Conditions: Infertility Controlled Ovarian Hyperstimulation
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 112
Healthy volunteers: yes

Brief summary:
Serum concentrations of the different hormones involved in follicular steroid genesis during a cycle of controlled ovarian stimulation with recombinant FSH or HMG will be compared in this study. Serum Progesterone (P) levels at the end of Controlled Ovarian Stimulation (i.e. the day of triggering) have been related to cycle outcome, in terms of ongoing pregnancy and live birth rates. Large cohort studies show that P levels above a certain threshold are associated with poorer cycle outcome. The mechanisms behind P elevation are not well understood yet. It has been shown that P levels are positively related to ovarian response and to the dose of FSH given during COS. Furthermore, it has been well documented that P levels at the end of stimulation are significantly higher when recombinant (r) FSH is used for COS when compared to HMG, either in a GnRH agonist long protocol or in a GnRH antagonist protocol. Some authors suggest that this finding is explained by the fact that COS with rFSH provides a higher oocyte yield than when hMG is given, so the higher P levels observed would be explained by the larger number of follicles developed when rFSH is used. On the other hand, other authors explain this event by a different follicular esteroidogenesis when HMG is used for COS compared to rFSH The hypotheisis behind this assumption is that rFSH enhances P synthesis from its precursor pregnenolone in the granulosa cells. This P is unable to be further metabolized into androgens because of the lack of 17-20 lyase in the human granulosa cells, and therefore is delivered into circulation. On the other hand, when HMG is given for COS, the ∆4 pathway is promoted, and pregnenolone will be catabolized in to Dehidroepiandrostenodione (DHEA), in the theca cells, and this one to Androstenodione, which will be finally aromatized in to estrogens. This mechanism will explain the lower P and higher E2 levels observed in HMG cycles in comparison to rFSH cycles.

Detailed description:

Arms:
arm 1: Controlled ovarian hyperstimulation with GnRH antagonists and rFSH in women with normal ovarian function. arm 2: Controlled ovarian hyperstimulation with GnRH antagonists and HP-HMG with normal ovarian function.

Interventions: intervention 1: COS with GnRH antagonists and rFSH intervention 2: COS with GnRH antagonists and HP-HMG
Intervention descriptions: intervention 1: Controlled ovarian hyperstimulation with GnRH antagonists and rFSH in women with normal ovarian function. intervention 2: Controlled ovarian hyperstimulation with GnRH antagonists and HP-HMG in women with normal ovarian function.

Protocol document:
Prot_SAP_000.pdf: 
 
 
 
 Follicular 
Esteroidogenesis 
in 
Controlled 
Ovarian 
Stimulation 
EudraCT: 2015-005762-28 
NCT:  NCT02738580 
 
PROTOCOL REVISION HISTORY
 
Version
Date
State
1
17-DEC-2015
Final
 
Confidentiality Agreement 
 
This document is a confidential communication of the Sponsor. Acceptance of this document 
constitutes the agreement by the recipient that no unpublished information contained within 
will be published or disclosed without prior written approval, except that this document may 
be disclosed to the appropriate Ethical Review Committees and Regulatory Authorities under 
the condition that they are requested to keep it.


 
Clinical Trial Protocol – [Sponsor]
[EudraCT Code] – Version ## on [Version date]
 
FPE-410/05 – Ed.4 (09.04.2019) 
[1/9] 
 
Project Title 
 Follicular Esteroidogenesis in Controlled Ovarian Stimulation 
 
Acronym:  
¡Error! No se encuentra el origen de la referencia.ESTEFOL 
EudraCT:  
2015-005762-28¡Error! No se encuentra el origen de la referencia. 
Internal Code:  
¡Error! No se encuentra el origen de la referencia.1512-VLC-066-EB 
Development Phase:  
Fase IV¡Error! No se encuentra el origen de la referencia. 
Investigational Medicinal 
Product (IMP):  
Recombinant FSH, Highly Purified HMG, Ganirelix, Triptorelin 
Research Area:  
Ovarian function and stimulation 
Report entry: 
12/17/2015 
key words: 
Estradiol, Pregnenolone, Progesterone, Testosterone, Androstenodione, ovarian stimulation
Sponsor Information 
Name 
IVI Valencia  
 
Adress 
Plaza de la policía Local, 3. 46015. Valencia 
 
e-mail 
ivivalencia@ivirma.com 
 
Phone / Fax 
[96 305 09 00 / 96 305 09 99] 
 
