Embryo Ploidy Selection by Nuclear Magnetic Resonance: a Fast, Low Cost and Non-invasive Technique to Increase the Success Rate of ART
Conditions: Aneuploidy, Infertility
Sponsor: Instituto Valenciano de Infertilidade de Lisboa

 
 
 
 
2004-LIS-037-SN 
Version:  1.1 
Version date: 20/Apr/2021 

Biomedical Research Protocol 
2004-LIS-037-SN 
Protocol version: 1.1 (20/04/2021) 
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Project Title 
Title: Embryo ploidy selection by Nuclear Magnetic Resonance: a fast, low cost and non-invasive 
technique to increase the success rate of ART 
Project Code: 
2004-LIS-037-SN  
Research Area: 
Non invasive embryo selection 
Date: 
20/04/2021 
Keywords: 
Embryo selection, metabolic profiling, nuclear magnetic resonance 
Sponsor information 
Name 
Instituto Valenciano de Infertilidade - Clínica de Reprodução Assistida, LDA 
(hereinafter IVI Lisboa) 
Address 
Avenida Infante Dom Henrique 333 H 1- 9, 1800-282 Lisboa, Portugal 
e-mail 
samuel.ribeiro@ivirma.com 
Phone / Fax 
+351 21 850 32 10  
Principal Investigator information 
Name and surname 
Sofia Gouveia Nunes 
Filiation 
IVI Lisboa 
e-mail 
sofia.nunes@ivirma.com 
Phone / Fax 
+351 218 50 32 10 
Responsible for the administrative management of the study 
Name and surname 
Carla Jorge  
Filiation 
IVI Lisboa 
e-mail 
carla.jorge@ivirma.com 
Phone/ Fax 
+351 218 50 32 10 

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Research team members 
Cassandra Santos 
ITQB 
cassandraossantos@live.com.pt 
Luís Gafeira 
ITQB 
lgafeira@itqb.unl.pt 
Samuel Santos-Ribeiro  
IVI Lisboa 
samuel.ribeiro@ivirma.com 
Sérgio Reis Soares 
IVI Lisboa 
sergio.soares@ivirma.com 
 
David Moreno Torrado 
IVI Lisboa 
David.moreno@ivirma.com 
 

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Responsibilities and Signatures 
By signing this protocol from the project entitled: Title: Embryo ploidy selection by Nuclear Magnetic 
Resonance: a fast, low cost and non-invasive technique to increase the success rate of ART 
 
Those undersigning state that:  
- 
This study 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. 
 
 
 
Center and/or Laboratory Director 
 
 
 
 
 
 
 
 
 
 
 
 
Dr. Sérgio Reis Soares 
IVI Lisboa Diretor 
 
Date 
 
 
 
 
Principal Investigator 
 
 
 
 
 
 
 
 
 
 
 
 
 
Dra. Sofia Gouveia Nunes 
IVI Lisboa 
 
Date 
 
 
 

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Table of contents 
Project Title................................................................................................................................................................................................ 2 
Sponsor information .................................................................................................................................................................................. 2 
Principal Investigator information ............................................................................................................................................................. 2 
Responsible for the administrative management of the study ................................................................................................................. 2 
Research team members ........................................................................................................................................................................... 3 
Responsibilities and Signatures ................................................................................................................................................................. 4 
Table of contents ....................................................................................................................................................................................... 5 
Abstract ..................................................................................................................................................................................................... 7 
List of acronyms ......................................................................................................................................................................................... 8 
AIM: main objective/research question .................................................................................................................................................... 9 
Introduction ............................................................................................................................................................................................. 10 
A. 
Background ............................................................................................................................................................................... 10 
B. 
Justification and translational potential to the clinic and impact on patient’s care improvement: .......................................... 10 
Methodology ........................................................................................................................................................................................... 11 
A. 
Study Design .............................................................................................................................................................................. 11 
B. 
Study period and context .......................................................................................................................................................... 11 
C. 
Reference Population ................................................................................................................................................................ 12 
D. 
Subject Inclusion/exclusion criteria and case-control matching ............................................................................................... 12 
E. 
Intervention and Follow-up ....................................................................................................................................................... 12 
F. 
Study restrictions ...................................................................................................................................................................... 13 
G. 
Participant withdrawal and protocol violation .......................................................................................................................... 13 
H. 
Potential advantages and financial incentives for the participating subjects ........................................................................... 13 
I. 
Collection, selection, analysis and destruction of biological samples and information ............................................................ 13 
Statistical methodology ........................................................................................................................................................................... 15 
A. 
Data base .................................................................................................................................................................................. 15 
B. 
Study Variables .......................................................................................................................................................................... 16 
C. 
Sample Size ............................................................................................................................................................................... 16 
D. 
Statistical data analysis ............................................................................................................................................................. 17 
Work plan ................................................................................................................................................................................................ 17 
Ethical issues ............................................................................................................................................................................................ 18 
A. 
Ethical conduct of the study ...................................................................................................................................................... 18 
B. 
Independent ethics committee /Institutional review board ..................................................................................................... 18 
C. 
Subject information and consent .............................................................................................................................................. 19 
D. 
Data Handling and Record Keeping ........................................................................................................................................... 19 
E. 
Quality Assurance and Confidentiality ...................................................................................................................................... 20 
Funding .................................................................................................................................................................................................... 20 
Insurance ................................................................................................................................................................................................. 21 

