Introduction

In December 2019, China has reported a group of pneumonia cases of unknown etiology to the World Health Organization. A novel β-coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was detected in samples taken from the patient's lower respiratory tract. According to the statistics of the World Health Organization, as of March 21, 2023, coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has led to 761 million confirmed cases and 6.88 million deaths, which has significantly stressed public health systems worldwide [1]. Like SARS-CoV-1, SARS-CoV-2 infects host cells with transmembrane serine protease 2 and receptor angiotensin-converting enzyme 2 (ACE2) [2]. Notably, ACE2 is expressed in many tissues including the cardiovascular system, gastrointestinal tract and liver in addition to the lungs. Correspondingly, damage to these organs has been observed in patients with COVID-19 [3,4,5].

The human male reproductive system is vulnerable to virus infection because the blood-testis barrier cannot completely prevent the virus. Up to now, a variety of viruses that can induce orchitis and lead to male infertility have been detected in human semen (e.g., mumps virus, Zika virus, human immunodeficiency virus etc.), but the picture is less complete for COVID-19 [6]. Seminiferous duct cells, renal tubular cells, Leydig and Sertoli cells show a high level of ACE2 expression, which suggests that the testis could be a potential target for direct damage by SARS-CoV-2 [7]. In addition, the testicular expression of ACE2 is age related. Specifically, ACE2 is highly expressed in patients aged 20-30 years, while it is extremely low in patients aged 60 years [8]. Thus, male reproductive health during the COVID-19 pandemic has aroused widespread concern. However, there have been some limitations such as small sample size and inadequate study design in some studies on the effects of COVID-19 on male reproduction [9]. In addition, most studies conducted in this area did not present pre-infection data as a reference point and, instead, rely on cross-sectional comparisons with uninfected controls. Moreover, there have been few studies on the mechanisms underlying the observed impacts of SARS-CoV-2 infection on semen quality. Here, based on the available evidence, we included controlled before-and-after trials to discuss the changes in sperm parameters and further explored the mechanism of the effects of COVID-19 on male reproduction in terms of fever, inflammation and oxidative stress (OS). This helps clinicians assess male fertility in COVID-19 positive or recovered patients and provides guidance for the andrology treatment.

Changes of semen parameters in COVID-19 cases

In our previous systematic review, we reviewed the effect of COVID-19 on male fertility [10]. However, majority of included studies were difficult to use the self-controlled study design to eliminate confounding factors and selection bias since most subjects in these studies underwent semen analysis only once. To obtain more accurate data, all the studies we included in this review were controlled before-and-after trials.

[Table 1 summarizes the changes in semen parameters of subjects before and after SARS-CoV-2 infection [11,12,13,14,15]. Most patients diagnosed with COVID-19 showed decrease in total motility of sperm. At the same time, the sperm progressive motility, concentration and volume also showed significant reduce in some semen samples. Furthermore, it appears to correlate to the disease severity. Specifically, all sperm parameters were significantly reduced in the moderate symptom group, while only the progressive and total motility were decreased in the mild symptom group [14].

Table 1 Pre-COVID-19 and post-COVID-19 semen parameters of the participants
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Possible mechanisms of COVID-19 affecting male reproduction

We have summarized that SARS-CoV-2 was not found in semen samples of COVID-19 patients in most studies [16]. Even several studies described the presence of SARS-CoV-2, it may be because the samples are not collected under strictly sterile conditions, and thus be contaminated with aerosols or other body fluids from the patients [17]. Therefore, the semen injury described above is probably caused by the coexistence of several biological mechanisms that synergistically interfere with the reproductive system instead of direct infection.

Fever

Fever has been widely reported as one of the most common clinical manifestations of COVID-19 patients. For example, a study of 4203 patients mostly from China identified that 80.5% were diagnosed with fever [18]. Moreover, the estimated duration of fever in patients with SARS-CoV-2 is longer than those with MERS and other virus diseases [19,20,21]. Thus, COVID-19-induced fever may have a greater impact.

