Update on art

Update on Assisted
Reproductive Techniques
Dr Akiseku A.K
Department of Obstetrics and Gynaecology.
OOUTH, Sagamu

OUTLINE
• History
• Introduction
• Indication
• Preparation for ART
• Intrauterine insemination
• In vitro fertilization and ET
• Intracytoplasmic sperm injection
• Preimplantation Genetic Diagnosis
• Frozen embryo replacement cycle
• Third party ART

History of ART
• 1978- first successful
birth using In Vitro
Fertilization
• 1984- first successful
birth using Gamete Intra
Fallopian Transfer
• 1986-first successful
birth using Zygote Intra
Fallopian Transfer

Robert edwardsPatrick step toe
Nobel prize in2010

Introduction {ART}:
• All treatments or procedures that include the handling of human
oocytes and sperms or embryos for the purpose of establishing a
pregnancy.
• ART includes fertility treatments in which both eggs and sperm are
handled in the laboratory (i.e., IVF and related procedures).
• ART does not include treatments in which only sperm are handled
(i.e., intrauterine insemination) or procedures in which a woman
takes drugs only to stimulate egg production without the intention of
having eggs retrieved. (CDC, 2009)

• The simplest ART procedure, IVF has been around for over 20 years
and is perhaps the most commonly recognized ART of all procedures.
• According to the European Society for Human Reproduction and
Embryology, more than three million babies have been born using
ART worldwide in the last 30 years, enabling infertile women and
men; single women and men; and lesbian, gay, and transgender
couples to form genetically-related families.

Indications of ART
• Tubal factor infertility
• Endometriosis
• Male factor infertility
• Unexplained infertility
• Ovarian failure and diminished ovarian reserve
• Pelvic malignancy
• Mullerian anomaly
• Genetic risk

ASSISTED REPRODUCTIVE TECHNOLOGIES
(ART)
•Intrauterine insemination (IUI)
• In vitro fertilization and embryo transfer (IVF-ET)
• Gamete intra-fallopian transfer (GIFT)
• Zygote intra-fallopian transfer (ZIFT)
• Intracytoplasmic sperm injection (ICSI)
•intracytoplasmic morphologically selected sperm injection (IMSI)
•Frozen embryo replacement cycle (FERC)

• Preimplantation genetic diagnosis (PGD)
• Preimplantation genetic screening (PGS)

Preparation for Assisted Reproductive
Technique (ART)
Determination of ovarian reserve:
 Measurement of FSH and estradiol level on Day 2/3 of menstrual
cycle.
 Measuring the level of AMH(most sensitive marker)
Value<5.4pmol/l(0.8ng/ml) predict a low response to ovarian stimulation,
value of 25pmol/l(3.6ng/ml) predicts a high response
Performing a clomiphene Citrate challenge test(CCT)
 Antral follicle count.

Evaluation of uterus (HSG,Hysteroscopy,Sonohysterography). Uterine
cavity abnormalities such as leiomyomas, polyps or septum may need
correction before ART.
Hydrosalpinx reduce IVF success, its advised that salpingectomy
should be performed before IVF
Seminal fluid analysis should be done before ART
Infectious disease, chlamydial ,HIV,HBV,HCV.

Intrauterine insemination
• Intrauterine insemination (IUI) is where a prepared sample
• of sperm is inseminated into the uterine cavity at the appropriate
time of the patient’s menstrual cycle.
• Approximately two weeks later a pregnancy test is performed to see
if the cycle has been successful.
• Success rates increase from unstimulated IUI through to stimulation
with Clomid and FSH.
• The overall success rate, as with any subfertile couple, depend on
multiple factors, most importantly female age and with IUI the quality
of the sperm.

INDICATIONS
• Unexplained infertility
• Mild male Factor
• Ejaculatory problems
• Cervical problems
• Ovulatory disorders
• Mild endometriosis
• To optimize the use of donor sperm

ADVANTAGES
• IUI is relatively a simple technique that is cost-effective
• Can be offered by both secondary and tertiary fertility centres.
• It is not as invasive as IVF and allows fertilization to occur within the
fallopian tubes
• Is generally acceptable to most religious groups.

DISADVANTAGES
• The success rates are lower than those with IVF.
• If the cycle fails less information is obtained than with an IVF cycle.
• It also requires at least one healthy fallopian tube and reasonable
sperm parameters.