Principal Investigator 
Name and surname 
Ernesto Bosch Aparicio 
Filiation 
Instituto Valenciano de Infertilidad 
e-mail 
Ernesto.Bosch@ivirma.com 
Phone / Fax 
963050900/963050999 
Responsible for the administrative management of the study  
Name and surname 
Ernesto Bosch Aparicio 
 
Filiation 
Instituto Valenciano de Infertilidad 
 
e-mail 
Ernesto.Bosch@ivirma.com 
 
Phone 
963050900/963050999 
 


 
Clinical Trial Protocol – [Sponsor]
[EudraCT Code] – Version ## on [Version date]
 
FPE-410/05 – Ed.4 (09.04.2019) 
[2/9] 
 
Research team members 
Pilar Alamá
IVI VALENCIA 
Pilar.Alama@ivirma.com 
Elena Labarta
IVI VALENCIA 
Elena.Labarta@ivirma.com 
Jose Luis Zuzuarregui
IVI VALENCIA 
JoseLuis.Zuzuarregui@ivi.es 
 
 
 


 
Clinical Trial Protocol – [Sponsor]
[EudraCT Code] – Version ## on [Version date]
 
FPE-410/05 – Ed.4 (09.04.2019) 
[3/9] 
 
Responsibilities and Signatures 
By signing this protocol from the project entitled:  Follicular Esteroidogenesis in Controlled Ovarian 
Stimulation 
Those undersigning state that:  
− This  clinical trial respects the ethical and legal rules and follows good clinical practice in its 
implementation 
− It has the material and human resources needed to carry out the study, without interfering in other 
studies or clinical tasks usually entrusted to them 
− They are committed that each subject is treated and controlled according the approval granted by 
the Ethics Committee for Clinical Research, Institutional Review Board, remaining committees and 
the involved authorities 
− Collaborators included in this study are adequately trained for its implementation, they will have 
an active participation, and they consent thereto. 
 
 
 
 
 
 
 
Sponsor
 
 
 
 
 
Clínica IVI Valencia
Dr/a. Amparo Ruiz 
Director/a IVI Valencia 
 
 
12/17/2015 
 
 
 
 
 
 
Principal Investigator
 
 
 
 
 
Ernesto Bosch Aparicio
Servicio Ginecología y Reproducción Humana 
Clínica IVI Valencia 
 
 
12/17/2015 
 


 
Clinical Trial Protocol – [Sponsor]
[EudraCT Code] – Version ## on [Version date]
 
FPE-410/05 – Ed.4 (09.04.2019) 
[4/9] 
 
 
Protocol Synopsis 
Protocol Title:  Follicular Esteroidogenesis in Controlled Ovarian Stimulation 
EudraCT Code: 2015-005762-28 
Sponsor´s protocol version and date: 1.0 of 12/1/2015 
Sponsor:  
IVI Valencia  
Plaza de la policía Local, 3. 46015. Valencia 
ivivalencia@ivirma.com 
[96 305 09 00 / 96 305 09 99] 
Principal Investigator: 
Ernesto Bosch Aparicio 
Instituto Valenciano de Infertilidad 
Ernesto.Bosch@ivirma.com 
963050900/963050999 
Phase: Fase IV 
Centros participantes: 
IVI Valencia 
Investigational Medicinal Product (IMP): Recombinant FSH, Highly Purified HMG, Ganirelix, Triptorelin  
Primary Objective: To compare the serum concentrations of the different hormones involved in follicular 
steroidogenesis during an EOC cycle between FSHr and hpHMG. 
Secundary Objectives:  
- Define the area under the curve for each of the follicular steroids along the EOC in cycles stimulated with 
FSHr and hpHMG. 
- Calculate the contribution to the circulation of each of the follicular steroids per unit of follicular volume. 
- To identify the metabolic pathways of follicular steroidogenesis as a function of the type of gonadotrophin 
used for COC. 
- To compare the concentration of steroids in follicular liquid in stimulated cycles with FSHr and hpHMG. 
- Correlate the hormone levels observed in serum with the concentration in follicular fluid] 
Methodology: 110 women will be included (55 in each study group, i) FSHr; ii) hpHMG) with normal ovarian 
function, who will follow ovarian stimulation in cycle with GnRH Antagonists. For greater uniformity in the 
study population, oocyte donors will be used. 
Serum extractions and transvaginal ultrasound for folliculometry will be performed on the days of 
stimulation 1, 4, 6, 8 and the day of administration of the triptorelin. In the follicular puncture for the 
extraction of the oocytes, the first follicle will be individually aspirated to obtain the follicular fluid. The 
samples obtained will be frozen and stored at -80ºC for later analysis.  
In each serum sample they will be determined: Pregnenolone, Progesterone, 17-OH-Progesterone, 
Dehydroepiandrostenodione, Androstenodione, Testosterone, Estrone and Estradiol. 


