Semen: A modulator of female genital tract inflammation and a vector for HIV‐1 transmission

Abstract In order to establish productive infection in women, HIV must transverse the vaginal epithelium and gain access to local target cells. Genital inflammation contributes to the availability of HIV susceptible cells at the female genital mucosa and is associated with higher HIV transmission rates in women. Factors that contribute to genital inflammation may subsequently increase the risk of HIV infection in women. Semen is a highly immunomodulatory fluid containing several bioactive molecules with the potential to influence inflammation and immune activation at the female genital tract. In addition to its role as a vector for HIV transmission, semen induces profound mucosal changes to prime the female reproductive tract for conception. Still, most studies of mucosal immunity are conducted in the absence of semen or without considering its immune impact on the female genital tract. This review discusses the various mechanisms by which semen exposure may influence female genital inflammation and highlights the importance of routine screening for semen biomarkers in vaginal specimens to account for its impact on genital inflammation.


| INTRODUC TI ON
Despite the advances made in the treatment of human immunodeficiency virus (HIV), the global HIV prevalence remains unacceptably high. 1 The primary determinants of HIV transmission include the accessibility of target cells for infection and viral characteristics such as quantity and fitness. Female genital inflammation contributes to both the availability of HIV target cells and reduced mucosal barrier integrity. 2,3 Genital inflammation, defined by elevated proinflammatory and chemotactic cytokines, has also been linked to a three-fold greater risk of acquiring HIV in women. 2 Additionally, microbial dysbiosis contributes to inflammation through increased cytokine production, mucosal barrier disruption and immune cell recruitment at the female genital tract (FGT). 4-7 These studies emphasise the role of genital inflammation in HIV acquisition in women and highlight the need to determine factors that contribute to genital inflammation and then limit their relative impact on HIV risk.
The immune altering capacity of semen is often overlooked in heterosexual HIV transmission and semen is merely considered a vehicle for viral transmission to women during condomless sex. 8,9 Semen induces mucosal changes at the FGT to increase the chances of pregnancy, 10-14 and also contains several immunologically active molecules known to both promote and inhibit female genital inflammation. [10][11][12][13][15][16][17][18][19][20][21][22] Initially, the presence of semen in the female reproductive tract results in an inflammatory response involving cytokine production and leukocyte recruitment for the removal of excess and abnormal sperm. 10,11,20,21 The alkaline pH of semen and the microbial content of the ejaculate also contribute to alterations in the vaginal microbiome which are known to promote genital inflammation and HIV risk in women. 4,5,7,23-28 A semen-induced proinflammatory immune response to prime the female reproductive tract for conception may also promote genital inflammation and HIV acquisition in women. 11,[20][21][22] Conversely, semen also contains factors to help regulate this pro-inflammatory response at the FGT since excessive inflammation may lead to adverse pregnancy outcomes. This results in the induction of a regulatory T-cell (Treg) immune response for tolerance to the paternal antigens and to facilitate embryo implantation. 16,[29][30][31][32] A semen-induced tolerogenic immune response may also inhibit the clearance of HIV and other pathogens at the FGT. Taken together, these studies suggest that semen directly alters the biology of the FGT and may have significant consequences for the risk of HIV infection in women. Here, we review the relationship between female genital immunity and male partner semen and its implications for HIV risk in women.  composed of water and mucin glycoproteins but also contains immunoglobulin (Ig)G, IgA and several antimicrobial agents which provide additional protection at the female genital mucosa. 49,50,[53][54][55][56][57] The lower FGT is populated by commensal microbes that can modify the CVM composition and influence its ability to defend against pathogens. Acidic CVM associated with Lactobacillus crispatus dominance and high levels of D-lactic acid can hinder HIV-1 mobility and prevent infection. 52,58,59 Conversely, HIV mobility is significantly increased in CVM derived from women with bacterial vaginosis (BV). 60 This is likely since Gardnerella vaginalis, a common BV-associated microbe secretes sialidase enzymes that degrade the CVM. 61 These findings highlight the complex interplay between the vaginal microbiome and host innate immunity.

