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1
pH, Temperature, and Light
Kimball O. Pomeroy, Ph.D.1
Michael L. Reed, Ph.D.2
1The World Egg Bank
7227 N. 16th Street, Ste. 160
Phoenix, Arizona 85020
Email: kopomeroy@gmail.com
2The Fertility Center of New Mexico
201 Cedar Street SE, Ste. S1-20
Albuquerque, New Mexico
Email: mleroyreed@gmail.com
This review has been approved by both authors. Neither of the authors have a conflict of
interest.
Contents
1.1 pH Homeostasis in Ova and Embryos
1.1.1 pH
1.1.2 Cell Response to pH Changes
1.1.3 pH of Oviduct and Uterus /pH of Ova and Embryos
1.1.4 pH of Culture Media
1.1.5 Adjuvant Media and pH
1.2 Temperature
1.2.1 In Vivo Temperatures
1.2.2 In Vitro Temperature Control
1.2.3 Sperm
1.2.4 Oocytes
1.2.5 Embryos
1.2.6 Conclusion
1.3 The Effect of Light on Embryos and Embryo Culture
1.3.1 Conclusion
2
1.1 pH Homeostasis in Ova and Embryos
In clinical IVF, the incubator is a surrogate oviduct and uterus. Its purpose is to
provide an ideal environment for fertilization and the growth of the developing embryo.
The embryo normally travels through a dynamic environment from the oviduct to the
uterus. Temperature, pH, osmolality and many other environmental factors change during
its journey. Current incubators and culture media are fairly static. The incubator must
often provide an environment that is a compromise - suitable for sperm, eggs and the
various stages of embryos. This system should not just address ideal growth, but should
also allow for ideal expression of embryonic genes. It should provide for the necessary
chemicals, growth factors, proteins, osmolality, temperature and pH. Almost all current
media have been developed for mice and then applied to humans. In addition, we often
are not aware of the ideal conditions for human gametes and embryos.
1.1.1 pH
pH is the measurement of the concentration of the hydrogen ion (H+) and
conversely the hydroxide ion (-OH) concentration in a solution. It is the negative
logarithm of the H+ activity. Because this is a logarithmic scale, the difference in
concentration from a pH 5 to 6 is 10-fold and the difference between pH 7.35 and 7.55 is
a 60% increase.
1.1.2 Cell Response to pH Changes
The maintaining of a proper internal pH (pHi) is important for the survival of all
cells. The activities of many intracellular enzymes are regulated by pH. Protein synthesis
(1), DNA and RNA synthesis (2) as well as contractility of myosin (3) are affected by pH.
Changes in pH have even been theorized to be important in the control of the cell cycle
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and cell division of several cell types (4). It is the production of proton gradients (H+)
that drives the ATP synthases to produce ATP, the “energy currency” of most cells. For
these processes to occur, pH must be precisely regulated.
All animal cells that have been examined, aside from non-nucleated erythrocytes,
vigorously regulate their pHi (5). They do this by sensing changes in pHi and then
appropriately speeding up or slowing down the activity of transporters that move acids
and/or bases across the plasma membrane. The vital process of pHi homeostasis is
regulated by a delicate balance between the rate of metabolic acid generation and the
activity of acid/base transporters in the plasma membrane. When an acid or base load is
applied in the cell, these transporter proteins will react and maintain homeostasis by
shuttling acids or bases into or out of the cell. Three transport proteins have been
identified in ova and embryos (Figure 1). Two of the transporters function to overcome
acid loads by increasing the pH and the third overcomes alkaline loads by decreasing the
pH (Figure 2).
Na+/H+ Antiporters (exchangers) play a major role in maintaining the pHi from
bacteria to humans. These proteins exchange Na+ for a H+. When the intracellular pH falls
(an acid load), this protein will absorb a Na+ molecule and extrude a H+, thus increasing
the pH inside of the cell. The second protein involved in alkalizing the cytosol is the
Sodium Dependent Chloride Bicarbonate Exchanger. Sodium and bicarbonate are
transported into the cell in exchange for the external transport of chloride. The major
transporter used to acidify the cytosol is the Anion Exchanger (HCO3-/Cl- Exchanger)
which pumps out bicarbonate in exchange for pumping in chloride.
