What is a follicle?

An ovarian follicle is a small fluid-filled sac that contains one egg (also known as an oocyte). At birth, each ovary contains hundreds of thousands of immature follicles, called primordial follicles.ⁱ A female will not create any more ovarian follicles (nor eggs) during her lifetime. What is important within the context of fertility is the number of eggs present at birth and their rate of decline.ⁱⁱ

Primordial follicles

Primordial follicles are in a dormant or resting phase at birth. They are small structures, about 15 to 40 micrometers (μm) in diameter, and consist of a single layer of flat granulosa cells surrounding a single oocyte.ⁱⁱⁱ

Primordial follicles can evolve in four different ways:^iv

1. Remain in their dormant state for the entire human lifespan
2. Undergo apoptosis (die off)
3. Begin to develop and then undergo apoptosis
4. Begin development and progress to ovulation, where the oocyte is released from the follicle

The oocyte inside a primordial follicle is called a primary oocyte. Primary oocytes are not yet mature and cannot be fertilized; they are paused in the prophase part of meiosis I, a cell division process that, when completed, leads to the development of a mature oocyte for reproduction.^v

Primary follicles

Primordial follicles stay dormant until puberty when menstruation starts.^vi At the beginning of each menstrual cycle, a number of primordial follicles are triggered to begin development toward becoming primary follicles. Like primordial follicles, each primary follicle has a single layer of support cells (granulosa cells) surrounding a central primary oocyte.^vii However, the primary follicles grow in size (to roughly 100 μm in diameter) and the granulosa cells become cuboidal (square in shape).

Each primary follicle also develops a zona pellucida, a thin shell made of glycoproteins (carbohydrates linked to proteins) that surrounds the oocyte. Development of this shell is essential for normal fertilization.^viii The primary oocytes inside the primary follicles start to grow dramatically, and the primary follicles develop receptors to follicle-stimulating hormone (FSH), which allow the follicles and oocytes to continue developing in response to FSH.^ix

Secondary follicles

Some primary follicles develop into secondary follicles, which are larger, contain a more developed oocyte,^x and have more granulosa cells.^xi These surrounding cell layers protect the oocyte and aid in ovarian follicle development through exposure to hormonal signaling from follicle-stimulating hormone (FSH), luteinizing hormone (LH), and insulin.^xii

Antral, tertiary, or Graafian follicles

During a process called cavitation, the secondary follicles develop a fluid cavity called an antrum.^xiii At this point, the follicles are called antral, tertiary, or Graafian follicles. FSH and LH signaling controls this process. The follicles continue to increase in size, up to approximately 20 to 24 mm, but the oocytes inside them (measuring around 120 μm) do not.^xiv One of these antral follicles becomes the dominant follicle and continues to grow during the menstrual cycle. The rest of the follicles (primary, secondary, and antral) then undergo apoptosis (programmed cell death).^xv

It takes more than 300 days for a primordial follicle to be selected into the follicular pool as a primary follicle and reach the final phase of development as an antral follicle before ovulation. At any given point during the menstrual cycle, there are follicles at all stages of development.^xvi

Typically, the dominant follicle reaches its maximum size and maturity and is ready to ovulate around day 12 to 16 of the menstrual cycle. The pituitary gland then releases luteinizing hormone (called the LH surge), causing final maturation of the oocyte in the dominant follicle and release of the oocyte (ovulation) into the pelvis to be picked up by the fallopian tube. The oocyte resumes meiosis following the LH surge, becoming a secondary oocyte, also called an MII oocyte (short for metaphase II of meiosis II), which is now capable of being fertilized.^xvii

After ovulation, the follicle from which the oocyte was released becomes a corpus luteum cyst,^xviii producing progesterone and estradiol (an estrogen derivative) to support implantation if fertilization occurs and an embryo is formed.^xix

If an embryo does not implant, the corpus luteum degenerates and progesterone levels drop, leading to a menstrual period.^xx If an embryo implants in the uterus, beta-hCG (human chorionic gonadotropin) is produced, which maintains the corpus luteum and ensures continued production of progesterone and estradiol to support the early pregnancy.^xxi

What are cysts?

Follicles are sometimes referred to as cysts, or functional cysts, which are normal follicles developing each month during a menstrual cycle.^xxii The term cyst is used clinically to describe a fluid-filled sac structure. This terminology can be confusing for patients, since people often think of cysts as abnormal or pathologic.

