Enhanced progesterone support during stimulated cycles of transvaginal follicular aspiration improves bovine in vitro embryo production

The in vitro production (IVP) of cattle embryos requires that germinal-vesicle stage oocytes undergo a period of maturation in vitro prior to fertilization and culture to the blastocyst stage. Success of IVP in taurine cattle is enhanced following ovarian stimulation prior to oocyte retrieval (OPU), particularly if preceded by a short period of FSH withdrawal (‘coasting’). However, evidence regarding the importance of progesterone (P4) support during OPU-IVP is equivocal. The current study, therefore, determined the effects of increased peripheral P4 concentrations during FSH-stimulated (‘coasted’) cycles of OPU. Progesterone support was provided by either an active corpus luteum (CL) and/or one of two intravaginal P4 releasing devices (i.e., CIDR® [1.38g P4] or PRID® Delta [1.55g P4]). Expt. 1 established an initial estrus prior to OPU, allowing CL formation (single luteal phase) spanning the first two of five cycles of OPU; the remaining three cycles were supported by either a CIDR® or PRID® Delta. Expt. 2 commenced with two cycles of dominant follicle removal (including prostaglandin F2α) undertaken seven days apart prior to six cycles of OPU. The absence of a CL meant that these cycles were supported only by a CIDR® or PRID® Delta. As each experiment involved several sequential cycles of OPU, the cumulative effects of device use on vaginal discharges were also assessed. Each experiment involved 10 sexually mature Holstein heifers. In the absence of a CL, peak plasma P4 concentrations were greater (P = 0.002) for the PRID® Delta (4.3 ± 0.22) than for the CIDR® (2.9 ± 0.22). In Expt. 1 there was an interaction (P < 0.05) between CL presence at OPU and P4 device on Day 8 blastocyst yields, indicating an effect of P4 device only when the CL was absent. The percentage hatching/hatched blastocysts of matured oocytes for the CIDR® and PRID® Delta was 44.3 ± 5.04 and 41.0 ± 5.40 in the presence, and 17.1 ± 3.48 and 42.2 ± 3.76 in the absence, of a CL (P = 0.018). Combined analyses of data from Expt. 1 and 2, when no CL was present, confirmed that Day 8 blastocyst yields were greater (P = 0.022) for the PRID® Delta than the CIDR®. Vaginal discharge scores were higher (P < 0.001) for the PRID® Delta than the CIDR® in Expt. 1 but not in Expt 2; however scores were low, did not increase with repeated use, and thus were deemed of no clinical or welfare concern. In conclusion, enhanced P4 support during FSH-stimulated cycles of OPU-IVP can improve in vitro embryo development

Developmental, cytogenetic and epigenetic consequences of removing complex proteins and adding melatonin during in vitro maturation of bovine oocytes

BackgroundIn vitro maturation (IVM) of germinal vesicle intact oocytes prior to in vitro fertilization (IVF) is practiced widely in animals. In human assisted reproduction it is generally reserved for fertility preservation or where ovarian stimulation is contraindicated. Standard practice incorporates complex proteins (CP), in the form of serum and/or albumin, into IVM media to mimic the ovarian follicle environment. However, the undefined nature of CP, together with batch variation and ethical concerns regarding their origin, necessitate the development of more defined formulations. A known component of follicular fluid, melatonin, has multifaceted roles including that of a metabolic regulator and antioxidant. In certain circumstances it can enhance oocyte maturation. At this stage in development, the germinal-vesicle intact oocyte is prone to aneuploidy and epigenetic dysregulation.ObjectivesTo determine the developmental, cytogenetic and epigenetic consequences of removing CP and including melatonin during bovine IVM.Materials and methodsThe study comprised a 2 x 2 factorial arrangement comparing (i) the inclusion or exclusion of CP, and (ii) the addition (100 nM) or omission of melatonin, during IVM. Cumulus-oocyte complexes (COCs) were retrieved from stimulated cycles. Following IVM and IVF, putative zygotes were cultured to Day 8 in standard media. RNAseq was performed on isolated cumulus cells, cytogenetic analyses (SNP-based algorithms) on isolated trophectoderm cells, and DNA methylation analysis (reduced representation bisulfite sequencing) on isolated cells of the inner-cell mass.ResultsRemoval of CP during IVM led to modest reductions in blastocyst development, whilst added melatonin was beneficial in the presence but detrimental in the absence of CP. The composition of IVM media did not affect the nature or incidence of chromosomal abnormalities but cumulus-cell transcript expression indicated altered metabolism (primarily lipid) in COCs. These effects preceded the establishment of distinct metabolic and epigenetic signatures several days later in expanded and hatching blastocysts.ConclusionsThese findings highlight the importance of lipid, particularly sterol, metabolism by the COC during IVM. They lay the foundation for future studies that seek to develop chemically defined systems of IVM for the generation of transferrable embryos that are both cytogenetically and epigenetically normal

