Jessica White NEUR493 Thesis

The Effects of Hormonal Contraception on Hippocampal Volume in Young Women: A Neuroimaging Study

Abstract

17b-Estradiol (E2) is a female sex hormone produced by the ovaries that is involved in women’s monthly menstrual cycles. E2 increases before ovulation in response to release of follicle stimulating hormone (FSH) from the anterior pituitary. However, when a woman is taking hormonal contraception (HC), this FSH release is prevented by the synthetic estradiol and progestin in the hormone-containing portion of the contraceptive regimen, which typically lasts three weeks, followed by an inactive week. During this inactive week, the lack of estradiol and progestin delivery allows endogenous E2 levels to rise. Because of this, and based on unpublished data from Dr. Ycaza Herrera, we predict E2 concentration should be highest during the inactive week in women using HC. Previous studies in naturally cycling women have shown that increased E2 concentration during the menstrual cycle is associated with increased hippocampal volume. However, no study has yet examined if there are similar estradiol-dependent structural changes during different HC phases. We hypothesized that, in women using HC, hippocampal volume should be largest during their inactive weeks, since this is when we predict their natural E2 concentration will be highest. To test this, we collected MRI structural images of the brain from thirteen women who were on monophasic HC. Hippocampal tracings and volume calculations were conducted using FSL during the active and inactive phases of HC. In contrast to prior work showing increased hippocampal volume during the period of the menstrual cycle when E2 levels are high, we failed to find a change in hippocampal volume from the active to inactive phase of HC. These results may indicate different regulation of hippocampal volume during HC and suggest that HC phase does not need to be taken into account methodologically in future studies that examine the effects of HC on hippocampal volume.

Introduction 17b-Estradiol (E2) is the primary form of estrogen, a female sex hormone that is

produced by the ovaries and critically involved in women’s monthly menstrual cycle. A synthetic form of estradiol, most often ethinyl estradiol, is one of the main ingredients in hormonal contraception. Women using monophasic hormonal contraception are typically prescribed three weeks of active doses containing synthetic forms of estradiol and progestin; the term “monophasic refers to the fact that the same amounts of synthetic hormones are given during all three weeks. This is followed by one week in which they receive no hormone exposure either via inactive, placebo-containing pills, or via the absence of contraception, i.e. removing the vaginal ring.

The normal menstruation process, which occurs in young to middle-aged women who do not take birth control, involves the hypothalamus acting on the anterior pituitary of the brain, causing cells there to release follicle stimulating hormone (FSH) and luteinizing hormone (LH), two hormones known as gonadotropins that are vital to the female reproductive system. Release of these hormones, which typically occurs near the beginning of the cycle, causes an increase in estradiol release from the ovaries between days 8-12 of their cycle (before ovulation, which

typically occurs around day 14), and an increase in progesterone around day 18 (after ovulation). However, when a woman is taking hormonal contraception, her exposure to synthetic estradiol and progestin prevents FSH secretion from occurring early in the cycle, which prevents the natural increases in E2 that lead to the LH surge, and thus prevents ovulation.

A relationship between E2 concentration and hippocampal volume has been discovered in studies of human females, for instance in a study with 21 women who experienced natural menstrual cycles (i.e., were not using any form of hormonal contraception; Protopopescu, Butler, Pan, Root, Altemus, Planescky, McEwen, Silberswig, & Stern, 2008). To see how the fluctuation in menstrual cycle phase affected their hippocampal volumes, these women underwent T1 MRI brain scans, which are structural scans used to identify anatomical brain structures based on proton density. Each woman completed one scan 1-5 days before they began menses (premenstrual), when their E2 levels were low. The second scan was completed 10-12 days after they began menses (preovulatory), when their E2 levels were high. Results showed that the volume of the right hippocampal grey matter (the regions of the brain that contain cell bodies of neurons) increases during the preovulatory phase of the menstrual cycle, when E2 concentration is high, in comparison to during the premenstrual phase, when it is lower. A similar study found this same correlation in both the left and right hippocampi (Lifosky, Eckert, Lindenberger, Gallinat, and Kuhn, 2015).

