Ultrastructural analysis of Lyophilized Human Spermatozoa

Abstract

These results were presented at the 35 th annual meeting of the European Society of Human Reproduction and Embryology ABSTRACT 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 Microscopy.Methods: From a total of 21 donated seminal samples, 30 aliquots were originated and divided into two aliquots so that one could have been submitted to cryopreservation/thaw and the other for lyophilization/rehydration. The liquefied aliquots were homogenized at room temperature. Samples assigned for cryopreservation were placed in straws and samples assigned for lyophilization were placed in the appropriate vials. Cryopreservation samples were placed at -30oC for 30 minutes subsequently for 30 minutes at vapour phase and then plunged into liquid nitrogen. Lately, were warmed in water bath at 37oC for 10 minutes followed by 10 minutes centrifugation. The pellet was resuspended and analysed in a Makler chamber. The semen vials assigned for lyophilization were loaded into a pre-fixed freeze-drying chamber. Following lyophilization, vials were removed from the freeze-drying chamber and kept at 4oC until rehydration. TEM was performed after rehydration and thawing. Sperm samples were fixed, rinsed in buffer, post fixed and dehydration was carried out in escalating concentrations of alcohol solution, acetone and then, embedding in Epon resin. Ultrathin sections were stained and examined in a Transmission Electron Microscope.Results: Analysis of sperm after freezing/thawing using Transmission Electron Microscopy showed lesions to the midpiece, with some mitochondria degeneration and random rupture of plasma membrane. In the head, we identified intact plasma membrane, nucleus and acrosome, as in the flagellum all main structures remained intact including the plasma membrane, the longitudinal columns of dense fibers and the semicircular fibers. Analysis by Transmission Electron Microscopy showed that spermatozoa heads had ruptured plasma membranes, absence of acrosomes, nuclei with heterogeneous and decompressed chromatin. Mitochondria were deteriorated in the midpiece. Longitudinal columns of dense fibers were absent in the flagellum. Axonemes, in cross-sections, were disrupted with disorganized structures.Conclusions: To our knowledge, our study demonstrated, for the first time, the structure of the human spermatozoa after lyophilization using Transmission Electron Microscopy. The use of a fixed lyophilization protocol with media containing cryoprotectants might explain the damage to the structures. More studies are necessary to improve the results of sperm lyophilization. In the future, the use of lyophilization of spermatozoa might reduce the costs of fertility preservation, since there will be no need for storage space and transportation is simpler.

Damages and stress responses in sperm cells and other germplasms during dehydration and storage at nonfreezing temperatures for fertility preservation

“…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).…”

“…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.…”

Human Sperm Vitrification: Review of Recent Progress

“…The possibility that vitrified sperm preserve their function at temperature of −80°C could simplify storage, optimizing the space and time as well as the operator's safety [3]. Lyophilization of spermatozoa is another method that requires additional investigation and validation [20]. Since the lyophilized spermatozoa are immobile, they can only be used in intracytoplasmic sperm injection (ICSI).…”

Update on techniques for cryopreservation of human spermatozoa

“… 69 Several reports on freeze-drying of animal spermatozoa have been published; however, because of ethical limitations or other unpublished reasons, ICSI has not been performed using freeze-dried human spermatozoa. 70 71 Studies on the dry preservation of human spermatozoa showed that the survival and maneuverability membrane of sperm were completely impaired after being freeze-dried; 72 however, the chromatin structure of spermatozoa and the protoplast structure in the sperm head were retained. 71 …”