Developmental, cytogenetic and epigenetic consequences of removing complex proteins and adding melatonin during in vitro maturation of bvovine oocytes

Background: In vitro maturation (IVM) of germinal vesicle intact oocytes prior to in vitro fertilization (IVF) is practiced widely in animals. In human assisted reproduction it is generally reserved for fertility preservation or where ovarian stimulation is contraindicated. Standard practice incorporates complex proteins (CP), in the form of serum and/or albumin, into IVM media to mimic the ovarian follicle environment. However, the undefined nature of CP, together with batch variation and ethical concerns regarding their origin, necessitate the development of more defined formulations. A known component of follicular fluid, melatonin, has multifaceted roles including that of a metabolic regulator and antioxidant. In certain circumstances it can enhance oocyte maturation. At this stage in development, the germinal-vesicle intact oocyte is prone to aneuploidy and epigenetic dysregulation. Objectives: To determine the developmental, cytogenetic and epigenetic consequences of removing CP and including melatonin during bovine IVM. Materials & methods: The study comprised a 2 x 2 factorial arrangement comparing (i) the inclusion or exclusion of CP, and (ii) the addition (100 nM) or omission of melatonin, during IVM. Cumulus-oocyte complexes (COCs) were retrieved from stimulated cycles. Following IVM and IVF, putative zygotes were cultured to Day 8 in standard media. RNAseq was performed on isolated cumulus cells, cytogenetic analyses (SNP-based algorithms) on isolated trophectoderm cells, and DNA methylation analysis (reduced representation bisulfite sequencing) on isolated cells of the inner-cell mass. Results: Removal of CP during IVM led to modest reductions in blastocyst development, whilst added melatonin was beneficial in the presence but detrimental in the absence of CP. The composition of IVM media did not affect the nature or incidence of chromosomal abnormalities but cumulus-cell transcript expression indicated altered metabolism (primarily lipid) in COCs. These effects preceded the establishment of distinct metabolic and epigenetic signatures several days later in expanded and hatching blastocysts. Conclusions: These findings highlight the importance of lipid, particularly sterol, metabolism by the COC during IVM. They lay the foundation for future studies that seek to develop chemically defined systems of IVM for the generation of transferrable embryos that are both cytogenetically and epigenetically normal

