The Effect of Constant 5% Versus Gradient 8%, 5%, 2% Oxygen Concentration on the Development of Sibling Human Embryos in Vitro
Phase: NA
Conditions: Embryonic Development, Embryo Morphokinetics, Embryo Morphometry
Sponsor: University Medical Centre Maribor

Title: 
The Effect of Constant 5% Versus Gradient (8%, 5%, and 2%) Oxygen Concentration on 
Development of Human Embryos to the Blastocyst Stage: A Sibling Oocyte Study 
 
 
 
Study Type: 
Prospective, Quasi-Randomized Sibling-Split Oocyte Study 
Study Site: 
Department of Reproductive Medicine and Gynecological Endocrinology 
University Medical Centre Maribor 
Slovenia 
Clinical Trial Registration Number: 
NCT05898178 
 
 
 
 
 
 
 
Document Date: 
April 24, 2025 
Principal Investigator: 
Dr. Borut kovačič 
Borut.KOVACIC@ukc-mb.si 
 
 

 
2. Study Design and Participants 
Prospective sibling-split oocyte study , conducted at the University Medical Centre Maribor and 
included a total of 658 cumulus–oocyte complexes (COCs) collected from 44 ICSI cycles 
between January 2022 and January 2023. A quasi-randomisation into control and intervention 
groups was performed immediately after oocyte retrieval. Embryonic outcomes between 
different oxygen culture conditions were compared. 
Eligibility Criteria 
• 
Inclusion: Women <35 years old, BMI 18–30 kg/m², normal uterine cavity, ≥6 MII 
oocytes retrieved, first or second ICSI cycle, male factor infertility (non-azoospermic), 
blastocyst culture, GnRH antagonist protocol. 
• 
Exclusion: Endometriosis, previous ovarian surgery, endocrine/metabolic disorders, 
PCOS. 
Ethical Approval 
• 
Institutional Ethics Committee of the University Medical Centre Maribor (No: UKC-MB-
KME-1/21) 
• 
National Medical Ethics Committee (No: 0120-405/2021/4) 
All participants provided informed consent. 
 
3. Objectives 
Primary Objective 
To compare the proportion of injected MII oocytes that developed into morphologically optimal 
blastocysts on day 5 between the control and intervention oxygen conditions. 
Secondary Objectives 
• 
Assessment of morphokinetic parameters (timing to key developmental stages) 
• 
Incidence of abnormal cleavage patterns 
• 
Morphometric characteristics of blastocysts (e.g., area, TE cell count) 
 
4. Sample Size Determination 
Sample size was calculated using the Sealed Envelope online tool (accessed January 2022). 
Based on a baseline blastocyst formation rate of 15% and a hypothesized improvement to 25% 
in the intervention group, 496 embryos (248/group) were required for 80% power at α = 0.05. 
With a buffer for potential fertilization failures, 47 patients were targeted. Ultimately, 44 
patients were enrolled, contributing 521 injected sibling MII oocytes. 
 
5. Randomization and Blinding 

A quasi-random allocation method was used post-oocyte retrieval. Group A (control) was 
cultured under 5% O₂; Group B (intervention) under a decreasing gradient from 8% to 2% O₂. 
Embryologists remained blinded through a coded system. 
 
6. Laboratory Procedures 
Sibling oocytes from each patient were divided into two groups: 
• 
Intervention Group (8–5–2% O₂): Cultured under a dynamic oxygen gradient to mimic in 
vivo conditions. Gas levels were adjusted by stage: 
o 
Day 0–3: 6% CO₂, 8% O₂, and 86% N₂ 
o 
Day 3: Oxygen concentration was reduced to 5% (6% CO₂, 5% O₂, 89% N₂). 
o 
Day 3–5: Oxygen was further reduced to 2% (6% CO₂, 2% O₂, 92% N₂). 
• 
Control Group (5% O₂): Cultured under fixed conditions (6% CO₂, 5% O₂) throughout. 
Common lab procedures for both groups: 
Sperm prep: Density gradient centrifugation 
ICSI: Standard protocol using a Nikon Eclipse TE 2000 microscope (200x magnification). 
Embryo culture: G-1 PLUS (Day 1–3) and G-2 PLUS (Day 3–5) (Vitrolife, Gothenburg, Sweden). 
Monitoring: Continuous time-lapse imaging with Primo Vision system (Vitrolife, Gothenburg, 
Sweden). 
 
