Jason E. Swain

ART failure: oocyte contributions to unsuccessful fertilization

BACKGROUND The complexity of fertilization failure during assisted reproductive technologies (ART) is often under-appreciated, as this failure can occur at any number of essential mechanistic and cellular events. Importantly, successful fertilization is heavily dependent upon inherent qualities of the oocytes, and thus reliant upon fidelity of oocyte maturation. METHODS Pubmed and medline were searched up to April 2008 for papers on oocyte fertilization and its mechanistic components. References to clinical/human studies were selected wherever possible. RESULTS Successful oocyte maturation cannot simply be determined via visual assessment of polar body extrusion, but rather entails coordination of numerous cytoplasmic processes not readily observed. Proper regulation of intra-oocyte signaling cascades is crucial for sufficient production and storage of carbohydrates and proteins, successful relocation of organelles and regulation of metabolic pathways required for an apparently mature metaphase II oocyte to complete subsequent fertilization events; such as cumulus penetration, sperm/oocyte binding, fusion, oocyte activation, sperm processing and pronuclear (PN) formation. Regulation of oocyte maturation begins during oocyte growth and is intimately connected with events influencing folliculogenesis. Therefore, the oocyte is subject to a multitude of potential effector impacting fertilization potential and developmental competence long before encountering the artificial environment of the IVF laboratory. CONCLUSIONS Although meticulous care and continued research is essential for future improvement, failure to fertilize and properly form PN following clinical ART is likely to be dependent on historical events in oocyte maturation, not easily explained or prevented through simple modification of contemporary laboratory protocols.

Direct effects of leptin on mouse reproductive function: regulation of follicular, oocyte, and embryo development.

Abstract Because body condition can affect reproduction, research has focused on the role of leptin, a body condition signal, in regulation of reproductive function. Objectives of this study were to determine if leptin supplementation directly affects 1) ovarian follicle growth and function, 2) oocyte maturation, or 3) preimplantation embryo development. Follicles cultured in the presence of recombinant mouse leptin resulted in a significant decrease in rate of follicle, but not oocyte, growth in a dose-dependent manner, with higher doses of leptin inhibiting growth. Leptin was also found to significantly increase stimulated progesterone, estradiol, and testosterone production/secretion by cultured follicles in a dose-dependent manner, with higher concentrations of leptin significantly increasing steroidogenesis. Culture of fully grown cumulus-enclosed germinal vesicle-intact (GV) mouse oocytes in the presence of increasing concentrations of leptin (0, 12.5, 25, 50, 100 ng/ml) had no effect on germinal vesicle breakdown (GVBD) or development to metaphase II (MII). Similarly, fully grown denuded oocytes showed no difference in GVBD at any concentration of leptin. However, maturation of denuded oocytes with 100 ng/ml leptin resulted in significantly reduced development to MII compared with oocytes matured with 0 or 12.5 ng/ml leptin. Culture of one-cell mouse embryos in increasing concentrations of leptin had no effect on cleavage or blastomere degeneration at 24 h of culture. Exposure of embryos for the first 96 h of development to increasing concentrations of leptin did not significantly affect total or expanded blastocyst development or hatching of blastocysts from zona pellucida. These results indicate leptin directly enhances insulin and gonadotropin-stimulated ovarian steroidogenesis, compromises denuded oocyte maturation, yet has no direct effect on preimplantation embryo development.

The great debate: Varicocele treatment and impact on fertility

OBJECTIVE To evaluate the current literature on the impact and potential mechanisms of varicocele repair on male fertility. DESIGN Pertinent articles were identified through computer PubMed search on varicocele repair and male factor infertility. References of selected articles were hand searched for additional citations. CONCLUSION(S) Varicocele repair has been shown to reverse a spectrum of effects contributing to men with impaired fertility. Clinical studies on the intervention have illustrated variable effects on postoperative sperm parameters and pregnancy rates (PR). Studies with conflicting results suffer from a significant number of confounding variables such as variable repair technique or lack of controls. Additional studies are warranted on the role of modern microsurgical varicocelectomy given the improvements in assisted reproductive technologies (ART).

