uterus . The classical form of PGS involves the biopsy at day 3 of
embryo development of a single cell of each of the embryos available in an
IVF cycle and analysis of this cell by Fluorescence In-Situ Hybridization (FISH)
for aneuploidies , for a limited number of chromosomes .Only embryo for
which the analyzed blastomere is euploid for the chromosomes tested are
transferred .

This decreases the risk of having a child with a serious

inherited disorder . Screening can detect a range of disorders, including
Down’s syndrome , Cystic fibrosis, Hemophilia A ,Tay-Sachs disease and
Turner syndrome .

[55]
STAGE 9 : EMBRYO TRANSFER

Embryo Transfer is not a complicated procedure – rather like a

pap smear – and can be performed without anesthesia . Two to five days
following egg pick up, the embryo is placed in a catheter (a soft tube) and
transferred to the uterus via the vaginal opening . Embryos are graded by the
embryologist based on the amount of cells, evenness of growth and degree
of fragmentation .

The number of embryo to be transferred depends on the

Number , Available Woman’s age , Cause of infertility , Pregnancy history and
other health & diagnostic factors . Generally one or occasionally two
embryos will be transferred to the uterus . In countries such as Canada, the
UK, Australia and New Zealand, a maximum of two embryos are transferred
except in unusal circumstances . In the UK and according to HFEA regulations,
a woman over 40 may have up to three embryos transferred, whereas in the
USA, the younger women may have many embryos transferred based on
individual fertility diagnosis .

It is important to note that the risk of multiple pregnancy

increases with the number of good quality embryos transferred . Most clinics
and country regulatory bodies seek to minimize the risk of pregnancies
carrying multiples, as it is not uncommon for more implantations to take
than desired . 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 .

[56]
[57]
STAGE 10 : LUTEAL PHASE SUPPORT

The luteal phase support is the two week period between the
embryo transfer and the pregnancy test . It is usually recommended that you
take it easy for a couple of days after the transfer . After 48 hours, you can
resume your normal activities – these will not affect implantation . The
corpus luteum ( the follicle after the egg is released) does not produce the
hormones estradiol and progesterone to prepare the uterus for embryo
implantation as it would in a natural cycle . This is due to the treatment prior
to egg collection and the collection process itself .

In order to ensure there is adequate progesterone present, you

will be prescribed progesterone as a vaginal gel or in the form of pessaries to
help keep the endometrium (the lining of the uterus ) in optimal condition
for implantation . After approximately 16 days, you will return to the clinic or
your doctor for a blood test to determine whether a pregnancy has occurred .
Progesterons, progestins and Gonadotrophin Releasing Hormone to increase
success rate of implantation . Giving preference during luteal phase (later
phase of menstrual cycle ) higher rate of live birth .

[58]
STAGE 11 : CRYOPRESERVATION

Cryopreservation can be performed as oocyte cryopreservation

before fertilization, or as embryo cryopreservation after fertilization . The
Rand Consulting Group has estimated there to be 400,000 frozen embryos in
the Untied stages . The advantage is that patients who fail to conceive may
become pregnant using such embryos without having to go through a full IVF
cycle . Or, if pregnancy occurred, they could return later for another
pregnancy .

[59]
 Spare oocyte or embryos resulting from fertility treatments
may be used for oocyte donation or embryo donation to another woman or
couple, and embryo may be created, frozen and stored specifically for
transfer and donation by using donor eggs and sperm . Also, oocyte
cryopreservation can be used for woman who are likely to lose their ovarian
reserve due to undergoing chemotherapy . The outcome from using
cryopreserved embryos has uniformly been positive with no increase in birth
defects or development abnormalities .

[60]
STAGE 12 : SURROGACY

Instead of IVF, the fertilized egg placed in womb of biological

mother . Surrogacy is the fertilized egg is not placed in the womb of
biological mother but a woman who has appeared to bear child for monetary
reason. Biological mothers don’t able to bear children for medical reason . So
mothers don’t able to go through the phase of pregnancy . Simply woman
may not be female i.e. gay and transgender .

