3.1 Viruses and the male reproductive tract

The male reproductive tract consists of (i) testes (or testicles, two oval-shaped organs contained in a thick-skinned sac or scrotum, consisting of seminiferous tubules or the spermatogenetic region of the testis, where the process of spermatogenesis occurs, and of the interstitium, where testosterone is produced), (ii) epididymis (a part of the paratesticular region, along with the rete testis and other stuctures of the testicular collecting system, the testicular tunics, and the spermatic cord, where sperm cells move from the testes, finish maturing, and are stored), (iii) vas deferens (ductus deferens, or sperm duct, a long muscular tube running from the epididymis into the pelvic cavity behind the bladder and connecting to the urethra), (iv) ejaculatory ducts (a paired excurrent duct system for sperm transport, maturation, and storage, which is the union of the duct of the seminal vesicles with the ampulla of the vas deferens), (v) accessory sex glandular organs (seminal vesicles, prostate, and bulbourethral glands or Cowper's Glands, which produce and release the seminal fluid, secreting complex substances and molecules into the final ejaculate), and (vi) the penis (an erectile organ for the penetration and delivery of the ejaculate).^{15, 16}

Several viruses can be detected in the human semen and in the male reproductive tract. Salam and Horby¹⁷ performed a literature review and found that at least 27 viruses, belonging to a broad range of virus families (from adenoviruses to anelloviruses, arenaviruses, bunyaviruses, filoviruses, flaviviruses, hepadnaviruses, herpesviruses, paramyxoviruses, parvoviruses, polyomaviruses, retroviruses, and togaviruses), can be found in human semen, even though for most of these viruses clear-cut data concerning sexual transmission are lacking.

Epidemiological, molecular/phylogenetical evidence of sexual transmission and/or semen isolation have been unambiguously demonstrated only for 12 viruses (44.4%): namely, Ebola virus,^18-22 Marburg virus,^{23, 24} human pegivirus (also known as GB virus C),²⁵ hepatitis B (HBV) and C (HCV) viruses,^26-28 Zika virus,^{29, 30} cytomegalovirus,^31-33 Epstein-Barr virus,³⁴ herpesviruses types 1 (HHV-1), 2 (HHV-2), and 8 (HHV-8, known as Kaposi's sarcoma-associated herpesvirus, KHV),^35-37 and human T-cell lymphoma virus type 1.³⁸ Survival in the testis and in sperm of these viruses may be related to their structural biology and stability, specific viral epitopes, immune evasion, and other factors. Many viruses have been found to interact directly with semen, impair its quality, or impact/disrupt spermatogenesis by causing local inflammation.^{17, 39, 40}

Besides these viruses, other viral agents could be potentially isolated from semen, including influenza virus, lymphocytic choriomeningitis virus, phlebotomus fever virus, coxsackie B virus, echovirus, dengue virus, “Severe Acute Respiratory Syndrome” (SARS) coronaviruses type 1 (SARS-CoV) and 2 (SARS-CoV-2), rubella virus, Lassa virus, Crimean-Congo hemorrhagic fever virus, and poxviruses (such as smallpox virus, and vaccinia virus).^41-53 However, for SARS-CoV-2 evidence related to sexual transmission is particularly ambiguous and contrasting. Evidence about vaccine-strain vaccinia viruses as sexually transmitted pathogens is scarce and anecdotal, with a few cases of genital lesions, including vaginal, vulvar, and, less frequently, perianal vaccinia infections, in women apparently healthy or with underlying conditions (like atopic dermatitis), reporting having sex with male civilian^41-44 or military smallpox vaccinees,^{45-51, 53} or cases of genital and perianal rash in men having sex with male civilian smallpox vaccinees.⁵² It can be hypothesized that “sexual intercourse provides the intimate contact required for heteroinoculation.”⁴¹ Of note, the cases reported in the MSM community include the first described instance of tertiary vaccinia transmission through sexual contact.⁵² Interestingly, the secondary vaccinia case reported a history of psoriasis and a possible history of eczema (atopic dermatitis), whilst the tertiary vaccinia case recalled a history of eczema: both exhibited systemic symptoms, requiring hospitalization and vaccinia immune globulin intravenous administration.⁵²

Recently, also the monkeypox virus has been detected in human male semen. In Italy, Antinori et al.⁵⁴ described four monkeypox cases in MSM in their thirties, with a past history of sexually transmitted diseases (including HAV, HBV, HCV, and syphilis), two were HIV-positive and the other two HIV-negative on HIV pre-exposure prophylaxis (PrEP). Interestingly, one of the four cases was a sex worker. The quantification of the viral load in the seminal fluid by means of the quantification cycle (Cq) technique yielded values comparable to those obtained for nasopharyngeal swabs (ranging from 27 to 30). Of note, the seminal sample was collected 5−7 days after symptoms onset. While, on the one hand, Cq values seem to exclude the hypothesis of biological sample contamination, on the other hand, they may be not enough to allow the virus to be isolated and cultured. However, virus infectivity cannot be ruled out and warrants further research.

