Natural Cycle Versus Hormone Replacement Therapy Cycle for a Frozen-thawed Embryo Transfer in PGT Patients: A Randomized Trial
Phase: PHASE4
Conditions: Miscarriage, Frozen Embryo Transfer, Natural Cycle, Hormone Replacement Therapy Cycle, Preimplantation Genetic Screening, Euploid Embryos
Sponsor: CRG UZ Brussel

                                         
 
 
HRT_NC-FET  
 
Protocol Version 4 -  26/03/2019 
 
Study Protocol  
Study Title: Natural cycle versus hormone replacement therapy cycle for a frozen-thawed  
embryo transfer in PGT patients. A randomized trial.  
Protocol Number: HRT_NC-FET 
Protocol Version and Date: Version 4; Date, 26/03/2019  
EudraCT Registry Number: 2018-003156-20 
ClinicTrials.gov Registry Number:   
 
 
 
 
 
Investigational product: 
 
 
 
 
 
 
Sponsor:  
 
 
Coordinating/Principal Investigator: Dr. Caroline Roelens,  Prof. Dr. Christophe Blockeel
 
 
 
The information contained in this document is the property of the Sponsor/Coordinating 
Investigator and may not be reproduced, published or disclosed to others without written 
authorization of the Sponsor/Coordinating Investigator. 
 
 

                                         
 
 
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PROTOCOL SIGNATURE PAGE 
Protocol Version and date: Version 1, 29/10/2018  
 
Protocol Title: Natural cycle versus hormone replacement therapy cycle for a frozen- 
thawed embryo transfer in PGT patients. A randomized trial.  
Sponsor:  
Coordinating/Principal Investigator: Dr. Caroline Roelens, Prof. Dr. Christophe Blockeel 
I agree: 
- 
to assume responsibility for the proper conduct of this study 
- 
to conduct the study in compliance with this protocol and any future 
amendments  
- 
not to implement any deviations from or changes to the protocol without prior 
review and written approval from the Ethics Committee, except where necessary 
to eliminate an immediate hazard to the subjects, or for administrative aspects of 
the study (where permitted by all applicable regulatory requirements) 
- 
that I am thoroughly familiar with the appropriate use of the investigational drug, 
as described in this protocol 
- 
to ensure that all persons assisting me with the study are adequately informed 
about the investigational drug and their study-related duties and functions as 
described in the protocol 
- 
that I am aware of and will comply with the current good clinical practice (GCP) 
guidelines and ethical principles outlined in the Declaration of Helsinki 
- 
to conduct the study in accordance with all applicable laws and regulations 
 
Printed name       Caroline Roelens 
 
 
     Christophe Blockeel   
 
Signature                                                             Date      
 
 

                                         
 
 
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1. Introduction:  
Cryopreservation of frozen-thawed embryos has become an indispensable procedure in 
reproductive medicine. Mainly, thanks to the development of the vitrification procedure and 
the improvement of embryo survival rates after thawing (Rienzi et al., 2017; Loutradi et al. 
2008), pregnancy rates have substantially increased. Likewise, the implementation of a 
single embryo transfer policy to reduce multiple pregnancies without lowering cumulative 
delivery rates (Peeraer et al., 2014) has led to an increase in surplus embryos deriving from 
an ovarian stimulation cycle.  Besides, freeze-all strategies have been adopted for an 
increasing number of indications, namely to avoid ovarian hyperstimulation syndrome 
(=OHSS), or for preimplantation genetic testing (=PGT) patients, or in case of late follicular 
progesterone (P) rise or in case of endometrium- embryo asynchrony (Bosch et al., 2016; 
Roque et al., 2015; Healy et al., 2016; Devroey et al., 2011; Shapiro et al., 2008; Racca et al, 
2018; Groenewoud et al., 2017).  These developments have led to an increase in the number 
of frozen-warmed embryo transfer (FET) cycles performed in fertility centres over the last 
decade.  
Despite this growing importance, little is known about the optimal endometrium 
preparation protocol prior to embryo transfer (Ghobara et al, 2017; Groenewoud et al., 
2017). The two most common used treatment regimens to prepare the endometrium for 
embryo transfer are the so called natural cycle (NC) and the hormone replacement therapy 
(HRT) cycle. In a natural cycle the endometrium undergoes structural changes caused by 
consecutive exposure to estrogens and progesterone derived from the growing follicle. The 
moment of embryo transfer is determined by the occurrence of the natural ovulation, 
meaning on the fourth day after luteinizing hormone (LH) surge for cleavage stage embryos 
and for blastocysts on the sixth day (Mackens et al., 2017).  HRT protocols are using 
exogenous administration of estrogens and progesterone in order to mimic a natural cycle 
and therefore endometrial development. The advantages of this latter protocol are the easy 
scheduling and minimal cycle monitoring. So far, no RCTs have been performed comparing a 
true NC (without hCG administration) and an HRT cycle for FET.  However, concerns are 
raising about the high miscarriage rates reported in HRT cycles (Tomás et al., 2012; 
Hreinsson et al., 2016; van de Vijver et al., 2016; van de Vijver et al., 2017).  

