Even brief exposure of preimplantation mouse embryos to high-osmolality culture medium (>300 mOsm/kg) in the absence of osmolytes resulted in impaired development [1-3]. The detrimental effect of hyperosmolality has also been reported in the development of various mammalian embryos, including murine [4-6], rat [7], porcine [8,9], and bovine [10,11] embryos. Moreover, in vitro two-cell blocks of mouse embryos were significantly alleviated by culturing them in low-osmolarity (250 mOsm) medium compared to high-osmolality (>300 mOsm) medium [12]. Biggers et al. [3] reported that when the NaCl concentration was increased in the medium, the intracellular Na⁺/K⁺ ratio dramatically increased, which was detrimental to mouse embryo development. The detrimental effects of hyperosmolality occur by triggering cell shrinkage, oxidative stress, protein carbonylation, mitochondrial depolarization, DNA damage, cell cycle arrest, and apoptosis [13,14].

From a different point of view, an increase in extracellular osmolality can promote water flux out of the cell, triggering cell shrinkage and intracellular dehydration [15]. Intracellular water loss interferes with many cellular functions, including DNA synthesis and repair, transcription, protein translation and degradation, and mitochondrial function. As a result, cell cycle progression and cell proliferation are arrested [13]. Dry-type incubators are now widely used instead of humid-type incubators due to the development of infrared CO₂ sensors [16] and the low possibility of microorganism overgrowth [17]. In addition, dry-type incubators have smaller culture chambers than humid-type incubators, is advantageous in terms of the short recovery time for gas and temperature after the door is opened. However, concerns about the possible change in the osmolality of the medium by evaporation remain a reason why clinicians may hesitate to use a dry-type incubator. Although overlying culture dishes with oil could inhibit the shift in the osmolality of the medium, whether it completely eliminates the change in osmolality remains controversial [18]. Recently, human embryos cultured in dry-type incubators showed significantly lower implantation and clinical pregnancy rates than those cultured in humid-type incubators [18].

The present study was performed to compare osmotic changes in culture media covered in oil in various types of culture dishes, and to investigate whether a beneficial effect on clinical outcomes in in vitro fertilization-embryo transfer (IVF-ET) cycles could be obtained by supplementation with outer-well medium to inhibit osmotic changes in culture media in a dry-type incubator.

This retrospective study was approved by the Institutional Review Board of Mamapapa and Baby Clinic (IRB No. 2019-10-01), and was conducted from August 2018 to August 2019.

When the osmolality of the medium increases above a certain threshold, embryo development is compromised [1,5] and apoptosis is increased [19]. Hyperosmolality (>300 mOsm) was shown to have a detrimental effect on preimplantation mouse embryo development in previous studies [4-6]. However, paradoxically, the osmolality of oviductal fluid is about 340 mOsm [2,20], which is high enough to impair mouse embryo development in vitro. However, unlike in vitro, mouse zygotes have no difficulty developing in vivo, which suggests that an unknown mechanism may exist that helps them overcome the detrimental effect of hyperosmolality in vivo [1].

One possible mechanism is the use of various amino acids as organic osmolytes by embryos. The addition of various organic osmolytes, including taurine [21-24], hypotaurine [25], and glutamine [26], were beneficial for embryo culture. Glycine has also been shown to protect mouse [3] and rabbit embryos [27] against the effects of high NaCl levels. Indeed, over 70% of zygotes developed to the blastocyst stage when 1 mM glutamine was present at 310 mOsm, but only about 10% did so in the absence of glutamine [1]. At 310 mOsm of KSOM medium in the absence of glycine, over 80% of outbred mouse zygotes were arrested at the two-cell stage. However, in the presence of glycine, 60% of the zygotes developed to the blastocyst stage [12]. Furthermore, more blastocysts formed when bovine zygotes were cultured in a 247 mOsm medium (34.6%) than in a 286 mOsm medium (17.0%) in the presence of 1 mM glycine [28]. The beneficial effect of organic osmolytes can be explained by previous findings that amino acids, including osmolytes, are present in oviductal and uterine fluid [29-31] and carry out various physiological functions in the preimplantation embryo, including ATP production [32], ammonium detoxification [33,34] and maintaining the redox balance [35].