In vitro fertilization- Indications
• Involves surgical removal of the mature oocyte from the ovary and
fertilization by sperm in the laboratory.
• • Severe tubal disease – tubal blockages
• • Severe endometriosis
• • Moderate male factor
• • Unexplained infertility
• • Unsuccessful IUI

Stages of in vitro fertilization
• Pituitary downregulation
• Ovarian stimulation
• HCG trigger
• Oocyte retrieval
• Insemination/ ICSI
• Embryo culture
• Embryo transfer
• Luteal support
• Transfer of fresh embryo/cryopreserved
• First trimester preg monitoring

Protocols
• Over the last 20 years protocols have been refined and these are now
broken down into three main categories.
1 Long protocol – Agonist cycles
2 Short protocol – Antagonist cycles

The benefits of antagonists versus
agonists:
• No menopausal side effects
• No cyst formation from the initial gonadotrophin surge
• Shorter cycle duration
• Less gonadotrophin required per cycle – therefore lower drug costs

OOCYTE RETRIEVAL
• Originally, this was done laparoscopically but with the advent of real-
time ultrasound this allowed a less invasive oocyte retrieval via
transvaginal (TV) ultrasound scanning,
• This has allowed both monitoring of the ovary during stimulation and
the actual retrieval itself to be done transvaginally .
• The laparoscopic route is still occasionally used if the ovaries are
inaccessible transvaginally.
• This can occasionally occur in frozen pelvises or when the ovaries
have been moved out of the pelvis prior to pelvic irradiation.

• Done 34- 36 hours after ovulation trigger
• Sedo-analgesia
• TIVA (Total intravenous anaesthesia)
• GA (General anaesthesia)
• Dark room

SPERM COLLECTION
• Masturbation
• Coitus interruptus
• Coitus with non spermicidal condom
• Surgical
• Prostatic massage, electro ejaculation etc

Surgical sperm retrieval
Percutaneous Epididymal sperm aspiration (PESA)
Microepididymal sperm aspiration (MESA)
Testicular sperm aspiration (TESA)
Testicular sperm extraction (TESE)

Testicular Sperm Aspiration
(TESA)
Sperm
Retrieval

Fertilization
• Sperm preparation
swim up
density gradient
• Incubation in high protien media for 0.5-4hr
• Each oocyte incubated
with 50-100 thousand motile sperm
in 5% CO2 in air,
98%humidity,37°C for 12-18hr

Intracytoplasmic sperm
injection
• Intracytoplasmic sperm injection (ICSI,
pronounced "eeksee") is an in vitro fertilization
procedure in which a single sperm is injected
directly into an egg.
• This procedure is used to overcome male
infertility problems, although it may also be
used where eggs cannot easily be penetrated by
sperm, and occasionally as a method of in vitro
fertilization, especially that associated with
sperm donation.

Procedure
• Single sperm is immobilized
• Drawn in to pipette
• Oocyte is stabilized
• Polar body is 6/12 o’ clock position
• Oocyte is entered 3 o’clock
• Pipette pierce zona and oolemma

Indication of ICSI
Male factor infertility- azoospermia, severe oligospermia (< 5million
sperm/ml), Asthenospermia (<5% progressive motility),
teratospermia (<4% normal forms by strict criteria).
Treatment involving pre-implantation genetic diagnosis as
conventional insemination may result in extra sperm attached to the
zona, which can contaminate the sample and affect the diagnosis.
In patients at risk of poor fertilization or fertilization failure.
Ejaculatory dysfunction such as retrograde ejaculation.

Immunogical factors with present of antisperm antibodies in seminal
plasma following vasectomy reversal or genital tract infection.
Testicular cancer patients in remission with semen cryopreserve prior
to chemotherapy or radiotherapy.

IMSI
• Latest advance in micromanipulative IVF
• Sperms are examined under high magnification microscopy x 6300
• Most “normal” sperms are identified for microinjection
• Indicated in severe male factor infertility
• Recurrent implantation failure
• Recurrent abortion

Abn sperms in high magnification

Advantages of IMSI
• Increases pregnancy rates by 50% over ICSI
• Reduces miscarriage rates by 75% over ICSI
• Shown to identify viable sperms in men who were considered
azospermic under normal magnification
• Treatment of choice in surgically obtained sperms

EMBRYO TRANSFER
• Eggs are fertilized either by routine insemination with a concentration
of approximately 100,000 normally motile sperm per ml or by ICSI.
• They are incubated in a commercially prepared culture medium
under strict laboratory conditions. Not only is the temperature
carefully controlled within the incubators but also the gas content
and pH.
• Most embryos are transferred at day 2 post egg collection.
• More embryos are now being transferred on day 5, at the blastocyst
stage

• At present the guidelines from the Humam Fertilization and
Embryology Authority (HFEA) state that only 2 embryos should be
transferred in people under the age of 40, unless exceptional
circumstances are present, but over the age of 40 then three
embryos can be transferred.
• In the USA, it would not be unusual for between 3 up to 5 embryos to
be transferred, depending on the age of the patient.
• In some Scandinavian countries, if the patient is 35 years old or
younger, then they are moving towards elective single embryo
transfer to reduce the incidence of twins or triplets and mobidity
associated with it.

