If you're curious about or are considering in vitro fertilization (IVF) as a way to add to your family, you might be wondering what exactly the process involves. The truth is that IVF is a multi-stage process and the exact steps of what is required (or what you choose) can vary between individuals. Treatment can differ depending on whether you are diagnosed with a certain condition like polycystic ovary syndrome (PCOS) or if the eggs involved are from a donor or a woman of advanced maternal age.

In general, the process of IVF involves retrieving eggs, fertilizing them in a laboratory, and transferring the resulting embryo(s) back to a uterus or freezing them for future use.

Here we'll take an in-depth look at IVF and explain the process and options that may be available to you in greater detail.

IVF Stimulation Protocols

Each menstrual cycle, most women produce multiple egg follicles, but only one follicle will become dominant and mature into an egg that is released through the process of ovulation. During an IVF cycle, fertility drugs are used to stimulate a woman's ovaries in a process known as superovulation, which causes the production of multiple follicles at once. More follicles generally translate to multiple eggs, which increases the potential for multiple embryos. The most important part of the IVF process is to have good-quality embryos to transfer back to a uterus.

A larger number of eggs is beneficial in an IVF cycle because it is common to see a dramatic reduction in the number of viable eggs and/or embryos from the time of the egg retrieval to an eventual embryo transfer. The more embryos that are available to choose from, the greater the chance of having good-quality embryos for transfer back into the body.

Follicle to Embryo

Follicle (Immature Egg) → Mature Egg → Embryo (Fertilized Egg)

Variation in IVF Stimulation Protocols

There are different ways medications are used to stimulate the ovaries during an IVF cycle. These are called IVF protocols. If a woman is a poor responder, meaning she has very few egg follicles, she will be on a different stimulation protocol than a woman with polycystic ovaries who has lots of egg follicles. (You can read more about this in IVF Stimulation Protocols.)

There are many possible stimulation protocols. It may take multiple cycles of fertility drugs to develop the proper response in terms of the number and size of follicles that develop. It might even be necessary to cancel a cycle after you have started the fertility medications and restart on a different protocol. Although this may be a disappointing result, remember that you are starting over with a better understanding of your body.

Triggering the Release of Mature Eggs

After fertility drugs stimulate the growth of multiple follicles, the follicles need additional "help" in order to mature into eggs. The growth of the follicles will be followed by ultrasound imaging known as follicle monitoring. Once the follicles in the ovary reach the appropriate size (generally around 10 days after fertility drugs were started), an injection of synthetic human chorionic gonadotropin (hCG) hormone called a trigger shot is given to mature the eggs and release them from the wall of the follicle. (The eggs will stay inside the follicle and not be ovulated) where it remains until egg collection.

The timing of this injection is critical as the eggs should be allowed to mature as much as possible before they are removed through an egg retrieval procedure. The shot is usually administered two days prior to an egg retrieval procedure.

The IVF Egg Retrieval Procedure

Once the follicles have matured into eggs, they need to be removed from the body. This process of removal is called an egg retrieval procedure and is performed by your IVF doctor and a team of nurses.

Most egg retrievals are done under light anesthesia and are usually painless; however, some women report feeling some discomfort for the remainder of the day. After the procedure is complete, it is advisable to rest and not plan on returning to work or other activity.

An egg retrieval process generally includes the following steps:

  • The doctor inserts a long hollow needle into the ovary through the wall of the vagina with the help of ultrasound images.
  • When the needle punctures a follicle, suction is applied and the contents of the follicle are drained and transferred to the embryologist.
  • The embryologist identifies and isolates the eggs from the follicular fluid.

Expectations After Your IVF Egg Retrieval

After the egg retrieval, it is only natural to have questions related to the degree of success of the procedure and what that means for your odds of achieving pregnancy. Here are a few tips to keep in mind to help adjust your expectations for the amount of post-retrieval info you can expect.

