Human Sperm Cryopreservation: Update on Techniques, Effect on DNA Integrity, and Implications for ART

Cryopreservation of human spermatozoa—introduced in the 1960's—has been recognized as an efficient procedure for management of male fertility before therapy for malignant diseases, vasectomy or surgical infertility treatments, to store donor and partner spermatozoa before assisted reproduction treatments and to ensure the recovery of a small number of spermatozoa in severe male factor infertility. Despite the usefulness of it, cryopreservation may lead to deleterious changes of sperm structure and function: while the effects of cryopreservation on cells are well documented, to date there is no agreement in the literature on whether or not cryopreservation affects sperm chromatin integrity or on the use of a unique and functional protocol for the freezing-thawing procedure. Therefore, sperm cryopreservation is an important component of fertility management and much of its successful application seems to affect the reproductive outcome of assisted reproduction technologies (ART): appropriate use of cryoprotectants before and sperm selection technologies after cryopreservation seem to have the greatest impact on preventing DNA fragmentation, thus improving sperm cryosurvival rates

The enigmatic morula: mechanisms of development, cell fate determination, self-correction and implications for ART

Background: Assisted reproduction technology offers the opportunity to observe the very early stages of human development. However, due to practical constraints, for decades morphological examination of embryo development has been undertaken at a few isolated time points at the stages of fertilisation (day 1), cleavage (day 2-3) and blastocyst (day 5-6). Rather surprisingly, the morula stage (day 3-4) has been so far neglected, despite its involvement in crucial cellular processes and developmental decisions. Objective and rationale: The objective of this review is to collate novel and unsuspected insights into developmental processes occurring during formation of the morula, highlighting the key importance of this stage for a better understanding of preimplantation development and an improvement of ART. Search method: PubMed was used to search the MEDLINE database for peer-reviewed English-language original articles and reviews concerning the morula stage in mammals. Searches were performed by adopting ‘embryo’, ‘morula’, ‘compaction’, ‘cell fate’ and ‘IVF/assisted reproduction’ as main terms, in association with other keywords expressing concepts relevant to the subject (e.g. cell polarity). The most relevant publications, i.e. those concerning major phenomena occurring during formation of the morula in established experimental models and the human species, were assessed and discussed critically.Outcomes: Novel live cell imaging technologies and cell biology studies have extended our understanding of morula formation as a key stage for the development of the blastocyst and determination of the inner cell mass (ICM) and the trophectoderm (TE). Cellular processes, such as dynamic formation of filopodia and cytoskeleton-mediated zippering cell-to-cell interactions, intervene to allow cell compaction (a geometrical requisite essential for development) and formation of the blastocoel, respectively. At the same time, differential orientation of cleavage planes, cell polarity and cortical tensile forces interact and cooperate to position blastomeres either internally or externally, thereby influencing their cellular fate. Recent time lapse microscopy (TLM) observations also suggest that in the human the process of compaction may represent an important checkpoint for embryo viability, through which chromosomally abnormal blastomeres are sensed and eliminated by the embryo.Wider implications: In clinical embryology, the morula stage has been always perceived as a ‘black box’ in the continuum of preimplantation development. This has dictated its virtual exclusion from mainstream ART procedures. Recent findings described in this review indicate that the morula, and the associated process of compaction, as a crucial stage not only for the formation of the blastocyst, but also for the health of the conceptus. This understanding may open new avenues for innovative approaches to embryo manipulation, assessment and treatment

Does the molecular and metabolic profile of human granulosa cells correlate with oocyte fate? New insights by Fourier transform infrared microspectroscopy analysis

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Ultrastructural features of human metaphase II oocytes subjected to slow freezing or vitrification in an IVF program: a comparative analysis

