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BASIC STEPS IN IN VITRO FERTILIZATION (IVF)
BY
ADESEJI, WASIU ADEBAYO
DEPARTMENT OF ANATOMY, UNIVERSITY OF ILORIN.
REPRODUCTIVE BIOTECHNOLOGY AND
ENDOCRINOLOGY
AUGUST, 2015.

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INTRODUCTION
The word “In vitro” was coined from Latin, meaning “in glass". In vitro fertilization (IVF) is a
process by which an egg is fertilized by sperm outside the body. The process involves monitoring
and stimulating a woman's ovulatory process, removing mature ovum or ova from the woman's
ovaries and letting sperm fertilize them in a liquid in a laboratory. The fertilized egg (zygote) is
cultured for 2–6 days in a growth medium and is then implanted in the same or another woman's
uterus, with the intention of establishing a successful pregnancy. It is a technique of assisted
reproductive technology (ART) for treatment of infertility. IVF techniques can be used in a
different types of situations such as gestational surrogacy, in which case the fertilized egg is
implanted into a surrogate's uterus, and the resulting child is genetically unrelated to the surrogate.
In some situations, donated eggs or sperms may be used for the process.
INDICATIONS FOR IVF
IVF is performed as a major treatment for infertility in male and female. Some of the indications
for IVF treatment are:
Tubal absence or disease
Endometriosis
Unexplained Infertility
Ovulatory failure
Oocyte donation
Gestational Carrier

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STEPS/PROCESSES IN IVF
Pituitary Suppression
Ovarian hyperstimulation
Follicular Aspiration or Oocyte Retrival/Collection
Sperm collection and assessment
Fertilization and Insemination (IVF/ICSI)
Embryo Culture
Embryo Transfer
PITUITARY SUPPRESSION AND OVARIAN HYPERSTIMULATION
Ovarian hyperstimulation is the stimulation to induce development of multiple follicles of the
ovaries. It should start with response prediction by e.g. age, antral follicle count and level of anti-
Müllerian hormone (La Marca and Sunkara, 2014). To retrieve multiple mature oocytes. The
pituitary gland is suppressed by a GnRH agonist or antagonist, according to the chosen protocol.
The ovaries are stimulated with gonadotropins. The response on the stimulation is followed
echographically and by examining hormone levels in the blood. The examined hormones are
oestradiol, luteinizing hormone, follicle stimulating hormone, and progesterone. When the
hormones have reached the expected levels and when there are at least three follicles larger than
17 mm, the final maturation of the oocytes is triggered by an injection of human chorionic
gonadotropin (hCG). Only one follicle will normally grow. When the follicle has reached the size
of 17 mm, hCG is also given to control the maturation of the oocyte. Natural managed cycles may
be beneficial for patients that poorly respond to stimulation protocols.

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Pituitary suppression of spontaneous ovulation is achieved by two main methods are: Using a
(usually longer) gonadotropin releasing hormone (GnRH) agonist protocol or a (usually shorter)
GnRH antagonist protocol (La Marca and Sunkara, 2014).
In a standard long GnRH agonist protocol the day when hyperstimulation treatment is started and
the expected day of later oocyte retrieval can be chosen to conform to personal choice, while in a
GnRH antagonist protocol it must be adapted to the spontaneous onset of the previous
menstruation. On the other hand, the GnRH antagonist protocol has a lower risk of ovarian
hyperstimulation syndrome (OHSS), which is a life-threatening complication (La Marca and
Sunkara, 2014).
For the ovarian hyperstimulation, injectable gonadotropins usually Follicle Stimulating Hormone
(FSH) analogues such Clomiphene citrate (trade names: clomid, serophene gonald-F, follistim)
are generally 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.
OOCYTE COLLECTION/ RETRIEVAL
Oocyte recovery is the process of collecting mature eggs directly from the ovaries prior to their
release from ovarian follicles (Taymor et al., 1992). The eggs are retrieved from the patient using
a transvaginal technique called transvaginal oocyte retrieval. This involves using an ultrasound-
guided needle piercing the vaginal wall to reach the ovaries. Through this needle follicles can be
aspirated, and the follicular fluid is passed to an embryologist to identify ova. It is common to
remove between ten and thirty eggs. The retrieval procedure usually takes between 20 to 40

