Immunobiology of genital tract trauma: Endocrine Regulation of HIV Acquisition in Women Following Sexual Assault or Genital Tract Mutilation

Abstract

Studies on HIV acquisition and transmission in women exposed to sexual trauma throughout their life cycle are lacking but some findings suggest that rates of HIV acquisition through coercive sex are significantly higher than that seen in consensual sex. Sexual trauma can also occur as a result of female genital mutilation, which makes sex extremely painful and can cause increased abrasions, lacerations and inflammation, which enhances the risk of HIV acquisition. This review presents an overview of the immune system in the human female reproductive tract from adolescence, through puberty to pregnancy and menopause. What is clear is that the foundation of information on immune protection in the female reproductive tract throughout the life cycle of women is extremely limited and at some stages such as adolescence and menopause are grossly lacking. Against this back backdrop, forced or coercive sexual intercourse as well as genital mutilation further complicates our understanding of the biological risk factors that can result in transmission of HIV and other sexually transmitted infections.

Introduction

The global HIV pandemic is now into its third decade with 30 million dead and 34 million currently living with HIV ¹. Globally, young women aged 15–24 are most vulnerable to HIV with infection rates twice as high as in young men, and accounting for 22% of all new HIV infections ². The reasons for this are both social and biological. Women, especially young women, may be less able to negotiate condom use and are more likely to experience coerced sex ^{1, 3–5}. Biologically, women are more likely than men to be infected in heterosexual encounters ^{6, 7} and critical endocrine and immunological parameters that are as yet ill-defined, play a major role in enhancing susceptibility in women and girls.

HIV can affect multiple dimensions of women’s sexual and reproductive health, fertility/pregnancy, breast-feeding, use of contraception, exposure to sexually transmitted infections (STI), and exposure to sexual violence ^{3, 5, 8–10}. Most women exposed to HIV and/or living with AIDS suffer from or fear stigmatization brought about through perceptions of promiscuity, blamed for bringing HIV in the family, transmitting HIV to her children, and violation of reproductive rights by forcible sterilization ^{5, 11}.

Sexual assault (SA) and female genital mutilation (FGM) constitute forms of sexual violence with important implications for HIV acquisition and transmission. Sexual assault is wide-spread all over the world including the USA ^{12, 13}. In a representative survey of over 9000 adults, 10.6% of women reported having coercive sex at some point of their lives. Among adolescents, 60.4% reported coercive sex before the age of 18 and 25.5% girls reported rape before the age of 12 ¹⁴. Sexual trauma alters a woman’s risk of HIV acquisition. Violence against women is strongly correlated with a woman’s risk of becoming infected with HIV ^{3, 5}. The epidemics of violence and HIV overlap intricately. Violence or the fear of it may interfere with a woman’s ability to negotiate safer sex or refuse unwanted sex. Forced or coercive sexual intercourse can result in transmission of HIV and other STIs. The risk of transmission increases as vaginal lacerations and abrasions occur during rape, especially if it occurs repeatedly. Further, violence against a woman can interfere with her ability to access treatment and care, maintain adherence to antiretroviral therapy or carry out her infant-feeding choice. Fear of violence is also a major barrier to disclosing HIV status. Studies in sub-Saharan Africa have found higher risks of disclosure-related violence compared with studies in the United States ^{5, 15, 16}.

The mucosa of the female reproductive tract (FRT) has evolved to meet the unique requirements of balancing immune protection against sexually transmitted pathogens, while being supportive to allogeneic spermatozoa and an immunologically distinct fetus ^{17, 18}. Accordingly, both the innate and adaptive immune system in the FRT is responsive to and precisely regulated by the female sex hormones, estradiol (E₂) and progesterone (P4) ¹⁸. The modulation by sex hormones results in altering a woman’s susceptibility to HIV and other STI through the course of her life cycle. The risks, therefore, are distinct during adolescence, stages of menstrual cycle, pregnancy, and menopause. In this review we aim to address the underlying immunobiology of HIV acquisition in women and how it might be affected by sexual trauma through her life cycle.

