Anatomic and Hormonal Changes in the Female Reproductive Tract Immune Environment during the Life Cycle: Implications for HIV/STI Prevention Research

Authors

  • Kartik K. Venkatesh,

    Corresponding author
    1. Department of Obstetrics and Gynecology, Brigham and Women's Hospital and Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA
    • Correspondence

      Kartik K. Venkatesh, Department of Obstetrics and Gynecology, Brigham and Women's Hospital, 75 Francis St, Boston, MA 02415, USA.

      E-mail: kvenkatesh@partners.org

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  • Susan Cu-Uvin

    1. Department of Obstetrics and Gynecology, Alpert Medical School, Brown University, Providence, RI, USA
    2. Division of Infectious Diseases, Department of Medicine, Alpert Medical School, Brown University, Providence, RI, USA
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Abstract

The female genital tract undergoes anatomic changes from birth through menopause. Concurrently, the relative levels and roles of estradiol and progesterone dramatically change during the female life cycle. This review provides a conceptual overview of clinically relevant anatomic and hormonal changes in the female genital tract, and how these changes may affect HIV-1 pathogenesis, transmission, and mucosal immunology and protection against HIV and STIs across the female life cycle.

Introduction

Women have increasingly been affected by the HIV epidemic worldwide and now account for over 33 million individuals living with HIV.[1] In sub-Saharan Africa, women account for nearly two-thirds of new HIV infections, the majority acquired through heterosexual sex. Strategies to prevent the sexual transmission of HIV in women depend on a better understanding of the interaction between immunologic mechanisms and the anatomic and hormonal milieu of the female genital tract.

The female genital tract undergoes anatomic changes from birth through menopause.[2] The female genital tract consists of five distinct anatomic sites (fallopian tubes, uterus, endocervix, ectocervix, and vagina), which function separately yet in coordinated fashion. Each of these anatomic sites is hormonally controlled by estradiol and progesterone. Multiple cell types in the female genital tract, including epithelial cells, macrophages, T cells, and dendritic cells, are regulated by estradiol and progesterone.[3] Concurrently, the relative levels and roles of estradiol and progesterone dramatically change during the female life cycle.[4] It is plausible that virological, immunologic, and microbiological parameters that affect HIV-1 pathogenesis and transmission may vary in the setting of evolving anatomic structures and fluctuations in female reproductive hormones through the life cycle.[3, 5, 6]

This review provides a conceptual overview of anatomic and hormonal changes in the female reproductive tract from adolescence through menopause to contextualize other articles in this special edition of AJRI (See Table 1). We highlight clinically relevant changes in the female reproductive tract that are mediated by estradiol and progesterone and the menstrual cycle, and how these changes may affect mucosal immunology and susceptibility to HIV and sexually transmitted infections (STIs) across the female life cycle. We first review relevant components of the menstrual cycle and then describe hormonally mediated morphological changes in the upper and lower genital tract across the female life cycle.

Table 1. Anatomic and Hormonal Changes in the Female Reproductive Tract Across the Life Cycle
Life cycleReproductive hormonesCervixUterusOvariesCo-factors mediating HIV susceptibility
InfancyMinimal levels of estradiol and progesterone

Morphological changes:

Lined by squamous epithelium

Morphological changes:

Low columnar/cuboidal epithelium without secretory or proliferative changes

Morphological changes:

400,000 primordial follicles are present at birth

Decrease of maternal reproductive hormones
Adolescence

Rise in lutenizing hormone

Rise is estradiol Beginning of menses

Morphological changes:

Ectopy: single layer of glandular cells in close association with underlying vascular stroma

Ectopy occurs when the columnar epithelium of the endocervical canal extends outwards into the ectocervix

Hormonal changes:

Ectopic changes promoted by reproductive hormones

Morphological changes:

Shedding of myometrium with each menstrual cycle

Hormonal changes:

Onset of menstrual cycles is hormonally mediated

Morphological changes:

90% of primordial follicles present at birth have undergone atresia

Increase in ovarian stroma with sparse distribution of primordial follicles

Hormonal changes:

Responsive to gonadotropins

Ovaries release estradiol in response to gonadotropins

1) Increased incidence of genital tract infections

2) Changes in sexual behavior

Reproductive yearsCyclic rise and fall of estrogen and progesterone with the menstrual cycle

Morphological changes:

Decrease in cervical ectopy

Hormonal changes:

Morphological changes:

Endometrial proliferation and degeneration as part of the menstrual cycle

Hormonal changes:

