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Transmission Electron Microscopy of Cryopreserved Sperm. A and C: Lyophilized in Sperm Freeze Medium: ruptured plasma membrane in head and flagellum, heterogeneous chromatin. Axoneme degenerated. B and D: Lyophilized in Freeze Medium: nuclei severed compromised, ruptured plasma membrane in head and flagellum, heterogeneous chromatin. a: axoneme, fl: flagellum, h: head, mp: midpiece, pm: plasma membrane.
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Objective:
Lyophilization is potentially more practical and cost-effective alternative for sperm preservation. However, there are no studies that evaluate the ultrastructure of human spermatozoa after lyophilization. Therefore, the aim of our study was to evaluate the ultrasctructure of lyophilized spermatozoa using Transmission Electron Microscop...
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Citations
... In this study, despite the damage to the sperm cell membrane and motility, the nucleus and its contents remained intact, and freeze-drying did not lead to a significant increase in the sperm DFI, which agrees with previous studies [9,18,19]. Also, in the comparison between the freeze-drying and freezing processes, the DFI was lower in the freeze-dried groups than in the freeze group. It can be concluded that the freeze-drying method causes less damage to sperm DNA than conventional freezing, which may be due to the chelating agents in the freeze-drying liquid, such as ethylene glycoltetraacetic acid (EGTA) and EDTA. ...
... Kaneko and Nakagata [20] observed the normal DNA structure of lyophilized mouse sperm at 4 °C [21]. Gianaroli et al. [22] reported that lyophilized human sperm were stored at 4 °C, with no damage to sperm DNA, which is similar to de Lima Bossi's findings [19]. The rate of blastocyst formation in large animals (horses, cows, and pigs) is between 10% and 12%, and in rabbits, it is about 24% [23]. ...
Objective: This study evaluated the effects of temperature and storage time on the quality and DNA integrity of freeze-dried sperm from individuals with normozoospermia.Methods: Normal sperm samples from 15 men aged 24 to 40 years were studied. Each sample was divided into six groups: fresh, freezing (frozen in liquid nitrogen), freeze-dried then preserved at room temperature for 1 month (FD-1m-RT), freeze-dried then preserved at room temperature for 2 months (FD-2m-RT), freeze-dried then preserved at 4 °C for 1 month (FD-1m-4 °C), and freeze-dried then preserved at 4 °C for 2 months (FD-2m-4 °C). The morphology, progressive motility, vitality, and DNA integrity of the sperm were evaluated in all groups.Results: In all freeze-dried groups, sperm cells were immotile after rehydration. The freeze-dried groups also showed significantly less sperm vitality than the fresh and frozen groups. Significantly more morphological sperm abnormalities were found in the freeze-dried groups, but freeze-drying did not lead to a significantly higher DNA fragmentation index (DFI). The DFI was significantly higher in the FD-2m-RT group than in the other freeze-dried groups.Conclusion: The freeze-drying method preserved the integrity of sperm DNA. The temperature and duration of storage were also identified as factors that influenced the DFI. Accordingly, more research is needed on ways to improve sperm quality in the freeze-drying process.
... Kusakabe et al. [173] demonstrated that only a low percentage of sperm showed chromosomal alterations and Gianaroli et al. [174] did not find increased DNA damage after dry storage with respect to the standard procedure. However, lyophilization may harm cell membranes [175] and produce detrimental effects on the sperm head [176]. Considering that after lyophilization spermatozoa do not preserve viability or motility, the fact that they can support embryo development and live births after ICSI in some mammalian species (see above) indicates that the maintenance of DNA integrity [173,174] is an important achievement of the freeze-dry procedure. ...