Lipid metabolism in in vitro produced embryos and epiblast-derived stem cells

Infertility is a disease of the reproductive system defined as the failure to achieve a clinical pregnancy after 12 months or more of regular unprotected sexual intercourse (PierreZegers-Hochschild et al., 2009). Between 9-15% couples will have infertility problems during their lifetimes and for these infertile people, the percentage searching for medical assistance is around 56% in more developed countries and 51% in less developed countries (Boivin et al., 2007). To treat infertility, Assisted Reproductive Technologies (ARTs) were developed beginning with artificial insemination (Jouannet, 2009). Today, ART also consists of in-vitro fertilization (IVF), intracytoplasmic sperm injection and cryopreservation of germ cells (i.e., sperm, oocyte and embryos) (Sejbaek et al., 2013). On the contrary, infertility is not common in cattle. However still ART is applied in these animals to increase food production, increase the birth of healthy offspring and store the genetic materials for future. On one hand, while high productive animals calve around eight or nine times during their lifetime, it is possible to take a few times more offspring than they will get through in their lives in a year by ART. On the other hand, ART plays a commercially important role while the import-export. Intercontinental transport of live animals cost more than transport of frozen embryos (Mapletoft and Hasler, 2005). The other fact, the ART gives us a chance to create new breeds as a productive race and expedite the genetic improvement (Wu and Zan, 2012). The human in vitro embryo production (IVP) protocols normally commence with in vivo maturation which takes place within the ovarian follicle during gonadotropin treatment. This is because IVM is not successful enough in human ART to encourage widespread uptake (Anckaert et al., 2012). In contrast, IVM of immature oocytes is routine practice prior to fertilization in cattle IVP programs. Recently there has been a trend towards transfer of Day 5/Day 6 blastocysts instead of Day 2 cleaved zygotes in human ART (Glujovsky et al., 2016), with many cycles embracing single transfer of frozen/thawed blastocysts (Tiegs et al., 2019). This change has been developed as a result of undesirable consequences of transferred cleaved zygotes such as low implantation and birth rate and much cumulative pregnancy. Limitations of human IVF research such as ethics and costs have led scientists to undertake animal studies. Mouse embryos have been used extensively to understand biochemical and physiology regulations of the human embryos. However, bovine and human preimplantation embryos have many similarities with respect to biochemical, paternal and maternal regulatory processes (Menezo and Herubel, 2002). Besides, bovine and human embryo development processes are completed almost at the same time. (Figure 1). Figure 1 Comparison of embryo development stages in cattle and humans. Embryos reach the 2- and 3-cell stage around the same time. However, while human embryos form a morula around Day 4, this stage is seen on Day 5/6 in cattle. Thereafter, bovine embryo stages occur typically 1 to 2 later than human embryo stages. Credit artwork: Gizem Guven Ates The number of cattle embryos transferred are steadily increased year on year as a consequence of improvements in bovine IVP (Figure 2A and B). The underlying reason of this development is comprehension of the metabolism of oocytes and embryos, thus determining the needs of cells viability culture. For instance, the human fluid composition of the oviduct where the zygote stays until formation of