A Novel Fertility Indicator Equation Using Estradiol Levels to Identify the Fertile Window
By: James Redmond
Editor’s Note: Could the fertility indicator equation using serum estradiol levels serve as an additional tool to improve currently available fertility awareness-based methods? Today’s research blog, written by former FACTS elective student James Redmond, summarizes an investigation by Usala S. et al., which aimed to address this question to offer patients more accurate information to identify their fertile window. In turn, this could also shorten the period of abstinence required with FABM use.
Urinary hormone home monitoring assays measuring urine LH and urine estrone-3-glucuronide (E3G) are available for use with fertility awareness-based methods (FABMs). These assays are utilized in the Clear Blue fertility monitor as a part of the Marquette method. However, as qualitative tests, they lack sensitivity and precision in determining the start of the six-day fertile window, which ranges from Day -4 to Day +1, with Day 0 defined as the LH peak. As a result, the Marquette method generally necessitates couples abstain from sexual relations for at least 11 to 12 days per cycle. In addition, there is more variance of day-specific urine E3G than serum estradiol (E2) levels, meaning the latter more accurately assesses the state of the preovulatory phase. Furthermore, fingerstick E2 measurements now have sensitivity and precision similar to serum E2 measurements. A fingerstick E2 monitor could be developed and integrated into various fertility awareness-based methods.
Investigators reviewed three studies (Dighe et al.  , Roos et al.  , Stricker et al.  ) with published day-specific serum E2 levels relative to the LH peak to determine a threshold E2 level that would determine the fertility start date (FSD) more precisely  . Dighe et al. used the Abbott AxSYM immunoassay platform, Roos et al. used the ADVIA Centaur XP Immunoassay system, and Stricker et al. used the Abott ARCHITECT i2000 system. During the review, the investigators devised a fertility indicator equation (FIE) that used the day-specific E2 levels to assess the FSD and transition to the luteal phase.
Investigators first tried to determine if there was a threshold serum E2 level that would accurately denote the FSD for all three studies. However, the data from the three studies precluded a reliable E2 value representing the FSD because 1) of the variance of Day -4 E2 values for each respective cohort and 2) the wide ranges of median E2 values on Day -4 across the three studies. Data from Dighe et al. had the least variance of E2 levels at any given day and the data suggested that an E2 of ~411 pmol/L (112 pg/mL) could possibly serve as the level for the FSD. However, this is highly dependent on the sensitivity and precision of the assay.
“Investigators first tried to determine if there was a threshold serum E2 level that would accurately denote the fertility start date (FSD) for all three studies. However, the data from the three studies precluded a reliable E2 value representing the FSD.”
Investigators sought to find a different way to determine the start of the fertile window. This led them to devise a novel fertility indicator equation (FIE) using day-specific serum E2 levels. The formula is the product of two sequential (normalized) changes of day-specific E2 levels with a sign to denote confidence in direction of change (+, -, indeterminate). Indeterminate (indFIE) denotes uncertainty in the direction of change. The FIE(D) plot showed a reproducible signature for the day-specific serum E2 median, upper limit, and lower limit values for each of the three studies (i.e., 9 total data sets). This reproducible pattern included the following:
- A significant increase in FIE at ~Day -4 or shortly before
- Marked maximum FIE between Day -4 and Day 0
- An inflection point with change in sign from +FIE to -FIE at Day 0 (w/ Day 0 indFIE sign)
- Short-lived, minor peak in FIE in the luteal phase at ~ Day +3 or Day +4
Using a FIE threshold of ≥2.5 as the start of the fertile phase, the FSDs were Day −5 or Day −6 for 8/9 data sets, and Day −4 for 1/9 data sets of E2 levels. The maximum FIE value ranged 9.5–27.8 and occurred most often on Day −2. An additional rule to validate the FSD states that the FSD must be confirmed by sequential + FIE values for at least two days with at least one value above the 2.5 threshold. This was devised to avoid discrepancy with the minor postovulatory E2 peak.