In another recent study, Thornhill et al.⁵⁵ analyzed an extensive series of monkeypox infection cases from 16 countries, suggesting a substantial likelihood of its sexual transmission, supported by findings of primary genital, anal, and oral mucosal lesions, which could represent the inoculation site. Still, according to these authors, virus DNA was PCR detectable in seminal fluid in 29 of the 32 tested cases (90.6%); however, it has to be determined yet if those viruses are competent for replication and transmission.

As previously said, the finding of a relatively high number of viruses across a span of such diverse viral families, including poxviruses and, in particular, monkeypox virus, seems to suggest that the presence of a virus in human male semen may depend on an array of parameters, including biochemical/biophysical features in terms of specific/conserved epitopes, the ability of a virus to replicate within the male reproductive tract and/or to exert direct cytopathic effects, and its structural stability, even though all this does not necessarily mean that the virus can be sexually transmitted.

Some factors can explain the presence of a virus in semen: for instance, (i) some mechanisms of immune evasion/escape, especially at the level of the male reproductive tract, (ii) high levels of viremia, (iii) some local/systemic inflammatory mediators that can alter the blood-testis barrier (or to be more precise, the blood-testis-deferens-epididymis barrier) permeability, (iv) systemic immunosuppression, and, finally, (v) a past history or the coexistence of sexually transmitted diseases, such as hepatitis or syphilis. Viruses could have also reached the testes, coming from accessory glands.^{56, 57}

Finally, viral persistence in human male semen does not imply its replicating capacity: viruses can persist even if they are unable to replicate since the testis is immunologically privileged (the so-called “sanctuary site theory”). Mammalian testes represent, indeed, a unique immunological milieu, which is restricted to enable the various steps and processes of spermatogenesis.^{56, 57}

3.2 Poxviruses and direct cytopathic effects at the level of the male reproductive tract

Smallpox can be subdivided into the following five clinical types: (i) ordinary, which has represented the most commonly observed type, characterized by fever and several vesiculopustular skin lesions, and affecting unvaccinated individuals, (ii) modified, without fever and few vesiculopustular skin lesions, affecting vaccinated individuals, (iii) variola sine eruptione, with fever, but without skin lesions, affecting vaccinated individuals, and two rare, but severe forms, namely, (iv) the flat form (with erythemas, and flat vesicles with little fluid), and (v) the hemorrhagic one (with erythemas, petechiae, and ecchymoses), both affecting unvaccinated individuals.⁵⁸ Smallpox, especially the hemorrhagic clinical form,⁵⁹ is a rare cause of acute inflammation and infection affecting the testicular interstitium (“multifocal interstitial orchitis”), with or without central necrosis, as well as of other smallpox-associated testicular and male reproductive lesions, including inflammation of the prostate gland (“prostatitis”), and of the epididymis. The latter inflammation is generally characterized by lymphohistiocytic infiltrates (“lymphohistiocytic epididymitis”).

A temporal correlation, even though loose, between the development of these lesions and the progression of the cutaneous ones has been described. According to Martin⁶⁰ and previous research,^61-65 the papular stage on the skin corresponded to hyperemia and infiltrates of large lymphocytes at the testicular interstitium level. During the pustulation stage, necrosis of the walls of the seminiferous tubules and the interstitium could be described. The picture may be age-specific, with a more ischemic picture predominating in children, because of their less-developed testicular vascularization.

From a histopathological perspective, the inflammatory infiltrate is generally histiocytic and lymphocytic, and less commonly eosinophilic, along with a seminiferous tubule degeneration.^{63, 66} Particularly advanced forms of smallpox can exhibit azoospermia,⁶⁷ as well as arteritis (including mononuclear endarteritis with endothelial hypertrophy), thrombosis, perivascular hemorrhage, and rather discrete infarcts.^{63, 68}

According to a report by Chowdhury et al.⁶⁹ azoospermia is quite a common finding in people with a history of smallpox. Out of 31 subjects with a past history of smallpox, 15 (48.4%) displayed complete azoospermia, while 1 (3.2%) showed sperm density of less than 10 million/ml.

In a case-control study carried out in Bombay, India, Phadke et al.⁷⁰ found that, while the incidence rates of severe and moderate oligospermia were, respectively, practically comparable in the smallpox and control series, the incidence of azoospermia was, however, higher in the smallpox series (42.57% vs. 17.87% in the control series). Similarly, the rate of normospermia was lower in the smallpox series (30.17% vs. 52.52% in the control series). Of note, the incidence rate of obstructive azoospermia was higher in the smallpox series (79.36% vs. 46.23% in the control series). On the other hand, the rates of testicular lesions and partial or complete arrest of spermatogenesis at various levels, as well as germinal cell aplasia, severe tubular atrophy, and hyalinization of the tubules were similar in both series.

