**6. Embryo monitoring**

*Induced Abortion and Spontaneous Early Pregnancy Loss - Focus on Management*

In recent years it was indicated that different etiologies of infertility arose as a result of the underlying genetic and epigenetic changes that contribute to the endometrial dysfunction and lead to implantation failure, miscarriage, and adverse outcomes. These epigenetic and genetic changes lead to placentation defects and contribute to the short- and long-term outcomes associated with infertility. One of the main causes for altered genetic and epigenetic regulation of embryo development and placentation was assigned to hormonal and nutrition-related changes in maternal environment. Embryos respond to the in vivo maternal environment during gestation or during cultivation in vitro in multiple ways that can influence their future growth and health. Developmental plasticity could be altered by the changes in imprinted gene expression, nutrient, and stress-related signaling pathways or cell cycling and apoptotic rates. Embryo phenotype changes through a complex network of interactions with a central role for maternal-fetal neuroendocrine signaling [55]. Maternal undernutrition during gestation alters maternal steroid hormone levels, including elevation of glucocorticoids (GC; corticosterone, cortisol), the stress hormones, which can alter the physiological condition of the conceptus and affect the intrauterine fetal and postnatal growth and cardiovascular and metabolic physiology and enhance the risk of adult-onset disease. This exposure of the embryo to glucocorticoids can alter the fetal hypothalamus pituitary adrenal (HPA) axis, leading to increased fetal GC activity which can, in turn, modify the expression of many downstreamregulated genes that control growth and metabolism, including cardiovascular

The influence of placental function and placental/fetal exchange on fetal programming has been in focus of the recent research. Now it has become widely accepted that maternal nutrition can have the long-term consequences on the offspring without necessarily affecting the size at birth. Altered embryo phenotypes induced by prenatal nutrition are associated with epigenetic modifications. Many imprinted genes contribute to placental function and nutrient exchange [56]. Early epigenetic effects in embryos caused by environmental conditions can lead to physiological impairment to growth due to reduced nutrient supply. There is now evidence from human studies and animal experiments that show the overnutrition and undernutrition during the prenatal period which have lifelong health effects for the offspring and induce the development of noncommunicable diseases during

Besides nutrition, the hormonal milieu at conception is known to affect a number of imprinted genes that are expressed during the preimplantation period. Hormonal status will be especially affected during fertility treatment, mostly during IVF. Because superovulation could lead to altered expression of endometrial genes critical to tissue remodeling and placentation, hyperstimulated hormonal status has been implicated in an increased risk for pregnancy complications related to abnormal placentation [58]. Although global methylation pattern was found to be similar among the IVF and spontaneous conceptions early during placentation in the first trimester, differential methylation has been identified in multiple loci between IVF and non-IVF fertility treatments pregnancies but not when compared with spontaneous conceptions. This suggests that there are differences in the infertile population that might be linked to specific treatments, including the hormonal hyperstimulation, that could affect gene imprinting [59]. Several studies have found that the use of ARTs is linked with irregular DNA methylation in human gamete, embryo, placenta, and umbilical cord samples [60, 61]. There were also studies that showed association between specific procedures with methylation differences in placenta, suggesting that specific fertility treatments affect the

**48**

placental epigenome and function [62].

and renal physiology [55].

postnatal life [57].

The embryo has, in addition to the endometrium, the crucial importance for the success and regularity of the implantation and then placentation. The morphological assessment of the embryos' quality is insufficient for the cognition of its biological resources. The new invasive and noninvasive techniques of embryo quality assessment have been developed. Nowadays, the invasive technology means preimplantation genetic testing (PGT), the aneuploidy screening, or diagnosis of specific genetic disorders of the embryo before the transfer by using nextgeneration sequencing (NGS). These tests include biopsy trophectoderm cells with blastocyst vitrification [63–65]. With trophectoderm biopsy, both maternal and paternal abnormalities can be studied. Possible disadvantages are the presence of mosaicism and the fact that the trophectoderm might not be a representative of the inner cell mass.

