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Cryopreservation of Gametes and Embryos

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Biology".

Deadline for manuscript submissions: closed (15 June 2021) | Viewed by 35896

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Dr. Marc Yeste

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Guest Editor

Biotechnology of Animal and Human Reproduction (TechnoSperm), Department of Biology, Institute of Food and Agricultural Technology, University of Girona, Pic de Peguera 15, 17003 Girona, Spain
Interests: cell biology; molecular biology; reproductive biology; cryobiology; sperm; oocyte; embryo; infertility; human repro-duction; animal reproduction
Special Issues, Collections and Topics in MDPI journals

Dr. Isabel Barranco

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Guest Editor

Department of Veterinary Medical Sciences, University of Bologna, Bologna, Italy
Interests: cryopreservation; semen evaluation; semen preservation; sperm fertility; sperm biology
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Cryopreservation is the most effective technique to preserve gametes and embryos for the long term. Cryopreservation includes slow freezing, mainly used in the case of sperm, and vitrification, mainly used in the case of oocytes and embryos. At present, this technology is combined with other assisted reproductive techniques (ART) and is also useful to preserve fertility. In addition, not only is cryopreservation of gametes and embryos used for humans, but also for livestock species and lab animals. Related to this, many efforts have been conducted in the last few years to improve cell survival upon post-thaw and post-warming, as well as to develop a more global picture of the impact of cryopreservation on cell function and integrity and how cells develop their resilience to withstand freezing and thawing.

Cryopreservation of gametes and embryos is a fascinating realm and has benefitted from outstanding contributions in the last few years. This Special Issue aims to tackle issues of sperm freezing and oocyte and embryo vitrification, mainly focused on humans. Papers dealing with other mammalian species, such as livestock species and laboratory animals, are also welcome if the emphasis is made on their usefulness as a biomedical model for humans rather than on animal production. We welcome both original research and review articles, especially those giving a mechanistic insight on how gametes and embryos withstand cryopreservation. This Special Issue will not consider pure clinical or descriptive manuscripts and case study reports.

Dr. Marc Yeste
Dr. Isabel Barranco
Guest Editors

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Keywords

cryobiology
slow freezing
vitrification
ART
sperm
oocyte
embryo
human species
livestock species
laboratory animals

Published Papers (10 papers)

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Research

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18 pages, 6170 KiB

Open AccessArticle

Optimization of Sperm Cryopreservation Formulation in Portunus trituberculatus

by Le Chang, Chengpeng Lu, Junquan Zhu, Yiner Chen, Chunlin Wang, Changkao Mu and Congcong Hou

Int. J. Mol. Sci. 2023, 24(5), 4358; https://doi.org/10.3390/ijms24054358 - 22 Feb 2023

Viewed by 1251

Abstract

Portunus trituberculatus is a very important marine economic species, and its aquaculture industry has been developing rapidly. However, the phenomenon of marine wild capture of P. trituberculatus and germplasm degradation has become increasingly serious. It is necessary to develop the artificial farming industry and carry out germplasm resource protection, for which sperm cryopreservation technology is an effective method. This research compared three methods (mesh-rubbing, trypsin digestion, and mechanical grinding) for acquiring free sperm, and the best method was mesh-rubbing. Then, the optimal cryopreservation conditions were selected, and the optimal formulation was sterile calcium-free artificial seawater, the optimal cryoprotectant was 20% glycerol, and the best equilibrium time was 15 min at 4 °C. The optimal cooling program was suspending the straws at 3.5 cm on the liquid nitrogen surface for 5 min and then storing them in liquid nitrogen. Finally, the sperm were thawed at 42 °C. However, the expression of sperm-related genes and the total enzymatic activities of frozen sperm were significantly decreased (p < 0.05), which showed that sperm cryopreservation damaged the sperm. Our study improves the sperm cryopreservation technology and the yield of aquaculture in P. trituberculatus. Additionally, the study provides a certain technical basis for the establishment of a sperm cryopreservation library of crustaceans. Full article

(This article belongs to the Special Issue Cryopreservation of Gametes and Embryos)

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17 pages, 2685 KiB

Open AccessArticle

Early Embryo Exposure to Assisted Reproductive Manipulation Induced Subtle Changes in Liver Epigenetics with No Apparent Negative Health Consequences in Rabbit

by Ximo García-Domínguez, Gianfranco Diretto, David S. Peñaranda, Sarah Frusciante, Victor García-Carpintero, Joaquín Cañizares, José S. Vicente and Francisco Marco-Jiménez

