Cryopreservation of human spermatozoa by vitrification versus conventional rapid freezing: Effects on motility, viability, morphology and cellular defects

To improve artificial insemination protocols in ovine species it is crucial to optimize sperm quality evaluation after preservation technologies. Emerging technologies based on novel biomolecules and related to redox balance and proteins involved in sperm motility such as ProAKAP4 could be successfully applied in ram sperm evaluation. In this work, a multiparametric analysis of fresh, cooled, and cryopreserved ram sperm was performed at different complexity levels. Samples were evaluated in terms of motility (total motility, progressive motility, and curvilinear velocity), viability, apoptosis, content of reactive oxygen species, oxidation‒reduction potential, and ProAKAP4 expression and concentration. As expected, cryopreserved samples showed a significant decrease of sperm quality (p < 0.05), evidencing different freezability classes among samples that were detected by ProAKAP4 analyses. However, in cooled sperm no differences were found concerning motility, viability, apoptosis, ROS content, and redox balance compared to fresh sperm that could explain the reported decrease in fertility rates. However, although the proportion of sperm ProAKAP4 positive-cells remained unaltered in cooled sperm compared to fresh control, the concentration of this protein significantly decreased (p < 0.05) in cooled samples. This altered protein level could contribute to the decrease in fertility rates of cooled samples detected by some authors. More importantly, ProAKAP4 can be established as a promising diagnostic parameter of sperm quality allowing us to optimize sperm conservation protocols and finally improve artificial insemination in ovine species.

https://www.mdpi.com/2218-273X/10/7/1046/pdf

ProAKAP4 as Novel Molecular Marker of Sperm Quality in Ram: An Integrative Study in Fresh, Cooled and Cryopreserved Sperm.

Introduction Sperm cryopreservation, more commonly referred to as sperm banking, has long been a fertility preservation technique for men but had been limited to those men whose post-thaw specimen after cryopreservation yielded sufficient motile sperm for intrauterine insemination. With the advances in assisted reproductive techniques (ART), and in particular intracytoplasmic sperm injection (ICSI), cryopreservation has become an essential component of fertility treatment for men with oligozoospermia, cryptozoospermia, and even azoospermia. It has also become standard practice to cryopreserve at the time of testis biopsy for azoospermia as well as when semen quality declines in the face of standard treatment paradigms. Over the past decade, sperm banking has become an essential component of the treatment of the subfertile couple. Impairment in male fertility can result from several causes. These include disease, anatomic, and/or functional issues (e.g., absence of the vas deferens, retrograde ejaculation or anejaculation), primary or secondary hormonal insufficiency, and frequently, from damage or depletion of the germinal stem cells resulting in impaired spermatogenesis. There are many potential threats to spermatogenesis with disease treatment, often resulting in compromised sperm number, motility, morphology, as well as DNA integrity. Chemotherapy and radiotherapy for various cancers easily disrupt biochemical processes that occur during spermatogenesis [1]. In addition, non-chemotherapeutic drugs and herbal as well as non-herbal supplements can and do affect sperm quantity, quality, and fertility potential. The popularity of sperm cryopreservation has been a direct result of patient demand. The majority of men who bank sperm have been afflicted by cancer at a young age and wish to preserve their future fertility. Although cancer survivors can become parents through adoption and gamete donation (using an anonymous or directed donor) most prefer to have biologic offspring [2]. In one study 48% reported that having children was an important issue to them after completing their treatment [3]. Similar results were reported by Schover et al. noting that over 50% of male cancer survivors in the reproductive age group desire to preserve their future fertility climbing to a rate of 77% in men who are childless at the time of cancer diagnosis [4,5]. Many of these men have concerns that their cancer or cancer treatment may result in birth defects or other health issues for their potential offspring [6]. Importantly, aside from inherited genetic syndromes, large registry studies have revealed no increased risk of genetic abnormalities, birth defects, or cancers in the children of cancer survivors [7,8,9,10,11,12]. This chapter follows the development of cryopreservation for mammalian sperm. It expands upon our earlier review of indications and protocols [13]. We also provide evidenced based answers to some of the questions our patients ask when seeking advice regarding sperm banking.

Sperm Cryopreservation

A novel approach for human sperm cryopreservation with AFPIII.

