A Complete Guide to In Vitro Fertilization (IVF)

The first step of an IVF cycle is called ovarian stimulation, which begins at the start of (or just prior to) your menstrual cycle.

At this time, medications are administered daily that stimulate the ovaries and control the timing of ovulation. These medications are important because they:

• Cause multiple eggs to mature in the ovaries

• Prevent ovulation from occurring too early (if ovulation occurs early, the eggs will be lost)

As you administer these medications, you will return to your IVF clinic frequently for monitoring. This monitoring often consists of:

• A transvaginal ultrasound, which counts and measures the diameters of the growing follicles (fluid-filled sacs that should contain eggs) inside the ovaries

• Bloodwork to determine if your hormone levels are rising appropriately in response to the medications

As you continue administering your medications, your follicles will continue to grow in size. Once some or all of your follicles reach a certain size (usually 16+ mm), your fertility specialist will prescribe a trigger medication and schedule your egg retrieval procedure.

The trigger medication must be administered at the proper time (usually 35-36 hours prior to the time of your scheduled egg retrieval) so ovulation does not occur too early.

Most egg retrievals are performed under some level of sedation, though some clinics do not offer sedation during retrievals (this is not common). Egg retrievals are low-risk procedures that are normally completed in 15-30 minutes.

During the procedure, the following steps occur:

• A thin needle is inserted through the vaginal wall and into the ovaries.

• Using an ultrasound as a guide, the needle is then pierced into each follicle in the ovaries and the fluid is aspirated (sucked up) using light vacuum pressure. This fluid, which hopefully contains one or more eggs, is collected into sterile test tubes.

• Each sterile test tube filled with fluid is sent to the IVF lab for inspection. An embryologist will inspect each tube, isolate any eggs, and discard the remaining fluid.

• Any eggs that are found are cleaned and graded based on their maturity (unless the eggs are undergoing conventional insemination, in which case they will not be graded). Mature eggs contain a polar body and can be inseminated, while immature eggs cannot.

For more information about the egg retrieval procedure, check out this video. 

If your plan is to freeze your eggs, any mature eggs will be frozen shortly after the egg retrieval procedure is completed. This process is similar to embryo freezing, and frozen eggs are stored in liquid nitrogen for future use. If you are thawing eggs for insemination, they will be thawed a few hours prior to insemination.

Insemination occurs on the same day as the egg retrieval or thaw. Insemination refers to the combination of a sperm and egg cell, which should ultimately result in fertilization. There are two primary types of insemination:

• Conventional insemination (IVF) consists of placing sperm and eggs into a drop of media together overnight in hopes that the sperm will fertilize the eggs. The primary downfall is that the eggs are not graded based on their maturity beforehand, and immature eggs will not properly fertilize. Therefore, you may have a lower fertilization rate with conventional IVF.

• Intracytoplasmic sperm injection (ICSI) consists of injecting a sperm into each mature egg. Only mature eggs undergo ICSI, and it tends to yield overall higher fertilization rates. However, ICSI is more invasive than conventional IVF and can cost more money. ICSI is indicated in cases of male factor infertility, or when a sperm sample is of poor quality.

For more information about IVF and ICSI, check out this video.

The day after insemination (referred to as day 1 in the IVF lab), the eggs are examined to determine if they are properly fertilized. Fertilized eggs (now called zygotes) consist of:

• Two pronuclei (one from the egg and one from the sperm)

• Two polar bodies (this signifies that the egg has completed its final stage of maturation, which requires fertilization to occur)

Eggs that do not show normal signs of fertilization are either discarded or isolated and observed at a later date. For more information about fertilization, check out this video. 

Over the next few days, the embryos undergo rapid cell division and development. Though each embryo grows at its own pace, there is general timeline that embryos are expected to follow:

• On day 2 (2 days after insemination), the embryo should consist of 2-4 cells.

• On day 3, we expect to see anywhere from 6-12+ cells. Some clinics observe embryos on day 3, while others do not. At this point, we hope to see 8+ cells, little to no fragmentation, and perfectly or moderately symmetrical cells. Some clinics also transfer embryos on day 3, while others wait until day 5.