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Publication and diffusion ......................................................................................................................................................................... 21 
References ............................................................................................................................................................................................... 21 
 
 
 

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Abstract 
Assisted reproductive technologies (ART) refers to treatments used to assist people in achieving a pregnancy. 
Over the last years, ART have been developed with efforts to deliver a healthy baby. However, the selection 
of the embryo that most likely results in pregnancy remains a critical step in ART. Currently, this selection is 
based on morphological assessment of the embryos, but up to 70% of those embryos display an abnormal 
number of chromosomes. Preimplantation genetic test for aneuploidy (PGT-A) is used to assess the ploidy of 
embryos, although this is an invasive, expensive and time consuming technique. Alternatively, in order to 
predict the ploidy of pre-implantation embryos (euploidy vs aneuploidy), we suggest to characterise the 
metabolic profile of the embryo culture medium by Nuclear Magnetic Resonance (NMR), which is a non-
invasive, low cost and fast technique. 
The aim of this study is to assess whether, using multivariable analysis to all the collected NMR data, we are 
able to identify a differential metabolic pattern between aneuploid and euploid embryos which could improve 
embryo selection. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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List of acronyms 
 
 
 
ART 
Assisted reproductive technologies 
DNA 
Deoxyribonucleic acid  
GCP 
Good clinical practice 
ECM 
Embryo culture medium 
ICH 
International Council for Harmonization of Technical Requirements for 
Pharmaceuticals for Human Use 
ICSI 
Intracytoplasmic sperm injection 
IEC 
Independent ethics committee 
IRB 
Institutional review board 
IVF 
In vitro fertilization 
IVI RMA 
Instituto Valenciano de Infertilidade / Reproductive Medicine Associates 
NMR 
Nuclear magnetic resonance 
PGT-A 
Preimplantation genetic test for aneuploidy 
SIVIS 
Software for medical record management  
 
 
 
 
 

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AIM: main objective/research question 
Main objective:  
To evaluate whether nuclear magnetic resonance (NMR) spectroscopy can assist in the prediction of ploidy of 
pre-implantation embryos (euploidy vs aneuploidy). 
 
 
Research question:   
Is NMR able to identify a differential metabolic pattern between aneuploid and euploid embryos in order to 
improve embryo selection? 
 
Is there a metabolic profile difference between an embryo with 3 days of culture and the same embryo after 
5 or 6 days of culture? 
 

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Introduction 
A. Background 
Worldwide, infertility affects around 15% of the couples in reproductive age. The ability to procreate 
is largely a female-age limited process, with one in eight women experiencing infertility, a proportion 
that is even higher among women aged 35 or older. Assisted reproductive technologies (ART) are of 
paramount importance for these couples, and in many they are the only solution to constitute a 
family (1). 
The optimal scenario in ART is a single-embryo transfer (SET) followed by the delivery of a healthy 
baby. For this reason, multiple laboratory technical approaches have been developed to improve the 
selection of the best embryo available. Currently, this selection process is largely based on 
morphologic assessment. This type of evaluation, however, appears to be insufficient since many 
apparently top quality embryos ultimately do not implant or eventually result in a miscarriage. One 
of the main reasons for this is the fact that up to 70% of the embryos considered viable from a 
morphological perspective present an abnormal number of chromosomes and are, therefore, 
generally not compatible with life (2). 
 