The process of spermatogenesis is temperature-dependent and occurs optimally when the temperature is slightly lower than that of the body. Although the testis contains heat shock proteins that resist high temperatures and chemical radiation, changes in its temperature occur when the body is in a state of high heat for a long time [22]. Jannes et al. observed that both relative and absolute testis weight decreased following scrotal heating [23]. Moreover, upon prolongation, the rate of cell cycle disruption is higher than the production of heat shock proteins, hence inducing spermatocyte apoptosis [24]. Several reports have found that an acute fever of 39-40 °C for 1-4 days could cause changes in the sperm parameters such as vitality, total sperm count, and DNA fragmentation [25, 26]. Thus, it is easy to understand that males showed a period of infertility for 10-32 days after testicular heating [27]. Temiz et al. demonstrated a significant decline in the percentage of normal morphology of the semen in COVID-19 patients and speculated that this was caused by fever [28]. Meanwhile, the fever-positive group showed significantly lower complete sperm motility and concentration than the fever-negative group [29]. However, Erbay et al. found that sperm motility and vitality decreased in both COVID-19 patients with and without fever. Moreover, it was observed that fever did not contribute to this condition [13]. Data derived from two other studies also supported this thought [12, 30]. There are several points to consider when interpreting these divergent results. First, the sample size was limited and most of the patients had only one semen sample collection performed after infection. In addition, there may have been differences in the degree of fever in the populations recruited for these studies. Thus, whether the effect of COVID-19 on sperm parameters is caused by fever remains controversial, and more detailed grouping studies of larger populations are necessary in the future.

Inflammation

The blood-testis barrier provides an isolated immune-privileged microenvironment for sperm, which can actively exclude immune cells and other factors from entering the seminiferous tubules and being exposed to developing germ cells [31]. However, despite the privileged immune status, testis cannot be protected from the general immune response. Histopathological changes including inflammatory damages of seminiferous tubules with interstitial edema, congestion, inflammatory cell infiltration and erythrocyte exudation were observed in COVID-19 patients in two separate reports, suggesting that COVID-19 might damage the male reproductive system by inflammatory [32, 33]. As a substantial systemic inflammatory syndrome, hemophagocytic lymphohistiocytosis is a recognized mechanism that appears to contribute to tissue damage and multiple organ dysfunction in COVID-19 patients. This immunologically induced multi-organ damage includes testicular damage, which may have a negative effect on spermatogenesis, thus adversely affecting the quality of semen and male fertility [34]. Moreover, infiltration of leukocytes, CD68+ macrophages and CD3+ T lymphocytes into the testicular interstitial tissue can produce interferons that may decrease the production of testosterone, which is also associated with abnormal spermatogenesis [35,36,37]. Notably, the plasma levels of cytokines (IL-2, IL-6, IL-7, IL-10, TNF-α and MCP-1) are higher in patients with severe SARS-CoV-2 infection [3, 38]. Available evidence suggests that the imbalance between these pro- and anti-inflammatory molecules, such as IL-6 and TNF-α, in testis cells can result in orchitis [39]. However, orchitis is not a common symptom in male patients with COVID-19 (8.82%, 50/567) (Table 2) [15, 29, 40,41,42,43,44,45,46,47,48,49,50]. This may be due to the immunosuppressive properties of Sertoli cells, which plays a key role in suppressing inflammation and limiting virus-associated testicular damage to a certain extent [51]. Notably, 74 (42.05%) showed an abnormal value for at least one sperm parameter among 176 male COVID-19 patients, which was much higher than the incidence of orchitis (Table 2). Thus, it is reasonable to speculate that orchitis is not the entire cause of abnormal sperm parameters.

