Fertility and bone mass in prior female athletes with a history of amenorrhea

Thesis

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the

Graduate School of The Ohio State University

By

Megan Rose Stanley, B.S.

Graduate Program in Allied Medicine

The Ohio State University

2019

Master’s Examination Committee:

Adviser: Jackie Buell, PhD, RDN, CSSD, ATC

Julie Kennel, PhD, RDN, LD

Holly Estes, MS, RDN, LD, CNSC

1

Copyrighted by

Megan Stanley

2019

2

Abstract

Background: The spectrum of menstrual dysfunction includes a wide domain from normal cycles to interrupted cycles of 3 months or more classified as amenorrhea. The disruption of hormones in amenorrhea can cause a cascade of issues such as low bone density, lowered resting metabolic rate, and potentially issues with fertility.

Objective/Hypothesis: 1. Identify the prevalence of fertility issues in prior Ohio State female athlete athletes who experienced amenorrhea. 2. Compare the prevalence of low bone density and low resting metabolic rate in prior athletes with a history of amenorrhea to those without a history of amenorrhea.

Methods: Past female athletes received a survey via an email through alumni services. The survey took about 30 minutes to complete and queried on sport participation, menstrual history, and fertility history. Local respondents could sign up for a lab visit to have an iDXA scan for bone density and a resting metabolic rate test.

Results: The survey was completed by 298 alumnae. Of those, 158 reported trying to conceive,

44 with a history of amenorrhea and 114 eumenorrheic. For respective menstrual groups, 5

(11.36%) and 11 (9.65%) were unable to conceive, suggesting no likely influence of a history of amenorrhea on fertility in this cohort. A total of 44 participants completed the lab visit. There were no significant differences in bone density or resting metabolic rate between amenorrheic and eumenorrheic groups.

Conclusions: These findings suggested that a history of amenorrhea does not lead to higher risk of infertility. When alumni who had experienced amenorrhea were compared with those who

ii reported only eumenorrhea, there was no statistical difference in bone density or resting metabolic rate in the lab visit volunteers.

iii Acknowledgements

I would like to thank my advisor, Dr. Jackie Buell, for the opportunity to work with her and develop this project. I have enjoyed learning from you and being able to share my thoughts and concerns with you comfortably. You always answered my questions, no matter how ridiculous, with such knowledge and acceptance. I would also like to thank my thesis committee members

Dr. Julie Kennel and Professor Holly Estes, for their encouragement and support. Additionally, I

would like to thank Nancy Clark and Dr. Nicola Rinaldi for helping develop this project and

sticking with me through the acceptance of this research.

I do not know where I would be without the support from my fellow MS/DI program members.

They were always there to help answer any question I had, to spend weekends working on our

papers, and pick me up with words of wisdom when I needed it.

I would finally like to acknowledge my family and friends for the many phone calls and pep talks over the last two years. Thank you all for encouraging me and letting me rant when I needed to.

You all inspire me with your hard work and dedication and I truly would not have made it here

without you.

iv Vita

March 14, 1995 ...... Born – Christiana, Delaware

May 2017 ...... B.S. Nutrition, Dietetics, The Pennsylvania State University

Field of Study

Major Field: Allied Medicine

Specialization: Medical Dietetics

v Table of Contents

Abstract ...... ii

Acknowledgements...... iv

Vita ...... v

List of Tables ...... viii

List of Figures ...... ix

List of Definitions ...... x

List of Abbreviations ...... xi

Chapter 1: Introduction ...... 1

Introduction ...... 1 Objectives...... 2

Chapter 2: Review of Literature ...... 3

Menstrual Classifications and Causes ...... 3 Causes of Secondary Amenorrhea ...... 7 Hypothalamic Amenorrhea ...... 10 Risks Associated with Amenorrhea...... 16 Measurement Tools ...... 23 Predicted Prevalence of Amenorrhea in Athletes ...... 24 Diagnosis of Amenorrhea ...... 24 Treatment for Amenorrhea ...... 26 What We Don’t Know ...... 27

Chapter 3: Methodology ...... 29

Research Design ...... 29 Recruitment and Description of Human Subjects ...... 29 Exclusion Criteria...... 30 Study Questionnaire ...... 30 Face Validity ...... 31 Laboratory Visit ...... 31 Anthropometrics and Bone Density ...... 31 Resting Metabolic Rate ...... 32 vi Lean versus Non-Lean Sports ...... 32 Statistical Analysis ...... 33

Chapter 4: Results and Discussion ...... 35

Results ...... 35 Amenorrhea and Fertility ...... 36 Lab Visit ...... 36 Bone Density ...... 37 Resting Metabolic Rate ...... 39 Sport Frequency ...... 40 Discussion...... 43 Limitations...... 46 Conclusion ...... 48 Implications for Practice ...... 48