3.3 Monkeypox virus and its atypical clinical presentation during the “2022 monkeypox epidemic”

According to a recent review of the literature,¹¹ anogenital lesions would be present in 31.4% of the monkeypox cases. Two recent publications describe a “pure” genital presentation of monkeypox, which is highly unusual for this disease, as it is classically known in endemic countries. In France, Davido et al.⁷¹ described a 37-year-old MSM on PrEP, with a past history of treated syphilis. The patient presented sudden fever, headache, mild diarrhea, and genital macular lesions without itching. Of note, face, trunk, and extremities (limbs, palms, and soles) were spared. Another 37-year-old self-defining MSM, with a past history of vitiligo, presented an initial series of itching, and vesicular lesions affecting the penis. Subsequently, the patient became febrile, with macular lesions and pubic erythema. Perianal lesions and inguinal adenopathy were also described. In Portugal, Patrocinio-Jesus and Peruzzu⁷² described the case of a 31-year-old MSM, HIV-positive on medications, with excellent virologic control, that presented with fever, sore throat, and painless genital rash. Ulcerated lesions and umbilicated pustules affecting his penis were noted, along with vasicopustular lesions affecting his face and hands. In the United Kingdom, Heskin et al.⁷³ have described monkeypox infection in a serodiscordant couple. The two subjects reported perioral white spots, perianal blistering lesions, anogenital, and pubic lesions, along with lymphadenopathy, fever, headache, and diarrhea. Interestingly, the authors noted a close correlation between the lesion site and the type of sexual practice, pointing to sexual transmission as a novel route of monkeypox infection. Finally, anogenital signs/symptoms had been rarely described in previous outbreaks, such as those occurring in the United Kingdom and in Israel.¹¹

All these findings have been confirmed by a recently published systematic review of the literature and meta-analysis, which synthesized data related to 4222 confirmed cases of monkeypox, of which 3876 (91.8%) were potentially deemed due to sexual transmission.⁷⁴ The most commonly reported clinical manifestations were painful perianal and genital lesions, together with fever, lymphadenopathy, headache, and malaise.⁷⁴

Furthermore, the origin of the present monkeypox epidemics, as previously said, has been associated with large sex-related parties among MSM, such as those at Antwerp (Belgium) and at Maspalomas (Spain), explaining the high prevalence of monkeypox infection in this group.^{11, 74} This is interesting since it could have impacted viral fitness, affecting endemic and epidemic circulation, as well as virulence, and facilitating transmission. Mass gatherings may have resulted in the geographic introductions of human-amplified monkeypox viruses and led to the emergence of human disease. Translating to infectious diseases a concept from the field of evolutionary genetics, this is known as the “founder effect.”⁷⁵

3.4 Poxviruses, the “imperfect nature” of the blood-testis barrier, and the testes as an immunologically privileged site

The blood-testis barrier is a unique, extremely complex, biologically, and physiologically fascinating ultrastructure in the mammalian testis.^{76, 77} It consists of Sertoli cells—the epithelial cells of the seminiferous epithelium—and, in rodents, also of peritubular myoid cells. It is located near the basement membrane of the seminiferous tubule segregating the seminiferous epithelium into the basal and the adluminal/apical compartments. Different types of junctions make the blood-test barrier particularly tight and sealed, restricting trans and paracellular flux of several biomolecules (from hormones to other endocrine and paracrine factors), electrolytes, water, and drugs/toxicants (Figure 1).

These various families of junctions include actin-based tight junctions, gap junctions, intermediate filament-based desmosomes, and basal ectoplasmic specializations. The latter represent a testis-specific, atypical adherens junction. Besides regulating the passage of molecular compounds, the blood-test barrier enhances and supports/promotes the various phases of the spermatogenesis process.^78-80

The Leydig cells are responsible for testosterone production and release and are located in the testicular interstitium, together with fibroblasts, macrophages, blood, and lymphatic vessels. The Sertoli cells along with the Leydig cells can secrete an array of multiple immunosuppressive molecules, including activin A, Transforming Growth Factor beta, and other growth factors, multifunctional cytokines, Programmed Death-Ligand 1 (PD-L1, also known as cluster of differentiation or CD-274 or B7 homolog 1), Growth arrest-specific protein type 6, and testosterone,^81-83 among others, which exert either direct or indirect effects, inhibiting/regulating immune cell activation. Since semen is highly immunogenic, these molecules enable its survival and maturation.⁸⁴

Moreover, the Sertoli cells are able to synthesize two membrane-associated proteoglycans containing, as glycosaminoglycan moieties, either chondroitin sulfate only or chondroitin sulfate and heparan sulfate; the latter proteoglycan is, therefore, known as the mixed proteoglycan.⁷⁹ Poxviruses can potentially exploit the binding to these molecules to effectively enter host cells. Other molecules are integrin β1 and CD98/CD98 receptor^{85, 86}: interestingly, these are also expressed at the level of Sertoli cells and are involved in various molecular events, such as testicular development and spermatogenesis.⁸⁰