Studies have also examined different structural brain changes in naturally cycling women versus women who are using hormonal contraception. Results from these studies suggest that women using hormonal contraception have larger hippocampal volumes on average than those who are not (Bondt, Jacquemyn, Van Hecke, Sijbers, Sunaert, and Parizel, 2013; Pletzer and Kerschbaum, 2014). However, it has not yet been investigated if estradiol-dependent structural changes similar to those observed during the menstrual cycle occur during different phases of the hormonal contraceptive cycle. The present study, designed by myself and Dr. Alexandra Ycaza Herrera in conjunction with Dr. Mara Mather, aimed to investigate this possible relationship between hormonal contraceptive phase and hippocampal volume.

The synthetic hormones that women using hormonal contraception are exposed to during their three active weeks, notably ethinyl estradiol, lead to the prevention of FSH release from the anterior pituitary, which in turn prevents their body’s natural increase in E2 (Chiapaikeo, 2016). When a female is no longer being exposed to the synthetic hormones and begins the inactive week, the FSH will once again be released from the anterior pituitary and cause a rise in the body’s production of natural E2. Consistent with this, unpublished data collected by Dr. Ycaza Herrera revealed that women who were evaluated during their inactive weeks tended to have higher levels of E2 than those who were evaluated during their active weeks. Although this difference was not statistically significant, variability that was present due to features of the study design may have reduced power. For instance, the previous design was a between-subjects design, leading to a much larger spread of contraceptive type and dosage. Furthermore, it did not control for the specific day of women’s hormonal contraception regimens, such that some women were seen on the first day of a new birth control pack or on the first day of the inactive phase. Women were also allowed to have a variable number of inactive days (e.g., oral pill packs with four versus seven placebo days).

This failure to control for the numerous varying factors present in different hormonal contraceptive regimens, such as those factors described above, is quite common in the literature. This likely has resulted in incorrect inferences about the effects of HC on some of the studied domains. For example, in some research domains, interpretation of results could depend on the

participants’ number of inactive days or the phase of the hormonal contraceptive cycle during which evaluation took place. However, to our knowledge, it has not yet been investigated if these factors are important for interpreting structural brain differences. In the current study, we used a within-subject design to compare hippocampal volume between active and inactive phases of hormonal contraception, and only accepted participants whose hormonal contraceptive regimens contained seven inactive days. Based on previous findings demonstrating the release of inhibition of FSH release by the anterior pituitary once the inactive week begins (Chiapaikeo, 2016), and on the preliminary results from Dr. Ycaza Herrera’s unpublished study, we expected participants in the current study to undergo an increase in natural E2 concentration during the inactive week due to the longer and more consistent decrease in inhibition of the ovaries. As a result of the predicted higher E2 levels during the inactive week compared with the active week, we in turn hypothesized that the hippocampus would be larger during the inactive week compared to during the active weeks, since in naturally cycling women, this enlargement accompanies the period of increased E2 levels.

Methods & Materials Participants

A group of thirteen healthy, right-handed women ages 18-28 was selected to participate in the study according to several criteria. Women had been using monophasic contraception (either the oral pill or a vaginal ring) for at least six months prior to their participation. None of the women suffered from any chronic illnesses or medical conditions, and none were prescribed medications such as corticosteroids, beta blockers, or psychoactive drugs. Each of the women was screened for safety and provided written informed consent in conformance with the University of Southern California University Park Campus Institutional Review Board Guidelines. Sessions

Participants were required to come in for two separate sessions to complete the study, once during their second or third week of hormones and once during days 3-7 of their inactive week. This was to ensure that their bodies would have a chance to adjust to the hormone changes that accompany these different phases. Participants completed one MRI brain scan during each session. The participants’ scan orders were randomly counterbalanced so that eight of the subjects were scanned first during their hormone weeks and five were scanned first during their placebo weeks. I personally conducted the sessions in conjunction with Dr. Ycaza Herrera, who supervised the imaging acquisition. Image Acquisition

Structural images were acquired using a Siemens MAGNETOM Prismafit MRI scanner to obtain high-resolution T1-weighted anatomical images, specifically, Magnestisation Prepared Rapid Gradient Echo (MPRAGE) images with the following scanning parameters: TR=2300 ms, TE=2.26 ms, slice thickness = 1.0 mm, flip angle = 9°, field of view = 256 mm, and voxel size = 1.0 x 1.0 x 1.0 mm. The scans were performed at USC’s Dana and Dornsife Cognitive Neuroimaging Center (DNI). The total acquisition time for the anatomical scan was 4 minutes.

