**4.1 Embryo storage**

*Embryology - Theory and Practice*

yields distinct results.

with the idea to overcome this problem.

**3.4 Artificial shrinkage**

cryopreservation process on the embryos. However, freezing an embryo also does not allow the inspection of other types of damages, which occur at the molecular level—DNA damage, altered gene expression, and protein function. These alterations require specific molecular biology methods in order to be assessed, and their impact on the embryo is not that clear. In contrast, when the survival rate after freezing is being assessed, we must say that this approach is straightforward and

When talking about the embryo survival rate nowadays, with the constantly improving cryopreservation techniques, a survival rate of more than 90% or even 95% could be observed depending on the vitrification protocol, carrier, embryologist experience, thawing process, and other variables. While this rate is indeed very high, unfortunately there is still a risk that a frozen embryo would not survive after thawing. The survival rate is different for the different stages at which the embryos are frozen. In fact, it is still unknown at which stage of development, the embryos are most suitable for freezing and therefore further research is needed. Moreover, the stage at which the embryo is frozen is connected to different types of cryodamage. At the PN stage, there is evidence that embryos may suffer integrity damage of the pronuclei after cryopreservation [39], and therefore, their developmental potential could be significantly impaired. At the cleavage stage, there is evidence of zona pellucida damage [40] and changes in metabolism [41]. Reduced implantation rates have been observed after the loss of blastomeres in day 2 grade 1 embryos with <10% fragmentation in a study with 363 thawing cycles [42]. Blastocyst cryopreservation represents a demanding task due to its size and the presence of blastocoel. Formation of ice crystals is probably the main factor affecting blastocysts survival rates, since the blastocoel contains large amounts of water. In order to reduce the negative effects of the blastocoel on survival rates, it was proposed that blastocysts should be frozen at the contraction stage or the blastocoel should be collapsed artificially before freezing [43] which can be done, for example, with an ICSI pipette. Despite all these difficulties, blastocyst survival rates seem to be higher compared to early cleavage embryos, as shown in a study by Cobo et al., where 6019 embryos were vitrified using Cryotop as a carrier [44]. In the study, 97.6% day 6 embryos survived compared to 95.7% day 5 embryos, 94.9% day 2, and 94.2% day 3 embryos. As a consequence of the freezing procedure, zona pellucida may become thicker, which could affect the implantation ability of the embryo, and this is why assisted hatching is performed

In a well expanded blastocyst, the large blastocoel may interfere with the permeation of CPAs during the vitrification procedure which in turn would decrease the survival rates after thawing. Mukaida et al. back in 2003 stated that blastocyst survival rate after vitrification/warming correlate negatively with the expansion of the blastocoel [45]. Artificial shrinkage (AS) of the blastocoel by different methods—laser pulse, microneedle, micropipetting, and 29-gauge needle was developed with the idea of overcoming this obstacle. A study by Gala et al. in 2014 encompassing 185 warming cycles reported a higher survival rate after AS (99.0%) compared to 91.8% survival rate in the control group without AS [46]. Darwish and Magdi in 2016 assessed clinical pregnancy rates, implantation rates, and blastocyst survival rates in more than 400 patients, divided into two groups—untreated expanded blastocysts and blastocysts undergone AS by laser pulse [47]. The study group found that after AS, there was significantly increased survival rates (97.3 vs. 74.9%),

implantation (39.1 vs. 24.5%), and clinical pregnancy rates (67.2 vs. 41.1%).

**110**

The main indication of embryo cryopreservation is for storage purposes. We have reviewed the basic cryopreservation methods. Our interpretation of this thought is: however, no matter how much they have improved recently, they could not be successful unless proper storage and thawing of the frozen objects are carried out. After freezing, the embryos are placed in storage tanks which are filled with liquid nitrogen. There is substantial variety of storage tanks and automated storage systems have been recently introduced, which offer optimal storage conditions and safety. It is not known for how long embryos can be stored in liquid nitrogen without affecting their potential, because embryo freezing was developed during the 1980s, which means that the longest time an embryo has been stored is around 35 years, and there is little chance that patients would come back for them after such a long period. There are some differences in the laws regarding embryo cryopreservation, and therefore, embryo storage limit varies between countries, for example, 3 years in Portugal, 5 years in Denmark, Norway, and many other countries, 10 years in Austria and Australia, 55 years in the UK, while in Venezuela, embryo freezing is prohibited [48]. However, a storage time of 5–10 years is most commonly observed.