1.1.3 pH of Oviduct and Uterus /pH of Ova and Embryos
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In trying to identify the optimal pH for culturing ova and embryos, the pH of the
oviduct and the uterus is a good start. There may be lots of data for the sheep, cow and
mouse, but whether these really pertain to the human reproductive tract is questionable.
Historically, the pH of media has been designed around the pH of blood – 7.35 to 7.45.
Only the oviduct of the human appears to be in this range (6).
During reproduction, the ova must leave the follicle where the pH is about 7.5 to
7.7 (7), it must then pass into the oviduct with a pH of 7.28 to 7.7 (6) where it may be
fertilized and then transverse to the uterus, where the embryo enters most likely as a
morula (8–10) and where the pH has been measured from 7.0 to 7.2 (11). As the embryo
navigates this wide range of external pHs it must be able to maintain the proper internal
pH demanded by the intracellular environment. The pHi of the ovum has been measured
to be about 7.0 to 7.1 and the pH of the cleavage embryo at 7.12 (12). It is the role of the
various transport proteins mentioned above to modulate the pH to a range that is
acceptable to the cell(s). It should also be appreciated that this is not without cost. Too
many fluctuations in external pH may tax the embryo’s energy stores, resulting in the
death of the embryo. It has been shown in mouse embryos, that raising the pHi by 0.1 to
0.15 pH units can result in increased glycolysis and lowered oxidative metabolism
(13,14).
Human cleavage-stage embryos appear to be able to respond to both alkaline and
acid loads and have active Na+/H+ Antiporters, Anion Exchangers and Sodium Dependent
Chloride Bicarbonate Exchangers (7,12). In contrast, human ova appear to be able to
regulate against alkaline loads but not acid loads. They appear to have an active Anion
Exchanger but impaired Na+/H- Antiporter and Sodium Dependent Chloride Bicarbonate
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Exchanger activity. Human ova thus appear to not to be able to regulate acid loads
effectively.
Bicarbonate is necessary to adequately control acid loads as the Na+/H+
Antiporter. It only kicks in below about pH 6.8. It is up to the Sodium Dependent
Chloride Bicarbonate Exchanger to perform the fine pH control around the internal pHi of
the embryo 7.1 (12). This is one of the reasons it is important that handling media (and
flushing media) should contain some bicarbonate. If the handling media did not contain
bicarbonate, it would rob the embryo of the ability to regulate its pH above 6.8. Because
oocytes do not have a fully active mechanism to modify their internal pH, care must be
used to ensure that 1) bicarbonate is present in the media and 2) the external pH does not
vary much from the pHi of the oocyte, about 7.1.
As regards vitrification, it has been shown that vitrified hamster 2-cell embryos
lose their ability to regulate pHi effectively for up to 6 hours after warming (14). The pHi
of these embryos goes from 7.24 to 7.34 after warming and results in reduced activity of
both the Na+/H+ Antiporter and Anion Exchanger systems. Whether this occurs in other
mammals, like humans, remains to be seen but caution should be exercised after the
warming of vitrified human embryos so that the external pH is optimized. pH excursions
should be avoided.
1.1.4 pH of Culture Media
A wide variety of media have been used for the successful culture of human
embryos. The manufacturers of these media recommend various pHs that range from 7.2
to 7.5. The first media used for human IVF was based on media used in tissue culture.
These were often simple salt solutions (like Earle’s) or complex media (like Ham’s F10).
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The pH of these media was set to that used in the cell line form which it was borrowed.
Later, more complex media were developed based mainly on research examining the
constituents of the human oviduct (15). Still, embryologists for the most part stuck with
the prior tissue culture pHs, around 7.35.
Later, media were developed that attempted to imitate some aspects of the
dynamic nature of the oviduct and uterus. This sequential media (16,17) used one media
for the first 3 days of embryo culture and another for the subsequent days up to day 7. By
adding amino acids, embryologists were finally able to routinely get human embryos to
the blastocysts stage. Of note, these newer media were formulated first for mouse
embryos and then applied to human embryos. To indicate the lack of concern with pH at
this time, none of these studies even mention the pH of the culture media.