morula, demonstrates pyruvate and lactate levels significantly higher than uterus where the pre-implanted blastocyst is located. This contrasts with glucose concentrations which is over five times higher in the uterus than the oviduct and until this statement was understood required pyruvate and glucose were not placed enough in the culture system (Leese et al., 2007). Successful production of embryo also depends on maternal and paternal factors such as age, environmental pollution and parenteral nutrition. Nowadays numerous studies have been done especially about parenteral nutrition (Sharma et al., 2020). The scientists associated parenteral nutrition with blastocyst quality, DNA methylation, metabolism of germ cells and embryos and even offspring disease risk over their lifetime. Attention was drawn to over- and undernutrition can affect sperm and seminal plasma in male (Sinclair and Watkins, 2013), metabolites in follicular environment (Robker et al., 2009), ovulation rate and embryo viability (Gonzalez-Añover et al., 2011) during preconception period. A. B. Figure 2 Number of cattle embryos transferred by year over the past 20 years (A) (IETS, 2016) and by geographical region in 2018 (B) (IETS, 2019). IVD = in vivo derived; IVP = in vitro produced Culture environment during in vitro maturation (IVM), fertilisation (IVF and culture (IVC) is a major limitation in the success of contemporary systems of both human and bovine IVP. An understanding of gamete/embryo metabolism is key to the successful development of these systems. There are major requirements for energy associated with biosynthetic processes and proliferation in these different cell types. Energy is provided by the breakdown of carbohydrates, proteins and lipids. Well-studied sources of energy relate to carbohydrates, such as pyruvate, glucose and lactate (Gray et al., 2014), and amino acids (Hemmings et al., 2012). The importance of lipid metabolism, however, has become increasingly appreciated in recent years (Leese, 2015). Oocytes tend to accumulate fatty acids during the terminal stages of oocyte growth prior to IVM (Sturmey et al., 2009). Fatty acid oxidation (FAO) was shown to be indispensable for oocyte meiotic maturation and developmental competence in mice (Dunning et al., 2010) although oocyte lipid content in mice is relatively low compared to that of farm animal species such as cattle and pigs (McEvoy et al., 2000). Despite differences in oocyte lipid quantity, inhibition of FAO with the carnitine palmitoyl transferase 1 (CPT1) activity inhibitor etomoxir during IVM delayed the completion of meiotic maturation in murine, bovine and porcine oocytes (Paczkowski et al., 2014). In contrast, increasing FAO by supplementation of culture media with lipid metabolism modulators such as L-carnitine, resveratrol, conjugated linoleic acid and forskolin significantly improve oocyte competence in terms of fertilization and embryo development in mice (Dunning et al., 2011) and cows (Sutton-Mcdowall et al., 2012). On the other hand, lipids are key structural components of cell membranes and form complexes with glycose and/or proteins (Muro et al., 2014). In addition, lipids are precursors of steroids which have important roles in cell signalling (Kusumi et al., 2012). With the foregoing discussion in mind, the aim of this thesis was to improve our understanding of lipid metabolism, and fatty acid oxidation in particular, during IVM of cattle oocytes, and to investigate the long-term consequences for embryo development and viability. The ultimate goal was to generate improved systems for cattle IVP that would lead to better pregnancy outcomes following embryo transfer

