**2. Organization's cultural sustainability**

Considering the conscious capitalism perspective, they insert that organizations must seek to promote social inclusion, improve income distribution, and reduce poverty through human and ethical values. Those organizations must contribute to the economy and society in which they are an integral part [28, 29]. Organizations establish contracts with society, legitimizing them whenever they act as expected. Organizations, even in a global capitalist context, must move in a way that adjusts to culture in search of legitimacy [30]. In this sense, organizations have been relevant actors in developing initiatives and policies oriented toward the entire community's sustainability [31]. The relationship between organizations and their stakeholders is not limited to business transactions. These organizations must help create value for everyone [32] and engage with ethical and cultural concepts [29], which must be made public.

Even if they try to maintain an independence degree, organizations must follow the uses, customs, rules, and legislation evolution of a standardized development in an eminently capitalist society. That means, in addition to governance structures suited to their constitution and strategic and democratic management form, they need to operate within a capitalist context, or they will be excluded [33]. Thus, the organization is a risky enterprise that depends on capitalist foundations and social construction [34]. However, considering the evolution of the capitalist concept, which migrates from global capitalism [35] to conscious capitalism [28], any organization type must have sustainability as its purpose [30]. This sustainability encompasses four dimensions. In addition to the three dimensions introduced by the triple bottom line: economic, social, and environmental [36], we included the cultural dimension as the focus of this book chapter [19, 37, 38].

Society is increasingly sensitive to issues related to sustainability. It is one of the modern society principles, valuing technologies, processes, products, and minimal impacts on the ecosystem, that is, organizations operating within sustainable limits [31, 39]. Sustainability is a concern that permeates all society levels and, on the premise that it must develop to meet the present needs without compromising the future generation's ability to meet their own needs. In addition, economic growth must concern the natural environment protection and humanity's social and cultural well-being [36, 37, 40, 41].

The term sustainability expanded over time, starting with the economic perspective in which an organization only views profitability terms, financial results, return on investment, or shareholder value [42]. More recently, the environmental sustainability perspective was incorporated, which considers an organization's environmental impact regarding the environmental resources' consumption, pollutant emissions, solid and water waste, recycling, and materials reuse, among others [43]. The social sustainability perspective was also incorporated, measured by the relationships' quality with employees, health and safety records, community impacts, and human rights in general [44], culminating in the sustainability triple bottom line [36, 45, 46]. That means organizations need to act in a way that provides social benefits, absorbing resources from society and giving social meaning to their existence.

Thus, we consider that these three aspects are respected, with equal importance, allowing the relationship between the organization and society to continue functioning. If one of the pillars is not strong, it compromises the entire system and makes it unsustainable. Therefore, there must be equity between people, the planet, and profit. Profitability must be socio-environmentally and economically correct, a constant challenge for managing organizational sustainability [36, 47, 48]. In this sense, economic and social development is essential, as well as environmental protection and social well-being. Immolating the environment and/or social well-being for economic growth's sake would lead to disastrous consequences for future generations [45, 47, 49]. This context makes the complex relationship between the sustainability pillars [45]. In short, economic, environmental, and social development is aimed at integrating all sustainability dimensions [19, 20].

Recently, policies and research with new demands began to guide organizations, which must align activities with broader sustainable development agendas, including cultural, economic, social, and environmental issues [19, 37]. It is necessary to integrate culture with sustainability because achieving its goals depends on human actions and behaviors that are culturally embedded and rooted [40].

The material and immaterial society attributes define culture. It incorporates social organizations, literature, religion, myths, beliefs, behaviors, social practices and methods, technologies and tools, and expressive art forms. Culture is a human knowledge set that depends on transmitting these characteristics to future generations [19, 26, 50, 51]. Culture understands as a constantly evolving procedure, a lifeway [40]. Therefore, cultural sustainability is a fundamental issue or a precondition to be fulfilled on the path to sustainable development. It made up the sustainability social pillar. However, with the recent development in this field and its growing importance, the creation of the sustainability cultural pillar has become eminent [50]. Cultural sustainability has become a priority in sustainable development agendas [37]. It is now often described as a fourth pillar, equal to the social, economic, and environmental sustainability issues [50]. The information representing the culture

is in family businesses, cooperative organizations, and others that often carry an identity confused with habits and customs.

Cultural sustainability recognizes the need to preserve and transmit culture to future generations, achieved through pluralistic and transformative learning to promote social and ecological changes in the capitalist system [19, 26]. Culture attributes an identity sense and determines the behavior of a society [3]. That is, it has to do with the ability to sustain or continue with cultural beliefs and practices, cultural heritage preservation as its entity, and attempts to answer whether any culture will exist in the future. Cultural sustainability's importance lies in its power to influence people since the decisions made in society are weight by its beliefs [50]. Thus, there can be no sustainable development without including culture [38].


**2. Frequently utilized corrosion inhibitors**

Corrosion inhibitors form a defensive barrier of one or several molecular layers against corrosive agent attack. The frequently used inhibitors can be divided into two main types, inorganic substances, and organic substances.

Indeed, inorganic inhibitors are those in which the active substance is an inorganic compound [10]. Their metallic atoms are enclosed in the film to improve corrosion resistance [11]. Many inorganic inhibitors are known for their excellent efficacy. In particular, chromium (Cr6+) or chromium compounds and chromium salts are among the passivating inhibitors par excellence, but they are environmentally unacceptable and their severe toxicity greatly reduces their use. As has been indicated, strontium chromate, zinc chromate, chrome phosphate, etc., are heavy-metal-based and highly carcinogenic. Small amounts of chromic acid or potassium dichromate can cause kidney failure, liver damage, DNA damage, and blood cell disorders. Chromate mists entering the lungs may eventually lead to lung cancer [12, 13]. Other inhibiting molecules in the form of salts have made it possible to obtain very good yields in terms of metal protection and thus corrosion prevention, such as sodium tungstate (Na2WO4), vandates (NaVO3), nitrites (NaNO2), and silicates (Na2Si2O5). However, these compounds today are highly toxic, leading to serious consequences for the environment and human beings. They can cause temporary or permanent

damage to the nervous system, and disrupt the biochemical process and the enzymatic system of our organism [9]. Molybdates (MoO3 − ) and phosphates (H2PO3 − ) also provide passivation protection to metallic surfaces by incorporating them into the oxide layer. Borates and arsenates are also known for their promising inhibitory activity against metal corrosion in various aggressive aqueous media. Apart from that, they have also proven to be intolerant because of the threat they pose to nature and social health in the long run [14, 15]. Pyrrole and derivatives exhibit good protection against metals corrosion, especially in acidic media. These inhibitors are also useful application in the formulation of primers and anticorrosive coatings, but the major disadvantage associated with them is their toxicity and as such their use has come under severe criticism [15].

On the other hand, the frequently synthetic organic molecules used as corrosion inhibitors in industrial environments include aliphatic or aromatic thioureas, amines, amides, pyrazoles, pyrimidines, acetylenic alcohols, aldehydes, benzylidenes, carbazones, azoles, Schiff's base, benzonitriles, dimeric and trimeric acids, etc. Indeed, most of these inhibitors are containing heteroatoms such as nitrogen, sulfur, and oxygen with lone pair of electrons and should have aromatic systems. These compounds can act on the metal surface by means of adsorption and there detracting of the active metallic surface area, leaving inactive sites on the surface exposed to corrosive media. The inhibition efficiency of these compounds is also related to its functional groups, steric effects, and π-orbital character of donating electrons [15]. Even though these organic compounds exhibit high inhibition efficiencies against the corrosion of many types of metals, they are toxic and non-environmentally friendly and their use causes toxic harm to humans, animals, and nature. In addition, the time of exposure is also a factor that can have a strong influence on the toxicity and which can increase the harmful effect of these molecules [13].

So, the environmental and health risks associated with the use of these inhibitors have prompted us to find or use non-toxic or green corrosion inhibitors that would offer maximum protection to metal structures but have minimal impact on human and nature. Because the choice of an effective corrosion inhibitor must not only be cost-effective, stable, compatible with the corrosive medium, and produce the desired effect at small concentrations; but it must also be compatible with the current standards for non-toxicity, biodegradability, bioaccumulation, and environmental protection.


**5. Environmental and social impacts of mismanaged waste**

Weak waste collection systems in Africa combined with uncontrolled and controlled dumping of waste, often associated with open burning, are causing significant economic, social and environmental impacts. Current waste management practices in Africa are resulting in the leakage of waste into the environment. Furthermore, the indiscriminate dumping of waste in urban areas creates risks of disease, flooding and environmental pollution. Research has shown a correlation between proximity to open dumpsites and incidence of disease, including increased risk of cholera, malaria, typhoid fever, dengue fever and Zika [19–22].

Open burning of waste causes significant air pollution which impacts human health and contributes to changing climates. These impacts are not always local, but can be far reaching.

Africa has become a dumping ground for waste, particularly hazardous waste, often from developed countries, with many African countries, particularly in west and north Africa, becoming a global destination for end-of-life EEE and vehicles exported from developed countries in North America, Europe and Asia [10, 16, 23, 24]. Current electronic waste recycling practices, often informal, pose potential risks to people and the environment [25, 26]. Children and women are heavily involved in WEEE recycling in Africa, constituting the most vulnerable group for this waste. Recycling of used lead-acid batteries in informal workshops in Africa is a major source of lead pollution, exposing considerable numbers of people and the environment to adverse effects [27, 28].

Plastic as a percentage of MSW for sub-Saharan Africa is higher than the global average, at 13% [9] and is a waste stream of growing concern for the continent. With weak waste collection systems and disposal of waste to uncontrolled and controlled dumpsites, the leakage of plastic into the environment poses a significant threat to the environment and economies [29, 30]. This is compounded by the lack of recycling in Africa.

Improper waste management has serious health and environmental consequences. If it persists, it will undermine Africa's efforts to achieve the sustainable development goals (SDGs).

*Regional Development in Africa*

produced, is unacceptable.

**Figure 3.**

**4. Waste governance in Africa**

*MSW composition in sub-Saharan Africa and global [9].*

food produced is lost or wasted [12]. For a continent with high levels of poverty, hunger and malnutrition, the loss and wastage of more than a third of all food

A number of policies (international, continental and regional) are already in place to address pollution and waste in Africa. However, it remains unclear as to how these policies have been translated into action and what progress, if any, has been made towards achieving their objectives and commitments. The inability of governments to enforce waste and environmental legislation in many African countries has given rise to a culture of impunity and has weakened the effectiveness of waste management in general [13]. What is clear, is the inability of governments and private industry to keep pace with growing waste streams and the timely

development of policies and strategies to effectively deal with it [14].