The pH of IVF media is determined mainly by the concentration of CO2 in the
incubator and the concentration of bicarbonate in the media. CO2, provided by tanks
attached to the incubator, must first permeate and equilibrate with any oil covering and
then with the actual culture media. The CO2 in solution then reacts with the bicarbonate
in the medium to form carbonic acid. The amount of carbonic acid formed depends
primarily on the amount of bicarbonate and CO2 in the medium. In the laboratory, pH of
the medium can be adjusted by changing the amount of CO2 delivered to the incubator –
more CO2 results in more carbonic acid and a lower pH. Proper quality control of the
incubator pH should include more than setting CO2 and measuring its concentration.
Fyrite is an inaccurate method of measuring CO2 (18) and is a poor substitute for actual
pH measurements (Figure 3). To ensure the proper pH of media, one must measure it
directly with a pH probe. This will especially be important when one is trying to
7
troubleshoot culture problems. A detailed discussion of how to perform pH measurements
can be found in Pool, 2004 (18). Briefly a calomel or a double junction silver/silver
chloride probe, less than one year old should be used. All media should contain the same
concentration of protein used in culture. Calibration and test measurements should be
measured at 37 C.
The addition of proteins and the elevation (altitude) of the laboratory are two
other factors that can affect the pH. This means that each laboratory will need to adjust
the CO2 of its incubators in order to produce the desired pH in the culture media. This pH
should be measured after protein supplementation as protein supplementation can change
the pH of media. Figure 4 shows two media with protein (Jason Swain unpublished). The
silver bar is for media supplemented with protein by the manufacturer (where pH is
adjusted after protein supplementation). The blue bars are the same media as in the gray
bars (without protein added by the manufacturer) but supplemented by the laboratory
with a protein source (10% v/v SSS). Note that the pH is lower when protein is added by
the laboratory by almost 0.1 pH point. Most likely this is due to simple dilution of
bicarbonate by the added protein solution.
Improper pH may not only affect the pHi of the cell, but it may have an indirect
effect on some of the properties of the major protein found in media, albumin. An
improper pH may affect the ability of albumin to act as a chelator, modifier of pH,
antioxidant, carrier of fatty acids, etc. (19).
What is not often appreciated is that pH is also affected by temperature. (This
affect is distinct from the effect of temperature on the pH probe’s ability to provide an
accurate pH). The pH of pure water at 0 C is 7.47, but at 25 C it is 7.00 and at 100 C it is
8
6.14. This is important to remember when considering use of non- CO2 -buffered media
(handling or modified media) and the use of these buffers where temperature is poorly
controlled or for vitrification.
As mentioned previously, pH deviations should be avoided in order to increase
viability of the embryo. This dictates a precise range for all media used in IVF. Retrieval
media (flush media), culture media, handling media, etc. should all follow this range. Any
exceptions to this range should be based on physiology. Care should be taken that
embryos and ova are not exposed to alkaline conditions above pH 7.45.
Currently, many embryologists are using media that is closer to the pHi of
embryos (pH 7.2). This has probably slowly evolved as embryologists observed better
embryo development with lower pHs than those previously recommended. It has been
proposed that a constant pH close to the pHi of the embryo will put less stress on the
embryo that may result in the use of limited supplies of energy in the form of ATP.
1.1.5 Adjuvant Media and pH
Not only must an optimum pH be maintained in culture medium, it is also
important in the use of adjuvant media – flush media, ICSI media, handling media and
vitrification media. Synthetic organic buffers, like HEPES or MOPS, are often used in
IVF for the handling of gametes or embryos outside of CO2 incubators. What is not
appreciated is that temperature can affect the pKa of these buffers as well as the pH of the
media (20). When these buffers go from 37 C to 25 C, their pH can decrease about 0.2 pH
units.
The pH of media during egg collection should also be examined to ensure that
during routine use the proper pH is obtained. One should pay particular attention to those
9
situations where the temperature of the medium may change. When one is making culture
dishes for IVF, it is important that the dishes be allowed sufficient time to equilibrate
with the incubator. One study (21), using four-well dishes with 50 ul or 500 ul of media
and 500 ul of oil, indicated that a minimum of 10 hours was necessary for the media to
equilibrate to pH. The size of the drop did not make a difference. If pre-equilibrated oil
was used, equilibration time was decreased to less than 1 hour. They also found that
removal of equilibrated dishes for up to 5 minutes had minimal effect on the pH of the
media.
