Bijlage 1.Wiley. COVID‐19 and human spermatozoa—Potential risks for infertility and sexual transmission

Received: 2 June 2020

| Revised: 29 June 2020 | Accepted: 1 July 2020

DOI: 10.1111/andr.12859

OPINION ARTICLE

COVID-19 and human spermatozoa—Potential risks for infertility and sexual transmission? Robert John Aitken Priority Research Centre for Reproductive Science, Faculty of Science and Faculty of Health and Medicine, University of Newcastle, Callaghan, NSW, Australia Correspondence Robert John Aitken, Priority Research Centre for Reproductive Science, Faculty of Science and Faculty of Health and Medicine, University of Newcastle, Callaghan, NSW 2308, Australia. Email: john.aitken@newcastle.edu.au

Abstract As COVID-19 infections wreak havoc across the globe, attention has rightly been focused on the vital organ systems (lung, kidney and heart) that are vulnerable to viral attack and contribute to the acute pathology associated with this disease. However, we should not lose sight of the fact that COVID-19 will attack any cell type in the body expressing ACE2 - including human spermatozoa. These cells possess the entire repertoire of receptors (AT1R, AT2R, MAS) and ligand processing enzymes (ACE1 and ACE2) needed to support the angiotensin signalling cascade. The latter not only provides COVID-19 with a foothold on the sperm surface but may also promote integration, given the additional presence of a range of proteases (TMPRSS2, TMPRSS11B, TMPRSS12, furin) capable of promoting viral fusion. This article reviews the roles played by these various cellular constituents in maintaining the vitality of human spermatozoa and their competence for fertilization. The reproductive consequences of a viral attack on these systems, in terms of fertility and the risk of sexual transmission, are currently unknown. However, we should be alive to the possibility that there may be reproductive consequences of COVID-19 infection in young males that go beyond their capacity to survive a viral attack. KEYWORDS

ACE2, COVID-19, male infertility, spermatozoa, TMPRSS2, viral fusion

The COVID-19 (SARS-CoV-2) virus has created a global pandemic re-

at the acute stage of infection and, alarmingly, two who were re-

sponsible for over 500 000 deaths and untold human and economic

covering) tested positive for the virus by RT-PCR.1 Importantly, at

suffering. Attention has rightly focused on the respiratory system,

this point, we have no idea whether the actual virus was viable and

because this is where a life-and-death battle is waged between the

infectious. Nevertheless, the possibility that this coronavirus could

viral horde and the host's immune system. However, other tissues are

have a pathophysiological impact on the testes was suggested by

also susceptible to viral attack including the kidney, heart, and brain.

additional data indicating that active COVID-19 infection dramati-

The purpose of this communication is to highlight the evidence that

cally reduced the testosterone-to-LH ratio, suggesting a significant

the male reproductive system, particularly spermatozoa, constitutes

impact on the responsiveness of Leydig cells to LH stimulation. 2

another vulnerable target, raising the possibility that COVID-19

In many ways, we should not be surprised by these observations

might ultimately induce male infertility and/or facilitate the sexual

because the blood-testes barrier is known to offer little defense

transmission of this virus, depending on the level of infection.

against viral invasion, given the wide range of pathogenic viruses

A recent report published in JAMA Network Open revealed that

(HIV, hepatitis, mumps, papilloma) that are known to be capable of

in an analysis 38 semen samples from COVID-19 patients, 6 (four

damaging the testes and rendering the host infertile. Furthermore,

© 2020 American Society of Andrology and European Academy of Andrology 48

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Andrology. 2021;9:48–52.

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F I G U R E 1 The angiotensin system plays a critical role in the survival and functionality of human spermatozoa but also creates a vulnerability to COVID-19 attack. Angiotensin 1 is a biologically inactive decapeptide that is cleaved by ACE1 to create angiotensin II, which in turn activates the AT1R and AG2R receptors, both of which are present in these cells. Angiotensin II is further processed by ACE2 to generate angiotensin 1-7 which binds the MAS receptor activating PI3K. The latter then phosphorylates AKT, which maintains cell viability by phosphorylating key regulators of sperm apoptosis such as BAD. As long as BAD is phosphorylated, it is held in abeyance by a 14-3-3 keeper protein. However, if the PI3/AKT pathway becomes compromised, BAD dephosphorylates, is released from its association with 14-3-3, and moves to the mitochondria where it inactivates anti-apoptotic factors and promotes the intrinsic apoptotic cascade. The spike protein on COVID-19 specifically targets ACE2 and in so doing removes an important stimulus for PI3K/AKT, thereby compromising sperm viability. Subsequent to COVID-19 binding, the ectodomain of ACE2 may be removed by ADAM proteases and shed from the sperm surface.