Int. J. Mol. Sci. 2021, 22(18), 9716; https://doi.org/10.3390/ijms22189716 - 8 Sep 2021

Cited by 5 | Viewed by 2115

Abstract

Embryo manipulation is a requisite step in assisted reproductive technology (ART). Therefore, it is of great necessity to appraise the safety of ART and investigate the long-term effect, including lipid metabolism, on ART-conceived offspring. Augmenting our ART rabbit model to investigate lipid metabolic outcomes in offspring longitudinally, we detected variations in hepatic DNA methylation ART offspring in the F3 generation for embryonic exposure (multiple ovulation, vitrification and embryo transfer). Through adult liver metabolomics and proteomics, we identified changes mainly related to lipid metabolism (e.g., polyunsaturated fatty acids, steroids, steroid hormone). We also found that DNA methylation analysis was linked to changes in lipid metabolism and apoptosis genes. Nevertheless, these differences did not apparently alter the general health status. Thus, our findings suggest that ART is likely to be a player in embryo epigenetic events related to hepatic homeostasis alteration in adulthood. Full article

(This article belongs to the Special Issue Cryopreservation of Gametes and Embryos)

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14 pages, 1940 KiB

Open AccessArticle

Effects of Vitrification on the Blastocyst Gene Expression Profile in a Porcine Model

by Cristina Cuello, Cristina A. Martinez, Josep M. Cambra, Inmaculada Parrilla, Heriberto Rodriguez-Martinez, Maria A. Gil and Emilio A. Martinez

Int. J. Mol. Sci. 2021, 22(3), 1222; https://doi.org/10.3390/ijms22031222 - 27 Jan 2021

Cited by 18 | Viewed by 2620

Abstract

This study was designed to investigate the impact of vitrification on the transcriptome profile of blastocysts using a porcine (Sus scrofa) model and a microarray approach. Blastocysts were collected from weaned sows (n = 13). A total of 60 blastocysts were vitrified (treatment group). After warming, vitrified embryos were cultured in vitro for 24 h. Non-vitrified blastocysts (n = 40) were used as controls. After the in vitro culture period, the embryo viability was morphologically assessed. A total of 30 viable embryos per group (three pools of 10 from 4 different donors each) were subjected to gene expression analysis. A fold change cut-off of ±1.5 and a restrictive threshold at p-value < 0.05 were used to distinguish differentially expressed genes (DEGs). The survival rates of vitrified/warmed blastocysts were similar to those of the control (nearly 100%, n.s.). A total of 205 (112 upregulated and 93 downregulated) were identified in the vitrified blastocysts compared to the control group. The vitrification/warming impact was moderate, and it was mainly related to the pathways of cell cycle, cellular senescence, gap junction, and signaling for TFGβ, p53, Fox, and MAPK. In conclusion, vitrification modified the transcriptome of in vivo-derived porcine blastocysts, resulting in minor gene expression changes. Full article

(This article belongs to the Special Issue Cryopreservation of Gametes and Embryos)

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16 pages, 5281 KiB

Open AccessArticle

Impact of Maturation and Vitrification Time of Human GV Oocytes on the Metaphase Plate Configuration

by Irene Peinado, Isabel Moya, Paula Sáez-Espinosa, Macarena Barrera, Laura García-Valverde, Raquel Francés, Patricia Torres and María José Gómez-Torres

Int. J. Mol. Sci. 2021, 22(3), 1125; https://doi.org/10.3390/ijms22031125 - 23 Jan 2021

Cited by 10 | Viewed by 3533

Abstract

The combination of in vitro maturation (IVM) techniques and oocyte vitrification (OV) could increase the number of useful oocytes in different types of patients. IVM and subsequent OV is the most widely used clinical strategy. Would the results improve if we reverse the order of the techniques? Here, we evaluated survival, in vitro maturation, time to extrude the first polar body (PB), and the metaphase plate configuration of human prophase I (GV) oocytes before or after their vitrification. Specific, 195 GV oocytes from 104 patients subjected to controlled ovarian stimulation cycles were included. We stablished three experimental groups: GV oocytes vitrified and IVM (Group GV-Vit), GV oocytes IVM and vitrified at MII stage (Group MII-Vit), and GV oocytes IVM (Group not-Vit). All of them were in vitro matured for a maximum of 48 h and fixed to study the metaphase plate by confocal microscopy. According to our results, the vitrification of immature oocytes and their subsequent maturation presented similar survival, maturation, and metaphase plate conformation rates, but a significantly higher percentage of normal spindle than the standard strategy. Additionally, the extension of IVM time to 48 h did not seem to negatively affect the oocyte metaphase plate configuration. Full article

