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What is Day 21 Fertility Testing and Why Is It Important?

January 4th, 2023 | 5 min. read

By Sarah Waters

When you begin your family building journey, one of the first orders of business is a comprehensive fertility workup. Bloodwork, hysterosalpingogram, saline sonogram...the list goes on. These assessments are vital to help your fertility specialist get the most accurate picture of what you might need to achieve fertility treatment success. 

Many of these tests need to be administered at a particular point in your menstrual cycle. One of those tests is appropriately named the "Day 21" test.

"Day 21” of your menstrual cycle can be a useful day to evaluate several different processes. But what if you don’t get a menstrual cycle every 28 days? Maybe you get your menses once every two or three months, or maybe it never seems to come at all? Perhaps your period is only regular when you are taking an oral contraceptive. Or maybe your menses are more frequent, like every 25 days? 

In this blog, we’ll review the so-called “Day 21” evaluation and why it's so important. For those of you who have irregular, more or less frequent, or rare to no menses, this blog includes what “Day 21” means for you, too. And by the end, you will understand why I put “Day 21” in quotes!

Let's Break Down The Menstrual Cycle

To understand the importance of cycle “Day 21," it helps to understand the menstrual cycle. People who have regular cycles presumably ovulate every month at a predictable time. In a typical natural 28 day cycle, it takes about 14 days to grow and develop a dominant follicle. If you are monitoring for ovulation, the LH surge comes roughly 24 to 44 hours before ovulation. 

In a 28 day cycle, ovulation of the oocyte (or egg) occurs around Day 14. The luteal phase starts once the follicle releases the oocyte, and generally lasts for 14 days. The area of ovulation on the ovary changes to form the “corpus luteum,” which secretes progesterone. Ovulation can be confirmed AFTER it has occurred by testing estrogen and progesterone hormone levels. 

If your progesterone level is high (above 4 ng/ml) this confirms that you have indeed ovulated and entered the luteal phase, the second half of the menstrual cycle. Progesterone rises after ovulation, reaching a peak around Day 21 of a 28-day cycle. Peak luteal phase progesterone levels can vary from cycle to cycle, and from person to person. Ideally, “Day 21” peak luteal progesterone levels should be 10ng/ml or higher.

The Importance of Progesterone

Progesterone is vital, as it changes the uterine lining into its secretory phase, making the uterine lining receptive and hospitable to the implantation of an embryo. Without implantation of an embryo, the corpus luteum and its secretion of progesterone will recede within 14 days. Thus, once ovulation has occurred, menses would come about 14 days later.

As you can see, Day 21 is meant to be a marker day for when you're in the middle of the luteal phase of your cycle, and when progesterone production is at a peak. For someone with a 28 day cycle, it takes 14 days to develop a follicle and ovulate the oocyte, and then 14 days of the luteal phase, ending with a menses on the 28th day of the cycle. So in this “textbook” 28 day cycle, day 21 is the middle of the luteal phase. 

If your reproductive endocrinologist is concerned about whether your luteal phase is adequate, (i.e. whether the corpus luteum makes enough progesterone to support a healthy secretory endometrium and implantation of an embryo), they may check your progesterone level on “Day 21,” at the luteal phase peak. If your reproductive endocrinologist is concerned that you may not be ovulating, “Day 21” is also a good day to check progesterone levels, as a level above 5 ng/ml will confirm that ovulation has taken place. 

Still with me? Let's talk about what happens for patients without "textbook" cycles.

Not All Cycles Are The Same

“Day 21” is in quotes because it doesn’t really make sense for the many people who don’t have 28 day cycles. Variability in cycle length is primarily due to the follicular phase. This means that the number of days it takes to grow and develop a dominant follicle that is ready to ovulate can be longer or shorter, depending on the person.

Remember, for those with 28 day cycles, the follicular phase is typically 14 days. For those with more frequent menses, the follicular phase is shorter. For those with longer cycles, the follicular phase is longer. The luteal phase is much less variable than the follicular phase. 

While the follicular phase can vary, the luteal phase still takes about 14 days and peak progesterone day should still be 7 days after ovulation and 7 days before your menses. If you have 35 day cycles, then you ovulate around day 21, and the peak progesterone level would be checked around day 28. If your cycle were 25 days, peak progesterone would be checked around day 18. 

Your reproductive endocrinologist will work with you and your personal cycle to determine the optimal time to perform "Day 21" testing or other fertility assessments. And remember: many people don't have a typical 28-day cycle! There's nothing wrong with you if you don't fit into that textbook 28-day cycle category. 

Prefer to see a video explanation? 

WATCH Monica Explain Day 21 Testing

What If I Don't Ovulate On My Own?

If you rarely have menses, it may be difficult to pinpoint when you ovulate, which means it will be difficult to determine when your peak progesterone level is. If you rarely or never get a natural menses on your own, you may be ovulating very rarely, or you may not ovulate on your own, which means that your natural day 21 progesterone levels can’t be checked. 

If you are not ovulating or are rarely ovulating, typically your doctor will prescribe medication to induce ovulation. For example: Clomiphene Citrate, Letrozole, or injectables (like Ovidrel, HCG, FSH).

Once ovulation has occurred in a treatment cycle, we can then assess your peak progesterone levels to confirm ovulation and whether your luteal progesterone and estrogen levels are adequate to support a healthy embryo implantation.

What If My Hormone Levels Are Low?

If your fertility specialist is concerned that you are not ovulating, or that your progesterone or estrogen levels might be too low to support a lining conducive to implantation, not to worry! There are many options to boost these hormone levels and make a receptive endometrium.

Progesterone levels can be supplemented with vaginal or injectable progesterone supplements. Estradiol levels can be supplemented with oral, vaginal or transdermal estrogen. Luteal phase hormones can also get a boost after a cycle of ovulation induction with Clomid, Letrozole, or injectables. Checking on “Day 21” can confirm whether the progesterone and estradiol levels have improved and are adequate to support a pregnancy.

During treatment cycles, your fertility specialist may also check your peak luteal progesterone and estrogen levels. For some treatment cycles, such as IVF cycles, we recommend estrogen and progesterone supplementation in the luteal phase to most of our patients. In ovulation induction and natural cycles, we typically only supplement if we check your estrogen and progesterone levels and have found them to be too low in the luteal phase of the cycle.

Fertility Treatment Success Begins With Knowledge

Performing fertility assessments like Day 21 testing gives you and your medical team the very best chance at achieving fertility treatment success. No matter which treatment path you're on, Day 21 fertility testing is a vital step in understanding your cycles, hormone fluctuations, and determining the best course of treatment for you.

If you're just getting started on your fertility journey, be sure to check out our ultimate guide to your first consultation and download the free worksheet!

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Sarah Waters

Sarah Waters: Sarah is a Nurse Practitioner and Patient Educator with Illume Fertility. She has been working with Illume since 2005. She graduated from University of Pennsylvania with her BSN/MSN and has over 20 years of experience working in the field of fertility.