Some laboratories use modified baby incubators (isolettes) to aid them during
retrievals or the transfer of embryos. Many of these incubators use a thermal conductivity
device to determine the CO2 concentration of the gasses inside the incubator. This type of
device will provide an inaccurate reading if the humidity of the chamber changes. The
device may read 6.2 % CO2, while in reality, the humidity has decreased due to improper
humidification or constant opening and closing of the hand ports. The embryologist may
actually be working in an environment with a much lower CO2 concentration and thus a
more alkaline external pH. An infrared CO2 controller will be more accurate in these
situations.
In order to have consistent results when working with embryos and ova, it is
important to always be aware of how the external pH can be affected by what you are
doing. All aspects of IVF should be characterized for pH fluctuations by first determining
the goal pH for media and then assuring that the goal is met with minimal excursions in
pH. pH should be verified when new lots of media are added to the system, when gas
tanks are changed or when any major change in the culture environment or methods
10
occur. One should empirically determine the maximum amount of time a dish can be left
on the bench top, by measuring pH changes in test media during mock procedures.
1.2 Temperature
The ideal temperature for human gamete and embryo handling has not been
clearly defined, but most embryologists will strive to maintain 37.0C for all surfaces,
media, and equipment to minimize ‘physiological and genetic stress’, and other factors
that could impact in vitro development (22–25).
Embryologist Gregory Pincus (26) said “In obtaining both unfertilized and
fertilized ova for culture in vitro the use of a warm washing solution is preferable. This is
often practically difficult and rabbit ova at least are not materially affected by handling at
room temperature over a period of several hours”. During this period Pincus was
studying oocyte cooling and parthenogenetic activation. Another embryologist, Ralph
Brinster (27) wrote “The cultivation temperature has not been studied to a great extent,
but Alliston (1965) has shown that rabbit ova cultivated at 40 C. for 6 hr. do not develop ⁰
as well as controls cultivated at 37 C. when transferred to foster mothers. In the absence ⁰
of contradictory evidence, it is generally considered that a temperature of 37 to 37.5 is ⁰ ⁰
the best temperature in which to maintain the cultures.” The actual control temperature
was 38.0C (28) but the context is correct.
It is important to realize that a lot of the information on embryo culture, including
temperature, has been translated from work with other species e.g. for mouse, rat, and
rabbit (29); as such, studies must be evaluated in context according to requirements and
findings for different species.
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1.2.1 In Vivo Temperatures
In vivo core temperatures for ovaries, oviducts, and the uterus during ovulation,
fertilization, and development, and eventual deposition of the embryo into the uterine
environment are discussed at length (30). Specific to female (non-human) physiology,
Hunter (31,32) describes temperature gradients across ovaries, and differential
temperatures between isthmus and ampulla oviduct, and has stated that deep rectal
temperature for any species could be misleading and may not translate to in vitro
conditions. Preovulatory follicles are cooler than surrounding tissue and isthmus and
ampulla temperatures differ by 0.9 to 1.6C in rabbit and 0.2 to 1.6C for the pig. One
possible explanation regarding the oviduct is the mounting evidence for mammalian
sperm thermotaxis (human included). Sperm appear to be uniquely sensitive to
temperature gradients (33). El-Sheikh Ali et al, (34) describes in cattle, that there is an
increasing thermal gradient from vagina to deep uterine horn relative to the steroid
hormone concentrations, albeit within a very narrow temperature range of less than 0.5C.
If human body temperature is accepted as 36.6 to 37.3C (rectal), which end of this
spectrum is appropriate for in vitro procedures, what temperature best represents
physiological reproductive temperature? Should in vitro temperature be static or
dynamic according to in vivo conditions? Hunter expressed concerns about accepted,
deep body and physiological temperatures in the human relevant to ART, and proposed
expanding research into effects of temperature on molecular aspects of reproductive
processes (31).
1.2.2 In Vitro Temperature Control
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In vitro, the temperatures for oocyte and embryo handling/maintenance are
controlled by equipment and technique. There have been many studies and discussions.