(This article belongs to the Special Issue Cryopreservation of Gametes and Embryos)

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17 pages, 4214 KiB

Open AccessArticle

Effect of Embryo Vitrification on the Steroid Biosynthesis of Liver Tissue in Rabbit Offspring

by Francisco Marco-Jiménez, Ximo Garcia-Dominguez, Marta Domínguez-Martínez, María Pilar Viudes-de-Castro, Gianfranco Diretto, David S. Peñaranda and José S. Vicente

Int. J. Mol. Sci. 2020, 21(22), 8642; https://doi.org/10.3390/ijms21228642 - 16 Nov 2020

Cited by 3 | Viewed by 2046

Abstract

Preimplantation embryo manipulations during standard assisted reproductive technologies (ART) have significant repercussions on offspring. However, few studies to date have investigated the potential long-term outcomes associated with the vitrification procedure. Here, we performed an experiment to unravel the particular effects related to stress induced by embryo transfer and vitrification techniques on offspring phenotype from the foetal period through to prepuberal age, using a rabbit model. In addition, the focus was extended to the liver function at prepuberal age. We showed that, compared to naturally conceived animals (NC), offspring derived after embryo exposure to the transfer procedure (FT) or cryopreservation-transfer procedure (VT) exhibited variation in growth and body weight from foetal life to prepuberal age. Strikingly, we found a nonlinear relationship between FT and VT stressors, most of which were already present in the FT animals. Furthermore, we displayed evidence of variation in liver function at prepuberal age, most of which occurred in both FT and VT animals. The present major novel finding includes a significant alteration of the steroid biosynthesis profile. In summary, here we provide that embryonic manipulation during the vitrification process is linked with embryo phenotypic adaptation detected from foetal life to prepuberal age and suggests that this phenotypic variation may be associated, to a great extent, with the effect of embryo transfer. Full article

(This article belongs to the Special Issue Cryopreservation of Gametes and Embryos)

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22 pages, 2811 KiB

Open AccessArticle

Glutathione Ethyl Ester Protects In Vitro-Maturing Bovine Oocytes against Oxidative Stress Induced by Subsequent Vitrification/Warming

by Tania García-Martínez, Meritxell Vendrell-Flotats, Iris Martínez-Rodero, Erika Alina Ordóñez-León, Manuel Álvarez-Rodríguez, Manel López-Béjar, Marc Yeste and Teresa Mogas

Int. J. Mol. Sci. 2020, 21(20), 7547; https://doi.org/10.3390/ijms21207547 - 13 Oct 2020

Cited by 38 | Viewed by 3272

Abstract

This study aimed to examine whether the addition of glutathione ethyl ester (GSH-OEt) to the in vitro maturation (IVM) medium would improve the resilience of bovine oocytes to withstand vitrification. The effects of GSH-OEt on spindle morphology, levels of reactive oxygen species (ROS), mitochondrial activity and distribution, and embryo developmental potential were assessed together with the expression of genes with a role in apoptosis (BAX, BCL2), oxidative-stress pathways (GPX1, SOD1), water channels (AQP3), implantation (IFN-τ) and gap junctions (CX43) in oocytes and their derived blastocysts. Vitrification gave rise to abnormal spindle microtubule configurations and elevated ROS levels. Supplementation of IVM medium with GSH-OEt before vitrification preserved mitochondrial distribution pattern and diminished both cytoplasmic and mitochondrial ROS contents and percentages of embryos developing beyond the 8-cell stage were similar to those recorded in fresh non-vitrified oocytes. Although not significantly different from control vitrified oocytes, vitrified oocytes after GSH-OEt treatment gave rise to similar day 8-blastocyst and hatching rates to fresh non-vitrified oocytes. No effects of GSH-OEt supplementation were noted on the targeted gene expression of oocytes and derived blastocysts, with the exception of GPX1, AQP3 and CX43 in derived blastocysts. The addition of GSH-OEt to the IVM medium before vitrification may be beneficial for embryo development presumably as the consequence of additional anti-oxidant protection during IVM. Full article