Baltz and Tartia [36] suggested a possible answer for the question “Why did lower osmolarity support embryo development in culture?” They proposed that the osmolality in the in vivo environment of early preimplantation embryos may be lower than in blood plasma (280 mOsm). This possibility conflicts with previous reports that the osmolality of oviductal fluid is about 340 mOsm [2,20]. Although the oviduct is the major in vivo environment for early-stage embryos, the follicle can be seen as the birthplace of the oocyte and embryo. In mice, a large amount of follicular fluid moves into the ampulla of the oviduct with all ovulated oocytes via the ovarian bursa. In human, immediately after ovulation, a small amount of follicular fluid flows weakly from the ovarian wall to the ampulla of the oviduct to help the sliding movement of the oocyte. This means that follicular fluid may play an important role in determining the oviductal environment for early-stage embryos. In our preliminary test (data not published), we compared the osmolality of human follicular fluid and blood serum. Interestingly, the osmolality of follicular fluid (275.3 mOsm) was lower than that of serum (282.0 mOsm). There are still no reports about the osmolality of human oviductal fluid, so further research on the osmolality of the in vivo environment of human preimplantation embryos is required. Nevertheless, interestingly, current commercial culture media already have lower osmolality (260-270 mOsm) than the older generation of media (280-295 mOsm) [36].

In the present study, after 3 days of incubation, even under an oil layer, the osmolality of media in the dry culture condition (301.1 mOsm) was higher than that of media in the humid culture condition (285.3–290.4 mOsm) in the dry-type incubator. Gasperin et al. [37] also reported that in the absence of an oil layer, the addition of water in the four-well dish central hole reduced the osmolality of medium (294 mOsm) after 24 hours of incubation compared to the osmolality of control dishes without water (305 mOsm) in a humid-type incubator. These results indicate that osmolality can increase in both dry- and humid-type incubators. Therefore, both an oil layer and extra medium or water supplementation are essential for maintaining the osmolality of the medium by inhibiting evaporation in in vitro culture conditions.

In the present study, in day 5 ET cycles, the rates of clinical and ongoing pregnancy in the humid culture group were higher than the rates in the dry culture group. Similarly, no meaningful difference was found in the clinical and ongoing pregnancy rates of the humid culture groups in day 5 ET cycles (57% and 52%) between the previous [18] and the present study (59.5% and 57.3%), even though there was a large difference in the mean age of female patients between the two studies (28 vs. 36 years, respectively). The first possible reason is medium renewal. We performed medium renewal by exchanging G1 to G2 medium on day 3, but Fawzy et al. [18] did not perform medium renewal for 5 days. Culture for 5 days without medium renewal could degrade the organic osmolytes, and this degradation may augment the negative influence of this osmolality shift on embryo development. In addition, medium renewal may not only reduce the accumulation of metabolites such as ammonium, but could also supply fresh nutrients for embryo development. The second possible reason relates to the humid culture condition. In the present study, for the preparation of the humid culture condition, we added 6 mL of medium into the outer well of GPS culture dishes (LifeGlobal), while Fawzy et al. [18] used petri dishes containing 10 mL of water. In the preliminary experiment, we also used petri dishes containing water, but this was not efficient to inhibit the increase of osmolality in the medium compared to direct medium supplementation in the outer well of the culture dish.

