**NEW** Read Our Most Recent White Paper Based on the Webinar
Title: pH Monitoring Practices and Benefits
Summary: Controlled embryo culture conditions are critical for optimal success rates within the in vitro fertilization (IVF) lab. The daily quality control required to track culture conditions in real-time is labor-intensive, costly, or misleading about true culture conditions. In this white paper, we discuss fundamentals behind the pH data point, the effects that laboratory staff and equipment have on media pH and discuss suggestions for pH value trending in embryo culture labs. pH trending data was collected using the SAFE Sens® TrakStation® pH monitoring product.
Check Out Our Most Recent Webinar With Embryology Product Specialist Susan Olds
Covered Questions Include
How Much pH Variation Is Harmful During Embryo Culture? What Degree Of Variation Can Be Acceptable For Quality Control Parameters?
In What Scenarios Should My Lab Take pH Measurements?
Are Regular CO2 Measurements Sufficient? Why do I also need to track pH if I am tracking CO2?
How Do Differences In Media Prep Setup Affect Embryo Culture System pH? Is There A Difference In pH When Using Microdroplet Versus Well Culture/Large Volume?
What Impact Do Daily Lab Functions And/or The HVAC Schedule Have On Culture pH?
How Does Daily Workflow Affect pH In Large Format Incubators Compared To Benchtop Incubators?
What Are Good Standard Practices To Maintain A Stable Culture Media In Large Format Incubators?
What is the difference between pH measuring and pH monitoring
How reliable are pH measurement devices? What kind of accuracy can I expect?
Where can I learn more about pH and pH monitoring?
Webinar Q&A Responses
Thank you for your interest in our webinar: “Top 10 Frequently Asked Questions on Embryo Culture.” See below for formal responses to the questions that weren’t covered in the audio Q&A section due to time constraints. If you have further questions, please feel free to reach out to me via my email at susan.olds@safesens.com.
1. In relation to the microdrop culture dish figure, what is responsible for the difference in pH between the culture dish and sv2 sensor?
The differing pH values between the trend lines are probably due to the differences in precision between the fluorescent membrane. The difference seen correlates with expected differences between SAFE Sens disposables (0.05 accuracy range). This figure is a great example that when using multiple monitoring devices, the recorded pH values may not be exaclty the same – however, these values give critical information on the trending of pH values and a better picture of how workflow affects pH fluctuation during embryo culture.
2. How much pH fluctuation would there be in an ESCO non-humidified incubator with an open four well dish in 24 hours? How bad would the impact be on embryo culture?
We do not have data to present effects on pH in a non-humidified incubator using an open culture 4-well dish setup- however, this setup type is not recommended. Even in a humidified incubator setting, open culture will have evaporation, causing rising values in culture media osmolality and pH values. An experiment using a multi-well dish filled with 0.5 mL of media in a humidified incubator showed significant increases in osmolality due to evaporation within 72 hours- the osmolality increased from 280 mOsm/kg to 321 mOsm/kg[1]. A non-humidified incubator setup would further exacerbate this effect, as the dry air and high temperature increases the rate of evaporation. For non-humidified incubator types, it is critical to have an oil overlay for embryo culture and to also verify with mass measurements that the optimal oil volume is being used to combat the effects of evaporation on culture media.
3. If using more than one type of medium in an incubator simultaneously (Fertilization and Cleavage stage medium) would you need more than one TrakPod in that incubator to keep track of multiple media?
There are several options for pH monitoring with multiple media types.
The lab can monitor each media and confirm that the environmental impact of that media remains optimal using multiple TrakPods to monitor each media type.
OR
Multiple sensors can be prepared (one with each media) and the sensors are exchanged when the embryos are moved to the next media or over a weeklong period to determine pH stability for QC purposes.
The lab should determine whether they wish to monitor media types simultaneously and continuously, or if they are O.K. with using one media type each week and observing data trends for out of range values or trend lines.
[1] Swain JE, Cabrera L, Smith GD (2012). Microdrop preparation factors influence culture media osmolality which can impair mouse preimplantation embryo development. RBM Online, 24(2), 142-7.
Susan Olds
Embryology Product Specialist
SAFE Sens / Blood Cell Storage Inc.
Tel: +1.425.654.8462 (D)
Email: susan.olds@safesens.com
4. As emerging ART methods such as follicle culture and ovarian tissue culture become more common, are there differences in culture condition environments that should be taken into account?
Follicle tissue and ovarian tissue culture is a great subtopic to discuss. Ovarian tissue cryopreservation is sometimes the only option available for infertility patients that must confront the gametotoxic effects of cancer treatment therapies, particularly younger patients or those that do not have the means to perform follicle stimulation and retrieval protocols before treatment. The purpose of follicle culture is to harvest gamete rich tissue and to create developmentally competent oocytes for use in conventional IVF protocols. Follicle culture systems are needed to maximize mature oocytes available most efficiently and face the challenge of supporting folliculogenesis and maturation of all immature oocytes types within a culture system. With the development of this new and novel subcategory of culture, the individual lab setup and culture system should be scrutinized very carefully. The success of live birth from these cases requires optimal culture conditions to increase the chance of prolonged viability of patient gametes and subsequent embryos. If a bicarbonate-based media is being used post-oocyte isolation from follicle culture, it is critical to reduce pH fluctuation during this time and reduce environmental stresses acting on the available oocytes.
5. Could the webinar be at a later time?
If you are interested in the webinar but were not able to attend, we have a recording available here. If you have any further questions on the topic matter or about the SAFE Sens TrakStation, feel free to email me at susan.olds@safesens.com.
