Influence of patient age on peak estradiol levels and ovarian follicle quantity with and without controlled ovarian hyperstimulation

Introduction

Hormones play an important role in reproductive processes across the lifespan, from sexual differentiation of the fetus, to development of secondary sexual characteristics at puberty, to mature ovarian and testicular function, pregnancy, parturition, lactation and reproductive ageing.  Therefore, the measurement of reproductive hormone levels can provide useful information about reproductive function for diagnostic, treatment and research purposes. Hormone levels are often measured from a blood sample – this is the ‘gold standard’ and that used in clinical situations.  However, some hormones can be measured in saliva and urine and these mediums are particularly useful for hormone determination in field research and non-clinical situations because subjects can collect and store the sample themselves (Zhang,Tian, Xie, Miao, Liu, and Wang, 2019).

Estradiol is the most biologically active of the estrogen family of steroid hormones.  In non-pregnant adult females, it is primarily produced by the ovarian follicle (Parida and Sharma, 2019).  In a clinical situation estradiol may be measured together with Luteinizing Hormone (LH) to predict the timing of ovulation (e.g. for timed intercourse, or artificial insemination).  Estradiol may also be measured together with FSH and/or AMH to determine the menopausal status of a female.  During IVF/ICSI treatment the female partner undergoes controlled ovarian hyperstimulation (COH) to induce the continued development of multiple ovarian follicles.  Measurement of blood estradiol levels gives an indication of the number of developing follicles and the risk of ovarian hyperstimulation (OHSS: a dangerous condition that can lead to stroke and death) (Rodgers, Reid, Koch, Deans, Ledger, Friedlander, Gilchrist, Walters, and Abbott, 2017). Typical reference ranges for estradiol are presented in Table 1.  However, as each method for measuring estradiol will generate slightly different values, each laboratory should establish their own reference range.

Table 1: Reference ranges for plasma estradiol

Enzyme linked immunosorbant assay (ELISA) is one method for measuring reproductive hormone levels.  In this assay a 96 well microtiter plate is coated with an antibody to estradiol. Estradiol in standards and unknowns competes with an estradiol-enzyme conjugate (estradiol – horse radish peroxidise) for the antibody binding sites.  After incubation unbound components are washed away and the bound estradiol-enzyme portion is measured by a color change reaction induced by adding the substrate tetramethylbenzidine (TMB).  This reaction produces a blue color.  The reaction is stopped by adding sulfuric acid, which turns the wells yellow. The optical density (absorbance of light through the well) is read on a plate reader at a wave length of 450 nm. The amount of estradiol-enzyme conjugate detected is inversely proportional to the amount of estradiol present in the sample or standard.  Optical density readings from the samples are compared to that of the standards to determine the level of hormone present in the sample.

Materials and Methods

Patients

Blood samples were collected for assaying between 7am and 9am from women undergoing fertility treatment. Of the 14 women, eight received controlled ovarian hyperstimulation (COH) drugs to induce the continued development of multiple ovarian follicles during in vitro fertilization (IVF) and Intracytoplasmic Sperm Injection (ICSI) treatments. The other six women underwent tracking to predict the time of ovulation. Blood samples continued to be taken throughout the menstrual cycle.

Ultrasound

A transvaginal Ultrasound (US) probe was used to count the number of follicles in the ovaries of each patient (Coelho Neto et al. 2018). The probe emits sound waves that are converted into images via a computer screen, from which the number of follicles can then be determined (Balen et al., 2003).

ELISA

The Enzyme linked immunosorbent assay (ELISA) method was used to measure estradiol levels. Following the aseptic technique to avoid contamination, Capture Antibody was pipetted into a 96 well microliter plate followed by Blocking Buffer (Guoning, Pengqi, Yan, Lu, Hua, Yunzhe, Wanghui, Chun, and Qiang, 2019). The centrifuged patient blood sample was then added to the wells following Hormone Enzyme Conjugate and incubation. The substrate tetramethylbenzidine (TMB) was then added to the wells to induce a color change which was then stopped via the addition of sulfuric acid. The optical density was then measured via a plate reader at a 450nm wave length. The amount of estradiol-enzyme conjugate measured in the sample is proportional to the estradiol present in the sample. Optical density readings from the samples are then compared to the density of the standards to calculate the level of estradiol present in the samples (Guoning et al., 2019).

Methodology

In this experiment the Interactive Virtual Endocrine Lab was used to carry out the study. The analysis using ELISA from the virtual lab used about one hour. However, in reality, ELISA analysis takes about 3 hours. After the virtual lab was completed, the integrated output was downloaded as a Microsoft Excel spreadsheet whose data was analyzed using Microsoft Excel 2016 software. The estradiol levels were recorded in the Table 2 and used for analysis for the data for each woman.

Statistical Analysis

A statistical Analysis of Variance (ANOVA) test was conducted using Microsoft Excel 2016 software to compare the mean values for estradiol in each category of treatment (IVF, ICSI, and Tracking). The results from the ANOVA analysis were as follows;

Results

Table 2.  Patient Data

Figure 1. Graphical representation of B/Bo vs. Log ₍₁₀₎ Concentration of Estradiol

Discussion

From table 2, it was seen that the concentration of estradiol was highest in patients treated with IVF than in the rest of the patients. Again, the concentration of estradiol was lowest for the patients treated with Tracking than the rest of the patients. The level of B/Bo decreased as the log of concentration of estradiol increased. Therefore, B/Bo indicated an inverse relationship with log of concentration of estradiol. In other words, an increase in the concertation of estradiol led to a decrease in the levels of B/Bo (see figure 1 above).

Conclusion

The results indicated that the number of follicles observed were highest when the concentration of estradiol was high. In this case, the number of follicles was directly proportional to the concentration of estradiol. Therefore, a control on the level of estradiol is essential for controlled fertilization such as in IVF and artificial insemination. In reality, the study results were subject to errors in the experiment such as contamination. Therefore, the experiment results would be improved by enhancing purity of the reagents and cleanliness working environment. Sterilization of the working apparatus would be essential to enhance the reliability and quality of the results.

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