The embryo non-invasive pre-implantation diagnosis era: how far are we?

Advancements in assisted reproduction (AR) methodologies have allowed significant improvements in live birth rates of women who otherwise would not be able to conceive. One of the tools that allowed this improvement is the possibility of embryo selection based on genetic status, performed via preimplantation genetic testing (PGT). Even though the widespread use of PGT from TE biopsy helped to decrease the interval from the beginning of the AR intervention to pregnancy, especially in older patients, in AR, there are still many concerns about the application of this invasive methodology in all cycles. Therefore, recently, researchers started to study the use of cell free DNA (cfDNA) released by the blastocyst in its culture medium to perform PGT, in a method called non-invasive PGT (niPGT). The development of a niPGT would bring the diagnostics power of conventional PGT, but with the advantage of being potentially less harmful to the embryo. Its implementation in clinical practice, however, is under heavy discussion since there are many unknowns about the technique, such as the origin of the cfDNA or if this genetic material is a true representative of the actual ploidy status of the embryo. Available data indicates that there is high correspondence between results observed in TE biopsies and the ones observed from cfDNA, but these results are still contradictory and highly debatable. In the present review, the advantages and disadvantages of niPGT are presented and discussed in relation to tradition TE biopsy-based PGT. Furthermore, there are also presented some other possible non-invasive tools that could be applied in the selection of the best embryo, such as quantification of other molecules as quality biomarkers, or the use artificial intelligence (AI) to identify the best embryos based on morphological and/or morphokitetic parameters.(AU)

The use of resveratrol decreases liquid-extend boar semen fertility, even in concentrations that do not alter semen quality.

In vitro and in vivo assays were conducted to investigate the effects of trans-resveratrol (RVT) on liquid-extended boar semen during 72 h of storage at 17 °C. Thirty-six ejaculates were collected from six boars, evaluated, and extended. RVT was then added at the indicated treatment concentration (0, 0.01, 0.1 or 1 mM), and the ejaculates were cooled to 17 °C and evaluated at 0, 24, 48, and 72 h. Samples were evaluated for sperm motility, kinetics, plasma and acrosome integrity, mitochondrial membrane potential, anion superoxide levels, lipoperoxidation, and antioxidant enzyme activity. In the follow-up experiment, twenty-eight gilts were fixed-time inseminated with 0 or 0.01 mM RVT liquid-extended boar semen. After five days, they were slaughtered, and their reproductive tracts were recovered. The embryos were collected, and the pregnancy, fertility, and viable embryo rates were calculated. In the in vitro assays, total motility, plasma and acrosome membrane integrity, mitochondrial membrane potential, anion superoxide levels, and lipoperoxidation did not change at any of the evaluation times with the use of RVT up to 0.01 mM. RVT decreased SOD activity without changes in GPx. RVT used at 1 mM showed harmful effects for almost all evaluated parameters. For the in vivo assay, the same pregnancy and fertility rates were observed for both groups, while the viable embryo rate was three-fold lower in the 0.01 mM group than in the 0 mM group. The results showed a dichotomous effect of RVT; a low concentration was not harmful in vitro but was catastrophic for embryo viability.

Evidence of Selection Against Damaged Mitochondria During Early Embryogenesis in the Mouse.

There is evidence of a purifying filter acting in the female germline to prevent the expansion of deleterious mutations in the mitochondrial DNA (mtDNA). Given our poor understanding of this filter, here we investigate the competence of the mouse embryo to eliminate dysfunctional mitochondria. Toward that, mitochondria were damaged by photoirradiation of NZB/BINJ zygotes loaded with chloromethyl-X-rosamine (CMXRos). The resultant cytoplasm was then injected into C57BL/6J zygotes to track the levels of NZB/BINJ mtDNA during the preimplantation development. About 30% of NZB/BINJ mtDNA was present after injection, regardless of using photoirradiated or non-photoirradiated cytoplasmic donors. Moreover, injection of photoirradiated-derived cytoplasm did not impact development into blastocysts. However, lower levels of NZB/BINJ mtDNA were present in blastocysts when comparing injection of photoirradiated (24.7% ± 1.43) versus non-photoirradiated (31.4% ± 1.43) cytoplasm. Given that total mtDNA content remained stable between stages (zygotes vs. blastocysts) and treatments (photoirradiated vs. non-photoirradiated), these results indicate that the photoirradiated-derived mtDNA was replaced by recipient mtDNA in blastocysts. Unexpectedly, treatment with rapamycin prevented the drop in NZB/BINJ mtDNA levels associated with injection of photoirradiated cytoplasm. Additionally, analysis of mitochondria-autophagosome colocalization provided no evidence that photoirradiated mitochondria were eliminated by autophagy. In conclusion, our findings give evidence that the mouse embryo is competent to mitigate the levels of damaged mitochondria, which might have implications to the transmission of mtDNA-encoded disease.

