The effect of microenvironmental free radicals on embryo development for assisted reproduction technology cycles
Date Issued
2012
Date
2012
Author(s)
Lee, Tsung-Hsien
Abstract
Subsequent to controlled ovarian stimulaiton and oocyte retrieval in assisted reproduction technology (ART) cycles, embryos are cultured in vitro of 2-5 days before transfer back into the uterus. During the process of embryonic development, some develop into good embryos, some features slow cleavage rates or arrest of development, while some appear cytoplasmic fragmentation. For the reason that the pregnancy rate in ART treatment is limited, many ART centers tend to transfer more number of embryos so as to enhance the pregnancy success rates. In Taiwan, since health insurance system does not pay for ART treatments, patients’ desire for pregnancy success is so strong that they consider multiple pregnancy acceptable. However, multiple gestation causes subsequent high risk pregnancy and prematurity, which may cost a great quantity of medical resources. Therefore, there is a need to establish a set of selection criteria for day 3 embryo transfer (D3ET) and blastocyst transfer (BT) cycles, so as to enable clinicians to provide infertile couples appropriate counseling.
This research analyzes the relevance of morphological parameters of embryo quality to the pregnancy success and multiple gestation in D3ET cycles or BT cycles. Among the common used criteria for embryo morphology, the embryo scores for the best three embryos (Top3) are the most crucial to predict the probability of pregnancy. Nevertheless, for women >35 years of age, it is female age that is the major factor for pregnancy outcome in D3ET cycles. After analysis of the BT program, the Top3 blastocyst scores can be utilized to decrease the number of embryos transferred in patients < 38 years of age. However, for women >38 years of age, it is female age acting the major factor for pregnancy outcome in BT cycles. The above-mentioned analysis revealed that the effect of embryo quality on pregnancy outcome in ART cycles is prominent. The permanent arrest of embryo development is viewed as cellular senescence, which is connected with mitochondrial reactive oxygen species (ROS). Furthermore, cytoplasmic fragmentation of embryos is related to apoptosis, which is also connected with intracellular ROS levels. Therefore, the correlation between microenvironmental ROS levels and embryo quality were subsequently investigated. The results revealed that the ROS levels in spent culture media are correlated to embryo fragmentation degree. Furthermore, culture media ROS was inversely relevant to implantation potential of day 3 embryos. The intracellular ROS levels of embryos are related to mitochondrial metabolism, which is regulated by nitric oxide (NO) in studies on somatic cells. Our previous reports indicated that, in patients with endometriosis, the expression of nitric oxide synthase increased in ectopic endometrial tissue. Furthermore, the elevated follicular NO levels are detrimental to embryo quality and may be related to failure of embryo implantation. Consequently, the in vitro development of mouse zygotes was utilized to elucidate the effect of microenvironmental NO on mitochondrial metabolism and blastocyst formation. The results of mouse embryo experiment demonstrated that the apoptotic effect of NO on embryo development is closely related to protein S-nitrosylation within lipid-membrane rich organelles, such as mitochondria and endoplasmic reticulum. Totally abolishing NO production by NOS inhibitor is deleterious to embryo development and results in 2-cell block. The supplement of reduced form glutathione to NO-exposed embryos was able to maintain development competence, adequate proliferation of blastomeres, and ATP production in blastocysts. In conclusion, the development potential is varied for the embryos in ART cycles. The best three embryos and the female age are both decisive to predict the pregnancy success and multiple gestation rates in D3ET and BT program, so that they could be utilized to help counseling for infertile couples and determine the number of transfer embryos. The fragmentation degree and implantation potential of embryos is correlated to the ROS levels in spent culture media. The mouse embryo experiment confirmed that NO is essential for embryo development. However, elevated microenvironmental NO levels may induce embryo degeneration and apoptosis. The addition of reduced glutathione is able to rescue the NO-exposed embryos. The effect of NO is intimately related to protein S-nitrosylation and mitochondrial activity. Further studies on the benefit of antioxidants in culture media on in vitro embryo development are deserved.
This research analyzes the relevance of morphological parameters of embryo quality to the pregnancy success and multiple gestation in D3ET cycles or BT cycles. Among the common used criteria for embryo morphology, the embryo scores for the best three embryos (Top3) are the most crucial to predict the probability of pregnancy. Nevertheless, for women >35 years of age, it is female age that is the major factor for pregnancy outcome in D3ET cycles. After analysis of the BT program, the Top3 blastocyst scores can be utilized to decrease the number of embryos transferred in patients < 38 years of age. However, for women >38 years of age, it is female age acting the major factor for pregnancy outcome in BT cycles. The above-mentioned analysis revealed that the effect of embryo quality on pregnancy outcome in ART cycles is prominent. The permanent arrest of embryo development is viewed as cellular senescence, which is connected with mitochondrial reactive oxygen species (ROS). Furthermore, cytoplasmic fragmentation of embryos is related to apoptosis, which is also connected with intracellular ROS levels. Therefore, the correlation between microenvironmental ROS levels and embryo quality were subsequently investigated. The results revealed that the ROS levels in spent culture media are correlated to embryo fragmentation degree. Furthermore, culture media ROS was inversely relevant to implantation potential of day 3 embryos. The intracellular ROS levels of embryos are related to mitochondrial metabolism, which is regulated by nitric oxide (NO) in studies on somatic cells. Our previous reports indicated that, in patients with endometriosis, the expression of nitric oxide synthase increased in ectopic endometrial tissue. Furthermore, the elevated follicular NO levels are detrimental to embryo quality and may be related to failure of embryo implantation. Consequently, the in vitro development of mouse zygotes was utilized to elucidate the effect of microenvironmental NO on mitochondrial metabolism and blastocyst formation. The results of mouse embryo experiment demonstrated that the apoptotic effect of NO on embryo development is closely related to protein S-nitrosylation within lipid-membrane rich organelles, such as mitochondria and endoplasmic reticulum. Totally abolishing NO production by NOS inhibitor is deleterious to embryo development and results in 2-cell block. The supplement of reduced form glutathione to NO-exposed embryos was able to maintain development competence, adequate proliferation of blastomeres, and ATP production in blastocysts. In conclusion, the development potential is varied for the embryos in ART cycles. The best three embryos and the female age are both decisive to predict the pregnancy success and multiple gestation rates in D3ET and BT program, so that they could be utilized to help counseling for infertile couples and determine the number of transfer embryos. The fragmentation degree and implantation potential of embryos is correlated to the ROS levels in spent culture media. The mouse embryo experiment confirmed that NO is essential for embryo development. However, elevated microenvironmental NO levels may induce embryo degeneration and apoptosis. The addition of reduced glutathione is able to rescue the NO-exposed embryos. The effect of NO is intimately related to protein S-nitrosylation and mitochondrial activity. Further studies on the benefit of antioxidants in culture media on in vitro embryo development are deserved.
Subjects
embryo quality
reactive oxygen species
nitric oxide
S-nitrosylation
apoptosis
mitochondria
assisted reproduction technology cycles
Type
thesis
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