                                         
 
 
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2. Study Flowchart and Design 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Group A: NC (awaiting spontaneous LH surge) 
 
 
 
 
      D1-2 
  
D10-12 
       B  
 
 B+US 
 
 
 
Group B: HRT1 
 
 
 
 
       D1-2          
      
 
 
D10-14 
       B  
               
 
 
  B+US 
 
 
                                                      
1 Progesterone supplementation is started when the endometrium is considered adequate after E2 administration. E2 
supplementation vary between 1 to 3 weeks depending on the adequate proliferation of the endometrium.  
ICSI  
+ PGT D5  
Freeze all 
FET NC  
FET HRT 
LH surge  
+1 
+2
+4 
+3 
+5 
+6 
FET 
Day 5 
+1 
+2
+3 
+4 
+5 
+6 
FET 
Day 5 
E2 supplementation 
P  supplementation 

                                         
 
 
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Legend: B: serum hormone analysis (estradiol (E2), P, LH, follicle stimulation hormone (FSH)); 
US: ultrasound scan; P: progesterone 
 
3. Study objectives and endpoints 
The aim of the current RCT is to compare miscarriage rates (before 8 weeks) between true 
NC (awaiting spontaneous LH surge) and HRT FET cycles in PGT patients, with biopsy on day 
5 of embryonic development. The advantage of performing the study in PGT patients is the 
exclusion of aneuploidy as a cause of miscarriage. 
 
3.1 Primary outcome:  
Miscarriage rate before 8 weeks of gestation, defined as a spontaneous loss of a clinical 
pregnancy before 8 weeks of gestational age, in which the embryo(s) is/are nonviable and 
is/are (not) spontaneously absorbed or expelled from the uterus (Zegers-Hochschild et al 
2017).  
 
3.2 Secondary outcomes:  
Miscarriage rate after 8 weeks of gestation, defined as a spontaneous loss of a clinical 
pregnancy after 8 weeks but before 22 completed weeks of gestational age, in which the 
embryo(s) or fetus(es) is/are nonviable and is/are not spontaneously absorbed or expelled 
from the uterus (Zegers-Hochschild et al 2017). 
Clinical pregnancy defined as a pregnancy diagnosed by ultrasonographic visualization of one 
or more gestational sacs or definitive clinical signs of pregnancy. In addition to intra-uterine 
pregnancy, it includes a clinically documented ectopic pregnancy (Zegers-Hochschild et al 
2017).  
Ongoing pregnancy rate defined as the number of pregnancies after 20 weeks of gestation.  
 
4. Methodology  

                                         
 
 
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4.1 Study design  
The current study will be an open label randomized trial using a two–arm design with 1:1 
allocation ratio.  The participating centre is the Centre for Reproductive Medicine, 
Universitair Ziekenhuis Brussel, Belgium. PGT patients who fulfil the inclusion criteria of the 
study will be recruited from this centre. The aim of the current study is to investigate the 
effect of the above-mentioned treatment regimen (NC and HRT) in FET cycles on miscarriage 
rate. 
  
