Serum Estradiol Level as a Predictor of Ovarian Response and Pregnancy Outcome During Controlled Ovarian Hyperstimulation in Women from Gaza Strip

Background: Currently, controlled ovarian hyperstimulation (COH) is monitored by serum estradiol (E2) levels which are believed to primarily detect functional activity of follicles. Objective: To evaluate estradiol level as a predictor of ovarian response and pregnancy outcome during COH in women from Gaza Strip. Methods: This prospective cohort study consisted of 75 women attending in vitro fertilization (IVF) at Al-Basma Fertility Center in Gaza City. Blood withdrawal for E2 hormone measurement was performed in all the patients and the number of oocytes and embryos were recorded for each female and the occurrence of pregnancy was followed for three months. Obtained data were computer analyzed using SPSS statistical package version 18. Results: The mean age of the study population was 29.2±5.9 years. Questionnaire interview showed that the cause of infertility was mostly referred to husbands. More than half of women seeking IVF had no children and had repeated IVF. The mean level of E2 showed the highest value of 2194.4 (pg/ml) at age group 26-35 years. However, the difference in E2 levels among the age groups was not significant (F=0.940 and P=0.395). When related to the number of oocytes retrieved, E2 level showed general increase with increase ovarian response, recording values of 1642.7, 1665.1, 2156.8 and 1798.7 pg/ml with <4, 4-8, 9-16 and >16 oocytes, respectively, but this change was not significant (F=0.219 and P=0.883). The mean level of E2 showed its maximum value of 2143.6 pg/ml in positive pregnancy. However the difference in E2 levels among the different categories of IVF outcome was not significant (F=0.423 and P=0.656). The numbers of total and mature oocytes, and embryo were significantly increased with increased levels of E2, showing a good response at E2 level=1000-2000 pg/ml. The number of mature oocytes showed positive correlations with E2 and number of embryo (r= 0.159, P= 0.177 and r= 0.890, P=0.000, respectively) and negative correlation with age (r=-0.276, P=.017). Conclusion: Estradiol level 1000-2000 pg/ml at mean age of 27.8±4.9 years, could be a predictor of ovarian response and pregnancy outcome during COH. Consequently, for women to undergo a more likely successful IFV program, it is recommended to optimize E2 level at 1000-2000 pg/ml.


Introduction
Controlled ovarian hyperstimulation is a standard practice in assisted conception programs.Monitoring of COH in IVF aims to evaluate whether the response to exogenous gonadotropins is sufficient to obtain an adequate number of mature oocytes.The second aim is to determine the optimal time for induction of final oocyte maturation by human chorionic gonadotropin (hCG) administration.Currently, COH is monitored by repeated pelvic ultrasonography or serum E2 measurement.It is believed that ultrasonographic findings reflect growth, whereas serum E2 levels primarily detect functional activity of follicles 1 .Estradiol is the 2nd predominant steroid sex hormone present in females when naturally produced, synthesized from cholesterol and produced mainly by the ovary, placenta, and in smaller amounts by the adrenal cortex 2 .There are two peaks of secretion: one just before ovulation and one during the midluteal phase.In blood stream, 98% of estrogen circulates bound to sex hormone binding globulin 3,4 .Estrogens facilitate the growth of the ovarian follicles and increase the motility of the uterine tubes.They increase uterine blood flow and have important effects on the smooth muscle of the uterus 5 .Globally several investigators have been focused on serum E2 level in COH to improve the outcome of IFV 1,6,7,8 .In the Gaza Strip only one study speculates the role of antimullerian hormone as a predictor of ovarian reserve and ovarian responses in IVF candidates 9 .However, there were no previous studies on the role of serum E2 in COH or even in the IVF process.Therefore, this study is the first to assess the E2 levels during COH influence the ovarian response and pregnancy outcome in IVF women from Gaza Strip.

Materials and Methods
This prospective cohort study consisted of 75 women undergoing COH program and aged between 20-40 years without history of other diseases.The subjects were recruited from Al-Basma fertility Center in Gaza City in the period September 2011 to November 2011.Each patient gave informed consent to participate in the study.The criteria for inclusion were as follows: (i) age 20-40 years, (ii) regular menstrual cycle, (iii) not on hormone therapy for three months and (iv) have not been subject to surgical operation in the reproductive system.This study was approved by the Ethical Committee of the institution.All participants were guaranteed confidentiality, and only the principal investigator has full access to the data.

