ASSISTED REPRODUCTIVE TECHNOLOGY (ART)

ART encompasses all the procedures that involve manipulation of gametes and embryos outside
the body for the treatment of infertility.

DIFFERENT METHODS OF ART

IVF-ET: In Vitro Fertilization And Embryo Transfer
GIFT :Gamete Intra-Fallopian Transfer
ZIFT : Zygote Intra-Fallopian Transfe

POST : Peritoneal Oocyte And Sperm Transfer

SUZI : Subzones Insemination

ICSI : Intracytoplasmic Sperm Injection

METHODS OF SPERM RECOVERY

TESE : Testicular sperm extraction

MESE : Microsurgical epididymis sperm aspiration

PESE : Percutaneous epididymis sperm aspiration

PRINCIPAL STEPS OF AN ART CYCLE

 Down regulation using GnRH agonist.

 Controlled ovarian hyper stimulation (COH)
 Monitoring of follicular growth.
 Oocyte retrieval.
 Fertilization in vitro (IVF, ICSI, GIFT)
 Transfer of gametes or embryos.
 Luteal support with progesterone.

IN VITRO FERTIILISATION AND EMBRYO TRANSFER (IVS-ET)

The field of reproductive medicine has changed forever with the birth of LouiseBrown in 1978
by IVF-ET. Patrick Steptoe and Robert Edwards of England are remembered for their
revolutionary Work.The past decade has witnessed two more a dramatic change in the technique
protocol of IVF-ET One such changes was from natural cycle to superovulation Protocol and the
other one was replacement of laparoscopy by vaginal zoography for ovum retrieval.

Indications of IVF-ET

 Tubal disease
  Unexplained infertility
 Endometriosis
 Male factor infertility
 Cervical hostility
 Failed ovulation induction
 Ovarian failure (Donor oocyte IVF)
 Women with normal ovaries but no functional uterus (mulleins agenesis).
 Women with genetic risk (IVF and PGD).

Patient selection (ideal)

 Age <35 years

 Presence of ovarian reserve (D-3, serum FSH <10 IU/L)
 Husband-normal seminogram
 Couple must be screen negative for HIV and hepatitis.
 Normal uterine cavity as evaluated by hysteroscopy / sonhysterograpy

GNRH analogues for down regulation

 Currently most ART procedures involve the use of GNRH agonists

 GHRH agonist therapy used for down regulation of pituitary,gives higher pregnancy
rates.
 GNRH agonist therapy is continued either subcutaneously or intranasalduring
thegonadotropin treatment phase.
 GNRH antagonists are currently tried along with gonadotropin stimulation to prevent
premature LH surge or premature ovulation.Cetrorelix and Ganirelex are the available
drugs.

Different schedules for GNRH agonist are available:

 Long follicular down regulation- When therapy is started in the follicular phaseof
previous cycle.
 Long luteal down regulation (most commonly used) therapy is begun on D-21 of the
previous cycle. Gonadotropin stimulation is started following the menses.
 Short protocol - therapy is started in the follicular phase (D-1) along with gonadotropin
stimulation. This is also called “flare” protocol, as gonadotropin can work over the
stimulatoryeffect of GNRH agonist.

Controlled ovarian hyper stimulation (COH)

In the first case, Steptoe and Edwards (1978) achieved the success from collecting the oocyte
from a natural cycle,36 hours after the onset of LH surge. But subsequently, it has been found
that the success rate is much higher when more embryos are transferred which is only possible
by COH. The other advantages of induction of superovulation are improved quality ofthe oocyte,
timing of ovulation can be controlled,suited to the personnel involved and extended to all cases
of ovulatory dysfunction. The drug regimens used differ in each center.

Monitoring of follicular growth

The follicular growth response is monitored by cervical mucus study, zoographicmeasurement of

the follicles and serum estradiol estimation, commencing on the 8th day of treatment cycle When
three or more follicles are greater than 18 mm in diameter and serum E2 levels >250 pg /ml/per
follicle, 5,000-10,000 IU of HCG(250 qg of recombinant HCG) is given intramuscularly.
Oocyte is retrieved 36 hours after the hCG is given. hCG induces oocyte maturation. The
individual woman may be a high responder or a poor responder. Depending upon the response
management is done.