During the past two decades important advances have been made in the field of assisted reproduction by using oocyte cryopreservation. However, mature (metaphase II) oocytes are very susceptible to cryodamage. In order to contribute to the identification of a cryopreservation protocol with minimal side effects on the oocyte structure and function, we evaluated and compared the subcellular features of human oocytes cryopreserved either with slow (controlled rate) freezing or vitrification (ultrarapid freezing). Supernumerary human metaphase II oocytes were donated by consenting patients enrolled in an IVF program. The age of these women ranged from 27 to 32 years old. The eggs were cryopreserved using slow freezing with 1.5M propanediol + 0.2M sucrose concentration or a closed vitrification system (Cryotip Irvine Scientific CA). Fresh oocytes were used as controls. Samples were fixed and prepared for light and transmission electron microscopy (LM and TEM) observations. By LM, all the oocytes were generally rounded, 90-100 microns in diameter, with regular ooplasm showing uniform distribution of organelles. By TEM, mitochondria-smooth endoplasmic reticulum (M-SER) aggregates were the most common structures found in all the oocytes fixed or cryopreserved within 3-4 hours after the retrieval. M-SER aggregates appeared instead partially replaced by mitochondria-vesicle complexes when oocytes were maintained in culture for a prolonged period of time. A slight to moderate vacuolization was found in the cytoplasm of the oocytes subjected to slow freezing. Slight microvacuolization was also found in vitrified oocytes, whereas vacuoles were almost completely absent in fresh controls. Amount and density of cortical granules (CGs) appeared abnormally reduced in all cryopreserved oocytes, irrespective of the protocol applied. In conclusion, it has been evidenced that prolonged stay in culture induces an intracellular membrane “recycling” in the oocytes, that causes the transformation of slender, anastomosed SER tubules into rounded vesicles surrounded by mitochondria, whose role is still uncertain. In addition, even though all cryopreservation protocols studied ensured a good overall preservation of the oocyte, vacuolization appears as a recurrent form of cell damage. This happens both during slow freezing and, at a lesser extent, during vitrification using a closed device. In addition, premature CG exocytosis was observed in both groups

Detailed investigation into the cytogenetic constitution and pregnancy outcome of replacing mosaic blastocysts detected with the use of high-resolution next-generation sequencing

Objective: To determine the pregnancy outcome potential of mosaic embryos, detected by means of preimplantation genetic screening (PGS) with the use of next-generation sequencing (NGS). Design: Retrospective study. Setting: Genetics laboratories. Patient(s): PGS cycles during which either mosaic or euploid embryos were replaced. Intervention(s): Blastocysts were biopsied and processed with the use of NGS, followed by frozen embryo transfer. Trophectoderm (TE) biopsies were classified as mosaic if they had 20%–80% abnormal cells. Main Outcome Measure(s): Implantation, miscarriage rates, and ongoing implantation rates (OIRs) were compared between euploid and types of mosaic blastocysts. Result(s): Complex mosaic embryos had a significantly lower OIR (10%) than aneuploidy mosaic (50%), double aneuploidy mosaic (45%), and segmental mosaic (41%). There was a tendency for mosaics with 40%–80% abnormal cells to have a lower OIR than those with 40% abnormal cells and those with multiple mosaic abnormalities (chaotic mosaics) are likely to have lower OIRs and should be given low transfer priority

Science and technology roadmap for graphene, related two-dimensional crystals, and hybrid systems

We present the science and technology roadmap for graphene, related two-dimensional crystals, and hybrid systems, targeting an evolution in technology, that might lead to impacts and benefits reaching into most areas of society. This roadmap was developed within the framework of the European Graphene Flagship and outlines the main targets and research areas as best understood at the start of this ambitious project. We provide an overview of the key aspects of graphene and related materials (GRMs), ranging from fundamental research challenges to a variety of applications in a large number of sectors, highlighting the steps necessary to take GRMs from a state of raw potential to a point where they might revolutionize multiple industries. We also define an extensive list of acronyms in an effort to standardize the nomenclature in this emerging field.Peer ReviewedPostprint (published version

Expatriates managers' cultural intelligence as promoter of knowledge transfer in multinational companies

This study analyzes the role of the Cultural Intelligence (CQ) of expatriate managers in the processes of Conventional (CKT) and Reverse Knowledge Transfer (RKT) in Multinational Companies (MNCs). The Partial Least Squares-Structural Equation Modeling (PLS-SEM) technique was adopted to analyze the data from a survey of 103 senior expatriate managers working in Croatia. The study reveals how CQ, in all of its four dimensions (metacognitive, cognitive, behavioral, and motivational), acts as a knowledge de-codification and codification filter, assisting managers in the Knowledge Transfer process. The study also reveals how previous international experience does not moderate the positive effect of CQ on both CKT and RKT, offering important theoretical and practical insights to support MNCs in the KT process