Advances in embryo culture platforms: novel approaches to improve preimplantation embryo development through modifications of the microenvironment

BACKGROUND The majority of research aimed at improving embryo development in vitro has focused on manipulation of the chemical environment, examining details such as energy substrate composition and impact of various growth factors or other supplements. In comparison, relatively little work has been done examining the physical requirements of preimplantation embryos and the role culture platforms or devices can play in influencing embryo development. METHODS Electronic searches were performed using keywords centered on embryo culture techniques using PUBMED through June 2010 and references were searched for additional research articles. RESULTS Various approaches to in vitro embryo culture that involve manipulations of the physical culture environment are emerging. Novel culture platforms being developed examine issues such as media volume and embryo spacing. Furthermore, methods to permit dynamic embryo culture with fluid flow and embryo movement are now available, and novel culture surfaces are being tested. CONCLUSIONS Although several factors remain to be studied to optimize efficiency, manipulations of the embryo culture microenvironment through novel culture devices may offer a means to improve embryo development in vitro. Reduced volume systems that reduce embryo spacing, such as the well-of-the-well approach, appear beneficial, although more work is needed to verify the source of their true benefit in human embryos. Emerging microfluidic technology appears to be a promising approach. However, along with the work on specialized culture surfaces, more information is required to determine the impact on human embryo development.

Regulation of spindle and chromatin dynamics during early and late stages of oocyte maturation by aurora kinases

Examination of factors regulating oocyte chromatin remodeling is crucial to circumvent embryonic aneuploidy and resulting defects. Aurora kinases (AURK) are involved in regulation of chromatin remodeling, however, little attention has been paid to AURKs in regard to oocyte maturation. Meiotically incompetent mouse oocytes contain transcripts for all three Aurk isoforms: A, B and C. Upon achieving meiotic competence, oocytes showed significant increases in transcript levels of all three Aurk isoforms and transcript levels remained unchanged as oocytes progressed through meiosis, with AurkA being the predominant isoform. Inhibition of oocyte AURKs during the prophase–metaphase I (MI) transition via inhibitor ZM447439 (ZM) had no effect on germinal vesicle breakdown. However, meiotic spindles were malformed, and microtubule organizing centers and chromatin were scattered. Chromosomal spreads of MI oocytes indicated AURK inhibition resulted in abnormal chromosome condensation. Furthermore, inhibition of AURK during prophase I–MII prevented completion of MII and extrusion of the polar body. Inhibition of AURKs during the MI–MII transition resulted in significantly fewer cells progressing to MII and induced aberrant chromatin remodeling. Further analysis indicated that inhibition of AURKs resulted in absence of histone-H3 phosphorylation at serine 10 and 28. These data suggest a ZM-sensitive AURK may be an oocyte histone-H3 kinase capable of regulating chromatin remodeling throughout oocyte meiosis, both pre- and post-MI.

Optimizing the culture environment in the IVF laboratory: impact of pH and buffer capacity on gamete and embryo quality

Supplying and maintaining appropriate culture conditions is critical to minimize stress imposed upon gametes and embryos and to optimize the in-vitro environment. One parameter that requires close scrutiny in this endeavour is pH. Though embryos have a limited ability to regulate their internal pH (pH(i)), oocytes lack robust mechanisms. Thus, careful attention to external pH (pH(e)) of culture media is imperative in IVF. Ability to withstand deviations in hydrogen ion concentration varies depending on culture conditions, as well as laboratory procedures. Cryopreserved--thaw--thawed embryos, as well as denuded oocytes, are especially susceptible to perturbations in pH(e). Therefore, proper setting, monitoring and stabilizing of pH(e) during IVF laboratory procedures is a crucial component of a rigorous quality control programme. Here, importance of both pH(i) and pH(e) in respect to gamete and embryo quality are discussed. Furthermore, factors influencing selection of pH(e), as well as emerging methods to stabilize pH(e) in the IVF laboratory are detailed.

Thinking big by thinking small: application of microfluidic technology to improve ART

In Vitro Fertilization (IVF) laboratories often carry a penchant to resist change while in the pursuit of maintaining consistency in laboratory conditions. However, implementation of new technology is often critical to expand scientific discoveries and to improve upon prior successes to advance the field. Microfluidic platforms represent a technology that has the potential to revolutionize the fundamental processes of IVF. While the focus of microfluidic application in IVF has centered on embryo culture, the innovative platforms carry tremendous potential to improve other procedural steps and represents a possible paradigm shift in how we handle gametes and embryos. The following review will highlight application of various microfluidic platforms in IVF for use in maturation, manipulation, culture, cryopreservation and non-invasive quality assessment; pointing out new insights gained into functions of sperm, oocytes and embryos. Platform design and function will also be discussed, focusing on limitations, advancements and future refinements that can further aid in their clinical implementation.