One means to overcome this issue is to perform preimplantation genetic testing of embryos to detect 
aneuploidy (PGT-A), which entails the removal of a small number of cells for analysis by either next-
generation screening or comparative genomic hybridization (3). However, this analysis has the 
disadvantage of being invasive, time-consuming and expensive. Thus, the search for an efficient 
alternative selection method during the preimplantation stage which could improve the success rate 
of ART while decreasing the cost and time required to select chromosomally normal embryos as best 
as possible continues. 
 
A suitable alternative to PGT-A could rely on the characterization of the metabolic profile of the 
embryo culture medium (ECM) by NMR, a non-invasive, rapid, economic and sensitive technique 
which is already currently clinically applicable. The conceptual basis of this approach has been 
described in a previous publication performed after a day 3 embryo biopsy, which demonstrated that 
embryos with chromosomal abnormalities presented a different metabolic pattern and, 
consequently, a different ECM composition (4). However, this study is of limited value, since it has 
been demonstrated that embryos are able to self-correct chromosomal errors found by day 3, 
whereby the current recommendation is for PGT-A to be performed on day 5 (or day 6). Further 
studies are needed to assess the usefulness of NMR for prediction of the ploidy of pre-implantation 
embryos. 
 
B. Justification and translational potential to the clinic and impact on patient’s care 
improvement:  
This research study aims to validate the ECM analysis by NMR spectroscopy, as a faster and less-
costly alternative to PGT-A which could significantly enhance embryo selection and the success rate 
of ART. 

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Methodology 
A. Study Design 
We propose a single-center prospective sibling case-control pilot study. In summary, all consenting patients 
will undergo in vitro fertilization (IVF) followed by PGT-A according to normal clinical practice. While the overall 
procedures towards PGT-A will not change, we will request that they consent additionally to donate the 
embryo culture medium, which otherwise would be discarded, for investigational purposes. The study will be 
conducted in conformance with Good Clinical Practices (GCP).  
 
B. Study period and context 
Women who will undergo IVF followed by PGT-A at the Reproductive Medicine Clinic of IVI RMA Lisboa will be 
screened each day by a study collaborator and treating physician. Eligible women will be invited to participate 
in the study and provided written patient information on the trial (pre-screening visit). Those who accept to 
participate will be requested to sign written informed consent (screening/inclusion visit). 
The on-study period is planned to have an overall duration of approximately 4 weeks with 2 visits for each 
subject, as detailed in Fig 1. The on-study period starts on the day that informed consent is signed, which can 
occur up to the day of patient oocyte retrieval, and ends when the results of the PGT-A procedure are known. 
More information regarding the overall recruitment plan, exposure, monitoring and data collection are 
described below. 
 
 
Procedures 
 
Provide information regarding the study 
 
 
 
Verification of eligibility criteria 
 
 
 
Informed consent form signature 
 
 
Collection of demographic and anthropometric data 
 
 
 
Collection of culture medium of embryos for assessment 
 
 
Centre visits 
Visit number 
 
0 
1 
 
 
Pre- Screening 
 
Screening/Inclusion 
Figure 1 – overall on-study period scheme 

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C. Reference Population 
Our target population are healthy women who will undergo a IVF followed by PGT-A. This study will evaluate 
embryo culture medium compositions of 50 aneuploid embryos, 50 euploid embryos, and 30 controls (no-
embryo culture medium). 
 
D. Subject Inclusion/exclusion criteria and case-control matching 
The subject inclusion and exclusion criteria are summarized in Table . 
 