Noninvasive time-lapse embryo monitoring allows continuous embryo observation without the need to remove the embryo from optimal culturing conditions. The information on the cleavage pattern, morphologic changes, and embryo development dynamics could help us identify embryos with a higher implantation potential. It has also been shown that imaging phenotypes reflect the molecular program of the embryo, where individual blastomeres develop autonomously toward embryo genomic activation [66].

This type of monitoring allows for the collection of much more information on the timing of the cleavages and the dynamics of the morphologic changes, with analysis of the kinetics of the events up until the blastocyst stage [67].

Various kinetic and morphologic markers have already been found that are associated with the minimal likelihood of implantation and others that are predictive of blastocyst development, implantation potential, genetic health, and pregnancy [68, 69].

#### **7. Gametes**

After the formation of the embryo, its fate is already determined. The gamete quality has the crucial part in the creation of the high-quality embryos. The conditions, in which oogenesis and spermatogenesis take place, have a crucial impact on the quality of embryos that is formed from these gametes.

#### **7.1 Oogenesis**

The evaluation of the oocyte quality based on morphological evaluation is not sufficient for an insight into the biological potential. It can identify those cells that have nuclear immaturity, significant degeneration, or major abnormalities. Recently, the developed strategies including the genomic, transcriptomic, and proteomic approaches have been applied in assisted reproduction. Their goal is to identify a "molecular profile" of embryo development by detecting the chemical components in the oocyte, granulosa cells, follicular fluid, and embryo culture medium [70].

Better predictors, the birefringence properties of the meiotic spindle, and the zona pellucida are indicative of good health of the oocyte [71]. A very useful data can be obtained from the application of studying gene expression from cumulus cells, using microarrays, as biomarkers for oocyte viability*.* The metabolomic profiling of oocyte spent culture media by mass spectroscopy has shown differences related to oocyte maturation, embryo development, and implantation

success [72]. Oocyte quality can be assessed by the measurement of oocyte oxygen consumption [73].

**Spermatogenesis:** the quality of spermatogenesis is the condition for the formation of a good embryo. The advanced sperm selection techniques are based not only on the morphological assessment (defragmentation, MACS) but also on the evaluation of specific cellular characteristics (membrane integrity, density, surface charge) that provide a choice of better quality sperm. The methods of improving conditions of gametogenesis, which are applied so far, do not provide a sufficient effect. They are mainly related to the balance correction of microelements and vitamins, as well as the oxydo-reductive processes in the body. The sperm chromatin and DNA integrity are necessary to ensure normal embryo development. It is now clear that DNA damage in spermatozoa has a negative influence on blastocyst development and the pregnancy outcome [74] . Similarly, centrosome integrity is critical for successful fertilization and embryo development*.* There are studies that have described the association between sperm with DNA damage and a history of recurrent miscarriage [75]*.*

#### **7.2 Advanced therapy**

Magnetic-activated cell sorting (MACS) technology for sperm could improve obstetric and perinatal outcomes compared with those achieved after swim up. Treatment of sperm with MACS procedure prior to IVF results in a marked improvement in pregnancy rate and cessation of the abortion rate in couples whose ejaculates initially had high levels of SDF [76].

A number of prerequisites are needed to create high-quality oocytes, those conditions are likely to be grouped into several parts: the existence of high quality responsive oogonia, its potential of the adequate number increase and quality of mitochondria, the presence of sufficient amounts and types of growth factors, orchestrated by the balance of blocking (Hippo) and activating (ACT) gene pathways [77].

For decades it was believed that the woman's reproductive potential is entirely dependent on the size of the stock (pool) of primordial follicles in the ovary. The paradigm that has prevailed for decades in the scientific world about the existence of a consistent number of primordial follicles, established during embryonic and fetal period, was in many ways changed by Tilly's group work. They practically demonstrated the existence of germline or oogonial stem cells [78].