Sonography, Blood Sampling and IVF Protocol
The vaginal sonography was performed in the second day of the menstrual cycle.Blood samples were collected on the third day of the menstrual cycle.Serum samples were then separated by centrifugation at 3500 rpm for 10 minutes and stored at -20°C until use.All serum samples were submitted to E2 determination using Diagnostic Systems Laboratories Inc ELISA kit for E2.There were two protocols used during ovarian hyperstimulation, the first one is long protocol, which relies on pre-stimulation pituitary down regulation using GnRH agonists in daily intermittent or depot formulations, the second one is short protocols using GnRH antagonist during the late follicular phase of the stimulation cycle have been utilized.Adding recombinant LH to recombinant FSH protocols, when starting antagonists, as a strategy to increase oocyte yield and improve pregnancy rates.Human chorionic gonadotropin (hCG) was injected at a dose of 5000 or 10,000 IU.Oocyte retrieval for IVF was then typically scheduled for 30-34 hr thereafter.Then the fertilized oocyte was placed in G1 media for 3-4 days then in G2 media before rewind.

Statistical analyses
Simple distributions of the study variables and cross tabulation were applied.One-way ANOVA test was used for evaluating the relation between hormone level and qualitative variables.Person correlation test was applied.The results in all the above mentioned procedures were accepted as statistically significant when the p-value was less than 5% (p<0.05).

Results
The study population comprised 75 females who had seeking in IVF at Al-basma fertility Center in Gaza City, Gaza Strip.Age classification of the study population revealed that more than half of woman 42 (56.0%)were 26-35 years old.The age was ranged from 20-42 years with mean of 29.2±5.9 years (Table I).Clinical data showed that most of the interviewed woman 46 (61.3%) claimed that their husbands were the main cause of infertility.Forty six (61.3%) women do not have children.Around half of population 38 (50.7%) had undergone repeated IVF; out of them 30 (40.0%) had repeated IVF 2-4 times.The result of IVF was positive in 27 (36.0%) of the study population.

Discussion
In the Gaza Strip, women seek IVF mostly when the man had fertility problems and the process of pregnancy is delayed.Data on IVF are rare in Gaza Strip.There is only one recent study on the role of antimullerian hormone as a predictor of ovarian reserve and ovarian response in IVF candidates in Gaza Strip 9 .The present study is the first to assess serum estradiol level and it is possible role as an indicator of ovarian response and pregnancy outcome during COH in women from Gaza Strip.The results presented in this study dealt with 75 women enrolled in IVF programs.The mean age of women (29.2 years) was close to that reported in Egyptian (29.0 years) and Iranian (29.1 years) studies 10,11 .But lower than that reported from the Maryland and South Korea (34.0 years) 8,12 .The younger age of women seeking IVF in developing countries, including Gaza Strip, could be explained in the context of social habits where most families have the desire to have children immediately after marriage and they encourage women to undergo IVF.This is supported by the finding that more than half of the study population had no children and had undergone repeated IVF.The outcome of IVF was positive in 27 (36.0%) of the study population.This finding was in agreement with the results of studies from the Netherlands and South Korea which showed that 36.0%and 32.5% of women undergoing IVF achieved viable pregnancy 8,13 6,8,12,17 .E2 level showed its maximum value of 2143.6 pg/ml in positive pregnancy.This reinforced the previous finding that such E2 level, which was postulated to be used as primary indicator of good ovarian response, could enhance the chance of pregnancy.Higher levels of E2 in pregnant women undergone IVF were recorded 9,7,13,18,19 .At E2 level 1000-2000 pg/ml, the highest number of mature oocytes and embryo were detected.Similar result was reported 6,8,12 .From clinical point of view, one can say that at E2 level of 1000-2000 pg/ml, good ovarian response and high chance of pregnancy could be achieved.Consequently, for women to undergo a more likely successful IFV program, it is recommended to optimize E2 level at 1000-2000 pg/ml.

Conclusion
Estradiol level 1000-2000 pg/ml at mean age of 27.8±4.9years, could be a predictor of ovarian response and pregnancy outcome during controlled ovarian hyperstimulation.Consequently, for women to undergo a more likely successful IFV program, it is recommended to optimize E2 level at 1000-2000 pg/ml.

Table I
Clinical data of the study population (n=75)As indicated in table II, the mean levels of E2 showed the highest value of 2194.4 (pg/ml) at age group 26-35 years.However the difference in E2 levels between the age groups was not significant (F=0.940 and P=0.395).According to the number of oocytes retrieved upon stimulation by menotrophin (FSH 75IU, LH 75 IU), the study population was divided into poor, normal, good and high responders.
* One case had no sperm

Table III
Estradiol level in relation to age, number of oocytes and embryoAs illustrated in TableIV, the number of mature oocytes showed positive correlations with E2 level and number of embryos (r=0.159, P=0.177 and r=0.890,P=0.000) and negative correlations with age (r=-0.276,P=0.017).