Estrogen promotes endometrial proliferation

After ovulation, progesterone promotes secretory tissue

Withdrawal of progesterone and estrogen leads to endometrial mucosal degeneration (i.e., menstruation)

Morphological changes:

Primordial follicles undergo maturation and atresia

Dominant follicle undergoes complete maturation and release of the oocyte (ovulation)

Hormonal changes: Maturation, atresia, and ovulation are controlled by reproductive hormones

1) Contraception use: thinning of vaginal epithelium due to progesterone in animal models, but not humans

2) Semen: alkaline substance, potent immune modulator of the cervix and uterus

PregnancyHigh systemic concentrations of estradiol and progesterone

Morphological changes:

Ectopy: Endocervical canal is everted into the ectocervix due to hyperplasia of columnar epithelium

Hormonal changes:

Hyperplasia promoted by reproductive hormones

Morphological changes:

Hypertrophic and hypersecretory features termed ‘gestational hyperplasia’

Hormonal changes:

Hyperplasia promoted by reproductive hormones

Morphological changes:

Stromal edema

Cessation of ovulation

Hormonal changes:

 
Menopause

Minimal levels of estradiol and progesterone

High levels of lutenizing hormone and follicle-stimulating hormone

Absence of ovarian estrogen feedback

Morphological changes:

Decreased cervical ectopy with squamous epithelium replacing columnar epithelium

Hormonal changes:

Morphological changes:

Few glands that are devoid of secretory or proliferative changes

Hormonal changes:

Morphological changes:

Ovaries no longer release ova

Devoid of primordial follicles

Decrease in stromal volume and cellularity, with an increase in collagen

Hormonal changes:

Cessation of follicular estrogen synthesis

Continued production of testosterone and androstenedione

 

Menstrual cycle

We first describe the stages of female sexual development from the onset of puberty to menopause and the role of estradiol and progesterone in inducing these changes.[4] We then describe the components of the menstrual cycle and the role of estradiol and progesterone in each part of the cycle. Finally, we highlight a period of the menstrual cycle that is associated with an enhanced susceptibility to HIV infection.

Female Sexual Development

The menstrual cycle with consequent changes in circulating levels of estradiol and progesterone has been implicated in modifying the disease prevalence and pathogenesis of many chronic non-infectious diseases.[7] Throughout childhood, both progesterone and estradiol are minimally present. Puberty is marked by a dramatic rise in secretions of lutenizing hormone followed by increased ovarian secretions of estradiol. A woman's first menstruation is termed menarche and occurs typically around age 12–13. In the setting of pregnancy, there are high systemic concentrations of both estradiol and progesterone. In the perimenopausal period, a chaotic relationship ensues between estradiol and progesterone, until menopause when levels of both hormones return to pre-adolescent levels. The end of a woman's reproductive phase is marked by menopause, which commonly occurs between the ages of 45 and 55. Menopause begins when the ovary can no longer release ova, and lutenizing hormone and follicle-stimulating hormone increase in the absence of ovarian estrogen feedback.

Components of the Menstrual Cycle

This cycle is conventionally defined as 28 days in length. Every month, the cyclic fluctuation of sex hormones prepares the uterus for the potential implantation of a fertilized ovum and induces the shedding of the endometrium if implantation does not occur.[8] The menstrual cycle, divided into the follicular phase (also known as the proliferative phase) and luteal phase (also known as secretory phase), is followed by menstrual bleeding.[9] During the follicular phase, estradiol levels progressively rise until lutenizing hormone secretion from the pituitary gland induces ovulation, and if no fertilization occurs, these levels sharply drop. Lutenizing hormone leads to increased progesterone levels throughout the luteal phase. If no implantation has occurred, progesterone levels will drop after 2 weeks and menstrual bleeding will occur.

This systematic progression of a 2-week estrogen-driven environment followed by a 2-week progesterone predominant environment affects immune cell numbers, activation, and activity fluctuation.[10] Immunoglobulin levels have been shown to vary during different phases of the menstrual cycle,[11, 12] and hence it is plausible that menses-mediated variations in the female reproductive tract may affect HIV transmission and susceptibility.[13, 14] Given that female reproductive tract immunity is suppressed by estradiol during the menstrual cycle, it has been proposed that engineering a commensal Lactobacillus to secrete the anti-HIV molecule elafin as estradiol levels increase could protect women from HIV infection.[15]

