Improving Embryo Quality by Strictly Controlling IVF Laboratory Environment

Abstract

In recent years, several changes have been made in different aspects of in vitro fertilization to improve embryo quality and ultimately the clinical outcomes in assisted reproduction technology (ART). These approaches include improvements in air quality inside the lab to ensure VOCs-free air, use of tri-gas incubator and embryo-tested devices and plastics, adequate control of pH and osmolarity of culture media, and strict quality control that allows an adequate development of the embryos until blastocyst stage. Other strategies to improve the embryo quality during in vitro culture include volume reduction of drop culture media, and individual or group culture of embryos. This work summarizes several strategies to improve embryonic quality during their in vitro culture in assisted reproduction procedures.

1. Introduction

Assisted reproduction technologies allow for better clinical outcomes for infertile couples to achieve a pregnancy and a liveborn baby compared to those obtained in the beginning of in vitro fertilization, and this goal is due a series of innovations made in different aspects of the in vitro fertilization laboratory that guarantee the quality of embryos obtained after an IVF procedure. pH is an essential environmental variable that should be under strict control in the culture media inside the incubators. Gametes and embryos are very sensitive to changes of pH, which calls for special attention to the elevation of the performing IVF laboratory to ensure adequate development and quality of embryos. Furthermore, according to different studies the strategy of physiological culture with low oxygen compared to the atmospheric environment showed excellent quality of blastocysts and higher clinical outcomes. Cultured embryos in atmospheric oxygen systems show damaged inner cell mass and are disorganized, diffuse and with low vacuolated cells compared to those blastocysts with large and compact inner cell mass cultured in lower concentrations of oxygen.

On the other hand, the autocrine and paracrine growth factors are very important and positive to the development and quality of embryos cultured in vitro. Strategies such as reduced culture volume and group embryo culture allow to increase the effect of embryotrophic factors on embryonic development. During an in vitro culture, a reduced volume allows for a balance between the beneficial effect of growth factors and the negative effect of embryotoxic substances; and similarly, culturing embryos in groups allowed those beneficial embryotrophic factors to positively influence the embryo development via paracrine signals. This review summarizes several strategies to improve embryonic quality during their in vitro culture in assisted reproduction procedures.

2. Oxygen tension in embryo culture

Oxygen during in vitro embryo culture is an essential chemical factor that improves development from gametes to the blastocyst stage, intervening in their metabolism, genetics and epigenetics [1, 2, 3, 4, 5]. In some mammals including humans, the oviduct and uterus oxygen concentrations are between 2–8% [6], showing lower concentrations of oxygen in the uterus [7], thus concentrations close to these should represent a more physiological environment. For many years, in most IVF laboratories worldwide embryonic culture has been made in atmospheric concentration of oxygen (20%); however, the current trend has turned to culture at reduced oxygen concentrations (5%) simulating the normal physiological environment.

During in vitro development, the embryos before and after compaction have completely different characteristics and this is one of the reasons why oxygen can affect the culture to a different extent before its transfer to the uterus [8, 9]. The atmospheric oxygen is potentially toxic through reactive oxygen species (ROS) which are a threat to gametes and embryos cultured in vitro, but a reduced oxygen concentration similar as possible to the in vitro environment improves the embryonic development. Bontekoe et al. [10] realized a meta-analysis of four studies analyzing the effect of low oxygen in human embryo cultures showed that a concentration of 5% had a benefit on clinical outcomes compared with those embryos cultured at atmospheric concentrations. Guo et al. [11] evaluating the effect of culturing embryos in 20% or 5 % oxygen concentration from zygotes to blastocyst showed that the low oxygen group had a significantly higher blastocyst formation rate during fresh cycle, and clinical pregnancy and implantation rates significantly higher in the subsequent warming blastocyst-transfer cycles.

Studies of Karagenc [12] culturing embryos in 20% of O₂ showed damage mainly in the embryonic inner cell (ICM) which was morphologically disorganized, diffuse and with few vacuolated cells unlike the blastocysts with large and compact IMC cultured in low concentration of O₂. Rho et al. [13] cultured bovine embryos and showed that low concentrations of oxygen produce higher rates of cleavage and blastocyst stages compared to embryos cultured in 20% of O2. Also, in mice there is a better development to blastocyst stage, bigger number and size of ICM, and a gene expression profile similar to that observed in embryos in vivo [14]. Studies of Adam et al. [15], Meintjes et al. [2], and Nanassy et al. [16] have concluded that culturing embryos in 5% of O₂ is beneficial to preimplantational embryo development especially in patients older than 40 years old, as demonstrated by García-Ferreyra et al. [17]. This last study compared the clinical outcomes in patients older than 40 years old whose embryos were cultured at blastocyst stage under two different oxygen environments (5% vs 20% O₂) and concluded that the women group of 5% of O₂ had implantation and pregnancy rates significantly higher compared to group of 20% of O₂ (Table 1).