[61]
ALTERNATE METHODS INSTEAD OF IVF

I. Intra Uterine Insemination (IUI)

Inter Uterine Insemination (IUI) is a relatively simple fertility

treatment . It may be done with or without drugs . It is an option for an IVF
alternative . In this treatment procedure, sperm is inserted through a
catheter, all the way into a woman’s cervix when all is due to ovulate . This
way the sperm do not have try to make it through all the way to the cervix on
their own . This also eliminate the problem of thin cervical mucus, since IUI
will completely bypass that step . It is less invasive than other forms of
fertility treatment, and it does not have to be used in combination with
fertility drugs, although it can be if needed .

IUI may be recommended to treat any of the following infertility situations :

 Hostile cervical mucus

 If a sperm donor is being used
 If sexual pain make intercourse not possible
 If treatment with fertility drugs alone is not successful
 Male infertility
 Unexplained infertility

[62]
II. Gamete Intra Fallopian Transfer ( GIFT )

Gamete Intra Fallopian Transfer (Gift ) is , in some ways , very

similar to IVF . In this procedure woman will take fertility drugs to stimulate
egg production, and the eggs will then be removed fromher body . Then male
sperm is collected and washed . The eggs then are mixed together with a
large number of sperm, but there is no attempt made by a laboratory to
fertilize the eggs . That is why GIFT is different from IVF .

It used multiple eggs collected from the ovaries . The eggs are
placed into a thin flexible tube (catheter) along with the sperm to be used .
The gametes (both eggs and sperm) are then injected into the fallopian tubes
using a surgical procedure called laparoscopy . The doctor will use general
anesthesia . The egg and sperm, pretty much, are on their own at that point .
GIFT allows conception to happen in its natural environment, with just a little
bit of help, without outside interference . Of course, with GIFT, there is no
way to be sure that fertilization will occur .

[63]
III. Zygote Intra Fallopian Transfer (ZIFT) & Tubal

Embryo Transfer (TET)

These both are pretty similar to GIFT in that the woman will
take a fertility drugs to stimulate her reproduction . Zift ( Zygote Intra
Fallopian Transfer ) and TET ( Tubal Embryo Transfer ) are both wherw the
eggs are removed and mixed with washed sperm . The difference is that the
eggs are fertilized in a laboratory setting .

In ZIFT , the eggs are transferred back into the fallopian tubes
the day after they are fertilized . In TET, the eggs are given a few extra
developmental days before they are placed in the fallopian tubes . Advocates
of ZIFT and TET think that by placing the eggs in the fallopian tubes rather
than the uterus, the eggs will follow the fertilization pathway .

[64]
IV. Pronuclear Stage Tubal Transfer ( PROST )

It is similar to ZIFT, uses in vitro fertilization . But it transfer the

fertilized egg to the fallopian tube before cell division occurs . These
procedure have higher cost and risks related to laparoscopy and they do not
provide as much useful information about embry development as IVF does .
For these reasons, these procedures are rarely used .

Comparative Treatment Outcomes for Female

Antisperm Antisperm Antibody Subfertility Category Managed by
IVF and PROST During the Same Frame

Program Cycles Transfers Pregnancies

(%)

IVF 20 20 4 (20.0)
PROST 8 8 3 (37.5)
x2 ( Yates correction ) = 0.9, not significant.

[65]
V. Intra Cytoplasmic Sperm Injection ( ICSI )
Intra Cytoplasmic Sperm Injection (ICSI) is an in vitro
fertilization in which a single sperm cell is injected directly into the cytoplasm
of an egg .This technique is used in order to prepare the gametes for the
obtention of embryos that may be transferred to a maternal uterus . With
this method, the acrosome reaction is skipped . This procedure is most
commonly used to overcome male infertility problems, and although it may
also be used where eggs can not easily be penetrated by sperm, and
occasionally in addition to sperm donation .

[66]
[67]
RESULTS

POSITIVE RESULTS

If the result is positive, you will have to continue the

medication and set a date for reexamination and later another visit, ehich
includes a blood test and ultrasound study, to make sure that the pregnancy
is normal. On the 5th to 6th week of pregnancy, an amniotic sac will already be
visible via US in the uterus, and in the 6th to 7th week an embryonic pulse
may already be visible . The pregnancy follow up at the unit will usually end
in the 7th week of pregnancy, at which time you will be referred for further
pregnancy follow up by your gynecologist .

