**1. Introduction**

Interest has increased in the use of arterial conduits for CABG significantly in most major cardiac surgery centers around the world, because the number of patients receiving arterial grafts and our knowledge about the biologic characteristics of arterial grafts have increased. In addition, more advanced clinical protocols for the use of grafts have been developed and midterm results with alternative arterial grafts are encouraging.

The internal mammary artery (IMA) has been shown to have greater long-term patency for coronary artery bypass grafting when compared with the saphenous vein graft. Because of the superior long-term results of the IMA, other arterial grafts which have recently been advocated include the radial artery (RA), the gastroepiploic artery (GEA), the inferior epigastric artery (IEA), the splenic artery, the subscapular artery, the inferior mesenteric artery, the descending branch of lateral femoral circumflex artery, the intercostal artery and the ulnar artery. One of the various manifestations clinically observed among these arterial grafts is a different tendency to develop spasm during surgical dissection and during the perioperative period which could be the cause of perioperative morbidity and mortalitiy [1-8]. For example, there are reports of vasoactive drugs altering IMA graft flow [3,4]. Moreover, there is accumulating evidence that blood flow in arterial grafts is insufficient in some circumstances [6,7]. Many vasoconstrictors (spasmogens) may cause arterial grafts spasm. Accordingly, antispastic therapy is important in the development of arterial grafts and the nature of constrictor substances that cause arterial graft spasm needs to be determined. In recent years, the problem of graft spasm has become more frequent with the increasing use of new arterial grafts. Therefore, it is essential for surgeons to understand the causes of vascular graft spasm, to improve patency rates and to use the optimal vasodilator in the most appropriate way to counteract vasospasm.

© 2013 Yildiz et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Surgeons have studied graft pharmacology by measuring the effects of vasodilators on blood flow through arterial grafts before they were attached to the heart [9]. Pharmacologists have also joined the study of graft pharmacology by evaluating endothelial and smooth muscle function of bypass grafts using their standard in vitro method, the isolated vessel ring preparation in the organ bath. However, results from these in vitro studies need to be carefully extrapolated to the clinical situations, where the conditions of the arterial grafts are compli‐ cated. Even so, the organ bath method can provide very useful information about the effects of vasoactive substances in the arterial grafts.

for tension measurement along the former circumferential axis. The solution is gassed with % 95 O2 and % 5 CO2 at 37 ºC. Changes in arterial tensions are recorded isometrically by a forcedisplacement transducer by using a recording system, preferably a computer software. The segments are allowed to equilibrate under final resting force of 1-2 g for at least 1 to 1.5 h and they were washed every 10-20 minutes. After the equilibration period, arterial strips were challenged with a vasoconstrictor, i.e. phenylephrine, prostaglandin F2α or potassium chloride (KCl) to test the viability of the vessel. After an additonal 30 min of equilibration period with repeated washing every 10 min, the tissues are challenged with increasing cumulative concentrations of the vasoconstrictor substance to be tested and responses are recorded.

Pharmacology of Arterial Grafts for Coronary Artery Bypass Surgery

http://dx.doi.org/10.5772/54723

253

**Figure 1.** A schematic diagram of a human arterial ring preparation in an organ bath.

Several vasodilators have been tested and various antispastic methods have been suggested to prevent graft spasm; including papaverine, phenoxybenzamine, calcium antagonists and nitrates etc. Choice of a pharmacological agent to overcome the vasospasm encountered in the arterial grafts must be on the basis of pharmacological studies. Accordingly, current state of knowledge based on experiments to study the pharmacological effect of a number of vaso‐ constrictor and vasodilator substances and the practical application of this knowledge can be outlined as following sections:


**Multi-Spacecraft Attitude Path Planning Using Consensus with LMI-Based Exclusion Constraints** Multi-Spacecraft Attitude Path Planning Using

DOI: 10.5772/intechopen.71580

Consensus with LMI-Based Exclusion Constraints


**6. Self-care**

### **6.1 Introduction**

#### *6.1.1 What is self-care?*

The WHO has defined self-care as 'the ability of individuals, families and communities to promote health, prevent disease, maintain health and cope with illness and disability with or without the support of a healthcare provider' [91].

