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MicroRNAs (miRNAs) are small non-encoding RNAs that actively regulate biological and physiological processes, and play an important role in regulating gene expression in all cells, especially in most animal cells, including oocytes and embryos. The expression of miRNAs at the right time and place is crucial for the oocyte’s maturation and the embryo’s subsequent development. Although assisted reproductive techniques (ART) have helped to solve many infertility problems, they cause changes in the expression of miRNA and genes in oocytes and preimplantation embryos, and the effect of these changes on the future of offspring is unknown, and has caused concerns. The relevant genomic alterations commonly imposed on embryos during cryopreservation may have potential epigenetic risks. Understanding the biological functions of miRNAs in frozen maturated oocytes may provide a better understanding of embryonic development and a comparison of fertility conservation in female mammals. With the development of new techniques for genomic evaluation of preimplantation embryos, it has been possible to better understand the effects of ART. The results of various articles have shown that freezing of oocytes and the cryopreservation method are effective for the expression of miRNAs and, in some cases, cause changes in the expression of miRNAs and epigenetic changes in the resulting embryo. This literature review study aimed to investigate the effects of oocyte cryopreservation in both pre-maturation and post-maturation stages, the cryopreservation method and the type of cryoprotectants (CPA) used on the expression of some epigenetic-related genes and miRNAs.
Since the birth of the first baby by in vitro fertilization in 1978, more than 9 million children have been born worldwide using medically assisted reproductive treatments. Fertilization naturally takes place in the maternal oviduct where unique physiological conditions enable the early healthy development of the embryo. During this dynamic period of early development major waves of epigenetic reprogramming, crucial for the normal fate of the embryo, take place. Increasingly, over the past 20 years concerns relating to the increased incidence of epigenetic anomalies in general, and genomic-imprinting disorders in particular, have been raised following assisted reproduction technology (ART) treatments. Epigenetic reprogramming is particularly susceptible to environmental conditions during the periconceptional period and non-physiological conditions such as ovarian stimulation, in vitro fertilization and embryo culture, as well as cryopreservation procedure, might have the potential to independently or collectively contribute to epigenetic dysregulation. Therefore, this narrative review offers a critical reappraisal of the evidence relating to the association between embryo cryopreservation and potential epigenetic regulation and the consequences on gene expression together with long-term consequences for offspring health and wellbeing. Current literature suggests that epigenetic and transcriptomic profiles are sensitive to the stress induced by vitrification, in terms of osmotic shock, temperature and pH changes, and toxicity of cryoprotectants, it is therefore, critical to have a more comprehensive understanding and recognition of potential unanticipated iatrogenic-induced perturbations of epigenetic modifications that may or may not be a consequence of vitrification.
One of the prominent peculiarities of nanoparticles (NPs) is their ability to cross biological barriers. Therefore, the development of NPs with different properties has great therapeutic potential in the area of reproduction because the association of drugs, hormones and other compounds with NPs represents an alternative for delivering substances directly at a specific site and for treatment of reproductive problems. Additionally, lipid-based NPs can be taken up by the tissues of patients with ovarian failure, deep endometriosis, testicular dysfunctions, etc., opening up new perspectives for the treatment of these diseases. The development of nanomaterials with specific size, shape, ligand density and charge certainly will contribute to the next generation of therapies to solve fertility problems in humans. Therefore, this review discusses the potential of NPs to treat reproductive disorders, as well as to regulate the levels of the associated hormones. The possible limitations of the clinical use of NPs are also highlighted.