• On day 4, the embryo’s cells should begin to condense to form a cluster (known as a morula). A small cavity (blastocoel) may also begin to form in the embryo. At this point, the embryo has reached the blastocyst stage of development.

• On day 5, the embryo should consist of over 100 cells, a large blastocoel, two distinct cell lines, and a thin shell (zona pellucida) surrounding it.

• On days 6/7 (some clinics culture to day 7, while others do not), the embryo should create a hole in this shell and begin to hatch from it. An embryo must hatch from its shell in order to implant into the uterine lining.

While some embryos that are slightly delayed in development can result in healthy live births, embryos that are severely delayed in development (arrested) often do not develop further and are discarded per your laboratory’s protocol.

Click here to watch a video of an embryo developing in a time-lapse incubator.

Many clinics begin to grade embryos when they reach the blastocyst stage of development. Each clinic may have its own grading method, but most clinics grade embryos based on three criteria:

• Stage of development. Many clinics utilize a numbering system, known as the Gardner scale, to record an embryo’s stage of development. This scale utilizes a number (1-6) to record the embryo’s stage of development.

• The inner cell mass (ICM). This cohort of cells eventually develops into the baby. Most clinics grade ICMs on an A, B, C (sometimes D) scale, with A being the best grade.

• The trophectoderm (TE). This cohort of cells lines the embryo’s shell and eventually develops into the placenta. The TE is also typically graded on an A, B, C (sometimes D) scale, with A being the best.

• On average, good quality embryos have a roughly 50% chance of resulting in a healthy live birth. Poor quality embryos have lower implantation rates but similar success rates if implantation does occur. Certain factors can increase or decrease an embryo’s success rate. For example, a genetically normal embryo (see below) has a higher chance of resulting in a successful pregnancy than an untested embryo.

Embryos that are considered suitable for transfer (this may also differ between clinics) have three possible fates that usually begin 5 days after insemination (on day 5):

• The embryo(s) can be transferred to the uterus via an embryo transfer

• The embryos can be biopsied for preimplantation genetic testing (PGT) and frozen and stored in liquid nitrogen for future use.

• The embryos can be frozen (with or without being biopsied) and stored in liquid nitrogen for future use.

An embryo transfer procedure is a minimally invasive, low-risk procedure. It is not normally performed under sedation, though there are exceptions, and it normally takes 5-15 minutes to complete.

During this procedure, the following steps typically occur:

• An abdominal ultrasound is used to visualize the uterus and cervix.

• A thin, flexible catheter, which may or may not contain the embryo (each clinic does this a little differently), is then inserted into the uterus through the cervix. This catheter consists of an inner piece (called a noodle) and outer plastic sheath. If the embryo is loaded into this catheter, it will be plunged into the uterine cavity once the tip of the catheter is in place on the ultrasound. 

• If the embryo is not loaded into this catheter, the noodle is removed from the catheter and the outer sheath remains in place. The embryo(s) is loaded into a different catheter, and the inner noodle of this catheter is thread through the outer sheath of the original catheter (which creates a “tunnel” for the catheter with the embryo). Once the tip of the new catheter is in view on the ultrasound, the embryo is injected into the uterine cavity.

Afterward, the catheter is flushed to ensure that the embryo has successfully transferred into the uterus. If the embryo is retained, the process is repeated (don’t worry, this hasn’t been shown to affect success rates!).

A blood test is normally scheduled 9-12 days after an embryo transfer to determine if embryo implantation occurred. This blood test is known as a quantitative test, which measures the level of the hormone hCG (human chorionic gonadotropin) in the blood. HCG is produced by placental cells, so the presence of hCG indicates that embryo implantation has occurred. 

Click here to see how an embryo transfer occurs.

Embryos that are of a certain developmental stage and grade can also be biopsied for preimplantation genetic testing (PGT). This screening test determines if an embryo has genetic abnormalities.