(This article belongs to the Special Issue Cryopreservation of Gametes and Embryos)

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14 pages, 873 KiB

Open AccessArticle

Metabolomic Analysis Reveals Changes in Preimplantation Embryos Following Fresh or Vitrified Transfer

by Ximo Garcia-Dominguez, Gianfranco Diretto, Sarah Frusciante, José Salvador Vicente and Francisco Marco-Jiménez

Int. J. Mol. Sci. 2020, 21(19), 7116; https://doi.org/10.3390/ijms21197116 - 26 Sep 2020

Cited by 6 | Viewed by 2523

Abstract

Although assisted reproduction technologies (ARTs) are recognised as safe, and most of the offspring seem apparently healthy, there is clear evidence that ARTs are associated with changes in the embryo’s developmental trajectory, which incur physiological consequences during the prenatal and postnatal stages of life. The present study aimed to address the influence of early (day-3 embryos) embryo transfer and cryopreservation on embryo survival, size, and metabolome at the preimplantation stage (day-6 embryos). To this end, fresh-transferred (FT) and vitrified-transferred (VT) embryos were compared using naturally-conceived (NC) embryos as a control reference. The results show that as in vitro manipulation was increased (NC < FT < VT), both embryo survival rate (0.91 ± 0.02, 0.78 ± 0.05 and 0.63 ± 0.05, for NC, FT, and VT groups, respectively) and embryo size (3.21 ± 0.49 mm, 2.15 ± 0.51 mm, 1.76 ± 0.46 mm of diameter for NC, FT, and VT groups, respectively) were significantly decreased. Moreover, an unbiased metabolomics analysis showed overall down-accumulation in 40 metabolites among the three experimental groups, with embryo transfer and embryo cryopreservation procedures both exerting a cumulative effect. In this regard, targeted metabolomics findings revealed a significant reduction in some metabolites involved in metabolic pathways, such as the Krebs cycle, amino acids, unsaturated fatty acids, and arachidonic acid metabolisms. Altogether, these findings highlight a synergistic effect between the embryo transfer and vitrification procedures in preimplantation embryos. However, the ex vivo manipulation during embryo transfer seemed to be the major trigger of the embryonic changes, as the deviations added by the vitrification process were relatively smaller. Full article

(This article belongs to the Special Issue Cryopreservation of Gametes and Embryos)

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22 pages, 6161 KiB

Open AccessArticle

In Vitro Maturation with Leukemia Inhibitory Factor Prior to the Vitrification of Bovine Oocytes Improves Their Embryo Developmental Potential and Gene Expression in Oocytes and Embryos

by Meritxell Vendrell-Flotats, Tania García-Martínez, Iris Martínez-Rodero, Manel Lopez-Bejar, Jonathan LaMarre, Marc Yeste and Teresa Mogas

Int. J. Mol. Sci. 2020, 21(19), 7067; https://doi.org/10.3390/ijms21197067 - 25 Sep 2020

Cited by 6 | Viewed by 3994

Abstract

Oocyte cryopreservation has a significant impact on subsequent embryonic development. Herein, we investigated whether supplementing in vitro maturation medium with Leukemia Inhibitory Factor (LIF) prior to vitrification affects embryo development and gene expression at different embryo developmental stages. A panel of genes including maternal effect, epigenetics, apoptosis and heat stress was relatively quantified. The results show reduced cleavage rates after vitrification, regardless of the LIF treatment. Although not statistically different from control-vitrified oocytes, oocyte apoptosis and the blastocyst yield of LIF-vitrified oocytes were similar to their non-vitrified counterparts. Vitrification increased oocyte ZAR1, NPM2 and DPPA3 gene expression while its expression decreased in LIF-vitrified oocytes to similar or close levels to those of non-vitrified oocytes. With a few gene-specific exceptions, vitrification significantly increased the expression of DNMT3A, HDAC1, KAT2A, BAX and BCL2L1 in oocytes and most stages of embryo development, while comparable expression patterns for these genes were observed between LIF-vitrified and non-vitrified groups. Vitrification increased HSPA1A expression in oocytes and HSP90AA1 in 2-cell embryos. Our data suggest that vitrification triggers stage-specific changes in gene expression throughout embryonic development. However, the inclusion of LIF in the IVM medium prior to vitrification stimulates blastocyst development and several other developmental parameters and induces oocytes and embryos to demonstrate gene expression patterns similar to those derived from non-vitrified oocytes. Full article