Life cycle of women: adolescence, reproductive age, and menopause

Sex hormones E₂ and P4 regulate various aspects of FRT immune system both directly and indirectly ^{19, 20}. Innate immune protection against FRT pathogens is multi-factorial and multi-layered. Soluble mediators, both pro-inflammatory and anti-inflammatory, are produced by cells of the FRT and many are regulated by E₂ and P4 ^{19, 21, 22}. Pro-inflammatory mediators can enhance HIV replication/infection whereas anti-inflammatory mediators and/or antimicrobials can be protective and inhibit HIV replication/infection ²³. Susceptibility to a pathogen for a given woman can therefore change, depending on her hormonal status (e.g. adolescence, stages of menstrual cycle, pregnancy, menopause ^22–24).

The first point of contact between HIV and host is the epithelial barrier of the FRT. Whereas all sexual encounters cause micro-trauma in the epithelium, repeated coercive sex enhances the trauma and compromises the epithelial lining to facilitate viral entry. Preexisting STI also create an inflammatory micro-environment which also facilitates HIV infection.

Adolescence

Epidemiological studies indicate that adolescent girls are particularly susceptible to HIV infection ^{25, 26}. Although much is attributed to high risk behavior, underlying biological mechanisms are poorly understood. Young girls are highly susceptible to sexual violence and sustain more injuries than adults especially in cases of repeated coercive sex ^{27, 28}. The anatomy of the FRT is a determinant for HIV acquisition. Whereas the vaginal and ectocervical epithelium is protected by multi-layer squamous epithelial cells, the cervical epithelium is composed of columnar epithelial cells which breach easily. In the human FRT, the point of entry for HIV is thought to be the transitional zone between the ectocervix composed of squamous cells and the endocervix composed of columnar epithelial cells ²⁹. During adolescence, the cervix is immature and characterized by ectopy, a condition in which the endocervical columnar epithelium protrudes through the cervix and onto the vaginal portion of the ectocervix, thereby enhancing the risk for acquiring HIV ²⁵. Recent studies show anal sex is highly prevalent in the adolescent population especially in cases of coercive sex and in girls who have undergone FGM ^{27, 28, 30, 31}. As in the endocervix, the rectal epithelium is also composed of a single layer of columnar epithelial cells which is easily damaged with sexual trauma. Adolescents who are victims of sexual assault have been reported to have more anogenital injuries than older women ^{27, 28}. This population is also characterized by high levels of STI, which in combination of cervical ectopy, further enhances risks for HIV acquisition. Oral contraceptive use has also been linked to increasing risk in this population. Finally, mucosal immune factors in the FRT are very different when comparing adolescent and adult women. As the menstrual cycle is irregular during adolescence, protection by hormonally-induced immune factors can be lacking. Hwang et al and Madan et al reported higher levels of inflammatory cytokines in the adolescent FRT compared to adults ^{25, 26}. Madan et al also reported reduced levels of protective factors such as defensins and SLPI as well as a difference in genital tract microbiome that enhances the risk of HIV acquisition even further ²⁶.

Reproductive Age and The Window of Vulnerability

Under the control of the hypothalamus and pituitary, ovarian production of E₂and P4 are low during menses. In response to Follicle Stimulating Hormone (FSH), follicle growth and maturation occurs with increased production of E₂. At midcycle, estradiol levels rise sharply to stimulate the release of Luteinizing Hormone (LH), which initiates ovulation. Following ovulation, LH stimulates the formation and maintenance of the corpus luteum, which produces estradiol and progesterone during the secretory phase of the menstrual cycle. These hormones, in turn, prepare the reproductive tract for fertilization, implantation and pregnancy ³². It is against this changing pattern of hormones that the immune system at each of site throughout the FRT is regulated to optimize conditions for reproductive success.