8. Statistical Analysis Plan (SAP) 
Statistical analysis was performed using SPSS (SPSS Inc., Chicago, Illinois, USA). Continuous 
variables were tested for normality using the Shapiro-Wilk test. Data are presented as mean ± 
standard deviation (SD) for normally distributed variables or as median with interquartile range 
(IQR) for non-normally distributed variables. Categorical data are expressed as frequencies and 
percentages. Group comparisons for embryological outcomes, irregular cleavage events, and 
clinical outcomes were carried out using the chi-square test or Fisher’s exact test, depending 
on the expected frequencies. Odds ratios (ORs) with 95% confidence intervals (CIs) were 
calculated using the Baptista-Pike method for categorical variables. Morphokinetic variables 
were reported as median hours post-insemination (HPI) with IQR. Differences in morphokinetic 
timings between oxygen groups were analyzed using a linear mixed-effects model (REML), and 
Šídák’s correction was applied for multiple comparisons at individual time points. 
Morphometric measurements of blastocysts graded Gardner ≥3 were compared using unpaired 
t-tests, with significance determined at p < 0.05. All statistical tests were two-sided; a p-value < 
0.05 was considered statistically significant throughout the study. 
9. Informed Consent Form (ICF) 
9.1 Original (Slovenian language): 
Obrazec izjave o zavestni in svobodni privolitvi 

OBRAZEC PROSTOVOLJNE IN ZAVESTNE PRIVOLITVE PO POUČITVI 
 
Podpisana _____________________________, rojena ___________________, in 
 
Podpisani ______________________________, rojen ___________________, 
 
sva bila pisno in ustno seznanjen/a s potekom, namenom in cilji raziskave z naslovom: 
 
“Vpliv različnih koncentracij kisika na presnovo in razvoj človeških zarodkov in vitro in na 
uspešnost programa oploditve z biomedicinsko pomočjo” 
 
Veva, kako bo poskrbljeno za najino varnost v raziskavi in da lahko kadar koli zaprosiva za 
dodatne informacije in jih tudi dobiva. Prav tako nama je bilo pojasnjeno, da lahko privolitev 
prekličeva, ne da bi morala preklic utemeljiti in ne da bi prenehanje sodelovanja v raziskavi 
okrnilo najino morebitno siceršnjo zdravstveno obravnavo. 
S podpisom prostovoljno potrjujeva svojo pripravljenost za sodelovanje v raziskavi. Dovoljujeva 
tudi, 
da se najini demografski in zdravstveni podatki uporabijo v anonimizirani obliki v znanstvene 
namene. Obrazec podpisujeva v navzočnosti raziskovalca/raziskovalke. 
 
Podpis: _____________________________ Podpis: _____________________________ 
 
Ime in priimek raziskovalca/raziskovalke: _____________________________ 
 
Podpis raziskovalca/raziskovalke: _____________________________ 
 
Datum: _____________________________ 
 
 
9.2 Translation (English language): 
 
We, the undersigned: 
 

Name: _____________________________, Date of Birth: ___________________ 
 
Name: _____________________________, Date of Birth: ___________________ 
 
have been informed in writing and verbally about the course, purpose, and objectives of the 
study titled: 
 
"The Effect of Different Oxygen Concentrations on the Metabolism and Development of 
Human Embryos In Vitro and on the sucess of assisted reproductive technology (ART) 
programs.« 
We understand how our safety will be ensured during the study and that we can request 
additional information at any time, which will be provided. We have also been informed that we 
can withdraw our consent at any time, without needing to provide a reason, and that 
discontinuing participation in the study will not affect our possible further medical care. 
 
By signing this form, we voluntarily confirm our willingness to participate in the study. We also 
allow our demographic and medical data to be used in anonymized form for scientific 
purposes. We are signing this form in the presence of the researcher. 
 
Signature: _____________________________  Signature: _____________________________ 
 
Name of Researcher: _____________________________ 
 
Researcher’s Signature: _____________________________ 
 
Date: _____________________________ 
 
 
10.  Ethical Evaluation of the Submitted Research 
 10.1 Original (Slovenian language): 
 
Štefanova ulica 5, 1000 Ljubljana 
T: 01 478 69 06, 01 478 69 20 
F: 01 251 77 55 
E: gp.mz@gov.si, kme.mz@gov.si 
www.mz.gov.si 