Image Preprocessing

Image preprocessing was performed using FSL Version 5.0. Using the Brain Extraction Tool (BET), I personally extracted each brain image (two per participant) from the images the MRI produces that contain non-brain tissue as well as brain tissue (see Figure 1).

Anatomical definition of hippocampus ROI Also using FSL, I performed hand-drawn, blind bilateral hippocampal tracings on each

participants’ scans in order to determine if the volumes of the participants’ hippocampi changed in response to the phase of hormonal contraception. Although I ran the sessions, I was able to remain blind to the session number and study ID number by using the independent DNI scan ID numbers prior to conducting any analyses.

The hippocampal tracings were done primarily in the coronal plane, and then verified in the sagittal and transverse planes, moving from anterior/medial slices to posterior/lateral slices. Moving anteriorly to posteriorly in the coronal view allows one to move from slices in which the hippocampal head is visible towards those in which the tail is visible. Although segmentation protocols vary greatly in the literature, I based my boundaries on those defined by Gurbani and Dimtchevka (2014) at the Ahmanson-Lovelace Brain Mapping Center at UCLA, as well as those defined by Konrad, Ukas, Nebel, Arolt, Toga, and Narr (2009). I also referenced Duvernoy’s atlas of the human hippocampus throughout the tracings, and to familiarize myself with the structure of the hippocampus in general (Duvernoy, 1988). I isolated the structures in order to accomplish accurate and uniform tracings.

Because the superior medial boundary of the hippocampus (the beginning of the hippocampal head) is difficult to identify in the coronal plane, I used the sagittal plane to identify the starting point for each trace by first finding the alveus, the white matter between the hippocampus and the amygdala (see Figure 3). The alveus consists of fibers of white matter that

Figure1:AnextractedbrainafterusingthebrainextractiontoolinFSL.Leftpanel:sagittalview;Middlepanel:coronalview;Rightpanel:transverseview.

connect to the fimbria (Konrad et al., 2009). I began my tracing on the most anterior coronal slice that was posterior to the alveus. In these more anterior slices, the hippocampal head is visible, and looks similar to a small gray triangle. Moving posteriorly, this triangle enlarges. Digitations are also visible in these anterior slices and can be identified as round dips in the superior border of the hippocampus.

The inferior boundary was easily identified throughout the coronal slices by using the white matter of the parahippocampal gyrus, which is in sharp contrast to the gray matter of the hippocampus (see Figure 4). As for the posterior boundary in the more medial coronal slices in which the hippocampal body is visible, I used the white border inferior to the inside of the lateral ventricle, the choroid plexus (see Figure 4).

Moving even more posteriorly, the hippocampal tail becomes visible. While the tail initially looks small and thin, as one moves posteriorly, and the superior and inferior horns of the lateral ventricle meet (see Figure 5), the tail grows larger and begins to tilt upward towards the midline of the brain. The posterior boundary of the hippocampus, in which the tail is visible in the coronal view, was identified as the slice at which the crura of the fornix can no longer be seen in full (see Figure 6). The crura can be identified as long, thin regions of white matter superior and lateral to the lateral horn (Duvernoy, 1988). This boundary was used as the last slice of the tracings in the coronal plane. After I reached this point in the coronal plane, I next went through the slices in the sagittal plane, and finally, in the transverse plane, to catch any outlying voxels and to verify my initial estimates of hippocampal size in general through these different views.

Figure2:Anexampleofahippocampaltracinginthesagittalplane(left)andinthecoronalplane(right).

Figure5:Leftpanel:Sectionbeforethesuperiorandinferiorhornsofthelateralventriclemeet.Rightpanel:After.

Figure3:Thearrowpointstothealveus,thewhitematterbetweenthehippocampusandtheamygdala,whichiseasilyidentifiedinthesagittalplane.

Figure4:Theredarrowdenotestheparahippocampalgyrus,whiletheblackarrowdenotesthechoroidplexus.

Figure6:Left:Thecruxofthefornix,denotedbythearrow,isstillvisible.Right:Thelastsliceselectedfortracingwasthesliceinwhichthecruxcannolongerbeseeninfull,asshownhere.