NEGATIVE RESULTS

If the result is negative, you will have to stop taking the

medications and arrange for an appointment with the doctor for
summarizing the current treatment cycle and planning another treatment
cycle . Remember that failing to achieve pregnancy may involve a feeling of
grief, disappointment and emotional difficulties . Do not hesitate to take
advantage the emotional support that the unit offers you . If you have
fertilized oocytes that have been frozen, you may plan the date of starting
the treatment for their thawing with the doctor and unit staff.

[68]
IVF SUCCESS RATES BY AGE AND NUMBER OF
EMBRYOS

IVF is a commonly referred fertilization treatment for couples,

single parents and also for a couple of the same gender who want to have a
baby. This procedure is generally 6-8 weeks long procedure as first the
person has to get ready mentally and physically , followed by taking
medicines for egg maturation , leading towards extraction of the eggs from
the ovary , eggs are then fertilized with the sperm in a laboratory and
implanted back into the womb when it turns into an embryo for further
developing into a baby.

Though it is impossible to predict the exact result of the

success of the treatment but definitely there are research and calculations
done by the specialist to find out approximate IVF success rate by age and
embryos. However , we would still suggest not to keep any false high hopes
as every human body has its own reaction and working agenda towards the
treatment, what may have worked for others may not work for you or vice-
versa .

1. IVF SUCCESS RATES BY AGE

One of the prominent factors that are most likely to affect the
success rate of the whole IVF treatment is "age" of the woman undergoing
IVF treatment. Age has prominence in women pregnancies since long , no
matter it is a natural pregnancy or an artificial pregnancy treatment .
Similarly , it plays a significant role in IVF treatment as well .

Although it had been noticed , that women of lower age group

, are most likely to have higher IVF success rates , whereas women of
comparatively older age group , tend to face a decrease in the success rates
of the treatment . We will further look into it by the age category .

I. IVF SUCCESS RATES UNDER 35

It is said that women under 35 and especially in their 20's have

higher success rates with regards to age as this the period when they are
most fertile. Women who don't have any identified fertility issues have a 20%
success rates of pregnancy but on the other hand if women are facing any
kind of infertility issues like less number of eggs , genetic diseases , partner's

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infertility or same sex relationship then they would take up IVF for which the
success rates are assimilated as per the research of CDC ( The Centre for
Disease Control ) below :-

 The success rates are higher in women under the age of 35 as compared
to those who are above 35 undergoing the IVF treatment .
 If the woman has never ever conceived before and is under 35 then it is
more likely for her that she would conceive in 32% of cases.
 If women have had a child before then there are 37% of chances for IVF
cycle to work and result in a successful pregnancy under the age of 35 .
 If a woman is in her 20's then there are 34% chances for IVF cycle to work
with two or more than two children .

Though the number seems to be comparatively low , further

research shows that the IVF success rates for women in their 20's are very
likely to result in success . Approximately there are 40-43% chances of
embryo transferring to turn into a successfully pregnancy . Also , IVF is
considered as one of the costly infertility treatments and patients except it to
work in one cycle , so as to save money . As per the CDC data analysis , it has
been calculated that woman falling in the category of under 35 have a 52%
chance of acquiring pregnancy by only one round of IVF .

II. IVF SUCCESS RATES OVER 40

Women are not likely to conceive when over in their 40's , in

earlier generations it was not even near possible to have a baby in the 40 or
more years of age for women but with emerging new technologies and
various innovation like Assisted Reproductive Technology (ART) , it is now
possible for women over the age of 40 to have a baby and enjoy motherhood
even they are hitting close to menopause .
As per the research done by the Centre of Disease Control (CDC) and the
Society for Assisted Reproductive Technology (SART) , it has been revealed
that if we use fresh embryos for the treatment then IVF success rates over 40
are as follows :-

 For 40 years old , there are 20% chances for IVF cycle to work and 14% if
going for live birth .

 For 41 years old , the chances are 15% in IVF cycles and 10% for live birth .

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 For 42 years old , it is 12% and 7% for both IVF cycles and live birth ,
respectively.

 For 43 years , the number decreases to 8% , there are only 3% chances of a

healthy delivery .

 For 44 years of age , it is 6% chance of IVF resulting in pregnancy and 3%

of a live birth chance .