The practice of self-care empowers people to take charge of their health and well-being, giving them the incentive to live a healthier and happier life. It is key to preventing certain conditions and improving recovery times when total prevention fails. In every stage of life, it is an essential part of well-being [92].

Self-care involved: [92].

#### *6.1.2 Importance of self-care*

The importance of self-care lies in the empowerment of people to take control of their health and well-being and to improve their quality of life for the better [92]. Self-care can help certain conditions and improve recovery times when total prevention does not work. In all stages of life, it is an integral part of well-being. As people's life expectancy continues to rise globally, there has been a proportional increase in the number of individuals living with chronic conditions [93]. This circumstance has prompted individuals to autonomously manage and seamlessly incorporate their health conditions into their daily routine [94]. To be able to manage this situation well, healthy individuals or those living with chronic conditions need to know how to conduct it. The achievement of effective self-care necessitates a proactive collaboration between consumers and healthcare providers (HCPs), including pharmacists, and is contingent upon well informed consumers and HCPs equipped with proficient communication abilities [95].

Upon discharge from the hospital, patients encounter a multitude of challenges. This transitional phase places patients at risk of unnecessary harm due to insufficient *Delivery of Pharmaceutical Care at Tertiary Level: From Admission to Home Care DOI: http://dx.doi.org/10.5772/intechopen.112503*

preparation before their discharge, thereby potentially giving rise to errors and compromising their well-being [96]. It is during the discharge phase that proper medication reconciliation and comprehensive education sessions between pharmacists and patients, and their caregivers should take place to prepare patients for self-care while at home.

There is the potential for substantial healthcare cost savings from enhancing the self-care options that are available to consumers. It was estimated that 10–25% of GP consultations in different European countries could be substituted by self-care, which varies from country to country, depending on the current level of self-care uptake. Further substitution of GP visits by responsible self-care could release 17.6 billion euros per year for society [97].

### **7. Conclusions**

Collaboration between healthcare providers, including pharmacists and well informed consumers is essential when it comes to managing medical conditions at the tertiary level. This is important to ensure that patients get the best possible care starting from admission to discharge. The practice of self-care can bring significant advantages to individuals and society by relieving the burden on medical resources. Pharmacists have a crucial role to play in ensuring proper medication management and continuity of care encompassing activities such as home medicines review (HMR) and discharge planning. Maintaining a seamless continuum of quality medicine use from hospitals to the community is paramount in guaranteeing patients receive the appropriate level of care.


**3.4 Summary**

288 Hydrodynamics – Advanced Topics

the case of the O radical, the density profile of Fig. 11 was averaged along the radial direction until rd=50µm and drawn in Fig. 16 with the magenta color. The light blue color curve represents the O radical profile obtained with the Fluent Software when the specific source term profile SOc(z) is injected between a pin and the cathode plane in the simulation

In the following results, the complete simulation of the successive discharge and postdischarge phases involves 10 neutral chemical species (N, O, O3, NO2, NO, O2, N2, N2 (A3∑u+), N2 (a'1∑u-) and O2 (a1∆g)) reacting following 24 selected chemical reactions. The pin electrodes are stressed by a DC high voltage of 7.2kV. Under these experimental conditions the current pulses appear each 0.1ms (i.e. with a repetition frequency of 10KHz). It means that the previous described source terms are injected every 0.1ms during laps time td or tp and only locally inside the micro-plasma filament located between each pin and the plane.

Pictures in Fig. 17 show the cartography of the temperature and of the ozone density after 1ms (i.e. after 10 discharge and post-discharge phases). One, two, three or four pins are stressed by the DC high voltage. Pictures (a) show that for the mono pin case, the lateral air flow and the memory effect of the previous ten discharges lead to a wreath shape of the

**(a)**

**(a)**

**(b)**

**(c)**

**(c)**

**(d)**

The lateral air flow is fixed with a neutral gas velocity of 5m.s-1.

space distribution of both the temperature and the ozone density.

