In the field of assisted reproductive technology (ART), human embryo culture plays a pivotal role in the success of in vitro fertilization (IVF) treatments. The delicate and intricate nature of preimplantation human development demands a meticulously controlled environment. During human embryo culture, chemical and physical parameters play a crucial role in embryo development and viability ^[1][2][3][1,2,3]. These parameters encompass a range of environmental conditions, including temperature, oxygen concentration, humidity conditions (HC), the use of oil overlay, and light exposure, all of which are carefully regulated within the laboratory setting. Moreover, these parameters directly influence the embryo metabolic activities ^{[4][5][6][7][8][9][10][11][12][13][14][15][16][17][18]}[4,5,6,7,8,9,10,11,12,13,14,15,16,17,18]. It is well established that temperature ensures proper enzymatic reactions and cellular functions [4]. In addition, oxygen plays a vital role in supporting embryo metabolism and development [5]. While a consensus has been reached regarding the utilization of 5% oxygen levels compared to atmospheric levels (20%) [6], conflicting results have emerged when employing biphasic oxygen conditions (5–2%). Although the use of biphasic oxygen conditions appears to offer advantages in terms of blastulation, inconsistent findings have been reported in relation to clinical pregnancy ^[7][8][7,8].

Oil overlay has several important functions and benefits: (i) gas exchange, (ii) temperature stability, (iii) pH regulation, (iv) preventing contamination, and (v) minimizing disturbance. The inherent chemical and physical properties of the oil exert a significant influence on this vital aspect. These properties play a crucial role in shaping and determining the outcome, emphasizing the importance of understanding and considering them when working with human embryo culture [9]. Light exposure during mammalian embryo culture has garnered significant interest. However, despite several investigations, the impact of light on embryos remains a subject of ongoing debate, with inconclusive findings ^[10][11][10,11]. Recently, due to the introduction of dry incubators, several studies have investigated the impact of humidity conditions (HC) and dry conditions (DC) on IVF outcomes. While basic research studies show increased osmolality in culture medium under DC ^[12][13][14][12,13,14], these conditions do not seem to have negative effects on biological and clinical outcomes such as blastulation and pregnancy rates ^[16][17][16,17]. By carefully controlling these parameters, embryologists create an environment that mimics the natural conditions required for healthy embryo development. Nevertheless, despite these efforts, our culture conditions are unlikely to mirror precisely the dynamic environment experienced by embryos in vivo. Concerns exist that sub-optimal culture conditions could affect embryo developmental competence. Therefore, the meticulous quality control of these parameters is critical in maximizing the efficiency of treatments.

2. Oxygen

Oxygen plays a vital role in supporting embryo metabolism and development. In the female reproductive tract, oxygen concentration is typically around 2–8% [18]. Thus, in vivo, the oxygen concentration is different from the atmospheric levels. Several studies have investigated oxygen concentration during human embryo culture. One study showed higher blastulation, pregnancy, and live birth rates using 5% oxygen concentration [19], in contrast to another study that showed no improvements on fertilization, blastulation, and pregnancy rates [20]. Previous studies showed no significant difference in terms of fertilization, pregnancy, and implantation rates between 5% and 20% oxygen concentrations at the cleavage stage ^[21][22][21,22]. On the other hand, several studies showed higher top quality embryos, blastulation rate, and live birth in favor of 5% oxygen than 20% ^[23][24][25][23,24,25]. No difference was found in fertilization rate between 5% and 20% oxygen tension, but an increased number of top quality embryos on day 3, higher blastocyst formation, clinical pregnancy, and implantation rates in favor of 5% [26], according to one study that showed an overall increase in live birth when embryos were cultured in low oxygen tension [27]. Finally, a meta-analysis showed an improvement in the live birth rate of 43% during embryo culture in 5% oxygen concentration [6]. Accordingly, the latest recommendations provided from the ESHRE guidelines suggest the use of low oxygen concentration [1]. Interestingly, recent studies investigated the use of sequential oxygen tension (5% until day 3 and, subsequently, 2% from day 3 to day 5). This is probably to mimic the natural conditions of in vivo embryo development. A sibling zygote randomized control trial showed, although a small sample size, better blastulation rate when oxygen tension is reduced from 5% to 2% on day 3 for extended embryo culture (day 5) [7], in contrast to two studies that showed a similar blastocyst formation rate between 2%, 5% and 20% oxygen tension ^[8][28][8,28]. One report showed that blastocyst utilization rate is higher in 2% oxygen tension group [29], according to another study that showed improvement in blastocyst formation but only in low-quality human embryos cultured with 2% oxygen [30]. No significant difference were found between 5% and 3% oxygen tension in fertilization, blastulation and euploid blastocyst [31]. Recently, two studies suggested that biphasic oxygen culture could be an alternative strategy to increase the euploid blastocyst [32], blastocyst formation, and cumulative live birth rate [33]. Researchers analyzed 18 studies for the LS calculation, 10 focused on comparing between 5% and a 20% oxygen concentration, resulting in a LS of 7. Additionally, eight studies examined the comparison between monophasic (5%) and biphasic (5–2%) culture oxygen tension, resulting in a LS of 5. These findings suggest there is no evidence that biphasic culture (5–2%) is better than monophasic culture (5%), especially in terms of clinical outcomes (Table 1).