  • The number of follicles seen on ultrasound is only the approximate number of eggs you can expect to retrieve; sometimes there are more, sometimes there are less.
  • Not all of the eggs retrieved will be mature and the immature eggs cannot be fertilized and will not be usable.
  • On the day of the egg retrieval, little information about the quality of the eggs is available. At most, we can tell the number of eggs and possibly how many are mature. Most eggs look identical at this stage. The difference in the quality of eggs becomes evident only in the days after fertilization when any fertilized eggs have started to divide and grow.

Semen Samples for an IVF Procedure

Once the eggs have been extracted, they need to be fertilized. This requires fresh or frozen sperm from a partner or donor. If the male partner is giving a fresh semen sample on the day of the egg retrieval process, he will likely be asked to do so around the time of the egg retrieval procedure. Alternatively, a sample may have been previously frozen.

The lab prepares the sperm by separating the sperm from the seminal fluid in a process known as sperm washing. In most cases, it’s spun in a centrifuge using a special solution that spins around quickly to allow the separation to occur. The eggs and sperm are kept warm in an incubator for the rest of the day until it’s time to inseminate the eggs.

Abstinence Prior to Producing a Semen Sample for IVF

The male partner is generally asked to abstain from ejaculating for two to three days before the egg retrieval procedure. Studies have shown that frequent ejaculation can improve sperm quality and reduce DNA fragmentation, which could lead to poor-quality embryos.

It is generally recommended that the male partner abstains from ejaculating for two or three days prior to the IVF cycle, but that he ejaculates frequently in the weeks leading up to the procedure.

IVF Egg Fertilization: Insemination or ICSI

There are two possible methods of fertilization that may occur as part of an IVF procedure. Either natural insemination or intracytoplasmic sperm injection (ICSI) is performed to fertilize the eggs, generally on the day of the egg retrieval.

  • Natural Insemination
    Insemination involves placing several thousand sperm around the eggs and allowing them to fertilize naturally. They are left overnight and examined the next morning for signs of fertilization.
  • Intracytoplasmic Sperm Injection (ICSI)
    If there is any concern about the sperm’s ability to fertilize the eggs, the embryologist may decide to perform intracytoplasmic sperm injection (ICSI). This procedure involves selecting a single, normal-appearing sperm with a high-power microscope and injecting it directly into an egg. This is considered to be a very effective way to fertilize eggs. (Learn more in An Intro to Intracytoplasmic Sperm Injection (ICSI).)

This procedure is used in cases where the male partner has:

Some clinics exclusively perform ICSI on all their patients to avoid the rare case where there is a fertilization failure and therefore no embryos for transfer.

Stages of Embryonic Development During IVF

Once fertilization has taken place, the wait is on to determine the quantity and quality of developing embryos. Here we provide an overview of what you might expect after the egg retrieval has taken place.

IVF Day 1: The Fertilization of Retrieved Eggs

The day of fertilization is counted as Day 1 of embryo development. On this day, your embryologist will check to see if any of the eggs were successfully fertilized overnight. On average, approximately 80% of the mature eggs fertilize normally. On this day, you should find out how many embryos you have. The lab staff or one of the nurses will likely call you with this information.

Fertilization is evident by the appearance of two circles inside the egg called pronuclei. One circle contains the genetic material from the sperm, and one contains the genetic material from the egg. These pronuclei fuse within a few hours. At this stage of development, the embryo is referred to as a zygote and is one single cell.

Sometimes more than one sperm enters the egg, and there are three or more pronuclei in a single egg. These are carefully isolated from the other fertilized eggs because they are not able to implant and grow normally.

Most patients are curious about the quality of the embryos. At this stage, the embryos look identical; it’s not yet possible to distinguish between one embryo and another. The embryologist will likely not have much additional information to share besides the number of fertilized eggs.

IVF Days 2 and 3: Cleavage Stage

On Day 2 of development, we expect the embryos to have subdivided into two or four smaller cells. This process of division is called cleavage.

On Day 3, embryos should have further divided to be six or eight cells.

Embryo Grading for IVF

Once the embryos start to divide and increase the total number of cells, it’s possible to grade their quality. In the lab, we usually see a wide range of quality within a batch of embryos. It’s rare to see all embryos looking identical once they start to divide.