Oocyte Retrieval

Oocyte retrieval is done aseptically through vaginal route under ultrasound guidance. With the
development of vaginal transducers, vaginal needle aspiration is done about 36 hours after hCG
administration but before ovulation occurs. Intravenous analgesia and sedation is adequate in
most of the cases. The oocyte is readily recognizable as a single cell surrounded by a mass of
cumulous cells. After recovery, the oocytes are maintained in culture in vitro for 4-6 hours.

Fertilisation

The sperm used for insemination in vitro is prepared by the wash and swim-up or density
gradient centrifugation technique. Approximately 50,000 to 100,000 capacitated sperm are
placed into the culture media containing the oocyte within 4-6 hours of retrieval. The eggs may
demonstrate signs of fertilization when examined 16-18 hours after insemination (presence of
two pronuclei in the presence of a second polar body). Sperm density and motility are the two
most important criteria for successful IVE. The semen is collected just prior to ovum retrieval.

Embryo transfer

The fertilized ova at the 4-8 cell stage are placed into the uterine cavity close to the fundus about
3 days after fertilization through a fine flexible soft catheter
transcervically. Not more than three embryos are transferred per cycle
to minimize multiple pregnancies.

The process of transfer should be accurate, a traumatic and aseptic.

Trial transfer is beneficial. The number of embryos to be transferred
depends mainly on maternal age and the embryo quality.
Excess oocytes and embryos can be cryopreserved for future use. This will reduce the cost of
ovulation stimulation as well as the risk of ovarian hyper stimulation.Luteal phase support is
maintained with progesterone. It is started on the day after oocyte retrieval. HCG is given in
supplemental doses (1,500-2,500 IU). Micronized progesterone 200 mg twice a day oral or as
vaginal suppository or progesterone in oil injection 50 mg IM daily is continued for about 14
days. By this time diagnosis of pregnancy by estimation of b-hCG (quantitative value) is
possible.

Result: The overall delivery rate varies from 25-35 percent per oocyte retrieval.

There is increased risk of abortion (18%), multiple pregnancy (30%), ectopic (0.9%), low birth
weight baby and prematurity. The risk of congenital malformation of the baby remains similar to
general population.

GAMETE INTRAFALLOPIAN TRANSFER (GIFT)

GIFT was first described by Asch and colleagues in 1984. It is a more invasive
procedure than IVF but the result seems better than IVF. In this procedure, both the sperm and
the unfertilized oocytes are transferred into the fallopian tubes. Fertilization is then achieved in
vivo.

The prerequisite for GIFT procedure is to have normal uterine tubes. The
indications are the same as that of IVF except the tubal factor. Best result is obtained in
unexplained infertility and the result is poor in male factor abnormality.

The superovulation is done as IVF. Two collected oocytes along with approximately 200,000-
500,000 motile sperm for each fallopian tube are placed in a plastic tube container. It is then
passed through laparoscope and inserted 4 cm. into the distal end of the fallopian tube where the
combination is injected.

Result: The overall delivered pregnancy rate is as high as 27-30 per cent.

GAMETE INTRAFALLOPIAN TRANSFER (GIFT)

ZYGOTE INTRAFALLOPIAN TRANSFER (ZIFT)

Zygote intra-fallopian transfer was first described by Defray et al. (1986).

The placement of the zygote (following one day of in vivo fertilization) into the fallopian tube
can be done either through the abdominal osmium by laparoscope or through the uterine osmium
under ultrasonic guidance.

This technique is a suitable alternative of GIFT when defect lies in the male factor or in case of
failed GIFT. Results are similar to that of IVF.GIFT or ZIFT is avoided when tubal factors for
infertility are present.

