Cryopreservation of Human Spermatozoa: Functional, Molecular and Clinical Aspects.

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.

Can We Cryopreserve the Sperm of COVID-19 Patients During the Pandemic?

An extreme strain has been placed on healthcare facilities in the COVID-19 era. Initial stage of the pandemic, national and international societies for reproductive medicine suggested the suspension of new IVF treatments and non-essential cryopreservation of gametes. Accordingly, the demands of cryopreservation of semen with COVID-19 patients also was suspended by some of cryobanks to protect staff and patients from unnecessary viral exposure at the acute stage. However, the pandemic may stay with us longer than expected. In addition, there will be some male COVID-19 patients with cancer or critically illness who needs to cryopreserve their semen before medical treatments, otherwise they might loss the chance of getting their own offspring. In this document, we summarize available evidence to deepen and expand awareness of feasibility of sperm cryopreservation and propose some suggestions to help cryobanks carry out sperm preservation procedure for COVID-19 male patients.

The Impact of SARS-CoV-2 on Sperm Cryostorage, Theoretical or Real Risk?

Cryopreservation of human gametes and embryos as well as human reproductive tissues has been characterized as an essential process and aspect of assisted reproductive technology (ART). Notably, sperm cryopreservation is a fundamental aspect of cryopreservation in oncological patients or patients undergoing gonadotoxic treatment. Given that there is a risk of contamination or cross-contamination, either theoretical or real, during the procedures of cryopreservation and cryostorage, both the European Society for Human Reproduction and Embryology (ESHRE) and the American Society for Reproductive Medicine (ASRM) have provided updated guidelines for preventing or reducing the contamination risk of sexually transmitted viruses. Given the ongoing and worldwide COVID-19 pandemic, there is considerable interest in what measures should be taken to mitigate SARS-CoV-2 contamination during cryopreservation and cryostorage of semen samples. The SARS-CoV-2 virus is the virus that causes COVID-19, and whose transmission and infection is mainly aerosol-mediated. Several ART professional societies, including ESHRE and ASRM have proposed measures to mitigate the spread of the SARS-CoV-2 virus. Whether the proposed safety directives are enough to mitigate the possible SARS-CoV-2-contamination of sperm samples during cryopreservation or whether the policies should be re-evaluated will be discussed in this review. Additionally, insights regarding the possible impact of COVID-19 vaccination on the safety of sperm cryopreservation will be discussed.

Easy and quick (EQ) sperm freezing method for urgent preservation of mouse strains.

Cryopreservation of mouse spermatozoa is widely used for the efficient preservation and safe transport of valuable mouse strains. However, the current cryopreservation method requires special containers (plastic straws), undefined chemicals (e.g., skim milk), liquid nitrogen, and expertise when handling sperm suspensions. Here, we report an easy and quick (EQ) sperm freezing method. The main procedure consists of only one step: dissecting a single cauda epididymis in a microtube containing 20% raffinose solution, which is then stored in a -80 °C freezer. The frozen-thawed spermatozoa retain practical fertilization rates after 1 (51%) or even 3 months (25%) with the C57BL/6 J strain, the most sensitive strain for sperm freezing. More than half of the embryos thus obtained developed into offspring after embryo transfer. Importantly, spermatozoa stored at -80 °C can be transferred into liquid nitrogen for indefinite storage. As far as we know, our EQ method is the easiest and quickest method for mouse sperm freezing and should be applicable in all laboratories without expertise in sperm cryopreservation. This technique can help avoid the loss of irreplaceable strains because of closure of animal rooms in emergency situations such as unexpected microbiological contamination or social emergencies such as the COVID-19 threat.

The effect of semen collection location and time to processing on sperm parameters and early IVF/ICSI outcomes.

PURPOSE: We aimed to assess whether home collection and increased time to semen processing are associated with altered sperm parameters, fertilization rates (FR), day 5 usable quality blastocyst development rates (D5-UQBR), or pregnancy rates (PR) in patients undergoing IVF/ICSI. METHODS: This was a retrospective cohort study of patients undergoing IVF/ICSI before the coronavirus disease 2019 (COVID-19) pandemic ("clinic" collection, n = 119) and after COVID-19 ("home" collection, n = 125) at an academic fertility practice. Home collection occurred within 2 h of semen processing. Patient sperm parameters, FR (#2PN/MII), D5-UQBR (# transferable and freezable quality blastocysts/# 2PN), and PR in fresh transfer cycles were compared between clinic and home groups with t-tests. The association between time to processing on outcomes was assessed with regression modeling, controlling for potential confounders. RESULTS: Mean male age was 37.9 years in the clinic group and 37.2 years in the home group (p = 0.380). On average, men were abstinent for 3.0 days (SD 1.7) in the clinic group and 4.1 days (SD 5.4) in the home group (p = 0.028). Mean time to semen processing was 35.7 min (SD 9.4) in the clinic group and 82.6 min (SD 33.8) in the home group (p < 0.001). There was no association between collection location and increased time to processing on sperm motility, total motile count, FR, D5-UQBR, or PR. CONCLUSIONS: Our data suggest that increased time to processing up to 2 h with home semen collection does not negatively impact sperm parameters or early IVF/ICSI outcomes.

Sperm cryopreservation during the SARS-CoV-2 pandemic.