Cryopreservation is an expanding strategy to allow not only fertility preservation for individuals who need such procedures because of gonadotoxic treatments, active duty in dangerous occupations or social reasons and gamete donation for couples where conception is denied, but also for animal breeding and preservation of endangered animal species. Despite the improvement in semen cryopreservation techniques and the worldwide expansion of semen banks, damage to spermatozoa and the consequent impairment of its functions still remain unsolved problems, conditioning the choice of the technique in assisted reproduction procedures. Although many studies have attempted to find solutions to limit sperm damage following cryopreservation and identify possible markers of damage susceptibility, active research in this field is still required in order to optimize the process. Here, we review the available evidence regarding structural, molecular and functional damage occurring in cryopreserved human spermatozoa and the possible strategies to prevent it and optimize the procedures. Finally, we review the results on assisted reproduction technique (ARTs) outcomes following the use of cryopreserved spermatozoa.
... However, this observation was not confirmed in microwave-dried cat sperm that kept normal centrosomal functions (Patrick et al., 2017). A recent study comparing cryopreservation versus lyophilization in human sperm demonstrated that freezing induces more lesions to the mid-piece with some mitochondria degeneration and rupture of the plasma membrane (Bossi et al., 2021). However, analysis by transmission electron microscopy showed that lyophilized sperm heads have ruptured plasma membranes, absence of acrosomes, and nuclei with heterogeneous and decondensed chromatin. ...
... However, analysis by transmission electron microscopy showed that lyophilized sperm heads have ruptured plasma membranes, absence of acrosomes, and nuclei with heterogeneous and decondensed chromatin. Mitochondrial degeneration was seen in the midpiece, and the organization of axonemes in the flagellum was also disrupted (Bossi et al., 2021). Regarding another specific sperm component, acrosome integrity after drying/ rehydration might not be as critical since ICSI is required. ...
... Overall damage of plasma membrane in lyophilized sheep lymphocytes (analyzed by transmission electron microscopy) or spin-dried human hepatoma cells (HepG2; analyzed by using Syto-13 and ethidium bromide viability dyes) had already been reported(Iuso et al., 2013;. Ruptures of the plasma membrane in dehydrated sperm from species as diverse as men(Bossi et al., 2021;Gianaroli et al., 2012), llamas(Carretero et al., 2020), or cattle(Sitaula et al., 2009) also have been observed. It is also interesting to note that lyophilization of horse sperm appears to induce less membrane damage (a higher percentage of cells with nonperoxidized lipid and nonpermeable membrane) than cryopreservation(Restrepo et al., 2019). ...
Long-term preservation of sperm, oocytes, and gonadal tissues at ambient temperatures has the potential to lower the costs and simplify biobanking in human reproductive medicine, as well as for the management of animal populations. Over the past decades, different dehydration protocols and long-term storage solutions at nonfreezing temperatures have been explored, mainly for mammalian sperm cells. Oocytes and gonadal tissues are more challenging to dehydrate so little to no progress have been made. Currently, the detrimental effects of the drying process itself are better characterized than the impact of long-term storage at nonfreezing temperatures. While structural and functional properties of germ cells can be preserved after dehydration, a long list of damages and stresses in nuclei, organelles, and cytoplasmic membranes have been reported and sometimes mitigated. Characterizing those damages and better understanding the response of germ cells and tissues to the stress of dehydration is fundamental. It will contribute to the development of optimal protocols while proving the safety of alternative storage options for fertility preservation. The objective of this review is to (1) document the types of damages and stress responses, as well as their mitigation in cells dried with different techniques, and (2) propose new research directions.
... El tratamiento con licopeno oral tiene un papel importante en el tratamiento de la infertilidad masculina idiopática, observándose aumento significativo en las concentraciones de espermatozoides posteriores al tratamiento. 25 Además, las células expuestas al licopeno secretan exosomas enriquecidos con este, dando como resultado degradación reducida del antioxidante y por lo tanto, aumentan la efectividad en los sitios de acción. Estos orgánulos actúan como vehículos de transporte y desempeñan un papel en la prevención de la infertilidad masculina. ...
... Las concentraciones iniciales más altas se asociaron con una mejoría mayor y significativa. 25 Otro estudio examinó el efecto del licopeno sobre la peroxidación lipídica y formación de 8-oxo-7,8dihidro-2-desoxiguanosina in vitro. Las células tratadas con licopeno mostraron reducción de 86% en la peroxidación lipídica y de 77% en las concentraciones de 8-oxo-7,8dihidro-2'-desoxiguanosina. ...