Fawzy et al. [18] reported that the blastulation rate of human embryos was significantly higher in the humid culture group (73%) than in the dry culture group (51%, p<0.05). Gasperin et al. [37] reported a similar trend, according to which the blastocyst rate of bovine embryos was higher in the humid culture group (29.7%, p<0.05) than in the dry culture group (16.2%). Unlike the above results, in the present study, there was no significant difference in the blastulation rate between the dry (56.5%) and humid culture (55.5%) groups. Of particular note, the blastulation rate of the humid culture group (55.5%) was markedly lower than the rate (73%) reported in the other study [18]. This difference may have resulted from the large gap in the mean age of female patients between the other study (28 years) and the present study (36 years). The age of female patients is widely considered as a critical factor for determining the quality of oocytes, the grade of embryos, and the success of pregnancy in IVF-ET cycles. Therefore, the relatively low blastulation rate in the humid culture group in the present study may be explained by the relatively old age of patients compared to the previous study [18].

There was no significant difference in the development of early-stage mouse embryos cultured between the high-osmolality (310–330 mOsm) and low-osmolality (270–290 mOsm) media. However, in the development of late-stage embryos, the embryos cultured in the low-osmolality medium showed a significantly higher blastocyst formation rate than those cultured in the high-osmolality medium [6]. A similar result was observed in porcine embryos cultured in vitro [8]. These results suggest that late-stage embryos are more susceptible to osmotic changes than early-stage embryos. In the present study, a similar pattern in response to osmotic changes in human embryos was observed, although we did not observe a significant difference in the blastocyst formation rate between the high-osmolality (56.5%, dry culture) and low-osmolality (55.5%, humid culture) culture groups. However, in day 3 ET cycles, involving relatively early-stage embryos compared to day 5 ET cycles, the transferred embryos showed a small difference in pregnancy outcomes between the high- and low-osmolality culture groups. The differences in the rates of pregnancy, abortion, and ongoing pregnancy were 4.8%, 6.8%, and 11.7%, respectively. In contrast, in day 5 ET cycles, the differences in these rates in the late-stage embryos were 9.5%, 22.8%, and 32.3%, respectively. This result is consistent with the previous finding that late-stage embryos are more susceptible to osmotic changes [6,8].

When a cell shrinks in hyperosmotic conditions, inorganic ions (Na⁺, K⁺) are accumulated via osmotically regulated ion transporters [38]. However, in this case, cells accumulate organic osmolytes intracellularly to replace a portion of the inorganic ions [39-41]. High ionic strength disrupts cell functions, while in contrast, a high concentration of organic osmolytes is not toxic [39,42]. The beneficial action of organic osmolytes was proven by a report that in a hyperosmotic medium (310 mOsm), the blastocyst formation rate of mouse embryos cultured in the presence of 1 mM glycine was significantly more favorable than that of embryos cultured in the absence of glycine [12].

We used G1/G2 medium (Vitrolife), which contains 135–145 µM glycine [43], a concentration that is much lower than the effective concentration of 1 mM in previous studies [12,28]. In the present study, the osmolality of G1 medium was 275 mOsm, which increased to 293.7 and 285.0 mOsm in the dry and humid culture conditions, respectively, after 3 days of incubation. The better clinical outcomes in the humid culture condition than the dry culture condition might have resulted from the low osmolality of the medium in the humid culture condition. Due to the maintenance of low osmolality in the culture medium, glycine probably did not have the chance to act as an organic osmolyte. Instead, the higher osmotic change in the dry culture condition might have increased the concentration of intracellular ionic osmolytes, which are detrimental for embryo development. Moreover, a portion of the ionic osmolytes could not be replaced completely with organic osmolytes because of the low concentration of glycine [43].

In conclusion, the dry culture condition showed a higher osmotic change in the medium than the humid culture condition, even though the media were covered in oil. Hyperosmotic changes showed a detrimental effect on clinical outcomes in human IVF-ET cycles. This hyperosmotic stress could be alleviated by supplementation with outer-well medium to maintain the optimal osmolality of medium, which resulted in the improvement of clinical outcomes in IVF-ET cycles. We are preparing a follow-up study to investigate relationships between the osmolality of culture media and specific patterns of gene expression in porcine embryos.