Alternatively proteases from the TMPRSS-family, either as intrinsic components of the sperm plasma membrane or delivered by seminal prostasomes, can facilitate fusion between the virus and the sperm surface by cleaving ACE2 and the viral spike proteins (S1 and S2) at the sites indicated by dashed lines, thereby completing the transformation of this cell from procreating gamete to viral vector

the spike protein that gives the COVID-19 virus its corona is known

the ACE gene; it lacks multiple 5' exons and has a distinct N-terminus:

to target ACE2 (angiotensin-converting enzyme 2), which is highly

biochemically however, it performs exactly the same function as

expressed by several cell types in the testes including Leydig cells,

somatic ACE1.10 Spermatozoa also express ACE2, which converts

Sertoli cells, and the germ line. As a result of these factors, several

angiotensin II to angiotensin (1-7). Reference to the human sperm

opinion pieces have been published already, raising the possibility

proteome also indicates that these cells possess the two known

of testicular damage and infertility consequent to COVID-19 infec-

receptors for angiotensin II: angiotensin II type-1 receptor (AT1R)

tion. 2-4 However, it is also possible that the virus could gain access to

and (angiotensin II type-2 receptor) (AT2R). Furthermore, a recent

male germ cells once they leave the testes, either in the epididymis

publication has revealed that human spermatozoa also express the

or following ejaculation. In this Opinion Article, I shall be focusing on

angiotensin (1-7) MAS receptor.9 These cells therefore possess the

this post-testicular route of infection pointing out, for the first time,

complete repertoire of ligand-processing enzymes and receptors

that the mature spermatozoon has all of the machinery needed to

needed to support angiotensin signaling pathways, raising questions

bind this virus, fuse with it, and even achieve reverse transcription of

about the physiological roles these pathways play and how they

the viral RNA into proviral DNA. Such considerations raise the pos-

might intersect with COVID-19 (Figure 1).

sibility that spermatozoa could act as potential vectors of this highly infectious disease. This happens in insects5—why not us?

ACE1 is a very ancient protein the appearance of which precedes the evolution of the angiotensin system. As a consequence, func-

It has been known for many years that the human sperm surface

tions other than angiotensin processing are likely. Indeed, spermato-

expresses ACE. Indeed, an examination of existing proteomic data-

zoa are a good example of the multiple roles of ACE1. This enzyme is

bases6-8 as well as surveys of the sperm surface with monoclonal an-

quite independent of ACE2 (the gene is located on chromosome 17,

tibodies,9 demonstrates that these cells, quite literally, hold all of the

while ACE2 is transcribed from the X chromosome); however, there is

ACEs. They have been known for some time to express a testicular

considerable sequence homology (~41%) between these peptidases

variant of ACE1, which converts the inactive decapeptide hormone,

in terms of their primary amino acid sequences. Notwithstanding

angiotensin I, to the active octapeptide, angiotensin II (Figure 1).

such similarities, COVID-19 does not interfere with the functions of

Testicular ACE corresponds to the ancestral non-duplicated form of

ACE1 and, as a result, one of the consequences of infection with this

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virus is to generate an increase in the availability of angiotensin II

relate to its capacity to enhance cAMP generation and tyrosine

relative to angiotensin (1-7). What does this mean in terms of sperm

phosphorylation. 24

cell biology?

The importance of ACE2 has recently been highlighted by dis-

ACE1 is most heavily expressed on the surface of viable, func-

covery of the MAS receptor on human spermatozoa.9 This receptor

tional spermatozoa and is concentrated in the neck and mid-piece

is localized in the principal piece of the sperm tail and the acrosomal

11

region of the cell.