Their dormant status is characterized by communication with surrounding granulosa cells and numerous mechanical and chemical factors controlling the progression of their cell cycle. These factors control signaling activation of the pathways included in the primordial follicle dormant status regulation, like Hippo and AKT signaling [77]. During the recent years, various programs have been developed to try to improve the quality of oocytes. It has been shown that it can be influenced on the activation of primordial cells and maturation to the mature oocyte. The stem cells can be influenced by the stem cell therapy in order to obtain the intracellular communication with the existing ovarian primordial oogonia. The therapy with mesenchymal stem cells has led to the recovery features of oocytes after the chemotherapy-induced insufficiency [79]. The animal experiments by the in vitro therapy with developed stem cells have led to the birth of live offspring without abnormalities [80]. Other groups of authors have tried to improve the ovarian function with the growth factors obtained from the plasma and enriched with platelets and leukocytes. The cases of childbirth after re-transplantation of ovaries with support of PRP have been published [81]*.* Our group has achieved a normal pregnancy outcome after the sonographically guided therapy with growth factors in a female patient aged 40 years, after 18 attempts of in vitro fertilization.

**51**

*Implantation and the Fetal Health*

the quality gametes [50, 84]*.*

**8. Conclusion**

**Author details**

Aleksandar Ljubic1

and Andjela Perovic4

1 Medigroup, Belgrade, Serbia

2 Renova Clinic, Belgrade, Serbia

4 Segova Biotechnology, Belgrade, Serbia

provided the original work is properly cited.

*DOI: http://dx.doi.org/10.5772/intechopen.87016*

high-intensity interval training (HIIT).

applying the latest biotechnological procedures.

, Dzihan Abazovic2

3 Special Gynecology Hospital Jevremova, Belgrade, Serbia

\*Address all correspondence to: andreapirkovic@segova.com

The role of the number and function of mitochondria in the development of quality oocytes is surely very important. The problems of mitochondrial heteroplasmy go with the complicated, technologically very complex methods of polar body transfer, spindle transfer, and pronuclear or oocyte transfer [82–84]. The augmentation of autologous mitochondria carries a potential treatment. Our team has inaugurated the attempt of the mitochondrial energy boosting with ovarian

The autologous growth factors that are intraovarian instilled are leading to the changes in the production and efficiency of the local growth factors. The influence on the genetic control of oogenesis, by the modification of the Hippo and AKT signaling pathways, is possible in different ways. The correction of the gene signaling or autologous tissue genetic bioengineering is certainly a step forward in obtaining

Implantation is one of the crucial periods in human reproduction. Increasing body of evidence suggests that the improper (dysfunctional) implantation and the formation of the placenta can endanger life and health of both the fetus and the mother, during prenatal life and decades after delivery. The changes that lead to the insufficient implantation should be sought in the preimplantation period, in relation between the embryo and the endometrium. It is possible that the time is approaching when the disorders of the pregnancy caused by dysfunctional implantation would be the indication for the application of a natural IVF (without ovarian stimulation) with the use of new biotechnological achievements. For better results of the perinatal medicine, it is necessary to apply earlier (in the preconception and preimplantation periods) the therapy based on the subcellular and genetic level by

, Dusica Ljubic3

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

, Andrea Pirkovic4

\*

#### *Implantation and the Fetal Health DOI: http://dx.doi.org/10.5772/intechopen.87016*

The role of the number and function of mitochondria in the development of quality oocytes is surely very important. The problems of mitochondrial heteroplasmy go with the complicated, technologically very complex methods of polar body transfer, spindle transfer, and pronuclear or oocyte transfer [82–84]. The augmentation of autologous mitochondria carries a potential treatment. Our team has inaugurated the attempt of the mitochondrial energy boosting with ovarian high-intensity interval training (HIIT).

The autologous growth factors that are intraovarian instilled are leading to the changes in the production and efficiency of the local growth factors. The influence on the genetic control of oogenesis, by the modification of the Hippo and AKT signaling pathways, is possible in different ways. The correction of the gene signaling or autologous tissue genetic bioengineering is certainly a step forward in obtaining the quality gametes [50, 84]*.*

#### **8. Conclusion**

*Induced Abortion and Spontaneous Early Pregnancy Loss - Focus on Management*

oxygen consumption [73].

recurrent miscarriage [75]*.*

ejaculates initially had high levels of SDF [76].