(This article belongs to the Special Issue Cryopreservation of Gametes and Embryos)

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Review

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17 pages, 762 KiB

Open AccessReview

Cryopreservation of Gametes and Embryos and Their Molecular Changes

by Enrique Estudillo, Adriana Jiménez, Pablo Edson Bustamante-Nieves, Carmen Palacios-Reyes, Iván Velasco and Adolfo López-Ornelas

Int. J. Mol. Sci. 2021, 22(19), 10864; https://doi.org/10.3390/ijms221910864 - 8 Oct 2021

Cited by 31 | Viewed by 9049

Abstract

The process of freezing cells or tissues and depositing them in liquid nitrogen at –196 °C is called cryopreservation. Sub-zero temperature is not a physiological condition for cells and water ice crystals represent the main problem since they induce cell death, principally in large cells like oocytes, which have a meiotic spindle that degenerates during this process. Significantly, cryopreservation represents an option for fertility preservation in patients who develop gonadal failure for any condition and those who want to freeze their germ cells for later use. The possibility of freezing sperm, oocytes, and embryos has been available for a long time, and in 1983 the first birth with thawed oocytes was achieved. From the mid-2000s forward, the use of egg vitrification through intracytoplasmic sperm injection has improved pregnancy rates. Births using assisted reproductive technologies (ART) have some adverse conditions and events. These risks could be associated with ART procedures or related to infertility. Cryopreservation generates changes in the epigenome of gametes and embryos, given that ART occurs when the epigenome is most vulnerable. Furthermore, cryoprotective agents induce alterations in the integrity of germ cells and embryos. Notably, cryopreservation extensively affects cell viability, generates proteomic profile changes, compromises crucial cellular functions, and alters sperm motility. This technique has been widely employed since the 1980s and there is a lack of knowledge about molecular changes. The emerging view is that molecular changes are associated with cryopreservation, affecting metabolism, cytoarchitecture, calcium homeostasis, epigenetic state, and cell survival, which compromise the fertilization in ART. Full article

(This article belongs to the Special Issue Cryopreservation of Gametes and Embryos)

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31 pages, 3102 KiB

Open AccessReview

Characteristics and Cryopreservation of Semen of Sex-Reversed Females of Salmonid Fish

by Sylwia Judycka, Joanna Nynca, Piotr Hliwa and Andrzej Ciereszko

Int. J. Mol. Sci. 2021, 22(2), 964; https://doi.org/10.3390/ijms22020964 - 19 Jan 2021

Cited by 10 | Viewed by 3653

Abstract

Sex reversal has been used as a breeding strategy by salmonid fish to produce genetically and phenotypically single sex populations. Production of all-female fish has great importance for the creation of monosex female triploids of salmonid fish, which are valued for their sterility, lack of female maturation, and larger commercial size. Among salmonids, the majority of rainbow trout (Oncorhynchus mykiss) production is based on all-female production with a high proportion of all-female triploid production in Europe. The main aim of this review is to present the recent knowledge regarding sex-reversed females (SRFs) of salmonid fish. We discuss the methods of sex reversal as well as their effects on the morphology and histology of the reproductive tract. We focus on the characteristics of SRF semen as well as the factors determining semen quality. The lower quality of SRF sperm compared to that of normal males has resulted in the need for the artificial maturation of semen. Most importantly, methods of semen storage—both short-term and long-term (cryopreservation)—that can improve hatchery operations are presented with the special emphasis on recent progress in development of efficient cryopreservation procedures and use of cryopreserved semen in hatchery practice. Moreover, we also address the emerging knowledge concerning the proteomic investigations of salmonid sperm, focusing primarily on the proteomic comparison of normal male and SRF testicular semen and presenting changes in SRF rainbow trout sperm proteome after in vitro incubation in artificial seminal plasma. Full article

(This article belongs to the Special Issue Cryopreservation of Gametes and Embryos)

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