Techniques Employed In Assisted Reproductive Technology

The important techniques employed in assisted reproductive technology are listed below:

i. Intrauterine insemination (IUI).

ii. In vitro fertilization and embryo transfer (IVF and ET).

iii. Gamete intra-fallopian transfer (GIFT).

iv. Zygote intra-fallopian transfer (ZIPT).

v. Intra-vaginal culture (IVC).

vi. Cytoplasmic transfer (CT).

vii. Micromanipulation (Intra-cytoplasmic sperm injection (ICSI), sub-zonal insertion (SUZI).

viii. Cryopreservation.

ix. Assisted hatching (AH).

Among these techniques, the most commonly used procedure is in vitro fertilization and embryo transfer. Important features of different types of ART are briefly described.

Intrauterine Insemination (IUI):

The infertile women (due to endometriosis, idiopathic infertility) without blockage or damage to fallopian tubes can be effectively treated by intrauterine insemination. The women with adequate ovulation and below the age of 40 years are considered for IUI.

The women are usually super-ovulated by administering gonadotrophins. This results in multiple egg development. The IUI is timed to coincide with ovulation. The semen is washed and the highly motile sperms are separated. By using a thin and soft catheter, the sperms are placed either in the cervix or in uterine cavity. The women subjects are advised to remain lying down for about 15-30 minutes following IUI.

Insemination should be carefully timed for good success. If it is done, a little before the expected time of ovulation, the chances for fertilization are much higher. IUI is usually successful in the first 3-4 attempts. In any case, this approach is not recommended for more than a maximum of 6 ovulation cycles. The success rates of IUI vary considerably and are in the range of 15-30%.

In Vitro Fertilization and Embryo Transfer (IVF and ET):

In vitro fertilization broadly deals with the removal of eggs from a women, fertilizing them in the laboratory, and then transferring the fertilized eggs (zygotes) into the uterus a few days later.

Indications for IVF:

Infertility due to the following causes may be considered for IVF.

i. Failed ovulation induction

ii. Tubal diseases

iii. Cervical hostility

iv. Endometriosis

v. Idiopathic infertility (in men and women).

Ideal Subjects for IVF:

Although it is not always possible to have a choice in the selection of subjects, the following criteria are preferred.

i. Woman below 35 years.

ii. Presence of at least one functional ovary.

iii. Husband with normal motile sperm count (i.e. normal seminogram).

iv. The couple must be negative for HIV and hepatitis.

Methodology of IVF:

The in vitro fertilization broadly involves the following steps.

1. Induction of superovulation.

2. Monitoring of ovarian response.

3. Oocyte retrieval.

4. Fertilization in vitro.

5. Embryo transfer.

Induction of Superovulation:

It is well known that the success rate IVF is much higher when more embryos (3-5) are transferred. This is possible only with controlled ovarian hyper-stimulation (COH). The other advantages of COH include improvement in the quality of oocyte, control of ovulation timing, besides overcoming the ovulatory dysfunction. The following drug regimens are in use to induce superovulation.

i. Clomiphene citrate (CC).

ii. CC + human menopausal gonadotrophin (hMG).

iii. CC + follicle stimulating hormone (FSH).

iv. Human menopausal gonadotrophin.

v. Follicle stimulating hormone.

vi. Gonadotrophin releasing hormone agonists (GnRHa) + hMG (or FSH).

It is now common to use GnRH agonists to induce ovulation. These compounds act through a process called down regulation of the physiologic hypothalamic- pituitary-ovarian feedback mechanism to effectively suppress spontaneous ovulation.

Monitoring of Ovarian Response:

The follicular growth or ovarian response can be monitored by increase in serum estradiol level, increase in follicular diameter and thickening of endometrial bed.

Oocyte Retrieval:

The most common method for oocyte retrieval is carried out through vaginal route under ultrasound guidance. This method is simple and less invasive, and can be performed with analgesics only. It is easy to recognize the oocyte as a single cell surrounded by a mass of cumulus cells. The recovered oocytes are maintained in vitro culture for 4-6 hours.

Fertilization in Vitro:

The semen specimens are collected (just prior to oocyte retrival) via masturbation, processed, and incubated in protein-supplemented media for 3-4 hours prior to fertilization. The incubation results in sperm capacitation.

The retrieved oocytes are also cultured in protein-supplemented media for about 6-8 hours. For the purpose of IVF, 50,000-1, 00,000 capacitated sperms are placed in culture with a single oocyte. The signs of fertilization may be demonstrated 16-20 hours later by the presence of two pronuclei within the developing embryo. There is no need to change the regime for a single failure of IVF. Many a times, success occurs in the subsequent cycles. The two most important criteria for the success of IVF are sperm density and motility.

Embryo Transfer:

Embryo at a stage between pronuclei and blastocyst stage are transferred. Conventionally, 4- 8 cell stage embryos are transferred between 48-60 hours following insemination. The transfer procedure is carried out by use of a catheter.