Lipid metabolism in in vitro produced embryos and epiblast-derived stem cells

Infertility is a disease of the reproductive system defined as the failure to achieve a clinical pregnancy after 12 months or more of regular unprotected sexual intercourse (PierreZegers-Hochschild et al., 2009). Between 9-15% couples will have infertility problems during their lifetimes and for these infertile people, the percentage searching for medical assistance is around 56% in more developed countries and 51% in less developed countries (Boivin et al., 2007). To treat infertility, Assisted Reproductive Technologies (ARTs) were developed beginning with artificial insemination (Jouannet, 2009). Today, ART also consists of in-vitro fertilization (IVF), intracytoplasmic sperm injection and cryopreservation of germ cells (i.e., sperm, oocyte and embryos) (Sejbaek et al., 2013). On the contrary, infertility is not common in cattle. However still ART is applied in these animals to increase food production, increase the birth of healthy offspring and store the genetic materials for future. On one hand, while high productive animals calve around eight or nine times during their lifetime, it is possible to take a few times more offspring than they will get through in their lives in a year by ART. On the other hand, ART plays a commercially important role while the import-export. Intercontinental transport of live animals cost more than transport of frozen embryos (Mapletoft and Hasler, 2005). The other fact, the ART gives us a chance to create new breeds as a productive race and expedite the genetic improvement (Wu and Zan, 2012). The human in vitro embryo production (IVP) protocols normally commence with in vivo maturation which takes place within the ovarian follicle during gonadotropin treatment. This is because IVM is not successful enough in human ART to encourage widespread uptake (Anckaert et al., 2012). In contrast, IVM of immature oocytes is routine practice prior to fertilization in cattle IVP programs. Recently there has been a trend towards transfer of Day 5/Day 6 blastocysts instead of Day 2 cleaved zygotes in human ART (Glujovsky et al., 2016), with many cycles embracing single transfer of frozen/thawed blastocysts (Tiegs et al., 2019). This change has been developed as a result of undesirable consequences of transferred cleaved zygotes such as low implantation and birth rate and much cumulative pregnancy. Limitations of human IVF research such as ethics and costs have led scientists to undertake animal studies. Mouse embryos have been used extensively to understand biochemical and physiology regulations of the human embryos. However, bovine and human preimplantation embryos have many similarities with respect to biochemical, paternal and maternal regulatory processes (Menezo and Herubel, 2002). Besides, bovine and human embryo development processes are completed almost at the same time. (Figure 1). Figure 1 Comparison of embryo development stages in cattle and humans. Embryos reach the 2- and 3-cell stage around the same time. However, while human embryos form a morula around Day 4, this stage is seen on Day 5/6 in cattle. Thereafter, bovine embryo stages occur typically 1 to 2 later than human embryo stages. Credit artwork: Gizem Guven Ates The number of cattle embryos transferred are steadily increased year on year as a consequence of improvements in bovine IVP (Figure 2A and B). The underlying reason of this development is comprehension of the metabolism of oocytes and embryos, thus determining the needs of cells viability culture. For instance, the human fluid composition of the oviduct where the zygote stays until formation of morula, demonstrates pyruvate and lactate levels significantly higher than uterus where the pre-implanted blastocyst is located. This contrasts with glucose concentrations which is over five times higher in the uterus than the oviduct and until this statement was understood required pyruvate and glucose were not placed enough in the culture system (Leese et al., 2007). Successful production of embryo also depends on maternal and paternal factors such as age, environmental pollution and parenteral nutrition. Nowadays numerous studies have been done especially about parenteral nutrition (Sharma et al., 2020). The scientists associated parenteral nutrition with blastocyst quality, DNA methylation, metabolism of germ cells and embryos and even offspring disease risk over their lifetime. Attention was drawn to over- and undernutrition can affect sperm and seminal plasma in male (Sinclair and Watkins, 2013), metabolites in follicular environment (Robker et al., 2009), ovulation rate and embryo viability (Gonzalez-Añover et al., 2011) during preconception period. A. B. Figure 2 Number of cattle embryos transferred by year over the past 20 years (A) (IETS, 2016) and by geographical region in 2018 (B) (IETS, 2019). IVD = in vivo derived; IVP = in vitro produced Culture environment during in vitro maturation (IVM), fertilisation (IVF and culture (IVC) is a major limitation in the success of contemporary systems of both human and bovine IVP. An understanding of gamete/embryo metabolism is key to the successful development of these systems. There are major requirements for energy associated with biosynthetic processes and proliferation in these different cell types. Energy is provided by the breakdown of carbohydrates, proteins and lipids. Well-studied sources of energy relate to carbohydrates, such as pyruvate, glucose and lactate (Gray et al., 2014), and amino acids (Hemmings et al., 2012). The importance of lipid metabolism, however, has become increasingly appreciated in recent years (Leese, 2015). Oocytes tend to accumulate fatty acids during the terminal stages of oocyte growth prior to IVM (Sturmey et al., 2009). Fatty acid oxidation (FAO) was shown to be indispensable for oocyte meiotic maturation and developmental competence in mice (Dunning et al., 2010) although oocyte lipid content in mice is relatively low compared to that of farm animal species such as cattle and pigs (McEvoy et al., 2000). Despite differences in oocyte lipid quantity, inhibition of FAO with the carnitine palmitoyl transferase 1 (CPT1) activity inhibitor etomoxir during IVM delayed the completion of meiotic maturation in murine, bovine and porcine oocytes (Paczkowski et al., 2014). In contrast, increasing FAO by supplementation of culture media with lipid metabolism modulators such as L-carnitine, resveratrol, conjugated linoleic acid and forskolin significantly improve oocyte competence in terms of fertilization and embryo development in mice (Dunning et al., 2011) and cows (Sutton-Mcdowall et al., 2012). On the other hand, lipids are key structural components of cell membranes and form complexes with glycose and/or proteins (Muro et al., 2014). In addition, lipids are precursors of steroids which have important roles in cell signalling (Kusumi et al., 2012). With the foregoing discussion in mind, the aim of this thesis was to improve our understanding of lipid metabolism, and fatty acid oxidation in particular, during IVM of cattle oocytes, and to investigate the long-term consequences for embryo development and viability. The ultimate goal was to generate improved systems for cattle IVP that would lead to better pregnancy outcomes following embryo transfer