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

A nurse practitioner explains why checking the levels of progesterone and estradiol hormones on Day 21 of your cycle is an essential step for those trying to conceive.

February 21st, 2023 | 8 min. read

By Sarah Waters, RN/WHNP

One of the first steps on any family-building journey is a comprehensive fertility workup. This assessment includes blood work and other tests performed at various points in your menstrual cycle. Day 21 fertility testing is an essential part of evaluating your hormone levels and ensuring your body is prepared for a healthy pregnancy.

Let's explore why!

In this article:

Why is fertility testing necessary?

Why is fertility testing necessary?

When you decide to seek help from a fertility specialist after trying to conceive with no success, your doctor's first priority is to get the most accurate picture of what you might need to achieve fertility treatment success.

As part of your fertility testing workup, your reproductive endocrinologist will order tests like a hysterosalpingogram (HSG) and saline sonogram (SHG) to evaluate the health of your uterus and fallopian tubes. Many of these tests need to be performed at a particular point in your menstrual cycle. One of those important tests is referred to as the "Day 21" test.

What is Day 21 fertility testing?

"Day 21" of your menstrual cycle can be a useful day to evaluate several different processes, particularly for those having trouble conceiving on their own. This evaluation involves checking the levels of certain hormones (like progesterone and estradiol) via bloodwork and measuring the thickness of your endometrium (uterine lining). 

For someone with a 28-day cycle, it takes 14 days to develop a follicle and ovulate the oocyte (egg), 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. 

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 more predictable than the follicular phase. So what happens if you don’t get a menstrual cycle every 28 days?

Maybe you get your menses once every two to 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 occur more frequently. Let's talk about how "Day 21" looks for those with irregular or absent cycles.

Day 21 Testing for Irregular Menstrual Cycles

We often refer to "Day 21" in quotes because it doesn’t really make sense for the many people who don’t have typical 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.

While the follicular phase can vary, the luteal phase always takes about 14 days. Your peak progesterone day should still be seven days after ovulation and seven days before your period begins.

How "Day 21" testing works if you have longer or shorter cycles:

If you have 35-day cycles, then you ovulate around day 21, and your peak progesterone level would be checked around day 28. If your cycle typically lasts 25 days, your peak progesterone level would be checked around day 18. 

Your reproductive endocrinologist will work with you and your individual 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. 

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What cycle day do you ovulate?

People who have regular cycles presumably ovulate every month at a predictable time. In a typical 28-day cycle, it takes about 14 days to grow and develop a dominant follicle. If you are monitoring for ovulation, the leutenizing hormone (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 via bloodwork. 

The Importance of Progesterone

Progesterone is a vital part of the conception process. 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. Once ovulation has occurred, your menses (or period) should begin about 14 days later.

As you can see, Day 21 is meant to be a marker for when you're in the middle of the luteal phase of your cycle, and when progesterone production is at its peak. 

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 at all, "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. 

What are normal progesterone levels in women?

If your progesterone level is high (above 5 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.

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!

Note: There are many options to boost these hormone levels and make a receptive endometrium (uterine lining). Your doctor will help you get your levels where they need to be so you can have the best chance at a healthy pregnancy!

Progesterone levels can be supplemented with vaginal or injectable progesterone supplements. Estradiol levels can be supplemented with oral, vaginal or transdermal estrogen.

Your fertility specialist may also check your peak luteal progesterone and estrogen levels during a treatment cycle. For certain treatments, such as in vitro fertilization (IVF), we recommend estrogen and progesterone supplementation in the luteal phase to most of our patients to boost hormone levels and ensure the best chances of embryo implantation.

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.

What are the effects of low progesterone?

Low progesterone levels can affect both the menstrual cycle and a person's overall fertility, since progesterone helps create a good environment for a pregnancy to develop. When progesterone levels are low, it’s harder for an embryo to implant and grow.

Low levels of progesterone can also contribute to:

  • miscarriage
  • absence of menstruation
  • poor ovarian function

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What if I don't ovulate on my own?

If you rarely or never get your menses (i.e. have a period), it means that you may be ovulating very rarely, or you may not ovulate on your own at all, which means that your natural "Day 21" progesterone levels can’t be checked.  

If you are not ovulating or are rarely ovulating, your doctor will prescribe medication to induce ovulation. The following are some commonly used medications:

  • Clomiphene citrate (i.e. Clomid)
  • Injectables (like Ovidrel, HCG, or FSH)
  • Letrozole

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.

Success Begins With Knowledge

Fertility assessments like Day 21 testing give 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 comprehensive guide to your first consultation and download your free worksheet.

We wish you all the best on your journey to baby!

Sarah Waters, RN/WHNP

Sarah Waters is a nurse practitioner at Illume Fertility focused on research, quality assurance and patient education. Sarah has been on the Illume Fertility team since 2005 and has over 20 years of experience working in the field of fertility.

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