Keeping embryos in liquid nitrogen raises some concerns about the safety of the procedure. First of all, liquid nitrogen that is used by the IVF laboratories has chemical standards of purity, not biological. That means that there might be some kind of contamination and we should think if there is any way to sterilize this liquid nitrogen. Bielanski et al. describes the potential for viral transmission from experimentally contaminated liquid nitrogen to vitrified embryos, stored in open freezing containers [49]. From a pool of 83 batches, 21% were positive for viral association. In contrast, vitrified embryos in sealed plastic cryovials and straws were free from viral contamination. These data support that sealing of the freezing container might prevent exposure to contaminants; however, that does not mean 100% safety, as the seal can be damaged. This information leads us back to the idea of sterilizing the liquid nitrogen. However, if this is possible, it should be evaluated if it can be applied practically.

Regarding the thawing process, it is very similar in both vitrification and slow freezing technique. The idea is to submerge the frozen object into a solution prewarmed at 37°C which is the core temperature in human body. Closed systems are usually plunged into water baths, while open systems could be put directly into a prewarmed medium. As mentioned before, CPAs are used for the cryopreservation of embryos and those CPAs must be removed during the thawing process and also

the cells must be rehydrated. This happens by incubating the embryo in decreasing concentrations of the CPAs and increasing concentrations of water.

#### **4.2 Embryo freezing in clinical practice**

Increasing the reliability of the embryo freezing/thawing procedure has enabled a wider application of the method in assisted reproduction.

Indications to why we freeze embryos and do not switch to fresh ET are: OHSS risk and significant increase in progesterone on the day of HCG administration during stimulation. Progesterone levels above 1.5 ng/ml in patients in advanced reproductive age are associated with lower success rates with fresh ET [50], low responders, inadequacy of the cervix, requiring hysteroscopy, areas affected with Zika virus, or the analysis of endometrial implantation "window."

When patients have cryopreserved embryos it provides them with additional embryo transfers thus increasing the chances of achieving a pregnancy from a single stimulated cycle. This also means that further cycles of hormonal stimulation are not required. Having supernumerary embryos frozen also facilitates the so-called single embryo transfer which helps in avoiding multiple pregnancies. Additionally, freezing of embryos enables patients to decide when is the most appropriate time to start their conceiving efforts.

For patients suffering from cancer, embryos can be frozen before the patient starts cancer treatment, which is done because chemotherapy may negatively affect one's reproductive ability, and this may be their only option of having offspring.

Freezing embryos also gives the opportunity for genetic testing (PGD/PGS) which is essential in couples with recurrent pregnancy loss and older women who possess higher risk for chromosomal abnormalities.

The frozen embryos may be donated for scientific purposes or they may be used in a donor program if permitted by law, which is also an advantage.

Embryo cryopreservation has many benefits; however, there are some potential risks, and we hope that they will be eliminated in the near future, as this area of research is further developed.

#### **5. Present and future perspectives**

Looking at SART statistics, we can find that the number of frozen embryo transfers has increased more than 2.5 times over the 2004/2013 period. Accordingly, for 2004, we have 15,474 frozen embryo transfers vs. 40,015 FETs for 2013, while the number of fresh embryo transfers remains relatively unchanged, respectively, for 2004—2087,089 fresh ET and 87,045 for 2013. On the other hand, there has been a significant increase in the success rate of FET compared to fresh ET. In 2004, the average success rate of FET was 27.8%, while that of fresh ET was at 33%. In 2013, the average success rate of FET was 40.1% and of fresh ET it was 36.3% [51]. Another interesting fact supporting the increased success rate of FET is data from the Japanese National Registry on the number of ART procedures and their success rate based on births. The most significant is in 2014 when the children born after ART were 47,292, with 77.4% of them being after FET [52].

