Ultralow Oxygen Tension (2%) Is Beneficial for Blastocyst Formation of In Vitro Human Low-Quality Embryo Culture

Objectives To investigate whether a reduction in the O2 tension from 5 to 2% during extended culture from day 3 onwards was beneficial to human blastocyst development in vitro. Methods We firstly identified 139 patients who had no low-quality embryos on day 3, and all the embryos were prolonged to culture on day 5 or 6. We mainly analyzed 188 patients receiving IVF/ICSI-ET for the first time, and no high-quality embryos were obtained on day 3 from January 2018 to December 2019. After transferred with one or two low-quality embryos, extended culture was performed under low O2 (5%) or ultralow O2 (2%) tension for surplus embryos. 296 embryos from 106 patients were continued to culture under 5% O2 tension, and 214 embryos from 82 patients were continued to culture under 2% O2 tension. Main outcomes compared were blastulation and high-quality blastulation rates. Results We observed no significant differences in the blastulation and high-quality blastulation rates for high-quality cleavage-stage embryos between 2% and 5% O2 groups (p > 0.05). For low-quality cleavage-stage embryos, we observed that the 2% O2 group showed a significantly higher blastulation (39.72 versus 31.08%; p = 0.043) rate than that in the 5% O2 group. The high-quality blastocyst formation rate (10.75 versus 8.45%; p = 0.380) was comparable between the 2% and 5% O2 groups. The blastulation rate reached 44.86% by culturing blastocysts an additional day under 2% O2 tension which was significantly higher than that (32.09%) under 5% O2 tension (p < 0.05). Conclusion A reduction in O2 tension from 5 to 2% after day 3 might be beneficial to the patients with no high-quality embryos. Extended culture to day 7 under 2% O2 tension increased the number of available blastocysts per IVF/ICSI cycle and was worth recommending especially for patients with few blastocysts.


Introduction
Culture of human embryos was traditionally performed under atmospheric O 2 tension of about 20%. In 1971, the scientists noticed the first efficient culture of a human embryo from day 2 to day 5 and emphasized that the O 2 in the culture system was not about 20% (atmospheric O 2 ), but that the gas phase was 5% O 2 (physiologic O 2 ), 5% carbon dioxide, and 90% nitrogen [1]. During this period, 20% and 5% O 2 were widely used in human embryo culture and showed similar developmental rates.
Multiple meta-analysis reviews demonstrated an increase in pregnancy and live birth rates at 5% O 2 embryo culture compared with that at 20% O 2 [2,3]. Human embryo culture under 2-8% O 2 was recommended by ESHRE revised guidelines for good practice in IVF labs. Thus, 5% O 2 embryo culture was widely adopted in most IVF labs.
It was generally accepted that O 2 tension was lower in the uterus than in the oviduct, and that the embryo crossed the uterotubal junction sometime on day 3. In 2017, Morin proposed that the optimal O 2 tension in embryo culture might depend on the stage of development [4]. Meanwhile, Kaser put forward the hypothesis which assumed that sequential O 2 exposure (5% from days 1 to 3, then 2% from days 3 to 5) would improve blastocyst yield and quality compared to continuous exposure to 5% O 2 among human preimplantation embryos [5]. Nevertheless, Munck et al. showed a reduction in O 2 tension from 5 to 2% O 2 after day 3 did not improve embryo development, quality, and utilization rate [6]. Therefore, it was controversial whether 2% O 2 tension was more suitable than 5% O 2 for extended culture of human embryos.
Embryonic genome activation always occurred at the 8-cell stage; so, it appeared that blastocyt-stage embryo transfer (BSET) had a higher success rate than cleavage-stage embryo transfer (CSET). Although the utilization of BSET had made as more IVF centers switched from day-3 cleavage stage to routine day-5/6 transfers of blastocyst-stage embryos, there were a majority of patients who had no opportunity to try blastocyst transfers especially for the patients with no high-quality embryos on day 3. It was confirmed that multifetal pregnancies carried high risks of poor clinical outcomes, and it could be effectively avoided by the adoption of elective single BSET [7]. Thus, it was significant to make some improvements in blastocyst formation and blastocyst quality by some technical advancements and revised professional guidance.
In this study, we aimed to investigate whether a reduction in O 2 tension from 5 to 2% during extended culture from day 3 onwards was beneficial to human blastocyst development in vitro.

Patients.
This study included 139 patients who had no low-quality embryos and 188 patients who had no highquality embryos on day 3 from January 2018 to December 2019. All the embryos were prolonged to culture on day 5 or 6 under low O 2 (5%) or ultralow O 2 (2%) tension for the patients with no low-quality embryos. After transferred with one or two low-quality embryos, extended culture was performed under 5% or 2% O 2 tension for surplus embryos of the patients with no high-quality embryos. Main outcomes compared were blastulation and high-quality blastulation rates. All female patients were not older than 38 years to eliminate possible age-related cycle characteristics, and all cycles were the first attempted downregulated ovarian stimulation cycles.
The study protocol was approved by the Ethics Committee of Northwest Women and Children's Hospital (No. 2022007). Patient consent was not required due to the retrospective nature of the study.