*African countries that have implemented local or national bans against waste plastics (dark blue shaded)*

**238**

**Figure 4.**

*(adapted from [15]).*


**5. Pooled analysis about intradialytic cardiovascular stability**

Since, most of the studies, addressed the intradialytic hypotension, we summarized the data about the cardiovascular instability (intradialytic hypotension and weight loss) in the following pooled analysis. We included all the studies of any type (random cross-over, random not cross over and not random), which reported available data in form of percentage of dialysis complicated by hypotensions or average frequency of hypotension events, disregarding this was or was not the primary response variable.

Fig. 4. Pooled analysis of intradialytic hypotensions. The figure shows the hypotension events expressed as mean ± SD over the total number of dialysis.

We did not include in the analysis those studies for which confounding factors like hemodiafiltration and/or acetate free biofiltration, cold dialysate temperature if not present both in the intervention and control treatments could interfere with data interpretation. For each study we extracted the total number of treatments performed both with standard and HBS hemodialysis and the total events (dialysis complicated by hypotensions or major hypotension events) for each one, or the meanSD hypotension events as reported in the paper.

Blood Volume Regulation 245

pressure. However, during the first half of the therapy time with in-line hemodiafiltration, HBS is also characterised by a high convective dialysis rate. The recommended increase of the dialysate calcium concentration to 1.5 mmol/L should be considered with caution because it may result in a significant elevation of the calcium x phosphate product. HBS works with ultrafiltration control and sodium management to realise better refilling of the

The short reaction time of the MiMo (Multi input - Multi output) controller (Figure 2) guarantees the prevention of a hypotensive event or reduces its intensity. The underlying mechanisms should be found both in the fast reaction of the system to rapid blood volume drops and in the pre-set pathway which avoid the achievement of risky patient dependent

The electrolyte implication of HBS have been included in some of the studies and deeply

In this study they compared the effect of sodium manual profiling and automatic profiling versus standard treatment on the sodium mass balance measure through its surrogate variable, the ionic mass balance. It is interesting the comparison between standard dialysis prescribed at 140 mMol/L and biofeedback technologies (HBS and biofeedback on plasma conductivity) prescribed at equivalent values of 14.0 mS/cm. The manual profiling was indeed, prescribed with a linear time course starting at 15.0 mS/cm and ending at 14.0 mS/cm. The ionic mass balance in each study session was 423166 mMol, 488179 mMol and 409109 mMol respectively in standard, HBS and plasma conductivity biofeedback. Other studies on HBS reported data about the post dialysis natremia. Despite they did not investigated the actual sodium mass removal, they found no differences between standard versus HBS sessions. Santoro in 1998 reported end dialysis natremia equal to 1461.5 mMol/L in standard hemodialysis and 146.21.1 in HBS. They did not showed the predialysis natremia then one cannot conclude that the ionic mass balance could have been similar in the two treatments. Moreover, this was a short term study and a potential sodium overload could not have been seen. Subsequently the same group published a multicenter study (Santoro et al, 2002) in which data about the pre and postdialysis natremia were shown. Pre and post-dialysis plasma sodium values were: 138.70.5 and 141.80.6 in standard HD and 138.8 .6 and 141.30.7 in HBS. Moreover, if we consider that actual weight loss, blood flow rate and treatment time were equal in both the treatment, we could argue that the sodium mass balance should have been similar in the two treatment. Similar results in short-term study were reported by Wolkotte (Wolkotte et al, 2002). Pre and Post dialysis sodium levels were 139.82.5, 141.21.8 in standard HD and 139.62.4, 141.31.8 in HBS respectively and corresponding to weight loss of 2.40.7 in HD and 2.50.8 in HBS. Mid-term results were reported only in the study by Dasselaar (Dasselaar et al., 2007). The follow-up lasted 12 week per group (HD and HBS) and the values were at the end of the follow-up were: 139.22.5, 139.82.1 in HD and 139.82.2, 139.92.1 in HBS. Then, they observed an increase of 1 mMol/L in the postdialysis natremia but, the actual wight loss

was 2.76.7L in HD while 3.16.5L in HBS even though not statistically significant.

used to regulate the sodium mass balance in the form of equivalent conductance.

In conclusion, the several authors who investigated this aspect did not find any potential sodium overload in HBS. This is due to the underlying mathematical sodium kinetics model

intravascular compartment.

blood volume thresholds.

**6. Electrolyte implications**

investigated in one paper by Moret (Moret et al., 2006).

Figure 4 shows the results of the pooled analysis about the intradialytic hypotension of the thirteen included studies in the analysis. The reported data refer to 2520 dialysis in HBS against 2130 dialysis in conventional dialysis.

The overall mean difference was nearly -12% favoring the HBS treatment with an estimated range between -15% and -8%. The range is almost narrow despite some studies showed wider ranges (Dasselaar et al., 2007).

Discrepancies between the single studies can be partially explained by: a) the difference in within study variance underlying the difference in the sample size (from 8 to 44 recruited patients), b) the difference in the study design (cross-over, parallel group, etc) c) the target population (hypotension prone, non-hypotension prone and hypertensive patients), d) the difference in the between study reflecting the difference in the primary response variable (blood pressure drop, fluid overload) leading sometimes to a bias in the dialysis complicated by hypotension variable, e) the length of follow up ranging from few weeks to two years.

These good results are emphasized by looking at the weight loss during dialysis in the two treatments as reported in Figure 5. The overall result shows a higher weight loss in HBS than in standard treatment equal to 160 g. All the authors reported this behavior even though some were not statistically significant. McIntyre indeed report the interdialytic weight gain, which could not properly reflect the weight loss during the follow-up.

Fig. 5. Body weight loss during dialysis. Data report the pre to post dialysis body weight change during dialysis (in Kg or L) expressed as mean SD over the total assessed dialysis.

Different alternatives are available to reduce or prevent the noticeable drop in blood pressure during the extracorporeal therapy. In this context, the time and frequency of dialysis play a significant role, but the time of dialysis cannot be infinitely expanded because of socioeconomic framework conditions. Regulating the blood temperature via dialysate temperature can also have a noticeable positive effect on the blood pressure management. To what extent the efficacy of the therapy is limited because of the higher peripheral overall resistance is the subject of further studies. On-line hemodiafiltration is another established possibility to prevent a drop in blood pressure during blood purification therapy. The high convective rate of this procedure is capable of achieving the stabilisation of the blood

Figure 4 shows the results of the pooled analysis about the intradialytic hypotension of the thirteen included studies in the analysis. The reported data refer to 2520 dialysis in HBS

The overall mean difference was nearly -12% favoring the HBS treatment with an estimated range between -15% and -8%. The range is almost narrow despite some studies showed

Discrepancies between the single studies can be partially explained by: a) the difference in within study variance underlying the difference in the sample size (from 8 to 44 recruited patients), b) the difference in the study design (cross-over, parallel group, etc) c) the target population (hypotension prone, non-hypotension prone and hypertensive patients), d) the difference in the between study reflecting the difference in the primary response variable (blood pressure drop, fluid overload) leading sometimes to a bias in the dialysis complicated by hypotension variable, e) the length of follow up ranging from few weeks to two years. These good results are emphasized by looking at the weight loss during dialysis in the two treatments as reported in Figure 5. The overall result shows a higher weight loss in HBS than in standard treatment equal to 160 g. All the authors reported this behavior even though some were not statistically significant. McIntyre indeed report the interdialytic

weight gain, which could not properly reflect the weight loss during the follow-up.

Fig. 5. Body weight loss during dialysis. Data report the pre to post dialysis body weight change during dialysis (in Kg or L) expressed as mean SD over the total assessed dialysis. Different alternatives are available to reduce or prevent the noticeable drop in blood pressure during the extracorporeal therapy. In this context, the time and frequency of dialysis play a significant role, but the time of dialysis cannot be infinitely expanded because of socioeconomic framework conditions. Regulating the blood temperature via dialysate temperature can also have a noticeable positive effect on the blood pressure management. To what extent the efficacy of the therapy is limited because of the higher peripheral overall resistance is the subject of further studies. On-line hemodiafiltration is another established possibility to prevent a drop in blood pressure during blood purification therapy. The high convective rate of this procedure is capable of achieving the stabilisation of the blood

against 2130 dialysis in conventional dialysis.

wider ranges (Dasselaar et al., 2007).

pressure. However, during the first half of the therapy time with in-line hemodiafiltration, HBS is also characterised by a high convective dialysis rate. The recommended increase of the dialysate calcium concentration to 1.5 mmol/L should be considered with caution because it may result in a significant elevation of the calcium x phosphate product. HBS works with ultrafiltration control and sodium management to realise better refilling of the intravascular compartment.

The short reaction time of the MiMo (Multi input - Multi output) controller (Figure 2) guarantees the prevention of a hypotensive event or reduces its intensity. The underlying mechanisms should be found both in the fast reaction of the system to rapid blood volume drops and in the pre-set pathway which avoid the achievement of risky patient dependent blood volume thresholds.

### **6. Electrolyte implications**

The electrolyte implication of HBS have been included in some of the studies and deeply investigated in one paper by Moret (Moret et al., 2006).

In this study they compared the effect of sodium manual profiling and automatic profiling versus standard treatment on the sodium mass balance measure through its surrogate variable, the ionic mass balance. It is interesting the comparison between standard dialysis prescribed at 140 mMol/L and biofeedback technologies (HBS and biofeedback on plasma conductivity) prescribed at equivalent values of 14.0 mS/cm. The manual profiling was indeed, prescribed with a linear time course starting at 15.0 mS/cm and ending at 14.0 mS/cm. The ionic mass balance in each study session was 423166 mMol, 488179 mMol and 409109 mMol respectively in standard, HBS and plasma conductivity biofeedback.