3. Temperature

Maintaining the correct temperature is essential for proper gamete function and/or embryo metabolism and development [4]. Deviation from the optimal temperature can have detrimental effects on gamete function and embryo development, resulting in reduced viability and lower success rates in ART. Typically, the temperature is set at approximately 37 degrees Celsius (°C) to emulate the natural temperature found within the female reproductive tract. However, certain studies have suggested that a temperature of 36 °C may be more suitable to mimic the conditions of the female reproductive tract, potentially leading to improved fertilization and implantation rates ^[34][35][34,35]. Several studies have investigated the impact of temperature on IVF outcomes, yielding contradictory results. There has been evidence supporting negative consequences on the stability of the oocyte’s meiotic spindle when the temperature decreases ^[36][37][36,37], resulting in delayed embryo development [38], lower fertilization, and pregnancy rates [37]. A particular study found that the temperatures measured in the oviducts of non-mated, pre-ovulatory, peri-ovulatory, and post-ovulatory rabbits ranged from approximately 34.8 to 35.8 °C and from 35.9 to 36.6 °C in the sperm storage and fertilization site, respectively. These findings suggest that working at these temperatures (around 36 °C) may better mimic the human female reproductive tract [34], according to Higdon and colleagues, who showed a higher pregnancy rate when the incubator environment was cooler than 37 °C [35]. On the contrary, one randomized control trial showed that 36 °C does not improve embryo developmental competence and implantation rate [39]. A recent prospective sibling oocyte study suggests that culture temperature at 36.6 °C or 37.1 °C did not affect embryo development. However, it was observed that the clinical pregnancy rate was higher when the culture temperature was set at 37.1 °C [40], according to Fawzy and colleagues, who showed improvement in embryo development when the incubator was set at 37 °C [41]. Finally, one meta-analysis [42] showed no evidence that embryo culture at a lower temperature than 37 °C improves biological and clinical outcomes. The researchers analyzed six studies for the LS calculation, obtaining a high LS of 8.3 (Table 1), suggesting a prevalence of studies in favor of 37 °C.