Impact of the Menstrual Cycle on HIV Susceptibility

The immune environment of the uterine endometrium fluctuates during the menstrual cycle, and sex hormones affect the expression of a variety of uterine chemokines.[16] At the time coinciding with ovulation, Wira and colleagues have identified a potential ‘window of vulnerability’ during the progesterone-dominant luteal phase for optimal viral infection.[17, 18] Through analysis of multiple immunologic parameters, they identified this 7-to 10-day period when important components of the immune response are suppressed by estradiol and/or progesterone, enhancing the potential for HIV infection. Saba et al.[19] showed in a recent study utilizing cervical tissue explants that HIV infection was associated with the luteal phase. White and colleagues found that cytotoxic activity in the upper reproductive tract was present during the follicular phase of the cycle prior to ovulation, but was absent in the consequent luteal phase.[20] In contrast, cytotoxic activity in the cervix and vagina persisted throughout the menstrual cycle.[21] In a recent study assessing cyclic changes in HIV shedding during the menstrual cycle, shedding frequency and magnitude were greatest during the follicular phase and lowest at the time of ovulation.[22] Of note, during the luteal phase, shedding rose significantly only in women with CD4 cell counts <350 cells/ul.

Understanding the association between cyclic changes in HIV shedding during the menstrual cycle is challenging across different studies due to methodological differences, heterogeneous study populations, varying sampling methods, and the vast number of biologic and behavioral variables that may influence shedding levels. Together these menstrual-dependent changes may contribute to a cycle-dependent ‘window of vulnerability’ to HIV infection coinciding with the luteal phase.

Upper genital tract

Although the upper reproductive tract may be a site of infection and virus production, particularly because the upper tract has been under-studied in the past, there is strong earlier evidence demonstrating that HIV/SIV enters readily, and rapidly, via the vagina and ectocervix.[23-25] Studies have suggested that the fallopian tube and uterus are potential entry sites for HIV-1 infection.[26] Upper genital tract epithelial cells express HIV-1 co-receptors, which are under hormonal regulation, and these cells can be infected with the HIV virus in vitro under certain conditions.[27, 28]

Morphologic Changes

At birth, the endometrium is lined by low columnar to cuboidal epithelium, which is devoid of secretory or proliferative changes.[29] It resembles the inactive endometrium in menopausal women. In the pre-pubertal period, the endometrial mucosa remains inactive, and the cervix comprises the primary portion of the uterus. In the reproductive years, the endometrium undergoes cyclic morphologic changes, which is most apparent in the upper two-thirds of the mucosa, the functionalis layer; morphologic changes are minimal in the lower third of the basalis layer. If pregnancy occurs, the luteal phase endometrium displays hypertrophic and hypersecretory features termed ‘gestational hyperplasia’ to provide an appropriate environment for the conceptus. In the post-menopausal period, the endometrium resembles that from the neonatal-fetal period with few glands that are devoid of proliferative or secretory activity.

Adult ovaries are pink-white smooth ovoid structures that increasingly become convoluted later in the reproductive period. Approximately, 400,000 primordial follicles are present at birth, and about 400 mature to ovulation.[30] By puberty, 90% of the primordial follicles present at birth have undergone atresia. The depletion of follicles with a consequent increase in ovarian stroma results in an increasingly sparse distribution of primordial follicles in adolescence and young adulthood compared with childhood. Their numbers continue to decrease progressively by atresia and folliculogenesis (continuous process of primordial follicles undergoing maturation during each menstrual cycle) until they eventually disappear. Folliculogenesis and atresia occur prenatally, throughout childhood, and during pregnancy. In women of reproductive age, the follicles are distributed on the superficial cortex of the ovary and each consists of a primary oocyte. During the reproductive years, follicular maturation begins early in the luteal phase and continues through the follicular phase of the next cycle. Each month, usually one developing follicle is dominant, leading to complete maturation and release of the oocyte (i.e., ovulation). The other follicles undergo atresia. After ovulation, which is typically on the 14th day of a 28-day menstrual cycle, and without fertilization, the collapsed ovulatory follicle becomes the corpus luteum of menstruation. In many women, during the later reproductive years and post-menopause, there is a decrease in stromal volume and cellularity, with an increase in collagen. The appearance of shrunken post-menopausal ovaries is devoid of primordial follicles.

Hormonal Changes

The proliferative, secretory, and degenerative changes of the uterine lining are controlled by the release of progesterone and estradiol.[29] Thus, the endometrium is a highly sensitive indicator of the hypothalamopituitary-ovarian axis. Estradiol promotes endometrial proliferation, and after ovulation, progesterone converts the estradiol-primed endometrium into secretory tissue. Post-ovulatory estradiol amplifies the progesterone effect. After withdrawal of progesterone and estradiol, the endometrial mucosa degenerates and regenerates during the period of menstruation.

Cortical and medullary stromal cells of the ovary are responsive to female sex hormones.