Other studies on HBS reported data about the post dialysis natremia. Despite they did not investigated the actual sodium mass removal, they found no differences between standard versus HBS sessions. Santoro in 1998 reported end dialysis natremia equal to 1461.5 mMol/L in standard hemodialysis and 146.21.1 in HBS. They did not showed the predialysis natremia then one cannot conclude that the ionic mass balance could have been similar in the two treatments. Moreover, this was a short term study and a potential sodium overload could not have been seen. Subsequently the same group published a multicenter study (Santoro et al, 2002) in which data about the pre and postdialysis natremia were shown. Pre and post-dialysis plasma sodium values were: 138.70.5 and 141.80.6 in standard HD and 138.8 .6 and 141.30.7 in HBS. Moreover, if we consider that actual weight loss, blood flow rate and treatment time were equal in both the treatment, we could argue that the sodium mass balance should have been similar in the two treatment. Similar results in short-term study were reported by Wolkotte (Wolkotte et al, 2002). Pre and Post dialysis sodium levels were 139.82.5, 141.21.8 in standard HD and 139.62.4, 141.31.8 in HBS respectively and corresponding to weight loss of 2.40.7 in HD and 2.50.8 in HBS. Mid-term results were reported only in the study by Dasselaar (Dasselaar et al., 2007). The follow-up lasted 12 week per group (HD and HBS) and the values were at the end of the follow-up were: 139.22.5, 139.82.1 in HD and 139.82.2, 139.92.1 in HBS. Then, they observed an increase of 1 mMol/L in the postdialysis natremia but, the actual wight loss was 2.76.7L in HD while 3.16.5L in HBS even though not statistically significant.

In conclusion, the several authors who investigated this aspect did not find any potential sodium overload in HBS. This is due to the underlying mathematical sodium kinetics model used to regulate the sodium mass balance in the form of equivalent conductance.

Blood Volume Regulation 247

Effects of the achieved cardiovascular stability were accompanied by an improved efficiency of treatment. A lower urea rebound was observed during the HBS sessions, resulting in a higher equilibrated Kt/V (eKt/V) as shown by several groups (Ronco et al., 2000, McIntyre

In hypotension-prone patients, total urea removed was significantly higher during HBS compared to HD sessions (Ronco et al., 2000). This was also demonstrated for nonhypotension-prone patients. The eKt/V ratio increased from 1.01 ± 0.03 to 1.13 ± 0.03 with HBS (*P*<0.01) (McIntyre et al., 2003) In this group of patients, the mass of urea removed

The effect of HBS on interdialytic symptoms, such as muscle cramps, headache, dizziness, thirst, dyspnoe, angina, vomiting, itching, the need to lie down, anorexia and asthenia, was analysed by Santoro (Santoro et al., 2002) They looked at the number of symptoms between 2 consecutive sessions within the first 6 hours from the end of dialysis (early symptoms) and later until the beginning of the next dialysis session (late symptoms) and observed a 10%

Déziel et al. investigated the impact of HBS treatment on health-related quality of life (Deziel et al., 2007). They included 44 patients who were partially hypertensive and/or hypotension-prone in a 6-month randomised controlled trial comparing standard HD and HBS. Quality of life was assessed using the Kidney Disease and Quality of Life Short Form (KDQOL-SF) questionnaire. There was a significant improvement in the burden of

Fig. 7. Decrease of antihypertensive drug and end dialysis body weight over 48 months of

et al., 2003, Dasselaar et al., 2007, Neshrallah et al., 2008, Winkler et al. 2008).

overall reduction (*P*<0.001) in symptoms after treatment with HBS.

**8. Efficiency of treatment**

increased from 24.9 to 32.7 (*P*<0.01).

**9. Interdialytic events**

**10. Quality of life**

follow-up.


*3.5. Antibacterial Proteins*

In addition to the previous examples, it is also possible to transport proteins bearing antimicrobial effects. On the delivery of antibacterial proteins with MSNs, most contributions were made with lysozyme, a 14.4-kDa enzyme known to damage bacterial cells by hydrolyzing the major component of Gram-positive bacterial walls, i.e., the β-1,4 bonds between *N*-acetylmuramic acid and *N*-acetyl-d-glucosamine. The first example, reported by Lin et al., employed negatively charged MCM-41 MSNs to bind lysozyme units through electrostatic interactions to provide a stable corona [82]. This model permitted having an antibacterial e ffect even on Gram-negative bacteria such as *E. coli*. Such an e ffect was a consequence of the high concentration of lysozyme released in bacterial surroundings. Although forthcoming models improved the antibacterial performance of this device, in this work, the authors set two important advances: the possibility of treating bacterial infections with nanotechnology, and the finding that even Gram-negative bacteria could be highly damaged by these glycoside hydrolase enzymes.

In light of these previous results, Yu's group attempted to improve the antibacterial capacity by using higher-loading carriers. In one contribution, they di fferently evaluated dendritic pore MSNs, finding that a bigger pore led to a faster release and, thus, higher antibacterial e ffects [83]. In the second contribution, they developed silica nanopollens, which are engineered non-porous hollow nanoparticles coated with nanosized silica spikes to provide a rough surface [84]. Similarly, to the former model, they evaluated di fferent roughnesses, finding that a rougher surface led to a better loading and the associated antibacterial e ffect. A comparison between models seemed to indicate that, in the case of large-pore MSNs, the antibacterial e ffect of lysozyme was a consequence of a faster release, while, in the case of nanopollens, it was a combination of a more sustained release together with an unknown nanoparticle-induced e ffect. Unfortunately, these models were only tested on planktonic bacteria. To address a more complex situation in which the bacteria form a biofilm, Ye and coworkers studied hollow MSNs as high-capacity nanocarriers (up to 350 mg per gram in the case of their enlarged pore HMSNs) [85]. In this work, the authors reported a threshold for free lysozyme activity (400 μg/mL), over which there was no therapeutic improvement. However, the use of MSN-based delivery raised that maximal e ffect, as they showed a more sustained release pattern able to induce a long-lasting e ffect.

Apart from lysozyme, antibacterial e ffects with other proteins were also reported such as ConA (in this case, in combination with levofloxacin) [86]. In this example, carboxylate-modified MCM-41 MSNs were functionalized with ConA upon drug loading. Herein, the combined action of both species permitted achieving complete biofilm destruction even at minimal concentrations (10 μg MSNs per mL), improving the precedent models due to combination therapy. In addition to these reported models, there are many more proteins that lead to successful combinations [97]. Such is the case of lactoferrin, known for having antibacterial properties [98], which was extensively employed in the preparation of glioblastoma-targeted nanodevices [99,100].

## **4. Delivery of Peptides with Therapeutic E** ff**ect**

Many of known bioactive peptides are simplified amino-acid sequences that replicate the active sites of proteins and enzymes. The success of these peptides is a consequence of two complementary aspects: (1) the ability to retain features from their parent proteins, and (2) an outstanding chemical profile that arises from their facile synthesis, significantly low cost, and chemical robustness. Those aspects permitted discovering a large number of peptides of di fferent nature and specificity. For example, regarding targeting, those sequences are selected for their a ffinity toward eukaryotic [101,102] or prokaryotic [103,104] cell receptors. However, applications of peptides go beyond targeting [105], as they are able to accomplish di fferent tasks in regulating metabolic cycles and signaling. In this section, we focus on the potential of MSN-carried peptides for the development of new therapeutic devices. As shown in Table 2 and Figure 4, their applications range from cancer treatment [106–108] to antibacterial e ffects [107,109], cell regulation processes [110], and immunostimulation.

**Figure 4.** Strategies for delivery of therapeutic peptides employing silica-based carriers.

nanoparticles; MS-HANs: mesoporous silica–hydroxyapatite nanoparticles; OGP: osteogenic growth peptide; PAMAM: polyamidoamine dendrimer.


*Edited by Carlos C. Perez-Marin*

Veterinary medicine is advancing at a very rapid pace, particularly given the breadth of the discipline. This book examines new developments covering a wide range of issues from health and welfare in livestock, pets, and wild animals to public health supervision and biomedical research. As well as containing reviews offering fresh insight into specific issues, this book includes a selection of scientific articles which help to chart the advance of this science. The book is divided into several sections. The opening chapters cover the veterinary profession and veterinary science in general, while later chapters look at specific aspects of applied veterinary medicine in pets and in livestock. Finally, research papers are grouped by specialisms with a view to exploring progress in areas such as organ transplantation, therapeutic use of natural substances, and the use of new diagnostic techniques for disease control. This book was produced during World Veterinary Year 2011, which marked the 250th anniversary of the veterinary profession. It provides a fittingly concise and enjoyable overview of the whole science of veterinary medicine.

ISBN 978-953-51-0031-7

A Bird's-Eye View of Veterinary Medicine

Photo by ElenaBelozorova / iStock


**5. Sensitivity analysis of power system harmonic response**

A sensitivity analysis of all variables involved in location of the parallel and series resonance is performed from (22). Thus, considering the range of the variables, Fig. 10 shows the contour plots of the harmonics where the parallel and series resonance is located. These harmonics are calculated from the expression of *kr*, a, (22), and the τ1 range is fixed from (4) considering the λ*L* value. From Fig. 10, it can be noted that

Fig. 10. Contour plots of *kr*, a.


*4.3. Annual Validation*

Mean annual values for the study period (see Figure 6) were calculated for both the set of 136 ground stations and the satellite precipitation products (mean values of the available corresponding grid cells within the study area). It is found that, overall, both SP data exhibit an underestimation, although it is lower for IMERG, with the exception of 2015, when a slight overestimation by the IMERG product was noticed. It should be noted, however, that the available precipitation estimates for 2014 were limited to the period April to December, while for 2018 they were limited to January to June.

**Figure 6.** Mean annual values of gauge stations and Satellite Precipitation (SP) data.

Figure 7 displays the variation of overestimation (>100%) or underestimation (<100%) of annual precipitation values at each of the corresponding SP grid cell, between the SP estimated and calculated mean annual gauge station records. We notice that, more or less, the SP estimates have the same behavior regarding the overestimation/ underestimation of annual rainfall. Underestimation is more evident in the central area of Cyprus (greater area of Troodos mountain range), where higher precipitation records are generally noticed, highlighting the known limitations of satellite products regarding heavy rainfall, while the overestimation is noticed in the coastal or rather flat areas, where again the estimation of precipitation still remains a challenge due to the difficulty in distinguishing between rain and non-rain pixels over a complex background [44]. Furthermore, there are no significant differences between the years, since the cells that generally overestimate/underestimate rainfall have a similar performance regardless of the year.