Not more than three embryos are transferred (per cycle) to minimize multiple pregnancies. However, in the women above the age of 40 years, higher number of embryo may be transferred. (Note: Excess oocytes and embryos are cryopreserved for further use. This will reduce the cost, besides the risk of ovarian hyper stimulation).

Luteal phase support is given by administration of progesterone for about two weeks. By this time, the diagnosis of pregnancy can be assessed by estimating human chorionic gonadotrophin (hCG).

Success Rates of IVF:

Success of IVF varies from programme to programme and within the same programme, the success rate is dependent on the correct diagnosis of the patient, and age. The overall pregnancy rate in IVF is in the range of 25-35% per oocyte retrival. The take home baby rate is about 15-20% per procedure.

The success rate of IVF is rather low due to the following reasons:

i. Increased risk of abortion

ii. Multiple pregnancy

iii. Ectopic pregnancy

iv. Low birth weight baby

v. Premature delivery.

The World’s Picture of Test Tube Babies:

By employing in vitro fertilization and embryo transfer, the world’s first test tube baby (Louise Brown) was born in UK on 28^th July 1978. The world’s second test tube baby (Kanupriya alias Durga) was born in Kolkata on 3^rd October 1978. A team led by Subhash Mukherjee carried IVF and ET in India. Scientists responsible for the “birth of test tube babies were severely criticized then.

In fact, IVF turned out to be one of the major achievements of medical sciences in the last century. It has become a novel way of treating infertility. Today, there are more than a million test tube babies born all over the world. In 2003, the world celebrated the silver jubilee of IVF with much fanfare.

Gamete Intra-Fallopian Transfer (GIFT):

Gamete intra-fallopian transfer involves the transfer of both sperm and unfertilized oocyte into the fallopian tube. This allows the fertilization to naturally occur in vivo. The prerequisite for GIFT procedure is that the woman should have at least one normal fallopian tube.

The induction of ovulation and the monitoring procedures for GIFT are almost the same as described for IVF. A couple of hours prior to oocyte retrieval, semen specimens are collected. Two oocytes along with 2-5 lakhs motile sperms for each fallopian tube are placed in a plastic tube container. It is then inserted (by laparoscopy) 4 cm into the distal end of the fallopian tube, and the oocyte sperm combination is injected.

The overall pregnancy rate is as high as 30- 40%. The take home baby rate is about 25%. This is much higher when compared to IVF. But the major limitation is the requirement of laparoscopy (a major surgical procedure) to transfer oocytes and sperms into the fallopian tubes.

Zygote Intra-Fallopian Transfer (ZIFT):

ZIFT is suitable when the infertility lies in men, or in case of failure of GIFT.

The wife’s oocytes are exposed to her husband’s sperms in the laboratory. The fertilized eggs (zygotes) within 24 hours are transferred to the fallopian tube by using laparoscopy.

ZIPT has an advantage over GIFT with male factor infertility. Further, it can be known whether the wife’s oocytes have been fertilized by her husbands’ sperms.

Intra Vaginal Culture (IVC):

The body’s own environment is appropriately utilized in intra-vaginal culture. The retrieved oocytes and sperms are placed in a culture medium inside a sealed container. This is inserted into the vagina. The container is held by a vaginal diaphragm. Thus, the oocytes and sperms are maintained at the normal body temperature (in contrast to any incubator in the laboratory). Two to 3 three days later, the container is opened, and the fertilized and dividing zygotes are transferred into the uterus. This procedure appears simple, but the success rate is very low. Only a few centers practice this.

Cytoplasmic Transfer (CT):

Cytoplasm includes many things, the most important being mitochondria which provide energy to the cell. It is possible that deficiency in the mitochondria may leave the oocyte without the necessary power for cell division, after fertilization. This may result in abnormal cell division and poor development of embryo.

It is therefore logical to think of the transfer of cytoplasm from a donor (with active mitochondria) into the oocyte of a woman. The advantage with cytoplasmic transfer is that the mother’s own genetic material is passed on to the offspring.

Two methods of cytoplasmic transfer have been developed:

1. Transfer of a small amount of cytoplasm by a tiny needle from a donor to a recipient oocyte.

2. Transfer of a large amount of cytoplasm which is fused with the recipient’s cytoplasm by applying electricity.

The procedure of cytoplasmic transfer is tedious and technically difficult, besides the cost factor. At least two viable pregnancies have been so far reported in literature by this approach.

Micromanipulation:

Micromanipulation involves in vitro micro-surgically assisted fertilization procedures. This is required when the sperms are unable to penetrate the zona pellucida of oocyte and fertilize. Micromanipulations are usually done in severe cases of male factor infertility.

A diagrammatic representation of micro­manipulation is depicted in Fig 18.3.

Intra-Cytoplasmic Sperm Injection (ICSI):

Intra-cytoplasmic sperm injection is a new and novel infertility treatment utilizing the micromanipulation technology. Many of the previous treatment processes for male infertility have been abandoned in favour of ICSI. The male factor infertility could be due to low sperm counts, poor sperm motility, and poor quality of sperm to penetrate oocyte.