Two excellent resources: McCulloh, 2012 (laboratory management and quality control
(35)), and Elder et al, 2015 (detailed descriptions of temperature control and equipment
management, among other topics in the book (36)).
1.2.3 Sperm
Testicles and epididymal structures for most mammals operate outside of the body
cavity (human, cattle, sheep, goats), or near to the body (rabbit, rodent, pig). Sperm are
generated and reside in organs slightly cooler than core body temperature. The
exceptions to this include hippopotamus, elephant, and aquatic (fresh and marine)
mammals which all have internalized testicles and storage structures.
Sperm tolerate and function at cooler-than-body temperatures; exposing germinal
and storage organs (externalized) to body and above body temperature can be damaging
to sperm, e.g. impact on in vitro acrosome function (37) and varicoceles in the human
(38). Sperm function (motility, capacitation, fertilization and post-fertilization events)
can be maintained and prolonged at room and colder temperatures with appropriate
technique and/or extenders (39–42). In fact, many protocols for human ART and sperm
processing for insemination involve room temperature handling of human sperm.
1.2.4 Oocytes
The oocyte spindle has been studied extensively in several species and
specifically in relation to temperature and oocyte competency. Pickering (43) cooled a
small number of human oocytes for observation of cytoskeletal changes. After warming
oocytes back to 37C after 10 or 30 minutes at room temperature, not all spindle structures
13
reformed with fidelity; the results were broadly accepted as critical to human IVF
procedures. Sathananthan (44) cooled mouse oocytes rapidly from 37 to 15, 4, 0, and
-7C for subsequent evaluation by light and electron microscopy. Cooling below 15C
induced major spindle depolymerization, and some reversible changes in cytoplasmic
components. A detailed study by Zenzes (45) demonstrated that human oocyte spindles
shortened after 2 to 3 minutes at 0C, and after 10 minutes, spindles depolymerized
completely. Yet chromosomes were not dispersed; two separate microtubule classes were
discussed, and the authors concluded that depolymerization was time-dependent and
tubulin reorganization could depend on the class of tubulin affected. In context, these
papers represent foundation studies, evaluating extreme oocyte cooling without
cryoprotective agents.
In one study, living (not fixed) spindle dynamics were evaluated using polarized
light microscopy after cooling oocytes. Microtubule reassembly was delayed after
warming after exposure to 25 and 28C, but not 33 or 37 (46), and down-stream metrics,
e.g. fertilization rates were higher when ICSI was performed at 37C (47).
Lenz (37) demonstrated in cows, that mishandling of sperm/oocyte co-incubation
or micromanipulation could impact outcomes. Acrosome function was impaired at 40C,
but at the lower temperatures (35 and 37C) fertilization was impaired when compared to
controls at 39C. In human oocytes, (48) a time-by- temperature interaction was observed,
involving oocyte spindle and chromosome competency. Sun (49) found that at 37C,
human oocyte spindles were stable for 20 minutes, but microtubules depolymerized at
39C (10 minutes) and 40C (1 minute). Cooler temperatures were not evaluated, but it
appears that human oocytes should not be exposed to temperatures beyond 38C.
14
There is credible (unpublished, Swain and Pool, personal communication)
evidence to suggest human oocytes may be cooled to room temperature during periods of
micromanipulation without compromising down-stream events. Data shared by Swain
and Pool (Table 1, 2) for fertilization and developmental metrics, for a very large number
of oocytes from two laboratories using similar protocols, are compelling. While no direct
comparisons were done (37C to room temperature ICSI) it appears oocyte competence
and meiotic spindle microtubule fidelity may be more robust than imagined.
Yang (50) found that porcine oocytes could be maintained at room temperature
for up to three days, as intact cumulus-oocyte-complexes, maintained meiotic and
cytoplasmic competence upon warming and fertilization. Immature horse cumulus-
oocyte-complexes could also be held under conditions simulating transport at room
temperature in buffered culture medium, with acceptable blastocyst rates following ICSI
(51).
Based on the available information, it would seem prudent to minimize
temperature extremes (below or above body temperature) for prolonged periods of time
during oocyte recovery, processing, and micromanipulation. Especially important,
exposing oocytes to temperatures above 38C for any length of time should be minimized.