Številka: 0120-405/2021/4 
Datum: 28. 9. 2021 
Zadeva: Ocena etičnosti predložene raziskave 
Zveza: Vaša vloga z dne 31. 8. 2021 
Spoštovani, 
Komisija Republike Slovenije za medicinsko etiko (v nadaljevanju KME RS) je dne 31. 8. 2021 
prejela vlogo za oceno etičnosti raziskave z naslovom  
“Vpliv različnih koncentracij kisika na presnovo in razvoj človeških zarodkov in vitro in 
nauspešnost programa oploditve z biomedicinsko pomočjo” 
Gre za: fiziološko – klinično raziskavo, ki bo potekala na Oddelku za reproduktivno medicino in 
ginekološko endokrinologijo, Klinike za ginekologijo in perinatologijo, Univerzitetnega kliničnega 
centra Maribor. 
Namen raziskave je določiti optimalno koncentracijo kisika in čim bolj posnemati fiziološke 
pogoje v kulturah človeških zarodkov ter posledično izboljšati uspeh programa oploditve z 
biomedicinsko pomočjo (OBMP). 
Spremljali bi fiziološki vpliv kisika na nivo optimalne blastulacije zarodkov, ki je trenutno 10% pri 
uporabljenem 5 % kisiku v atmosferi inkubatorjev. Ob hipotezi, da gojenje zarodkov pri 
atmosferi z 2 % izboljša optimalno blastulacijo zarodkov na 20 %, bi potrebovali skupno 398 
zarodkov. Pri tem upoštevate statistično moč 80 % in a = 0,05. V raziskavo bi torej vključili 
največ 100 preiskovanih parov (ciklusov OBMP z vsaj 4 jajčnimi celicami). Povprečno minimalno 
število jajčnih celic na pacienta v Mariborski IVF enoti, je okoli 8 jajčnih celic (6 zrelih M II jajčnih 
celic). Stopnja oploditve po ICSI je okoli 70 %. To pomeni da boste v raziskavi razpolagali s 
približno 400 zarodki. Vsaka raziskovalna skupini bo vsebovala 200 zarodkov. 
KME RS je na seji 21. septembra 2021 obravnavala prejeto vlogo in ugotovila, da je vloga 
popolna ter ocenila, da je raziskava etično sprejemljiva. S tem vam za njeno izvedbo izdaja svoje 
soglasje. 
Peter Slatinšek, mag. biol. in ekol. 
Oddelek za reproduktivno medicino in ginekološko endokrinologijo 
Klinika za ginekologijo in perinatologijo 
UKC Maribor 
Ljubljanska 5 
2000 Maribor 
e-naslov: peter.slatinsek.ps@gmail.com 
Pri nadaljnjih dopisih v zvezi z raziskavo se obvezno sklicujte na številko tega dopisa. 
S spoštovanjem, 
dr. Božidar Voljč, dr. med. 
predsednik KME RS 
Vročiti: 
– naslovniku – po e-pošti 

 
10.2 Translation (English language): 
 
Republic of Slovenia Ministry of Health 
Štefanova ulica 5, 1000 Ljubljana 
T: +386 1 478 69 06, +386 1 478 69 20 
F: +386 1 251 77 55 
E: gp.mz@gov.si, kme.mz@gov.si 
www.mz.gov.si 
Reference No.: 0120-405/2021/4 
Date: September 28, 2021 
Subject: Ethical Evaluation of the Submitted Research 
Reference: Your application dated August 31, 2021 
Dear Sir/Madam, 
The National Medical Ethics Committee of the Republic of Slovenia (hereinafter: NMEC RS) 
received your application on August 31, 2021, for the ethical assessment of the research titled  
"The Effect of Different Oxygen Concentrations on the Metabolism and Development of Human 
Embryos In Vitro and on the sucess of assisted reproductive technology (ART) programs." 
This is a physiological-clinical study, which will be conducted at the Department of 
Reproductive Medicine and Gynecological Endocrinology, Clinic for Gynecology and 
Perinatology, University Medical Centre Maribor. 
The purpose of the research is to determine the optimal oxygen concentration and to best 
simulate physiological conditions in human embryo cultures, thereby improving the success of 
medically assisted reproduction (MAR) programs. 
The study will observe the physiological impact of oxygen on the level of optimal blastulation of 
embryos, currently at 10% under a 5% oxygen atmosphere in incubators. Based on the 
hypothesis that culturing embryos at 2% oxygen may improve optimal blastulation to 20%, a 
total of 398 embryos would be required. This calculation assumes a statistical power of 80% 
and α = 0.05. Accordingly, a maximum of 100 participating couples (MAR cycles with at least 
four oocytes each) would be included in the study. The average minimum number of oocytes 
per patient at the Maribor IVF unit is about 8 oocytes (6 mature MII oocytes). The fertilization 
rate following ICSI is around 70%. Therefore, the study is expected to involve approximately 400 
embryos, with 200 embryos per research group. 
At its meeting on September 21, 2021, the NMEC RS reviewed the application, found it to be 
complete, and assessed the study as ethically acceptable. Therefore, the Committee grants its 
approval for the study to be conducted. 
Sincerely, 
Dr. Božidar Voljč, M.D. 
Chair, National Medical Ethics Committee of the Republic of Slovenia 

Delivered to: 
– Recipient via e-mail 
Contact: 
Peter Slatinšek, M.Sc. in Biology and Ecology 
Department of Reproductive Medicine and Gynecological Endocrinology 
Clinic for Gynecology and Perinatology, UKC Maribor 
Ljubljanska 5, 2000 Maribor 
Email: peter.slatinsek.ps@gmail.com