IN VITRO FERTILIZATION AND EMBRYO TRANSFER

IN HUMANS:TECHNIQUES, UTILITIES

AND ETHICAL ISSUES

PRESENTED BY-HASNAHANA CHETIA

Infertility is a common and serious problem in reproduction now-a-days. Animal culture and tissue techniques provide a solution to this problem through the techniques of IVF and Embryo Transfer (ET).

Invitro fertilization( IVF) refers to the union between egg cell and sperm outside body in a culture vessel involving their collection and fusion under appropriate conditions in vitro to give rise to zygotes which are cultured in vitro to obtain young embryos.

DEFINITION

The implantation of young embryos developed in vitro or obtained from the uterus of females into the womb of selected females is termed as Embryo Transplantation or Embryo Transfer.

Collection of oocytes Collection of sperms IVF of the oocytes Implantation of the resulting zygotes in the

uterus.

STEPS INVOLVED-

COLLECTION OF OOCYTES

Collected from females desirable of having baby.

Cannot be collected from females with non-functional ovaries.

Can be collected during a natural or induced menstrual cycle.

Time for this is determined by monitoring rise in the level of Luteinizing Hormone(LH) in urine or blood.

Ovulation may be stimulated by administration of Clomiphene or Human menopausal Gonadotropin (hMG).

Follicle development maybe arrested at the optimum stage by administering Human chorionic gonadotropin (hCG) so that ova are not released.

Recovery of oocytes can be done most conveniently by a laparoscopic instrument that allows the visualization of ovary through a monitor, aspiration (suction) of the follicular fluid containing the oocytes and the necessary surgical manipulation of ovary using sensors, laproscopic scissors and an aspirating apparatus inserted into the abdomen of the female via a suitable tube.

Fig. Collection of oocytes from ovary

COLLECTION OF SPERMS

Collected about 60-90 minutes prior to fertilization, liquefied and centrifuged.

Resuspended in culture medium, and incubated for 30-60 mins at 37⁰ C.

The most active sperms are located in the surface of the medium.

If sperm collection from the prospective father is not possible due to Oligospermia or Azoospermia, semen is collected from a suitable donor.

Fig. Procedure of collection, Centrifugation and Suspension of sperms in culture medium

FERTILIZATION OF THE OOCYTES BY SPERMS IN VITRO

Oocytes are identified by microscopic examination of the follicular fluid aspirated during laparoscopy, and are incubated for 10-15 hours depending upon the expected time of maturation.

Following mediums maybe used for serving the purpose-

1. Modification of Ham’s F10 medium 2. Earl’s solution 3. Modified Whitten’s medium 4. Whittingham’s T6 medium

All these media has the following in common- Utilizes a bicarbonate/CO2 buffer system to

keep PH in the range of 7.2-7.4. Osmolarity range of 275-290 osmol/kg.  Temperature setting of 37.0-37.5°C  Cultured under paraffin oil, which prevents

evaporation of the medium preserving a constant Osmolarity and minimizes fluctuations of pH and temperature.

Media must have 99% water,. Albumin or synthetic serum in concentrations

of  5 to 20% w/w or v/v as protein source. Carbohydrates- Pyruvate, lactate and high

conc. of glucose.

Amino acids- all 20 amino acids. Salt solutions- NaCl, KCl, KH₂PO₄ etc. Antibiotics- Penicillin, Streptomycin etc. Chelators like EDTA to prevent abnormal

glycosis.

Fertilization is started by adding 10,000-50,000 motile sperms to about 100 µl to 1 ml culture medium in which the oocytes is being incubated.

The oocytes is examined after 12-13 hrs for detection of –

1. Number of pronuclei and polar bodies 2. Granulation of the oocytes and 3. Shape of the oocytes

A normally fertilized oocyte (actually a

zygote now) contains two pronuclei and two polar bodies. Any zygote other than this and having abnormalities and granulation of any kind are rejected.

