FABBS Doctoral Dissertation Research Excellence Award

Stephanie Koebele, Arizona State University, Department of Psychology

“Hysterectomy and Cognition: A Novel Preclinical Assessment of the Longitudinal Cognitive Effects Resulting from Gynecological Surgery in Adulthood”

Stephanie Koebele, Arizona State University, Department of Psychology

Abstract

Hysterectomy (surgical uterus removal) is a common gynecological surgery. In pre-menopausal women, efforts are made to conserve the ovaries in order to prevent abrupt surgical menopause; however, oophorectomy occurs alongside hysterectomy in ~50% of cases. Thus, women who undergo surgery in adulthood may live a substantial number of years without a uterus and/or ovaries. It is well-established that a premature loss of ovarian hormones negatively impacts cognition in women and animal models. Pre-menopausal hysterectomy is also associated with a higher relative risk of dementia, though underlying mechanisms are unknown. For this dissertation, I developed a rat model of hysterectomy to systematically evaluate the role of hysterectomy in the context of cognition. Findings revealed that following hysterectomy, spatial working memory deficits were apparent at short-term and long-term post-surgical timepoints, suggesting that hysterectomy has long-lasting detrimental effects on cognition. Furthermore, endocrine and ovarian assessments did not differ between Hysterectomy and controls, collectively indicating a principal role for the uterus regulating working memory that is not secondary to ovarian influence. Translationally, these results highlight the necessity to better understand how gynecological surgery impacts the trajectory of cognitive aging in order to develop strategies to prevent or postpone cognitive decline in at-risk women.

Introduction/Background

The surgical removal of the uterus, or hysterectomy, is the most common non-obstetrical gynecological procedure in the US [1–3]. Despite declining procedure rates due to advancements in alternative therapies for benign uterine conditions, in 2008, it was projected that lifetime prevalence of hysterectomy neared 48% [4]. Hysterectomy is often performed in adulthood, prior to age 52, which is the average age of natural menopause onset [5,6]. If women undergoing hysterectomy are pre-menopausal and have a low cancer risk, ovaries are typically retained so as to prevent abrupt surgical menopause; however, oophorectomy (i.e., surgical ovary removal) occurs in conjunction with hysterectomy in about 50% of total cases. Although several studies indicate that the ovaries function normally following hysterectomy until natural menopause onset [7–10], it is generally thought that compromised ovarian blood flow occurs after hysterectomy, leading the observed earlier menopause onset than reproductive-tract intact women [11–16]. Ovarian hormones are intricately linked with the normal function and regulation of many peripheral body systems as well as with the central nervous system. It is well-established that a premature loss of ovarian hormones, as is the case with oophorectomy, negatively impacts cognition in women and animal models [17,18,27,28,19–26]. However, research from the Mayo Clinic and others has suggested that hysterectomy –with and without ovarian conservation– is also associated with an increased relative risk of developing neurodegenerative diseases such as dementia [19,23,29] and Parkinson’s disease [30] compared to age-matched reproductive-tract-intact women. The magnitude of this increased relative dementia risk rises with a younger age at surgery [29]. The dogma in the field is that any cognitive changes that present following gynecological surgery are a result of altered ovarian function; however, the association between hysterectomy alone and neurodegenerative disease has piqued interest into how the non-pregnant uterus —which is typically considered an endocrine target but otherwise dormant independent of the ovaries— may influence non-reproductive functions, including cognition.

Basic science and clinical research has predominantly focused on the role of oophorectomy in the context of cognitive changes. As such, there was a significant gap in the literature regarding the impact of hysterectomy on cognition. To address this, our laboratory recently developed a novel rat model of hysterectomy to systematically evaluate effects of variations in gynecological surgery on cognition. We reported that hysterectomy with ovarian conservation resulted in a unique, detrimental effect on spatial working memory two months after surgery [31]. The aim of the current experiment was to extend these findings by investigating whether effects were transient and reversed with time, or if the observed cognitive changes were the beginning of a long-term, possibly global, cognitive impairment. Rats underwent cognitive testing on a series of behavior tasks to elucidate the long-term impact of variations in gynecological surgery on cognition. Serum hormone levels and ovarian follicle count estimates were evaluated to determine if ovarian steroidogenesis and follicular development were impacted by hysterectomy, and whether the observed cognitive effects were primarily driven by ovarian changes, or if these changes were uniquely driven by uterus removal.

Methods

Three cohorts of forty 5-month-old female, virgin, reproductively-intact Fischer-344-CDF rats from the National Institute on Aging colony at Charles Rivers Laboratories (Raleigh, NC) were utilized (N=120). All experimental procedures were approved by the ASU IACUC and adhered to NIH standards.