PURPOSE: Sperm cryopreservation is fundamental in the management of patients undergoing gonadotoxic treatments. Concerns have risen in relation to SARS-CoV-2 and its potential for testicular involvement, since SARS-CoV-2-positive cryopreserved samples may have unknown effects on fertilization and embryo safety. This study therefore aimed to analyze the safety of sperm cryopreservation for cancer patients after the onset of the pandemic in Italy, through assessment of the risk of SARS-CoV-2 exposure and viral RNA testing of semen samples. METHODS: We recruited 10 cancer patients (mean age 30.5 ± 9.6 years) referred to our Sperm Bank during the Italian lockdown (from March 11th to May 4th 2020) who had not undergone a nasopharyngeal swab for SARS-CoV-2 testing. Patients were administered a questionnaire on their exposure to COVID-19, and semen samples were taken. Before cryopreservation, SARS-CoV-2 RNA was extracted from a 150 µl aliquot of seminal fluid in toto using QIAamp viral RNA kit (Qiagen) and amplified by a real time RT PCR system (RealStar SARS-CoV2 RT PCR, Altona Diagnostics) targeting the E and S genes. RESULTS: The questionnaire and medical interview revealed that all patients were asymptomatic and had had no previous contact with COVID-19 infected patients. All semen samples were negative for SARS-CoV-2 RNA. CONCLUSION: This preliminary assessment suggests that a thorough evaluation (especially in the setting of a multidisciplinary team) and molecular confirmation of the absence of SARS-CoV-2 in seminal fluid from asymptomatic cancer patients may assist in ensuring the safety of sperm cryopreservation.

[SARS-CoV-2 infection: implications for sexual and reproductive health. A position statement of the Asociación Española de Andrología, Medicina Sexual y Reproductiva (ASESA)]. / Infección por SARS-CoV-2: implicaciones para la salud sexual y reproductiva. Una declaración de posición de la Asociación Española de Andrología, Medicina Sexual y Reproductiva (ASESA).

OBJECTIVE: The main objective of this revision is to summarize the current existing evidence of the potential adverse effects of SARS-CoV-2 on the male reproductive system and provide the recommendations of the Asociación Española de Andrología, Medicina Sexual y Reproductiva (ASESA) concerning the implications of COVID-19 infection in the management of male infertilty patients and testicular endocrine dysfunction. METHODS: A comprehensive systematic literature search of the databases of PubMed, Web of Science, Embase, Medline, Cochrane and MedRxiv, was carried out. RESULTS: The presence of orchitis as a potential complication of the infection by SARS-CoV-2 has not yet been confirmed. One study reported that 19% of males with COVID-19 infection had scrotal symptoms suggestive of viral orchitis which could not be confirmed. It is possible that the virus, rather than infecting the testes directly, may induce a secondary autoimmune response leading to autoimmune orchitis. COVID-19 has been associated with coagulation disorders and thus the orchitis could be the result of segmental vasculitis. Existing data concerning the presence of the virus in semen are contradictory. Only one study reported the presence of RNA in 15.8% of patients with COVID-19. However, the presence of nucleic acid or antigen in semen is not synonyms of viral replication capacity and infectivity. It has been reported an increase in serum levels of LH in males with COVID-19 and a significant reduction in the T/LH and FSH/LH ratios, consistent with subclinical hypogonadism. CONCLUSIONS: The findings of recent reports related to the potential effects of COVID-19 infection on the male reproductive system are based on poorly designed, small sample size studies that provide inconclusive, contradictory results. Since there still exists a theoretical possibility of testicular damage and male infertilty as a result of the infection by COVID-19, males of reproductive age should be evaluated for gonadal function and semen analysis. With regard to the sexual transmission of the virus, there is not sufficient evidence to recommend asymptomatic couples to abstein from having sex in order to protect themselves from being infected by the virus. Additional studies are needed to understand the long-term effects of SARS-CoV-2 on male reproductive function, including male fertility potential and endocrine testicular function.

Influence of seasonal differences on semen quality and subsequent embryo development of Belgian Blue bulls.

Belgian Blue bulls are more susceptible to high temperature and humidity index (THI) than most other cattle breeds. Here, we investigated whether high ambient temperature during summer affected semen quality and subsequent embryo development in Belgian Blue cattle. For this purpose, semen samples were collected from six healthy mature Belgian Blue bulls in March (Low THI group; THI between 30.6 and 56.4) and August 2016 (High THI group; maximum THI of 83.7 during meiotic and spermiogenic stages of spermatogenesis; 14-28 days prior to semen collection) respectively. Motility, morphology, acrosome integrity, chromatin condensation, viability, and reactive oxygen species production were assessed for frozen-thawed semen. Moreover, the efficiency of blastocyst production from the frozen-thawed semen samples of the two groups was determined in vitro. Blastocyst quality was determined by assessing inner cell mass ratio and apoptotic cell ratio. Fresh ejaculates showed a higher sperm concentration in low THI when compared to the high THI group (P ≤ 0.05), whereas semen volume, subjective motility, and total sperm output were not affected (P > 0.05). In frozen-thawed semen, total and progressive motility, viability, and straight-line velocity were lower in high THI compared to the low THI group (P < 0.05), while H2O2 concentration, aberrant chromatin condensation, and abnormal spermatozoa were higher in the high THI group (P < 0.05). Blastocyst rates were significantly higher when low THI samples were used (P < 0.05). Moreover, the total cell number and trophectoderm cells were significantly higher (P < 0.05) in blastocysts derived from low THI samples, whereas the apoptotic cell ratio was significantly higher (P < 0.01) in blastocysts derived from high THI spermatozoa. In summary, our data show that elevated ambient temperature and humidity during summer can decrease the quality of frozen-thawed spermatozoa in Belgian Blue bulls and also affect subsequent embryo development.