**Figure 7.** *Cont*.

**Figure 7.** Underestimation/Overestimation of the annual satellite product estimates as compared with gauge station records at the corresponding grid cell: (**a**) TRMM—2014 (April–December); (**b**) GPM—2014 (April–December); (**c**) TRMM—2015; (**d**) GPM—2015; (**e**) TRMM—2016; (**f**) GPM—2016; (**g**) TRMM—2017; (**h**) GPM—2017; (**i**) TRMM—2018 (January–June); (**j**) GPM—2018 (January–June).

The performance of the IMERG product was slightly better than 3B43V7 with an R higher value (0.82 and 0.81, respectively), lower Bias values (−17.9 mm/month and −30.5 mm/month, respectively) and rBias values (3.1 and −3.0, respectively), while presenting worst performance in terms of RMSE (108.8 mm/month and 94.7 mm/month, respectively) and MAE (96.1 mm/month and 83.4 mm/month, respectively) values, as presented in Table 4 (minimum and maximum respective values are provided in brackets).

**Table 4.** Performance evaluation metrics of annual data from 3B43V7 and IMERG and (the range of values is given in parentheses).


**4. Conclusions**

An experimental study of the phase behavior of chiral 3-(3,4-dimethylphenoxy) propane-1,2-diol **1** showed that enantiopure samples exist in a single stable crystalline form. In contrast, racemic **1** during crystallization under different conditions can exist in at least three modifications, namely stable racemic conglomerate (*R*+S)-**1** and two metastable racemic compounds α-*rac*-**1** and β-*rac*-**1**. With rapid crystallization from racemic solutions, all three phases can crystallize simultaneously.

Using the DSC approach, methods have been developed to obtain individual metastable phases, and all identified homochiral and racemic crystal modifications were ranked by energy. The IR spectroscopy and PXRD methods demonstrated the identity or similarity of all forms except β-*rac*-**1**, which is significantly different from the rest. The crystal structure of the forms (*R*)-**1** and α-*rac*-**1** was established by the SC-XRD method. It was found that supramolecular crystal-forming motifs in both forms are fundamentally the same and only symmetries differ. Preliminary information on the structure of β-*rac*-**1** phase was obtained by the PXRD approach.

Based on the information received, the experimental conditions for a successful direct resolution of racemic **1** into individual enantiomers by a preferential crystallization procedure were selected.

**Author Contributions:** Conceptualization, writing—review and editing, A.A.B.; methodology, DSC investigation, thermodynamics calculations, D.V.Z.; methodology, proofread the manuscript, resolution experiments, Z.A.B.; samples preparation and resolution, A.V.K.; XRD investigation, A.I.S.; XRD investigation, proofread the manuscript, A.T.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors thank I.I. Vandyukova for valuable help with IR spectra analysis. The authors are grateful to the Assigned Spectral-Analytical Center of FRC Kazan Scientific Center of RAS for technical assistance in research.

**Conflicts of Interest:** The authors declare no conflicts of interest.


**14.3. DNA damage and cell cycle**

*p53 inhibitors:* Since p53 is frequently mutated in tumours, therapeutic approaches have been made to restore p53 function. Since p53 is targeted to degradation through interaction with mdm2, some drugs such as RITA, Nutlin-3 or HLI198 have been designed to disrupt p53 mdm2 interaction. These compounds bind to the p53 binding site on mdm2 or inhibit mdm2 ubiquitin ligase activity. This approach is valid in cancers with wild type p53, such as haematological malignancies [56]. A second approach is to rescue wild-type 53 function in p53-mutated tumours. PRIMA-1 restores sequence-specific DNA binding and active conformation of mutant p53 proteins. PRIMA-1 can synergize with conventional chemotherapeutic drugs and inhibit tumour growth in mice with no apparent toxicity and has recently entered clinical trials [78].

*Chk1/2 inhibitors:* The concept of "synthetic lethality" coined by Kaelin, by which two molecular lesions combine to have a lethal effect on the cell, although neither of them is harmful individually, has recently gained interest [193,194]. In this context, Chk inhibitors may have a therapeutic potential in p53-mutated tumours. UCN-01, the first Chk inhibitor evaluated in humans, has limited clinical value due to its toxicity. Other Chk inhibitors functioning as checkpoint abrogators that are being evaluated in clinical trials are: AZD7762, LY26303618, CBT501, PF-00477736, SCH 900776, XL844, and the wee-1 inhibitor MK-1775 [195,196].

*PARP inhibitors:* Other example of synthetic lethality is BRCA mutant breast cancer. BRCA1 and BRCA2 play a role in homologous recombination, an important repair pathway for DNA double strand breaks. The current hypothesis is that since PARP is involved in repairs in DNA single strands breaks, inhibition of PARP leads to accumulation of single strand unrepaired breaks and becomes synthetically lethal in BRCA-mutated cancers [197]. Several PARP inhibitors are being evaluated in clinical trials: AG014699, AZD2281 (olaparib), ABT888 (veliparib), BSI-201 (iniparib), INO-1001, GP121016, CEP-9722 and MKI4827 [197,198]. PARP inhibitors may be useful in tumours bearing other types of genomic or functional defects in DNA damage response pathways.


*Review* **A Review of Delayed Delivery Models and the Analysis Method in Mice**

**Hiroshi Yomogita 1,2 , Naoyuki Miyasaka 1 and Masami Kanai-Azuma 2,\***

**\*** Correspondence<EMAIL_ADDRESS>Tel.: +813-3813-6111

**Abstract:** In humans, the incidence of post-term delivery is 1–10%. Post-term delivery significantly increases the risk of cesarean section or neonatal intensive care unit (NICU) admission. Despite these serious challenges, the cause of prolonged delivery remains unclear. Several common factors of delayed parturition between mice and humans will help elucidate the mechanisms of pregnancy and labor. At present, gene modification techniques are rapidly developing; however, there are limited reviews available describing the mouse phenotype analysis as a human model for postterm delivery. We classified the delayed-labor mice into nine types according to their causes. In mice, progesterone (P4 ) maintains pregnancy, and the most common cause of delayed labor is luteolysis failure. Other contributing factors include humoral molecules in the fetus/placenta, uterine contractile dysfunction, poor cervical ripening, and delayed implantation. The etiology of delayed parturition is overexpression of the pregnancy maintenance mechanism or suppression of the labor induction mechanism. Here, we describe how to investigated their causes using mouse genetic analysis. In addition, we generated a list to identify the causes. Our review will help understand the findings obtained using the mouse model, providing a foundation for conducting more systematic research on delayed delivery.

**Keywords:** prolonged delivery; delayed delivery; progesterone; luteolysis

## **1. Introduction**

Placental–fetal–maternal close signal interactions are essential for pregnancy and labor. Imbalances between these factors cause abnormalities during the gestation period. Recently, several mice with delayed labor have been reported due to abnormal development of the fetus and placenta. In humans, the incidence of post-term delivery is 1–10% [1]. Postterm delivery significantly increases the risk of cesarean section or neonatal intensive care unit (NICU) admission. Despite these serious challenges, the cause of prolonged delivery remains unclear. Primates have similar-evolved villous placentas. In many studies, mouse pregnancy has been used as a model of human pregnancy. At present, gene modification techniques are rapidly developing; however, there are limited reviews to describe mouse phenotype analysis as a human model for post-term delivery. To elucidate, the effects of fetal and placental developmental abnormalities on delayed labor, we reviewed genetically modified mice that cause delayed labor. We reviewed the causes of delayed births and investigated them using mouse genetic analysis.

#### **2. Normal Progression of Pregnancy and Labor Onset in Mice**

Parturition is divided into two phases, pregnancy and labor, which correlate with changes in the cervix and myometrium (Figure 1). The cervix, a cylindrical tissue, is the lower part of the uterus and is used as the birth canal. During pregnancy, the cervix is firm and closed. It protects the fetus from microorganisms and acts as a barrier between

**Citation:** Yomogita, H.; Miyasaka, N.; Kanai-Azuma, M. A Review of Delayed Delivery Models and the Analysis Method in Mice. *J. Dev. Biol.* **2022**, *10*, 20. https://doi.org/ 10.3390/jdb10020020

Academic Editors: Tsutomu Nohno, Hideyo Ohuchi and Simon J. Conway

Received: 7 April 2022 Accepted: 17 May 2022 Published: 20 May 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

intrauterine and extrauterine conditions. The cervix gradually softens as pregnancy progresses. The uterine myometrium is quiescent and does not respond to natural stimuli. In the labor phase, the cervix is soft and elastic, which is referred to as 'cervical ripening'. Cervical ripening is caused by the decomposition of collagen and glycosaminoglycans (the main material of the cervix). The uterine myometrium contracts collaboratively and provides sufficient pressure to eliminate the fetus and placenta. Cervical ripening and uterine contractions are essential delivery processes, hence if either is missing, labor does not progress. The pregnancy-maintenance mechanism suppresses cervical ripening and uterine contractions. In the labor phase, the pregnancy-maintenance mechanism weakens, while the labor-induction mechanism is strengthened. Therefore, cervical ripening and uterine contractions occur. To deliver at term, the two mechanisms must switch at the right time. Delayed delivery occurs when the pregnancy-maintenance mechanism is excessively long or when the labor-induction mechanism is weak. A proper balance between the two systems is essential for normal pregnancy and delivery. intrauterine and extrauterine conditions. The cervix gradually softens as pregnancy progresses. The uterine myometrium is quiescent and does not respond to natural stimuli. In the labor phase, the cervix is soft and elastic, which is referred to as 'cervical ripening'. Cervical ripening is caused by the decomposition of collagen and glycosaminoglycans (the main material of the cervix). The uterine myometrium contracts collaboratively and provides sufficient pressure to eliminate the fetus and placenta. Cervical ripening and uterine contractions are essential delivery processes, hence if either is missing, labor does not progress. The pregnancy-maintenance mechanism suppresses cervical ripening and uterine contractions. In the labor phase, the pregnancy-maintenance mechanism weakens, while the labor-induction mechanism is strengthened. Therefore, cervical ripening and uterine contractions occur. To deliver at term, the two mechanisms must switch at the right time. Delayed delivery occurs when the pregnancy-maintenance mechanism is excessively long or when the labor-induction mechanism is weak. A proper balance between the two systems is essential for normal pregnancy and delivery.

firm and closed. It protects the fetus from microorganisms and acts as a barrier between

*J. Dev. Biol.* **2022**, *10*, x 2 of 17

**Figure 1.** Pregnancy-maintenance mechanism and labor-induction mechanism. During pregnancy, uterine contractions do not occur and the cervical canal is immature. During labor, uterine contractions are activated and cervical ripening is promoted. To deliver at term, the pregnancymaintenance mechanism and labor-induction mechanism need to work at the right time. Red ar-**Figure 1.** Pregnancy-maintenance mechanism and labor-induction mechanism. During pregnancy, uterine contractions do not occur and the cervical canal is immature. During labor, uterine contractions are activated and cervical ripening is promoted. To deliver at term, the pregnancy-maintenance mechanism and labor-induction mechanism need to work at the right time. Red arrows: contraction, blue arrows: cervical dilation.