Maternal transmission of mitochondrial diseases.

Given the major role of the mitochondrion in cellular homeostasis, dysfunctions of this organelle may lead to several common diseases in humans. Among these, maternal diseases linked to mitochondrial DNA (mtDNA) mutations are of special interest due to the unclear pattern of mitochondrial inheritance. Multiple copies of mtDNA are present in a cell, each encoding for 37 genes essential for mitochondrial function. In cases of mtDNA mutations, mitochondrial malfunctioning relies on mutation load, as mutant and wild-type molecules may co-exist within the cell. Since the mutation load associated with disease manifestation varies for different mutations and tissues, it is hard to predict the progeny phenotype based on mutation load in the progenitor. In addition, poorly understood mechanisms act in the female germline to prevent the accumulation of deleterious mtDNA in the following generations. In this review, we outline basic aspects of mitochondrial inheritance in mammals and how they may lead to maternally-inherited diseases. Furthermore, we discuss potential therapeutic strategies for these diseases, which may be used in the future to prevent their transmission.

Intrafollicular barriers and cellular interactions during ovarian follicle development

Follicles are composed of different interdependent cell types including oocytes, cumulus, granulosa, and theca cells. Follicular cells and oocytes exchange signaling molecules from the beginning of the development of the primordial follicles until the moment of ovulation. The follicular structure transforms during folliculogenesis; barriers form between the germ and the somatic follicular cells, and between the somatic follicular cells. As such, communication systems need to adapt to maintain the exchange of signaling molecules. Two critical barriers are established at different stages of development: the zona pellucida, separating the oocyte and the cumulus cells limiting the communication through specific connections, and the antrum, separating subpopulations of follicular cells. In both situations, communication is maintained either by the development of specialized connections as transzonal projections or by paracrine signaling and trafficking of extracellular vesicles through the follicular fluid. The bidirectional communication between the oocytes and the follicle cells is vital for driving folliculogenesis and oogenesis. These communication systems are associated with essential functions related to follicular development, oocyte competence, and embryonic quality. Here, we discuss the formation of the zona pellucida and antrum during folliculogenesis, and their importance in follicle and oocyte development. Moreover, this review discusses the current knowledge on the cellular mechanisms such as the movement of molecules via transzonal projections, and the exchange of extracellular vesicles by follicular cells to overcome these barriers to support female gamete development. Finally, we highlight the undiscovered aspects related to intrafollicular communication among the germ and somatic cells, and between the somatic follicular cells and give our perspective on manipulating the above-mentioned cellular communication to improve reproductive technologies.

Intrafollicular barriers and cellular interactions during ovarian follicle development

Follicles are composed of different interdependent cell types including oocytes, cumulus, granulosa, and theca cells. Follicular cells and oocytes exchange signaling molecules from the beginning of the development of the primordial follicles until the moment of ovulation. The follicular structure transforms during folliculogenesis; barriers form between the germ and the somatic follicular cells, and between the somatic follicular cells. As such, communication systems need to adapt to maintain the exchange of signaling molecules. Two critical barriers are established at different stages of development: the zona pellucida, separating the oocyte and the cumulus cells limiting the communication through specific connections, and the antrum, separating subpopulations of follicular cells. In both situations, communication is maintained either by the development of specialized connections as transzonal projections or by paracrine signaling and trafficking of extracellular vesicles through the follicular fluid. The bidirectional communication between the oocytes and the follicle cells is vital for driving folliculogenesis and oogenesis. These communication systems are associated with essential functions related to follicular development, oocyte competence, and embryonic quality. Here, we discuss the formation of the zona pellucida and antrum during folliculogenesis, and their importance in follicle and oocyte development. Moreover, this review discusses the current knowledge on the cellular mechanisms such as the movement of molecules via transzonal projections, and the exchange of extracellular vesicles by follicular cells to overcome these barriers to support female gamete development. Finally, we highlight the undiscovered aspects related to intrafollicular communication among the germ and somatic cells, and between the somatic follicular cells and give our perspective on manipulating the above-mentioned cellular communication to improve reproductive technologies.(AU)