Chapter 5: Bone Density and Resting Metabolic Rate in Prior Female Athletes with a History of Amenorrhea ...... 50

Introduction ...... 50 Objective ...... 51 Methods ...... 52 Results ...... 55 Discussion...... 59

References: ...... 62

Appendix A: Recruitment Email ...... 74

Appendix B: Questionnaire ...... 76

vii List of Tables

Table 1: Comparison of Duke radiation estimator to GE Lunar iDXA actual radiation ...... 32

Table 2: Survey Participant Descriptive Data ...... 35

Table 3: Ethnicity of Participants ...... 35

Table 4: Lab Participant Descriptive Data ...... 37

Table 5: Bone Density Measurements ...... 38

Table 6: Frequency of Low BMD for age ...... 39

Table 7: Frequency of Low Bone Mass by Skeletal Z-score for Amenorrhea and Eumenorrheic

Participants ...... 40

Table 8: Resting Metabolic Rate Values ...... 40

Table 9: Differences in Resting Metabolic Rate Based on Menstrual History ...... 40

Table 10: College Sport Frequency ...... 42

Table 11: Lab Participant Descriptive Data ...... 55

Table 12: RMR Values ...... 56

Table 13: Differences in RMR Based on Menstrual History ...... 57

Table 14: Bone Density Measures ...... 59

Table 15. Frequency of Low Bone Mass by Skeletal Z-score for Amenorrhea and Eumenorrhea

Participants (3 subjects excluded due to OCP amenorrhea) ...... 60

viii List of Figures

Figure 1: The spectrum of menstrual disorders, eumenorrhea on the left, amenorrhea on the right adopted from De Souza et al 2004 ...... 4

Figure 2: The changes in hormone levels throughout a normal menstrual cycle. adopted from

Cromer and colleagues 1996 ...... 5

ix List of Definitions

Eumenorrhea: normal, regular menstruation

Oligomenorrhea: cycles more than 35 days in length

Amenorrhea: 3 or more months without a period

Energy Availability: (calories consumed – calories expended in exercise)/kilograms (kg) of lean body mass

Fertility: natural capability to produce an offspring

Resting Metabolic Rate: how many calories a person burns at rest

Bone Mineral Density: amount of bone mineral in bony tissue

Low Bone Mineral Density: low bone mass for age – a Z-score of -1 or less

Urine Specific Gravity: tests the density of urine to measure hydration

x List of Abbreviations

EA Energy Availability

RED-S Relative Energy Deficiency in Sport

FSH Follicle Stimulating Hormone

LH Luteinizing Hormone

GnRH Gonadotropin Releasing Hormone

PCOS Polycystic Ovary Syndrome

FHA Functional Hypothalamic Amenorrhea

HPA Hypothalamic Pituitary Amenorrhea

TSH Thyroid Stimulating Hormone

T3 or TT3 Triiodothyronine

CRH Corticotropin-Releasing Hormone

IGF-1 Insulin-Like Growth Factor

IGFBP-1 Insulin-Like Growth Factor Binding Protein

PYY Peptide YY

DXA Dual X-Ray Absorptiometry

CT Computed Tomography pQCT Peripheral Quantitative Computed Tomography

QUS Quantitative Ultrasound

RMR Resting Metabolic Rate

REE Resting Energy Expenditure

LPD Luteal Phase Defects xi FAT Female Athlete Triad

ED Eating Disorders

AN Anorexia Nervosa

USG Urine Specific Gravity

DXA Dual X-ray Absorptiometry, Lunar iDXA is the specific General Electric machine used in this study

xii

Chapter 1

Introduction

The female menstrual cycle is a complex relationship between hormonal signals, energy availability, and reproductive organs. When any one of these factors is altered or not functioning optimally, the menstrual cycle can be affected leading to disruption or even cessation1. When the menstrual cycle is interrupted, there may be effects on many other related bodily functions such as bone health, cardiovascular health, and fertility2.

Although exercise and athletics pose many health benefits for those who participate, training habits, such as intense exercise paired with inadequate energy intake, can be detrimental to health. Insufficient supply of fuel presents risks for many body systems, including the reproductive system in women2. When the body has minimal energy available, it prioritizes where to direct its fuel supply and, in turn, ceases to provide energy for those processes not critical for survival, such as menstruation1.