It is worth noting that each clinic has its own grading system, so you will need to ask what system is used at your clinic to fully understand how grading may be applied to your embryos.

Embryo grading is generally based on:

  • The number of cells: The number should be appropriate for the day of development.
  • Size, shape, and degree of diversity in the size of cells: An even number of equally sized cells is best. The clarity of the cytoplasm inside the cells will also be examined. The interior of the cells should be free of inclusions and dark areas.
  • The extent of fragmentation: Fragmentation occurs when a cell divides and parts of the cell break off. This causes the embryo to have smaller buds or fragments of cells surrounding the larger cells.

For example, a lab might have a grading system from 1 to 4, where 1 is the best quality embryo and 4 is the poorest quality embryo. For cleavage stage embryos (days 2 and 3 of development), the grade includes two numbers:

  1. The first is the number of cells
  2. The second reflects the overall quality of the embryo

An 8/1 would be an eight-celled embryo of excellent quality; a 6/3 would be a six-celled embryo of poor quality, and so on.

Although low-grade embryos have a lower chance of implanting than their more handsome counterparts, it’s still possible to achieve a pregnancy from embryos that are not ideal when viewed under a microscope.

IVF Day 4: Transition and Morula Stage

Following the eight-cell stage, the cells of the embryo merge to form what is called a morula. Viewed through a microscope, the embryo looks almost like a single cell again because the cells have merged together. To merge, the cells have to express the correct molecules on the surface of the cells. It is the good-quality embryos that have a greater ability to undergo this process of merging.

Why did my embryos stop growing?
Between Day 3 and Day 5 of development, many important changes happen. This is the stage when an embryo is most likely to stop growing, or "arrest development." One reason for this is that after Day 3, the male chromosomes begin to contribute to the development of the embryo. Before this stage, the cell divisions are powered by only the egg’s energy. Any abnormalities from the sperm may slow down or stop the embryos from growing past Day 3.

IVF Days 5 and 6: Blastocyst Development

Following the morula stage is the all-important blastocyst stage, when the embryo takes in fluid to form a cavity and the cells begin to differentiate into two different types. These two types of cells are called:

  1. Trophectoderm (T) - The T cells are a single layer of cells around the circumference of the embryo that gives rise to the placenta and embryonic sac.
  2. Inner cell mass (ICM) - The ICM is a distinct clump of cells that form the actual baby.

The overall structure of the blastocyst is important, as is the presence of these two different cell types. Because the appearance of the embryo at this stage is dramatically different from earlier stages of development, blastocysts have their own separate grading system.

Blastocyst Grading for In Vitro Fertilization

Blastocyst grading is more complicated because both cell types in the embryo are graded. The following is one example of a blastocyst grading system; your clinic may use a different system.

Each egg is enclosed in a shell known as the zona pellucida. A blastocyst continues to take on fluid until there is enough expansion to allow it to hatch out of this protective casing in preparation for implantation in the uterine wall. (Read more about the process of implantation.) The blastocyst is designated a numerical value between 1 and 6 based on the degree of expansion, or how much fluid the embryo has taken into the cavity.

A Grade 1 embryo has just started to take in fluid and show signs of expansion and is still in the early stages. As an embryo takes in more fluid, the grading system reflects this with a 2, 3, or 4 grade, depending on how much fluid is inside the cavity and how big the embryo has grown. Grade 5 describes an embryo hatching out of its shell. Grade 6 describes an embryo that is fully hatched.

Next come two letters, A, B, or C. The first letter describes the quality of the inner cell mass (or baby making) cells. If there are many tightly packed cells, the embryo gets an A grade. If there are fewer cells more loosely packed, it gets a B grade. If there are few cells, it receives a C grade.

The grading system for the T cells follows a similar pattern, with A being the best and B and C being poorer quality, meaning the cells are less organized. For example, an embryo with a grade of 4AA is fully expanded with good-quality and well-organized ICM and T cells. A grade of 3AB reflects a full blastocyst with a good inner cell mass and less well-organized, or slightly poorer quality trophectoderm cells. (For more insight, read Embryo and Blastocyst Grading - How Does It Work?)