#### **3. Factors of Pregnancy Maintenance and Labor Induction**

rows: contraction, blue arrows: cervical dilation

**3. Factors of Pregnancy Maintenance and Labor Induction** In rodents, progesterone (P₄) has the strongest pregnancy-maintenance effect. P₄ is secreted by the corpus luteum in pregnant mice, and suppresses labor-induction substances, such as prostaglandins (PGs) and oxytocin receptor (OTR). At 18.5 days post coitum (dpc), serum P₄ declines rapidly because of luteolysis and P₄ withdrawal increases In rodents, progesterone (P4) has the strongest pregnancy-maintenance effect. P4 is secreted by the corpus luteum in pregnant mice, and suppresses labor-induction substances, such as prostaglandins (PGs) and oxytocin receptor (OTR). At 18.5 days post coitum (dpc), serum P4 declines rapidly because of luteolysis and P4 withdrawal increases the levels of the labor-inducing substances (Figure 2).

the levels of the labor-inducing substances (Figure 2). Other substances that maintain pregnancy include relaxin and placental lactogen (PL), a part of the PRL family, which can maintain the corpus luteum. Human placental lactogen (hPL) does not maintain pregnancy. In contrast, mouse placental lactogen 1 (mPL1) and mouse placental lactogen 2 (mPL2) prevent luteolysis.

Labor-inducing substances include oxytocin, PGE2 (prostaglandin E2), PGF2α (prostaglandin F2α), their receptors, and connexin (Cx). These are collectively referred to as contractile-associated proteins (CAPS).

**Figure 2.** Pregnancy-maintenance and parturition mechanisms in mice. The luteolytic factor maintains the corpus luteum during pregnancy. P₄ suppresses CAPS. Luteolysis at 18.5 dpc causes a sharp decrease in P₄. With an increase in CAPS, uterine contractions and cervical ripening pro-**Figure 2.** Pregnancy-maintenance and parturition mechanisms in mice. The luteolytic factor maintains the corpus luteum during pregnancy. P4 suppresses CAPS. Luteolysis at 18.5 dpc causes a sharp decrease in P4 . With an increase in CAPS, uterine contractions and cervical ripening progress rapidly.The red Arrows indicate increased effect.

gress rapidly. The red Arrows indicate increased effect. Other substances that maintain pregnancy include relaxin and placental lactogen (PL), a part of the PRL family, which can maintain the corpus luteum. Human placental lactogen (hPL) does not maintain pregnancy. In contrast, mouse placental lactogen 1 (mPL1) and mouse placental lactogen 2 (mPL2) prevent luteolysis. Oxytocin, a hormone secreted by the pituitary gland, has a uterine contraction effect and is used as a labor-inducing agent in humans; however, *oxytocin* knockout (*OT* KO) mice can normally deliver [2]. Oxytocin is not essential for delivery. In oxytocin-administration experiments in *OT* KO mice, low concentrations of oxytocin maintained the corpus luteum and caused delayed labor and high concentrations of oxytocin enhanced uterine contractions and caused preterm delivery [3]. It is, therefore, difficult to elucidate the mechanism of labor since these molecules show different functions depending on the site and timing.

Labor-inducing substances include oxytocin, PGE₂ (prostaglandin E2), PGF2α (prostaglandin F2α), their receptors, and connexin (Cx). These are collectively referred to as contractile-associated proteins (CAPS). PG is a bioactive lipid produced from free arachidonic acid. PG is produced by phospholipase A (PLA) 2 and Cox; Cox1 and Cox2 are rate-limiting enzymes. There are various types of PG, such as D2, E2, F2α, and GI2, all of which have a different function. PGF2α and PGE2 are used as labor-inducing agents in humans.

Oxytocin, a hormone secreted by the pituitary gland, has a uterine contraction effect and is used as a labor-inducing agent in humans; however, *oxytocin* knockout (*OT* KO) mice can normally deliver [2]. Oxytocin is not essential for delivery. In oxytocin-PGF2α also has a luteolytic effect in mice. PGE2 has a cervical-ripening effect and causes weak uterine contractions. However, PGE2 may also have an indirect luteotropic effect [4]. Since PG has a short half-life and acts locally around the secretory site, it is characterized by different functions depending on the site of action.

administration experiments in *OT* KO mice, low concentrations of oxytocin maintained the corpus luteum and caused delayed labor and high concentrations of oxytocin enhanced uterine contractions and caused preterm delivery [3]. It is, therefore, difficult to Cx is a protein that forms gap junctions, which are cell–cell conjugations. Gap junctions play a role in the exchange of calcium ions between cells. When Cx43 is abundantly expressed in the myometrium, gap junctions also increase. Gap junctions are important for cooperative uterine contractions.

#### elucidate the mechanism of labor since these molecules show different functions de-**4. Classification by Cause of Labor Delay**

PGF2α and PGE₂ are used as labor-inducing agents in humans.

characterized by different functions depending on the site of action.

for cooperative uterine contractions.

**4. Classification by Cause of Labor Delay**

according to the cause of delayed labor (Table 1).

pending on the site and timing. PG is a bioactive lipid produced from free arachidonic acid. PG is produced by We searched the literature published between 1970 and 2022 and classified 26 mice according to the cause of delayed labor (Table 1).

causes weak uterine contractions. However, PGE₂ may also have an indirect luteotropic

tions play a role in the exchange of calcium ions between cells. When Cx43 is abundantly expressed in the myometrium, gap junctions also increase. Gap junctions are important

phospholipase A (PLA) 2 and Cox; Cox1 and Cox2 are rate-limiting enzymes. There are various types of PG, such as D2, E2, F2α, and GI2, all of which have a different function.

PGF₂α also has a luteolytic effect in mice. PGE₂ has a cervical-ripening effect and

Cx is a protein that forms gap junctions, which are cell–cell conjugations. Gap junc-

We searched the literature published between 1970 and 2022 and classified 26 mice

**Table 1.** Classification of genetically modified mice that exhibit delayed delivery (official name: abbreviation). We searched the literature published between 1970 and 2020, and classified the 26 mice according to the cause of delayed labor.

#### *4.1. Failure of Luteolysis*

The most common cause of delayed delivery in genetically modified mice is the absence of P4 withdrawal due to the failure of luteolysis. The decrease in serum P4 can be confirmed by measuring approximately 18.5 dpc. Ovariectomy and administration of RU486 (a P4 antagonist) are methods to determine whether P4 maintains pregnancy [5,33]. Wild-type mice are delivered within 24 h using these methods [5,33].

There are two phases in the degeneration of the corpus luteum: functional and structural degeneration. Functional degeneration is associated with P4 withdrawal. The enhanced action of 20α-hydroxysteroid dehydrogenase (20αHSD), which inactivates P4, reduces serum P4 levels. Meanwhile, in structural degeneration, apoptosis of pregnant luteal cells is enhanced, replacement of capillaries and connective tissue occurs, and the size and weight of the corpus luteum decreased rapidly. Structural degeneration can be confirmed by exenteration of the ovaries after 18.5 dpc and observing them with hematoxylin–eosin staining and/or tunnel staining.

#### 4.1.1. *Prostaglandin F Receptor* Knockout Mouse: *Fp* KO

One of the most famous genetically modified mice with delayed delivery is *Fp*-KO. In 1997, Sugimoto et al. produced mice to investigate the physiological function of PGF2α [5]. *Fp* KO mice that lack the prostaglandin F receptor (FP) do not deliver and absorb their fetuses in utero [5]. The P4 level of *Fp* KO mice did not decrease at 18.5 dpc [5]. *Fp* KO mice with both ovaries removed at 19.5 dpc delivered within 24 h [5]. Luteolysis failure is the cause of the delayed delivery. This report suggests that PGF2 leads to luteolysis in pregnant mice.

In *Fp* KO at 19.5 dpc, ovariectomy leads to the expression of oxytocin, OTR, and Cox2 [6]. When Cox2 selective inhibitor was administered to these mice, labor did not occur [6]. Labor after luteolysis is caused by COX-2-derived PGs in the myometrium [6]. In wild-type mice, Cox2 selective inhibitor leads to prolonged delivery [34]. Conversely, in humans, Cox2 selective inhibitors restrict uterine contractions and have been investigated as a treatment to prevent preterm delivery [35].

#### 4.1.2. *Cox1*-Deficient Mouse: *Cox1* KO

The average gestation period of *Cox1* KO mice is 21.6 ± 0.2 days [7]. These mice showed no sharp P4 decline and had a luteal regression disorder [7]. *Cox1* KO in late pregnancy results in a decreased amount of PGF2α in the uterus [7]. The administration of PGF2α to mice improved delayed delivery [7]. This research suggests that the cause of delayed parturition in mice is a failure of luteolysis due to a decrease in PGF2α levels [7]. The uterine contractions of *Cox1* KO mice were the same as those of wild-type mice, but the cervix was immature [33]. The cause of delayed labor in mice may also be related to poor cervical ripening due to impaired PG production.

#### 4.1.3. *20α Hydroxysteroid Dehydrogenase*-Deficient Mice: 20α *Hsd* KO

20αHSD is an enzyme that inactivates P4. *20α Hsd* KO caused delayed labor without P4 withdrawal [8]. Administration of PGF2α did not induce labor in these mice [8]. 20αHSD is presumed to act downstream of PGF2α [8].