 Lastly , women ageing 45 or above have only 3% chances of pregnancy by

an IVF treatment and only 1% for the live birth .

If you want to undergo just one round of IVF cycle to acquire

pregnancy at the age of 40 then the chances are of 8% that you would result
in getting pregnant . So , it is recommended that you should go to more than
one round atleast . Also it is important to state that doesn't presume your
personal results according to this data because every human body has its
own tenderizes and there many other factors that play a considerable role in
the success rate of this treatment .

2. IVF SUCCESS RATES BY NUMBER OF EMBRYOS

There are plenty of factors affecting the success rate of IVF

treatment but one of the most important factors which efficiently decide the
success rate is an embryo , the number and the quality of the embryo . Most
of the failure in the treatment occurs due to false embryo used in the
treatment .

There are technique like PGD ( Pre-implantation Genetic

Diagnosis ) which helps the doctor to carry out the most efficient embryo for
IVF treatment . There are embryos that look efficient by outer appearance
but are lacking factors needed for pregnancy but on the other hand embryos
which don't look efficient on the appearance are most likely to have all the
efficiency required for successfully and to determine , differentiate and
further select the appropriate embryo the PGD technique is used by the
doctors .

SINGLE VS. MULTIPLE EMBRYOS

It is recommended that when carrying out IVF treatment only

single embryo transfer is considered to be healthy for the pregnancy results .

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 The most important factor for a mother is the healthy of a
baby , so if we transfer multiple embryos the chances of having twins and
multiple births increases along with which the risk factors for the baby also
increase its level simultaneously .

Also the success rate for both single embryo transfer and
multiple embryos transfer is more or less similar . No matter how many
numbers of the embryo for IVF are used , one should not neglect the risk
factors . As per one of the studies of "Fertility Reasearch and Practice" it had
been calculated that the success rate of "Single Embryo Transfer" was 45%
whereas for "Double Embryo Transfer" was 42% . This clearly shows that it is
more likely to be more or less similar to each other . So single embryos , is
most likely to be safe and sufficient number of embryos for IVF treatment .

IVF SUCCESS RATES IN INDIA

It has been greatly developing in the field of medicine and

technology as well. It had been years since IVF treatment came in India and
was widely accepted and adopted by couples facing infertility problems .

The success rate as talked about earlier greatly varies and

depends upon a wider range of factors in IVF, the age, the lifestyle, the cause
of infertility and of course the most important factor "embryo" . IVF Success
Rates In India , fails between 30-35% , as calculated by the Obstetrics and
Gynecology Development under the guidance of Dr. Alka Kriplani of one of
the prominent hospitals of India , situated in New Delhi , known as All India
Institute of Medical Science (AIIMS) .

During a good run of treatment , the success rate tend to

increase up to 40% . The resulting calculations of success rates by AIIMS are
highly comparable with many other nations as the number being quite high
for women under 35 years of age .

Apart from AIIMS , there are many private clinics in India that
are more likely to claim about their success rates being 2 to 20 minutes
higher . They have specified the 70 - 80% success rates for women under 35
and 40 , 50% of chances for over 40 years of age .

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[73]
ADVANTAGES OF IVF
 A boon to couple who can't give birth to babies due to some
defect in them ( either parent ) .

 The desire of getting baby fulfilled .

 For most children conceived by IVF there are no long term

problems .

 It can be helpful for successful pregnancy and healthy baby .

 Block tubes:- blocked fallopian tubes women can be helped

through IVF .

 Older patients with low ovarian reserve can avail thin

opportunity .

 Male infertility problem can overcome .

 Unexplained infertility couple can get benefit .

 Poly Cystic Ovary Syndrome ( PCOS ) , a common condition ,

the hormonal imbalance leading to irregular menstrual cycle .

 Premature ovarian failure - women with menopause can have

IVF using donors egg .

 It helps sterile women or some sex couples .

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RISKS OF IVF
 Multiple births:- IVF increases the risk of multiple births if
more than one embryo is transferred to your uterus . A
pregnancy with multiple fetuses carries a higher risk of early
labour and low birth weight than pregnancy with a single
fetus does .

 Premature delivery and low birth weight:- Research

suggests that IVF slightly increase the risk that the baby will
be born early or with a low birth weight .