Numerous studies have shown that the reproductive tract of women is periodically exposed to a number of pathogens, commensals, allogeneic sperm and the developing semi-allogeneic fetus. To meet these challenges, the immune system has evolved to maintain a level of protection that distinguishes between these challenges. Studies from our laboratory led to the conclusion that, during a normal menstrual cycle, there is a window of vulnerability (7–10 days) during which the potential for HIV and possibly other STI in the FRT is optimized, as a result of E₂ and P4 suppression of aspects of the innate, humoral and cell-mediated immune systems ³³. As discussed in detail elsewhere ²⁴, immune suppression occurs in the upper (Fallopian tubes and uterus) and lower (cervix and vagina) reproductive tract as a part of the physiological process that prepares the reproductive tract for reproduction. Acting directly and indirectly through growth factors and cytokines, E₂ and P4 both suppress and enhance immune cell migration, cytotoxic T cell activity, coreceptor expression, antibody and antimicrobial secretion and innate immune cell function. These changes coincide with ovulation and persist thoughout most of the secretory phase of the cycle. One example is the activity of cytotoxic T lymphocytes (CTL), which kill cancer cells and virally-infected cells present in the FRT. CTL activity is suppressed in the uterus but not in the cervix and vagina during the secretory stage of the menstrual cycle and occurs without changes in the number of CD8+T cells in reproductive tract tissues ^{34, 35}. Of equal importance are the findings of others showing that immature and mature dendritic cells (DC), when cultured with TGFβ, up-regulate coreceptor expression (CXCR4, CCR5) ³⁶. Given that estradiol stimulates FRT secretion of TGFβ ³⁷, these studies suggested that estradiol acts indirectly to alter coreceptor expression on immune cells in the FRT. These and other cycle dependent changes are presented in greater detail in other sections of this chapter.

Evidence in support of a “window” has recently been demonstrated in the primate model ³⁸. Using repeated, low-dose Simian-human immunodeficiency virus (SHIV) vaginal exposure, Vishwanathan et al. concluded that a “window” of most frequent virus transmission exists between days 24 and 31 of the menstrual cycle (late luteal phase). These findings provide the first proof of concept that susceptibility to vaginal SHIV exposure is elevated in the second half of the menstrual cycle. Further studies are needed to determine whether susceptibility to infection during the “window” is unique to HIV or a characteristic of the FRT that includes other sexually transmitted pathogens. What is clear is that immune protection varies with the site studied in the FRT and hormonal balance during the menstrual cycle.

Although no studies have investigated the association between incidence of genital injury and stage of the menstrual cycle, one study reported that all nine women having their menstrual period at the time of the assault sustained injury ³⁹. Since sex hormones regulate the immunobiology of the FRT ²¹, hormonal status at the time of the assault may be relevant for HIV acquisition.

Pregnancy

Studies have indicated higher rates of HIV acquisition in women during pregnancy ⁸. The mechanisms are likely to be social as well as biological. Pregnant women are vulnerable to sexual violence from intimate partners ⁴⁰. Pregnant women who are HIV positive or at a high risk for HIV transmission through their partners might not get tested, disclose status, or access health care system, due to fear of violence and stigmatization ⁵. From an immunological perspective, the FRT in pregnant women is distinct from that seen in non-pregnant women. Traditionally, pregnancy has been defined as a general state of immune suppression. However, this notion has been challenged recently with an evolution of our understanding. Pregnancy seems to be both a pro-inflammatory and an anti-inflammatory state depending on the stage of gestation ⁴¹. Recent publications by Ghartey et al ⁴² and Anderson et al ⁴³ have demonstrated alterations in soluble mediators in genital tract secretions in pregnant compared to non-pregnant women. Both studies reported higher levels of pro-inflammatory cytokines and lower levels of protective endogenous antimicrobials in pregnant compared to non-pregnant women. These findings may have important clinical implications since a more inflammatory and less protective microenvironment has the potential to attract HIV target cells and thereby increase risk of acquisition.

Menopause

Older women are also susceptible to sexual violence ⁴⁴. Although post-menopausal women do not have to worry about pregnancy following an assault, they are more vulnerable to injury and infections ⁴⁴. There is considerable thinning of the vaginal epithelium following menopause, which, coupled with vaginal dryness, leads to greater trauma during sex. Trauma is more severe in cases of non-consensual sex. Other severe injuries are also sustained in older women due to osteoporosis, which requires a longer time for healing ⁴⁴. As multiple immune factors of the FRT are estrogen responsive, the loss of estrogen with aging results in loss of TLR function, secretory antimicrobial components, commensal lactobacilli, and acidity of vaginal microenvironment ²². There is also reduced production of cervical mucus, which itself is a protective barrier against pathogens ²². Post-menopausal women show higher chronic levels of proinflammatory cytokines IL-6, MCP-1, and TNFα as well as a reduced ability to respond to pathogens or stimuli ^45–47. This inflammatory microenvironment devoid of protective antimicrobials can enhance risk of HIV acquisition in post-menopausal women.