	5% O2	20% O2
Cycles	341	217
Fertilization rate (%)	83.5	81.6
Blastocyst development rate (%)	34.0	31.4
Implantation rate (%)	25.0*	2.7
Pregnancy rate (%)	41.4*	5.6
Transferred embryos (%)	1.93 ± 0.05*	2.06 ± 0.01

Table 1.

Effect of atmospheres of 5% and 20% of O₂ on clinical outcomes in older women.

*

P < 0.05 in relation to 20% O₂ group.

García-Ferreyra et al. Beneficial effect of reduced oxygen concentration with transfer of blastocysts in IVF patients older than 40 years old. Health 2010.

3. Importance of CO₂ to control the pH value in culture media at sea level and high altitude

The control of pH during in vitro culture of human embryos is very important to achieve an excellent preimplantational embryonic development. Intracellular pH of the human oocytes and embryos is ~7.1–7.2 (usually accepted to be 7.12), this value could be affected by changes in the extracellular pH of the culture media, and variations in their values cause cell stress during in human embryo cultures [18]. During the manipulation of gametes and embryos in in vitro procedures, it is crucial to minimize stress factors to achieve adequate clinical outcomes. Intracellular pH regulates cell processes such as enzymatic reactions, cell division, differentiation, membrane calcium concentration, cell communication, protein synthesis, cytoskeleton formation; and slight variations can have detrimental effects on the developing embryo. It is important to highlight that even slight variations in the intracellular pH can significantly impact blastocyst development, alter gene expression profiles, induce relocalization of mitochondria and actin cytoskeletal elements, and alter glycolytic activity and oxidative metabolism [19]. Previous studies showed that changes in the culture media pH (extracellular pH) directly affects the intercellular pH in the embryos during IVF procedures, altering their homeostasis and quality of development [20].

Routinely, a bicarbonate/carbon dioxide (CO₂) buffering system has been employed during in vitro culture to regulate the pH of fluids, and manipulating the concentration of CO₂ and the concentration of bicarbonate is readily feasible to obtain the required pH. This system has the advantage that changing the CO₂ concentration permits the manipulation of the pH value in the culture media. Calibrating the percentage of carbon dioxide gas in the incubators can then allow for precise pH control in the culture medium to adequately support embryo development. Given that factors as altitude will affect the partial pressure of CO₂ in the medium, it is imperative to increase the percentages of gas to reach a physiological pH that allows for an adequate embryonic development in vitro, and IVF labs at high altitude will need higher percentages of carbon dioxide to achieve good embryo development. At high altitude, hypoxia affects male and female reproductive health at hormonal level and gene expression [21], which in turn could affect key events during embryo development [García-Ferreyra, 2022; Personal communication]. However, there are not many reports on IVF and altitude. García-Ferreyra and collaborators carried IVF procedures in a city at high altitude (Huancayo-Perú; 3300 m.a.s.l.) and needed a minimum concentration of 11,9% of CO₂ in incubators to have a suitable pH to obtain a good development and quality in human embryos compared to those observed at sea level (data unpublished). Finally, it is necessary to measure and calibrate CO2 concentrations to ensure that the pH of the culture medium inside the incubator will be adequate to support the embryonic development, and always keep in mind the height above sea level of our IVF laboratory.

4. Strategies of culture to improve embryo quality in IVF

4.1 Dry versus humidified incubator

The maintenance of stable and optimal conditions during in vitro culture of human gametes, zygotes, and embryos is critical to achieve excellent clinical outcomes; and some variables as the pH of the culture media, temperature, and media osmolality, that directly depend the type of incubator used, could be affected by presence of humidity inside the incubators in the embryology laboratory. Media osmolality, a measure of osmotic pressure of a solution, is one of the main factors in cell volume regulation and osmolality shift during extended culture, as it will affect the embryonic development [22, 23]. In animal models like the mouse, it has been demonstrated that hyperosmotic stress is associated to reduced blastocysts formation rates [24, 25], low cell number, apoptosis [26, 27], and altered epigenetics and gene expression processes [28, 29].