**7.2 Advanced therapy**

success [72]. Oocyte quality can be assessed by the measurement of oocyte

tion of a good embryo. The advanced sperm selection techniques are based not only on the morphological assessment (defragmentation, MACS) but also on the evaluation of specific cellular characteristics (membrane integrity, density, surface charge) that provide a choice of better quality sperm. The methods of improving conditions of gametogenesis, which are applied so far, do not provide a sufficient effect. They are mainly related to the balance correction of microelements and vitamins, as well as the oxydo-reductive processes in the body. The sperm chromatin and DNA integrity are necessary to ensure normal embryo development. It is now clear that DNA damage in spermatozoa has a negative influence on blastocyst development and the pregnancy outcome [74] . Similarly, centrosome integrity is critical for successful fertilization and embryo development*.* There are studies that have described the association between sperm with DNA damage and a history of

**Spermatogenesis:** the quality of spermatogenesis is the condition for the forma-

Magnetic-activated cell sorting (MACS) technology for sperm could improve

A number of prerequisites are needed to create high-quality oocytes, those conditions are likely to be grouped into several parts: the existence of high quality responsive oogonia, its potential of the adequate number increase and quality of mitochondria, the presence of sufficient amounts and types of growth factors, orchestrated by the

For decades it was believed that the woman's reproductive potential is entirely dependent on the size of the stock (pool) of primordial follicles in the ovary. The paradigm that has prevailed for decades in the scientific world about the existence of a consistent number of primordial follicles, established during embryonic and fetal period, was in many ways changed by Tilly's group work. They practically

Their dormant status is characterized by communication with surrounding granulosa cells and numerous mechanical and chemical factors controlling the progression of their cell cycle. These factors control signaling activation of the pathways included in the primordial follicle dormant status regulation, like Hippo and AKT signaling [77]. During the recent years, various programs have been developed to try to improve the quality of oocytes. It has been shown that it can be influenced on the activation of primordial cells and maturation to the mature oocyte. The stem cells can be influenced by the stem cell therapy in order to obtain the intracellular communication with the existing ovarian primordial oogonia. The therapy with mesenchymal stem cells has led to the recovery features of oocytes after the chemotherapy-induced insufficiency [79]. The animal experiments by the in vitro therapy with developed stem cells have led to the birth of live offspring without abnormalities [80]. Other groups of authors have tried to improve the ovarian function with the growth factors obtained from the plasma and enriched with platelets and leukocytes. The cases of childbirth after re-transplantation of ovaries with support of PRP have been published [81]*.* Our group has achieved a normal pregnancy outcome after the sonographically guided therapy with growth factors in

obstetric and perinatal outcomes compared with those achieved after swim up. Treatment of sperm with MACS procedure prior to IVF results in a marked improvement in pregnancy rate and cessation of the abortion rate in couples whose

balance of blocking (Hippo) and activating (ACT) gene pathways [77].

demonstrated the existence of germline or oogonial stem cells [78].

a female patient aged 40 years, after 18 attempts of in vitro fertilization.

**50**

Implantation is one of the crucial periods in human reproduction. Increasing body of evidence suggests that the improper (dysfunctional) implantation and the formation of the placenta can endanger life and health of both the fetus and the mother, during prenatal life and decades after delivery. The changes that lead to the insufficient implantation should be sought in the preimplantation period, in relation between the embryo and the endometrium. It is possible that the time is approaching when the disorders of the pregnancy caused by dysfunctional implantation would be the indication for the application of a natural IVF (without ovarian stimulation) with the use of new biotechnological achievements. For better results of the perinatal medicine, it is necessary to apply earlier (in the preconception and preimplantation periods) the therapy based on the subcellular and genetic level by applying the latest biotechnological procedures.

#### **Author details**

Aleksandar Ljubic1 , Dzihan Abazovic2 , Dusica Ljubic3 , Andrea Pirkovic4 \* and Andjela Perovic4


\*Address all correspondence to: andreapirkovic@segova.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