Inclusion criteria 
Exclusion criteria 
▪ 
Age: ≥18 and <49 years old. 
▪ 
Body Mass Index (BMI) ≥18.5 Kg/m2 and <30 
Kg/m2. 
▪ 
Planned for IVF followed by PGT-A. 
▪ 
Oocytes retrieval and fertilization by ICSI 
▪ 
Six follicles over 14 mm on the day of the 
triggering. 
▪ 
Signed and dated informed consent.  
▪ 
Previous history of poor ovarian response (<4 oocytes retrieved) with a 
maximal dose of ovarian stimulation (≥300 IU/day). 
▪ 
Presence of a medical condition which is known to affect ART outcome 
(e.g. thyroid dysfunction). 
▪ 
Current use of anti-depressants, anti-psychotics, steroids, antiepileptics 
or chemotherapy. 
▪ 
Those unable to comprehend the investigational nature of the proposed 
study.  
Table 1 – Study inclusion and exclusion criteria 
 
E. Intervention and Follow-up 
Ovarian stimulation, oocyte retrieval, all embryology procedures and PGT-A will be performed according to 
how they are normally performed in IVI Lisboa. Women will start ovarian stimulation with either recombinant 
follicle-stimulating hormone (Gonal F, Puregon, Pergoveris, Bemfola, Ovaleap or Elonva) or highly purified 
human menopausal gonadotrophin (Menopur). Women will be submitted to pituitary down regulation, 
through daily administrations of either cetrorelix or ganirelix (GnRH antagonist) or oral progestogens 
(desogestrel) in either a fixed or flexible protocol. Cycles will be monitored by vaginal ultrasound scans and 
serum determination of estradiol and progesterone. Once three follicles of ≥17 mm are observed, oocyte 
maturation will be triggered with a GnRH agonist (0.3 mg decapeptyl). Cumulus-oocyte complexes will be 
collected by transvaginal aspiration 35-36 h after hCG administration (5). Oocytes will be denudated and ICSI 
will be performed at least 4 hours after the pick up to all mature oocytes. Fertilized oocytes will be cultured in 
the IVF laboratory incubators until they reach blastocyst stage. The ones presented good morphology will be 
biopsied, according to the ASEBIR embryo classification (6). After biopsy embryos will be frozen and the 
trophoectoderm cells will be analyzed by next generation sequencing (NGS) to identify aneuploidies. Culture 
media where each embryo developed will be analyzed via NMR Spectroscopy in order to evaluate the 
metabolomic profile and compare it with the chromosomic analysis.  
The study period includes the following visits to the clinic and procedures: 

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VISIT 0 (PRE-SCREENING VISIT) 
 
The study coordinator will contact potential participants in our center. Information regarding the study 
will be provided followed by an invitation to participate in this study. 
 
VISIT 1 (SCREENING AND STUDY INCLUSION) 
 
Participants will sign and date the informed consent.  
 
Collection of demographic and anthropometric data, medical and surgical history 
 
Verification of eligibility criteria check list. 
 
As described in more detail below, the culture medium from euploid and aneuploid embryos will be 
collected and then analyzed by NMR. 
F. Study restrictions 
 
During the study, patients are restricted to use any type of medication that may interfere with ovarian 
stimulation or embryology. Specifically, current use of anti-depressants, anti-psychotics, steroids, 
antiepileptics or chemotherapy constitute an exclusion criterion for study participation. 
 
G. Participant withdrawal and protocol violation 
Participants may withdraw from the study at any time. Eligible participants who gave adequate consent to the 
study and later withdraw will be replaced. Whenever a participant does not follow the planned protocol, this 
will be considered as a protocol violation. Given the pilot and explanatory nature of the study, these subjects 
will be excluded from the analysis with justification. 
 
H. Potential advantages and financial incentives for the participating subjects 
Beyond the potential clinical advantage of the study in the future, no other clinical or financial incentive will 
be provided for the participating subject. 
 
I.  Collection, selection, analysis and destruction of biological samples and 
information 
BIOLOGICAL SAMPLE GATHER PROTOCOL AND OBJECTIVE OF SAMPLE COLLECTION 
 
ECM collection 
After embryo biopsy, a minimum quantity of 25 uL of embryo culture medium will be collected at Day 3 and 
Day 5 (or Day 6) of embryos development. Collection of the media, will be carried out by pipetting culture 

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media into Eppendorf tubes which will be immediately centrifugated in a mini centrifuge during 1 minute to 
remove cell contamination. Supernatant will be collected to a new tube with phosphate buffer, D20 and 
sodium azide to prevent bacterial and fungi development. Samples will be immediately anonymized, as 
described below, and frozen in liquid nitrogen and then stored at -20 ºC at IVI RMA Lisboa. Samples will be 
transferred in dry ice to ITQB, which is located at Av. da República, 2780-157, Oeiras, Portugal. Dr Sofia Nunes 
will be responsible for the collection and storage of samples at IVI Lisboa. This facility has a 24-hour security 
system in place and access to the storage area is restricted to authorized personnel only. 
 