Contributions from the ovarian follicular environment to oocyte function

The magnitude of oocytes role for embryo development is categorical. This unique cell contains the machineries and cellular components necessary to remodel male and female chromatin, to sustain early development and to, ultimately, generate a complete and complex individual. However, to gain these competences before fertilization, the oocyte undergoes several morphological, cellular and molecular changes during its lifetime enclosed in the ovarian follicle. This review will briefly revisit how the oocyte orchestrate the follicular cells, and how molecules transit to the oocyte from the innermost (cumulus) and outermost (antrum and granulosa cells) layers surrounding the follicle-enclosed oocyte. Finally, we will discuss the interferences of in vitro culture conditions in the communication of the oocyte with its surrounding cells and the potential strategies to modulate these communication systems to increase oocyte competence.(AU)

Contributions from the ovarian follicular environment to oocyte function

The magnitude of oocyte’s role for embryo development is categorical. This unique cell contains the machineries and cellular components necessary to remodel male and female chromatin, to sustain early development and to, ultimately, generate a complete and complex individual. However, to gain these competences before fertilization, the oocyte undergoes several morphological, cellular and molecular changes during its lifetime enclosed in the ovarian follicle. This review will briefly revisit how the oocyte orchestrate the follicular cells, and how molecules transit to the oocyte from the innermost (cumulus) and outermost (antrum and granulosa cells) layers surrounding the follicle-enclosed oocyte. Finally, we will discuss the interferences of in vitro culture conditions in the communication of the oocyte with its surrounding cells and the potential strategies to modulate these communication systems to increase oocyte competence.

Effects of melatonin during IVM in defined medium on oocyte meiosis, oxidative stress, and subsequent embryo development.

Melatonin may have beneficial effects when used in oocyte maturation and embryo development culture. The effect of melatonin during IVM on meiosis resumption and progression in bovine oocytes and on expression of antioxidant enzymes, nuclear fragmentation and free radicals, as well as on embryo development were assessed. Cumulus-oocyte complexes were matured in vitro with melatonin (10(-9) and 10(-6) M), FSH (positive control), or without hormones (negative control) in defined medium. Maturation rates were evaluated at 6, 12, 18, and 24 hours. Transcripts for antioxidant enzymes (CuZnSOD, MnSOD, and glutathione peroxidase 4 (GPX4)) in oocytes and cumulus cells, nuclear fragmentation in cumulus cells (TUNEL) and reactive oxygen species levels in oocytes (carboxy-H2 difluorofluorescein diacetate) were determined at 24 hours IVM. Effect of treatments on embryo development was determined after in vitro fertilization and culture. At 12 hours, meiosis resumption rates in FSH and melatonin-treated groups were similar (69.6%-81.8%, P > 0.05). At 24 hours, most oocytes were in metaphase II, with FSH showing highest rates (90.0%, P < 0.05) compared with the other groups (51.6%-69.1%, P > 0.05). In cumulus cells, MnSOD expression was higher in FSH group (P < 0.05) whereas Cu,ZnSOD transcripts were more abundant in melatonin group (10(-6)M; P < 0.05). Nuclear fragmentation in cumulus cells was highest in controls (37.4%/10,000 cells; P < 0.05) and lower in FSH and 10(-6)M melatonin (29.4% and 25.6%/10,000 cells, respectively). Reactive oxygen species levels were lower in oocytes matured with 10(-6)M melatonin than in control and FSH groups (P < 0.05). Embryo development from oocytes matured only with melatonin was similar to those matured in complete medium (P > 0.05). In conclusion, although melatonin during IVM in a defined medium does not stimulate nuclear maturation progression it does stimulate meiosis resumption and such treated oocytes support subsequent embryo development. Melatonin also shows cytoprotective effects on cumulus-oocyte complexes.