But when discussing frozen embryos and their clinical practice use, the first question arising is the risk to the offspring, when we are applying that technology. The risk for at least one major congenital abnormality of the children born after FET was not increased compared to the children born after fresh ET [53]. On the contrary, the increase in blastogenesis birth defects appears greater for fresh ET than for FET, and the frequency of Down syndrome was statistically more likely

**113**

*The Present and Future of Embryo Cryopreservation DOI: http://dx.doi.org/10.5772/intechopen.80587*

after a spontaneous pregnancy [54, 58].

etic reprogramming?

bility in ART children [62].

(n = 29,760 cases).

is performed.

part reflect parental subfertility [63].

the epigenetic aberrations induced by IVF or ICSI [64].

after 39 years, 34.9% and 22.7%, respectively [68].

in the children born after fresh ET than FET [54]. On the other hand, FET has advantages in that it decreases the risk of low birth weight (LBW) (<2500 g), very low birth weight (<1500 g), very preterm birth (VPTB) (<32 gw), placenta previa, small, placental abruption, gestational age, antepartum hemorrhage, ectopic pregnancy, and perinatal mortality [55, 56]. If we look at the weight indicator of the newborn after ART, the results of the studies show that ART-born children have a lower weight, and the risk of LBW in newborns is 2.6 higher than those spontaneously conceived [57]. However, after FET, children are being born 90–190 g heavier than those after fresh ET. This brings them statistically closer to the children born

Here a question arises: is the change in the weight of the newborn due to epigen-

Human studies have shown that different ART methods, such as ovarian stimulation and supraphysiological levels of sex steroid hormones, culture media, and embryo cryopreservation, may be associated with intrauterine growth change, resulting in altered birth weight profiles, which may be caused by epigenetic modifications [59]. Furthermore, some reports indicate that children conceived by ART have an altered lipid profile, fasting glucose, body fat distribution, and cardiovascular function [60, 61]. ICSI/IVF children showed a significantly decreased DNA methylation at birth. Studies suggest an impact of ICSI on the offspring's epigenome(s), which may contribute to phenotypic variation and disease suscepti-

Differences in DNA methylation between IVF and non-IVF twins on a genomewide scale and their results show evidence for epigenetic modifications that may in

The methylation profiles of ART and IUI newborns were different from those of naturally conceived newborns. But the profiles of ICSI-frozen (FET) and IUI infants were very similar, suggesting that cryopreservation may eliminate some of

In addition to the above mentioned advantages, frozen ET, compared to fresh ET, also has some drawbacks, such as: macrosomia (OR 1.64), large for gestation age (OR 1.54), post-term birth (OR 1.40), and placenta accrete (OR 3.20) [65]. In cases with preeclampsia, the risk after FET in twin pregnancies is 19.6% with risk difference 5.1% in fresh ET, while in singleton pregnancies, the risk is 7.0% after FET with risk difference 1.8% in fresh ET [66]. Probably, the main reason for the increase of these complications is the protocol of the endometrium preparation for FET. Analyzing the Japanese assisted reproductive technology registry in 2014, Saito et al. [67] found that pregnancies following FET after hormone replacement cycle (n = 75,474 cases) have significant higher risks of hypertensive disorders and placenta accrete compared to FET in natural ovulatory cycle

Due to the many advantages of FET compared to fresh ET, a number of clinics have begun to implement the "freeze all" policy, so they do not perform ET in the stimulated cycle, but rather freeze all embryos. In a subsequent or later cycle, with or without HRT (hormone replacement therapy), the embryos are thawed, and ET

The first publications of results show optimism, because the FET success rate is significantly higher than that of the classic ET approach. In support of these results, it was reported by Lopez et al. in a retrospective study with 1697 IVF cycles that the FET versus fresh ET rate for women up to 39 years was 44.5–38% and for women

Similar results were presented from another retrospective study made by Santistevan et al. [69]. It encompassed more than 16,000 IVF cases, with

#### *The Present and Future of Embryo Cryopreservation DOI: http://dx.doi.org/10.5772/intechopen.80587*

*Embryology - Theory and Practice*

start their conceiving efforts.

research is further developed.