1.2.5 Embryos
Most embryologists agree that human embryos should be maintained at a
(relative) constant temperatures, with minimal environmental excursions from stage to
stage during culture, and during routine evaluations, micromanipulation, preparation for
transfer, and so on. But for context, before cryopreservation was an industry standard,
15
storage and transport of gametes and embryos of various species, laboratory and
livestock, was an important topic (52).
Mouse embryos enclosed in oviducts were transported successfully at 4C (53),
and cleavage-stage bovine embryos (54) were stored for 30 minutes at 0C. Cattle
embryos could be held at 4C for up to 7 days, without cryopreservation, yielding 24/32
pregnancies (55). Grau (56) maintained human tri-pronucleate cleavage and blastocyst-
stage embryos at 4C for 48 hours. Development to blastocyst, or blastocyst re-expansion,
was reduced after 48, but not after 24 hours. Lastly, human blastocysts were cooled
during transport to another facility for cryopreservation (57). After warming, clinical
pregnancy and delivery rates were improved for the transported group (note that after 30
minutes, medium temperature dropped from 33C to approximately 24C).
Regarding in vitro culture of human embryos, Hong (58) describes a well-
controlled study where sibling human oocytes (with-in patient, prospective
randomization) were incubated at two temperatures, 36C and 37C; multiple incubators
were utilized, and care was taken at all steps to minimize study variation. Incubator
stability was 36 ± 0.07C and 37 ± 0.04C. Fertilization and embryonic aneuploidy rates
were not significantly different; however, there were significantly higher cleavage-stage
cell numbers, higher blastocyst formation rates, and ‘usable’ blastocyst numbers were
greater with incubation at 37C compared to 36C. There were no differences in per
embryo implantation rates.
Higher-than-body temperatures were not addressed by Hong et al, 2014, but in
vivo and in vitro heat stress does elicit concern (59–62). McCulloh, in his chapter on
laboratory quality control (35) described an experience where fertilization, poly-
16
pronuclear rate, and subsequent embryo cleavage was affected due to an incubator
operating out of control – an inaccurate thermometer reported 37C, when the actual
temperature was 41C.
Choi (63) exposed 1-cell mouse embryos to elevated temperatures (37C, 39C,
40C, and 41C) for short (8hr) and long-term (96hr) intervals. Severe, short-term heat
stress compromised early cleavage, while trophectoderm cell number and quality was
diminished with long-term heat stress, despite formation of blastocyst-stage embryos.
Gene expression was also altered, as were post-transfer fetal metrics. Youssef (64)
examined the ideal culture temperature for mouse embryos. They found variable
blastocyst and hatching blastocyst conversion rates with culture at 36, 37, 37.5, 38, and
39C; blastocyst hatching was highest at 37.5C, but combined blastocyst and blastocyst
hatching rate was higher at 37C.
Post-fertilization ova appear to be more tolerant of cooler temperatures, especially
later-stage embryos. In the human IVF laboratory, this may be comforting, regarding 1)
planned events (e.g., evaluations on various days (without time-lapse), assisted hatching,
transfer, biopsy, or cryopreservation), or 2) unplanned events (e.g. power loss, embryo
transfers that take longer than usual).
1.2.6 Conclusion
Oocyte cytoskeletal and cytoplasmic components appear to be sensitive to cooler-
than-body temperature when there are excursions below 33C for longer periods of time,
unless the bolstered by (cryo)protective agents. Cumulus-oocyte-complexes may be
more tolerant of cooling.
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Human oocytes and embryos are sensitive to temperatures above accepted body
temperature. In vitro temperature excursions (unplanned) can be largely avoided by
having a series of defined and dedicated equipment and technique protocols in place.
Consider that human culture media and products are performance-validated at
37C; bioassays, and human IVF procedures alike might not perform as expected at
different temperatures.
Quality management is tied directly to regulatory compliance and medical/legal
concerns, no matter the biological (outcomes) performance of a laboratory. As such,
acceptable temperature targets for each phase of the human IVF process should be
selected, and steps should be taken to maintain and monitor those targets.