Hormonal regulation of immunity in secretions (CVL)

FRT has unique requirements for the regulation of immune protection as it must deal with sexually transmitted bacterial and viral pathogens, allogeneic spermatozoa, and the immunologically distinct fetus. The FRT has evolved immune mechanisms to protect against pathogens without compromising fetal survival. The mucosal immune system of the FRT is under hormonal control that regulates the transport of immunoglobulins (Igs), the levels of cytokines, the distribution of various cell populations, and antigen presentation in the genital tissues during the reproductive cycle ¹⁸. In addition to protecting against infectious agents, it must adapt to a spectrum of physiological events that includes fertilization, implantation, pregnancy, and parturition. A balance is maintained by sex hormones, especially estradiol and progesterone, throughout the menstrual cycle to respond to the challenges of pathogenic invasion without interfering with events that surround conception. During pregnancy, in addition to protecting against infection, the mucosal immune system adapts to support a fetal placental unit that is immunologically distinct. To meet these challenges, the reproductive tract has evolved into separate compartments consisting of ovaries, the Fallopian tubes, uterus, cervix, and vagina that are precisely regulated by estradiol and progesterone. Failure of the immune system either to rid the reproductive tract of pathogens or to resist attacking allogeneic sperm and fetus significantly compromises procreation as well as the health of the mother ¹⁸.

Soluble mediators in the FRT that are hormonally regulated have been described. Each stage of the life-cycle of women is characterized by changes in sex hormones which result in altered protection against HIV acquisition. As discussed above, in healthy adult women, stages of menstrual cycle are characterized by differential levels of soluble mediators thereby creating a “window of vulnerability” during the secretory phase of the menstrual cycle when women are most vulnerable to STI and HIV acquisition ²⁴. Cervical-vaginal lavage (CVL) contain soluble mediators that have intrinsic anti-HIV and anti-HSV activity, these can change with disease progression ^{23, 48, 49}. In adolescent girls where sex hormone levels are fluctuating, more inflammatory mediators and less protective mediators have been reported ²⁶. In the same study, CVL from adolescent girls had diminished ability to inhibit E.Coli, but enhanced ability to inhibit HIV when compared to CVL from adult women. Levels of soluble mediators change during pregnancy depending on the week of gestation ⁴¹. Anderson et al demonstrated that inflammatory mediators increase and protective mediators decrease in CVL from 14–26 week pregnant women compared to non-pregnant women ⁴³. Interestingly, no difference was detected in intrinsic anti-HIV activity in this cohort indicating other immune factors at play must be taken into consideration. As women age, reduction of estradiol correlates with reduction of protective immune mediators in the FRT such as SLPI ¹⁹. Preliminary data from our laboratory shows a reduction in anti-HIV activity in post-menopausal women compared to pre-menopausal women (Ghosh and Wira unpublished).

The role of sex hormones in the case of sexual assault is unknown. It can be postulated that trauma will significantly alter the local immune microenvironment by changing levels of cytokines, chemokines, and antimicrobials. Coercive sex is likely to induce an immune response that will result in generalized local immune activation and an increased the risk of HIV acquisition.

Hormonal control of HIV-target cells in the FRT

In the context of sexual assault, the antiviral effects of secretions, mucus and epithelial barriers may be overcome by the presence of genital trauma, which provides easy access of HIV virions to susceptible immune cells present in the submucosa. Susceptible target cells are present all along the FRT and under hormonal control. These include CD4⁺Tcells, macrophages, dendritic cells (DC) and Langerhans cells (LC). While the characteristics and distribution of these cell types in premenopausal adult and postmenopausal women are progressively better understood, no information is available for adolescents or pre-puberal girls. How differences attributable to hormonal changes and an immature immune system may contribute to the increased prevalence of HIV-infection in girls and very young women is unknown.

CD4⁺T cells are present in the vagina, cervix and endometrium, localized both in the sub-epithelial stroma, and as intraepithelial lymphocytes between the epithelial cells lining the lumen ²¹. Several studies strongly suggest that the initial main target cell for HIV-replication are CD4⁺T cells ^{50, 51, 52}. However, not all CD4⁺T cells are equally susceptible to HIV-infection, and the location and phenotype of these initial CD4⁺T cell targets in the human FRT remains unknown. Expression of CCR5 can be detected in a subset of CD4⁺T cells from endocervix obtained from cytobrush ⁵³ and this expression is higher in postmenopausal women compared to premenopausal controls ⁵⁴ suggesting a possible hormonal regulation of CCR5 expression. CCR5 expression levels in CD4⁺ T cells from girls and adolescents are unknown.