Selection process of case-control sibling embryo culture media 
In order to minimize potential confounding, paired-sibling embryo culture media (two samples derived from 
the same patient and treatment cycle) will be allocated in pairs and in a 1:1 ratio to each study group. The 
pair-sibling allocation will be performing after the results of the PGT-A procedure are known. Pairing will be 
performed preferably using embryos with similar developmental staging and morphological grading.  
 
Finally, each patient can include up to 3 pairs of sibling culture media in the analysis. The remaining samples 
will be immediately destroyed. 
 
 
NMR procedure  
ECM samples will be thawed at room temperature before analysis, will be transferred to a 1.7-mm NMR 
capillary shigemi tube for analysis. The NMR spectra will be acquired in a BrukerAvance II 500 spectrometer 
(BrukerBiospin) equipped with a prodigy TCI cryoprobe. All experiments will be acquired at 298 K. For each 
sample, two 1H-NMR spectra will be obtained: a Carr-Purcell-Meiboom-Gill (CPMG) spin echo pulse sequence, 
which generates spectra edited by T2 relaxation times to reduce signals from high molecular weight species 
and to facilitate the detection of low molecular weight species, and a 1D NOESY pulse sequence, which 
generates an unedited spectrum with improved solvent peak suppression (extremely useful for the 
observation of signals near the water resonance). The spectra will be processed by a Fourier Transform and 
multiplied by an exponential function using Topspin 4.0. Phase correction, baseline correction and spectra 
calibration (methyl group signal of the lactate at a chemical shift 1.32 ppm) will be performed before further 
analysis. Metabolites will be quantified with ChenomxNmrSuite 8.0.2 using the Human Metabolites Database 
as reference. This software allows the deconvolution of the NMR spectra and the determination of the 

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metabolite concentrations. From the previous studies it is expectable that we will be able of determine around 
20 to 30 metabolites including aminoacids, fatty acids and carbohydrates like sucrose, glucose and pyruvate.  
The samples analysed in this study will be destroyed immediately upon analysis. 
 
SAMPLE IDENTIFICATION PROTOCOL 
All consenting subjects will be given a unique screening number (eg. 037-SN-0X). Each participant will be 
assigned only one screening number. Screening numbers will not be re-used for different participants. 
Samples (ECM) will be collected and identified immediately using sticker labels. These labels contain a single 
anonymized identifying code with the following format: <STUDY CODE> <SUBJECT NUMBER> <TYPE OF 
SAMPLE> <SUBJECT YEAR OF BIRTH> <SAMPLE DATE>. For example, the identifying code 2004-LIS-037-SN-01-
ECM_D3-1983-30042020 provides the following information:  
 
<STUDY CODE>: internal reference code of study (2004-LIS-037-SN) 
 
<SUBJECT NUMBER>: 0X 
 
<TYPE OF SAMPLE>: ECM_DX (X = day 3 or day 5/ day 6) 
 
< SUBJECT YEAR OF BIRTH>: coded as YYYY; 
 
<SAMPLE DATE>: date the sample was collected, coded as DDMMYYYY. 
 
Statistical methodology 
A. Data base 
Data will be collected in a secure and encrypted case report form system created specifically for the study 
hosted on a dedicated server firewall-protected serve. The database will be rigorously defined with the 
variables destined to be analyzed according to the described study objectives and will have inbuilt validation 
procedures to ensure the correct introduction of data and avoid cases of missing information where such is 
not applicable. The study team collaborating in the study will be responsible for the data collection. These 
anonymized data will be stored for at least an additional 25 years.  
In accordance with Portuguese law, all data exported for statistical analysis will be anonymized to protect the 
clinical and personal information of the subjects. Finally, and prior to the statistical study, an exploratory data 
analysis will be carried out to review the quality of the information extracted. 
 

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B. Study Variables 
MAIN-OUTCOME MEASURES (DEPENDENT) 
The primary outcome of this study is the assessment of different metabolic profiles between culture medium 
of euploid versus aneuploid embryos. 
 
EXPOSURE VARIABLES (INDEPENDENT) 
The metabolic profile of culture medium of euploid and aneuploid embryos will be compared to culture 
medium without embryo (control). 
 