**5. Present and future perspectives**

**4.2 Embryo freezing in clinical practice**

the cells must be rehydrated. This happens by incubating the embryo in decreasing

Increasing the reliability of the embryo freezing/thawing procedure has enabled

Indications to why we freeze embryos and do not switch to fresh ET are: OHSS risk and significant increase in progesterone on the day of HCG administration during stimulation. Progesterone levels above 1.5 ng/ml in patients in advanced reproductive age are associated with lower success rates with fresh ET [50], low responders, inadequacy of the cervix, requiring hysteroscopy, areas affected with

When patients have cryopreserved embryos it provides them with additional embryo transfers thus increasing the chances of achieving a pregnancy from a single stimulated cycle. This also means that further cycles of hormonal stimulation are not required. Having supernumerary embryos frozen also facilitates the so-called single embryo transfer which helps in avoiding multiple pregnancies. Additionally, freezing of embryos enables patients to decide when is the most appropriate time to

For patients suffering from cancer, embryos can be frozen before the patient starts cancer treatment, which is done because chemotherapy may negatively affect one's reproductive ability, and this may be their only option of having offspring. Freezing embryos also gives the opportunity for genetic testing (PGD/PGS) which is essential in couples with recurrent pregnancy loss and older women who

The frozen embryos may be donated for scientific purposes or they may be used

Embryo cryopreservation has many benefits; however, there are some potential risks, and we hope that they will be eliminated in the near future, as this area of

Looking at SART statistics, we can find that the number of frozen embryo transfers has increased more than 2.5 times over the 2004/2013 period. Accordingly, for 2004, we have 15,474 frozen embryo transfers vs. 40,015 FETs for 2013, while the number of fresh embryo transfers remains relatively unchanged, respectively, for 2004—2087,089 fresh ET and 87,045 for 2013. On the other hand, there has been a significant increase in the success rate of FET compared to fresh ET. In 2004, the average success rate of FET was 27.8%, while that of fresh ET was at 33%. In 2013, the average success rate of FET was 40.1% and of fresh ET it was 36.3% [51]. Another interesting fact supporting the increased success rate of FET is data from the Japanese National Registry on the number of ART procedures and their success rate based on births. The most significant is in 2014 when the children born after

But when discussing frozen embryos and their clinical practice use, the first question arising is the risk to the offspring, when we are applying that technology. The risk for at least one major congenital abnormality of the children born after FET was not increased compared to the children born after fresh ET [53]. On the contrary, the increase in blastogenesis birth defects appears greater for fresh ET than for FET, and the frequency of Down syndrome was statistically more likely

concentrations of the CPAs and increasing concentrations of water.

a wider application of the method in assisted reproduction.

Zika virus, or the analysis of endometrial implantation "window."

possess higher risk for chromosomal abnormalities.

in a donor program if permitted by law, which is also an advantage.

ART were 47,292, with 77.4% of them being after FET [52].

**112**

in the children born after fresh ET than FET [54]. On the other hand, FET has advantages in that it decreases the risk of low birth weight (LBW) (<2500 g), very low birth weight (<1500 g), very preterm birth (VPTB) (<32 gw), placenta previa, small, placental abruption, gestational age, antepartum hemorrhage, ectopic pregnancy, and perinatal mortality [55, 56]. If we look at the weight indicator of the newborn after ART, the results of the studies show that ART-born children have a lower weight, and the risk of LBW in newborns is 2.6 higher than those spontaneously conceived [57]. However, after FET, children are being born 90–190 g heavier than those after fresh ET. This brings them statistically closer to the children born after a spontaneous pregnancy [54, 58].