Macrophages are predominantly found in the endometrial stroma and myometrial connective tissue ¹⁸ with limited numbers present in the endo- or ectocervix. Sex hormones regulate the influx of macrophages into the endometrium, increasing their presence just prior to menstruation ⁵⁵. In contrast, vaginal macrophage numbers are not hormonally controlled ¹⁸. The role of macrophages in initiating HIV infection in the FRT mucosa is controversial. Initial infection supported by macrophages from women was demonstrated in vitro ^56–58 but primate models of SIV infection did not support these findings. Macrophages may be involved in the initial steps of HIV-infection in women either as cells supporting viral replication or through capture and transmission of virus to T cells ⁵⁹.

DCs sample antigens at mucosal surfaces and potentially are one of the first cells to encounter HIV ^60–63. As antigen-presenting cells, DCs generate and regulate adaptive immune responses ^{64, 65}. In the vagina, DCs reside predominantly within the epithelial layer ^{66, 67} while in endometrium DCs are located in the sub-epithelial stroma. The movement of CD1a+DCs into the FRT is regulated by cyclical hormonal changes with gradual increases from the proliferative to the secretory phases that peak at menses ⁶⁸. HIV can exploit the biological properties of DCs ^{69, 70} to spread virus from mucosal surfaces to lymph nodes, the main site for HIV replication. Although DC-SIGN can mediate HIV transmission from DCs to CD4⁺T cells in vitro ^{71, 72}, the role of DC-SIGN⁺ DCs in sexual transmission in vivo is not well characterized. DC-SIGN⁺ DCs remain relatively constant in the human endometrium throughout the proliferative and secretory stages of the menstrual cycle ⁷³. Studies from our laboratory demonstrate that DC phenotype is modulated by uterine epithelial cells through secreted soluble factors, resulting in decreased sensitivity to Toll-like receptor (TLR) 3 and TLR4 stimulation and reduced expression of co-stimulatory molecules and DC-SIGN ^{74, 75}. Importantly, down-regulation of DC-SIGN was associated with decreased HIV trans-infection by immature DCs ⁷⁴. DCs also exert antiviral activity, which is susceptible to hormonal regulation as well. For example, increased production of α-defensins by immature DC was associated with slower disease progression in HIV-infected subjects ⁷⁶ and α-defensin production by immature DC can be inhibited by high doses of estradiol ^{77, 78}.

Langerhan cells constitute a subset of DCs only present in tissues. LCs display very different characteristics from one tissue to another, they express Langerin, a specific C-type lectin, and are predominantly found in stratified squamous epithelia in the lower FRT. Although LC can be productively infected in vitro, it has been speculated that their main role is capture and transfer of virions to susceptible cells ⁶³. Epidermal LCs demonstrated viral up-take through Langerin, which mediated internalization of HIV into Birbeck granules and degradation of the virus ⁷⁹. In contrast, in vaginal LCs viral internalization occurred primarily by endocytosis, resulting in the presence of intact virions in the cytoplasm for days ⁸⁰, allowing migration of HIV-loaded LCs from the exposed vaginal epithelium and transfer of HIV to CD4⁺T cells ⁸¹.

Biological implications of sexual assault and female genital mutilation for HIV acquisition