The most common type of PGT is called PGT-A (aneuploidy), which determines if the embryo has the right number of chromosomes (for example, those with Down Syndrome have an extra 21st chromosome). Other types of PGT include PGT-M (monogenic, for single gene mutations) and PGT-SR (structural rearrangement, for chromosomal rearrangements such as translocations and inversions).

To perform PGT, roughly 5-10 cells are removed(biopsied) from an embryo’s trophectodermand are sent for PGT. The embryo is frozenat the IVF clinic and remains stored there in liquid nitrogen. PGT examines the DNA of the cells in the biopsy sample and determines what percentage of those cells have the correct number of chromosomes (or if the embryo has a genetic mutation or structural rearrangement). Embryos with a high percentage of abnormal cells, a genetic mutation, or certain structural arrangements, are not recommended for transfer. Embryos that are suitable for transfer can be thawed the day of the transfer. 

PGT-A is most commonly recommended for women over 36 years of age. Keep in mind that this does not apply to donor eggs or eggs that were frozen before a woman reached 36 years of age. As a woman ages, the quality and quantity of her eggs decline, which can result in an increased incidence of embryos with the wrong amount of DNA. Read more about maternal age and egg quality here. 

PGT-A results usually fall into one of the following categories:

• An embryo is deemed chromosomally normal (euploid) if most (usually >80%) of its cells have the correct number of chromosomes.

• An embryo is deemed abnormal (aneuploid) if most of its cells (usually >80%) do not have the correct number of chromosomes.

• Mosaic embryos do not have enough normal cells to be considered euploid, but they also do not have enough abnormal cells to be considered aneuploid. It sounds confusing, but what it breaks down to is that these embryos have normal and abnormal cell lines in them. Mosaic embryos result from nondisjunction (an uneven disbursement of DNA during cell division) after fertilization occurs.

• In other words, some cells end up with the right amount of chromosomes, while other cells end up with the wrong amount. Mosaic embryos can be low level (usually <50% abnormal cells) or high level, and new research has found that low level mosaic embryos have the potential to result in healthy pregnancies through a process known as self-correcting. However, mosaic embryos do tend to have lower success rates compared to euploid embryos.

• In some cases, there may not be enough DNA present for the PGT machine to analyze the DNA. In these cases, the results may be inconclusive. It’s possible to thaw the embryo with these results, biopsy it, and refreeze it. The new biopsy sample can then be analyzed.

It’s possible to perform PGT on an embryo that has already been frozen. This process involves thawing the embryo, performing the biopsy, and re-freezing the embryo. The biopsy is then analyzed in the same manner described above. There is a slight risk of damage to the embryo during these processes due to the additional manipulation involved, but most embryos (>90%) do tend to survive the processes without any issues.

There are many reasons why embryos are frozen or cryopreserved. For example, if multiple embryos are created during an IVF cycle, they can be frozen for future use. Embryos that are frozen remain stored in liquid nitrogen, where all cellular activity is halted, for future use.

Almost all embryos are frozen through a technique known as vitrification, or rapid freezing. This process involves:

• Placing the embryo(s) in a drop of “equilibrating” media for a specific amount of time (it can vary depending on the manufacturer). This media pulls water from the embryo, which prevents ice crystals from forming during the vitrification. Ice crystals can damage the embryo’s cells, so it’s important to have water removed from the embryo prior to vitrification.

• Moving the embryo into a viscous “vitrification” media for a few seconds, where it is coated in a cryoprotectant.

• Placing the embryo at the tip of a labeled cryo device and plunging it into liquid nitrogen. 

This process is repeated for all embryos that are frozen that day. Afterward, the devices are placed in a plastic holding device (goblet) under liquid nitrogen, which is attached to a metal cane. Everything is then quickly transferred to a cryotank (Dewar), where the embryos are stored and consistently monitored.

Click here to see how embryos are frozen.

Embryo thawing is basically the same thing as embryo freezing, but in reverse. The embryo is quickly removed from liquid nitrogen and plunged into a warm “thawing” solution. After about a minute (this can vary between manufacturers), the embryo is moved through a series of drops that safely warm the embryo and move water back into it. Most embryos are collapsed when they are thawed (this is normal) and will expand after an hour or so in culture.