L-carnitine has an important role in the control of oxidative stress and lipid β-oxidation during in vitro culture and cryopreservation of ovarian follicles, oocytes and embryos. This substance balances the acetyl-CoA/CoA ratio, maintains glucose metabolism and increases energy production in mitochondria. It also plays a key role in reducing endoplasmic reticulum stress, by transferring palmitate to mitochondria or eliminating it to avoid toxicity. By eliminating reactive oxygen species, L-carnitine increases the percentages of mature oocytes with uniform mitochondrial distribution and improves embryo post-thaw cryotolerance. Therefore, L-carnitine controls lipid β-oxidation and oxidative stress during in vitro culture of ovarian follicles, oocyte maturation, embryonic development and cryopreservation.

Testicular biopsies (9 mm3) from domestic cats (n = 10) submitted to orchiectomy were submitted to equilibrium vitrification in the presence of ethylene glycol (EG) alone or combined with dimethylsulfoxide (DMSO) as intracellular cryoprotectants, and sucrose or trehalose as extracellular cryoprotectants. The samples were vitrified with 40% EG or 20% EG + 20% DMSO, plus 0.1 M or 0.5 M of sucrose or trehalose. The study was divided into Step 1 and Step 2. In Step 1, intratubular cells (spermatogonia, spermatids, spermatocytes, and Sertoli cells) were quantified and classified as intact or degenerated (pyknotic and/or vacuolated cells). Cryodamage of seminiferous cords was determined by spermatogonia and Sertoli cell scoring of nuclei alterations, tubular basement membrane detachment, epithelium shrinkage, and tubular measures (total area, epithelium area, larger and smaller diameter, and height of the epithelium). In Step 2, Hoechst 33342 stain and propidium iodide (PI) fluorescent stain were used to assess the cell viability of the four best experimental groups in Step 1. The effect of treatments on all analyses was accessed using analysis of variance (ANOVA), and Fisher’s post hoc test at P < 0.05 significance was considered. In Step 1, the mean percentage of spermatogonia and Sertoli cells morphological integrity did not show a difference when using both sugars at different concentrations, but their morphology was more affected when DMSO was used. EG use associated with 0.1 M of sucrose or trehalose positively affected spermatocyte and spermatid morphology, respectively. The larger diameter and epithelium height of seminiferous tubules were increased using DMSO plus 0.5 M sucrose and DMSO plus 0.1 M trehalose. The changes in spermatogonial/Sertoli nucleoli visualization were best scored in the EG groups, while the nuclei condensation was lower with sucrose. The basement membrane was satisfactorily preserved with 0.1 M sucrose. In Step 2, the percentage of cell viability was higher when EG plus 0.1 M sucrose was used. Therefore, DMSO’s negative effect on the vitrification of testicular biopsies of adult domestic cats was evident. The EG plus 0.1 M of sucrose or trehalose associations are the most suitable CPAs to preserve the testicular histology structure of adult domestic cats in vitrification.

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.

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.

In a landmark Fatwa, Saudi Arabia’s highest religious authority—The Council of Senior Scholars—declared the Islamic permissibility of oocyte cryopreservation. The fatwa sanctioned the retrieval, preservation, and future use of oocytes, ovarian tissue, and whole ovaries from cancer patients receiving gonadotoxic interventions. Although momentous, the fatwa’s specification of cancer patients effectively rendered this technology unavailable to others to whom it may be similarly beneficial, including patients with other medical conditions or patients seeking elective cryopreservation. This article argues in favor of widening reproductive choices through expanded access to oocyte cryopreservation in Saudi Arabia—regardless of the underlying cause of infertility—on three grounds: the technology’s compliance with Islamic law, as a matter of fairness in medical practice, and as a means to support the well-being and flourishing of Saudi women within the context of a national societal and economic transformation strategy closely linked to their success.