Ten rats per cohort were assigned to undergo Sham surgery (control; reproductive tract intact), Ovariectomy (Ovx; ovary removal only), Hysterectomy (uterus removal only), or Ovx-Hysterectomy (ovary plus uterus removal). Following surgery, subjects underwent a series of cognitive tasks assessing spatial memory. The Adult cohort was tested 6 weeks after surgery (7mo at test), the Middle-Aged cohort was tested 7 months after surgery (12mo at test), and the Aged cohort was tested 12 months after surgery (18mo at test). The water radial-arm maze (WRAM) was an eight-arm apparatus with a circular center arena that evaluated spatial working memory (a form of short-term memory that requires updating) and reference memory (a form of long-term memory that stays constant). Rats received four trials/day for twelve days, and must locate hidden platforms submerged beneath the water in four of the eight arms using spatial cues. Once found, each platform was subsequently removed from the WRAM within a day. Thus, working memory load increased as trials progress, making this a complex cognitive task. After WRAM, rats were tested on the Morris water maze (MM), a spatial reference memory task. Rats received four trials/day for five days. Rats were dropped off at various spatial locations into a round tub that contained a single hidden platform. Swim distance (cm) to the platform was calculated across days and trials as the dependent measure. On the last day of MM, a probe trial was conducted wherein the platform was removed from the maze, and spatial localization of the prior platform location was assessed. Following MM, rats underwent a one-day control swim task assessing motor and visual competency (Visible Platform). Following the behavioral battery, rats were euthanized; brains, blood, ovaries, and uteruses were collected. Ovaries from Sham and Hysterectomy groups were assessed for estimated ovarian follicle counts and blood serum was profiled for a panel of reproductive hormone levels. Vaginal cytology and body weights were periodically collected throughout the experiment as peripheral markers of hormone status.

Cognitive data for WRAM and MM were analyzed using two-group planned comparison repeated measures ANOVA between Hysterectomy and each other group. Ovary and serum data were assessed using ANOVA two-group comparisons. WRAM data were divided into the Learning Phase (Days 2-7) and Asymptotic Phase (Days 8-12), and quantified by counting the number of unplatformed arm entry errors made before locating a platform on a given trial. WRAM errors were divided into two subtypes; working memory correct (WMC) errors were entries into arms that were previously platformed within a day, and working memory incorrect (WMI) errors were repeat entries within a day into arms that never contained platforms. Trial 3 and Trial 4 (the moderate and maximum working memory load trials, respectively) were assessed independently for the Learning and Asymptotic Phases.

Results

Adult Cohort: During the Learning Phase of WRAM, Hysterectomy rats made more WMC errors than Ovx-Hysterectomy rats on Trial 3 (p<0.05). Furthermore, Hysterectomy rats made more WMI errors than Sham rats on Trial 3 (p<0.01) and tended to make more errors than Ovx-Hysterectomy rats (p=0.09), replicating our previously published work [31]. There were no main effects of Surgery for Trial 4 alone during this block for any planned comparison. Moreover, there were no significant differences during the Asymptotic phase of WRAM for any comparison. All rats learned to solve the MM across days and performance did not differ by Surgery type. All Sham and Hysterectomy rats (i.e., ovary-intact groups) had normal estrous cycles, did not differ in body weight, ovarian follicle counts, or serum hormone levels.

Middle-Aged Cohort: During the Learning Phase of WRAM, Hysterectomy rats made more WMC errors than Ovx rats (p<0.05), Ovx-Hysterectomy rats (p<0.05), and tended to make more errors than Sham rats (p=0.09). For WMI errors on Trial 3, Hysterectomy rats made more errors than Sham rats (p<0.05) and Ovx rats (p<0.01). On Trial 4, the maximum working memory load trial, Hysterectomy rats made more WMI errors than Ovx-Hysterectomy rats (p<0.05), collectively indicating that rats that underwent Hysterectomy surgery in adulthood exhibited spatial working memory impairments in Middle-Age compared to reproductive-tract intact rats as well as other variations in surgical menopause during learning. There were no significant differences during the Asymptotic Phase of WRAM for any comparison. All rats learned to solve the MM across days and performance did not differ by Surgery type. All Sham and Hysterectomy rats (i.e., ovary-intact groups) had normal estrous cycles, did not differ in body weight, ovarian follicle counts, or serum hormone levels.