 
Clinical Trial Protocol – [Sponsor]
[EudraCT Code] – Version ## on [Version date]
 
FPE-410/05 – Ed.4 (09.04.2019) 
[5/9] 
 
Each day's hormone concentration will be compared between the two groups, so that the profile of each of 
the hormones can be defined for both stimulation protocols, and thus the follicular steroidogenesis profile. 
The area under the curve during ovarian stimulation will be calculated for each of the hormones included in 
the study, and both groups will be compared. The proportion of patients with elevated Progesterone on the 
last day of stimulation (>1.5 ng/mL) will be compared between both groups. 
Follicular fluid concentrations will be defined for each of the steroids analyzed, and the values between both 
groups will be compared, as well as their correlation with the levels observed in serum. 
Clinical Trial Prospective randomized, open, parallel groups. 
 
Number of subjects: 110 
A previous study conducted by our group (Bosch et al, 2008) in which these two same protocols were 
compared in terms of clinical outcomes, showed that the serum P on the last day of stimulation was 0.73 ± 
0.42 in the HMG group vs 0.99 ± 0.48 in the FSH group (p<0.001). According to these data, to determine a 
difference of this magnitude with a 95% confidence level (5% alpha error) and 80% power (20% beta error), 
48 patients per group are required. Assuming a loss rate of 15%, 55 patients should be randomly assigned 
to each group, i.e. 110 patients in total 
Inclusion criteria: 
- Women 18-35 years old 
- Good state of psycho-physical health 
- Normal menstrual cycle (25-35 days) 
- Normal ovarian reserve, determined by an AMH=10-30 pMol/L) 
- BMI < 25.0 
- Other criteria to be met by oocyte donors 
Exclusion criteria:  
- Kidney failure 
- Polycystic ovary 
- BMI >=25.0 
- Any systemic or metabolic disorder that contraindicates the use of gonadotrophins. 
- Any reason for exclusion from the oocyte donation program. 


 
Clinical Trial Protocol – [Sponsor]
[EudraCT Code] – Version ## on [Version date]
 
FPE-410/05 – Ed.4 (09.04.2019) 
[6/9] 
 
Test Product, Dose and Mode of Administration:  
Arm 1:  
MEDICATION: Recombinant FSH 
Therapeutic group: Gonadotrophins for ovarian stimulation 
Route of administration: subcutaneous 
Dose: 225 IU/day 
Arm 2:  
MEDICATION: Highly purified HMG 
Therapeutic group: Gonadotrophins for ovarian stimulation 
Route of administration: subcutaneous 
Dose: 225 IU/day 
Reference Therapy, dose and Mode of administration: 
 
Duration of study: 
- Recruitment period: April 2016-July 2016 
- Estimated end-of-treatment date of the last patient: October 2016 
- End of study date: December 2016 
Schedule of Assessments: 
The patients will be recruited from the population of oocyte donors of the IVI Valencia programme. Patients 
will come to the clinic on their 2nd day of menstruation of the cycle in which ovarian stimulation is to be 
performed. At that time patients will be randomly assigned 1:1 to start COCs with 225 IU/day of FSHr (Gonal 
F) or hpHMG (Menopur). On the 6th day of stimulation, all patients will start a daily dose of 0.25 mg of the 
antagonist (Orgalutran), until the day of the final induction of oocyatric maturation. When at least 8 follicles 
reach a diameter of at least 17 mm, a bolus of 0.2 mg of triptorelin (Decapeptyl) should be administered. 
Follicular puncture will be performed 36 hours later, according to usual clinical practice.  
Serum extractions and transvaginal ultrasound for folliculometry will be performed on the days of stimulation 
1, 4, 6, 8 and the day of administration of the triptorelin, and the follicular liquid of the first aspirated follicle 
will be individually aspirated. The samples obtained will be frozen and stored at -80ºC for later analysis.  
Each serum sample shall be determined:  
- Pregnenolone 
- Progesterone 
- 17-OH-Progesterone 
- Dehydroepiandrostenodiona 
- Androstenodione 
- Testosterone 
- Strona 
- Estradiol 
- FSH 
- LH 
 
 


 
Clinical Trial Protocol – [Sponsor]
[EudraCT Code] – Version ## on [Version date]
 
FPE-410/05 – Ed.4 (09.04.2019) 
[7/9] 
 
Statistical Methods and Data Analysis 
1. 
Population 
It will include 110 women (55 in each study group) with normal ovarian function, aged between 18 and 
35 years, who will follow ovarian stimulation in cycle with GnRH antagonists. For greater uniformity in 
the study population, oocyte donors will be used. 
 
