# Temperature Dynamics in Early Pregnancy: Implications for Improving In Vitro Fertilization Outcomes
## 4 Discussion
### Limitations and Future Studies  
This study successfully extracted characteristic CBT features in pregnant mice over the five days following successful mating, providing reference values for temperature variations in in vitro culture. However, because body temperature varies by individual, anatomical site, and over time, it must be carefully considered whether these temperature fluctuations are optimal for all embryos. Additionally, this study had several limitations, which are discussed below. First, transient increases in CBT, frequently observed in the light period (Figure 1), were analyzed not as random and stochastic events but as part of irregular fluctuations and complexity. These increases occurred approximately three times during the light phase and were not approximated by the Fourier analysis (Figure 2). Awakenings during sleep may cause a transient increase in CBT. While transient temperature fluctuations are considered detrimental to embryonic development, it remains unclear whether these transient temperature rises in vivo adversely affect embryonic development. Moreover, while door openings can cause a drop in culture temperature, CBT does not drop but increases inversely. Therefore, rapid decreases in CBT may negatively impact embryonic development, whereas rapid increases in CBT may not necessarily have a negative effect. Second, this study assumes a uniform intraperitoneal temperature. However, it is known that there is a temperature gradient within the fallopian tubes and that the temperature changes during tubal transport may also impact embryonic development. The isthmus is consistently cooler than the ampulla, with the temperature difference between these regions being most pronounced during ovulation. Rabbits have evidenced a temperature difference of 1.4 \({}^{\circ}\)C [6], while differences of 0.2 \({}^{\circ}\)C to 1.6 \({}^{\circ}\)C have been reported in pigs [7]. [PERSON] et al. conducted experiments using pig oocytes to replicate this temperature gradient within the fallopian tubes. They cultured the oocytes at 37.0 \({}^{\circ}\)C, simulating the ampulla for 20 h post-fertilization, then they increased the temperature to 38.5 \({}^{\circ}\)C--a standard for pig embryo culture--until 168 h post-fertilization. This protocol caused the blastocyst formation rate to increase 1.6-fold, although no differences were observed in morphological quality or blastocyst cell numbers [33]. These findings suggest that the temperature gradient within the fallopian tube is greater than the circadian temperature variation, indicating the need to incorporate periodic circadian rhythms and also a trend of increasing temperature into in vitro culture conditions. Third, the sample size must be increased to enhance the mean accuracy. A larger sample size would also strengthen the statistical tests by comparing changes in mean CBT and standard deviations. However, since all the pregnant mice in this study demonstrated an increase in their mean CBT during the light phase and a decrease in standard deviation, and the number of mice tested met the minimum sample size, these trends appear to be consistent in pregnant mice. A statistical test between the pregnant and non-pregnant groups would be possible with a larger sample size. Fourth, the precise timing of fertilization remains unknown. While mating occurred between 6 PM and 6 AM in the dark phase, the exact times of mating and subsequent fertilization were not determined, resulting in a possible 12 h discrepancy. Given that embryos undergo cell division approximately once a day, the phase difference between circadian temperature fluctuations and the stages of cell division could be significant. In future studies, it will be important to adjust the in vitro culture temperature to align with the stages of cell division. Finally, we will discuss the potential application of the results of this study to the in vitro culture of human embryos. [PERSON] et al.