Impact of Serum Progesterone Levels in GnRH Antagonist Assisted Reproduction Cycles on Pregnancy Outcomes : A Prospective Cohort Study

Background: With controlled ovarian hyperstimulation (COH) with gonadotrophin releasing hormone (GnRH) antagonists, sometimes it is associated with incomplete luteolysis leading to elevated serum progesterone in early follicular phase. Persistence of this elevation might reduce the chance for clinical pregnancy. Objective: To assess the effect of elevated early and late follicular progesterone (P) levels during gonadotrophins releasing hormone (GnRH) antagonist cycles on pregnancy outcome. Design: Prospective single center study. Setting: North-western Military hospital, Kingdom of Saudi Arabia. Patients: 302 in vitro fertilization/intra-cytoplasmic sperm injection (IVF-ICSI) patients. Intervention(s): Recombinant follicle stimulating hormone (r-FSH), (150 300 IU) started daily from cycle day 2; GnRH antagonist treatment started on day 6 of the cycle. The serum progesterone (P) measured twice on cycle day 2 and human chorionic gonadotrophin (hCG) day. Main Outcome Measures: Clinical pregnancy and live birth rates per started cycle. Results: The incidence of elevated serum P on day 2 was (5.3%) and on hCG day was (17.5%), statistically significant differences in clinical pregnancy rate (32.3% versus 13.0%) and in live birth rate (23.4% versus 11.1%) were present between the normal and high serum progesterone groups on hCG day, but these differences were not statistically significant in the groups of elevated basal progesterone. Conclusion: Follicular phase progesterone rise either on day 2 or the day of hCG trigger was associated with lower clinical pregnancy and live birth rates. This impact was more prominent with trigger day elevation. How to cite this paper: Abdelaziz, A. and Atia, H. (2019) Impact of Serum Progesterone Levels in GnRH Antagonist Assisted Reproduction Cycles on Pregnancy Outcomes: A Prospective Cohort Study. Open Journal of Obstetrics and Gynecology, 9, 42-53. https://doi.org/10.4236/ojog.2019.91005 Received: December 14, 2018 Accepted: January 11, 2019 Published: January 14, 2019 Copyright © 2019 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access DOI: 10.4236/ojog.2019.91005 Jan. 14, 2019 42 Open Journal of Obstetrics and Gynecology A. Abdelaziz, H. Atia


Introduction
The usage of controlled ovarian stimulation created the necessity for continuous research efforts to adjust the physiological performance of the stimulated cycle and increase the chance for pregnancy after intra-cytoplasmic sperm injection (ICSI) cycles.The cellular and molecular impacts of progesterone rise on the human chorionic gonadotrophin (hCG) trigger day and consequently reproductive outcomes are of these concerns [1] [2].
With controlled ovarian hyperstimulation (COH) with gonadotrophin releasing hormone (GnRH) antagonists, sometimes it is associated with incomplete luteolysis leading to elevated serum progesterone in early follicular phase [3] [4].
If the elevated progesterone levels persisted or for any other reasons became high pre-ovulatory, this might reduce the chance for clinical pregnancy.Some studies confirmed in their findings the detrimental impact of this elevation [5] [6] [7], while others failed to appreciate this negative effect [8].Third group confirmed the negative effect for cases with reduced or normal ovarian response for induction, while high responders were exempted from that effect [1] [4] [9].
Most of the concerned studies neglected other peri-ovulatory parameters like number of retrieved follicles, oocyte maturation and endometrial thickness and receptivity that might modify the outcome and act as confounders for the real impact of elevated serum progesterone on the clinical pregnancy and live birth rates [10] [11] [12].
We aimed to study the impact of elevated basal serum progesterone and at the time of ovulation triggering by hCG on the success of achieving clinical pregnancy, in patients receiving controlled ovarian hyperstimulation with GnRH antagonist with normal response and in the light of other possible confounders.