4.2 Selection of study population 
The study population consists of all PGT patients who fulfil the following inclusion criteria:  
- 
Age between 18 and 42 years  
- 
BMI under 35 kg/m2 
- 
Regular menstrual cycle pattern (i.e. 24-35 days cycle) 
- 
First, second and third ICSI-PGT cycle 
- 
First frozen embryo transfer cycle following a fresh ICSI-PGT attempt 
- 
PGT with trophectoderm biopsy on day 5 of embryonic development  
- 
Signed informed consent  
 
The exclusion criteria involve: 
- 
Oligo-amenorrhea 
- 
Age above 43 years  
- 
BMI above 35  
- 
Contraindications for the use of hormonal replacement therapy 
 

                                         
 
 
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4.3 Screening 
On the first consultation, the treating physician will inform the eligible PGT patients about 
the study. Patients willing to participate and fulfilling the inclusion criteria will be 
randomized to one of the two treatment arms following the fresh ICSI cycle if non-affected 
embryos are available. Informed consent forms will be signed either on the first day of the 
following menstrual cycle or on the moment of frozen-warmed embryo transfer.  
 
4.4 Randomization  
Patients will be randomized to either natural cycle frozen-thawed embryo transfer (group A), 
or hormonal replacement therapy cycle frozen-thawed embryo transfer (group B). As 
mentioned above, patients will only be randomized if they meet the complete eligibility 
criteria. Randomization will be ensured using a computer-generated randomization list and 
allocation concealment by the use of sealed opaque envelopes in the centre (physician will 
neither have access to the randomization list nor to the envelopes, which will be opened by 
study nurses involved in the study). 
 
4.5 Interventions 
- 
Natural cycle FET (Group A) 
Patients are asked to perform a blood sample, with evaluation of serum estradiol 
(E2), progesterone (P), luteinizing hormone (LH) and follicle stimulating hormone 
(FSH), on the first or second day of the menstrual cycle (day 1 is considered as the 
first day of fresh red blood loss before 12AM). If these serum hormonal values are 
considered basal for the beginning of the follicular phase, patients are asked to come 
back on day 10 to 12 of the cycle for blood sample and transvaginal ultrasound scan 

                                         
 
 
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in order to assess follicular growth.  Depending on the values on day 1 or 2, the 
physician performing the daily monitoring can decide to perform an earlier 
examination if necessary. 
The timing of ovulation is determined based on a combination of ultrasonography 
features (the presence of a dominant follicle and adequate endometrium) and 
endocrine hormonal values in serum blood samples. Ovulation is generally defined as 
an, at least, 180% increase of LH compared to the mean level in the previous 24h 
(Frydman et al., 1982). 
Frozen-warmed embryo transfer will take place six days following the spontaneous 
LH surge.  Another blood sample will be performed 12 days after embryo transfer 
measuring human chorionic gonadotrophin (hCG), progesterone and estradiol in 
order to assess the outcome of the FET cycle.  
- 
Hormonal replacement therapy cycle FET (Group B)  
Patients are asked to perform a blood sample, with evaluation of serum estradiol 
(E2), progesterone (P), luteinizing hormone (LH) and follicle stimulating hormone 
(FSH) on the first or second day of the menstrual cycle (day 1 is considered as the 
first day of fresh red blood loss before 12AM). If these values are considered basal 
for the beginning of the follicular phase, estrogen supplementation (Estradiol 
valerate, Progynova® 3x2mg/day) is started to induce proliferation of the 
endometrium.  Blood sample and transvaginal ultrasound are thereafter performed 
ten to fourteen days later. If the endometrium is considered adequate (generally 
considered if triple line and above 6,5 mm thickness)(Liu et al., 2018), embryo 
transfer is scheduled on the sixth day of progesterone (vaginal micronized 
progesterone, Utrogestan® 2x200mg twice a day) supplementation.  
If the endometrium remains inadequate on day 10-14, estrogen priming will be 
prolonged with a maximum of 14 days of additional estrogen supplementation. If 
hereafter the endometrium stays below 6,5mm the cycle will be cancelled.  

