IVF Process Step by Step: Stimulation, ICSI and Transfer

The IVF process takes roughly four to five weeks from first injection to pregnancy test. About two weeks of injections stimulate the ovaries, the eggs are collected through the vagina under anaesthesia and fertilised in the laboratory (by standard IVF or by ICSI, injecting one sperm into each egg), embryos grow to day three or five, and an embryo is transferred into the uterus. The pregnancy test follows two weeks later.

Dr. Parul Prakash, Head - Reproductive Medicine (IVF), Artemis Hospitals, Gurugram, India, walked through each of these steps with procedure video and embryo images. This article follows her sequence, then sets out when ICSI is needed and how the injection itself is done. For the causes that lead a couple to IVF, see causes of female and male infertility and the workup; for the overall decision between IVF, ICSI and donor eggs, see the complete guide to IVF, ICSI and egg donation.

What IVF changes about natural conception

In a natural cycle, the ovary releases one egg at ovulation. It is picked up by the fallopian tube and meets sperm there after intercourse. Fertilisation, the first cell divisions and the journey to the uterus all happen inside the body, unobserved.

IVF moves three of those events into the laboratory. Instead of one egg, the ovaries are stimulated to mature as many eggs as are available. Instead of meeting in the tube, egg and sperm meet in a dish. Instead of travelling down the tube, the embryo is placed directly in the uterus. Each change removes an obstacle: a blocked tube no longer matters, a low sperm count is concentrated, and fertilisation that would have failed unseen can be watched and, with ICSI, assisted.

The IVF process step by step

1. Starting the cycle

Dr. Prakash asks the woman to come in on the second or third day of her menstrual cycle. Treatment begins at that point, timed to the start of the natural cycle, when the ovaries are hormonally quiet and a new group of follicles is available to be recruited.

2. Ovarian stimulation

The woman then receives about two weeks of injections to stimulate the ovaries. These are gonadotrophins, the same hormones the pituitary gland uses to drive the ovary, given at higher doses so that many follicles grow at once rather than the single follicle of a natural cycle. Progress is tracked by ultrasound scans and blood tests.

Dr. Prakash's aim is to stimulate all the eggs visible in the ovaries and grow them to about 18 mm. On ultrasound, the small follicles seen at the start of the cycle enlarge into mature ones. When they reach that size, a final trigger injection matures the eggs, and the collection is scheduled for around 34 to 36 hours later.

3. Egg retrieval

The eggs are collected under anaesthesia through a vaginal ultrasound probe. Dr. Prakash described the technique: a needle is attached to the probe, and under ultrasound guidance it punctures each follicle in turn, drawing out the follicular fluid together with the egg it contains. The procedure needs no abdominal incision. The fluid is passed to the embryologist, who identifies and isolates the eggs.

4. Fertilisation in the laboratory

In standard IVF, the eggs are exposed to sperm in the laboratory. The sperm are processed first: the semen is washed and concentrated so that only the most motile sperm are used. This step is what the term in vitro fertilisation literally describes, fertilisation outside the body, and it produces the embryo in the laboratory rather than in the tube.

Where the sperm are too few or too poor to fertilise on their own, or fertilisation has failed before, ICSI replaces this step (see below).

5. Embryo development and grading

Dr. Prakash showed the stages through which an embryo is assessed in the laboratory:

• Day one: the fertilised egg. Fertilisation is confirmed by seeing two pronuclei (2PN), one from the egg and one from the sperm.
• Day two: four cells.
• Day three: eight cells.
• Day five: the blastocyst. By now the embryo has formed a fluid-filled cavity, an inner cell mass that will become the fetus, and an outer layer that will form the placenta. Dr. Prakash showed a day-five blastocyst as her example of a best-quality embryo.

Growing embryos to day five also shows which ones are developing well. Not every embryo that looks healthy on day three reaches the blastocyst stage.

6. Embryo transfer at day three or day five

Depending on embryo quality, Dr. Prakash transfers embryos at day three or day five. The transfer itself is brief: the embryo is loaded into a thin, soft catheter, passed through the cervix and placed inside the uterine cavity. Implantation, the embryo attaching to the lining of the uterus, then has to happen on its own, as in a natural pregnancy.

7. The pregnancy test

A pregnancy test is done two weeks after the transfer. It measures human chorionic gonadotrophin (hCG), the hormone produced once an embryo has implanted. A positive result means the embryo has attached to the uterine lining.

ICSI: when IVF needs help at fertilisation

Intracytoplasmic sperm injection (ICSI) is the deposition of a single sperm inside the cytoplasm of the egg. It replaces the natural process in which sperm must bind to and penetrate the egg on their own. Everything before it (stimulation, retrieval) and after it (embryo culture, transfer) is the same as in IVF.

When ICSI is needed

Dr. Prakash listed the situations in which she uses ICSI:

• Azoospermia, where sperm are completely absent from the ejaculate and have to be retrieved surgically.
• Low sperm count, poor motility or poor morphology, where too few functional sperm are available for standard IVF.
• Surgically retrieved sperm, from a testicular biopsy or aspiration, which are too few and too immature for standard IVF.
• Pre-implantation genetic testing, where embryos are tested for a genetic condition before transfer. ICSI is used so that no stray sperm attached to the outside of the egg contaminates the genetic test.
• Repeated fertilisation failure in previous IVF cycles.

Surgical sperm retrieval: TESA vs PESA

When a man has no sperm in the ejaculate, sperm can often still be found in the testis or the epididymis. Dr. Prakash described two needle techniques.

TESA (testicular sperm aspiration). A needle is passed into the testis and a small amount of testicular tissue is drawn out. The tissue is processed in the laboratory, and if sperm are found, they are frozen for use in ICSI.

PESA (percutaneous epididymal sperm aspiration). Sperm are drawn from the epididymis, the coiled duct behind the testis where sperm are stored and mature.

Epididymal retrieval suits obstructive azoospermia, in which sperm are produced normally but cannot get out; testicular retrieval is used when sperm have to be sought where they are made. The difference between these two forms of azoospermia is covered in the causes and workup article.

How the injection is done

ICSI is micromanipulation under a high-powered microscope, and Dr. Prakash took the audience through the technique on video:

1. Immobilise the sperm. The embryologist breaks the sperm's tail, stopping it from moving.
2. Load the injection needle. The immobilised sperm is drawn into a fine injecting needle.
3. Hold the egg. A second instrument, the holding (stabilising) pipette, holds the egg steady with gentle suction.
4. Orient the egg. The egg is turned so that the polar body, a small cell cast off as the egg matures, sits at the 12 o'clock position.
5. Inject. The needle enters at the 3 o'clock position and deposits the sperm inside the cytoplasm.

The orientation is not arbitrary. The polar body roughly marks the region where the egg's chromosomes are organised, and entering at 90 degrees from it keeps the needle away from that structure.

IVF vs ICSI: how the two differ

How long an IVF cycle takes for travelling couples

For couples travelling for treatment, the sequence matters for logistics: the cycle is anchored to day two or three of a period, stimulation takes about two weeks, and the transfer follows retrieval by three or five days. Where sperm must be retrieved surgically, TESA or PESA can be performed ahead of the cycle, with the sperm frozen until the eggs are ready. Treatment costs are set out on the IVF and fertility treatment cost page, and Dr. Prakash's practice is described on her doctor profile.