1.3 The Effect of Light on Embryos and Embryo Culture
To work safely with embryos, monitor embryo quality and manipulate embryos,
embryos must be exposed to light - a potentially damaging environmental factor that is
not relevant during in vivo culture; specifically, the embryo may not have developed any
protective measures during the evolution of the species. During ART procedures
embryos, sperm and oocytes are exposed to light during oocyte retrieval, placement of
dishes into and out of incubators, exposure during observation during microscopy for
ICSI, fertilization checks, morphological assessment and during the embryo transfer
(Figure 5).
In the early days of IVF, Sir Robert Edward was concerned about the affect of light on
embryos.
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“Light has also been one of my major concerns ever since IVF began. We were aware of
the many papers on mammals published by embryologists on the evolution of reactive
oxygen species in response to light exposure, and its deleterious effects on embryo
growth. We could not afford any risks with human embryos to be replaced into the
mother, so we used green filters routinely to remove some of the light radiation, lower the
light intensity and produce a more acceptable colour for the eye by modifying the harsh
artificial light from the microscope. The potential effects of light concerned me in another
way. During transfer, gynaecologists often used an intense operating theatre light to shine
on the cervical os. Yet this was where the embryo is passed during the transfer process. At
the last moment, after hormone stimulation, oocyte collection, fertilization and cleavage
in vitro, these precious embryos could be exposed to an intense light which might impair
their ongoing development. We therefore dimmed this light during transfer to avoid any
damage to the embryos in the last stage of their ex-utero existence. Several investigators
have disparaged my attitude, and they may even be right when they claim that human
embryos can tolerate this degree of intense light exposure. But I have never seen any
evidence on this point from these investigators, and it is surely better to be safe than
sorry. So I still use many of these precautions.” (65).
There are no well-designed studies utilizing human gametes and embryos in vitro
to evaluate the impact, if any, of the type of lighting, duration of exposure, or exposure to
specific wavelengths; rather the information available on this topic is derived from
animal model studies.
Several variables are important when measuring light. They are duration of
exposure, intensity of exposure and finally, the wavelength of light. Often, light intensity
19
is measured in lux, but this measurement is the intensity as measured by the human eye
and is not suitable for non-visible wavelengths. Lux also is a poor measurement as it does
not take into account the length of exposure. A better measure of intensity is the
irradiance (w/m2) which, as a measure of power, includes also the measurement of the
duration of the exposure. Unfortunately, most of the studies done on the influence of light
on culturing of cells do not include this measurement of irradiance and so it is difficult to
even determine the amount of light the cells were exposed to.
There are several ways that light might affect a cell. There may be a direct effect
where light “stresses” the cell, activates stress genes or even damages DNA directly via
ionization. Light may also indirectly affect cells by oxidation of components in the media
or oil, changing a neutral or even beneficial component into a toxicant. This indirect
method can occur via photooxidation - a chemical reaction between light and components
of culture medium and oil. Light has been implicated in the oxidation of oil used in the
culture of human embryos (66). The mechanisms involved in photooxidation of media
components and oil may also work in photooxidation of sperm and oocyte membranes
(which are also lipids), producing changes in these membranes that could potentially
inhibit fertilization. Light has also been shown to induce production of hydrogen
peroxide, a substance toxic to cells, when media containing HEPES and riboflavin are
exposed to light (67–71).
One of the first observations to indicate light could be damaging to cells was the
observation that light exposure killed protozoans placed in an acridine dye solution (67).
Light modified the acridine, resulting in decreased photo synthesis, inhibition of
20
replication and cessation of growth. This affect, where light modifies media components
that then become toxic, has also been shown by others (68,69,71).
Light has been implicated in harming gametes or embryos of rabbits (72), hamster
(73–76) and mouse embryos (77). Although bovine embryos show no negative growth
effects from light exposure, they do show higher levels of the inducible stress protein,
HSP70 (78).
Most toxic effects appear to occur from light exposure in the visible blue to ultra-
violet range of 445 nm to 500 nm (Figure 6). There have been no studies to date looking
at wavelengths that might damage DNA in human gametes or embryos, but UVB
radiation (290 to 300 nm) has been shown to damage the DNA of sea urchin embryos
(79) and damage proteins and membrane lipids (80). Peroxides can form in a cascade
reaction because of exposure to light and/or heat, resulting in the transfer of water-soluble
toxicants to the culture drops (81–83).