Discarding the first ejaculate is recommended as an alternative for improving seminal quality after long sexual resting, especially when semen should be used for cryopreservation. However, when the males are not in sexual resting the necessity to discarding the first ejaculate is still unknown. Therefore, this study aimed to compare by flow cytometry the quality of the first and second ejaculates. Ten kids and uniform goats between 5 and 6 months of age were used in a completely randomized design. Semen collection was carried out every 4 days, until a total of five ejaculates per animal in each treatment was completed. The fresh and frozen semen collected were processed and analyzed using macroscopic and microscopic parameters, resistance test, hypo-osmotic medium test, and flow cytometry (FC). The FC parameters were production of reactive oxygen species, plasma and acrosomal membrane integrity, and lipid peroxidation of the plasma membrane. The ejaculates did not differ for the resistance test, the reactivity in the hypo-osmotic medium and for the macroscopic and microscopic seminal parameters, except for sperm volume and concentration. The first ejaculate had a higher percentage of minor and total defects. None of the FC parameters analyzed differed between the first and second ejaculates. The first and second ejaculates demonstrated similar seminal qualities, so for Alpine kid goats without a sexual resting period, discarding the first ejaculate it is not recommended.

The aim of this study was to assess the consequences of treatment with pentoxifylline (PTX), an inducer of sperm motility, on sperm DNA fragmentation (SDF) and clinical characteristics in non-obstructive azoospermia (NOA) patients. The pilot study included 15 NOA patients. Half of each sperm sample before and after rapid freezing, was treated with PTX (3.6 mM /l, 30 min) as the PTX group and the remaining samples were considered as the control. SDF and sperm motility were assessed in each group. The clinical study comprised 30 fresh testicular sperm extractions (TESE) and 22 post-thawed TESE intracytoplasmic sperm injection cycles. Half of the mature oocytes from each patient were injected with PTX-treated spermatozoa and the remaining oocytes were injected with non-treated spermatozoa. Fertilization was assessed at 16 h post injection. Embryo transfer was carried out on day 2 after fertilization. Chemical pregnancy was assessed 2 weeks after transfer. PTX was found to significantly increase (P < 0.05) sperm motility. There was an insignificant difference in SDF rates between the groups (P > 0.05). In patient ovaries given fresh TESE, there was not any significant difference in clinical characteristics (P > 0.05). In patient ovaries given post-thawed TESE, there was a significant difference in the number of 2PN and in embryo formation (P < 0.05). Differences in the results of chemical pregnancy were insignificant (P > 0.05) between the groups. In addition, there was not any correlation between DNA fragmentation index and sperm motility and laboratory outcomes. Therefore, obtaining viable spermatozoa using PTX was more effective in post-thawed TESE regime patients in terms of 2PN and in embryo formation, deprived of damaging effects on sperm DNA integrity.

The objective of this study was to investigate the effects of adding β-mercaptoethanol (βME) to culture medium of bovine in vitro-produced (IVP) embryos prior to or after vitrification on embryo development and cryotolerance. In Experiment I, Day-7 IVP blastocysts were vitrified and, after warming, cultured in medium containing 0, 50 or 100 μM βME for 72 h. Embryos cultured in 100 μM βME attained higher hatching rates (66.7%) than those culture in 0 (47.7%) and 50 (52.4%) μM βME. In Experiment II, IVP embryos were in vitro-cultured (IVC) to the blastocyst stage in 0 (control) or 100 μM βME, followed by vitrification. After warming, embryos were cultured for 72 h (post-warming culture, PWC) in 0 (control) or 100 μM βME, in a 2 × 2 factorial design: (i) CTRL–CTRL, control IVC and control PWC; (ii) CTRL–βME, control IVC and βME-supplemented PWC; (iii) βME–CTRL, βME-supplemented IVC and control PWC; or (iv) βME–βME, βME-supplemented IVC and βME-supplemented PWC. βME during IVC reduced embryo development (28.0% vs. 43.8%) but, following vitrification, higher re-expansion rates were seen in βME–CTRL (84.0%) and βME–βME (87.5%) than in CTRL–CTRL (71.0%) and CTRL–βME (73.1%). Hatching rates were higher in CTRL–βME (58.1%) and βME–βME (63.8%) than in CTRL–CTRL (36.6%) and βME–CTRL (42.0%). Total cell number in hatched blastocysts was higher in βME–βME (181.2 ± 7.4 cells) than CTRL–CTRL (139.0 ± 9.9 cells). Adding βME to the IVC medium reduced development but increased cryotolerance, whereas adding βME to the PWC medium improved embryo survival, hatching rates, and total cell numbers.