Ovarian Stimulation
Protocol. The ovarian stimulation protocol was described previously [8]. In brief, stimulation protocols were used with a combination of GnRH agonist/ GnRH antagonist and recombinant FSH. The ovarian response was monitored by serial ultrasound examination and hormone measurement. Ten thousand units of human chorionic gonadotrophin (hCG) were administered to patients when three follicles were >18 mm. Oocyte retrieval was performed 36 h later by transvaginal ultrasonographyguided aspiration.
2.3. Embryo Culture and Assessment. The OCCs were cultured in the medium (IVF; Vitrolife, Sweden) after retrieval. Fertilization was performed 39 to 40 hours (39-42 hours for ICSI) after HCG administration while incubated in fertilization medium (IVF; Vitrolife). The zygotes were shifted to cleavage medium (G-1; Vitrolife) 5 hours after IVF fertilization. The embryos to culture blastocysts on day 3 were transferred to blastocyst medium (G-2; Vitrolife) until day 6. All media were covered with paraffin oil in a humidified atmosphere at 37°C for prior 24 h.
The same brands of incubators (Model c200; Labotect, Germany) were used for both 2% and 5% O 2 tension to culture embryos. The 5% O 2 tension was to culture in 6% CO 2 , 5% O 2 , and 89% N 2 . The 2% O 2 culture condition also had 6% CO 2 in air. All other conditions were equal except O 2 tension between the two systems. In our center, we had two IVF labs with equal conditions. After transferred with one or two low-quality embryos, extended culture was performed under 2% O 2 in one lab and 5% O 2 in the other lab.
The scoring system for blastocyst evaluation was a combination of the stage of development from 1 to 6 (early, blastocyst, full blastocyst, expanded, hatching/hatched) and of the grade of the inner cell mass (ICM; A, tightly packed, many cells; B, loosely grouped, several cells; or C, very few cells.) and of the trophectoderm (TE; A, many cells forming a cohesive epithelium; B, few cells forming a loose epithelium; or C, very few large cells.) [9]. In this study, the blastocyst scored ≥3BB was defined as high-quality blastocyst.

Statistical Analysis.
Comparisons of the results between groups in the case of continuous variables were analyzed by using Student's t-test for data with normal distribution and nonparametric Mann-Whitney U-test for data with skewed distribution. Comparisons of the results between groups in the case of categorical variables were expressed as number and percentage and analyzed using the chi-square test or Fisher's exact test. The statistical analysis was performed with SPSS version 21 (IBM Corp., USA). Differences were considered statistically significant when p < 0:05.

Demographic and Baseline
Characteristics. The study flow chart was shown in Figure 1. A total of 1552 cleavagestage embryos were prolonged for extended culture to blastocyst stage. 474 high-quality embryos from 63 patients were cultured under 2% O 2 tension, and 568 high-quality embryos from 76 patients were cultured under 5% O 2 tension. A total of 188 patients who had no high-quality embryos on day 3 underwent IVF/ICSI-ET during the study period were included. After transferred with one or two lowquality embryos, extended culture was performed under 5% or 2% O 2 tension for surplus embryos. A total of 296 embryos from 106 patients were cultured under 5% O 2 tension, and 214 embryos from 82 patients were cultured under 2% O 2 tension. The embryos for extended culture to blastocyst stage were divided into four subgroups according to the different numbers of blastomeres as follows: 4-5 C (embryos with 4-5 blastomeres), 6-7 C, 8-10 C, and >10 C groups. The demographic and baseline characteristics of the study population were described in Tables 1 and 2, no significant differences were observed in the female age, BMI, basal FSH value,

Blastocyst Outcomes of Extended
Culture with Low-Quality Cleavage-Stage Embryos to Day 7. We observed that 11 embryos and 3 embryos achieved blastulation on day 7 under 2% and 5% O 2 tension, respectively. The blastulation and high-quality blastulation rates were improved from 39.72% and 10.75% to 44.86% and 13.08% under 2% O 2 tension, respectively. The blastulation and high-quality blastulation rates showed no obvious improvement from 31.08% and 8.45% to 32.09% and 8.78% under 5% O 2 tension, respectively ( Figure 2).