• Although this would have a slight effect on success rates, the other
normal embryos are frozen and hence if a cycle is unsuccessful, then
the patient can undergo repeated single embryo transfers from
frozen embryo replacement cycles.

The benefits of a day 2 transfer are
I. A single stage Culture medium can be used
II. Most normal embryos survive to this stage.
III. After two or three embryos have been replaced, there may be
surplus embryos of a satisfactory quality that are suitable for
cryopreservation.

Downside of a day 2 transfer
I. In a normal menstrual cycle, the day 2 embryo is still in the
fallopian tube and not in the uterine cavity.
II. The grading system utilized by the embryologist is not totally
accurate and therefore it can sometimes be difficult to judge the
best two embryos out of potentially 6 or 7 to transfer.

Benefit for a day 5/blastocyst
I. The transfer is made when the embryo would physiologically be in
the uterine cavity and this may have some benefits regarding
certain growth factors which can improve embryo development.
II. Blastocyst transfer also allows better selection of the embryos as
the majority of abnormal embryos perish between day 2 and day 5
III. Allows for preimplantation genetic diagnosis

Downside of blastocyst transfer
I. Is that it requires a 2 stage culture medium as the blastocyst
metabolic requirements change after day 2 and that there are
generally no embryos left over that would be suitable for freezing.
II. The other potential downside is in some patients all of the embryos
may perish before day 5 and hence the patient may have nothing
to transfer at all.
III. For this reason majority of centres will only do a blastocyst
transfer if the patient has five or more good quality embryos.

Preimplantation Genetic Diagnosis
• It offers couples who carry serious genetic disordes the opportunity to
have healthy child
• Aneuploidy
• Structural abnomalites (translocations,invertions)
• Inherited gene disorder(single gene defect)
Cystic fibrosis
Thalassemia
Haemophelia
Duchne muscular dystrophy

• It allows many genetically heritable diseases to be identified using a
variety of molecular biologic techniques.
• These techniques include but are not limited to polymerase chain
reaction (PCR) and fluorescent in situ hybridization (FISH).
• Recent advances in embryo manipulation have made possible the
removal of 1 or 2 cells, or blastomeres, from a developing 8-cell
human embryo without harm to the embryo.

• Biopsy of the first and/or second polar bodies can also be done for
several single-gene defects.
• In patients at risk of passing along a heritable genetic disease, PGD
has made possible the identification of normal embryos (those with
no risk of passing the heritable disease). These normal embryos are
then transferred back to the patient.

PREIMPLANTATION GENETIC DIAGNOSIS
(PGD)

• Preimplantation genetic diagnosis (PGD) refers specifically to when
one or both genetic parents has a known genetic abnormality and
testing is performed on an embryo to determine if it also carries a
genetic abnormality.
• In contrast, preimplantation genetic screening (PGS) refers to
techniques where embryos from presumed chromosomally normal
genetic parents are screened for aneuploidy.

• At present, no specific list of indications for preimplantation genetic
screening (PGS) is available.
Primary candidates for PGS can include the following:
• Women of advanced maternal age
• Couples with history of recurrent pregnancy loss
• Couples with repeated IVF failure
• Male partner with severe male factor infertility

Frozen embryo replacement cycle
• Freezing surplus morphologically normal embryos allows the use of
those embryos which otherwise may have been wasted.
• It therefore gives double benefit from one fresh cycle and
significantly adds the cumulative conception rate per cycle when
surplus embryos are frozen.
• Normally embryos are frozen on day 2 after the selected ones have
been replaced fresh, but can be frozen anytime from day 1 through to
day 5 if excess blastocysts are obtained.

• The use of day 1 freezing is normally confined to elective freezing of
all embryos when there is a high risk of ovarian hyperstimulation
syndrome occurring.
• At day 2 any morphologically normal embryos of suitable quality are
selected and through specific cryopreservation protocols are frozen
and stored in liquid nitrogen in specially monitored tanks.
• The success rates of day 1 and day 2 embryos are cited as 20.4% live
birth rate per transfer.