4. Humidity Conditions

Humidity plays a significant role in the incubator environment. Maintaining optimal humidity levels is crucial to prevent excessive evaporation from the culture medium, which can affect embryo development by altering osmolality and pH [13]. However, it is important to acknowledge that humidity conditions in the incubator can have drawbacks as well. One notable concern is the increased risk of microbial contamination ^[12][43][12,71]. Advancements in IVF technology have led to significant improvements in incubator design. The latest generation of incubators now feature smaller individual chambers, specifically designed to minimize oscillations that may occur when the chambers are opened. However, the introduction of these new incubators, with their smaller individual chambers, has initiated a shift towards utilizing a DC atmosphere, as opposed to the conventional humidified environment. While this innovation offers advantages in minimizing oscillations during the opening of the chambers ^[44][72], there are concerns among scientists regarding the potential negative impact of DC on embryo developmental competence and clinical outcomes ^[15][45][15,43]. Two studies showed that significant evaporation occurs during single-step medium culture after 6 days in a dry incubator ^[14][46][14,44]. The humidity levels within incubators have a significant impact on the stabilization of osmolality ^[47][45], according to a previous study ^[48][46], suggesting that incubating the medium in a non-humidified environment leads to an increase in osmolality. The osmolality and pH of the culture media increase significantly over the course of 6 days of culture in both DC and HC, although the change was less with HC [13]. Nonetheless, evidence relevant to the impact of HC on biological and clinical outcomes are scarce and conflicting. A randomized controlled trial revealed a statistically significant decrease in implantation rates, as well as clinical and ongoing pregnancy rates, in DC [15], while another study found a difference in terms of ongoing pregnancy in the day 3 but not in the day 5 transfer policy ^[45][43]. Embryos developing under DC produced lower blastulation rates ^[49][47], in contrast to Valera and colleagues, who showed a comparable blastocyst formation rate and usable blastocyst [16]. The same authors showed a higher clinical pregnancy rate under HC in PGT cycles, but not in egg donation or autologous cycles. Moreover, the authors observed a negative impact of DC only on clinical pregnancy but not on ongoing pregnancy and live birth with use of single-step medium [16], according to a previous report that showed similar pregnancy and miscarriage rates ^[49][47]. Interestingly, another recent study using sequential medium yielded similar results. The authors suggest that HC do not enhance the rate of ongoing pregnancy and several embryological outcomes when employing a day 3 medium change-over [17]. These recent results ^[16][17][16,17] are reinforced by the control approach (the same incubator under two different conditions). In conclusion, while basic research studies consistently indicate alterations in pH and osmolality of the culture medium under DC (although a relatively large volume of medium and a thick oil overlay cooperate in reducing evaporation), it is important to note that this consensus does not align with the clinical evidence. For the LS calculation, researchers analyzed four studies and obtained a LS of 5 (Table 1), suggesting no evidence of superiority for HC over DC.

5. Oil Overlay

In human embryo culture, an oil overlay is often used as a covering layer on top of the culture medium to create a specific environment for the embryos. One of the primary purposes of an oil overlay is to facilitate appropriate gas exchange within the embryo culture system, to minimize evaporation, and help maintain a stable environment. Despite this, evaporation could also occur with the use of a mineral oil overlay ^[46][48][50][44,46,73]. A study discovered that the osmolality of the medium (microdrops ranging from 50 to 200 μL) increased significantly when it was covered with mineral oil during a 5-day incubation period in a dry incubator. However, no such increase was observed when the incubation took place in a humidified atmosphere [14]. Furthermore, one study showed that one particular oil (oil B) exhibited a greater increase in osmolality compared to the three other oils (oils A, C, and D), which displayed similar increases in osmolality. This discrepancy can be attributed to the distinct physical oils composition. Specifically, oil B had lower viscosity and density, while its water content and activity were significantly higher [14]. Furthermore, denser oils have been observed to effectively reduce evaporation. In this context, a slight density difference of 0.04 g/mL can have a considerable influence on the rate of evaporation ^[48][46]. Another report indicated that using a 5 mL oil overlay resulted in lower osmolality compared to when only 3 mL was used ^[48][46]. Interestingly, a comparison of various brands of oil proposed that commercial oils exhibit variations in their ability to maintain the stability of osmolality and pH. Furthermore, the authors found differences in the total number of cells and the number of inner cell mass (ICM) of the obtained blastocysts across different oils ^[51][48]. To mitigate evaporation and prevent an increase in osmolality, employing a large volume of oil can effectively counteract these phenomena ^[47][45]. Indeed, higher evaporation occurs when using 3 mL of oil compared to using 5 or 7 mL in the same type of dish ^[50][73]. In this scenario, the volume of oil used to prepare the culture dishes plays a significant role in preventing medium evaporation and ensuring temperature stability. Using higher volumes of oil and ensuring a thicker layer can effectively minimize evaporation and maintain stable medium osmolality, particularly in single-step medium culture. Due to the specific inclusion criteria, calculating LS in relation to oil was not feasible.