Maintenance of eukaryotic microalgae strains for the long term is generally carried out using serial subculture techniques which require labour, time and cost. Cryopreservation techniques provide long-term storage of up to years for numerous microorganism strains and cell cultures. Ssu930ijn vbvbhnn8;l,n is related to a successfully designed mass and heat transfer balance throughout the cell. In this study, optimization of the cryopreservation process was carried out for two commercially used microalgal strains. The parameters to be optimized were DMSO percentage (0–25%), incubation time (1–15 min) and cryopreservation term (7–180 days) using a central composite design (CCD). Long-term storage up to 123.17 and 111.44 days corresponding to high cell viabilities was achieved for Chlorella vulgaris and Neochloris texensis, respectively. Generated models were found to be in good agreement with experimental results. The study also revealed holistic results for storage of microalgal strains in a stable state for industrial applications.

This study aimed to evaluate the effect of the cryopreservation duration (up to 160 months) on the clinical and neonatal outcomes of slow-frozen early-cleavage human embryos. Clinical data collected between February 2013 and August 2017 were included in this retrospective study. Cases were classified into five groups by the duration of cryopreservation: Group 1, 6–12 months; Group 2, 13–36 months; Group 3, 37–60 months; Group 4, 61–84 months; and Group 5, >84 months. The embryo survival rate, implantation rate, clinical pregnancy rate, live-birth rate, newborn sex ratio, singleton gestational age, singleton birth weight and malformation rate were compared between the groups. The cryopreservation duration did not significantly affect the rates of clinical pregnancy (P = 0.119) and live birth (P = 0.354), the newborn sex ratio (P = 0.614) or the singleton gestational age (P = 0.212) and birthweight (P = 0.212). Although decreases in the embryo survival and implantation rates were observed in groups 4 and 5 compared with those in groups 1–3, these differences were not statistically significant (P = 0.329, P = 0.279, respectively). Long-term cryopreservation does not appear to adversely affect the clinical and neonatal outcomes of slow-frozen early-cleavage human embryos.

The genus Lilium contains a number of ornamental crop species, which are commercially important in many countries. As they are vegetatively propagated, maintaining genetic stability is essential for their efficient conservation. In this study, we investigated the genetic stability of regenerated plants of three cultivars (L. bolanderi ‘Lenora’, L. bolanderi ‘Mount Duckling’ and L. bolanderi ‘Mount Dragon’) and one variety (L. callosum var. flavum) after cryopreservation, compared with fresh (donor) and non-cryopreserved plants using morphological traits and ISSR markers. No differences in morphological parameters including flower, stigma and pollen colour, floral spots, floral direction or polymorphic bands were observed between control (fresh and non-cryopreserved) and cryopreserved plantlets. In addition, based on the resulting UPGMA dendrogram, the four taxa were divided into different clusters. All cryopreserved, non-cryopreserved and fresh plants in each group could be grouped together in a single cluster with more than 97 or 100% similarity. The results suggest a very low level or the absence of genetic variation in terms of morphological and genetic stability among the plants regenerated after cryopreservation.