 Ovarian hyperstimulation syndrome:- Use of injectible

fertility drugs , such as human chorionic gonadotropin (
HCG), to induce ovulation can cause ovarian hyperstimulation
syndrome , in which your ovaries become swell and painful .

 Symptoms typically last a week and include mild abdominal

pain , bloating , nausea , vomiting and diarrhea .

 Miscarriage :- The rate of miscarriage for women who

conceive using IVF with fresh embryos is similar to that of
women who conceive naturally — about 15% to 25% but the
rate increases with maternal age .

 Egg-retrieval procedure complications :- Use of an

aspirating needle to collect eggs could possibly cause
bleeding, infection or damage to the bowel, bladder or a blood
vessel . Risks are also associated with sedation and general
anesthesia , if used .

 Ectopic pregnancy :- About 2% to 5% of women who use

IVF will have an ectopic pregnancy — when the fertilized egg
implants outside the uterus, usually in a fallopian tube . The
fertilized egg can't survive outside the uterus , and there's no
way to continue the pregnancy .

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 Birth defects :- The age of the mother is the primary risk
factor in the development of birth defects , no matter how the
child is conceived . More research is needed to determine
whether babies conceived using IVF might be at increased
risk of certain birth defects.

 Cancer :- Although some early studies suggested there may

be a link between certain medications used to stimulate egg
growth and the development of a specific type of ovarian
tumor, more-recent studies do not support these findings .
There does not appear to be a significantly increased risk of
breast , endometrial , cervical or ovarian cancer after IVF .

 Stress :- Use of IVF can be financially , physically and

emotionally draining . Support from counselors , family and
friends can help you and your partner through the ups and
downs of infertility treatment .

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[77]
CONCLUSION
In vitro fertilization can be very safe alternative to
natural child bearing if used under regulation . Often times, couples
with infertility problems turn to this process for help with
conceiving . It is used by mixing a woman's egg with a man's
sperm outside the body and letting it fertilize, making it an embryo .
If successful , the women become pregnant . If she will not have to
pay another ten thousand for a second cycle . The history began in
Great Britain when the world's first successful test tube baby was
born, Louise Joy Brown . Today if not used with care and
consideration , in vitro fertilization can take an unexpected turn .
Nyada Suleman , whether it was her intensions or not , found this
out the hard way . Many people frown upon her decision of placing
all her frozens egg back into her uterus . Others , perhaps those
who are pro-life , think Nyada did the right thing by not wasting life
. It doesn't matter who's at fault : All that does matter is that
Americans are playing for Nyada's mistake and IVF is not regulated
properly, there will soon be people repeating her mistakes.

IVF can be a successful method in treatment of

infertility in women aged 40 - 45 years . Our results suggest that the
treatment regimen used in this study results in a reasonably good
pregnancy rate in both groups . Abnormal fertilization rate was
similar in both groups . However evidence of higher pregnancy rate
in ICSI cycle is still vague and more studies are needed .

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 PHOTO
GALLERY

[79]
 With Dr. Pragyan Dash

With the laboratory assistants

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Indira IVF Hospital, Bhubaneswar

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[82]
[83]
Indira IVF Hospital, Bhubaneswar

Indira IVF Hospital, Cuttack

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REFERENCES

[85]
 REFERENCES

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Reuters . 3 may 2012 . Retrieved 5 November 2015 .

 European society of Human Reproductive and

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Daily . Retrieved 8 December 2018 .

 Fertility : assessment and treatment for people with

fertility problems . NICE clinical guideline :- issued
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 Golden R ( June 2018 ). " Jean Mardian purdy

remembered - the hidden life of an IVF pioneer ". Human
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 "In Vitro Fertilization (IVF) Results - Mayo

Clinic". www.myoclinic.org . Retrieved 5 November
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S.K.(2019) . Clinical adjuncts in vitro fertilization : a
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 Moreton C (14 January 2007) . "World 's first test tube
baby Louise Brown has a child for own". Independent.
London. Retrieved 21 May 2010 . The 28-Years-old ,
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impregnating it with sperm and planting the resulting
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 Weiss NS , Braam S , Konig TE , Hendriks ML , Hamilton

CJ , Smeenk JK et al. (November 2014). "How long
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 Wade JJ , MacLauchlan V , Kovacs G (October 2015).

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