Participation in women’s collegiate athletics has grown exponentially by at least 545% since the 1970s due, in part, to Title IX3. The continued increase in participation is paving the way for more elite athletes, but is also putting more women at risk for the negative concomitants of intense exercise without adequate nutritional intake. Each sport requires a different set of physical capabilities, body types, and energy needs. For those sports that require long bouts of endurance exercise such as cross country running, or those where athletes are encouraged to maintain lean body types, the sequelae of low energy availability may be an even greater risk. Much has been learned about how training habits and nutritional

1 status impact athletic women while they are highly active, but less is known about the long- term impact these factors may have as these athletes continue into adulthood.

The Female Athlete Triad demonstrates the negative influence of poor energy availability on reproductive and skeletal systems2. Low energy availability (EA) can lead to lack of menstrual cycles, or amenorrhea. Amenorrhea not only causes problems in the present, but has the potential to affect a woman’s fertility in the future. By altering hormone levels and reproductive function for long periods of time, the body may lose the capacity to conceive and carry a fetus4. It is purported that prolonged amenorrhea not only impacts reproductive ability, but may also affect bone health through the influence of many hormones on bone metabolism2,4,5.

The relationship between amenorrhea in young adulthood and bone density in adulthood has not yet been well-established. As a relatively recent extension to the triad,

Relative Energy Deficiency in Sport (RED-S) contends that low energy availability may also adversely impact other physiological processes such as the regulation of energy metabolism.

Assessment of the relationships between menstrual function during athletic participation and the potential outcomes throughout adulthood will inform future research investments.

Objectives:

1. Identify the prevalence of fertility issues in prior Ohio State female athlete alumni who

experienced amenorrhea.

2. Compare the prevalence of low bone mineral density and low resting metabolic rate in

prior athletes with a history of amenorrhea to those without.

2 Chapter 2

Literature Review

Menstrual function is described as a continuum of eumenorrhea to amenorrhea.

Amenorrhea in athletic populations is best known for its role in the “female athlete triad”, which includes amenorrhea, low energy availability, and low bone density. The current triad paradigm considers low energy availability as the initiating factor. Energy availability is defined as:

(calories consumed – calories expended in exercise)/kilograms (kg) of lean body mass

However, this condition has recently been re-evaluated and labeled relative energy deficiency in sport (RED-S)6. This classification comprises impaired normal physiological function including changes in metabolic rate, menstrual function, immunity, and other aspects of health6. Nonetheless, even with the new proposed classification of RED-S, many researchers still prefer the term female athlete triad6.

In sports medicine, issues with the menstrual dysfunction are of concern as these issues can be an outward sign of inadequate fueling in female athletes. Given the potential impact of poor energy availability on bone and other body systems, menstrual function as a marker of energy availability is gaining more attention as an issue that should be addressed promptly by healthcare professionals.

Menstrual Classifications and Causes

The spectrum of menstrual dysfunction has been studied for many years and includes a wide domain of menstrual patterns. These patterns range from normal or eumenorrheic cycles to cycles that are absent for more than three months at a time, or amenorrhea. There is also a 3 range of subclinical disorders within the spectrum, often classified as oligomenorrhea. DeSouza and Williams delineate the continuum of menstrual issues for athletes as shown in Figure 1.

Ovulatory Luteal Phase Anovulation Oligomenorrhea Amenorrhea (Eumenorrhea) Defect (FHA)

Figure 1. The spectrum of menstrual disorders, eumenorrhea on the left, amenorrhea on the right, adopted from De Souza et al 2004 2

Eumenorrhea

Normal menstrual function is classified as eumenorrhea7. A normal menstrual cycle is about 21-35 days in length, consisting of luteal and follicular phases where the endometrium prepares for implantation of a fertilized egg8. If no fertilized egg implants, the lining of the uterine wall sheds, which causes what is known as the “period” of bleeding that lasts about 2-7 days8.

The menstrual cycle is tightly regulated by communication between the hypothalamus, pituitary gland, and the ovaries. During the follicular phase (days 1-14 in Figure 2 below) ovarian follicles are being developed and prepared for fertilization. In the early follicular phase, follicle-stimulating hormone (FSH) levels decline through a negative feedback loop of estrogen from the ovaries back to the hypothalamus pituitary. This leads to luteinizing hormone (LH) secretion in pulses. The frequency and amplitude of the LH secretion regulates the functions of the luteal phase (days 15-28 in Figure 2 below). During the luteal phase, gonadotropin-releasing 4 hormone (GnRH), estrogen, progesterone, and FSH levels increase. Estrogen levels rise and fall during this time in response to a rise in FSH, activating the pathway to convert androgens to estrogen.

Figure 2. Changes in hormone levels throughout a normal menstrual cycle. Adopted from Cromer and colleagues 19969.

Luteal Phase Defect and Anovulation

The earliest changes in the menstrual cycle are not obvious and require in-depth medical testing such as blood tests for hormone levels10. Luteal phase defects result from insufficient progesterone production, which affects the function of the corpus luteum. LPD is often not diagnosed until after issues of infertility or recurrent abortion have presented

5 themselves. This diagnosis is thought to affect 3-4% of couples who have issues with fertility10.