1.3.1 Conclusion
No conclusive data exists to indicate that light is harmful to human gametes or
embryos, but there is substantial evidence that light can be harmful to non-primate
mammalian gametes and embryos. It is also known that light can affect the quality of oil
and culture media, including buffers such as HEPES. Light in the blue visible and
ultraviolet spectrum (<500) appears to have the highest potential for harm. There may be
ways to reduce the effects of light on embryos and media by the inclusion of antioxidants
or the exclusion of photooxidative media components. Light exposure can also be
reduced by reducing the amount of harmful wavelengths in our laboratories via limited
21
exposure to any light, use of ambient light filters, and avoidance of most fluorescent
lighting.
Embryology laboratories should not be located in areas where direct sunlight
might damage them. Care should be taken with hood lights, ambient lights, headlamps
and microscope lamps. A green bypass filter may be prudent when viewing gametes and
embryos. With new methods for determining embryo quality and suitability for transfer
with frequent monitoring using in-incubator optics, it is prudent to try to understand more
regarding the role light might play in the production and growth of human embryos.
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28
Abbrevia
tion
Transporter Ova Embry
o
Recovers From
NHE Sodium Hydrogen
Exchanger
(Antiporter)
X Acidosis
NDCBE Sodium Dependent
Chloride-
Bicarbonate
Exchanger
X Acidosis
AE Anion Exchanger
(Bicarbonate-
Chloride
Exchanger)
X X Alkalosis
Fig. 1 List of transporters used in pH homeostasis, cells in which they function and their
roles.
29
Fig. 2 Schematic of the function of three transporter molecules.
Recovery from Acidosis
Na+
HCO3-
Na+
NHE NDCBE
HCO3-
Cl-
H+
Cl-
AE
Recovery from Alkalosis
30
Fig. 3 Plots of CO2 concentration as measured by the incubator’s digital read out and
Fyrite, with the measured pH of the media. Note lack of correlation between media pH
(upper line), Fyrite CO2 measurements (middle line) and incubator readout (bottom line).
(From Pool, 2004).
31
Fig. 4 pH of media. #1 is media where 10% protein (SSS) was added by the laboratory
and #2 is media with protein added during its manufacture. (Personal communication
Jason Swain 2017).
32
Table 1. Fertilization rates over a 10-year time frame for IVF and ICSI procedures
IVF&ICSI ICSI*
Main lab 12,545/18,002 (69.7%) 7,076/10,124 (69.9%)
Satellite lab 6,688/9,172 (72.9%) 2,499/3,589 (69.6%)
Combined 19,233/27,174 (70.8%) 9,575/13,713 (69.8%)
*Room temperature
Swain and Pool, unpublished, with permission
Table 2. Clinical outcomes following room temperature ICSI; day three transfer
33
Patient age
23-45
Patient age
≤35
Clinical pregnancy 407/740 (55.0%) 244/389 (62.7%)
Delivery 348/740 (47.0%) 214/289 (55.0%)
Implantation rate 531/1607 (33.0%) 333/804 (41.4%)
Swain and Pool, unpublished, with permission
FIGURE 5. Typical sources of visible light in the laboratory
Windows
34
Filtered, curtained, shades, blinds
Ceiling lights
Fluorescent; cold white, warm white
Incandescent; dimmable and single intensity
Lamps
Floor, desktop
Hoods/cabinets
Microscopes
Inverted
Dissection
Time-lapse
Direct effects of light:
Mineral oil overlay
Culture medium components
Plasticware
Indirect effect of light:
ROS
Gene transcription
Culture medium breakdown products
Egg retrieval
Direct and indirect room lighting, hoods, other
Surgery lamps
Microscope
Manipulation and routine handling
Direct and indirect room lighting, hoods, other
Microscope
Embryo transfer
Direct and indirect room lighting, hoods, other
Microscopes
Headlamps, floor lamps, surgery lamps
380 450 495 570 590 620 750
Potential Danger
Fig. 6 Visible Light Wavelength nm