Commercial application of embryo transfer in pig breeding is dependent on the storage of embryos. The aim of this study was to assess the embryo quality of in vitro-produced blastocysts after 3 h liquid storage at 37°C in CO2-free medium by evaluating morphology, in vitro developmental capacity and apoptosis. Blastocysts at days 5 and 6 post-fertilization were randomly allocated to the storage group (HEPES-buffered NCSU-23 medium including bovine serum albumin in a portable embryo transport incubator at 37°C) or a control group (porcine blastocyst medium in a conventional culture incubator). Thereafter, blastocysts were evaluated for morphology and stained to assess apoptosis straight after the 3 h storage period or after a further 24 h conventional incubation. There was no significant difference between the storage and control group after 3 h storage and the further 24 h conventional incubation for any of the parameters, nor for apoptosis straight after the 3 h storage. Embryos that reached the blastocyst stage at day 5 showed less apoptosis (6.6% vs 10.9%, P = 0.01) and a trend for a higher rate of developmental capacity (70.6% vs 51.5%, P = 0.089) than embryos reaching the blastocyst stage on day 6. In conclusion, in vitro-produced porcine blastocysts can be stored for 3 h at physiological temperature in transportable incubators using a CO2-independent medium without compromising quality.
Mammalian oocytes not fertilized immediately after ovulation can undergo ageing and a rapid decline in quality. The addition of antioxidants can be an efficient approach to delaying the oocyte ageing process. Onion peel extract (OPE) contains quercetin and other flavonoids with natural antioxidant activities. In this study, we investigated the effect of OPE on mouse oocyte ageing and its mechanism of action. The oocytes were aged in vitro in M16 medium for 16 h after adding OPE at different concentrations (0, 50, 100, 200, and 500 μg/ml). The addition of 100 μg/ml OPE reduced the oocyte fragmentation rate, decreased the reactive oxygen species (ROS) level, increased the glutathione (GSH) level, and improved the mitochondrial membrane potential compared with the control group. The addition of OPE also increased the expression of SOD1, CAT, and GPX3 genes, and the caspase-3 activity in OPE-treated aged oocytes was significantly lower than that in untreated aged oocytes and similar to that in fresh oocytes. These results indicated that OPE delayed mouse oocyte ageing by reducing oxidative stress and apoptosis and enhancing mitochondrial function.
The inability to support the growth and development of a mature fetus up to delivery results in significant human suffering. Current available solutions include adoption, surrogacy, and uterus transplantation. However, these options are subject to several ethical, religious, economic, social, and medical concerns. Ectogenesis is the process in which an embryo develops in an artificial uterus from implantation through to the delivery of a live infant. This current narrative review summarizes the state of recent research focused on human ectogenesis. First, a literature search was performed to identify published reports of previous experiments and devices used for embryo implantation in an extracorporeally perfused human uterus. Furthermore, studies fitting that aim were selected and critically evaluated. Results were synthesized, interpreted, and used to design a prospective strategy for future research. Therefore, this study suggests that full ectogenesis might be obtained using a computer-controlled system with extracorporeal blood perfusion provided by a digitally controlled heart–lung–kidney system. From a clinical perspective, patients who will derive significant benefits from this technology are mainly those women diagnosed with anatomical abnormalities of the uterus and those who have undergone previous hysterectomies, numerous abortions, and experienced premature birth. Ectogenesis is the complete development of an embryo in an artificial uterus. It represents the solutions for millions of women suffering from premature deliveries, and the inability to supply growth and development of embryos/fetuses in the womb. In the future, ectogenesis might replace uterine transplantation and surrogacy.