The first division in the zygote occurs about

24-30 hrs after insemination, but each subsequent division takes about 10-12 hrs. Therefore, if an oocyte fails to divide by 30 hrs after insemination, it should not be used for implantation.

Fig. In Vitro Fertilization

EMBRYO TRANSFER

The best stage of transfer of embryos to uterus is 2-4 celled stage.

Fig. Different stages of development of a blastocyte

The patient is orally administered with Valium before embryo transfer.

The whole process is negotiated through cervical canal.

TECHNIQUE

The patient is placed in lithotomy (knee chest) position.

A sterile bivalve speculum is inserted to visualize the cervix.

The cervical canal and uterine cavity are aligned.

The embryo is drawn into a Teflon catheter in tissue culture medium. Teflon is used due to its low adhesiveness.

STEPS INVOLVED-

The catheter is inserted into the uterine cavity just short of the fundus.

The embryo is gently inserted in culture medium and the catheter and cannula are gently withdrawn.

Catheter is examined under the microscope to ensure that the embryo has been expelled.

Fig. Steps involved in Embryo Transfer

The babies produced using these approaches are called test tube babies.

The first test tube baby was born on July 25, 1978 and was named Louise Joy Brown.

The Nobel Prize of Medicine for the year 2010 was awarded to Robert G. Edwards for this outstanding invention.

Fig. Louise Brown with her parents Fig. Robert G. Edwards with Louise

IVF success rates are the percentage of all IVF procedures which result in a favourable outcome, which implies Pregnancy rate (Number of confirmed pregnancies) or Live birth rate (Number of live births).

Due to advancement in reproductive technology, the IVF success rates are substantially better today than they were just a few years ago.

SUCCESS RATE

Maternal age Duration of infertility or sub-fertility FSH Number of oocytes Tobacco smoking High body mass index Alcohol/caffeine intake

FACTORS AFFECTING SUCCESS RATE

Pre-implantation genetic screening or diagnosis (PGS or PGD)-

Preimplantation genetic screening (PGS) or preimplantation genetic diagnosis (PGD) has been suggested to be able to be used in IVF to select an embryo that appears to have the greatest chances for successful pregnancy.

Embryo splitting- It can be used for twinning to increase the number of available embryos.

EXPANSIONS TO THE TECHNIQUE

Intra cytoplasmic sperm injection- It  is an invitro fertilization procedure in which a single sperm is injected directly into an egg.

Assisted zona hatching- Assisted zona hatching (AZH) is a procedure of assisted reproductive technology in which a small hole is made in the zona pellucida, using a micromanipulation, thereby facilitating for zona hatching to occur. Zona hatching is where the blastocyst gets rid of the surrounding zona pellucida to be able to implant in the uterus.

Laboratory mix-ups (misidentified gametes, transfer of wrong embryos) have occurred, leading to legal action against the IVF provider and complex paternity suits.

An example is the case of a woman in California who received the embryo of another couple and was notified of this mistake after the birth of her son.

This has led to many authorities and individual clinics implementing procedures to minimise the risk of such mix-ups. 

ETHICAL ISSUES

The Catholic Church opposes all kinds of in vitro fertilisation because, as with contraception, it separates the procreative purpose of the marriage act from its unitive purpose, i.e. to produce a new life.

Another concern is that people will screen in or out for particular traits.

A deaf British couple have petitioned to create a deaf baby using IVF. Medical ethicists are against this form of Pre-implantation genetic screening or diagnosis as “intentionally culling out blind or deaf embryos might prevent considerable future suffering, while a policy that allowed deaf or blind parents to select for such traits intentionally would be far more troublesome.”

Elements of Biotechnology by P.K. Gupta; pg.112

Biotechnology Expanding Horizons by B.D. Singh; pg.282

www.ivf-worldwide.com www.ivf.com www.advancedfertility.com Wikipedia

REFERENCES