Volumetric Calculations Volume calculations were also performed blindly for the left and right hippocampi for each session using the “fslstats” tool in FSL. Statistical Analyses

To examine the potential relationship between different hormonal contraceptive phases and hippocampal volume, a 2 (contraceptive phase: active vs. inactive) x 2 (laterality: left hippocampus vs. right hippocampus) repeated-measures analysis of variance (ANOVA) test was conducted using the statistical software SPSS. This general linear model calculates the means for each specified group and compares them to each other. A value of p < 0.05 was used to determine the statistical significance of the results.

Results We found no main effect of hormonal contraceptive phase, F(1,12) = .063, p = .806,

𝜂"# = .005 (see Figure 7), suggesting that hormonal contraceptive phase has no effect on hippocampal volume. We also found no interaction between hormonal contraceptive phase and laterality, F(1,12) = .007, p = .937, 𝜂"# = .001 (see Figure 8), suggesting that hormonal contraceptive phase does not affect the volume of the right hippocampi differently than the left. However, we did find a main effect of laterality, F(1,12) = 4.950, p = .046, 𝜂"# = .292 (see Figure 9) such that the right hippocampus was larger than the left on average. This finding is consistent with results of prior studies, which will be addressed in the Discussion section.

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Figure7:Thisgraphillustratestherelationshipbetweenhormonalcontraceptivephaseandmeanhippocampalvolume,independentoflaterality.

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Figure8:Thisgraphdepictstheleftvs.righthippocampalvolumesofparticipantsduringtheiractiveweekscomparedtoduringtheirinactiveweek.

Discussion

Effect of Hormonal Contraceptive Phase

This study aimed to identify the effects of the different phases of the hormonal contraceptive cycle on hippocampal volume. In naturally cycling women, E2 concentration typically increases before ovulation, and this increase has been shown to be associated with an increase in hippocampal volume (Protopopescu et al., 2008; Lifosky et al., 2015). In women using hormonal contraception, however, the synthetic forms of estradiol and progestin used in these medications prevent ovulation by inhibiting the increased production of E2 that begins shortly after the onset of menses (Chiapaikeo, 2016). Based on this mechanism of HC action, along with trends observed in Dr. Ycaza Herrera’s unpublished data, we hypothesized that the cessation of synthetic hormones during the inactive week would lead to increases in endogenous E2, and we would observe an associated increase in hippocampal volume similar to that observed during the preovulatory phase of naturally cycling women by Protopopescu et al. (2008). However, our results revealed no main effects of hormonal contraceptive phase on hippocampal volume. One possible reason for the difference between our results and those of Protopopescu et al. (2008) is the sample size. Their study examined 21 women, while ours only examined 13. This small sample size could explain why we did not find effects of hormonal contraceptive phase. On the other hand, our effect sizes are also very small, so it is unlikely that a larger sample size would have given us an effect (𝜂"#= .005 for HC phase; 𝜂"#= .001 for the interaction between HC phase and laterality). In addition to calculating 𝜂"#, we also calculated Cohen’s d to verify effect size, and confirmed no effect size (d = .102) for hormonal contraceptive phase, and no effect size (d = .034) for the interaction between hormonal contraceptive phase and laterality (Cohen’s d conventions: 0.0 to 0.2: no effect; 0.2 to 0.5: small effect; 0.5 to 0.8: intermediate effect; 0.8 - > 1.0: large effect).

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Figure9:Thisgraphcomparesthemeanhippocampalvolumesfortheleftandrighthippocampi,independentofcontraceptionphase.

The different outcomes may instead be related to different segmentation methodology, since Protopopescu et al. (2008) performed their hippocampal tracings using automated segmentation software, while I performed them manually. Additionally, the hippocampal tracings performed by Protopopescu et al. (2008) focused on the anterior portion of the hippocampus and therefore did not include the posterior hippocampal tail, while ours did. Results from previous studies suggest that the posterior tail of the hippocampus is the region with the most significant volume differences between normal individuals (Pruessner, Li, Serles, Pruessner, Collins, Kabani, and Evans, 2000; Videbech and Ravnkilde, 2004; Maller, Reglade-Meslin, Anstey and Sachdev, 2006). If the posterior tail is the major source of individual differences in hippocampal volume, including the posterior tail may have increased overall variability in our study. Due to time constraints, I was not able to repeat my analysis without including the posterior tail, but it would be interesting for future studies to examine this potential variable.