In the present study, the cryoprotective effects of Lolium perenne antifreeze protein (LpAFP) on the vitrification of bovine embryos were evaluated. In vitro-produced blastocysts were divided into two groups: the control group (CG) without the addition of LpAFP and the treatment group (TG) with the addition of 500 ng/ml of LpAFP in the equilibrium and vitrification solution. Vitrification was carried out by transferring the blastocysts to the equilibrium solution [7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO)] for 2 min and then to the vitrification solution (15% EG, 15% DMSO and 0.5M sucrose). The blastocysts were deposited on a cryotop device and submerged in liquid nitrogen. Warming was carried out in three steps in solutions with different sucrose concentrations (1.0, 0.5, and 0.0 M, respectively). Embryos were evaluated for re-expansion/hatching, the total cell count, and ultrastructural analysis. There was no significant difference in the re-expansion rate 24 h after warming; however, there was variation (P < 0.05) in the hatching rate in the TG and the total number of cells 24 h after warming was higher in the TG (114.87 ± 7.24) when compared with the CG (91.81 ± 4.94). The ultrastructural analysis showed changes in organelles related to the vitrification process but, in the TG, there was less damage to mitochondria and rough endoplasmic reticulum compared with the CG. In conclusion, the addition of 500 ng/ml of LpAFP during the vitrification of in vitro-produced bovine embryos improved the hatching rate and total cell number of blastocysts after warming and mitigated intracellular damage.
Diabetes mellitus (DM) is a common metabolic disease characterized by high blood sugar levels. It is well known that men with diabetes frequently experience reproductive disorders and sexual dysfunction. In fact, sperm quality has a significant effect on fertilization success and embryo development. The current study aimed to investigate the effect of Stevia rebaudiana hydroalcoholic extract on serum testosterone levels, sperm parameters, in vitro fertilization (IVF) success, and in vitro embryonic developmental potential to reach the blastocyst stage in a streptozotocin (STZ)-induced mouse model of diabetes. In this research, 30 male mice were distributed randomly into control, diabetic (streptozotocin 150 mg/kg) and diabetic + Stevia (400 mg/kg) groups. The results revealed a decrease in body and testis weight and elevated blood fasting blood sugar (FBS) levels in the diabetic group, compared with the control. However, Stevia treatment significantly increased body and testis weight, while serum FBS levels were decreased compared with the diabetic group. In addition, Stevia significantly increased blood testosterone levels compared with the diabetic group. Moreover, sperm parameters were improved considerably by Stevia treatment compared with the diabetic group. Furthermore, Stevia administration significantly promoted IVF success rate and in vitro development of fertilized oocytes compared with the diabetic group. In summary, our data indicated that Stevia enhanced sperm parameters, IVF success, and in vitro embryonic developmental competency in diabetic mice, probably because of its antioxidant effects. Therefore, Stevia could ameliorate sperm parameters that, in turn, increase fertilization outcomes in experimental-induced diabetes.
The influence of the method of evaluating developmentally competent oocytes on their viability after cryopreservation still needs to be better understood. The objective of this study was to determine the cleavage and embryo developmental rates after parthenogenetic activation of cumulus–oocyte complexes (COCs) selected by different concentrations of brilliant cresyl blue (BCB) and cryopreservation. In the first experiment, COCs were separated into groups and incubated for 1 h in medium containing BCB (13 μM, 16 μM, or 20 μM). The control group was not exposed to BCB staining. In the second experiment, COCs were divided into four groups: 13 μM BCB(+), 13 μM BCB(−), fresh control (selected by morphologic observation and immediately in vitro matured) and vitrified control (selected by morphologic evaluation, vitrified, and in vitro matured). In the first experiment, the 13 μM BCB group displayed greater development rates at the morula stage (65.45%, 36/55) when compared with the other groups. In the second experiment, cleavage (47.05%, 72/153) and morula development (33.55%, 51/153) of the control group of fresh COCs were increased compared with the other groups. However, when comparing morula rates between vitrified COC control and BCB(+) groups, the BCB(+) group had better results (19.23%, 5/26 and 64.7%, 11/17, respectively). Our best result in rat COC selection by BCB staining was obtained using a concentration of 13 μM. This selection could be a valuable tool to improve vitrification outcomes, as observed by the BCB(+) group that demonstrated better results compared with the vitrified COC control.