Male infertility is responsible for 50% of men’s health problems and has always been a concern for personal and social issues. A survey of global statistics suggests an increase in infertility rate as one of the critical issues documented in studies. There are different ways of maintaining fertility in men, depending on their age. In this paper, we review the preservation methods used for fertility treatment in Iran and other countries. Available data were reviewed from Google Scholar, PubMed, Scopus, Web of Science, IranMedex, MEDLIB, IranDoc and Scientific Information Database and searched for articles published up to 2018, using the medical subject heading (MeSH) terms for cryopreservation, sperm, testicular, spermatogonia stem cell, male infertility and/or Iranian and in the world, to provide evidence from evaluation of fertility preservation the methods. Based the search strategy, 274 manuscripts were found. After reviewing the titles, abstracts and manuscripts in their entirety, 119 articles were obtained and selected according to the eligibility criteria. The 85 studies mentioned above were divided into three categories (sperm, testis, and spermatogonia stem cells (SSCs)), and methods of fertility preservation were investigated. Ways to maintain male fertility were different depending on age, and included sperm, testicular, and SSC freezing. The number of studies on testicular tissue and SSCs was low for human samples, and more studies are still needed. Sperm freezing at infertility centres is the top for male fertility preservation.

Lipid accumulation occurs in cultured embryos and is associated with reduced cryotolerance. Here we report the use of a multiple pathway lipid modulator cocktail (l-carnitine, linoleic acid and forskolin) to improve cryosurvival. First, we stained oocytes and embryos with Oil Red to examine the time course of lipid accumulation during in vitro fertilization (IVF) and embryo culture. Then we evaluated the effects of the lipid modulators cocktail on lipid content, developmental rates and survival after vitrification. In our conditions, lipid accumulation was detected (P < 0.05) at the end of in vitro maturation (IVM) and after 4 days of embryo culture (D4-D5). In experiment 1, we used lipid modulator cocktail during IVM. Reduced (P < 0.05) lipid accumulation was detected in oocytes (Control: 49.9 ± 1.6, Lip. Mod. IVM: 45.0 ± 1.8) but no changes were present at blastocyst stage (Control: 62.4 ± 2.6, Lip. Mod. IVM: 66.8 ± 2.7). Treated oocytes presented decreased (P < 0.05) blastocyst rates and lower (P < 0.05) re-expansion after vitrification. In experiment 2, lipid modulators cocktail was used during embryo culture (from D4–D7 or D6–D7). Treatment had an effect on lipid metabolism, as lipid content was increased (P < 0.05) in D7 blastocysts in treated groups (Control: 52.7 ± 3.1a, D4: 65.9 ± 2.6b, D6: 78.1 ± 2.7b). However, no effect was present for cleavage, blastocyst and cryosurvival rates. No difference was detected in mean cell number comparing the three groups (Control: 78.9 ± 9.6, D4: 82.6 ± 16.5, D6: 68.3 ± 7.8), but apoptosis rate was increased (P < 0.05) in vitrified-warmed blastocysts from treated groups (Control: 14.77*, D4: 22.28, D6: 22.22). We concluded that the combined use of lipid modulators was efficient to promote changes in lipid content of oocytes and embryos in bovine, but those changes did not reflect positively on embryo development or cryosurvival.

This study examined the effect of a cryoprotectant with and without pentoxifylline supplementation on the motility and viability of human testicular sperm, both before and after freezing. Testicular samples were obtained from 68 patients with azoospermia who came to the Andrology Service of West China Second University Hospital, Sichuan University, for testicular biopsies from December 2019 to April 2020. All patients were assigned randomly to two groups: experimental, whose testicular sperm were added to the cryoprotectant with pentoxifylline, and the control, whose testicular sperm were added to the cryoprotectant without pentoxifylline. Both groups used the same freezing and thawing methods. Testicular sperm motility in the experimental group was significantly higher than that of the control group, both before and after cryopreservation. The recovery rate of sperm motility in the experimental group was significantly higher than that of the control group. The percentage of samples with motile testicular sperm in the experimental group was significantly higher than that of the control group after thawing. Sperm viability was unchanged between the experimental and control groups, both before and after freezing. Overall, a pentoxifylline-supplemented cryoprotectant can significantly improve the motility of testicular sperm before and after cryopreservation.