Sexual assault (SA) and female genital mutilation (FGM) constitute forms of sexual violence with important implications for HIV acquisition and transmission. While SA and HIV are coexisting problems, little information is available regarding followup of victims of SA and seroconversion rates ⁸². Nevertheless, it seems generally accepted that SA is a risk factor for HIV- acquisition. In countries with high rates of HIV prevalence, sexual assault rates are also high. In Kenya, rape accounts for 4% of HIV infection in adolescents and, over their lifetime, 24% of women are raped at least once ⁸³. In countries with low HIV prevalence, while the underlying community risk for HIV exposure is lower than 1%, victims of SA are likely exposed to higher risk than the community baseline ⁸⁴. Following SA, pregnancy and sexually transmitted diseases, including HIV may be prevented with prompt treatment and, when given within 72 h after SA, postexposure prophylaxis was shown to reduce by 81% the risk of HIV seroconversion ⁸⁵. Prophylactic treatment after SA for hepatitis B, gonorrhea, chlamydia and HIV is recommended by the centers for disease control and prevention (CDC) ⁸⁶. Strikingly, however, these treatments are often not offered to women victims of SA ⁸⁴. Regarding HIV prophylaxis, a recent study found that while 89% of emergency department physicians interviewed offered prophylaxis for other STIs, only 45% offered counseling or HIV prophylaxis, and one third of physicians working in hospitals with HIV prophylaxis included in their protocols did not typically offer this treatment ⁸⁴.

Possible explanations for how SA may increase the risk of HIV infection include genital injury, systemic extragenital trauma, forced anal intercourse and concurrent transmission of other STIs as well as inability to negotiate condom use. Medico-legal reasons rather than a medical-treatment interest are the current driving force for research on SA ⁸⁷, so little information is available comparing genital injury rates after consensual and non-consensual sexual intercourse. Genital injury after consented sex is rare, estimated in about 5–6% of cases, and more likely to occur in postmenopausal women ^87–89. All studies agree that genital injury after non-consensual sexual intercourse is significantly higher compared to consented sex, but incidence of injury vary depending on the method used to determine it ^{87, 90}. Gross visualization includes only bruising, abrasions and lacerations as injuries and is seen in 40% of reported cases, while inspection by colposcopy, which includes “subjective” injuries such as swelling and redness, results in up to 87% incidence ^{87, 90}. Presence of “subjective” injuries in 87–92% of victims within 48 hours after sexual assault ³⁹ appears of extreme relevance as a risk factor for HIV acquisition, since it results in active inflammation and the increased presence of HIV-target cells at the site of exposure. Although the specific inflammatory response after trauma due to SA has not been studied, it is well known that the immune system promptly reacts to tissue damage after traumatic injury ⁹¹. Tissue damage releases inflammatory mediators which activate innate immune cells, particularly macrophages, while CD4⁺ T cell responses are suppressed, probably due to increased regulatory T cells, resulting in T cell anergy and increased risk of infections ⁹¹. Studies of healing after genital injury in prepuberal and adolescent girls show variability in resolution time. Depending on the type of lesion, healing can last from 2 days for superficial injuries up to more than 20 days for deep lacerations ⁹². These findings imply that active inflammation can be present for an extended period of time after SA, which may have implications for increased HIV-susceptibility after the event.

When comparing post- and pre-menopausal women, some studies suggest that post-menopausal women are more likely to be genitally injured following sexual assault ^{93, 94} and more likely to suffer vaginal laceration, possibly due to atrophy of connective tissue and vaginal epithelium ⁹³. Genital injuries are also more likely to occur in very young females ^{88, 93, 94}, although one study did not find any correlation ⁹⁵.

Female genital mutilation (FGM) represents a major form of child and female abuse ⁹⁶. FGM is defined as removal of the external female genitalia for non-therapeutic reasons and includes 4 different types: 1) clitoridectomy, 2) excision (removal of clitoris and labia minora with or without labia majora), 3) infibulations (narrowing of the vaginal opening with creation of a covering seal), 4) other harmful procedures for non-therapeutic reasons. While this practice is widespread in Africa, it also occurs in immigrant communities in North America and Europe ⁹⁶. The consequences of FGM are serious and multiple including death, shock, tetanus, reduced fertility, vesicovaginal fistulae and the potential enhanced risk of HIV and other STIs. Very few studies have analyzed the relationship between FMG and HIV infection ⁹⁷, but the risk of HIV acquisition is likely increased considering the extensive trauma, inflammation and increased vaginal epithelial damage. Moreover, it is common practice that the procedure will be performed with shared instruments not properly cleaned, increasing even more the trauma and complicating infections ⁹⁶.

In conclusion, in the context of sexual assault and female genital mutilation, the mucosal microenvironment will be profoundly altered, and danger signals will attract and modify the phenotype of immune cells that are also target cells for the HIV, likely influencing HIV-susceptibility. The role that sex hormones play in this process is completely unknown.