Proper Chromatin Condensation and Maintenance of Histone H3 Phosphorylation During Mouse Oocyte Meiosis Requires Protein Phosphatase Activity

Abstract We have shown okadaic acid (OA) and calyculin-A (CLA) inhibition of mouse oocyte phosphoprotein phosphatase 1 (PPP1C) and/or phosphoprotein phosphatase 2A (PPP2CA) results in aberrant chromatin condensation, as evidenced by the inability to resolve bivalents. Phosphorylation of histone H3 at specific residues is thought to regulate chromatin condensation. Therefore, we examined changes in histone H3 phosphorylation during oocyte meiosis and the potential regulation by protein PPPs. Western blot and immunocytochemical analysis revealed histone H3 phosphorylation changed during mouse oocyte meiosis, with changes in chromatin condensation. Germinal vesicle-intact (GV-intact; 0 h) oocytes had no phospho-Ser10 but did have phospho-Ser28 histone H3. Oocytes that had undergone germinal vesicle breakdown (GVBD; 2 h) and progressed to metaphase I (MI; 7 h) and MII (16 h) had phosphorylated Ser10 and Ser28 histone H3 associated with condensed chromatin. To determine whether OA-induced aberrations in chromatin condensation were due to alterations in levels of histone H3 phosphorylation, we assessed phosphorylation of Ser10 and Ser28 residues following PPP inhibition. Oocytes treated with OA (1 μM) displayed increased phosphorylation of histone H3 at both Ser10 and Ser28 compared with controls. To begin to elucidate which OA-sensitive PPP is responsible for regulating chromatin condensation and histone H3 phosphorylation, we examined spatial and temporal localization of OA-sensitive PPPs, PPP1C, and PPP2CA. PPPC2A did not localize to condensed chromatin, whereas PPP1beta (PPP1CB) associated with condensing chromatin in GVBD, MI, and MII oocytes. Additionally, Western blot and immunocytochemistry confirmed presence of the PPP1C regulatory inhibitor subunit 2 (PPP1R2) in oocytes at condensed chromatin during meiosis and indicated a change in PPP1R2 phosphorylation. Inhibition of oocyte glycogen synthase kinase 3 (GSK3) appeared to regulate phosphorylation of PPP1R2. Furthermore, inhibition of GSK3 resulted in aberrant oocyte bivalent formation similar to that observed following PPP inhibition. These data suggest that PPP1CB is the OA/CLA-sensitive PPP that regulates oocyte chromatin condensation through regulation of histone H3 phosphorylation. Furthermore, GSK3 inhibition results in aberrant chromatin condensation and appears to regulate phosphorylation of PPP1R2.

Rethinking In Vitro Embryo Culture: New Developments in Culture Platforms and Potential to Improve Assisted Reproductive Technologies

ABSTRACT The preponderance of research toward improving embryo development in vitro has focused on manipulation of the chemical soluble environment, including altering basic salt composition, energy substrate concentration, amino acid makeup, and the effect of various growth factors or addition or subtraction of other supplements. In contrast, relatively little work has been done examining the physical requirements of preimplantation embryos and the role culture platforms or devices can play in influencing embryo development within the laboratory. The goal of this review is not to reevaluate the soluble composition of past and current embryo culture media, but rather to consider how other controlled and precise factors such as time, space, mechanical interactions, gradient diffusions, cell movement, and surface interactions might influence embryo development. Novel culture platforms are being developed as a result of interdisciplinary collaborations between biologists and biomedical, material, chemical, and mechanical engineers. These approaches are looking beyond the soluble media composition and examining issues such as media volume and embryo spacing. Furthermore, methods that permit precise and regulated dynamic embryo culture with fluid flow and embryo movement are now available, and novel culture surfaces are being developed and tested. While several factors remain to be investigated to optimize the efficiency of embryo production, manipulation of the embryo culture microenvironment through novel devices and platforms may offer a pathway toward improving embryo development within the laboratory of the future.

Biological pH buffers in IVF: help or hindrance to success

PurposeMinimizing environmental stress helps maintain cellular homeostasis and is a crucial component in optimizing embryo development in vitro and resulting ART success. One stressor of particular interest is pH. Biologic buffers, such as HEPES and MOPS, are valuable tools for stabilizing pH. The objective of this manuscript is to summarize efficacy and impact of various pH buffers used during IVF lab proceduresMethodsKeyword searches were performed using Pubmed and Medline and relevant literature reviewed.ResultsVarious pH buffers have been used with varying degrees of success for gamete and embryo processing in a variety of animal species, as well as in human.ConclusionThough biologic buffers off a means to improve pH stability, not all buffers may be appropriate for use with gametes and embryos. Specific buffers may have undesired effects, and these may be buffer, species, cell type or concentration dependent. Continued research is needed to further refine and improve the use of biologic buffers for use in human ART.