Here a question arises: is the change in the weight of the newborn due to epigenetic reprogramming?

Human studies have shown that different ART methods, such as ovarian stimulation and supraphysiological levels of sex steroid hormones, culture media, and embryo cryopreservation, may be associated with intrauterine growth change, resulting in altered birth weight profiles, which may be caused by epigenetic modifications [59]. Furthermore, some reports indicate that children conceived by ART have an altered lipid profile, fasting glucose, body fat distribution, and cardiovascular function [60, 61]. ICSI/IVF children showed a significantly decreased DNA methylation at birth. Studies suggest an impact of ICSI on the offspring's epigenome(s), which may contribute to phenotypic variation and disease susceptibility in ART children [62].

Differences in DNA methylation between IVF and non-IVF twins on a genomewide scale and their results show evidence for epigenetic modifications that may in part reflect parental subfertility [63].

The methylation profiles of ART and IUI newborns were different from those of naturally conceived newborns. But the profiles of ICSI-frozen (FET) and IUI infants were very similar, suggesting that cryopreservation may eliminate some of the epigenetic aberrations induced by IVF or ICSI [64].

In addition to the above mentioned advantages, frozen ET, compared to fresh ET, also has some drawbacks, such as: macrosomia (OR 1.64), large for gestation age (OR 1.54), post-term birth (OR 1.40), and placenta accrete (OR 3.20) [65]. In cases with preeclampsia, the risk after FET in twin pregnancies is 19.6% with risk difference 5.1% in fresh ET, while in singleton pregnancies, the risk is 7.0% after FET with risk difference 1.8% in fresh ET [66]. Probably, the main reason for the increase of these complications is the protocol of the endometrium preparation for FET. Analyzing the Japanese assisted reproductive technology registry in 2014, Saito et al. [67] found that pregnancies following FET after hormone replacement cycle (n = 75,474 cases) have significant higher risks of hypertensive disorders and placenta accrete compared to FET in natural ovulatory cycle (n = 29,760 cases).

Due to the many advantages of FET compared to fresh ET, a number of clinics have begun to implement the "freeze all" policy, so they do not perform ET in the stimulated cycle, but rather freeze all embryos. In a subsequent or later cycle, with or without HRT (hormone replacement therapy), the embryos are thawed, and ET is performed.

The first publications of results show optimism, because the FET success rate is significantly higher than that of the classic ET approach. In support of these results, it was reported by Lopez et al. in a retrospective study with 1697 IVF cycles that the FET versus fresh ET rate for women up to 39 years was 44.5–38% and for women after 39 years, 34.9% and 22.7%, respectively [68].

Similar results were presented from another retrospective study made by Santistevan et al. [69]. It encompassed more than 16,000 IVF cases, with

**Figure 2.**

*Age characteristics and fertility in nondonor IVF program after ET of fresh and thawed embryos (SART 2013).*

repeated success rates following FET versus fresh ET in women up to 35 years of age, being 50.240.3%. In women after the age of 35, the ratio was 46–33%, respectively. Another interesting study was published by Zhu et al. [70], which included 20,687 women who started their first IVF cycles using a "freeze-all" strategy. The authors report an average success rate of 50.74% live birth rates, establishing different success rates depending on collected oocytes and the age of the woman.

However, despite these preliminary positive results, it must be emphasized that these are retrospective studies. We cannot find enough proof to convince us to change our treatment strategy toward a "freeze all" policy, as the studies cannot give us an answer when it is better to freeze the embryos and when to apply fresh ET.

For this reason, a number of prospective studies are currently being carried out which are intended to answer those questions. What is the place of "freeze all" policy in treatment of infertility? Some of these studies have already published their results, such as Coates et al., who for the first time compared the success rate of FET to that of fresh ET of euploid embryos. In a study encompassing 179 cases, the authors found a significant increase in the success rate of frozen ET, based on developing pregnancy and delivery [71].