Third party ART
Production of an offspring through the gametes /uterus of an
individual who will not be involved in parenting
sperm donation
Oocyte donation
Embryo donation
Gestational surrogate
THESE IS ASSOCIATED WITH SOCIOCULTURAL/RELIGIOUS
IMPLICATIONS

Oocyte donation: indication
• Ovarian failure – either premature or physiological.
• Patients with very poor ovarian function where previous IVF has
repeatedly failed.
• Patients over the age of 45 and with severe male factor disease
necessitating ICSI.
• Patients with hereditary genetic disease where using the patients
own gametes is not advisable.

problems
• The main problem with oocyte donation is obtaining eggs.
• In the UK it is illegal at present to pay donors for their eggs and
they are only allowed to be compensated minimally for their time and
inconvenience.
• Since 1 April 2005 anonymity for the donors has also been repealed
and any resultant offspring can trace their genetic mother from the
age of 18.

• Not surprisingly, there is therefore a great paucity of suitable egg
donors in the UK
• Most programmes at present rely on altruistic donors bought in by
the recipients themselves. These are generally either family members
or friends.
• Elsewhere in the world, particularly in the United States, donors are
paid and therefore there tends not to be a lack of donors, but cycle
costs are considerably higher.

Donor sperm
• Donor sperm is obtained by masturbation from healthy screened
donors.
• All donor sperm in the UK has to be stored for 6 months, and then the
donor screened again.
• Sperm can then only be released for use after both sets of screening
have been found to be negative.

INDICATIONS
• Azoospermia
• Carriers of Severe Genetic Disease
• Lesbian/single women

Surrogacy
• Generally surrogates are women who have already had children
themselves and are recruited either by the patients or through an
organization such as COTS (Childlessness Overcome Through
Surrogacy).
• Surrogacy is legal in the UK and the surrogate can be compensated
for time lost away from employment during the pregnancy.
• However, the child’s legal mother is the woman who delivers the
child and therefore the patient and the husband have to undergo
formal adoption procedures to become the legal parents of their
genetic offspring which the surrogate has delivered.

Surrogacy
• A pregnancy may be carried by the egg donor (traditional surrogate) or by
another woman who has no genetic relationship to the baby (gestational
carrier).
• If the embryo is to be carried by a surrogate, pregnancy may be achieved
through insemination alone or through ART. The surrogate will be biologically
related to the child.
• If the embryo is to be carried by a gestational carrier, the eggs are removed
from the infertile woman, fertilized with her partner’s sperm, and transferred
into the gestational carrier’s uterus. The gestational carrier will not be
genetically related to the child.
• All parties benefit from psychological and legal counseling before pursuing
surrogacy or a gestational carrier.

Gestational surrogacy
Indication
Absence of uterus
Irreparable uterus
Congenital
Asherman Syndrome
life threatening medical disorder
Person
related/nonrelated
parous
healthy

Guidelines for the Number of Embryos to
Transfer Following In Vitro Fertilization
1.In women under the age of 35 years with excellent prognoses, the
transfer of a single embryo should be considered. Women with
excellent prognoses include those undergoing their first or second IVF-
ET cycle or one immediately following a successful IVF-ET cycle, with at
least two high-quality embryos available for transfer.

2 . In women aged 35 to 37 years, no more than three embryos should
be transferred in a fresh IVF-ET cycle. In those with high-quality
embryos and favourable prognoses, consideration should be given to
the transfer of one or two embryos in the first or second cycle.

3. In women aged 38 to 39 years, no more than three embryos should
be transferred in a fresh IVF-ET cycle. In those with high-quality
embryos and favourable prognoses, consideration should be given to
the transfer of two embryos in the first or second cycle.
4. In women over the age of 39 years, no more than four embryos
should be transferred in a fresh IVF-ET cycle. In those older women
with high-quality embryo in excess of the number to be transferred,
consideration should be given to the transfer of three embryos in the
first IVF-ET cycle.

5. In donor–recipient cycles, the age of the oocyte/embryo donor
should be used when determining the number of embryos to transfer.
6 . Couples should be adequately counselled regarding the obstetrical,
perinatal, and neonatal risks of multifetal gestation to facilitate
informed decision making regarding the number of embryos to
transfer. Emphasis on healthy singleton live birth as the measure of
success in IVF-ET may be beneficial in promoting a reduction in the
number of embryos transferred.

References
• Dewhurst’s Textbook of Obstetrics amd Gynaecology, Eight Edition.
Edited by D. Keith Edmonds. 2012: 580-594.
• Criteria for number of embryo to transfer: a committee opinion. The
practice committee of the American society of reproductive medicine
and society for assisted reproductive technologies . Fertil Steril
2013;99:44-46.
• Guidelines for the Number of Embryos to Transfer Following In Vitro
Fertilization. J Obstet Gynaecol Can 2006;28 (9)799–813.
• American Society for Reproductive Medicine. ASSISTED
REPRODUCTIVE TECHNOLOGY A Guide for Patients Revised 2015

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