Blastocyst Hatching

The zona pellucida (protective shell around an egg) has two important functions:

  1. It allows only one sperm to enter the egg during fertilization, thereby maintaining the genetic integrity of the embryo (one sperm and one egg combine to make the perfect number of chromosomes).
  2. It holds together all the cells at the early cleavage stages (cell dividing) of development.

In order for a blastocyst to properly implant within the uterine lining, it must "hatch" from its shell. Usually, this happens on late day 5 or day 6 of development when the embryo takes fluid into the cavity and becomes too big for the shell to take the strain of the growing embryo. The blastocyst pulses, contracting and expanding until it gradually squeezes out of a hole in the zona pellucida.

The hatched blastocyst now has molecules on its surface that recognize and bind to molecules on the cell surface of the uterine cells to aid in implantation of the embryo into the uterus. Normal blastocysts implant around day 6 of development by coming into contact with the cells of the uterine lining.

Assisted Hatching for IVF

If the shells of the embryos are thicker than usual, it may be difficult for blastocysts to hatch naturally. In such a case, the embryonic cells are not able to come into contact with and stick to the uterine lining. As a result, implantation fails.

Assisted hatching is a procedure where a hole is made mechanically or chemically in the shell of the embryo before embryo transfer. It can be done in several ways but is most often done using a laser that is mounted on a microscope. Once the embryos are "hatched" in the lab, there is no chance of their being trapped inside their shells after they are transferred to a uterus.

Several studies have shown the benefits of assisted hatching, including an increased chance of achieving pregnancy.That said, assisted hatching is not a procedure that is well-suited for everyone. These same studies have shown no benefit from hatching embryos when compared two similar groups (age, diagnosis) of patients in a study.

Assisted hatching is standard practice at most clinics and is used in all cases of frozen/thawed embryos and with patients who meet the following criteria:

  • Have a raised follicle-stimulating hormone (FSH) level
  • Are over 38 years of age
  • Have embryos graded as poor-quality
  • Have thickened shells around the embryos
  • Have previously failed attempts at IVF

Embryo Transfer During an In Vitro Fertilization Procedure

Embryo replacement, or transfer, is performed by loading the embryos into a very fine catheter (or tube) and inserting it through the cervix into the uterus. The embryos are gently expelled from the catheter with a syringe and placed at the top of the uterine cavity. This is done with or without ultrasound.

Embryo transfer is painless; anesthesia is not necessary for the majority of women. However, you may be offered a muscle relaxant, such as Valium, to minimize uterine contractions following the procedure.

Will my embryos fall out?

A common question asked is, “Will my embryos fall out?” The answer is no; they will not fall out. Perhaps you are thinking of textbook illustrations that show uterine cavities as big open spaces. This is inaccurate. In reality, the two walls of the cavity are pressed tightly together, and there are microscopic folds in the surface. This, coupled with sticky secretions inside the uterus, makes it a safe place for your embryos to stay firmly put.

How is it decided when embryos are ready for transfer?

Embryo transfer can be carried out on any day of development, although most clinics do so on either day 3 or day 5 of development. The primary benefit of growing the embryos in the lab as long as possible is that with each passing day, there is a greater variation in the quality of the embryos, allowing the best to be more easily identified. To better understand this concept, think of embryos like runners in a race.

Imagine that there are 20 runners in this race. At the starting line, they are all at the same place - there are no winners or losers. This is equivalent to day 1 when fertilized eggs all look the same. The runners stay quite close together at the beginning. As the race goes on, the stronger runners stream ahead of the slower ones. Eventually, there is a winner. Running the whole race is equivalent to leaving the embryos until day 5 and choosing the "winning" blastocysts for transfer.

In other words, when there is a large pool of embryos to choose from, letting them develop in the lab until the blastocysts stage at Day 5 can help to sort the most desirable embryos from the least desirable, thus allowing for the selection the best embryos to transfer.