Routinely, humified incubators have been used to culture human embryos, and this has allowed optimal osmolality control, but an important disadvantage of the humidity is risk of contamination by microorganisms which can negatively impact embryonic development. However, in the last years this problem has been overcome with the development of dry benchtop incubators for cultivating human embryos, but the use of dry atmospheres and uninterrupted culture systems leads to continued increases in the media osmolality [30]. A strategy to avoid the significant water evaporation in a dry atmosphere culture, and changes in temperature, pH (by increased concentration of bicarbonate), and osmolality (elevated organic and inorganic osmolytes) involves overlaying the culture dishes with mineral oil. Nonetheless, this plan has not been efficient according several studies published in the recent years.

Studies of Swain et al. [31] that compared media osmolality changes in uninterrupted cultures during 7 days in non-humidified and humified incubators, showed a significative increase of the osmolality over time in dry atmosphere. Osmolality in humidified incubators always remained unaltered and comparable to controls during the experiments. Similar results were observed by Yumoto et al. [29] who investigated the stability of osmolality in culture media microdrops covered with mineral oil incubated for 5 or 6 days incubated in a dry or humidified atmosphere. They observed a significant and linear increase in the osmolality during 5 days of incubation in dry benchtop incubators; concluding that mineral oil alone may not adequately prevent gradual changes in the osmolality of low-volume microdrops during extended in vitro culture of human embryos in non-humidified incubators.

In regards to clinical outcomes, Fazwy et al. [22] compared randomized and prospectively dry versus humid incubators and showed significantly lower blastocyst formation, clinical and ongoing pregnancies rates in the dry culture compared to those observed in humidified culture, indicating that human embryo development may be compromised in culture without humidity. On the other hand, a study done to investigate whether adding or not outer-well medium to inhibit osmolality changes in culture media during IVF cycles showed higher ongoing pregnancy and low miscarriage rates in embryo transfer at day 3 and similar pregnancy rates but significantly lower miscarriages in day 5 transfers [32]. Additionally, Mestres at al. [23] in an excellent work analyzed which culture system factors affect the media evaporation and osmolality, concluding that humidity levels inside the incubators, the volume of mineral oil, and the type of culture media, play a crucial role in osmolality stabilization. Finally, since osmolality is one of the critical parameters of in vitro culture systems that can affect embryonic development, the use of humidified incubators, and adequate techniques (preparation speed and preparing one dish at a time) are highly recommended to avoid evaporation and shifts in osmolality.

4.2 Volume of culture media drop

One of the conditions to achieve an excellent human embryo culture system is to provide the optimal medium to the embryos, guaranteeing accumulation of autocrine factors that act upon the embryo itself or upon neighboring embryos, without affecting an adequate blastocyst development by embryotoxic metabolic products as ammonium; in such a way the medium volume will be an important factor in a successful blastocyst culture program.

Routinely, culture human embryos in larger medium volume have been the standard practice in worldwide clinics, but is expected that nutritional and autocrine factors will be diluted in these culture methods. Studies of O′Doherty et al. [33], and Gopichandran and Leese [34] using murine and bovine models suggested that culturing embryos in a reduced volume can increase the blastocyst development rates, and Lane and Gardner [35] showed that decreasing the volume from 320 to 20 μL had a positive effect on cell number of morulae and blastocysts. Studies by Gardner and Lane [36] in humans, suggested that the minimum amount of medium should be 6.26 to 12.5 μL per embryo as a way to avoid the depletion of nutrients and possible effects of negative factors. However, it is important to consider the embryo density by media drop because culturing embryos in droplets of small volumes could lead to the accumulation of detrimental factors such as ammonium and/or oxygen-derived free radicals [37, 38]. Carolan et al. [39] cultured individually bovine embryos but did not observe blastocyst development in 1, 2 or 5 μL drops covered with oil, requiring a minimum of 10 μL medium to achieve the full developmental potential in vitro. In mouse embryos, 2μL drop medium may provide appropriate conditions for individual cultures and less than 1 μL is enough for two mouse zygotes to achieve blastocyst stage [40].

Rijenders and Jansen [41] cultured individual human embryos in two medium culture volumes (160 μL vs 5 μL) and showed improvement, albeit not significant, in the blastocyst formation rate when the embryos were cultured in small volumes. A study performed by Melin et al. [42] showed that reducing culture volume from 20 to 5μ L affected the mouse embryo development with a reduction of blastocyst development rate from 86.6% to 50%. On the other hand, Minasi et al. [43] considered that a “reduced volume” of 5 μL adopted by previous studies is not an ideal balance between the negative effect of toxic metabolites and the positive effect of beneficial autocrine factors; and in this way, they decided increase to 7 μL as “reduced volume” sustaining that 2 μL may be important to prevent evaporation and changes in osmolarity which can significantly affect embryo development, and when they reduced the medium volume from 35 to 7 μL they observed a significantly higher blastocyst development rate at day 5 (50.5% vs 70%, respectively).