MICROMANIPULATION

ICSI was first described by van Steirteghem and colleagues in Belgium (1992)

Indications:

 Severe oligospermia(<5 million sperm/ml)

Asthenopermia, Teratospermia
Presence of sperm antibodies
 Obstruction of efferent duct system (male)
 Congenital absence of vas (bilateral)
 Failure of fertilization in IVF
 Fertilization of cryopreserved oocytes (with hardened sonar pellucid)
 Unexplained infertility
  Sperm is recovered from the ejaculate otherwise sperm is retrieved by TESE
(testiculasperm extraction) or by MFSA (microsurgical epididymis sperm aspiration)
procedures.
 Technique

Oocyte is injected during ICSI 8-cell embryo for transfer

Blastocyst for transfer

One single spermatozoon or even a spermatid is injected directly into the

cytoplasm of an oocyte by micro puncture of the sonar pellucid. This procedure is carried out
under a high quality inverted operating microscope. Micropipette is used to hold the oocyte
while the spermatozoon is deposited inside the opals by an injecting pipette.

ICSI is found to be very effective compared to other micromanipulation methods like subzone
insemination (SUZI). ICSI is very effective to reduce the need of AID

Results : Fertilization rate is about 60-70 per cent. Pregnancy rate is 20-40 per cent per embryo
transfer.

EMBRYO OR OOCYTE DONATION

Ovum donation and IVF can help women with successful pregnancy. The
essential requirements for successful outcome are :(i)successful ovum donation and IVF . (ii)
Embryo endometrial synchronization and (iii) Exogenous hormonal support until luteal-
placement shift

Indication
 Women with premature ovarian failure.
 Women with removed ovaries
 Older women (poor oocyte quality)
 Failure to respond with superovulation regimen (poor ovarian reserve)
 Women with repeated failure of ART cycle.
 Genetic disease

The oocyte are collected from:

  Sister or a friend (age between 21 and 34 years)
 Those for IVF candidates, excess oocytes following retrieval andcryopreservation
(see above)
 One undergoing laparoscopic sterilization (with financial compensation)
 The oocyte donor like the semen donor must be screened for infection and genetic
diseases.

Successful implantation needs a perfect coordination of embryo and the endometrium. Estrogen
therapy (in the recipient) is started at the same time when the donor gets cycle stimulation.
Progesterone treatment in the recipient generally begins on the day the donor undergoes ovum
retrieval. Generally D3 embryos are transferred on the fourth day of progesterone therapy.

Luteal support: As the recipient has no corpus lutein, exogenous luteal support is needed.
Exogenous estrogen and progesterone treatment should therefore be continued until 10 weeks of
gestation. Oocytes and embryos can be cryopreserved (at-196 under liquid nitrogen) for
restoration of fertility in future. Survival of cryopreserved embryo is more than that of oocytes.

Results: Live birth rate is approximately 47 per cent.

GESTATIONAL SURROGACY

Women without a functional uterus can have her genetic offspring with the help of ART.
Embryos are transferred to the uterus of another woman who is willing to carry the pregnancy
on behalf of the infertile couple.

Cryopreservation of ovarian tissue

Restoration of reproductive function of a women undergoing chemotherapy is
possible these days with the help of cryobiology. Cryopreservation of ovarian tissue or auto
transplantation may allow natural pregnancy later on. With this method, ovulation using
exogenous gonadotropins can be achieved.

Oocyte cryopreservation by freezing is an alternative method. Verification, using

high concentration of cry protectant can solidify cells without ice formation Human
pregnancies and deliveries from verified mature oocytes have been recorded.
Health hazards of ART

 Most of the ART procedures are not associated with any increased risk of fetal congenital
malformations or birth defects. ICSI is often done due to male factors for infertility and
it eliminates the natural process of sperm selection. It is no yet certain whether ICSI is
associated with increased chromosomal abnormalities of the offspring.
 Peri-implantation genetic diagnosis (PGD) is done by blastomeric biopsy. Genetic
screening can avoid transferring embryos with aneuploidy and autosomal recessive
or autosomal dominant gene mutation.
 Increased number of pregnancy loss, multiple pregnancy and ectopic pregnancy have
been reserved.
 Perinatal mortality and morbidity are high.
 Ovarian hyper stimulation syndrome though rate but is a known health risk.
 Fertility drugs and cancer –no association have been found between ovulation induction
drugs and ovarian cancer.
 Psychological stress and anxiety of the couple are server. It is specially so when there is
failure in the treatment or with a pregnancy loss.