Follicular cysts from a previous menstrual cycle are not uncommon and occur when follicles fail to resolve and instead fill with fluid. These appear as simple cysts measuring 1 to 4 cm on ultrasound and usually resolve on their own in four to six weeks. The exact frequency of these cysts is not known since the majority cause no symptoms and go unnoticed. They are usually discovered incidentally on examination for another reason, such as on ultrasound during fertility treatment.^xxiii In rarer cases, the cyst can be larger or cause pelvic pain and should be assessed by a physician.

Hemorrhagic cysts are blood-filled cysts, usually from a follicle that ovulated an oocyte (corpus luteum) in a previous cycle. This type of cyst also typically resolves on its own in four to six weeks. Hemorrhagic cysts are different than endometriomas, which are cysts filled with old blood that are associated with endometriosis and do not resolve on their own. These two types of blood-filled cysts usually look different on ultrasound.^xxiv

Other benign cysts include cystadenomas, cystadenofibromas, and dermoid cysts. These each have characteristic findings on ultrasound and do not originate from follicles in a previous cycle. Cysts can also be pre-cancerous (borderline tumors) or cancerous (ovarian cancer). It is important to speak with your physician about the type of cyst noted on ultrasound and any recommended follow-up.

What is an egg (oocyte)?

An oocyte, often called an egg, is the female reproductive cell, or gamete, located in the ovaries. Typically, females are born with 1 to 2 million primary oocytes. This number drops to between 300,000 and 500,000 oocytes at puberty and about 25,000 at age 37.^xxv Only around 400 oocytes will be ovulated from dominant follicles throughout the reproductive lifespan.^xxvi That means 99.9 percent of follicles and oocytes will undergo apoptosis (programmed cell death), also referred to as follicular atresia. Only 0.1 percent will mature to ovulation.^xxvii

Every oocyte is contained in a follicle, as described above. An oocyte found inside a primordial follicle is called a primary oocyte. Primary oocytes are not mature and cannot be fertilized. Genetic material (DNA) in a primary oocyte is susceptible to damage over time. This sustained damage is one reason why the chromosomes in oocytes may not always separate correctly, causing an abnormal chromosome number (aneuploidy) in embryos. As the oocytes age, reactive oxygen species (ROS) can also damage DNA and impair the oocytes’ ability to mature and function normally.^xxviii

As mentioned above, during a normal menstrual cycle, hormonal signaling from the brain triggers development of a dominant follicle. The LH surge causes final maturation of the oocyte, in which meiosis resumes, and the oocyte progresses to become an MII oocyte (metaphase II of meiosis II). This maturation is required for normal fertilization.

Oocyte retrieval for IVF

During in vitro fertilization (IVF), physicians prescribe medication to stimulate follicle growth of multiple dominant follicles to retrieve as many mature (MII) oocytes as possible. A trigger shot is given in IVF cycles to promote final oocyte maturation, similar to the LH surge in a natural menstrual cycle. Even with a trigger shot, however, not all oocytes retrieved are in the MII stage; typically, some immature oocytes are retrieved as well.^xxix Only mature oocytes can become fertilized and develop into embryos.

How is ovarian reserve measured?

Ovarian reserve is the reproductive potential left in a female’s ovaries based on the number of remaining follicles and eggs. Ovarian reserve naturally declines as a female ages.

Several factors influence ovarian reserve:

• Age
• Genetics
• Certain health conditions
• Environmental influences
• Gonadotoxic (damaging to the ovaries) treatments, such as radiation or chemotherapy

Ovarian reserve decreases at different rates throughout a female’s life. Oocyte depletion starts occurring at a faster rate when individuals reach their mid-30s.^xxx Ovarian reserve can be measured in three ways:

• Anti-Müllerian hormone (AMH) levels
• Follicle-stimulating hormone (FSH) levels
• Antral follicle count

Anti-Müllerian hormone (AMH) levels

AMH is a glycoprotein (carbohydrate linked to a protein) made by the surrounding granulosa cells of the preantral and antral follicles. AMH can be measured in the blood with a laboratory test.^xxxi,xxxii