Methodology
The current research is a single-center prospective cohort study of 302 cases underwent ICSI antagonist cycles, conducted at North-Western Armed Forces Hospital, Kingdom of Saudi Arabia.Recruitment of cases started from January 2015 till January 2018.Ovarian stimulation was carried out using only recombinant follicle-stimulating hormone (rFSH; Gonal-F®: Merck Serono, Geneva, Switzerland).Gonadotrophins were initiated on day 2 of the cycle.Doses varied from 150 to 300 IU/day subcutaneous injections, according to patient's age, body mass index (BMI), menstrual cycle characteristics, profile of basal hormones, and response to previous COH.Follicular growth was evaluated by vaginal sonography.A daily dosage of 0.25 mg of GnRH antagonist (Cetrorelix®; Cetrotide: MerckSerono, Geneva, Switzerland) subcutaneous administration was started on day 6 of the cycle and sustained till the day of hCG trigger.Oocyte maturation was initiated by subcutaneous administration of hCG (rhCG; Ovidrel®: 250 micrograms Merck Serono, Geneva, Switzerland) when at least one follicle had a mean diameter of 18 mm and two had a mean diameter of 16 mm.Transvaginal sonographic guided oocyte retrieval was performed 34 to 36 hours after trigger.Number of replaced embryos (1 -3 embryos) depended on the patient age, developmental stage and quality of the embryos and previous IVF cycle outcome [2].Luteal phase support began on oocyte retrieval day and was composed of vaginal progesterone applied intravaginally, in two separate doses (Crinone 8% gel; Merck Serono, Geneva, Switzerland ), and was continued till performance of the serum ß-hCG assay and, if positive, it was maintained till the 12 th gestational week.
All 302 cycles have been categorized in accordance to their progesterone serum levels at the start of COHand on hCG trigger day by usage of cutoff value of 1.5 ng/ml.Four research groups:  Group A: cases with both basal progesterone and on hCG day ≤ 1.5 ng/ml. Group B: cases with basal progesterone ≤ 1.5 ng/ml and > 1.5 ng/ml on hCG day. Group C: cases with basal progesterone > 1.5 ng/ml and ≤ 1.5 ng/ml on hCG day. Group D: cases with both basal progesterone and on hCG day > 1.5 ng/ml. These groups were reassigned into two main groups according to basal serum progesterone then its level on the hCG trigger day either below or more than 1.5 ng/ml.

Hormonal Measurement
Early in the follicular phase of GnRH antagonist cycle, serum estradiol (E2), progesterone, luteinizing hormone (LH) and follicle stimulating hormone (FSH) were measured on the second day of cycle, which was the day of initiation of therapy.Serum progesterone and estradiol were repeated on the day of triggering of ovulation by hCG.The result of progesterone did not affect the decision for hCG administration.

Primary Outcomes
Clinical pregnancy and live birth rates.Clinical pregnancy is defined by sonographic gestational sac visualization at 6 weeks of gestation with presence of embryonic heart beat on ultrasound.Live birth is birth beyond 24 weeks with any sign of life [13].

Secondary Outcomes
The dosage and duration of gonadotrophin administered; endometrial lining thickness, number of oocytes retrieved, number of metaphase II (M II) oocytes, rates of fertilization and cleavage, and available number of embryos.
All cases recruited have signed an informed written consent to allow usage of their medical data for scientific research, before beginning their own management cycles.Ethical approval was obtained from the local institutional research board of the hospital.

Sample Size
We used G*Power software (version 3.1.9.2, Heinrich-Heine-Universitat, Dusseldorf, Germany) to calculate the sample size.We calculated the required sample size sufficient for detection of difference between cases with low and high progesterone regarding clinical pregnancy and live birth rates with effect size d 0.5, α error 0.05% and power 90%.Least required sample size was 172 cases.

Statistical Methods
Statistical analysis was performed using SPSS version 21 [IBM Corp., Armonk, NY].Shapiro-Wilk test was used to examine the numerical data for normality of distribution.Normally distributed data were presented as mean ± standard deviation (SD) and in-between groups' differences were calculated using independent samples t-test and one-way ANOVA test as appropriate, Game-Howel post hoc test to estimate significance in-between groups.Categorical data were presented as number and percentage (%) and in-between groups' differences were calculated using Chi-squared test or Fisher's exact test as appropriate.Multivariable binary logistic regression was used to determine independent predictors for successful clinical pregnancy and live birth.All the potential factors were adjusted for, including: basal progesterone, FSH, number of follicles, trigger day progesterone, number of oocytes retrieved, oocyte maturation and fertilization and endometrial thickness.The "enter" method was used to force all explanatory variables into the model.

Results
At the end of study period, data of 302 participants were statistically analyzed.
Cases with both basal progesterone and on hCG day ≤ 1.5 ng/ml (group A), cases with basal progesterone ≤ 1.5 ng/ml and > 1.5 ng/ml on hCG day (group B), cases with basal progesterone >1.5 ng/ml and ≤ 1.5 ng/ml on hCG day (group C) and cases with both basal progesterone and on hCG day > 1.5 ng/ml (group D).All groups were comparable in demographic criteria including age, body mass index (BMI), parity and infertility duration.No statistically significant difference in basal FSH and estradiol serum levels, stimulation duration and endometrial thickness on the day of embryo transfer.While the differences were statistically significant between groups in basal progesterone, progesterone level on trigger day, the total amount of gonadotrophins used to accomplish ovarian hyperstimulation and the number of follicles on hCG day (Table 1).
According to basal serum progesterone levels then at the day of hCG triggering of ovulation, these four groups were coalesced into two main groups with the cutoff 1.5 ng/ml to study the outcome of ovarian stimulation, fertilization and embryo transfer for normal responders [1].The number of follicles on day of hCG trigger, endometrial thickness on day of embryo transfer and total number of retrieved and M II oocytes were also sig-