The expression of ACE1 appears to be important

domain of the sperm head, in exactly the same place as phospho-

for sperm function because patients exhibiting complete absence of

inositide 3-kinase (PI3K). 25 Activation of the MAS receptor by an-

12

The precise role played

giotensin (1-7) leads to PI3K phosphorylation, which in turn leads

by ACE1 is not certain but a synthesis of the data suggests a role

this enzyme are incapable of fertilization.

to the phosphorylation of AKT. 25 While AKT is phosphorylated,

in cell-cell interaction. ACE1 is important for the expression of tes-

downstream phosphorylation targets such as the apoptosis regula-

tis-expressed gene 101 (TEX101) which is, in turn, linked to the sta-

tor, BCL2-associated agonist of cell death (BAD), are also phosphor-

bilization of disintegrin and metallopeptidase domain 3 (ADAM3)

ylated. Phospho-BAD forms a heterodimer with its 14-3-3 keeper

on sperm plasma membrane.13 Mice lacking ADAM3 possess motile

protein, leaving Bcl-2 free to inhibit Bax-triggered apoptosis, thereby

spermatozoa that are incapable of negotiating the utero-tubal junc-

maintaining spermatozoa a viable motile state (Figure 1). However, if

tion and exhibit an impaired capacity to bind to the zona pellucida.

PI3K activity is disrupted, AKT and its downstream targets become

Interestingly, the importance of ACE1 in mediating sperm transport

dephosphorylated, thereby initiating a truncated apoptotic cascade

and interaction with the oocyte may not necessarily be related to its

characterized by rapid motility loss, mitochondrial ROS generation,

angiotensin-processing function but rather to its ability to catalyze

caspase activation in the cytosol, annexin V binding to the cell sur-

the release of glycosylphosphatidylinositol (GPI)-anchored proteins

face, cytoplasmic vacuolization and oxidative DNA damage. 25 Thus,

14

from the sperm surface.

ACE2 plays an essential role in sperm biology; by stimulating PI3K/

This observation does not, however, preclude the possibility that ACE1 also plays an important role in spermatozoa as a com-

AKT phosphorylation, it actively prevents spermatozoa from engaging the intrinsic apoptotic pathway (Figure 1).

ponent of the renin-angiotensin system. By analogy with somatic

The fate of ACE2 following interaction with COVID-19 binding

cells, a COVID-19 attack on human spermatozoa would be ex-

is complex and dependent on cell type. If the cells are capable of

pected to impact ACE2 activity leading to a buildup of angiotensin

endocytosis, then ACE may become internalized within the endo-

II (Figure 1). An increased local availability of angiotensin II would

cytotic vesicle. However, as spermatozoa are generally regarded as

be expected to enhance sperm phagocytosis by neutrophils and

non-endocytotic, the more likely fate of ACE2 is cleavage (shedding)

possibly increase the local availability of reactive oxygen species

of its ectodomain through the action of surface proteases such

(ROS) generated during the phagocytic process.15 Excessive stim-

as ADAM17. 26,27 This may be a protective strategy because shed

ulation of AT1R by angiotensin II might also enhance the genera-

ACE2 ectodomain should bind the virus and prevent it gaining ac-

tion of ROS in association with the induction of apoptosis and cell

cess to the cell surface. On the other hand, proteases recruited by

senescence, given its apoptosis-inducing role in other cell types.16

the virus to facilitate membrane fusion, notably TMPRSS2 (a type

Curiously, AT1R has been identified as one of a handful of proteins

II transmembrane serine protease), can also cleave ACE2 at amino

that are thought to be actively generated in the mature gamete as

acids 697 to 716, resulting in the shedding of 13 kD ACE2 fragment

a result of de novo translation on mitochondrial-type ribosomes,

from the cell surface and facilitating viral entry. 28 Cleavage of ACE2

suggesting that this receptor must play a critical role in the lead-up

following exposure to COVID-19 would be expected to induce a

to fertilization.17 A role in the induction of the acrosome reaction

decrease in sperm viability and function, leading to a loss of fertil-

has been suggested on the basis of the ability of the AT1R antag-

ity. Furthermore, the processing of ACE2 on the surface of human

onist, losartan, to inhibit the induction of acrosomal exocytosis in

spermatozoa may effectively turn these cells into viral vectors, ca-

bovine spermatozoa.18 As angiotensin II also stimulates the acro-

pable of sexually transmitting COVID-19 to other individuals in the

some reaction in human spermatozoa, it is possible that prolonged

community.