Aged Cohort: During the Learning Phase of WRAM, there were no differences on the moderate working memory load trial, Trial 3. For Trial 4, the maximum working memory load trial, Hysterectomy rats tended to make more WMC errors than Sham rats (p=0.06) and Ovx rats (p=0.06). For WMI errors on Trial 4, Hysterectomy rats made more errors than Sham rats (p<0.05) and Ovx rats (p<0.05), and tended to make more WMI errors compared to Ovx-Hysterectomy rats (p=0.06), collectively indicating that rats that underwent Hysterectomy surgery in adulthood exhibited spatial working memory impairments one year after surgery compared to reproductive-tract intact rats as well as other variations in surgical menopause during learning. There were no significant effects of performance during the Asymptotic Phase of WRAM. All rats learned to solve the MM across days and performance did not differ by Surgery type. All Sham and Hysterectomy rats (i.e., ovary-intact groups) were in persistent diestrus at the end of the experiment, did not significantly differ in body weight, ovarian follicle counts, or serum hormone levels.

Discussion

This series of dissertation experiments was the first systematic evaluation of the short- and long- term effects of hysterectomy and other variations in gynecological surgery on cognition using a rodent model. Remarkably, when Hysterectomy surgery occurred in adulthood, Hysterectomy rats exhibited impaired spatial working memory performance during the learning phase of the WRAM compared to reproductive-tract intact rats as well as rats that received Ovx with or without hysterectomy at all three time points assessed. These working memory impairments manifested when memory load was sufficiently taxed. That Ovx-Hysterectomy did not have similar detrimental effects as Hysterectomy alone is a matter of substantial interest for future investigations; we hypothesize that disruption to the brain-reproductive-tract axis via hysterectomy results in dysregulated feedback mechanisms, whereas complete reproductive tract removal may result in neural communication pathway reorganization that does not alter memory processes in a similar fashion. Overall, these observed behavioral effects provide convincing evidence that Hysterectomy with ovarian conservation has long-term deleterious consequences for spatial working memory, and that the intact uterus likely plays a role in normal cognitive function.

The currently-accepted idea in clinical practice is that the ovaries become compromised following hysterectomy in adulthood, potentially leading to an earlier menopause onset in women. However, the data presented herein indicate that following surgery in adulthood, Hysterectomy and Sham rats continued to have normal estrous cycle patterns until the average age of rodent reproductive senescence. Furthermore, there were no morphological changes in the estimated number of healthy ovarian primordial follicles, growing follicles, or corpora lutea between Hysterectomy and Sham rats. Additionally, there were no statistically significant changes in serum hormone profiles between ovary-intact rats. Taken together, these findings suggest a unique role for the uterus itself in mediating cognitive function that is not secondary to alterations in ovarian function. Thus, the purported dogma that the uterus is by-and-large a quiescent organ independent of the ovaries must be overturned, and moving forward, further methodical investigations into the brain-uterus connection should be a central focus in the field of behavioral neuroendocrinology and women’s health.

Collectively, these findings have significant translational implications for the role of hysterectomy and its effect on cognition and quality of life during aging, particularly when the surgery occurs during a woman’s reproductive years. It is well-accepted that intricate feedback loops exist between the reproductive system and the brain. Variations in gynecological surgery likely result in reorganizational processes of important neural circuits, including those associated with learning and memory. Future directions of this research include determining brain regions impacted by age and gynecological surgery manipulations. Identifying the neurobiological underpinnings of the observed cognitive effects will aid in developing novel and targeted therapies with the goal of delaying or preventing the onset of deleterious cognitive effects associated with hysterectomy and other variations in surgical menopause. In the future, it will also be critically important to explore whether the age at which hysterectomy occurs differentially impacts cognitive outcomes, such that women and their physicians can make informed decisions about their lifelong health outcomes.

Impact Statement

Many thousands of reproductive-age women undergo hysterectomy with ovarian conservation for benign uterine conditions each year in the US alone; although the nonpregnant uterus has traditionally been considered a quiescent organ, in recent years, an association between hysterectomy and an increased relative risk of dementia has been reported in the scientific literature. The dissertation research described here was the first systematic and comprehensive evaluation of the short- and long- term cognitive, ovarian, and endocrine effects of gynecological surgery, including hysterectomy, in adulthood using a rat model. Results revealed a unique, detrimental impairment of hysterectomy with ovarian conservation at several time points— even up to one year after surgery— without significant ovarian or reproductive hormone changes, indicating that the uterus itself likely plays a key role in moderating cognition via the brain-reproductive tract axis. This research has the potential for significant societal impact, such that this series of experiments has uncovered a novel role for the uterus and its direct connection to the brain, whereby this fundamental contribution to the field will charter new pathways toward efforts to prevent or postpone potential cognitive decline in women as they age, and improve the general health and quality of life of women throughout the lifespan.

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