The ovarian stroma in both pre- and post-menopausal women can produce steroids and is responsive to gonadotropins.[30] Ovarian stromal tissue secretes androstenedione, as well as smaller quantities of testosterone and dehydroepiandrosterone (DHEA). At the time of puberty, the rise in gonadotropins stimulates the production of follicular estrogens that are responsible for the development of secondary sexual characteristics. In the post-menopausal period, there is a cessation of follicular estrogen synthesis with continued production of testosterone and androstenedione by the ovaries.

It is possible that the hormonally mediated immune environment that is under ovarian and uterine control could affect HIV susceptibility. The implications of these hormonal changes mediated by the upper reproductive tract on HIV risk in women largely remain to be investigated.

Lower genital tract

Morphologic Changes

The uterine cervix superior to the vagina is termed the endocervix, whereas the inferior portion is termed the ectocervix. The external os is where the endocervical canal joins the vagina, and the internal os is where the endocervical canal joins the endometrium.[31] Squamous epithelium lines the vagina and part of the ectocervix. Relative to vaginal tissue, it has been hypothesized that the cervix is more susceptible to HIV because of its fragility, frequent compromise by classical STIs, and the presence of HIV receptor sites.[32] Other immunologic factors that may facilitate HIV infection, including CD4 positive T-helper cells and the expression of the chemokine receptor CCR5, are also found more frequently with columnar epithelium of the endocervical mucosa compared with the squamous epithelium of the ectocervix and vagina.[33-36] Increased cervical CCR5 expression in post-menopausal women may increase their risk for HIV-1 acquisition, and further studies are needed to understand whether increased CCR5 expression is related to hormonal effects of aging.[37] Finally, cervical mucus produced by the endocervix may impede HIV-1 to enhance mucosal barrier function.[38]

Hormonal Changes

Estradiol and progesterone affect the types of epithelia that line the two parts of the cervix. Estradiol induces proliferation, complete maturation, and desquamation of all layers of the squamous epithelium of the cervix, whereas progesterone causes thickening of intermediate layers but does not lead to complete maturation.[39] When the ratio of progesterone to estradiol is increased such as during pregnancy or during the secretory phase of the menstrual cycle, intermediate cells predominate; when this ratio is reversed such as during the proliferative phase of the menstrual cycle or when exogenous estradiol is administered, superficial cells predominate.[31] The squamous epithelium remains undifferentiated and atrophic without hormonal stimulation.[40] The original position of the squamocolumnar junction changes as a result of hormonal influences in utero, at puberty, during pregnancy, and after menopause. Movement of the endocervix onto the ectocervix occurs under the influence of reproductive hormones. This results in exposure of the endocervical mucosa to the vaginal environment (i.e., cervical ectopy).

Cervical ectopy has been implicated in some epidemiological studies as a risk factor for HIV acquisition.[41] ‘Ectopy’ occurs when the columnar epithelium of the endocervical canal extends outwards into the ectocervix.[39] This appears as a single layer of glandular cells that reside in close association with the underlying vascular cervical stroma. Due to its thin, vascularized epithelium, ectopic tissue is fragile. The prevalence of ectopy ranges widely from 17 to 50%.[42] Post-mortem studies from female infants suggest that in the neonatal period, the fetus is exposed to maternal hormones, which leads to hyperactivity of columnar epithelium of the cervix leading to ectopy and metaplasia.[43] At the time of puberty, due to a rise in ovarian release of estradiol, an enlarged endocervix is pushed down and out, thereby exposing the endocervical columnar cells on the ectocervix. Hence, adolescents are more likely to have immature epithelium or larger areas of ectopy that could facilitate the acquisition of HIV and other STIs.[44] A recent study also found higher levels of cervicovaginal inflammatory and regulatory cytokines and chemokines in healthy young women with immature cervical epithelium.[34] Again during pregnancy, the endocervical canal becomes everted into the ectocervix due to hyperplasia of the columnar epithelium, which is stimulated by reproductive hormones. The area of cervical ectopy decreases with aging in which squamous epithelium replaces columnar epithelium,[39] as well as with sexual activity.[44] It is likely that most, if not all, women will develop ectopy at some point during their lifetimes.

Co-factors that mediate hormonal and menstrual influences on the female genital tract

Multiple factors can mediate the influence of menstrual and hormonal changes on the female genital tract and its microbiological and immunologic environment, including genital tract infections, pregnancy, contraception, and semen.