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minutes, depending on the number of mature follicles, and is usually done under conscious
sedation or general anaesthesia.
Figure 1: Illustration of oocyte retrieval from ovary.
Oocytes are extremely sensitive to changes in temperature, especially declines. Temperature
fluctuations have been shown to be detrimental for the meiotic spindle. The maintenance of 37°C
during in vitro manipulation is required. Therefore, all disposables and media that come in direct
or indirect contact with the retrieved oocytes are prewarmed to 37°C. Heating devices such as table
incubators, heating blocks, and plates of microscope are calibrated on a regular base and their
temperature is monitored using calibrated thermometers (Dale et al., 1998; Swain and Pool, 2009).

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pH oscillations also have detrimental consequences. The cytoskeletal dynamics and cellular
metabolism are highly dependent on pH. In order to avoid pH fluctuations of the external pH and
also indirectly the intracellular pH, oocytes are collected in HEPES (2-hydroxyethyl)-1-piperazine
ethane sulfonic acid) buffered or bicarbonate buffered media at pH 7.3. HEPES buffered media do
not require a CO2 atmosphere, whereas bicarbonate buffered media do and therefore, have to be
equilibrated beforehand in an incubator. Dishes containing bicarbonate buffered media are
prepared one day before oocyte retrieval and kept in an incubator overnight. Dishes containing
HEPES buffered medium are prepared the day of oocyte retrieval. All materials that are used
during the retrieval are sterilized (Dale et al., 1998; Swain and Pool, 2009).
Finally, oocyte quality can also be affected by changes in osmolarity which result from the
evaporation of medium. Changes in osmolarity cause shrinking or swelling of the oocytes.
Evaporation can be avoided by covering the media with oil (Dale et al., 1998; Swain and Pool,
2009).
Figure 2: Mature oocyte

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Procedures
1. Oocyte retrieval is planned 36 hours after triggering ovulation, 40 hours after hCG
injection.
2. The procedure takes place in the Oocyte retrieval room and is performed by the fertility
doctor and nurses.
3. The procedure is carried out with a vaginal transducer equipped with a needle guide.
4. Once the follicle is entered, suction is gently applied to aspirate follicular fluid and with it,
cellular material including the Oocyte is collected using a vacuum pump.
5. Transferred to bicarbonate or HEPES buffered media.
6. Flushing of follicles with NaCl (0.9 %) and Heparin (5,000 IU) is optional.
7. The follicular fluid is captured in a pre warmed tube and put at 37°C.
8. Immediately thereafter, the aspirate is examined for the presence of COC in the laboratory.
9. The other follicles are aspirated. Once the ovarian follicles have been aspirated on one site,
the needle is withdrawn, and the procedure is repeated on the contralateral site.
10. The process takes about 30 minutes and is usually carried out under conscious sedation or
light general anesthesia.
11. After the procedure, patients are given progesterone to support the luteal phase
(Killick, 2006).

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SPERM COLLECTION AND ASSESSMENT
Sperm is usually obtained from the male partner by masturbation or in a special condom used
during intercourse. Alternatively, sperm may be obtained from the testicle, epididymis, or vas
deferens from men whose semen is void of sperm either due to an obstruction or lack of production.
Sperm is prepared for fertilization by removing inactive cells and seminal fluid from the semen in
a process called sperm washing. There are two methods of sperm washing:
Swim up method
Density gradient separation method.
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.
EGG AND SPERM PREPARATION
In the laboratory, the identified eggs are stripped of surrounding cells and prepared for fertilization.
An oocyte selection may be performed prior to fertilization to select eggs with optimal chances of
successful pregnancy. In the meantime, semen is prepared for fertilization by removing inactive
cells and seminal fluid in a process called sperm washing. 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.
FERTILIZATION AND INSEMINATION
Fertilization may be accomplished by insemination, or by intracytoplasmic sperm injection (ICSI).
In insemination sperms are placed together with the oocytes and incubated for 12-14 hours usually