| Role of the vaginal microbiome in innate immune defence
An optimal vaginal microbiome is dominated by Lactobacilli spp., which exists in a mutualistic relationship with the host and contributes to the immune defences at the FGT. 62 Commensal microorganisms such as L. crispatus prevent pathogen colonisation by inhibiting their growth, preventing biofilm formation, lowering the vaginal pH, competing for nutrients and adherence to the epithelium, and by producing antimicrobial agents such as lactic acid, hydrogen peroxide (H 2 O 2 ) and bacteriocin. [63][64][65][66][67] Lactobacilli metabolise glycogen secreted by vaginal epithelial cells to produce L-and D-isomers of lactic acid. 67,68 Physiological concentrations of vaginal lactic acid are sufficient to inactivate BV-associated microbes and other sexually transmitted agents of infection, including HIV. 58,59,[69][70][71] Lactic acid lowers the vaginal pH, enhances the activity of other antimicrobial factors and upregulates the production of anti-inflammatory cytokines. 67,72 Taken together, these data suggest that a Lactobacillus-dominant vaginal microbiome is highly beneficial and less vulnerable to HIV infection.

| Adaptive immune responses at the female genital mucosa
Adaptive immunity at the FGT involves either cell-mediated or humoral immunity. Cell-mediated immunity involves the removal and destruction of intracellular pathogens and virus-infected cells by T lymphocytes. Antigen-presenting cells process and display antigens to T cells to trigger a pathogen-specific immune response and promote immunological memory. This adaptive immune response is  73 Conversely, CD4+ T cells recognise antigens presented on MHC class II molecules and respond by secreting cytokines to activate CD8+ T cells, macrophages, and B cells to destroy infected cells. 74,75 Humoral immunity is mediated by B cells and their secreted antibody products. Antibodies prevent and fight infections by binding to antigens on the pathogen and preventing their entry into host cells, coating the pathogen for phagocytosis, inducing antibodydependent cell-mediated cytotoxicity, and by activating the complement pathway. 76,77 IgG is the predominant immunoglobulin isotype found in genital secretions of both HIV-infected and uninfected women. 78,79 T-cell immunity and the abundance of immunoglobulins at the FGT are highly regulated by sex hormones. 73,80 One to two weeks after infection, effector CD4+ and CD8+ T cells die, leaving behind antigen-specific memory T cells that persist long after infection. Memory T cells mount a rapid immune response upon reinfection with the same pathogen and can be subdivided into central memory cells that circulate between the blood and lymph nodes, and resident and recirculating effector memory cells in nonlymphoid tissue. 75,81,82 Tissue-resident memory T cells (TRMs) reside in mucosal tissues and rapidly respond to local infections by producing cytokines to induce immune cell activation and recruitment at the FGT. 75,[83][84][85] Although the physiological role of TRMs is to defend against infections, these cells have also been identified as major targets for HIV at the lower FGT. 86,87

| G ENITAL INFL AMMATION IN CRE A S E S HIV ACQU IS ITI ON RIS K IN WOMEN
Although the female genital mucosa has several defences to prevent infection and the probability of heterosexual HIV transmission is relatively low, 9,88 inflammation can increase the risk of HIV acquisition at this site. This is supported by observations of infection by less fit HIV variants in women with genital inflammation than without. 89 Inflammation is the body's natural response to injury or infection and