Genital Tract Infections

Genital tract infections not only facilitate HIV susceptibility, but also alter the mucosal immune environment. Prior observational epidemiological studies have suggested that cervical ectopy can increase the risk of acquiring some STIs, such as C. trachomatis,[45] human papilloma virus,[46] and cytomegalovirus,[47] but not N. gonorrhoeae.[48] The varying histology of the cervix also leads to preferential susceptibility to different organisms. While the columnar epithelium, typically of the endocervix, is susceptible to C. trachomatis and N. gonnorheae, the squamous epithelium is susceptible to human papillomavirus. Additionally, asymptomatic infections of the lower genital tract, namely bacterial vaginosis and trichomoniasis, enhance HIV susceptibility through altering mucosal immunity, altering normal vaginal flora and pH, and weakening the cervico-vaginal mucosa.[49] Recent data suggest the mechanism by which Herpes simplex virus Type-2 (HSV-2) increases the risk of HIV acquisition may be through HSV-2-stimulated HIV replication in cervical tissue.[50, 51]

Pregnancy

Multiple observational studies conducted in sub-Saharan Africa have demonstrated that the risk of HIV acquisition among at risk women is increased almost twofold during pregnancy compared with the post-partum period[52-54]; however, a recent cohort study conducted in Zimbabwe and Uganda did not find an association between pregnancy and HIV acquisition.[55] It is postulated that higher levels of progesterone and estrogen mediate increased susceptibility to HIV infection in pregnancy through inducing structural changes in the genital tract mucosa and immunologic effects. Pregnancy itself induces hyperplasia of the columnar epithelium and glands, hyperemia, and stromal edema, which could influence HIV susceptibility.[56, 57] Vaginal cytokine profiles differ between pregnant and non-pregnant women, and it has been postulated that these fluctuations in inflammatory markers during pregnancy may account for an increased risk of HIV infection.[58, 59] This finding suggests that the lower genital tract secretions of pregnant women are capable of potent anti-HIV activity. However, another study comparing cervicovaginal secretions between pregnant and non-pregnant women found that suppression of HIV-1 activity was similar between the two groups during the second and third trimesters.[60] In summary, immunologic and hormonal changes during pregnancy could increase susceptibility to HIV infection in gravid women, but the evidence is inconclusive.

Contraception

Given that hundreds of millions of women worldwide are exposed to hormonal contraceptives, particularly in regions with high HIV prevalence, any modification of hormonal contraceptives on HIV susceptibility could have major implications for the global HIV epidemic. Hormonal contraception has been associated with an increased risk of HIV acquisition in some but not all epidemiological studies,[61] which is discussed in further depth in another review paper in this series. Recent data suggest that hormonal contraception may alter cervical immunity via increased levels of pro-inflammatory cytokines, including RANTES, or decreased levels of secretory leukocyte protease inhibitor (SLPI).[62] The thinning of the vaginal epithelium is a known effect of natural progesterone during the luteal phase of the normal menstrual cycle. Although studies using a rhesus macaques model have shown that subcutaneous progesterone administration by implants resulted in vaginal epithelial thinning and increased simian immunodeficiency virus vaginal transmission, studies using human vaginal biopsies have generally not shown an association between hormonal contraception and significant changes in vaginal epithelial thickness and the number of cell layers from biopsy, Langerhans' cell count, or maturation index.[63-65] Hence, humans may not respond to exogenous progestins with the dramatic vaginal thinning noted in primates, which is a reassuring finding.

Semen

The sexual transmission of HIV often occurs in the context of semen. Semen is an alkaline substance and has many immunosuppressive aspects that could promote the progression of genital infections in the female genital tract.[66] It is possible that semen in vaginal fluids may interact with microbicides reducing the efficacy of these agents in preventing HIV acquisition.[67] Seminal fluid is a potent immune modulator in the cervix and uterus, and its effects on impacting HIV susceptibility in women remain largely undefined.[68]

Conclusions

This review provides a clinical framework to contextualize immunologic changes occurring in the female reproductive tract in the setting of HIV exposure, which are described in further detail in other reviews in this issue of the Journal. Hormonally-driven changes in the female reproductive tract have implications for the future development of HIV vaccines, microbicides, and other biologically based HIV prevention modalities for women.[3, 69] Future areas for clinical research involve assessing and controlling for phase of the menstrual cycle, measuring levels of female reproductive hormones, and histopathological assessment of morphological changes in the genital tract when assessing HIV transmission and susceptibility in at risk women. The role of anatomic and hormonal changes on the immune environment of the female reproductive tract and its clinical consequences for HIV acquisition and transmission have only begun to be understood and involves a complex, dynamic interplay of multiple biologic and clinical factors that generally cannot be assessed in isolation.

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