In another prospective study, however, Vuong et al. [72] and Shi et al. [73] did not establish a statistically significant difference between FET and fresh ET. The results in the first study were 36.3 and 34.5% in 782 IVF/ICSI cases of non-PCOS patients, while in the second study, the results were 48.7 and 50.2%, respectively, in 2157 women who were undergoing their first in vitro fertilization cycle. The main conclusions of those articles are that freezing embryos do not decrease pregnancy rates, and the "freeze all" policy only raises expenses. But there were some limitations to those studies. The methodology is based on the embryo cultivation to an early cleavage stage, and if the effect of the freezing procedure is sought, the embryos should be cultured and frozen during the blastocyst phase. In support of that opinion is the analysis of the success rates of 236,191 cases after FET. Holden et al. [74] found that there is a 49% increase of live births after blastocyst-stage FET, compared to cleavage-stage FET. The authors examined only a group of patients up to 35 years of age. However, if we were to analyze the success rate after in vitro procedures in different age groups, we would find that in females more than 37 years of age, the results after frozen transfer are significantly higher than in fresh transfer [51] (**Figure 2**).

**115**

and over 42 years (**Figure 3**).

**6. Conclusion**

**Figure 3.**

*protocols.*

*The Present and Future of Embryo Cryopreservation DOI: http://dx.doi.org/10.5772/intechopen.80587*

In spite of controversial results, FET has its own place in treatment of couples with infertility. To date, the main explanation for the high FET success rate is the so-called "Hormonal Theory." According to it, high levels of estrogen during stimulation have a detrimental effect on embryos and placentation, a negative effect on the preparation of the endometrium for implantation of the embryo. If we accept this hypothesis, we would not be able to explain why women in advanced reproductive age achieve FETs with higher success rates than fresh ET. The results of the studies show that, as women age, there is a higher possibility of a displacement of the implantation "window." In this situation, it is only logical that the FETs have a lower success rate. Continuing the discussion on this issue, Vladimirov et al. [75] researched the age distribution of cases with a displacement of the implantation "window." This is a study with 402 women to whom we have applied the endometrial receptivity analysis (ERA) test due to different indications. The results show that, with the increase of the woman's age, there are increased number of cases of displacement of the implantation "window" with a statistically reliable difference between the groups of up to 35 years

*Age distribution of cases with endometrial non-receptivity (endometrial receptivity analysis) in LH/HRT* 

According to the "Theory about the Embryo-Cryo treatment," the procedure of freezing/thawing of the embryo probably has a positive "therapeutic" effect on embryos [6]. In fact, results show that FET has a high delivery rate, and the resulting offspring has a better perinatal outcome, compared to children born after fresh ET. The frequency of obstetric complications during pregnancy and children born with congenital abnormalities is lower in FET. However, we still do not have a clear answer to the question, what are the effects of hormonal stimulation and laboratory conditions of cultivation on the normal development and embryo implantation, as well as on pregnancy, birth and development of the individual. These scientific questions and many more still await their answers (**Figure 4**). We believe that applying this freezing approach can reduce the negative effects of the in vitro

We would like to close this chapter with Mahatma Gandhi's thought: "A nation's greatness is measured by how it treats its weakest members." Our interpretation of this thought is: "The level of human societal development is measured based on

procedures on embryo development and implantation.

how we take care about our unborn children, i.e., the embryos."