CONTROL VARIABLES 
The following variables will be recorded during the analysis (as continuous variables, unless mentioned 
otherwise):  
 
Female subject age (in years). 
 
Male subject age (in years) 
 
Female body mass index. 
 
Menstrual cycle length. 
 
Number of preceding ART cycles. 
 
Duration of infertility (in years). 
 
Infertility diagnoses (tubal, ovulatory, endometriosis, male factor or otherwise unexplained). 
 
Baseline AMH. 
 
Baseline AFC. 
 
Number of follicles >11 mm on the day of ovulation triggering. 
 
Total dose of exogenous gonadotropins. 
 
Total duration of OS. 
 
Number of oocytes retrieved. 
 
Number of fertilized oocytes. 
 
Number of embryos available for biopsy. 
 
Quality of each embryo available for biopsy. 
 
Number of euploid and aneuploid embryos. 
 
C. Sample Size 
Owing to the exploratory nature of the study, we are unable to determine an accurate sample size for this 
study hypothesis. Hence, we elected to recruit a pre specified number of a maximum of 50 subjects (donating 
a minimum of a pair of sibling culture media). 
 

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D. Statistical data analysis 
EXPLORATORY DATA ANALYSIS 
Basic demographic characteristics will be compared amongst the study who did and did not have a 
spontaneous miscarriage using the t-student/Mann-Whitney test (for continuous variables, according to the 
normality of the distribution) or Fisher’s Exact test (for categorical variables). 
 
HOMOGENEITY ANALYSIS 
In order to minimize potential confounding, paired-sibling embryo culture media (two samples derived from 
the same patient and treatment cycle) will be allocated in pairs and in a 1:1 ratio to each study group. The 
pair-sibling allocation will be performing after the results of the PGT-A procedure are known. 
OBJECTIVES ASSESSMENT 
Considering that we will be able to identify around 20 to 30 metabolites, the minimum number of samples per 
group for a False Discovery Rate (FDR) of 5% is 40 samples, this value was calculated using the default 
parameters of MetSizeR (7), a software that allows sample size calculation for metabolomic studies. Assuming 
that a pre-specified proportion of at least 10% metabolites will be predictive of the outcome, the estimated 
power to detect a statistically significant difference among the groups is >90%. 
Metabolite concentration will be used for univariate and multivariate analysis. In the univariate analysis, data 
normality will be checked by Shapiro test. Two-sample t-test, if the data has normal behavior, or 
Wilcoxon-rank test, if the data has not a normal behavior. Comparisons will be performed to confirm the 
metabolite differences significance. In multivariate analysis, sample classification will be performed by 
Principal Component Analysis (PCA), Partial Least Squares Discriminant Analysis (PLS-DA), and Orthogonal 
Projections to Latent Structures (OPLS-DA) using R and Simca. 
Pathway analysis will be performed in Metaboanalyst (www.metaboanalyt.ca) to identify which metabolic 
pathways are affected in aneuploid embryos. Pathway Analysis combines pathway enrichment analysis with 
pathway topology analysis, allowing identification of the most relevant pathways involved in aneuploidy. For 
that, it will be used the metabolite concentrations determined previously for the aneuploid embryos and 
controls; and human pathway library as reference.   
 
Work plan 
The pilot study workplan of the pilot phase includes three phases (ECM collection, ECM analysis by NMR and 
multivariable analysis), as detailed below.  

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Months 
  
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
Phase I - ECM collection 
  
  
  
  
  
  
  
  
  
  
Phase II - ECM analysis by NMR 
   
  
  
  
  
  
  
  
  
  
Phase III - Multivariable analysis 
 
 
 
 
  
  
  
  
 
 
The responsible persons for each phase are: I- Sofia Nunes; II and III – Luis Gonçalves 
 
Ethical issues 
A.  Ethical conduct of the study 
This study will be submitted for approval by the Local Ethics Committee of IVI Lisboa. For the inclusion of all 
patients in the study, a signed informed consent is required, which must first be approved by this Ethics 
Committee. 
This study respects the fundamental principles of the Declaration of Helsinki, the Council of Europe Convention 
on Human Rights and Biomedicine, the UNESCO Universal Declaration on the Human Genome and Human 
Rights, as well as the requirements of Portuguese law in the field of biomedical research, the protection of 
personal data and bioethics. 
This study will be conducted in accordance with the ethical principles that have their origins in the Declaration 
of Helsinki and its most recent amendments, in compliance with the approved protocol, ICH-GCP guidelines, 
the EU Clinical Trial Regulation, and other national law(s) or regulatory requirements applicable in the 
countries where the study takes place. 
 