By partial zona dissection (PZD), the zona pellucida is opened using either chemical dissolution or a sharp instrument. A single spermatozoon can be directly injected into the cytoplasm of the oocyte through the micro-puncture of zona pellucida. A micropipette is used to hold the oocyte while the spermatozoon is deposited inside the ooplasm of the oocyte. Besides using normal sperms, round-headed sperms, sperms collected directly from the epididymis and previously cryopreserved sperms can be used in ICSI.

Among the micromanipulation techniques ICSI is the most successful one with a fertilization rate of about 65%. Attempts are on to improve this further. In fact, ICSI has revolutionized assistant reproductive technology by utilizing the sperms of husbands who were once considered to be unsuitable for fertilization process.

Sub-zonal Insertion (SUZI):

In sub-zonal insertion, the zona pellucida is punctured and sperms (1-30 in number) are injected into an area between the zona and the egg. It is expected that one of the sperms will fertilize the egg. The major limitation of SUZI is polyspermy since it is not possible to control the number of sperms that enter the egg.

Round Spermid nucleus Injection (ROSNI):

There are a few men who cannot manufacture sperms, and therefore they have a zero sperm count. For these men, it is possible to take out the round spermatids (immature cells) directly from the testicle, isolate the nucleus (containing the genetic material) and inject it into the partner’s eggs. ROSNI is a recent exciting breakthrough to solve the problem of male infertility through micromanipulation.

Cryopreservation:

Preservation in a frozen state is regarded as cryopreservation. Cryopreservation is very useful in assisted reproductive technology.

i. Semen can be cryopreserved. This may be from the donors, cancer patients (before the commencement of treatment).

ii. Fertilized eggs after IVP or ICSI can be preserved.

iii. Embryos can also be preserved for transfer at a later stage.

Human embryos have been successfully preserved in the presence of cryoprotectants (1, 2-propanediol/dimethyl sulfoxide/glycerol) and stored at -196°C under liquid nitrogen. At appropriate time, the embryos are thawed, cryoprotectants removed and then transferred. Many test tube babies in fact have been born as a result of application of freezing technology.

Assisted Hatching (AH):

Improper implantation of the embryo in the uterus is one of the limiting factors in the success of ART in humans. Assisted hatching is a novel approach for the proper implantation of the embryo in the endrometrium.

The embryos in the uterus possess an outer coating namely zona pellucida (the shell). These embryos must be hatched to remove the shell, a step necessary for implantation. In certain women, particularly above 40 years age, natural hatching does not occur, and requires outside assistance.

Assisted hatching is carried out by using a Laser to make a small hole in the shell of the embryo. These embryos when transferred into the uterus hatch and get implanted.

During the course of AH for 3-4 days, the women are kept on steroids (to suppress mother’s immunity) and antibiotics (to counter infections). Better results are reported with this approach.

Pre-implantation Genetic Diagnosis (PGD):

The genetic defects in ovum before fertilization or in the embryo before implantation can be identified by a new medical tool namely pre-implantation genetic diagnosis. It is estimated that about 60% of the ART driven pregnancies are lost due to chromosomal abnormalities. This can be minimized or prevented by using PGD.

A direct determination of chromosomal abnormalities prior to implantation ensures a successful pregnancy and ultimate delivery of a healthy baby. One group of workers has reported an increase in the pregnancy rate from 15 to 30% by employing pre-implantation genetic diagnosis.

DNA Amplification and Analysis:

The latest in PGD is the direct DNA analysis. This can be carried out by removing a single cell from 6-8-cell embryo. The DNA is removed and amplified by employing polymerase chain reaction.

Direct DNA analysis is useful for the diagnosis of several genetic diseases e.g. cystic fibrosis, sickle-cell anemia, hemophilia, Duchene’s muscular dystrophy, Tay-Sachs disease.

Ethical Advantages of PGD:

PGD is highly advantageous from the ethical point of view, since the embryos with genetic disorders can be discarded in the very stages without the formation of offspring’s with undesirable characteristics.

The Negative Aspects of Art:

There are certain limitations/disadvantages associated with assisted reproductive technology in humans. Some highlights are given. It must however, be noted that the advantages of ART outweigh the disadvantages.

Ovarian Hyper-stimulation Syndrome (OHSS):

Due to administration of hormones and drugs, ovarian hyper-stimulation is frequently associated with complications, sometimes even life- threatening. OHSS is more severe in women who conceived in the same cycle, and received hCG as luteal support (following embryo transfer).

Risks Associated with Pregnancy:

ART is associated with multiple pregnancy, increased risk for anemia, gestational diabetes and premature labour. Low birth weight and prematurity are closed linked with mortality and morbidity.

Premature Menopause:

Controlled ovarian hyper-stimulation (COH) causes multiple follicular utility. There is a risk of premature menopause as COH may reduce the ovarian follicles, besides faster aging. Sometimes, a single COH may use ovarian follicles, which in the normal course are equivalent to two years of ovulation during the natural menstrual cycle.

Ovarian Cancer:

The use of fertility drugs and injuries to epithelium increase the risk of ovarian cancer at least by three times when compared to normal women.