**Figure 3.**

*Embryology - Theory and Practice*

repeated success rates following FET versus fresh ET in women up to 35 years of age, being 50.240.3%. In women after the age of 35, the ratio was 46–33%, respectively. Another interesting study was published by Zhu et al. [70], which included 20,687 women who started their first IVF cycles using a "freeze-all" strategy. The authors report an average success rate of 50.74% live birth rates, establishing different success rates depending on collected oocytes and the age of

*Age characteristics and fertility in nondonor IVF program after ET of fresh and thawed embryos (SART 2013).*

However, despite these preliminary positive results, it must be emphasized that

In another prospective study, however, Vuong et al. [72] and Shi et al. [73] did not establish a statistically significant difference between FET and fresh ET. The results in the first study were 36.3 and 34.5% in 782 IVF/ICSI cases of non-PCOS patients, while in the second study, the results were 48.7 and 50.2%, respectively, in 2157 women who were undergoing their first in vitro fertilization cycle. The main conclusions of those articles are that freezing embryos do not decrease pregnancy rates, and the "freeze all" policy only raises expenses. But there were some limitations to those studies. The methodology is based on the embryo cultivation to an early cleavage stage, and if the effect of the freezing procedure is sought, the embryos should be cultured and frozen during the blastocyst phase. In support of that opinion is the analysis of the success rates of 236,191 cases after FET. Holden et al. [74] found that there is a 49% increase of live births after blastocyst-stage FET, compared to cleavage-stage FET. The authors examined only a group of patients up to 35 years of age. However, if we were to analyze the success rate after in vitro procedures in different age groups, we would find that in females more than 37 years of age, the results after frozen transfer are significantly higher than in fresh

these are retrospective studies. We cannot find enough proof to convince us to change our treatment strategy toward a "freeze all" policy, as the studies cannot give us an answer when it is better to freeze the embryos and when to apply fresh ET. For this reason, a number of prospective studies are currently being carried out which are intended to answer those questions. What is the place of "freeze all" policy in treatment of infertility? Some of these studies have already published their results, such as Coates et al., who for the first time compared the success rate of FET to that of fresh ET of euploid embryos. In a study encompassing 179 cases, the authors found a significant increase in the success rate of frozen ET, based on

**114**

transfer [51] (**Figure 2**).

the woman.

**Figure 2.**

developing pregnancy and delivery [71].

*Age distribution of cases with endometrial non-receptivity (endometrial receptivity analysis) in LH/HRT protocols.*

In spite of controversial results, FET has its own place in treatment of couples with infertility. To date, the main explanation for the high FET success rate is the so-called "Hormonal Theory." According to it, high levels of estrogen during stimulation have a detrimental effect on embryos and placentation, a negative effect on the preparation of the endometrium for implantation of the embryo. If we accept this hypothesis, we would not be able to explain why women in advanced reproductive age achieve FETs with higher success rates than fresh ET. The results of the studies show that, as women age, there is a higher possibility of a displacement of the implantation "window." In this situation, it is only logical that the FETs have a lower success rate. Continuing the discussion on this issue, Vladimirov et al. [75] researched the age distribution of cases with a displacement of the implantation "window." This is a study with 402 women to whom we have applied the endometrial receptivity analysis (ERA) test due to different indications. The results show that, with the increase of the woman's age, there are increased number of cases of displacement of the implantation "window" with a statistically reliable difference between the groups of up to 35 years and over 42 years (**Figure 3**).

## **6. Conclusion**

According to the "Theory about the Embryo-Cryo treatment," the procedure of freezing/thawing of the embryo probably has a positive "therapeutic" effect on embryos [6]. In fact, results show that FET has a high delivery rate, and the resulting offspring has a better perinatal outcome, compared to children born after fresh ET. The frequency of obstetric complications during pregnancy and children born with congenital abnormalities is lower in FET. However, we still do not have a clear answer to the question, what are the effects of hormonal stimulation and laboratory conditions of cultivation on the normal development and embryo implantation, as well as on pregnancy, birth and development of the individual. These scientific questions and many more still await their answers (**Figure 4**). We believe that applying this freezing approach can reduce the negative effects of the in vitro procedures on embryo development and implantation.

We would like to close this chapter with Mahatma Gandhi's thought: "A nation's greatness is measured by how it treats its weakest members." Our interpretation of this thought is: "The level of human societal development is measured based on how we take care about our unborn children, i.e., the embryos."

#### **Figure 4.**

*Possible future directions aiming to improve our understanding of embryo cryopreservation and its place in the field of ART.*