Objective To investigate the effects of in vitro myo-inositol (Myo-Ins) supplementation of cryopreserved human semen on the cryo-survival rate (CSR). Methods Semen samples were obtained from 41 infertile men. Following routine semen analysis, each sample was divided into two equal aliquots (0.5 mL each). One aliquot was treated with 1 mg of Myo-Ins dissolved in 10 µL of sperm preparation medium. The second aliquot was treated with 10 µL of the same medium (control). Both aliquots were incubated for 20 minutes prior to freezing to slow the freezing process. The frozen samples were examined for post-thaw percentages of total motility (TM), progressive motility (PM), and the CSR, defined as the percentage of post-thaw TM divided by the percentage of pre-freeze TM and multiplied in 100. The results were expressed as median and interquartile range (25th and 75th percentiles). Results The pre-freeze TM (50% [30%-50%]) and PM (35% [20%-35%]) were significantly higher than the post-thaw TM and PM in the Myo-Ins group (15% [10%-35%] and 10% [5%-20%]; p<0.001 and p<0.001, respectively) and the control group (10% [6%-30%] and 5% [3%-15%]; p<0.001 and p<0.001, respectively). The CSR of the 41 semen aliquots supplemented with Myo-Ins (40% [25%-70%]) was significantly higher than that of the control samples (30% [13%-58%], p=0.041). The CSR of the 26 abnormal semen samples that were supplemented with Myo-Ins (38% [20%-50%]) was significantly higher than that of the control samples (23% [12%-30%], p=0.031). Conclusion In vitro Myo-Ins supplementation of ejaculated human sperm from infertile men resulted in a significant increase in the CSR in samples with abnormal pre-freeze sperm parameters.

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Increased cryo-survival rate in ejaculated human sperm from infertile men following pre-freeze in vitro myo-inositol supplementation

Purpose
The aim of this study is to evaluate which cryopreservation protocol, freezing before or after swim-up, optimizes cryopreservation outcomes in terms of motile sperm count, motility, morphology, and viability, and also to establish whether sperm viability could be assessed based on sperm motility.

/pdf/sperm-preparation-after-freezing-improves-motile-sperm-count-4fwzgtlap6.pdf

Sperm preparation after freezing improves motile sperm count, motility, and viability in frozen-thawed sperm compared with sperm preparation before freezing-thawing process

Semen cryopreservation is the only available method to preserve the fertility in young and adult men. Semen freezing is the first line option for the group of patients in which fertility preservation is required. Conventional slow freezing of spermatozoa is commonly used for cryopreservation of both ejaculated and surgically retrieved spermatozoa for preservation of fertility before cancer treatment, in severe male factor infertility (obstructive azoospermia) and for establishment of donor banks. Cryopreservation of spermatozoa is therefore an important part of a successful assisted reproductive technology program. Sperm cryopreservation should be considered in cases of azoospermia so repeated surgical sperm retrieval techniques are avoided; also, regarding male patients who present impaired semen parameters, sperm cryopreservation is indicated to prevent the risk of azoospermia [1]. In addition, fertility preservation is a major concern for male cancer patients who are undergoing chemotherapy or radiotherapy because these gonadotoxic agents threaten their reproductive potential [2]. Moreover, some studies suggest that not only malignancies but also other non-malignant diseases including autoimmune disorders affect male fertility. Nowadays, there is an increasing interest in fertility preservation of patients with cancer or systematic diseases, since survival rates are increasing due to the new therapeutic options [3, 4]. Therefore, as long-term quality of life after cancer treatment has become a significant fact, semen cryopreservation before chemotherapy is seen as a real possibility of maintaining male reproductive potential after cancer treatments. Unfortunately, semen cryopreservation has a great impact on the quality of thawed samples. According to several studies in this field, viability of semen samples after freezing and thawing process only raise 50% [5]. The drastic decrease in viability of thawed spermatozoa is due to cell damage caused by intracellular ice formation, and osmotic and oxidative stress during the freezing process [6–8]. During the freezing process, freezing rates and concentration of cryoprotectant agents have to be balanced, to minimize mechanical and osmotic damage. Despite all the research done to date, there is no standardized freezing protocol leading consistently to higher viability rates. Moreover, in the literature, there are a wide range of sperm parameters that are thought to be affected by sperm freezing, but only a few are really useful in clinical practice.

A. Palomar Rios

Papers

Sperm preparation after freezing improves motile sperm count, motility, and viability in frozen-thawed sperm compared with sperm preparation before freezing-thawing process

Sperm parameters that play a major role in the assessment of semen quality after cryopreservation.