Discussion
Follicular phase progesterone rise affects 12.3% to 46.7% of the fresh IVF-ICSI cycles depending on the progesterone cut-off value used.This wide variation may be attributed to the type of stimulation protocol, gonadotrophins used and patients' characteristics such as age, body mass index, ethnicity, ovarian response and history of recurrent IVF failure [14] [15] [16].
In or its effect on oocyte/embryo quality or the synchrony between the two (advancement of implantation window) [19].
The present literature presents conflicting evidence on its effect on oocyte and embryo quality.While some studies demonstrated an adverse effect on oocyte and top quality embryos rates [20] [21] [22] [23], others showed comparable oocyte quality, fertilization, cleavage rates and embryo quality between high and normal progesterone groups [9] [24]- [29].When it comes to clinical pregnancy and live birth rates, the dilemma continues.Some reports showed evidence of improved outcome [30] [31], while others concluded no significant effect [32] DOI: 10.4236/ojog.2019.91005 nificant independent predictors for clinical pregnancy occurrence in the study group [B = −1.502,−1.073, 0.979 and 0.966 respectively].The same variables were significant predictors for live birth [B = −0.995,−0.419, 0.417 and 0.412 respectively] beside the trigger day progesterone [B = −1.961].These can function as modifiers of the real impact of high trigger day serum progesterone on clinical pregnancy and live birth rates (

Table 1 .
Baseline and stimulation parameters among the studied groups.
Cases with higher basal serum DOI: 10.4236/ojog.2019.91005 a Group A: cases with both basal progesterone and on hCG day ≤ 1.5 ng/ml.b Group B : cases with basal progesterone ≤ 1.5 ng/ml and > 1.5 ng/ml on hCG day.c Group C : cases with basal progesterone > 1.5 ng/ml and ≤ 1.5 ng/ml on hCG day.d Group D : cases with both basal progesterone and on hCG day > 1.5 ng/ml.*Statistically significant result.BMI: body mass index; FSH: follicle stimulating hormone; ET: embryo transfer.binary logistic regression model, despite progesterone level on day of ovulation trigger was the strongest independent reverse predictor [regression coefficient (B) = −4.915] of clinical pregnancy occurrence, it was not the only predictor.DOI: 10.4236/ojog.2019.91005

Table 2 .
Outcome of ovarian stimulation, fertilization and embryo transfer between cases with basal serum progesterone ≤ 1.5 ng/ml and those with > 1.5 ng/ml.

Table 3 .
Outcome of ovarian stimulation, fertilization and embryo transfer between cases with serum progesterone ≤ 1.5 ng/ml and those with > 1.5 ng/ml on hCG day.

Table 4 .
Multivariable binary logistic regression model showing different parameters as independent predictors for occurrence of clinical pregnancy.95% confidence interval; B, regression coefficient; OR, odds ratio; SE, standard error.Hosmer and Lemeshow goodness of fit test was insignificant.χ 2 (8, N = 302) = 150.69,P < 0.016.36.4% to 52.0% of the variance in the dependent variable can be explained by independent variables in the model.Percentage of accuracy (PAC) of the model is 78.8%.Statistically significant result.

Table 5 .
Multivariable binary logistic regression model showing different parameters as independent predictors for occurrence of live birth.
[4]the study done by Hamdine et al.[4]incorporating their results with the above mentioned studies, their meta-analysis showed that the chance of achieving ongoing pregnancy decreases by 15% if serum progesterone was high in early follicular phase in GnRH antagonist cycle.In view of available evidence, elevated serum progesterone in follicular phase may be a contributing factor to lower clinical pregnancy and live birth rates.In this study elevation of serum progesterone on day of hCG trigger was associated with higher number of retrieved oocytes (15.81 ± 1.56 versus 10.79 ± 1.23 oocytes) and metaphase II oocytes (8.93 ± 2.57 versus 6.86 ± 1.51 oocytes) compared to the group with normal serum progesterone on day of trigger.However, maturation and fertilization rates were higher in group with normal serum progesterone on day of trigger when compared to the group with the elevated serum progesterone (63.6% versus 56.5% and 66.5% versus 59.3% respectively).These differences in outcomes between both groups continued to be in favor of normal serum progesterone in embryo cleavage rate (98.2% versus 92.5%), clinical pregnancy rate (32.3% versus 13.0%) and live birth rate (23.4% versus 11.1%) compared to elevated progesterone group on day of trigger.The lower clinical pregnancy and live birth rates in group with high basal or late follicular progesterone may be explained by its effect on endometrium (altered receptivity)