Figure 1. Benchtop incubators in an IVF laboratory (1)

Every procedure carried out in an IVF laboratory, from ovarian puncture and semen capacitation to the embryo transfer, must be performed under specific safety conditions. These standards must be followed to avoid a decrease in gamete/embryo viability (2).

From the in vitro culture, gametes and embryos are exposed to diverse artificial situations that do not take place in nature. In vivo, both fertilization and embryo development in the female reproductive tract occurs in the complete absence of light. In this environment, other conditions include oxygen (O2) concentration of 2-8%, pH 7.2-7.4, 37⁰C and gradients of diverse nutrients to which the embryo is exposed (3). Changes in temperature, CO2 and O2 pressure, light exposure or volatile organic compounds may adversely affect embryo quality.

Also, the manipulation of embryos by embryologists is as important as air quality and culture conditions. Each human being is covered by about 1012 bacteria (4), which could contaminate embryo cultures if embryologists do not use the necessary clothing, such as laboratory cap, footwear and mask.

Therefore, daily quality control at different levels should be carried out to obtain good results in IVF cycles. We are going to analyze the effect of some of the elements that can affect germ cells and embryos.

LIGHT EFFECT 

In vivo, mammalian germ cells and embryos are not exposed to light, which might explain why they do not exhibit any protection mechanism against this factor (5,6). In addition, sperm does not have the capacity to repair DNA, unlike oocytes and embryos, which do present some mechanisms for DNA repair (7).

Light variables to be considered are intensity, duration, and wavelength. It seems clear that photooxidation increases along with light intensity and duration. However, what type of wavelength would be the most harmful for embryos and germ cells? Energy increases when the wavelength is shorter (8). Accordingly, artificial cool white fluorescent light has been demonstrated as the most stressful in mouse embryos. Incandescent light, in turn, seems to be less harmful, and the best outcomes are achieved when warm white fluorescent light is used (7).

HOW CAN LIGHT AFFECT THE QUALITY OF THESE CELLS

Indirect effect: Culture and oil photooxidation can affect embryo development (8). In this case, modified components will damage the lipid membranes. Also, if HEPES- or riboflavin-containing media is exposed to light, it results in the formation of hydrogen peroxide, a highly cytotoxic substance (9). Additionally, light can heat up both the plasticware and the oil, resulting in more toxic and damaging components (8,9).

Direct effect: Light can potentially compromise the quality of gametes and embryos, by activating stress-related genes or by ionization, which may also damage the DNA. This phenomenon would cause DNA fragmentation and mutation, as well as an increase in the apoptotic index and change in the number of mitochondria levels (10).

HOW CAN WE AVOID THIS EFFECT? (7)

1) Reducing the exposure time.
2) Using warm white fluorescent light in the lab and green filters on microscopes.
3) Adding antioxidants in the media in order to mitigate damages from ROS.
4) Avoiding riboflavin, which is responsible for the phototoxicity in the media.

VOLATILE PARTICLES EFFECT

Since the 1990s, IVF laboratory indoor air quality has taken a high relevance. Thence, focused on creating an optimal environment, laboratories have become clean rooms where filtration of particles is performed by using high-efficiency particle arresting (HEPA) filters, and successful chemical air filtration is achieved by removing volatile organic compounds (VOCs) with solid-phase filtration (e.g., potassium permanganate-impregnated, carbon filters) (11).

Focusing on VOCs (hydrocarbon-based compounds that are emitted by industries, cleaning products, computers, and microscopes among others), several studies have demonstrated their harmful effect on embryos, initially reported by Boone laboratory on mouse embryo development (11). Moreover, VOCs have been shown to increase DNA fragmentation in human sperm, and they can also have detrimental effects on pregnancy rates (12).

Recently, a retrospective study by Munch et al. concluded that, without solid carbon filtration, fertilization, cleavage, and blastocyst conversion rates declined in fresh IVF cycles. Even more, results were found to be even worse in ICSI cycles, probably due to the lack of protective barrier provided by the cumulus cells (13). However, the authors did not observe the same results when embryos had been cryopreserved in an environment with carbon solid filtration but thawed in a laboratory deprived of such systems. The absence of significant changes in cleavage and blastocyst conversion rates, as well as in the proportion of good quality blastocyst developed after thawing suggests that embryos are affected in the peri-fertilization period  (13).