Now what would happen if there were only three runners in the race and you only had to choose the top three? To accomplish this, you do not need to wait until the finish line. In other words, if there are only three embryos in total and you want to transfer three embryos, they can be transferred on any given day. If you end up with the exact number of embryos that you plan on transferring or some embryos clearly stop growing early on, it becomes possible to choose the best embryos for transfer on day 2 or day 3. In fact, they might grow better inside the body than they would if they had been kept in the laboratory incubator; we just don't know for sure.

Hopefully, the race analogy helps you to understand why embryos are transferred at different times. Let's review some of the pros and cons to transfers on each day.

Pros of Embryo Transfer on Day 5

By leaving the embryos in the lab until Day 5, the embryologist is better able to choose the better quality ones, which will provide a higher implantation rate while transferring fewer embryos. This offers the best chance of a pregnancy while reducing the chances of a high-order multiple pregnancy (triplets or more, which are high-risk pregnancies).

In an attempt to reduce the number of multiple or high-order pregnancies from IVF, more clinics are turning exclusively to blastocyst (day 5) transfer. The lab has to have an exceptionally good culture system that it trusts to grow embryos to the blastocyst stage as well as they would grow inside the body. If this is the case, it allows the embryo selection to be made at the end of the “race” and increases the pregnancy rates for those who have an embryo transfer.

Cons of Embryo Transfer on Day 5

The downside to waiting until Day 5 to do blastocyst transfer is that it is possible that all embryos stop growing before day five, leaving no embryos for transfer. This a devastating situation to face when a couple has made it so far through the IVF cycle. This does not alter the outcome of the cycle. It is simply that the outcome is known prior to the embryo transfer. The positive aspect of this scenario is that the woman doesn’t have to wait two weeks until the pregnancy test, and potentially endure painful injections, only to find out that the embryos did not grow.

Genetic Screening and In Vitro Fertilization

An option for couples undergoing IVF is to have all the embryos genetically tested before embryo transfer. This can be done to reduce miscarriage rates by transferring only genetically normal embryos or to eradicate a genetic disorder from a family. More detailed information: An Intro to Preimplantation Genetic Screening.

How are embryos selected for transfer during an IVF cycle?
The easy answer to this question: the best embryos are chosen!

There are a number of factors that determine which embryos are deemed best. On any given day of culture, the embryos are expected to have a specific appearance:

  • On Days 2 and 3, they should be two, four, or eight cells
  • They should be a morula on Day 4
  • They should be at blastocyst stage by Day 5 or Day 6

With an embryo transfer, it’s important to choose only embryos that look viable given the day of culture. For example, we don’t want to transfer an embryo on Day 5 that looks like a Day 3 embryo, which has surely stopped growing and won’t implant and grow.

An additional factor is when to transfer the embryos. Embryos generally look similar on Day 3 and become more diverse in appearance by Day 5. By waiting until Day 5, the embryos with a better chance of success are more evident and can be selected. There are two issues to consider:

  1. Whether to transfer the embryos on Day 3 without knowing if they are going to make it to a blastocyst.
  2. Whether to wait until Day 5 to see if they make it to the blastocyst stage, risking the chance of not having a transfer at all.

If the lab has a good culture (embryo growing) system, the outcome will probably be embryos (and not blastocysts) if you transfer on either day. The embryos will, hopefully, continue to develop, but may not in either case.

Keep in mind that the difference is the disappointment of not having a transfer versus knowing sooner and stopping the medication and restrictions. It’s not always an easy decision to make, and it is suggested that you follow the guidance of your doctor and embryologist.

How many embryos should you transfer during an IVF cycle?
Because of age-related changes in fertility, the number of embryos transferred is directly related to the age of the mother or egg donor. There is a balance to strike between achieving a pregnancy and reducing the chances of high-order multiples - triplets or more.