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overnight. In intracytoplasmic sperm injection (ICSI), a single sperm is directly injected into each
mature egg.
Figure 3: Illustration of ICSI
ICSI is usually performed when there is a likelihood of reduced fertilization, e.g., poor semen
quality, history of failed fertilization in a prior IVF cycle. Overall, pregnancy and delivery rates
with ICSI are similar to the rates seen with traditional IVF. Genetic counseling is advisable before
ICSI if inherited abnormalities are identified that may be passed from father to son (ASRM, 2011).
EMBRYO CULTURE
Culture of embryos can either be performed in an artificial culture medium or in an autologous
endometrial coculture (on top of a layer of cells from the woman's own uterine lining). With
artificial culture medium, there can either be the same culture medium throughout the period, or a
sequential system can be used, in which the embryo is sequentially placed in different media. For
example, when culturing to the blastocyst stage, one medium may be used for culture to day 3, and
a second medium is used for culture thereafter (Evans et al., 2014). Single or sequential medium
are equally effective for the culture of human embryos to the blastocyst stage. Artificial embryo
culture media basically contain glucose, pyruvate, and energy-providing components, but the

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addition of amino acids, nucleotides, vitamins, and cholesterol improve the performance of
embryonic growth and development (Nargund, 2009). Methods to permit dynamic embryo culture
with fluid flow and embryo movement are also available (Heijnen et al., 2007). A new method in
development uses the uterus as an incubator and the naturally occurring intrauterine fluids as
culture medium by encapsulating the embryos in a permeable intrauterine vessel (Fauser et al.,
2010).
A review in 2013 of studies on individual culture media came to the result that the G3 medium
confers a significantly higher live birth rate and a lower risk of low birth weight than the Sydney
IVF medium.
Usage of low oxygen concentrations of 5% rather than about 20% in the atmosphere has been
shown to increase live birth rate to a relative probability of 1.24, without any evidence of increased
risk for multiple pregnancies, miscarriages or congenital abnormalities (Kovacs, 2004).
The duration of embryo culture can be varied, conferring different stages of embryogenesis at
embryo transfer. The main stages at which embryo transfer is performed are cleavage stage (day 2
to 4 after co-incubation) or the blastocyst stage (day 5 or 6 after co-incubation).
Embryo culture until the blastocyst stage confers a significant increase in live birth rate per embryo
transfer, but also confers a decreased number of embryos available for transfer and embryo
cryopreservation, so the cumulative clinical pregnancy rates are increased with cleavage stage
transfer. Transfer day 2 instead of day 3 after fertilization has no differences in live birth rate (La
Marca and Sunkara, 2014).

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EMBRYO SELECTION
Laboratories have developed grading methods to judge oocyte and embryo quality. In order to
optimize pregnancy rates, there is significant evidence that a morphological scoring system is the
best strategy for the selection of embryos (Rebmann et al., 2010). Since 2009 where the first time-
lapse microscopy system for IVF was approved for clinical use, morphokinetic scoring systems
has shown to improve to pregnancy rates further (Meseguer et al., 2012).
During embryo selection prior to embryo transfer, can be tested for chromosomal or specific
genetic defects by preimplantation genetic diagnosis (PGD).
EMBRYO TRANSFER
Embryo transfer takes place after eggs have been collected and fertilized in the laboratory. Embryo
transplant involves three process:
Uterine preparation
Transplant proper
Follow up
Uterine preparation: In the human, the uterine lining (endometrium) needs to be appropriately
prepared so that the embryo(s) can implant. In a natural cycle the embryo transfer takes place in
the luteal phase at a time where the lining is appropriately undeveloped in relation to the status of
the present Luteinizing Hormone. In a stimulated or a cycle where a "frozen" embryo is transferred,
the recipient woman could be given first estrogen preparations (about 2 weeks), then a combination
of oestrogen and progesterone so that the lining becomes receptive for the embryo. The time of
receptivity is the implantation window. A scientific review in 2013 came to the conclusion that it