                                         
 
 
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Another blood sample will be performed 12 days after embryo transfer measuring 
hCG, P and E2 in order to assess the outcome of the FET cycle. 
In case of a positive pregnancy test, estrogen and progesterone supplementation will 
be continued until 8 weeks of gestation (in line with the onset of the luteo-placental 
shift) (Scott et al., 1991).  
In case of escape spontaneous ovulation embryo transfer will be performed 
considering the presumable time of ovulation.  
5. Treatment 
- 
Group A: NC FET 
In this group, no medication will be administered to the patients. The aim of this 
protocol is to detect the occurrence of a spontaneous LH surge in order to calculate 
the day of embryo transfer.  
 
 
 
 
- 
Group B: HRT FRET  
Estradiol valerate (Progynova®, Bayer-Schering Pharma AG, Berlin, Germany) will be 
started on the first or second day of the menstrual cycle, in case of basal hormonal 
conditions, at a regimen of 2mg thrice a day. After ten to fourteen days of estrogen 
supplementation a check-up is planned with a blood sample and transvaginal 
ultrasound. If the endometrium is adequate (as above mentioned) transfer of the 
frozen-thawed embryo is planned on the sixth day of progesterone supplementation. 
We will use micronized progesterone vaginally (Utrogestan®, Besins International) 
two capsules of 200 mg twice a day.  

                                         
 
 
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HRT cycles, using a consecutive administration of estrogen and progesterone, are 
used worldwide especially due to the advantage of easy and flexible scheduling and 
minimal monitoring which reduces the visits to the hospital compared to NC FRET.  
Although the universally use of HRT protocols in fertility centres no consensus is yet 
established concerning the ideal duration, route of administration or type of drugs 
used in order to prepare the endometrium for embryo transfer (Glujovsky et al., 
2010).  
We will adopt the above-mentioned protocol which is in line with the local protocol 
applied in the Centre of Reproductive Medicine in Brussels.  
6. Data collection and Management 
5.1 Statistical analysis 
Continuous variables will be analyzed using the independent t-test or Mann–Whitney U-test 
depending on the normality of the distribution. Normality will be examined by the use of the 
Shapiro–Wilk test. Categorical variables will be analyzed by Pearson's chi-squared test or 
Fisher's exact test, as appropriate. To identify characteristics that may be related with the 
miscarriage rate, multivariate logistic regression analysis will be performed with the 
miscarriage rate as the dependent variable and transfer policy (NC versus HRT) as the main 
independent variable. Candidate variables considered for the analysis are: age, BMI, 
indication of infertility, percentage of patients with at least one top quality embryo and 
outcome of the previous fresh attempt. All candidate variables will be simultaneously 
entered into the logistic regression model. All tests will be two-sided with a level of 
significance set at p < 0.05. Analyses will be performed using STATA 15.0. 
5.2 Sample size calculation 
Sample size calculation was performed using internal data of our centre considering early 
miscarriage rates (defined as miscarriage before 8 weeks of gestation). We observed in our 
PGT population in 2017 an early miscarriage rate per started FET cycle of 9,9% in the natural 
cycle group compared to 18,4% in the group using HRT.  

                                         
 
 
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Based on these results, 522 patients are required (261 in each treatment arm) to have an 
80% chance of detecting, as significant at the 5% level, an increase in the primary outcome 
measure (early pregnancy loss) from 9,9% in the NC group to 18,4% in the HRT group.  
 
7. Ethical consideration 
The protocol will be submitted to the local Ethics Committee of UZ Brussel. Strict 
confidentiality of all personal and research data will be ensured. The recruiting centre will be 
required to sign a clinical trial agreement document detailing their commitment towards 
complying with the relevant laws, regulations, codes of practice and obligations to 
publication. There are no conflicts of interest. 
No specific reimbursement will be given to patients participating in the study.  
 