La evidencia sobre la asociación entre dieta y fertilidad humana ha aumentado en forma exponencial en la última década, lo que ha permitido identificar algunos hallazgos claros. La infertilidad masculina es un problema común que está incrementando. Aunque algunos pacientes tienen causas hormonales anatómicas o funcionales reconocidas, la etiología exacta en un gran número de casos es desconocida. En diferentes condiciones de infertilidad masculina, generalmente asociadas a anomalías morfofuncionales de los espermatozoides, existen pruebas claras de daño celular causados por el estrés oxidativo. En las últimas 5 décadas, varias investigaciones han intentado comprobar si la suplementación de micronutrientes puede tener efectos positivos en los parámetros cualitativos/cuantitativos del semen y la frecuencia de embarazo. Las vitaminas C y E, junto con la L-carnitina, han demostrado ser eficaces. Sin embargo, otros micronutrientes han sido menos estudiados. Existe la necesidad de realizar más investigaciones con estudios controlados aleatorios para confirmar la eficacia y seguridad de los suplementos antioxidantes en el tratamiento médico de la infertilidad masculina idiopática. El objetivo de esta revisión fue evaluar los efectos de los micronutrientes en el tratamiento de la infertilidad masculina.
Spermatozoa cryopreservation is an effective method for maintaining male
fertility in humans. Nevertheless, there are some limitations of sperm
cryopreservation, which is called as cell injury by cryoprotectant, that cannot
be avoided. This process will affect embryo quality. Therefore, it is mandatory
to modify cryoprotectant in spermatozoa cryopreservation, to improve embryo
quality. This review aimed to summarize the modification of cryoprotectant that
can damage the cell, thereby improving embryo quality. To this purpose, a
computerized search of EMBASE, PubMed, Scopus and Google Scholar databases from
2008 to 2022 were performed on general term such as “sperm cryopreservation”,
“cryoprotective agent”, “modified cryoprotectant”, “cell injury”. Of these, 1847
publications were screened and 38 articles were obtained and evaluated.
Although no formal conclusions can be drawn regarding the cryopreservation of
spermatozoa to obtain good embryo quality, our results suggest that modified
cryoprotectants can be an alternative cryoprotectant compared to commercial
cryoprotectants. In addition, the use of antioxidant in spermatozoa
cryopreservation can also prevent cell damage due to the negative effects of
cryoprotectants. However, further researches
still need to be performed to investigate the cellular mechanisms.
Sperm vitrification has been used in the field of assisted reproductive technology (ART) for years and has resulted in many healthy live births. Compared to the conventional sperm slow freezing method, vitrification is simpler, quicker, and less expensive, and some vitrification methods are also cryoprotectant free, which has the potential to become an alternative cryopreservation method for human sperm. Human sperm vitrification has been the most commonly used and valuable way to preserve the fertility of males with small numbers of spermatozoa. Recently, new sperm vitrification devices have been developed to help improve volume control. Direct contact during the vitrification process with liquid nitrogen increases the risk of cross-contamination. New strategies have been implemented to minimize the contamination risk. Depending on the variety of semen parameters and patients’ purposes at ART clinics, specific sperm cryopreservation approaches should be personalized to achieve the optimal results for each case.
In the 1960s, sperm cryopreservation was developed as a method to preserve fertility. Currently, techniques for the cryopreservation of human spermatozoa have been widely used in assisted reproduction. However, although sperm cryobiology has made notable achievements, the optimal method for the recovery of viable spermatozoa after cryopreservation remains elusive. Postthawing sperm quality can be affected by cryoprotectants, ice formation, storage conditions, and osmotic stress during the freezing process. This review discusses recent advances in different cryopreservation techniques, cryoprotectants, and freezing and thawing methods during cryopreservation and new indications for the use of cryopreserved spermatozoa.