The number of embryos transferred should be decided under the guidance of the individuals’ doctor. When deciding how many to transfer, your doctor will take the following into account:

  • The age of the female partner or egg donor
  • The stage at which the embryos are transferred
  • The quality of the embryos
  • The number of embryos available
  • The woman's history and diagnosis
  • The outcome of previous treatments

The American Society for Reproductive Medicine (ASRM) has set guidelines for the number of embryos to transfer, depending on age and situation. These are not laws, but they are used to help counsel patients. It is possible that more than the recommended number of embryos can be transferred depending on the specific circumstances of each case. There are no guidelines for women over the age of 42.

Recommended Limits for IVF Embryo Transfer by Age

PrognosisUnder 3535-3738-4041-4242+
Cleavage-stage embryos (Day 3)

Favorable1-2235No guideline
All others2345No guideline
Blastocysts (Day 5)

Favorable1223No guideline
All others2233No guideline

Favorable conditions for embryo transfer include:

  • First IVF cycle
  • Good embryo quality
  • Excess embryos available for freezing
  • Previously successful IVF cycle

All other (less-favorable) conditions include any other situation.

After the Embryo Transfer

After the embryo transfer, you should receive instructions before leaving the clinic. These instructions will include:

  • What medication to take
  • When your pregnancy test will be
  • Any lifestyle restrictions such as bed rest, exercise, or abstaining from alcohol or caffeine

Clinics vary in the extent of restrictions following the transfer. As a general rule, take it easy at least until you hear your results from the pregnancy test.

Your Pregnancy Test After IVF

You will most likely receive results of a pregnancy test around two weeks after your egg collection. This time period is often referred to as the two week wait. (See our best tips for surviving the two week wait after infertility treatment.) On the day of your pregnancy test, you will most likely have a blood (or potentially urine) test to detect the level of human chorionic gonadotropin (hCG).

A positive level indicates that the embryos have implanted. A follow-up test called beta testing will be performed two days later to check to see if the pregnancy is progressing normally; the hCG level should approximately double every 48 hours.

Unfortunately, not all embryos develop normally and some pregnancies end in miscarriage. Your reproductive endocrinologist will follow the hCG blood levels early in the pregnancy, followed by one or two ultrasounds at between six and 10 weeks of pregnancy.

Whatever the outcome of your fertility treatment, it’s very important to stay on all prescribed medication and follow your doctor’s instructions. A normal pregnancy will be followed until approximately 10 weeks gestation at the IVF clinic, at which time you will be referred to a regular obstetrician for prenatal care.

Early Outcomes From an IVF Cycle

IVF outcomes are individual. Personal circumstances include maternal age, the quality of embryos, and other physiological considerations. These have a considerable impact on the outcome of an IVF cycle. Early outcomes that may be expected from an IVF cycle include the following:

PregnantNormal levels of hCG doubling appropriately followed by an ultrasound at 6-7 weeks detecting a heartbeat and growing gestational sac.
Not pregnantA negative or undetectable level of hCG. You stop all medications and meet for a follow-up appointment to discuss the cycle and formulate a plan for the future.
Chemical pregnancyA chemical pregnancy or missed miscarriage is when an initial positive hCG level is followed by an abnormal rise or fall in hCG levels. Pregnancy loss happens before a gestational sac is detectable by ultrasound. (Read more: Experiencing a Missed Miscarriage)
Blighted ovumA blighted ovum involves initial positive hCG level followed by a normal or abnormal rise in hCG levels. Ultrasound reveals an empty gestational sac and no heartbeat. Follow-up ultrasounds confirm pregnancy is nonviable.
Ectopic PregnancyAn ectopic pregnancy is when the embryo grows outside of the uterus, usually in the fallopian tube, but possibly also in the cervix, ovary, or bladder. This is a potentially dangerous situation and must be followed carefully by the doctor. The hCG level may start out low and double slowly, but this is not always the case. Ectopic pregnancies can be detected via ultrasound and treated with medication or surgery. Ectopic pregnancies are nonviable pregnancies.

Is IVF Right for you?

Hopefully the information we have provided will help you to better understand what is involved in an IVF cycle. As detailed above, it's an involved process that can be both physically, emotionally, and financially taxing. Get educated and consult with medical professionals to decide what's right for you.