 
 
 
 
 
 
 
 
 
 
 
8. References  
Bosch E, Labarta E, Kolibianakis E, Rosen M, Meldrum D. Regimen of ovarian stimulation 
affects oocyte and therefore embryo quality. Fertil Steril 2016;105:560-570 
Devroey P, Polyzos NP, Blockeel C. An OHSS-Free clinic by segmentation pf IVF treatment. 
Hum Reprod 2011; 26:2593-2597.  
 

                                         
 
 
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Frydman R, Testart J, Fernandez H, Arvis P, Belaesch JC. [Prediction of ovulation]. J Gynecol 
Obstet Biol Reprod (Paris) 1982;11:793-799. 
 
Ghobara T, Gelbaya TA, Ayeleke RO. Cycle regimen for frozen-thawed embryo transfer 
(Review). Cochrane Database Syst Rev 2017;7:Art. No.:CD003414.  
 
Glujovsky D, Pesce R, Fiszbajn G, Sueldo C, Hart RJ, Ciapponi A. Endometrial preparation for 
women undergoing embryo transfer with frozen embryos or embryos derived from donor 
oocytes. Cochrane Database Syst Rev 2010;1:Art. No.:CD006359.  
 
Groenewoud E, Cantineau A, Kollen B, Macklon N, Cohlen BJ. What is the optimal means of 
preparing the endometrium in frozen-thawed embryo transfer cycles? A systematic review 
and meta-analysis. Hum Reprod 2017; 23:255-261. 
 
Groenewoud E,Cohlen BJ, Al-Oraiby A, Brinkhuis EA, Broekmans FJM, de Bruin JP, van Dool 
G7, Fleisher K, Friederich J, Goddijn M, Hoek A, Hoozemans DA, Kaaijk EM, Koks CAM, Laven 
JSE, van der Linden PJQ, Manger AP, van Rumste M, Spinder T, Macklon NS. Influence of 
endometrial thickness on pregnancy rates in modified natural cycle frozen-thawed embryo 
transfer. Acta obstet Gynecol Scand 2018;97:808-815. 
 
Healy MW, Patounakis G, Connell MT, Devine K, DeCherney AH, Levy MJ, Hill MJ. Does a 
frozen embryo transfer ameliorate the effect of elevated progesterone seen in fresh transfer 
cycles? Fertil Steril 2016;105:93-9.el.  
 
Hreinsson J, Hardarson T, Lind A-K, Nilsson S, Westlander G. Perspectives on results from 
cryopreservation/thawing cycles. Hum Reprod 2016;31:2894-2894. 
 
Liu KE, Hartman M, Hartman A, Luo Z-C, Mahutte N. The impact of a thin endometrial lining 
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Reprod 2018;33:1883-1888.   
 
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Peeraer K, Debrock S, Laenen A, De Loecker P, Spiessens C, De Neubourg D, D’Hooghe TM. 
The impact of legally restricted embryo transfer and reimbursement policy on cumulative 
delivery rate after treatment with assisted reproduction technology. Hum Reprod 
2014;20:267-275.  
 

                                         
 
 
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Racca A, Santos-Ribeiro S, De Munck N, Mackens S, Drakopoulos P, Camus M, Verheyen G, 
Tournaye H, C Blockeel. Impact of late-follicular phase elevated serum progesterone on 
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Roque M, Velle M, Guimarães F, Sampaio M, Geber S. Freeze-all policy: fresh vs. frozen-
thawed embryo transfer. Fertil Steril 2015;103:1190-1193. 
 
Scott R, Navot D, Liu HC, Rosenwaks Z. A human in vivo model for the luteoplacental shift. 
Fertil Steril 1991a;56:481-484.  
 
Shapiro BS, Daneshmand ST, Garner FC, Aguirre M, Ross R. Contrasting patterns in in vitro 
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