The expression of 20αHSD is controlled by various pathways (Figure 3). For example, PRL activates a transcription factor called Stat5b (signal transducer and activator of transcription 5 B) in the ovary [36]. Since Stat5b reduces the expression of 20αHSD, PRL has a luteotropic function. In mice, the lack of Stat5b is associated with early abortion [8]. *J. Dev. Biol.* **2022**, *10*, x 6 of 17

**Figure 3.** Functional luteolysis. Functional luteolysis is caused by the inactivation of P₄ by 20αHSD. There are various pathways for regulating the expression of 20αHSD, such as the FP and prolactin-receptor pathways. **Figure 3.** Functional luteolysis. Functional luteolysis is caused by the inactivation of P4 by 20αHSD. There are various pathways for regulating the expression of 20αHSD, such as the FP and prolactinreceptor pathways.

branes and transmits signals to cells. It has been speculated that FP activates the PLC pathway via Gαq/11 and activates 20αHSD in luteal cells [9]. *Gq/11* cKO labor was prolonged without P₄ withdrawal [9]. The mice showed decreased expression of *Akr1c18* (a gene encoding 20αHSD) in the ovary [9]. Therefore, FP was presumed to stimulate *Akr1c18* expression and increase 20αHSD via the Gq/11 phospholipase C pathway [9].

Mamld1 is a gene that causes hypospadias in humans and its KO can delay parturition by approximately 50% [10]. In *Mamld1* KO, P₄ withdrawal does not exist, and Akr1c18 expression is decreased in the ovary [10]. Mamld1 suppresses *Akr1c18* by increasing Stat5 expression [10]. Therefore, these mice suffer from functional luteal regression disorders. Mamld1 is speculated to regress the corpus luteum by a different route to

There are many regressive pathways in the corpus luteum, which are extremely

The progesterone receptor (PR) has two different nuclear receptors, PRA and PRB, with different functions. PRA knockout mice are infertile, whereas PRB knockout mice suffer from mammary gland dysgenesis [37,38]. In humans, PRB suppresses Cx43, Cox2, OTR, and NFκβ pathways and maintains pregnancy [39]. However, PRA suppresses

Kruppel-like factor 9 (KLF9) is a transcription factor for PRA and PRB (Figure 4). KLP9 deficiency in early pregnancy suppresses PR expression and reduces fertility [40]. The serum P₄ level of *Klf 9* KO mice were the same as that of wild-type mice, but the gestation of *Klf 9* KO mice was longer than that of wild-type mice [11]. In these mice, PRB

complex. Further research is needed on the maintenance and disappearance of CL.

4.1.5. *Mastermind-like Domain-Containing 1* Knockout Mouse: *Mamld1* KO

*;Cre+* Mice: *Gq/11* cKO

4.1.4. *Gαqf/f;Gα11-/-*

the FP pathway [10].

PRB and induces labor [39].

*4.2. Abnormalities in P₄ Metabolism and Receptors*

4.2.1. *Kruppel-like Factor 9* Knockout Mouse: *Klf* 9 KO

#### 4.1.4. *Gαq f/f;Gα11*−*/*−*;Cre+* Mice: *Gq/11* cKO

Gαq/11 is a G protein that binds to receptors on the surface of activated cell membranes and transmits signals to cells. It has been speculated that FP activates the PLC pathway via Gαq/11 and activates 20αHSD in luteal cells [9]. *Gq/11* cKO labor was prolonged without P4 withdrawal [9]. The mice showed decreased expression of *Akr1c18* (a gene encoding 20αHSD) in the ovary [9]. Therefore, FP was presumed to stimulate *Akr1c18* expression and increase 20αHSD via the Gq/11 phospholipase C pathway [9].

#### 4.1.5. *Mastermind-Like Domain-Containing 1* Knockout Mouse: *Mamld1* KO

Mamld1 is a gene that causes hypospadias in humans and its KO can delay parturition by approximately 50% [10]. In *Mamld1* KO, P4 withdrawal does not exist, and Akr1c18 expression is decreased in the ovary [10]. Mamld1 suppresses *Akr1c18* by increasing Stat5 expression [10]. Therefore, these mice suffer from functional luteal regression disorders. Mamld1 is speculated to regress the corpus luteum by a different route to the FP pathway [10].

There are many regressive pathways in the corpus luteum, which are extremely complex. Further research is needed on the maintenance and disappearance of CL.

#### *4.2. Abnormalities in P4 Metabolism and Receptors*

The progesterone receptor (PR) has two different nuclear receptors, PRA and PRB, with different functions. PRA knockout mice are infertile, whereas PRB knockout mice suffer from mammary gland dysgenesis [37,38]. In humans, PRB suppresses Cx43, Cox2, OTR, and NFκβ pathways and maintains pregnancy [39]. However, PRA suppresses PRB and induces labor [39].

#### 4.2.1. *Kruppel-Like Factor 9* Knockout Mouse: *Klf* 9 KO

Kruppel-like factor 9 (KLF9) is a transcription factor for PRA and PRB (Figure 4). KLP9 deficiency in early pregnancy suppresses PR expression and reduces fertility [40]. The serum P4 level of *Klf 9* KO mice were the same as that of wild-type mice, but the gestation of *Klf 9* KO mice was longer than that of wild-type mice [11]. In these mice, PRB expression is presumed to increase owing to the decreased expression of PRA [11]. KLF9 is expressed in humans and decreased in patients with delayed delivery [41]. This factor may be involved in the mechanisms of pregnancy and delivery in humans [41]. *J. Dev. Biol.* **2022**, *10*, x 7 of 17 expression is presumed to increase owing to the decreased expression of PRA [11]. KLF9 is expressed in humans and decreased in patients with delayed delivery [41]. This factor may be involved in the mechanisms of pregnancy and delivery in humans [41].

**Figure 4.** The function of P₄ receptors. There are two types of PRs. PR-B suppresses CAPS and maintains pregnancy. PR-A induces labor induction by suppressing the PR-B. **Figure 4.** The function of P4 receptors. There are two types of PRs. PR-B suppresses CAPS and maintains pregnancy. PR-A induces labor induction by suppressing the PR-B.

In humans, the corpus luteum disappears during the second trimester of pregnancy. Therefore, P₄ withdrawal was not observed. The switching of progesterone receptors

Cyp11a1 (P450scc) is an enzyme that produces P₄ from cholesterol. Cyp11a1 knockout mice are fatal because of their inability to produce steroids and their underdeveloped fetal adrenal glands [43]. Chien et al., created a *Cyp11a1* Tg that overexpresses Cyp11a1 [12]. The mice showed delayed maturation of the corpus luteum during early pregnancy, causing implantation abnormalities, placental dysplasia, and fetal development abnormalities [12]. The serum P₄ of *Cyp11a1* Tg mice was similar to that of wildtype mice in early pregnancy, but remained high in late pregnancy [12]. *Cyp11a1* Tg mice

In autosomal dominant inheritance, crossing genetically modified females with male wild-type mice can determine whether the cause of delayed labor is the mother or the placenta/fetus. If the cause was the mother, the mice showed delayed delivery, while this does not occur if the cause was the placenta or the fetus. This can also be examined

Lung surfactant protein (Sp) is a lipoprotein produced in the lungs that assists breathing. There are four types of SP, A to D. SP-A is the most abundant and structurally similar to SP-D. SP is produced in the fetal lungs and then flows into the amniotic fluid. In humans, fetal lung maturity can be estimated by examining the SP in the amniotic fluid. SPA acts as the trigger of labor, because administration of Sp-A to the amniotic fluid of mice results in preterm birth [13]. *Sp A/D* dKO showed delayed delivery for approximately 12 h in the second and subsequent pregnancies [13]. Mice at 18.5 dpc had de-

frequently show delayed parturition [12].

by transplanting KO embryos into wild-type mice [16].

4.3.1. *Surfactant Protein A and D* Double-Deficient Mice: *Sp A/D* dKO

*4.3. Fetal Factors*

4.2.2. *Cytochrome p450 Family 11a1*-Overexpression Mouse: *Cyp11a1* Tg

In humans, the corpus luteum disappears during the second trimester of pregnancy. Therefore, P4 withdrawal was not observed. The switching of progesterone receptors from PRB to PRA in late pregnancy is presumed to trigger labor induction [39,42].

#### 4.2.2. *Cytochrome p450 Family 11a1*-Overexpression Mouse: *Cyp11a1* Tg

Cyp11a1 (P450scc) is an enzyme that produces P4 from cholesterol. Cyp11a1 knockout mice are fatal because of their inability to produce steroids and their underdeveloped fetal adrenal glands [43]. Chien et al., created a *Cyp11a1* Tg that overexpresses Cyp11a1 [12]. The mice showed delayed maturation of the corpus luteum during early pregnancy, causing implantation abnormalities, placental dysplasia, and fetal development abnormalities [12]. The serum P4 of *Cyp11a1* Tg mice was similar to that of wild-type mice in early pregnancy, but remained high in late pregnancy [12]. *Cyp11a1* Tg mice frequently show delayed parturition [12].

#### *4.3. Fetal Factors*

In autosomal dominant inheritance, crossing genetically modified females with male wild-type mice can determine whether the cause of delayed labor is the mother or the placenta/fetus. If the cause was the mother, the mice showed delayed delivery, while this does not occur if the cause was the placenta or the fetus. This can also be examined by transplanting KO embryos into wild-type mice [16].

#### 4.3.1. *Surfactant Protein A and D* Double-Deficient Mice: *Sp A/D* dKO

Lung surfactant protein (Sp) is a lipoprotein produced in the lungs that assists breathing. There are four types of SP, A to D. SP-A is the most abundant and structurally similar to SP-D. SP is produced in the fetal lungs and then flows into the amniotic fluid. In humans, fetal lung maturity can be estimated by examining the SP in the amniotic fluid. SPA acts as the trigger of labor, because administration of Sp-A to the amniotic fluid of mice results in preterm birth [13]. *Sp A/D* dKO showed delayed delivery for approximately 12 h in the second and subsequent pregnancies [13]. Mice at 18.5 dpc had decreased expression of CAPS in the uterine muscle and decreased secretion of inflammatory cytokines from macrophages in the amniotic fluid [13]. The hypothesis that the Sp of the fetal lung can be the starting point for labor induction is particularly important since the fetus itself is involved in determining the time of labor.

#### 4.3.2. *Steroid Receptor Coactivator 1 and 2* Double-Deficient Mice: *Src1/2* dKO

Steroid receptor coactivator 1 and 2 (Src1/2) (transcription factor of Sp-A) knockout mice showed prolonged delivery by 38 h [14]. These mice exhibited a reduction in Sp-A, PGF2α, Cx43, and OXTR, a restricted NF-κB pathway, and P4 withdrawal did not occur [14]. Interestingly, the higher the proportion of *Src1/2* KO fetuses, the higher the serum P4 concentration in late pregnancy [14].