6. Is your system adaptable to ESCO bench tops “MIRI” incubators and embryoscopes?
Our benchtop incubator system is available in the Esco Miri, Esco Mini Miri, and the Esco Time-Lapse incubator types. To learn more about integrating SAFE Sens into Esco incubators, please see here.
7. Can you correlate your pH findings to biological outcomes? I would also like to expand into incubation systems – dry vs humid again with biological outcomes.
Correlating pH to biological outcomes is an area of study that is needed in the field. It is a particularly challenging experiment setup to perform, as there are so many confounding variables that can affect biological outcomes that are not directly related to the effects of media pH on embryo development. The effects of pH directly on embryo development are difficult to isolate due to the variable composition of culture media types, the variability of recommended pH ranges found in media types, and the lack of transparency by media companies to release the composition details of media.
An example of the difficulties in this experimental setup is properly altering pH without indirectly associating results with another culture system component. For example, changing pH values by changing the sodium bicarbonate concentration or through CO2 gas values will not inherently reflect direct effects from pH on embryo development. The embryo uses the carbon from CO2 for the biosynthesis of multiple compounds, and bicarbonate plays a critical role in membrane transporters and internal pH regulation.
For further reading on biological outcomes associated with dry versus humid incubator usage, I recommend a recent publication from Fertility and Sterility by Fawzy Et al. (2017). A summary of the publication can be found here with the full publication here.
References
See below for the List of References used during the Webinar.
1. Lane, M., Baltz, J.M., et al. (1999a). Bicarbonate/chloride exchange regulates intracellular pH of embryos but not oocytes of the hamster. Biol. Reprod. 61, 452–457.
2. Lane, M., Baltz, J.M., et al. (1999b). Na+ /H+ antiporter activity in hamster embryos is activated during fertilization. Dev. Biol. 208, 244–252. Lane, M., Bavister, B.D., 1999. Regulation of intracellular pH in bovine oocytes and cleavage stage embryos. Mol. Reprod. Dev. 54, 396–401.
3. Lane, M., Bavister, B.D. (1999). Regulation of intracellular pH in bovine oocytes and cleavage stage embryos. Mol. Reprod. Dev. 54, 396–401.
4. Leclerc, C., Becker, D., et al. (1994). Low intracellular pH is involved in the early embryonic death of DDK mouse eggs fertilized by alien sperm. Dev. Dyn. 200, 257–267.
5. Zhao, Y., Baltz, J.M. (1996). Bicarbonate/chloride exchange and intracellular pH throughout preimplantation mouse embryo development. Am. J. Physiol. 271 5 Pt. 1, C1512–C1520.
6. Zhao, Y., Chauvet, P.J., et al. (1995). Expression and function of bicarbonate/chloride exchangers in the preimplantation mouse embryo. J. Biol. Chem. 270, 24428–24434.
7. Lane, M., Baltz, J.M., et al. (1998). Regulation of intracellular pH in hamster preimplantation embryos by the sodium hydrogen (Na+ / H+ ) antiporter. Biol. Reprod. 59, 1483–1490.
8. John, D.P., Kiessling, A.A. (1988). Improved pronuclear mouse embryo development over an extended pH range in Ham’s F-10 medium without protein. Fertil. Steril. 49, 150–155.
9. Dale, B., Menezo, Y., et al. (1998). Intracellular pH regulation in the human oocyte. Hum. Reprod. 13, 964–970.
10. Edwards, L.J., Williams, D.A., et al. (1998a). Intracellular pH of the mouse preimplantation embryo: amino acids act as buffers of intracellular pH. Hum. Reprod. 13, 3441–3448.
11. Edwards, L.J., Williams, D.A., et al. (1998b). Intracellular pH of the preimplantation mouse embryo: effects of extracellular pH and weak acids. Mol. Reprod. Dev. 50, 434–442.
12. Lane, M., Lyons, E.A., et al. (2000). Cryopreservation reduces the ability of hamster 2-cell embryos to regulate intracellular pH. Hum. Reprod. 15, 389–394.
13. Nishigaki T. et al. (2014). Intracellular pH in sperm physiology. Biochem. Biophys. Res. Commun. 450, 1149–1158.
14. Dale B, Menezo Y, Cohen J, DiMatteo L, Wilding M., (1998). Intracellular pH regulation in the human oocyte. Hum Reprod. 13:964–970.
15. Ying Liu, Deng-Ke Wang, Li-Ming Chen.(2012). The Physiology of Bicarbonate Transporters in Mammalian Reproduction, Biology of Reproduction, 86:4:1:99 1–13.
16. Hamamah, S., Magnoux, E., Royere, D., Barthelemy, C., Dacheus, J.-L. and Gati, J.-L., (1996). Internal pH of human spermatozoa: effect of ions, human follicular fluid and progesterone. Mol Human Reprod. 2:219.
17. Swain, JE. (2010) “Back to Basics: pH for the ARTisan.” Journal of Clinical Embryology. 13(2):9-28.
18. Ramirez et al (2016). Noninvasive pH monitoring for bacterial detection in platelet concentrates. Transfusion 56, 1348-55.
19. Montag, T et al., (2008). Time course of pH in platelet concentrates after bacterial contamination. Poster presented at AABB DGTI.
20. Miller KF, Goldberg JM, Collins RL(1994). Covering embryo cultures with mineral oil alters embryo growth by acting as a sink for an embryotoxic substance. J Assist Reprod Genet.;11:342-5.
21. Quinn, Patrick MJ. (2014). Culture Media, Solutions, and Systems in Human ART. Cambridge CB2 8BS (UK): Cambridge University Press. Chapter 11, pH control in the embryo culture environment; pg. 150.