AMH begins to rise during puberty and typically peaks around age 25. It then declines until a few years prior to menopause.^xxxiii Levels generally remain consistent during the menstrual cycle, so AMH can be measured on any cycle day.^{xxxiv,xxxv,xxxvi} Hormonal medication, such as birth control pills and progesterone intrauterine devices (IUDs), can slightly lower the AMH level, so it is important for patients to mention this information to their doctor if they check AMH while on these medications.^xxxvii

AMH correlates with the primordial follicle pool and is used to predict how well individuals will respond to ovarian stimulation during assisted reproductive technology (ART). A 2022 meta-analysis of 27 studies (including 27,029 females) found that a more favorable AMH level predicted improved outcomes, including live birth rates.^xxxviii Similarly, a retrospective study found that high AMH was associated with higher cumulative live birth rates following IVF, compared to individuals with normal AMH.^xxxix

Overall, AMH higher than 1.7 ng/mL is linked to good IVF outcomes and to acceptable ovarian reserve.^xliii,xliv Importantly, AMH is not a specific marker for fertility and does not account for egg quality. Some studies have shown equivalent fertility rates for people with low and normal AMH levels when conceiving naturally (not using assisted reproductive technology).^xlv,xlvi

Follicle-stimulating hormone (FSH) levels

Early follicular phase FSH level is a blood test that measures FSH in the bloodstream during days 2 to 4 of the menstrual cycle.^xlvii Ovaries in females with normal ovarian reserve make enough of the ovarian hormones estradiol and inhibin B to keep FSH low in the first few days of the menstrual cycle. FSH levels rise early in the cycle if the ovaries fail to produce enough estradiol and inhibin B, which occurs in females with diminished ovarian reserve.^xlviii

A normal early follicular phase FSH level is typically under 10 IU/L. When levels are consistently higher than that, it is a marker of lower ovarian reserve. It is important to also check estradiol level along with the FSH level. If the estradiol level is high (over 80 to 100 pg/mL), FSH will be artificially lowered. A high estradiol level early in the cycle is also a marker of low ovarian reserve. IVF patients with high early follicular FSH levels may not respond well to ovarian stimulation medication.^xlix,l

Antral follicle count (AFC)

AFC is an ultrasound measurement of ovarian reserve that is often used in addition to blood AMH and FSH measurement. The number of antral follicles helps predict the ovaries’ responsiveness to ovarian stimulation medication in IVF.

The test is typically conducted between day 2 and day 5 of the menstrual cycle. A transvaginal ultrasound is used to count the number of antral follicles (small fluid-filled cysts measuring 2 to 10 mm) in each ovary.^li,lii

How many follicles are considered normal?

On average, females are born with 1 to 2 million primordial follicles, and each contains a primary oocyte.^liii The number of ovarian follicles declines over time as follicles go through development cycles, and over 99 percent of them eventually undergo apoptosis (programmed cell death).

The number of antral follicles (follicles recruited for that specific menstrual cycle) can be measured on ultrasound by performing an antral follicle count (AFC). The definition of normal AFC varies by study, and there is a large variation based on age. For example, females aged 20 to 34 can generally expect a higher AFC (average 14 to 15 antral follicles) than females aged 38 to 46 (average four to five antral follicles).^liv,lv

There is also a great deal of variation between individuals of the same age. Even in the same person, AFC can vary from one cycle to the next. AFC also can vary if the patient is diagnosed with certain conditions. For example, patients with polycystic ovary syndrome (PCOS) have much higher AFC than patients of the same age without PCOS.^lvi

Although normal AFC cut-off values vary between studies, most providers agree that fewer than five total antral follicles is a sign of diminished ovarian reserve and predicts poor ovarian response to IVF medications.^lvii It is important to note, however, that even if a female has fewer than five antral follicles seen on ultrasound, that does not necessarily mean she will not respond to ovarian stimulation or be able to conceive.^lviii

How many follicles and eggs are needed for IVF?