Table 2 Clinical characteristics of semen or testicular tissue among patients with COVID-19
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Oxidative stress

Most mechanisms of tissue damage caused by SARS-CoV-2 infection are directly associated with OS, which occurs when the balance between oxidants and antioxidants is disrupted towards an overabundance of reactive oxygen species (ROS) [52, 53]. Indeed, ROS production was significantly elevated in COVID-19 patients with a median of nine times higher than in healthy control group and was particularly high in severe cases [54]. At normal physiological levels, ROS mediate important physiological mechanisms such as sperm maturation, capacitation, acrosome reaction, and fertilization by regulating intracellular signaling cascades [55]. However, excessive ROS have adverse effects on the proteins and lipids of the sperm plasma membrane as well as induce the breakdown of sperm DNA [56]. This probably because the cell membranes of sperm are rich in polyunsaturated fatty acids that are extremely vulnerable to ROS-induced lipid peroxidation [57]. Any damage to the cell membrane consequently disturbs its fluidity, leading to impaired sperm motility and dysregulated events of membrane fusion during the acrosome reaction and fertilization [58]. OS status in the COVID-19 patients was remarkably changed as evidenced by increased levels of lipid peroxidation [59]. It promotes destruction of membrane fluidity and rapid loss of sperm adenosine triphosphate, leading to midpiece defects, reduced sperm motility and viability [60]. In addition, ROS-induced DNA damage was found in both the nuclear and mitochondrial genomes of sperm [61]. Human telomeres contain many repeats of the guanine rich hexamer, which are preferential targets of free-radical attack owing to the fact that guanine is more susceptible to OS than other nucleotides. Oxidative damage to telomeres usually results in the generation of highly mutagenic 8-hydroxy-2-deoxyguanosine adducts [62]. When paired with adenine, it forms a stable Hoogsteen mispair containing two hydrogen bonds by adopting the syn conformation around the N-glycosylic bond. This results in G: C to T: A inversion during DNA replication and leads to single-strand and double-strand breaks [63,64,65]. Moreover, mitochondrial DNA is susceptible to oxidative damage caused by OS due to the lack of protective histones, DNA-binding proteins and effective repair systems [66]. In the meanwhile, dysfunctional mitochondria produce less adenosine triphosphate levels which can inhibit the progression of stem cell precursors during the cell cycle, leading to hypospermatogenesis or maturation arrest [67]. These OS-induced damages, especially sperm DNA fragmentation may adversely affect reproductive outcomes because the inherent integrity of DNA determines the success rate of fertilization and embryonic development [68]. Generally speaking, DNA fragmentation index <30% is considered a necessary prerequisite for optimal fertility. Falahieh et.al detected that it was 33.10% in samples from COVID-19 patients on day 14 after diagnosis. Moreover, sperm total motility and progressive motility were below the normal range. Fortunately, the ROS concentrations decreased at 120 days after the COVID-19 diagnosis compared with the acute phase, accompanied by improvements in DNA fragmentation index and sperm motility [69]. Thus, the increases in semen ROS levels play a key role in the adverse effects of SARS-CoV-2 infection on sperm parameters, but the damage will gradually decrease over time.

Conclusions

Given the limitations of previous studies, we included controlled before-and-after studies to get more accurate data on the effects of SARS-CoV-2 infection on male reproduction. The results showed that patients diagnosed with COVID-19 had a significant decrease in sperm parameters (sperm concentration, volume, progressive motility and total motility). Moreover, the magnitude of the decline is related to the severity of the disease. Given that SARS-CoV-2 viral RNA was not found in semen samples from most COVID-19 patients, these changes may be caused by several biological mechanisms that synergistically interfere with the reproductive system rather than direct viral infection. COVID-19-induced fever was once thought to be one of the important causes, but the extent to which fever plays a role in this remains to be further investigated. Systemic or local inflammation in COVID-19 patients can cause hemophagocytic lymphohistiocytosis and upregulation of cytokines, thus leading to testicular damage and abnormal spermatogenesis. Notably, the prevalence of abnormal sperm parameters was higher than the incidence of orchitis in COVID-19 patients. Thus, the orchitis is not the only cause of abnormal sperm parameters. Another concern about how COVID-19 affects male fertility is OS. Recent study has indicated that excessive ROS can result in decreased sperm motility and sperm maturation arrest by lipid peroxidation of sperm membrane as well as sperm DNA damage. Therefore, infected males should be examined for fertility as soon as possible after recovery, particularly if they are married and wish to have children. Moreover, further studies are needed to assess the exact mechanisms by which COVID-19 affects the male reproductive system and fertility and to estimate the reversibility of its long-term effects.