| THE S TRUC TURE OF THE MALE G ENITAL TR AC T AND HIV INFEC TI ON
The male genital tract (MGT) is comprised of the penile urethra and the testes (Figure ). In uncircumcised males, the foreskin provides both physical and immunological protection to the glans 103 but is also highly susceptible to HIV infection. 104,105 The outer surface of the foreskin is lined by a double layer of keratinised stratified squamous epithelium that covers the glans/corona and the opening of the penile urethra (meatus). 104,106 The epithelium of the foreskin is relatively resistant to HIV infection unless microabrasions are induced during condomless sex, which may facilitate access to target cells within the underlying epithelium. 104,106,107 The subpreputial cavity, which is the inside of the foreskin, provides an anoxic and moist microenvironment that harbours a diverse array of anaerobic microbes. 27, [108][109][110] The presence of these anaerobic microbes increases the susceptibility of the neighbouring epithelium and the urethral opening to HIV infection via activation of target cells. [108][109][110][111][112][113] Additionally, when the penis is erect, the foreskin retracts, exposing the glans and inner foreskin, which are more susceptible to viral infection. 114 The inner foreskin contains HIV target cells that are directly exposed to the vagina during sexual intercourse. 105,[114][115][116][117][118] Medical male circumcision involves the surgical removal of the foreskin resulting in a dry keratinised epithelial surface that is more resistant to HIV infection. [119][120][121] Circumcision also reduces the diversity of the penile microbiota and may decrease HIV acquisition risk in both men and women. 108,[122][123][124][125][126][127][128] Urine and semen are secreted from the penile urethra, which originates at the bladder and is approximately 20 cm in length and 1-2 cm in diameter. 106,117 In contrast to the foreskin, the urethra is lined with non-keratinised pseudostratified glandular columnar epithelium, which is less resilient to HIV infection. 117,129,130 Given that the epithelium of the penile urethra confers reduced protection against HIV entry and contains a high density of intraepithelial immune cells, this serves as a primary site for infection by sexually transmitted infections (STIs), including HIV. 106,107,117,[130][131][132][133] The epithelium of the urethra also contains several deep invaginations called the periurethral glands of Littre. 117 These Littre glands are responsible for pre-ejaculate secretion that neutralises residual urine in the urethral lumen and acts as lubrication during condomless sex. 117 The testes can be divided into two main regions; these are the interstitial spaces between the tubules and the seminiferous tubules. 131,134 The testes are responsible for the production of testosterone 134,135 and spermatogenesis, which occurs in the coiled seminiferous tubules. [136][137][138] The seminiferous tubules connect to the head of the epididymis and then to the vas deferens via the rete testes. 137 The seminiferous tubules are made up of Sertoli cells that F I G U R E 1 Structure of the male genital tract. The male genital tract is made up of the penile urethra and the testes. The penile urethra is lined with a less resilient non-keratinised pseudostratified glandular columnar epithelium and is a primary site for infection in men. The testes can be divided into two main regions, the seminiferous tubules and the interstitial spaces between the tubules. The testes are responsible for the production of testosterone and spermatogenesis surround the spermatogenic cells and provide essential nutrients to the spermatozoa. 134,135 The peritubular myoid cells are smooth muscle cells that surround the seminiferous tubules of the testis and provide structural integrity to the tubules. 137

| Impact of semen on immune cells at the female genital mucosa
Since semen is initially recognised as foreign in the FGT an immune response is mounted, resulting in cytokine upregulation and the chemotaxis of immune cells. In reproduction, this pro-inflammatory immune response is necessary for the removal of excess and abnormal sperm. 29, 169 However, these semen-induced alterations may also increase susceptibility to HIV infection in women. Semen-derived PGE2 has been associated with the recruitment and activation of HIV target cells. 162 In addition, studies have demonstrated that prior and prolonged exposure to the same donor's semen improved fertility and reduced preeclampsia rates in women, highlighting the importance of immune tolerance to semen in these contexts. 176

cells and reduced infection by Simian Immunodeficiency
Virus. 181 These findings suggest that semen exposure to new or multiple concurrent partners may induce a greater and prolonged inflammatory response, which is associated with adverse pregnancy outcomes and possibly an increased risk of HIV transmission. [176][177][178]180,181 Immune tolerance may be lost on exposure to semen from a new partner, resulting in a more pronounced immune response and suggests a biological link for the relationship between partner concurrency and HIV risk in South African women. 182