Also, products like cosmetics emit VOCs, especially perfumes, colognes, and deodorants. They are highly toxic to embryo development in vitro, primarily due to evaporation of their solvent bases (14,15). After analyzing the results of studies determining the toxicity of VOCs, ideal levels should be below 0.2 ppm but preferably zero (12). Personnel must understand the principles of air quality control, including the function of airflows and airlocks, hygiene, dress code and the use of cleaning agents (16).

pH EFFECT

pH level depends on bicarbonate concentration of culture media and the CO2 concentration of the incubator. However, other factors like altitude and composition of culture media could affect the pH level, too (17,18). Embryos are able to develop over a range of media pH, considering that they possess an intracellular mechanism to regulate its internal level (17,18). However, it is important to control pH variations because they affect development (17). To control pH level outside the incubator some culture media contain buffers like HEPES or MOPS, but long exposure of embryos to these buffers is not recommended (17). Thawed denuded oocytes and embryos are especially sensitive to pH variations because they do not have an inner system to regulate pH (17). So, an increase in the pH of the medium can affect the physiology and development of oocytes and embryos. Thus, acidification of the medium can even affect fetal weight and size (18).

COEFFECT

As previously mentioned, CO2 is necessary to control the pH level of culture media (17,18). The importance of CO2 was demonstrated in 1985, in a study carried out on hamsters (19). The author’s cultured hamster embryos in different CO2 concentrations (5% and 10%). They found a higher rate of blastocysts in those cultured at 10% compared to 5%, which demonstrated differences in embryonic development. This results showed that CO2 level is an important factor for embryo culture (19). The capacity of CO2 to get through cell membranes allows for regulation of the inner pH levels in blastomeres. In other studies, it has been shown that the required CO2 concentration to achieve the optimal pH varies in different species. For instance, the required CO2 level in rats is 7.5%, whereas for humans it is 6.5% (19).

O2 EFFECT

Some studies have compared different values of O2 concentration in the incubator and they show that a low level (5-6%) improves results when compared to an ambient level (21%). It has been shown that low O2 levels increase implantation, pregnancy and live birth rates (17,20). It seems that a low O2 level reduces ROS in the culture and the presence of volatile particles in the air, although the exact mechanism of action is still unknown (17).

TEMPERATURE EFFECT

Standard temperature generally used in IVF laboratories is 37⁰C (17,18). However, the optimal temperature is unknown because in the female reproductive tract it could be slightly lower, about 36⁰C. On average, the temperature of the Fallopian tube is about 1.5⁰C less, whereas the follicular liquid temperature can reach 2-3⁰C lower than core body temperature (17). It is important to control and prevent temperature variations because it can affect meiotic spindle stability and alter embryonic metabolism. It has been shown that an increase of 2⁰C during 20 minutes potentially alters the integrity of the meiotic spindle, which cannot be completely repaired when the temperature is set back to 37⁰C. As a consequence of this increase in temperature, embryos express some stress-response genes that compromise development (18). Interestingly, a small decrease in temperature does not have any effect on oocytes, whereas a large difference can be severely harmful to the meiotic spindle (18).

CULTURE MEDIA EFFECT 

Nowadays, there exist two kinds of culture media: one-step media and sequential media (with different compositions for days 0-3 and 3-6) (17,21). All culture media are similar in composition; they contain energy substrates like glucose, pyruvate or lactate, and both organic and inorganic salts, which must be balanced accordingly. Culture media also contain amino acids in different proportions. The exact composition of amino acids in culture media is unknown. One of the most important problems related to the presence of amino acids is the ammonium generated as a product of metabolism. Ammonium has negative effects on embryo and fetal development. To avoid this problem, some culture media contain glutamine, which reduces ammonium production (17,21,22). Also, culture media can be supplemented with macromolecules and other components like HSA, α and β globulins, growth factors, vitamins, lipids, nucleotides, cytokines and hormones (17,22).

WHAT CAN WE CONCLUDE? 

There are many parameters that should be kept in mind in order to maintain the optimal conditions for both gamete and embryo development in an IVF laboratory. In vitro, cells and embryos are exposed to different stress situations that must be minimized. Therefore, a routine control at different levels needs to be performed, so that the environment in the laboratory is adapted to resemble the reproductive tract and the intrauterine conditions.