In these mice, platelet-activating factor (PAF) in the fetal lung and amniotic fluid was also reduced [14]. PAF is involved in several physiological events, such as platelet aggregation, inflammation, and anaphylaxis, and is produced in the fetal lung; its concentration in the amniotic fluid increases during late pregnancy [44]. Therefore, PAF was presumed to play an important role in the onset of labor and preterm delivery. Administration of PAF to SRC-1/2 dKO increased NF-κB pathway activation and CAPS expression in the myometrium and improved delayed labor [14]. The results of this study suggest that PAF and SP-A may contribute to delivery.

#### *4.4. Placental Factors*

When the placenta is the cause, abnormal placental development often occurs, such as in Nrk KO and *Sirh7/Ldoc1* KO mice [15,16]. The lentiviral vector (LV) or tetraploid chimera can determine whether the cause is the placenta or fetus. When LV is administered to mouse blastocysts, the gene is transferred only to the placenta [45]. LV rescue delayed parturition due to placental factors. The tetraploid chimera utilizes the property that tetraploid embryonic cells contribute only to the placenta [46]. In chimeric mice established using wild-type diploid embryos and nuclear-transplanted cloned tetraploid embryos, delayed labor occurs when the cause is the placenta, but not when the cause is the fetus. In chimeras of wild-type tetraploid embryos and diploid embryos of nuclear-transplanted clones, the opposite result was observed.

#### 4.4.1. *Solute Carrier Organic Anion Transporter Family Member 2A1* Knockout Mouse: *Slco2a1* KO

*Slco2A1* encodes OATP2A1, an organic anion-transporting polypeptide. OATP2A1 is a PG transporter that takes up extracellular PGs. Mating between *Slco2A1* KO mice resulted in prolonged labor in approximately 50% of mice [4], and this did not occur after mating with wild-type male mice. This suggests that the cause is fetal or placental factors, hence P4 withdrawal does not occur [4]. The level of mPL2 is elevated in the placenta [4]. mPL-2 is a member of the prolactin family and is known to have a luteotropic effect. In cultured placental cells of the sponge layer (one of the layers of the placenta), PGE2 enhances the secretion of mPL-2 [4]. *Slco2a1* KO maintains the corpus luteum by mPL-2 from the sponge layer of the placenta and causes delayed labor [4].

#### 4.4.2. *Sushi Ichi Retrotransposon Homolog 7/Leucine Zipper, Downregulated Cancer 1* Knockout Mice: *Sirh7/Ldoc1* KO

*Sushi ichi retrotransposon homolog 7/leucine zipper* (*Sirh7/Ldoc1*) is a member of the long terminal repeat (LTR) retrotransposon-like gene family [15]. *Sirh7/Ldoc1* is an X-linked gene that is unique to eutherians; therefore, it is highly conserved in both mice and humans. *Sirh7/Ldoc1* KO showed a delayed delivery of approximately 1–4 days in 49% of mice due to residual P4 at 18.5 dpc [15]. RU486 and ovariectomy improved prolonged delivery, suggesting that P4 was the cause of prolonged delivery [15]. The mice had elevated levels of mPL1 as well as placental P4 production and exhibited a delayed switch from mPL1 to mPL2 during mid-pregnancy [15]. Similar to mPL-2, mPL-1 also has a luteotropic function. MPL-1 has been proposed to be one of the causes of delayed delivery of *Sirh7/Ldoc1* KO [15]. The *Sirh7/Ldoc1* KO placenta also had an irregular boundary between the sponge and labyrinth layers.

#### 4.4.3. *Nik-Related Kinase* Knockout Mouse: *Nrk* KO

In 1996, *Nik-related kinase* (*Nrk*) was named by Kanai-Azuma et al. [47]. Nrk is another X-linked gene that is highly conserved in mice and humans, and its KO mice exhibit three phenotypes: overgrowth of the spongy layer of the placenta, high frequency of breast tumors, and prolonged parturition [16].

Wild-type dams with transplanted *Nrk* KO blastocysts showed delayed parturition, which suggests that the *Nrk* KO fetus or placenta is the cause of delayed delivery [16]. The P4 and estrogen levels of *Nrk* KO mice were about the same as those of the wild-type at 17.5 and 19.5 dpc [16]. The report postulated that an unidentified soluble factor from the spongy layer protracts parturition [16]. However, they could not identify leading candidates.

#### *4.5. Autoimmune Disorder*

Toll-like receptors (TLRs) are receptors on surface of cells such as macrophages and dendritic cells, that bind to different ligands. TLRs recognize pathogens and activate innate immunity. There are 1 to 13 types, each of which is a pattern-recognition receptor that binds to different ligands.

The model of preterm delivery of lipopolysaccharide (LPS) into mice at midgestation has been previously used. TLRs induce labor by activating the NF-κβ pathway and increasing CAPs by recognizing inflammatory substances such as LPS [48]. Therefore, TLR is presumed to be a trigger for pathological preterm delivery due to bacterial infection. TLRs bind to non-inflammatory substances such as Sp-A and damage-associated molecular

patterns (DAMPs) emitted by stress, such as uterine stretch [49]. TLRs are also involved in the onset of normal labor.

#### 4.5.1. *Toll-Like Receptor 2*-Deficient Mice: *Tlr2* KO

Compared with wild-type mice, *Tlr2* KO mice showed a significant delay in the timing of labor during the first pregnancy, and a decrease in the amniotic fluid macrophages and Cx43 expression in the myometrium [13]. Sp-A/D diffuses from the fetal lung into the amniotic fluid, and binds to TLR2 in amniotic fluid macrophages [13]. Amniotic fluid macrophages secrete IL1b and IL6, which in turn activate the NF-κB pathway in the uterus and induce labor [13].

#### 4.5.2. *Toll-Like Receptor 4*-Deficient Mice: *Tlr4* KO

Since TLR4 binds to LPS and DAMPs, it is associated with both bacterial infection and aseptic preterm delivery [48,49]. The average gestation length of *Tlr4* KO mice was extended by 13 h [17]. The timing of FP, OTR, and Cx43 expression was delayed [34], and P4 withdrawal did not occur [17]. Administration of a TLR4 antagonist to wild-type mice resulted in delayed delivery [17]. Therefore, TLR4 is presumed to play an important role in gestation.

#### 4.5.3. *Interleukin 6* Null Mutant Mice: *Il6* KO

*Il6* KO mice delivered 24 h after wild-type mice, yet the P4 level of the mice was equivalent to that of the wild-type [18]. *Il6* KO delayed the expression of FP and Cox2 in the myometrium, while administration of IL6 to *Il6* KO improved delayed delivery. IL6 is involved in the pathway after P4 withdrawal and induces PG [18].

#### *4.6. Uterine Contractile Dysfunction*

Uterine contractile dysfunction refers to the lack of intrauterine pressure sufficient to push a fetus out of the uterus. It is caused by a weak contraction force of the uterine muscles and impairment of coordinated contractions. The intrauterine pressure should be measured directly to accurately observe labor.

There are in vitro and in vivo methods for measuring uterine contractile force. The contractile force of the removed uterine muscle can directly be measured in vitro [33]. Pure muscle contraction force can be measured, but the reproducibility of the in vivo environment is poor.

In vivo, there are two methods: surgical implantation of a pressure transducer and transvaginal insertion into the uterus. For surgical implantation, a pressure transducer is surgically implanted in the myometrium after laparotomy under anesthesia [50]. The advantage of this method is that intrauterine pressure can be continuously observed without anesthesia from pregnancy to delivery. However, special techniques are required, and the operation may cause fetal loss [50]. The transducer insertion method, which has also been used in humans, is less invasion of the dams [51]. This method had three disadvantages: limited insertion time, anesthesia was required at the time of the insertion, and the position of the inserted transducer was not identified [51].

## 4.6.1. *Connexin 43fl/fl:SM-CreERT2* mice: *Sm-CreERT2* KO

Delayed delivery was observed in 82% of mice [19]. Uterine contractions were not directly measured. The author reported that the cause of the abnormal delivery was insufficient contraction due to the loss of coordinated contraction of the uterine muscle [19].

## 4.6.2. *Kcnn3tm1Jpad/Kcnn3tm1jpad*: *Sk3T/T* mice

The small-conductance Ca2+-activated potassium channel isofrom-3 (SK3 channel)is a selective potassium channel. *SK3T/T* mice overexpress SK3 channels [20]. The contractile force in these mice was attenuated in vivo [20]. Although the mechanism is unclear, the investigation showed that mice also experience abnormal labor due to weak uterine

contractions [20]. It has been reported that the SK3 channel is involved in delivery even in humans [52]. This research is expected to be applied to the treatment of preterm births.

#### *4.7. Poor Cervical Ripening*

Almost 90% of the cervix is composed of an extracellular matrix, such as collagen and glycosaminoglycans. Cervical ripening is the decomposition of collagen and glycosaminoglycans during late pregnancy. In humans, cervical ripening is evaluated using pelvic examination. In mice, cervical ripening is often evaluated using pathological and biomechanical examinations. There are three pathological examinations: Masson trichrome staining (evaluation of collagen degradation), immunostaining (evaluation of neutrophils and monocytes), and Mayer's mucicarmine (evaluation of epithelial mucus volume) [22–24]. The pressure generated can be determined by mechanically dilating the removed cervix [22,24].

#### 4.7.1. *5α-Reductase Type 1* Knockout Mice: *5αR1* KO

5α-reductase (5αR1) is an enzyme involved in the production of dihydrotestosterone and the decomposition of testosterone. Thirty-three percent of *5αR1* KO mice did not start delivery [23], while 25% of mothers had died [23]. These mice showed a decrease in blood P4, but administration of RU486 improved delayed labor [23]. Uterine contractions of *5αR1* KO mice were measured by surgical transducer implantation and were not different from those of wild-type mice [22]. The elasticity of the cervix decreased and pathologically, the cervix was poorly mature [22]. It has been speculated that the amount of P4 in the cervix is increased, resulting in poor cervical ripening and delayed delivery [22].

#### 4.7.2. *TgN(hApoB) 1102SY* Line: *Tg/Tg* Mouse

The *Tg/Tg* mouse is a strain that occurs by the insertion, by chance, of the human apolipoprotein B gene [24]. Mice exhibited delayed labor when the mother was a *Tg/Tg* mouse, regardless of the fetal genotype [24]. P4 withdrawal was prolonged by one day compared to wild-type mice; administration of a P4 receptor antagonist (ZK98299) and ovariectomy did not improve prolonged delivery [24]. Uterine contractions were similar to those observed in wild-type mice, the cervix was not elastic, and pathological evaluation showed poor cervical ripening [24].