2. 
Sample size 
A previous study conducted by our group (Bosch et al, 2008) in which these two same protocols were 
compared in terms of clinical outcomes, showed that the serum P on the last day of stimulation was 
0.73 
± 
0.42 
in 
the 
HMG 
group vs 
0.99 
± 
0.48 
in 
the 
FSH 
group 
(p<0.001).                 
According to these data, to determine a difference of this magnitude with a 95% confidence level (5% 
alpha error) and 80% power (20% beta error), 48 patients per group are required. Assuming a loss rate 
of 15%, 55 patients should be randomly assigned to each group, i.e. 110 patients in total. 
 
3. 
Study Variables and Database 
3.1. 
 Main variable: 
 Area under the serum progesterone curve during ovarian stimulation 
3.2. 
Secondary variables: 
- Area under the serum estradiol curve during ovarian stimulation 
- Area under the serum Pregnenolone curve during ovarian stimulation 
- Area under the 17-OH serum progesterone curve during ovarian stimulation 
- Area under the serum Androstenodione curve during ovarian stimulation 
- Area under the serum Dehydroandrostenodione curve during ovarian stimulation 
- Area under the serum Testosterone curve during ovarian stimulation 
- Area under the curve of Serum Strone during ovarian stimulation 
- Area under the serum FSH curve during ovarian stimulation 
- Area under the serum LH curve during ovarian stimulation 
- Concentration of P in follicular liquid 
- Concentration of Estradiol in follicular liquid 
- Concentration of Pregnenolone in follicular liquid 
- Concentration of 17-OH Progesterone in follicular liquid 
- Concentration of Androstenodione in follicular liquid 
- Concentration of Dehydroepiandrostenodione in follicular liquid 
- Concentration of Testosterone in follicular liquid 
- Concentration of Estrone in follicular liquid 


 
Clinical Trial Protocol – [Sponsor]
[EudraCT Code] – Version ## on [Version date]
 
FPE-410/05 – Ed.4 (09.04.2019) 
[8/9] 
 
- Concentration of FSH in follicular liquid 
- Concentration of LH in follicular liquid 
 
3.3. 
 Control variables 
- Total number of oocytes 
- Number of mature oocytes (metaphase II) 
- Number of follicles > 10 mm on the last day of stimulation 
- Total follicular mass defined by the sum of all follicular volumes (defined by the formula 4/3 x πr3 , 
where r=radius=diameter/2) in each of the ultrasound controls. 
 
3.4. 
 Descriptive variables: 
- Age 
- Body Mass Index 
- Stimulation days 
- Total dose of gonadotrophins 
- Days with antagonist GnRH 
 
4. 
Statistical analysis 
 
4.1. 
Descriptive data analysis:  
Description of continuous quantitative variables (all study variables are) in their central parameters 
(mean and median) and dispersion values (standard deviation, p10, p25, p75, p90, maximum and 
minimum). 
 
4.2. 
Intergroup homogeneity analysis: 
 Comparison of study groups from the point of view of basal and demographic characteristics using 
Student Test "t" after checking the assumption of normality using Levéne Variance Equality Test. In the 
case of non-parametric distribution, the Mann-Whitney test will be used. 
 
4.3. 
 Analysis of the working hypothesis: 
Each hormonal determination of each day of stimulation in which it is determined will be compared 
by means of Student "t" for independent samples, once the normal distribution of the values by means 
of the tests of Kolmogorov-Smirnov and Shapiro-Wilks has been verified. Otherwise, comparisons will 
be made with the Mann-Whitney test. The area under the curve during ovarian stimulation will be 
calculated for each of the hormones included in the study, and both groups will be compared equally. 
The proportion of patients with elevated Progesterone on the last day of stimulation (>1.5 ng/mL) will 


 
Clinical Trial Protocol – [Sponsor]
[EudraCT Code] – Version ## on [Version date]
 
FPE-410/05 – Ed.4 (09.04.2019) 
[9/9] 
 
be compared between both groups by Chi-square test. For this particular event the relative risk and its 
95% confidence interval will also be calculated.