**T (°K)**

300

Fig. 17. Temperature and ozone density profile at 1ms i.e. after ten discharge and postdischarge phases. The number of high voltage pin is respectively (a) one, (b) two, (c) three

The temperature and the ozone maps are very similar. Indeed, both radical and energy source terms are higher near the pin (i.e. inside the secondary streamer area expansion as it was shown in section 3.2). Furthermore, the production of ozone is obviously sensitive to the gas temperature diminution since it is mainly created by the three body reaction *OO M O M* + +→ + 2 3 (having a reaction rate inversely proportional to gas temperature).

0

0.20

0.51

0.91

1.12

1.73

1.32

2.03

x 1022

**O3 (m-3 )**

305

313

323

333

341

338

351

conditions of Fig. 14.

**(d)**

and (d) four. The lateral air flow is 5m.s-1.

**(a)**

**(b)**

**(c)**

The complete simulation of all the complex phenomena that are triggered by microdischarges in atmospheric non thermal plasma was found to be possible not as usually done in the literature only for 0D geometry but also in multidimensional geometry. In DC voltage conditions, a specific first order electro-hydrodynamics model was used to follow the development of the primary and secondary streamers in mono pin-to-plane reactor. The simulation results reproduce qualitatively the experimental observations and are able to give a full description of micro-discharge phases. Further works, already undertaken in small dimensions or during the first instants of the micro-discharge development (Pancheshnyi 2005, Papageorgiou et al. 2011 ), have to be achieved in 3D simulation in order to describe the complex branching structure for pulsed voltage conditions. Nevertheless, the micro-discharge phase simulation gives specific information about the active species profiles and density magnitude as well as about the energy transferred to the background gas. All these parameters were introduced as initial source terms in a more complete hydrodynamics model of the post-discharge phase. The fist obtained results show the ability of the Fluent software to solve the physico-chemical activity triggered by the micro-discharges.


**3.6 Host immune system modulators**

Also, some drugs that modulate host immune system have reported antibacterial activity against GNB. Tamoxifen, a SERM used for breast cancer treatment, can reduced the migration of immune cells from bone marrow to blood through the reduction of monocytes chemoattractant protein 1 (MCP-1) and IL-18 in a murine model of sepsis by *A. baumannii*, *P. aeruginosa* and *E. coli* [36]. Moreover, tamoxifen

has been shown to enhance the killing activity of macrophages and neutrophils against *A. baumannii* and *E. coli in vitro* [36]. Calcitriol, a bioactive form of vitamin D3 used to treat hypocalcemic conditions and renal osteodystrophy, has a similar mechanism of action which it has been described to enhance the killing activity of monocytes and macrophages towards *P. aeruginosa* [37]. Moreover, GTS-21, an anti-inflammatory drug, has presented therapeutic efficacy against *P. aeruginosa in vivo* by enhancing macrophage function via inhibiting the release of nuclear protein high mobility group box-1 (HMGB1) [39]. When GTS-21 is combined with M1 muscarinic acetylcholine receptor agonist and α7n-acetylcholine receptor agonist against *E. coli in vivo*, the blood concentrations of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 were reduced significantly [38].


Improving Embryo Quality by Strictly Controlling IVF Laboratory Environment

*Javier García-Ferreyra and Alfonso Sánchez-Pavón*

### **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 embryotested 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.

**Keywords:** in vitro culture, embryo quality, culture system, IVF, IVF laboratory environment

### **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 [1–5]. 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**).

*\*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.*

### **Table 1.**

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

## **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

### *Improving Embryo Quality by Strictly Controlling IVF Laboratory Environment DOI: http://dx.doi.org/10.5772/intechopen.106782*

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].

### **Table 2.**

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

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.

*Embryology Update*