#### 4.7.3. *Anthrax Toxin Receptor 2* Knockout Mice: *Antxr2* KO

Antxr2 has been postulated to be involved in the proliferation and development of the vascular endothelium, but its function is unknown. *Antxr2* KO mice exhibited a sharp drop in P4 but did not deliver [21]. The cervix was immature with no collagen degradation, while the myometrium had lost cricothyroid and longitudinal muscle cells [21]. The cause of delayed labor in the mice was poor cervical ripening and uterine contraction failure.

#### *4.8. Delayed Implantation*

A common method of observing the implantation time is to inject a pigment such as Chicago blue into the tail vein 5 and 6 days after confirmation of the vaginal plug and direct observation of the implantation site [25]. If implantation is delayed, the number of fetuses changes. Delayed implantation often causes a reduction in litter size, fetal dysgenesis, and abnormal spacing.

#### 4.8.1. *Cytosolic Phosphatase A2* Mutant Mouse: *Pla2g4a* KO

Cytosolic phosphatase A2 (Pla2g4a) is an enzyme that produces PGs. *Pla2g4a* KO mice showed prolonged delivery, decreased fetal population, and increased stillbirth rate [26]. The number of implantations in mice increased at 6 dpc compared to that at 5 dpc [25]. The experiment suggested that the implantation period of the mice was delayed [25]. Administration of PGI2 and PGE2 to mice improved delayed implantation and prolonged delivery [27].

#### 4.8.2. *Lysophosphatidic Acid Receptor 3*-Deficient Mouse: *LPA3* KO

LPA is a lipid that has a signaling effect and is present in the ovaries, testes, and uterine muscles. There are six types of LPA receptors that have various functions in the reproductive process. Mating between *LPA3* KOs resulted in reduced litter size, delayed implantation, abnormal spacing, and prolonged delivery. In these mice, Cox2, PGE2, and PGI2 are decreased in the uterus [28]. Administration of PGE2 + PGI2 to mice improved delayed implantation and prolonged parturition; similar to *Pla2g4a* KO, delayed implantation of *LPA3* KO is caused by a decrease in PGs [28].

In cattle, LPA promotes the secretion of P4 and PGE2 and maintains pregnancy [53]. Therefore, LPA3 may be directly involved in prolonged parturition. It has been reported that in humans, LPA is associated with the maintenance of pregnancy and preterm birth [54]. Therefore, future studies on LPA are required.

#### *4.9. Unknown Cause*

There are some mice for which the cause is unknown, such as when a delay in labor occurs by chance.

#### 4.9.1. *Erk1 and Erk2 Conditional* Knockout Mouse: *Erk1/2* cKO

Extracellular signal-regulated kinase (ERK) is an intermediate of the MAPK pathway. The MAPK pathway is involved in cell differentiation and cell division. *Erk1* KO was reproductive with no apparent abnormalities, but *Erk2* KO was fatal [55]. *Erk1/2* cKO mice showed abnormal placental development, delayed delivery on average of approximately 2.3 days, and death of offspring [29]. This may have been due to the abnormal placental development [29]. However, the cause of this phenomenon is unknown.

#### 4.9.2.  *Leptin* KO mice were infertile because of an abnormality in the hypothalamic–pituitary system [56]. One study recovered pregnancy and childbirth by administering leptin [30]. *Leptin* KO mice that received leptin throughout gestation increased food intake and became obese compared to the mice that received leptin after that point [30]. The mice showed delayed parturition for approximately 1–3 days; the authors claimed that the cause may have been abnormal obesity [30]. However, to date, no detailed studies have been conducted.

#### 4.9.3. *MT-mER* Mouse

In 1994, Davis et al. published a study in which the estrogen receptor (ER) was forcibly expressed throughout the mouse body. These mice expressed ER in cells expressing the metallothionein-1 promoter. *MT-mER* mice showed delayed delivery for 4 days or more [31]. The *MT-mER* mouse strongly expressed ER in the gonads, but its expression in other tissues was weak. The authors concluded that the cause may have been the difference in the time and place of the ER [31]. However, there is no further description of this cause.

#### 4.9.4. *Activin/Inhibin βB Subunit-Deficient* Mouse: *Activin/Inhibin βB Subunit* dKO

Activin and inhibin are peptides produced in the gonads, pituitary gland, and placenta. The β subunit of activin is common to activin. Previously, activin was presumed to be a substance that induces the mesoderm. However, the *activin/inhibitor βB subunit* dKO showed morphological abnormalities only in the eyelids [32]. The mice showed mildly delayed labor of approximately half a day to a day, while in rats, the administration of activin to the pituitary gland increases blood oxytocin concentrations. Therefore, the authors concluded that a decrease in oxytocin may have been involved [32].

#### **5. How to Identify the Cause of Delayed Labor in Mice**

If genetically modified mice exhibit delayed labor, we recommend investigating the items presented in Table 2. This list can help to identify the cause of delayed labor more accurately and efficiently.

**Table 2.** List for finding the cause of delayed delivery. If genetically modified mice exhibit delayed labor, we recommend investigating the items.

#### *5.1. Measurement of the Landing Time*

Delayed implantation often results in fetal stunting and reduced litter size. Since delayed-labor mice often miscarry or absorb their fetus in utero, measurement of landing time is useful for measuring the true litter size. Some studies have reported that small litter sizes tend to delay delivery in some mouse strains [57].

#### *5.2. Investigation of the Following Factors: Dam, Fetus, and Placenta*

If the dam is abnormal, mice will exhibit delayed delivery, regardless of the sire genotype. If the placenta or fetus is abnormal, wild-type female transplanting KO embryos, such as *Nrk* KO, will exhibit delayed delivery (16). Investigating the expression of the target gene in the fetus and placenta is also useful to determine whether the cause is the fetus or placenta. Placental dysplasia often occurs if the placenta is the cause. In addition, the method using the above-mentioned LV vector and tetraploid chimera is also useful.

#### *5.3. Presence or Absence of P4 Withdrawal during Late Pregnancy*

Abnormal luteolysis is the most common cause of delayed labor; examining P4 at 16.5 dpc and 18.5 dpc is especially useful. Luteolysis can also be examined by observing the ovaries pathologically.

#### *5.4. Evaluation of Uterine Contractions and Cervical Ripening*

It is also important to assess uterine contractions and cervical ripening in late pregnancy to directly assess the status of labor. In delayed delivery, there must be an abnormality in either, or both.

#### *5.5. Comparison of Wild-Type Mice*

The gestation period of mice varies depending on the strain [58]. However, the variability within the strain is small [58]. Comparison with wild-type mice is necessary to accurately prove the delayed labor. In some mice strains, fetal count and gestational duration are inversely correlated [57,58]. Extension of gestation due to a decrease in the number of fetuses should also be considered.

#### **6. Conclusions and Future Perspectives**

The etiology of delayed parturition is overexpression of the pregnancy-maintenance mechanism or deficiency of the labor-induction mechanism. Overexpression of the pregnancymaintenance system is attributed to the excessive secretion of humoral factors, such as P4 and

PL, which have a pregnancy maintenance effect [4,33]. Suppression of the labor-induction mechanism is caused due to uterine contraction disorder and/or cervical ripening [20,21,24,59]. Understanding both mechanisms is important for elucidating the mechanisms of pregnancy and delivery.

The biggest difference between rodents and humans in pregnancy and labor is the trigger of labor. In rodents, P4 withdrawal triggers labor. In contrast, in humans, P4 withdrawal does not occur in late pregnancy, suggesting that the trigger for labor may be a PR subtype change [39,42]. P4 administration to high-risk patients with preterm birth reduces preterm birth rates [60]. The human corpus luteum disappears at approximately 20 weeks of gestation, and P4 is produced in the placenta. Luteolysis in humans does not appear to be involved in delayed labor. Mice with delayed parturition due to abnormal luteolysis may be useful to understand human early pregnancy maintenance.

Human PL, which is only one type of PL, does not affect pregnancy [61]. There are several types of mouse PLs, and mPL-1 and mPL-2 maintain the corpus luteum and pregnancy [4,15]. The functions of other PLs are unknown, and further research is required.

However, few studies have evaluated the effects of cervical ripening and uterine contractions in mice. In humans, cervical ripening is evaluated on the basis of the degree of cervical dilatation. Uterine contractions are evaluated using cardiotocography. Similarly, examining these two factors in mice will help to clarify the cause of labor disorders.

The length of the gestation period is controlled not only by the maternal signals but also by fetal and placental signals [4,13–15]. The amount of mPL changes with placental and fetal growth [4,15]. The concentration of Sp-A in amniotic fluid increases with fetal maturation and promotes labor [13]. In humans, corticotropin-releasing hormone secreted from the fetal hypothalamus promotes labor induction [42]. The mother controls these two systems by sensing fetal development.

Studies on preterm birth in humans, which has a greater effect on infants, are more abundant than those on delayed labor. However, findings from studies on delayed delivery can also be applied to the prevention and treatment of preterm births. For example, *Fp* KO and Cox1 KO has been used to study cox-selective inhibitors [35]. P4-related delayed delivery mice such as *Klf 9* KO and *Cyp11a1* Tg provide clues to the potential of P4 as a treatment for preterm birth. P4 has been used in the treatment of imminent preterm births in humans [60]. Delayed delivery mice due to autoimmune disorders may be associated with surrogate mothers and pregnancy due to uterine transplantation. In humans, delayed labor has a family accumulation and is likely to involve a genetic background. In the future, if the genetic background of women who are prone to delayed delivery is clarified, it may be possible to find an association with the gene related to these mice.

Elucidation of the causes of delayed parturition in humans and animals is essential to understand the mechanisms underlying pregnancy and delivery. Summarizing the causes and examination methods of delayed delivery in mice in this review will help to focus on future research and provide new insights into novel treatment strategies.

**Author Contributions:** Writing—original draft preparation, H.Y.; writing—review and editing, N.M. and M.K.-A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI (grant numbers 21H02387 and 18H02361).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** The authors thank the Center for Experimental Animals, Tokyo Medical and Dental University for supporting our research. The authors also thank H. Itoh, Y. Hirate, T. Endo, and F. Ishino for their critical reading.

**Conflicts of Interest:** The authors declare no conflict of interest directly relevant to the content of this article.

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