According to clinical studies, the average number of eggs retrieved during an IVF cycle varies from seven to nine eggs. This results in an average of three to five embryos.^lix The number of eggs retrieved is variable and depends on the patient’s age and ovarian reserve. It is important to note that not all retrieved eggs are mature, not all mature eggs will successfully fertilize to become embryos, and not all embryos will continue to develop and be able to be transferred into the uterus.^lx

Not surprisingly, research shows that the more eggs retrieved (up to around 15), the higher the likelihood of pregnancy success.^lxi,lxii Based on findings from several studies conducted between 2010 and 2014, retrieving between six and 15 eggs during a cycle is best to reach optimal pregnancy outcomes.^{lxiii,lxiv,lxv,lxvi,lxvii} These studies also showed a plateau effect beyond approximately 15 retrieved eggs. In other words, retrieving more than 15 eggs offered no additional improvement in live birth rate for that cycle.^lxviii,lxix  Of note, higher estradiol levels, a marker of egg development, on the day of the trigger shot are associated with a higher number of eggs retrieved.^lxx

How big should follicles be before triggering during fertility treatment?

Follicle size is continually monitored during an IVF cycle using transvaginal ultrasound. The size of the follicles on the day of the ovulation trigger shot correlates with the number of oocytes that are successfully retrieved.^lxxi,lxxii Many researchers suggest waiting to trigger until at least two follicles are over 16 to 18 mm in size.^lxxiii

However, there is debate around the optimal follicle size for ovulation trigger. Many studies have observed that follicles measuring less than 12 mm on the day of trigger are less likely to yield mature (MII) oocytes.^{lxxiv,lxxv,lxxvi} According to a 2017 study in Ultrasound in Obstetrics and Gynecology, the authors measured follicle size at the time of 118 egg retrievals and found that follicle sizes from 13 mm to 23 mm had higher oocyte recovery rates and potential to yield good quality blastocysts.^lxxvii Similar results were found in a 2022 Fertility and Sterility study that looked at results from 157 oocyte retrievals.^lxxviii

Regardless, some researchers recommend that even oocytes in small follicles (8 mm to 12 mm) at the time of retrieval should be collected, since eggs in these follicles may still lead to live births in some cases.^lxxix

Is it possible to have an empty follicle?

In a typical IVF cycle, the number of eggs retrieved can be lower than the number of follicles observed at the time of retrieval. That means an egg is not recovered from every aspirated (emptied) follicle.^lxxx The oocyte recovery rate refers to the percentage of aspirated follicles that result in a retrieved oocyte.^lxxxi Based on published literature, depending on the size of follicles aspirated and aspiration technique, the average oocyte recovery rate ranges from 40 to 80 percent.^lxxxii In general, the larger the follicle, the more likely an oocyte will be aspirated from it.^lxxxiii

What is empty follicle syndrome (EFS)?

Empty follicle syndrome (EFS) is the extreme end of low oocyte yield during egg retrieval. It is rare, occurring in 0.2 to 7 percent of IVF cycles.^lxxxiv With this condition, adequate ovarian stimulation is evident, the ovarian follicles appear normal, and estradiol production is appropriate. However, no oocytes (eggs) are obtained from the follicles that are successfully punctured and aspirated during the egg retrieval process.^lxxxv

Why EFS occurs is not exactly known. The most common theories suggest that aspiration fails to yield an oocyte because it has undergone apoptosis, the follicle is dysfunctional, or the oocyte failed to develop and mature normally. It is also possible that the trigger shot was not successful or premature ovulation occurred, meaning the oocyte was released prior to retrieval. Other potential causes include genetically abnormal oocytes or abnormal hormonal receptors on the follicles.^lxxxvi

How is empty follicle syndrome (EFS) treated?

For patients who have genuine EFS — meaning they correctly administered the trigger shot but have no retrieved oocytes — few treatments are available to prevent it from happening in the future. However, there are some techniques that are worth investigating:^lxxxvii

• Increasing the time from hCG trigger to retrieval
• Giving a second hCG trigger treatment prior to retrieval
• Using dual hCG and GnRH agonist to trigger

Using these techniques, 86 percent of patients in one 2017 Reproductive BioMedicine Online study were able to obtain mature oocytes in a subsequent cycle.^lxxxviii

Conclusion

The terminology and processes involved in IVF can be daunting to individuals who are new to the world of fertility treatment. In particular, ultrasound monitoring of follicles and the egg retrieval process itself are often challenging to understand, given the precision of measurements and procedures at each stage. Having a better grasp of the differences between follicles and eggs can provide clarity in terms of how an IVF cycle is progressing, thereby making it easier to set expectations during each step.