| Impact of semen exposure on the vaginal microbiota
Bacterial vaginosis is a state characterised by a shift in the vaginal microbiome from Lactobacillus dominance to a more diverse spectrum of facultative anaerobes. 62,183 Condomless sex has been associated with BV occurrence, 28,184-186 and increases in Escherichia coli at the FGT. 185,[187][188][189] Semen contains a diverse array of bacteria that are introduced into the vagina during condomless sex. 24-26 Additionally, the MGT itself (including the penile skin, meatus, glans/corona and the subpreputial cavity) also contains a diverse array of bacterial taxa that may be transferred to the FGT in the absence of ejaculation and semen exposure. [24][25][26][27][28]187,190,191 A high level of concordance has been observed between the MGT microbiome composition and BV incidence in female partners. 27,28,190 In addition, semen has an alkaline pH range between 7.2 and 7.8, capable of buffering the acidic pH of vaginal fluid. 23,192,193 This neutralisation of the vaginal pH may promote a shift in the vaginal microbiome to a BV-associated state that is conducive to HIV-1 infection. 4,5,7,52,69,193,194 Several factors in semen may also inhibit the activity of extracellular H 2 O 2 produced by Lactobacilli species and thus promote the growth of BV-associated microbes. 195 We have demonstrated that recent semen exposure is associated with increased detection of BVAB-2, Prevotella bivia, and G. vaginalis and reduced detection of Lactobacillus jensenii in vaginal specimens (Jewanraj et al, 2021;accepted). 165 Increases in other gut-associated microbes have also been observed in the FGT after protected sexual intercourse, suggesting that these alterations in the vaginal microbiota may also be associated with mechanical contamination rather than just semen itself. 185,187 These studies suggest that semen exposure and sexual intercourse may promote a shift in the microbial environments of the FGT that may facilitate HIV infection in women. 4,5,7,165

| Impact of sexual intercourse and semen exposure on the vaginal epithelial barrier
An intact vaginal epithelial barrier is the primary host defence against HIV entry and infection. Reduced epithelial barrier integrity may facilitate HIV access to target cells at the FGT. Colposcopic examination of the vaginal mucosa revealed that friction during consensual sexual intercourse might cause microabrasions in the epithelial barrier. [196][197][198] Additionally, pro-inflammatory cytokines within semen may also increase the permeability of the vaginal epithelium.
Interferon-gamma in semen may increase epithelial permeability by inducing macropinocytosis of tight junction proteins. 199

| THE ROLE OF S E XUAL INTERCOUR S E AND S EMEN E XP OSURE ON TOPI C AL PrEP EFFI C AC Y
In addition to its role in female genital inflammation and immune activation, semen exposure and sexual intercourse may also undermine topical pre-exposure prophylaxis (PrEP) efficacy [202][203][204] and has additional implications for HIV susceptibility in women. The physiological changes that occur during coitus may alter PrEP efficacy by changing the surface area of the vagina and redistributing cervicovaginal fluid and topically applied microbicides. 205,206 In clinical trials, vaginal microbicide gels PRO 2000 and cellulose sulphate failed to confer protection against HIV-1 transmission in women. 207,208 In vitro assays demonstrated a significant reduction in the antiviral activity of PRO 2000 gel following sexual intercourse. 204 Tenofovir gel concentrations were also significantly reduced in cervicovaginal lavage and vaginal and cervical tissues after coitus. 203 These findings were likely due to the redistribution of the microbicide gels in the vagina during sexual intercourse.
Semen and SP itself contains several bioactive molecules and may also alter the antiviral activity of microbicides. 202,204,209,210 SP was shown to interfere with the HIV-1 and herpes simplex virus (HSV)-2 inhibitory activity of PRO 2000 and cellulose sulphate microbicides. 202,209,210 Seminal proteins, fibronectin and lactoferrin competitively inhibited the binding of the microbicides to their target on the HSV envelope. 210 The reduced antiviral activity of these microbicides may also be due to electrostatic interactions be-  212,213 These studies suggest that sexual intercourse and semen itself may also reduce the efficacy of topical PrEP in women and highlights the need to assess and control for these factors.

| B I OMARK ER S OF S EMEN E XP OSURE
Research primarily relies on self-reports of condom use and sexual behaviour, which may lead to inaccurate data interpretation due to reporting bias. [214][215][216][217][218] Although biomarkers of semen exposure were developed for use in forensics, they also have several useful applications in HIV prevention research. Semen biomarkers can be used to control for semen-induced alterations at the FGT, assess condom use in clinical trials and determine the efficacy of barrier contraceptives and microbicides. 165

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing not applicable to this article as no datasets were generated or analysed during the current study.