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is not possible to identify one method of endometrium preparation in frozen embryo transfer as
being more effective than another (Groenewoud et al., 2013). New evidences suggest better
reception of the embryo if there is an imploratory reaction in the uterus prior to transfer (Nava et
al., 2010).
Embryo Transfer Procedure: The embryo transfer procedure starts by placing a speculum in the
vagina to visualize the cervix, which is cleansed with saline solution or culture media. A fine tube
(catheter) loaded with embryos is passed through the cervix, normally using ultrasound guidance.
The embryos are passed down the tube into the uterine cavity. After insertion of the catheter, the
contents are expelled and the embryos are deposited. The subject lie supine for few minutes after
the transfer.
Figure 4: Stages of development of fertilize egg.

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Figure 5: Illustration of embryo transfer procedure
Follow up: About two weeks after the embryo transfer, a pregnancy blood test is done. If it is
positive, a scan will be taken about two weeks later. If pregnancy does not occur, the results at
each stage of cycle will be evaluated such as response to the medications, the number and condition
of eggs retrieved, whether they fertilized, and how the embryos grew in culture.
The number to be transferred depends on the number available, the age of the woman and other
health and diagnostic factors. In countries such as Canada, the UK, Australia and New Zealand, a
maximum of two embryos are transferred except in unusual circumstances. In the UK and
according to HFEA regulations, a woman over 40 may have up to three embryos transferred,
whereas in the USA, younger women may have many embryos transferred based on individual
fertility diagnosis. 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.

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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.
SUCCESS RATES OF IVF
IVF success rates are the percentage of all IVF procedures which result in a favorable outcome.
Depending on the type of calculation used, this outcome may represent the number of confirmed
pregnancies, called the pregnancy rate, or the number of live births, called the live birth rate. Due
to advances in reproductive technology, IVF success rates are substantially higher today than they
were just a few years ago.
Live birth rate
The live birth rate is the percentage of all IVF cycles that lead to a live birth. This rate does not
include miscarriage or stillbirth and multiple-order births such as twins and triplets are counted as
one pregnancy. A 2012 summary compiled by the Society for Reproductive Medicine which
reports the average IVF success rates in the United States per age group using non-donor eggs
compiled the following data (Society for Reproductive Medicine, 2012).
Figure 4: Report of IVF success rate
Because not each IVF cycle that is started will lead to oocyte retrieval or embryo transfer, reports
of live birth rates need to specify the denominator, namely IVF cycles started, IVF retrievals, or

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embryo transfers. The Society for Assisted Reproductive Technology (SART) summarised 2008-
9 success rates for US clinics for fresh embryo cycles that did not involve donor eggs and gave
live birth rates by the age of the prospective mother, with a peak at 41.3% per cycle started and
47.3% per embryo transfer for patients under 35 years of age.
IVF attempts in multiple cycles result in increased cumulative live birth rates. Depending on the
demographic group, one study reported 45% to 53% for three attempts, and 51% to 71% to 80%
for six attempts.
Pregnancy rate
Pregnancy rate may be defined in various ways. In the United States, the pregnancy rate used by
the Society for Assisted Reproductive Technology and the Centers for Disease Control (and
appearing in the table in the Success Rates section above) are based on fetal heart motion observed
in ultrasound examinations.
The 2009 summary compiled by the Society for Reproductive Medicine included the following
data for the United States (Society for Reproductive Medicine, 2011).
Figure 5: Report of IVF success rate

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