Discussion
O 2 tension was lower in the uterus than in the oviduct, and the human intrauterine O 2 tension had been measured to be around 2% when the human embryo reached the uterine cavity on day 3.5. Kaser et al. analyzed the blastocyst development of normally and abnormally fertilized embryos under 2 or 5% O 2 tension from day 3 onwards. Culture under 2% O 2 tension led to a higher blastulation rate (40.2 versus 22.5%) and a higher utilization rate (36.8 versus 21.3%) [10]. A recent study showed that the total and usable blastocyst rates (44.4 versus 54.8% and 21.8 versus 32.8%, respectively) were significantly higher under 2% O 2 tension from day 3 onwards [11].
For patients with no high-quality embryos, cleavagestage embryo transfer was always recommended in cases of blastocyst formation failure. It was difficult to select an optimal embryo from several low-quality embryos by morphology evaluation for the embryologists. Extended culture of low-quality cleavage-stage embryos might be an alternative strategy. In this study, we mainly aimed to explore whether a reduction in the O 2 tension from 5 to 2% during from  Our results indicated that a reduction in O 2 tension from 5 to 2% after day 3 did not improve the blastulation rate and blastocyst quality of high-quality cleavage-stage embryos. It was significant that we observed that embryo culture in biphasic (5-2%) O 2 concentration significantly increased the total blastocyst formation rate of low-quality cleavagestage embryos compared with culture in monophasic (5%) O 2 concentration. This suggested that mimicking the physiological O 2 concentrations was beneficial to the blastocyst development of low-quality cleavage-stage embryos.
It was well known that the blastulation rate was associated with the number of blastomeres. Kong et al. showed that the blastocyst formation rate and high-quality blastocyst formation rate increased with cell number and reached high levels in the 7-8 C and >10 C groups [12]. Luna et al. observed that human blastocyst morphological quality was significantly improved in embryos classified as fast on day 3 (>or = 10 cells) [13]. In this study, both the 2% and 5% O 2 groups showed a similar proportion of day 3 lowquality embryos with different blastomeres. Embryos with a similar number of blastomeres were cocultured to the blastocyst stage to identify from which blastocyst was formed. Our results showed that a reduction in the O 2 tension from 5 to 2% during from day 3 onwards improved the blastulation rate beyond 10% for the embryos with ≥8 blastomeres; although, no significant differences were observed. It suggested that the problem of slow embryonic development might not be improved by extended culture under ultralow O 2 tension.
Another important factor affecting embryo quality was the homogeneous degree of blastomeres. The embryos with uneven cell cleavage had a lower developmental capacity in comparison with evenly cleaved embryos, and uneven cleavage might result in embryos with a higher degree of aneuploidy and/or multinuclear rate [14]. The problem of uneven cell cleavage might not be improved by ultralow O 2 tension. We suspected that the extended culture under ultralow O 2 tension was most likely to improve the blastulation rate of low-quality embryos with normal blastomere number and more cytoplasmic fragmentation. Embryo cytoplasmic fragmentation was associated with oxidative stress in embryos, and the presence of fragmentation was harmful to the subsequent development of embryos due to the loss  Figure 2: Blastocyst development of extended culture with lowquality cleavage-stage embryos to day 7 between the 2% and 5% O 2 groups. * was significantly different; NS: no significant differences.   [15]. Kim et al. showed that early fragment removal on in vitro fertilization day 2 significantly improved the subsequent development and clinical outcomes of fragmented human embryos [16]. Fragmentation removal was needed to a laser hole in the zona pellucida and used a hand-made micropipette with a mouthpiece to suction out the fragments from around and between the blastomeres. The ultralow O 2 tension culture seemed safer for fragmentation removal in comparison with manual removal.
High-quality cleavage-stage embryos showed a higher opportunity of excellent blastocyst formation compared to day 3 low-quality embryos [17]. For low-quality cleavagestage embryos, it should be less likely to form high-quality blastocysts. Our results showed a similar high-quality blastulation rate between 2% and 5% O 2 groups. Thus, the ultralow O 2 tension culture could improve the blastulation rate but not the high-quality blastulation rate. It was demonstrated that the blastocyst from the low-quality embryo had a comparable implantation rate [18]. Therefore, it might be helpful for such patients with bad prognosis to obtain more blastocysts.
Selection of available blastocysts typically occurred on days 5/6, and some embryos with slow development formed blastocysts on day 7. Should extended culture to blastocyst stage include day 7? Whitney et al. indicated that extended culture to day 7 was beneficial by achieving viable euploid embryos that would have otherwise been discarded [19]. A review concluded that culturing embryos an additional day increased the number of useable embryos per cycle and provided further opportunities for pregnancy for patients, especially those who had only a few or low-quality blastocysts [20]. In this study, we observed that the blastulation rate of extended culture with low-quality embryos to day 7 showed better improvement under 2% O 2 tension than 5% O 2 tension. Thus, it might be more worthy of consideration to culture blastocysts to day 7 if no blastocysts were obtained on day 5/6 for patients with no high-quality embryos on day 3.
In conclusion, extended culture of low-quality cleavagestage embryos under 2% O 2 tension might be an alternative strategy for patients with no high-quality embryos on day 3. There was no doubt that a prospective study was needed to further confirm our findings.

Data Availability
The data used to support the findings of this study were available from the corresponding author upon request.

Ethical Approval
The study protocol was approved by the Ethics Committee of Northwest Women and Children's Hospital (No. 2022007).