The FIE developed by these investigators showed remarkable plot signature consistency across all nine data sets. In light of this, the equation may be utilized to precisely determine the fertile start date (FSD). The FSD is set at a FIE > 2.5 with at least one subsequent FIE value of higher magnitude. In terms of a threshold E2 level denoting the FSD, Roos et al. and Stricker et al. had too much variance of day-specific E2 levels to find a reliable value. The Dighe et al. assay had sufficient sensitivity and precision to indicate a serum E2 level of 411 pmol/L (112 pg/mL) as the FSD with 99% of the cohort reaching this level between Day -6 and Day -4. Nonetheless, this E2 threshold value was not as precise or sensitive as the FIE in determining the FSD.
“The fertility indicator equation (FIE) developed by these investigators showed remarkable plot signature consistency … In light of this, the equation may be utilized to precisely determine the fertile start date (FSD).”
The FIE’s precision comes from its ability to detect changes in E2 that correlate with follicular development using prospective, quantitative data. Most current FABMs rely on retrospective (i.e., an individual’s previous cycles) and qualitative (e.g., threshold urinary E3G levels) data to determine the start of the potential fertile window. This potential start is the reason these methods have abstinence periods starting at much earlier than Day -4. With the FIE, an FABM could be developed that significantly decreases the required period of abstinence each cycle and approaches the range of the actual fertile window (i.e., Day -4 to Day +1). This decreased period of abstinence could lower a potential barrier to entry for some couples. While a market for fingerstick E2 remains to be seen, the FIE could likely be used for daily urinary E3G data as well, which future studies could evaluate. The use of prospective, quantitative data represents the implementation of personalized medicine in FABMs.
“With the fertility indicator equation, an FABM could be developed that significantly decreases the required period of abstinence each cycle and approaches the range of the actual fertile window (i.e., Day -4 to Day +1). This decreased period of abstinence could lower a potential barrier to entry for some couples.”
The study authors also note that the FIE could be tested with irregular cycles. They think that it could be particularly helpful for patients with polycystic ovarian syndrome, given extended intervals of anovulation. It could also be used in postpartum cycles, as serum hormone levels tend to represent impending ovulation more accurately than other biomarkers during the postpartum period. 
 Usala SJ, Trindade AA. A Novel Fertility Indicator Equation Using Estradiol Levels for Assessment of Phase of the Menstrual Cycle. Medicina (Kaunas). 2020 Oct 22;56(11):555. doi: 10.3390/medicina56110555. PMID: 33105641; PMCID: PMC7690440.
 Dighe, A.S.; Moy, J.M.; Hayes, F.J.; Sluss, P.M. High-resolution reference ranges for estradiol, luteinizing hormone, and follicle-stimulating hormone in men and women using the AxSYM assay system. Clin. Biochem. 2005, 38, 175–179. [CrossRef]
Roos, J.; Johnson, S.; Weddell, S.; Godehardt, E.; Schiﬀner, J.; Freundl, G.; Gnoth, C. Monitoring the menstrual cycle: Comparison of urinary and serum reproductive hormones referenced to true ovulation. Eur. J. Contracept. Reprod. Health Care 2015, 20, 438–450. [CrossRef]
 Stricker, R.; Eberhart, R.; Chevailler, M.C.; Quinn, F.A.; Bischof, P.; Stricker, R. Establishment of detailed reference values for luteinizing hormone, follicle stimulating hormone, estradiol, and progesterone during diﬀerent phases of the menstrual cycle on the Abbott ARCHITECT analyzer. Clin. Chem. Lab. Med. 2006, 44, 883–887. [CrossRef]
 Bouchard T, Blackwell L, Brown S, Fehring R, Parenteau-Carreau S. Dissociation between Cervical Mucus and Urinary Hormones during the Postpartum Return of Fertility in Breastfeeding Women. The Linacre Quarterly. 2018;85(4):399-411. doi:10.1177/0024363918809698
ABOUT THE AUTHOR
James Redmond is a fourth-year medical student at the Keck School of Medicine of USC in Los Angeles, CA. He is applying to residency in Obstetrics and Gynecology. He took the FACTS elective because he wanted to learn about the ways fertility awareness-based methods could help with the work-up and management of various gynecological pathologies. His interests include restorative reproductive medicine, global health, and the effects of diet and exercise on gynecological health.