6. Light

In vivo embryos, which develop inside the female reproductive system, are not directly exposed to light. However, during IVF treatments, embryos may be exposed to light, albeit in a controlled and regulated manner. There is scientific literature available on light exposure and its potential effects on embryo development; nevertheless, contradictory results have been obtained. In a study focusing on pre-implantation rabbit embryos, researchers found that subjecting the embryos to 24 h of visible light exposure did not lead to a significant increase in DNA ploidy abnormalities ^[52][49], in contrast to another study that showed how exposure to light for 24 h induced vacuolization, lamellar bodies, and increased electron density in the cytoplasm ^[53][50]. Moreover, the same authors suggested that the susceptibility of embryos to light might vary depending on their developmental stage ^[53][50]. Several studies have shed light on the potential effects of direct and prolonged exposure to visible light on oocyte’s rabbit. Light exposure does not interfere with the normal oocyte’s maturation process, embryos implantation, and cleavage rate ^[54][55][56][51,52,53]. A study examining pre-implantation rabbit embryos at different developmental stages investigated the effects of a 24 h exposure to light. The results of this study revealed contrasting outcomes for day 1 and day 3 embryos. In the case of day 1 embryos, exposure to light for 24 h led to noticeable cell degeneration, indicating a negative impact on their viability. On the other hand, day 3 embryos showed signs of apoptosis, albeit to a lesser extent compared to day 1 embryos. This suggests that the vulnerability to light-induced damage varies between different stages of embryo development ^[57][54]. Interestingly, one study, conducted on hamster and mouse embryos, showed that with just 3 min of exposure to microscope light, there was a significant increase in hydrogen peroxide levels ^[58][55]. Increased cytoplasmic electron density and fragmentation were found after an 8 h exposure to light ^[59][56]. Recently, white light has been reported to potentially decrease the implantation capacity of mouse embryos [11], in contrast to another two studies that showed no compromised fertilization rate, embryo development as well as clinical pregnancy with the use of a red filter light protection ^[60][61][57,64]. Moreover, prolonged exposure to light reduced the cleavage ability of rabbit embryos in a time-dependent manner, suggesting the use of red filtered light for prolonged exposure ^[62][58]. On the other hand, the use of a green filter on a microscope did not significantly improve bovine embryo development ^[63][59]. Two studies have investigated the probable harmful effects of blue light, showing that it has a negative impact on the blastulation rate of hamster and mouse embryos ^[64][65][60,61]. More recently, there has been evidence supporting the beneficial effects of yellow light irradiation on preimplantation development of mouse embryos during in vitro blastocyst production, regardless of the stage of the embryo ^[66][62]. Two studies investigated the potential detrimental effects of laser light on embryos, demonstrating no negative impact on embryo development, survival, and blastulation rates ^[10][67][10,63]. There exist differing perspectives regarding the potential adverse effects of light and the data suggest low scientific evidence for negative impacts with prolonged exposure to light. Moreover, researchers analyzed seven studies for the LS calculation, resulting in a low LS of 4.3 (Table 1).

7. pH

Culture media pH is a critical factor in human embryo culture. pH is closely correlated with carbon dioxide (CO₂) levels due to the bicarbonate buffering system, in which changes in CO₂ concentrations impact the production of carbonic acid, consequently leading to variations in pH. Adjusting the percentage of CO₂ gas in the incubator is a fundamental method for precise pH control in the culture medium, which is essential for embryo development ^[68][65]. While embryos exhibit an impressive capacity to tolerate a wide range of pH values, it is crucial to note that deviations from the optimal pH range can have adverse effects on developmental competence ^[68][65]. In zygotes and embryos, intracellular pH (pHi) plays a pivotal role in maintaining cellular homeostasis, governing a myriad of cellular processes, including enzymatic reactions, cell division, and differentiation ^[68][65]. Fluctuations in the extracellular pH of the culture media directly influence the pHi of embryos, consequently affecting their homeostasis and developmental competence [69]. Although human embryos possess several intracellular mechanisms to regulate their pHi ^[70][66], any fluctuations can lead to cellular stress, impairing embryo developmental competence ^[71][67]. In comparison to embryos, oocytes exhibit heightened fragility due to their limited intrinsic capacity for robust pHi regulation, rendering them more susceptible to pH fluctuations ^[72][68]. Mammalian embryos at the morula and blastocyst stages appear to exhibit enhanced capabilities in regulating their pH levels due to the presence of tight junctions that are less permeable to H+ ions ^[69][73][69,70]. The optimal extracellular pH (pHe) was determined to be slightly higher than the pHi. Deviations in either direction, whether towards higher or lower pHe values, were observed to have inhibitory effects ^[70][66]. An ideal pH range of approximately 7.30 was identified for the pronuclear stage, followed by a lower pH value of 7.15 for cleaving embryos ^[43][71]. The pH of the culture medium pH can also be influenced by various additional factors, such as the laboratory’s geographical altitude. Altitude and air pressure can influence pH levels in embryo culture media due to variations in the solubility of CO₂. Therefore, it is essential to consider altitude and air pressure to maintain a stable and optimal pH for embryo development.