B. Independent ethics committee /Institutional review board 
Prior to the start of the study, the investigator is responsible for ensuring that the protocol and consent form 
have been reviewed and approved by a relevant IEC/IRB. The IEC/IRB shall be appropriately constituted and 
perform its functions in accordance with ICH-GCP guidelines and local requirements as applicable. 
The IEC/IRB shall approve all protocol amendments (except for logistical or administrative changes), written 
informed consent documents updates, patient recruitment procedures (e.g. advertisements), written 
information to be provided to the patients, available safety information, information about payment and 
compensation available to patients, the investigator curriculum vitae and/or other evidence of qualification 
and any other documents requested by the IEC/IRB and Regulatory Authority (Competent Authority) as 
applicable. 

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C. Subject information and consent 
The investigator will obtain a freely given written consent from each subject after an appropriate explanation 
of the aims, methods, anticipated benefits, potential hazards, and many other aspects of the study, which are 
relevant to the subject´s decision to participate. The subject must be given ample time to consider 
participation in the study, before the consent is obtained. The informed consent form must be signed and 
dated by the subject and the investigator who has provided information to the subject regarding the study 
before the subject is exposed to any study-related procedure. 
The investigator will explain that the subject is completely free to refuse to enter the study or to withdraw 
from it any time, without any consequences for her further care and without the need to justify her decision. 
The subject will receive a copy of the informed consent documents. 
The subjects will be informed about a new information and re-consent will be obtained. 
 
D. Data Handling and Record Keeping 
CASE REPORT FORMS/SOURCE DATA HANDLING 
The study data must be verifiable with the source data, which requires access to all original records, laboratory 
reports and subject records. Therefore, the investigator must agree to allow access to subject records and the 
source data must be available for all study data. The subjects or their legal representatives must also allow 
access to the medical records of the subjects and they will be informed of this need and will express their 
agreement when they provide the informed consent. 
The principal investigator will be responsible of maintaining the accuracy and authenticity of the clinical and 
laboratory data entered in the eCRF. Subject´s source data will be data recorded by the embryologist, which 
will be kept in the clinical center where the study is carried out. 
The investigator/center must allow direct access to the data / source documents (originals) for the activities 
related to the monitoring study, audit, review by the ethics committees (IEC/ IRB) and inspection by the 
regulatory authorities. 
 
RETENTION OF ESSENTIAL DOCUMENTS 
All records relating to the conduct of this study are to be retained by the investigator according to ICH, local 
regulations, or as specified in the clinical study agreement, whichever is longer. Prior to transfer or destruction 

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of these records, the Sponsor must be notified in writing and given the opportunity to further store such 
records. The investigator will allow representatives of Sponsor´s monitoring members (and of the applicable 
regulatory authorities) to inspect all study records, eCRFs, and corresponding portions of the study patient´s 
office and/or hospital medical records at regular intervals across the study. These inspections are for the 
purpose of verifying the adherence to the protocol, the completeness and accuracy of the data being filled in 
the eCRF, and compliance with applicable regulations. 
 
E. Quality Assurance and Confidentiality 
CONDUCT OF THE STUDY 
The sponsor or designee will perform the quality assurance and quality control activities of this study, 
however, responsibility for the accuracy, completeness, and reliability of the study data presented to the 
Sponsor lies with the principal investigator generating data. 
CONFIDENTIALITY 
Any material, information (oral or written), unpublished documents provided by the investigators, including 
this protocol are owned by the sponsor, and will be regarded as confidential, even when used for review and 
for exclusive use in this study. This confidentiality clause, as well the obligation to comply during the study, 
applies to both the investigator and any collaborators. The investigator shall guarantee that the confidentiality 
of the data by the collaborators will be maintained. The data and/or any material shall not be disseminated, 
totally or partially, by the principal investigator and/or his/her team, to any unauthorized party, without 
previous written consent by the sponsor and it is not intended for any use other than the purposes of this 
study. The investigator and/or his/her team will treat as confidential all documents and results arising during 
the study, except for the information classified as releasable, pursuant to the law. The investigator and/or 
his/her team will ensure that anyone involved with the study will adhere to the same confidentiality 
obligations concerning the information about the participants. All parties involved with the study will maintain 
the strictest confidentiality so that they do not violate any confidentiality rights of the participants and their 
families. They will ensure that access to the research data is protected from unauthorized individuals. 
Considering the confidential nature, the data of a personal nature on subjects included in the study shall 
comply with the General Data Protection Regulation effective as of 25 May 2018 (Regulation (EU) 2016/679) 
and other national law(s) or regulatory requirements applicable in Portugal. Furthermore, the Investigator and 
the Sponsor agree to adhere to the principles of personal data confidentiality in relation to the subjects, 
Investigator and its collaborators involved in the study. 
 