Based on the fact that the follicular phase in the menstrual cycle has length variation, it has been assumed that the duration of oestrogen (E2) administration could also be variable; therefore, for the first time, this randomized clinical trial study was conducted to investigate and compare the duration of estradiol administration and the effect on pregnancy outcomes in the cleavage-stage frozen embryo transfer (FET) cycle. We included women aged 20–40 with a normal uterus on hysteroscopy between September and December 2022 and who were divided randomly into three groups: group A [n = 79; 8–11 days of oestrogen before progesterone (P4) supplementation], group B (n = 78; 12–14 days of oestrogen before P4 supplementation), and group C (n = 76; 15–18 days of estrogen before P4 supplementation). Serum levels for E2 on the initial progesterone day and P4 on the transfer day were measured. The effect of the duration of E2 administration on clinical pregnancy and pregnancy loss was investigated. We found no significant differences between the three groups in the clinical pregnancy rate (P = 0.696) and clinical abortion rate (P = 0.925) according to the duration of the E2. There was no significant difference in the E2, P4 levels, and endometrial thickness in pregnant vs. non-pregnant women. The mean of the E2 and P4 levels was 300.03 ± 22.21 and 25.36 ± 5.78, respectively. Our findings suggest that variation in the length of E2 administration (8–18 days) before progesterone initiation in day 3 FET cycles does not affect pregnancy outcome and transfer time can be flexibly arranged.
Placental trophoblastic cells play important roles in placental development and fetal health. However, the mechanism of trophoblastic cell fusion is still not entirely clear. The level of Tspan5 in the embryo culture medium was detected using enzyme-linked immunosorbent assay (ELISA). Fusion of BeWo cells was observed by immunofluorescence. Cell fusion-related factors and EMT-related factors were identified by qRT-PCR and western blotting. Notch protein repressor DAPT was used to verify the role of Tspan5 in BeWo cells. The expression of Tspan5 was significantly increased in embryo culture medium. The fusion of BeWo cells was observed after treatment with forskolin (FSK). Cell fusion-related factors (i.e. β-hCG and syncytin 1/2) and Tspan5 were significantly increased after FSK treatment. In addition, FSK treatment promoted EMT-related protein expression in BeWo cells. Knockdown of Tspan5 inhibited cell fusion and EMT-related protein levels. Notch-1 and Jagged-1 protein levels were significantly upregulated, and the EMT process was activated by overexpression of Tspan5 in FSK-treated BeWo cells. Interestingly, blocking the Notch pathway by the repressor DAPT had the opposite results. These results indicated that Tspan5 could promote the EMT process by activating the Notch pathway, thereby causing cell fusion. These findings contribute to a better understanding of trophoblast cell syncytialization and embryonic development. Tspan5 may be used as a therapeutic target for normal placental development.
Wagyu bulls are known to have a highly exacerbated libido, as shown by the intense sexual interest of young calves. Therefore we believe that Wagyu male animals have specialized Sertoli and Leydig cells that are directly involved with the sexual precocity in this breed as mature bulls have a small scrotal circumference. This study aimed to evaluate whether there were differences in the hormone and sperm characteristics of Wagyu bulls compared with the same characteristics of subspecies Bos indicus and Bos taurus sires. Frozen–thawed semen from Wagyu, Nellore, and Angus sires were analyzed for sperm kinetics (computer-assisted sperm analysis), plasma membrane integrity, chromatin integrity, acrosome status, mitochondrial activity, lipid peroxidation and hormone [luteinizing hormone (LH) and testosterone] serum concentration. The results showed that Wagyu had lower total motility and an increased number of sperm with no motility when compared with Nellore and Angus bulls. Wagyu breed did not differ from those breeds when considering plasma and acrosome membranes integrity, mitochondrial potential, chromatin resistance, sperm lipid peroxidation or hormone (LH and testosterone) concentrations. We concluded that Wagyu sires had lower total motility when compared with Nellore and Angus bulls. Wagyu breed did not differ from these breeds when considering plasma and acrosome membranes integrity, mitochondrial potential, chromatin resistance, sperm lipid peroxidation, or hormone (LH and testosterone) concentrations.