 

REFERENCES

  1. A practical guide for setting up an IVF lab. Assessment of embryo culture systems and running the unit. (2013). 1st ed. New Delhi: Jaypee Brothers Medical Publishers.
  2. Blockeel C, Mock P, Verheyen G, Bouche N, Le Goff P, Heyman Y, Wrenzycki C, Höffmann K, Niemann H, Haentjens P, de Los Santos MJ, Fernandez-Sanchez M, Velasco M, Aebischer P, Devroey P, Simón C. An in vivo culture system for human embryos using an encapsulation technology: a pilot study. Hum Reprod. 2009; 24(4):790-6.
  3. Wale PL, Gardner DK. The effects of chemical and physical factors on mammalian embryo culture and their importance for the practice of assisted human reproduction. Hum Reprod Update. 2016; 22(1):2-22.
  4. Sender R, Fuchs S, Milo R.Revised Estimates for the Number of Human and Bacteria Cells in the Body. PLOS Biology. 2016; 14(8): e1002533.
  5. Takenaka M, Horiuchi T, Yanagimachi R. Effects of light on development of mammalian zygotes. Proc Natl Acad Sci USA. 2007;104(36):14289-14293.
  6. Wale PL, Gardner DK. The effects of chemical and physical factors on mammalian embryo culture and their importance for the practice of assisted human reproduction. Hum Reprod Update. 2016 Feb;22(1):2–22.
  7. Pomeroy K, Reed M. The Effect of Light on Embryos and Embryo Culture. J Reproductive Biotechnology Fertility. 2012;3(2):46-54.
  8. Otsuki J, Nagai Y, Chiba K. Damage of embryo development caused by peroxidized mineral oil and its association with albumin in culture. Fertil Steril. 2009;91(5):1745-1749.
  9. Zigler JS, Lepe-Zuniga JL, Vistica B, Gery I. Analysis of the cytotoxic effects of light-exposed hepes-containing culture medium. In Vitro Cell Dev Biol. 1985;21(5):282–7.
  10. Otsuki J, Nagai Y, Chiba K. Peroxidation of mineral oil used in droplet culture is detrimental to fertilization and embryo development. Fertil Steril. 2007 Sep;88(3):741–3.
  11. Morbeck D. Air quality in the assisted reproduction laboratory: a mini-review. J Assist Reprod Genet. 2015; 32(7):1019-1024.
  12. Khoudja R, Xu Y, Li T, Zhou C. Better IVF outcomes following improvements in laboratory air quality. J Assist Reprod Genet. 2012; 30(1):69-76.
  13. Munch E, Sparks A, Duran H, Van Voorhis B. Lack of carbon air filtration impacts early embryo development. J Assist Reprod Genet. 2015; 32(7):1009-1017.
  14. Lawrence C. VOC levels in a new IVF laboratory with both central and in-laboratory photocatalytic air purification units. Alpha Scientists Reproduct Med. 2007;36:1–5.
  15. Khoudja R, Xu Y, Li T, Zhou C. Better IVF outcomes following improvements in laboratory air quality. J Assist Reprod Genet. 2012;30(1):69-76.
  16. Esteves S, Bento F. Implementation of cleanroom technology in reproductive laboratories: the question is not why but how. Reprod Biomed online. 2016; 32(1): 9-11.
  17. Swain J, Carrell D, Cobo A, Meseguer M, Rubio C, Smith G. Optimizing the culture environment and embryo manipulation to help maintain embryo developmental potential. Fertil Steril. 2016;105(3):571-587.
  18. Wale P, Gardner D. The effects of chemical and physical factors on mammalian embryo culture and their importance for the practice of assisted human reproduction. Hum Rep Update. 2015;22(1):2-22.
  19. Bavister BD. The mammalian preimplantation. Embryo. Regulation of growth and differentiation in vitro. New York. Plenum Press. 1987.
  20. Gomes Sobrinho D, Oliveira J, Petersen C, Mauri A, Silva L, Massaro F et al. IVF/ICSI outcomes after culture of human embryos at low oxygen tension: a meta-analysis. Reprod Biol Endocrinol. 2011;9(1):143.
  21. Sunde A, Brison D, Dumoulin J, Harper J, Lundin K, Magli M et al. Time to take human embryo culture seriously. Hum Reprod. 2016;31(10):2174-2182.
  22. Marianowski P, Dąbrowski F, Zyguła A, Wielgoś M, Szymusik I. Do we pay enough attention to culture conditions in context of perinatal outcome after in vitro fertilization? Up-to-Date Literature Review. BioMed Research International. 2016;2016:1-6.