Funding 
Needs funding 
Yes 

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Externally Funded project 
No 
Funding Institution 
Sponsor 
External Funding plan (if no 
funding is available yet) 
None 
IVI RMA Funding requested Yes 
Internally Funded project 
Yes 
Funding time range 
Start: 03/05/2021 
Finish: 31/01/2022 
Budget 
1085,28 Eur 
 
Insurance 
IVI Lisboa has an Insurance Policy in force that conforms to current legislation and with coverage to 
compensate and indemnify cases of ill health or injury, which may arise in connection with their participation 
in the study, within routine clinical practice. 
 
Publication and diffusion 
Upon conclusion, the results of the study will be submitted for presentation at the annual congress of the 
European Society of Human Reproduction and Embryology (ESHRE). Moreover, the study will be publicly 
disseminated following submission in peer-reviewed scientific journals, targeting the upper quartile in terms 
of impact factor according to Web of Science in Q1 of the preceding year in the field of Obstetrics and 
Gynecology, such as in the “Molecular Human Reproduction”. 
 
 
 
References 
1. Datta J, Palmer MJ, Tanton C, Gibson LJ, Jones KG, Macdowall W, et al. Prevalence of infertility and 
help seeking among 15 000 women and men. Human Reproduction. 2016;31(9):2108-2118. 
2. Minasi MG, Colasante A, Riccio T, Ruberti A, Casciani V, Scarselli F, Spinella F, Fiorentino F, Varricchio 
MT, Greco E. Correlation between aneuploidy, standard morphology evaluation and morphokinetic 
development in 1730 biopsied blastocysts: a consecutive case series study. Hum Reprod. 2016, 
31(10):2245-2254. 
3. Vaiarelli A, Cimadomo D, Capalbo A, Orlando G, Sapienza F, Colamaria S, Palagiano A, Bulletti C, Rienzi 
L, Ubaldi FM. Pre-implantation genetic testing in ART: who will benefit and what is the evidence? J 
Assist Reprod Genet. 2016, 33(10):1273-1278. 
4. Sánchez-Ribas I, Riqueros M, Vime P, Puchades-Carrasco L, Jönsson T, Pineda-Lucena A, et al. 
Differential metabolic profiling of non–pure trisomy 21 human preimplantation embryos. Fertility and 
Sterility. 2012, 98(5):1157-1164. 

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5. Santos-Ribeiro S, Polyzos NP, Haentjens P, Smitz J, Camus M, Tournaye H and Blockeel C.  Live 
birth rates after IVF are reduced by both low and high progesterone levels on the day of human 
chorionic gondotrophin administration. Human reproduction. 2014; 29(8):1698-1705. 
6. Cuevas-Sáiz, Irene & Pons, Maria Carme & Vargas, Muriel & Mendive, Arantzazu & Enedáguila, Natalia 
& Solanes, Marta & Carrasco, Beatriz & López, José & Bonet, Ana & Acosta, Mª. (2018). The 
Embryology Interest Group: updating ASEBIR's morphological scoring system for early embryos, 
morulae 
and 
blastocysts. 
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y 
Embriología 
Clínica. 
5. 
10.1016/j.medre.2017.11.002. 
7. Nyamundanda G, Gormley IC, Fan Y, Gallagher WM, and Lorraine Brennan. MetSizeR: selecting the 
optimal sample size for metabolomic studies using an analysis based approach. BMC Bioinformatics. 
2013; 14: 338.