Another possible reason we did not observe an effect of hormonal contraceptive phase on hippocampal volume could be that the ovaries simply do not have adequate time to release enough of their own E2 during the inactive week to influence hippocampal volume. Perhaps the increase in hippocampal size during the preovulatory phase of the menstrual cycle that was found by Protopopescu et al. (2008) and Lifosky et al. (2015) was able to occur because the increase in E2 concentration in naturally cycling women continues for more than a week, while in women using hormonal contraception, the only days they are not receiving synthetic estradiol and progestin are the seven days of placebo pills, or the seven days in which they remove their vaginal ring. This might not be a long enough window to affect hippocampal volume. If this reasoning is correct, then it also might be possible that we would have seen an effect if we had waited until the last day of the participants’ placebo weeks, or the last day in which their vaginal ring was removed, to run the scans for their inactive sessions. To test this, we again conducted a 2 (contraceptive phase: active vs. inactive) x 2 (laterality: left hippocampus vs. right hippocampus) repeated-measures ANOVA, but limited to women who were evaluated on the last two days of their inactive week (n=9). We still found no effect of hormonal contraceptive phase on hippocampal volume, F(1,12) = .354, p = .568, 𝜂"# = .042, suggesting that this is not the reason behind our failure to find an effect. Interestingly, however, when we ran this second ANOVA, the main effect of laterality on hippocampal volume was abolished, F(1,12) = 4.610, p = .064, 𝜂"#= .366. This could mean that during the last two days of the placebo week, either the right hippocampus decreases in volume, the left hippocampus increases in volume, or both of these occur. Another interesting finding we observed upon running this additional ANOVA was that both left and right hippocampal volumes were found to be numerically larger in this subset of women who were seen on days 6-7 of their inactive week. In order to make sure that this was not due to differences in overall skull size, we calculated hippocampal volumes as percentages of whole-brain volumes. Upon doing so, we still failed to find any main effects.

Another possibility as to why we found no effect of hormonal contraceptive phase on hippocampal volume is suggested by the finding that volume increases in rodents exposed to oral contraceptives, compared to controls, which may be caused by an increase in synaptic spine density mediated by estrogen receptors (Pletzer and Kerschbaum, 2014). Consistent with the rodent studies, human females using hormonal contraception have larger hippocampi than non-users (Bondt et al., 2013; Pletzer and Kerschbaum, 2014). These findings might help explain why we did not observe any changes in hippocampal volume between the phases. The hippocampal volume in women using hormonal contraception may have already been larger as a

result of the hormonal contraception itself, and therefore may not be susceptible to additional volume increases over the short course of natural E2 release that occurs during the inactive week. In other words, rather than preventing an effect of E2, as suggested in the previous paragraph, there might be a ceiling effect of hormonal contraception itself that makes it impossible to detect any effects of endogenous E2.

A slightly different potential explanation is that the use of hormonal contraception abolishes the effect that natural E2 normally has on the size of the hippocampus. If this occurs and if it requires the long-term use of hormonal contraception, it is possible that we would have observed volume changes had our participants been using hormonal contraception for a shorter time period prior to their sessions, before long-lasting effects took place (our criteria excluded women who had been using hormonal contraception for less than six months).

Another reason we might have failed to find an effect is due to the potentially conflicting influence of other hormones, such as progesterone, on the effects of E2. Rodent studies have shown that high levels of progesterone are associated with a decrease in hippocampal spine density as well as a decrease in hippocampal volume (Woolley and McEwen, 1993). Furthermore, it has also been shown in rodent studies that E2 and progesterone have interacting effects that lead to mitigation and even reversal of structural brain changes induced by E2 (Woolley and McEwen, 1993). If a similar pattern occurs in humans, this could explain why we were unable to observe volume changes that were related to predicted variations in E2 levels. Effect of Laterality