Therefore, it is important to consider media volume and embryo density during an in vitro embryo culture system to achieve that the autocrine and paracrine factors act on embryonic development and allow an increase in the embryo quality and finally the clinical outcomes.

4.3 Individual versus group embryo culture

Routinely, individual culture of human embryos has been standard method in fertility clinics around the world mainly as a way of evaluating the embryo development during the extended culture to blastocyst stage; however, this strategy is not necessarily related to an improvement in development and quality in embryos, or to the clinical outcomes.

It is important to highlight that in vivo, early embryonic development occurs in the absence of signalling factors because mammalian zygotes develop to the blastocysts stage free-floating in a dynamic fluid environment and without direct cell to cell contact; however, Lonergan [44] and O’Neill et al. [45] suggested that signaling factors are necessary to modulate cell growth and cell division, or have anti-apoptotic functions, during preimplantation embryo development in vivo. When embryos are cultured in group, developmental rates and embryo quality are improved compared to those embryos individually cultured [46, 47], and this beneficial nature is related to embryotrophic factors that support or promote their development in vitro, such as insulin-like growth factor-I [48], insulin-like factor-II [49], platelet-activating factor (PAF) [50], phospholipase C (PLC), protein kinase B/Akt, and 3-phosphoinositide-dependent kinase 1 (PDKS1) [51]. Cultured embryos in group produce and release trophic factors that act on the embryo itself and neighboring embryos through paracrine/autocrine actions improving their development, quality, and their implantation probability in the uterus [50].

Several authors have evaluated the effect of group embryo culture strategy compared to individual culture on several parameters of preimplantational embryonic development and clinical outcomes. In regard to embryonic parameters, Ebner et al. [52] prospectively studied the effect of individual or group embryonic culture and showed that the group culture strategy was superior in terms of compaction, development and quality of blastocysts; and Tao et al. [53] evaluated the influence of the same culture strategy but grouping embryos after day 3 according on embryo quality, showing that culturing embryos in group promoted blastocyst development and blastocyst utilization rate compared to those embryos individually cultured. Similar results were observed by Ruíz et al. [47], Glatthorn et al. [54], and García-Ferreyra, 2022 [personal communication].

With regard to clinical outcomes of group and individual strategies, some studies (Ebner et al. [52], Tao et al. [53], and Glatthorn et al. [54] did not observed significant differences in the pregnancy and implantation rates between group and individual culture. However, Ruíz et al. [47] carried out a prospective study to evaluate the effectiveness of group embryo culture using undergoing IVF one hundred forty-eight women data and the result showed a significantly higher implantation rate and live birth delivery rate with the first fresh embryo transfer. Our team used donor oocytes cycles to analyze the effect of group culture on embryo development and clinical outcomes and our results showed significantly more blastocysts, higher pregnancy, implantation ans live birth rates and lower miscarriage rates compared to individual culture (Table 2) [García-Ferreyra, data unpublished].

	Group culture	Individual culture
Cycles	70	70
Blastocyst development rate (%)	51.6*	46.7
Pregnancy rate (%)	88.6*	62.9
Implantation rate (%)	64.4*	45.9
Live birth rate per embryo transfer (%)	84.3*	54.3
Miscarriage rate (%)	4.8*	13.6

Table 2.

Effect of group culture compared to individual culture strategies on clinical outcomes in oocyte donor cycles.

*

P < 0.05 in relation to the individual culture

García-Ferreyra, personal communication.

At present, most laboratories worldwide using the individual embryo culture method as a manner to assess the embryo development and principally induced by the freeze-all and PGT-A practice, nonetheless this strategy has a detrimental impact on some embryonic parameters and inclusive on clinical outcomes as has been shown by several authors. Therefore, culturing embryos in group will be a good strategy because effectively improves the embryonic development to blastocyst stage, increasing the embryo utilization rate, and principally arise the clinical outcomes as pregnancy, implantation, and live birth rates.

5. Conclusions

Strategies like cultivating embryos in a humid atmosphere, decreasing the volume of culture medium, and group embryo culturing are appropriate to increase the effect of autocrine and paracrine factors that promote the quality and development of embryos cultured in vitro and increase overall success of clinical outcomes. Additionally, strict pH control to maintain intracellular pH levels during embryo culturing, and maintaining oxygen levels to mimic physiological conditions are crucial to increase success rates in infertile patients, especially those patients are 40 years or older. Finally, it is of utmost importance to highlight that any strategy to be used in an embryo culturing system must be adapted correspondingly to the performing laboratory in order to increase the pregnancy, implantation, and live birth rates.