exposure to elevated levels of angiotensin II as a consequence of

Actual fusion between the virus and human spermatozoa re-

COVID-19 infection might lead to premature acrosomal exocyto-

quires the presence of the above-mentioned protease, TMPRSS2,

sis and sperm senescence in our species.19 Such effects might be

to cleave the viral spike proteins (S) at the S1/S2 boundary or within

related to the ability of angiotensin II to stimulate nitric oxide gen-

S2 subunit, thereby removing the structural constraint of S1 on S2

20

and releasing the internal membrane fusion peptide (Figure 1). This

This reactive nitrogen radical would be expected to stimulate ca-

protease is known to be present in prostasomes that are released

pacitation and the acrosome reaction in the short term but gen-

into seminal fluid from the prostate gland at ejaculation. 29 As one

erate a state of oxidative stress and senescence if the exposure

of the major functions of these exosome-like structures is to trans-

eration via the AT1R pathway in a losartan-inhibitable manner.

is prolonged.

21

The AT2R receptor, on the other hand, appears

fer their contents, including proteins, to the spermatozoa following

to be important for the regulation of sperm motility in response

ejaculation, the incorporation of TMPRSS2 from this source seems

to angiotensin II generated by ACE1. 22,23 The stimulatory impact

probable.30 Furthermore, a close examination of the human sperm

of the angiotensin II/AT2R axis on sperm movement may, in turn,

proteomic databases reveals the presence of related proteases

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AITKEN

TMPRSS11B and TMPRSS127 as well as furin,6 in these cells, all of

capacity of these cells to fuse with viruses, suggests that we should

which are thought to serve as activating proteases for viral infec-

at least be open to the possibility.

tion including coronaviruses.31-33 The presence of these activating proteases as well as ACE2 in the sperm plasma membrane would be

ORCID

expected to allow the COVID-19 virus to bind to the cell surface and

Robert John Aitken

https://orcid.org/0000-0002-9152-156X

ultimately fuse, either in the testes or during the prolonged sojourn of these cells in the epididymis. In contrast, oocytes appear to be

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germ line highly unlikely—unless, of course, they are fertilized by a COVID-19 carrying spermatozoon. In this context, it should be emphasized spermatozoa have a demonstrable capacity to carry viral infections from the male to the female reproductive tract, as happens during the sexual transmission of the Zika virus, for example.34 They also have a proven capacity to fuse with enveloped viruses35 and possess reverse transcriptase activity capable of generating proviral DNA,36 as is apparently the case for human immunodeficiency virus 1.37

1 | S U M M A RY A N D FU T U R E D I R EC TI O N S At present, there have been few opportunities to study the reproductive competence of patients who have recovered from COVID19 infection. In order to prepare for the moment that we do, there are pieces of information that we should acquire and studies that we should contemplate. At the moment, we do not know the conditions regulating the relative activation of AT1R vs AT2R by angiotensin II in spermatozoa, nor do we fully understand the biological consequences of activating these specific receptors. Given the current availability of AT1R vs AT2R receptor antagonists exhibiting high levels of target specificity, it may be timely to exploit these compounds to gain a deeper understanding of the possible consequences of angiotensin II upregulation consequent to COVID-19 infection at different stages of the spermatozoon's journey from the seminiferous epithelium to the ejaculate.38 We shall also need to assess the extent to which COVID-19 triggers the anticipated release of ACE2 from the sperm surface and whether the loss of this enzyme leads to an increased rate of sperm senescence and cell death.

Most importantly, we shall need to investigate the competence of spermatozoa, at different stages of epididymal maturation and post-ejaculatory activation, to fuse with this coronavirus and reverse transcribe its genome to create the corresponding DNA. What subsequently happens, when viral RNA and proviral DNA are released into the oocyte at fertilization is a crucial question that will have to be addressed particularly if, as projected, post-COVID-19 patients end up requesting assisted reproductive technology to resolve their infertility problems. We clearly still have much to learn about COVID-19. Its arrival has been relatively recent and there are still no definitive data to indicate its impact on male fertility or the potential of spermatozoa to serve as vectors for the sexual transmission of this disease. Nevertheless, the evident importance of the renin-angiotensin and TMPRSS-family proteases in sperm cell biology, as well as the clear

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How to cite this article: Aitken RJ. COVID-19 and human spermatozoa—Potential risks for infertility and sexual transmission?. Andrology. 2021;9:48–52. https://doi. org/10.1111/andr.12859