While there was no main effect of hormonal contraceptive phase on hippocampal volume, we did observe a main effect of laterality on hippocampal volume. This is consistent with previous findings in the field (Jack, Twomey, Zinsmeister, Sharbrough, Peterson, and Cascino, 1989; Hou, Yang, and Yuan, 2012; Woolard and Heckers, 2012.) In these studies and ours, the right hippocampus was found to be larger than the left. It has been proposed that this difference is due to a neurodevelopmental model in which a growth vector exists that progresses from the right anterior to the left posterior hemispheric regions (Woolard and Heckers, 2012; Best, 1988). According to this reasoning, handedness should not alter this trend in R>L hippocampal volume. While most of the studies in the literature have looked only at the laterality differences in right-handed individuals due to the difficulty in comparing right and left handed MRI scans, one study that compared laterality in left-handed vs. right-handed participants found a similar, although less pronounced, effect of R>L in left-handed individuals (Bernard, Walker, Baudouin-Poisson, Giroud, Fayolle, Dumas, Martin, Binnert, and Brunotte, 1996), while another found no significant volume differences in left-handed individuals (Szabo, Xiong, Lancaster, Rainey, and Fox, 2001). More research should be done to evaluate potential laterality differences in left-handed individuals, since few studies have examined this issue and those that have provide conflicting data.

Additionally, because the study performed by Protopopescu et al. (2008) found an effect of menstrual phase on only right hippocampal volume, we conducted follow up analyses to see if there was an effect of hormonal contraceptive phase on the right and left hippocampi alone. However, we still did not see any main effects of hormonal contraceptive phase when we performed these follow-up analyses.

Future Directions While our results found no significant relationship between hippocampal volume and

hormonal contraceptive phase, previous findings in the field have shown that on average, women using hormonal contraception exhibit increased gray matter volume in certain brain structures, including the hippocampus, compared to naturally cycling women (Bondt et al., 2013; Pletzer and Kerschbaum, 2014). However, this result does not necessarily indicate a direct causal relationship. Instead, the increased hippocampal volume might be due to an effect of education level, since women with higher levels of education are more likely to use hormonal contraception than those with lower levels of education (Jones, Mosher, and Daniels, 2012; Shartzer, Courtot, McMorrow, Benatar, and Kenney, 2016). Further studies could examine this possibility by controlling for this factor in their methods.

These findings highlight how important it is to consider the many different factors involved in various hormonal contraceptive regimens when interpreting data collected from women using hormonal birth control. For example, it is possible that the relationship between E2 levels and phases of hormonal contraception differed for our subjects versus those subjects in Dr. Ycaza Herrera’s unpublished study. This might be due to the factors we controlled for in this current study that the unpublished one did not; our current study used a within-subjects design, and was restricted to participants who had the same duration of the inactive phase, i.e., seven days. While hormone concentration data were collected in our study, they were not processed in time to include in this report. It would be interesting to incorporate this data once it becomes available.

Despite these limitations, our failure to find any statistically significant differences in hippocampal volume in active versus inactive weeks of the hormonal contraceptive cycle still provides significant contributions to the field. First, our study points to the importance of controlling for multiple factors in studies of women using hormonal contraception. Perhaps controlling for certain additional factors in our study contributed to the discrepancy between our results versus what we predicted our results to be based on Dr. Ycaza Herrera’s unpublished data. Second, our results could mean a great deal methodologically for future studies. For example, if a study aimed to compare brain structure differences between naturally cycling women and women using hormonal contraception, the results of our study suggest that it would be unnecessary to control for whether the women using hormonal contraception were on active versus inactive weeks during participation, resulting in a larger variety of possible participants and an easier scheduling process. Although in this instance our results reveal that controlling for hormonal contraceptive phase when testing for structural differences is not important, it has been shown to be important to take into account in other domains, such as resting state activation (Peterson, Kilpatrick, Goharzad, and Cahill, 2014). Regardless, our study points to the importance of taking these factors into consideration when performing research on women using hormonal contraception, and especially when interpreting data from such studies. Doing so could result in different findings, or even the need to alter previously accepted conclusions. Finally, studies of the relationship between menstrual cycle phase and hippocampal volume have been important for supporting other work on the relationship between reproductive hormones and memory, given the role of the hippocampus in memory (Bird and Burgess, 2008; Bondt et al., 2013; Fortin, Agster, and Eichenbaum, 2002). Our results suggest that these relationships may be altered by hormonal contraception, information which would be important considering that 100 million women worldwide have used or are currently using hormonal contraception (Pletzer and Kerschbaum, 2014).

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