Anovulation or anovulatory cycles are characterized by the absence of a LH peak adequate enough to induce ovulation2. Luteal phase defects and anovulation are not typically diagnosed until the spectrum of menstrual disturbances provides a warning sign such as unexplained infertility of recurrent abortion10.

Oligomenorrhea

Next along the spectrum of menstrual abnormalities is oligomenorrhea. This classification is characterized by abnormally infrequent menstruation, with cycles lasting more than 35 days, and is the most commonly reported menstrual issue11,12. The alterations in hormones that lengthen the cycle are a warning sign in the cascade of alterations. Most causes of oligomenorrhea are the same as those of amenorrhea (discussed later), without the same degree of severity. However, oligomenorrhea is also a distinguishing characteristic in polycystic ovary syndrome (PCOS), thus exploration of the cause of oligomenorrhea should be fully evaluated as a warning signal of potential issues13.

Amenorrhea

On the opposite end of the spectrum from eumenorrhea is amenorrhea. There are two classifications of amenorrhea, primary and secondary. While the causes can be similar, they can also be quite different and some causes may be more serious than others.

Primary Amenorrhea

Primary amenorrhea is when a female does not begin her period by the age of fifteen14.

The cause of primary amenorrhea is thought to be genetic or due to chromosomal abnormalities or issues with the hypothalamus or pituitary gland15. The prevalence of primary 6 amenorrhea has not been well-reported. However, this classification of amenorrhea may be less common or more underreported than other cases of amenorrhea. Similar to the etiology of secondary amenorrhea, it is possible that primary amenorrhea can be due to insufficient fueling practices combined with intense training. It is important to identify the underlying causes of primary amenorrhea determined to rule out organic causes unrelated to fueling issues.

Secondary Amenorrhea

Secondary amenorrhea is the term used to describe the condition where a female who was previously having normal menstrual cycles experiences 3 or more months of no menstrual cycle1. The slight differences in menstrual disruptions between secondary amenorrhea and oligomenorrhea help demonstrate the similar mechanisms along a spectrum of dysfunction.

There are other descriptors of amenorrhea such as chronic anovulation. As it is thought to be more easily remedied, secondary amenorrhea is the focus of this study.

Causes of Secondary Amenorrhea

Secondary amenorrhea has many possible causes. These include natural occurrences, contraceptives, and lifestyle alterations. Mountjoy et al reported in the International Olympic

Committee consensus statement, that the prevalence of secondary amenorrhea was 2-5% in all collegiate women, including athletes, exercising, and non-exercising women6.

There are life stages where it is considered “normal” to be amenorrheic. During pregnancy, when an egg is fertilized the body no longer needs to ovulate or shed the lining of the uterus, therefore a woman typically does not experience her period. The menstrual cycle is also delayed while breastfeeding as menstrual hormones are suppressed by high prolactin levels16. Finally, menopause, when a woman reaches the age where her menstrual cycles stop 7 all together, is considered a form of secondary amenorrhea. All of these states cause a woman’s menstrual cycles to cease but are normal to female health.

If a woman chooses to use hormonal-based birth control methods, such as the pill, hormonal IUD, or subcutaneous implants or injections, the menstrual cycle may be suppressed.

Some brands/types of birth control or contraceptives cause women to not have a bleed each month, such as Depo Provera17. While normally stopping a period via birth control does not raise health concerns, some forms can lead to infertility or bone density issues if taken incorrectly or for an extended period of time outside medical recommendations. For example, bone loss of 7.7% in the hip, 6.4% in the spine, and 3.2% in the total body has been demonstrated with Depo Provera use after 2 years18,19. It is important for women to have regular medical monitoring of reproductive choices and menstrual health.

Another issue that arises with contraceptives inducing or ceasing menstrual cycles is that they can mask underlying issues that may affect reproductive and bone health20. For example, if a woman is getting a false period induced by the synthetic hormones of birth control, it may be masking underlying amenorrhea which then will not be treated for an extended period of time. This false period or artificial and pill-induced changes of the menstrual cycle may prevent discovery of underlying issues with bone loss as well.

There are also several lifestyle behaviors that can lead to secondary amenorrhea. These behaviors are concerning as they can be harder to treat and are generally coupled with other problems. Lifestyle behaviors that can cause amenorrhea include: nutritional imbalance, excessive exercise, stress, and weight loss1,2. Most of these are linked to low energy availability, meaning there are not enough calories being consumed to support activity. When this happens, 8 the body preserves the energy that it does have available for the necessary processes of survival, and no longer provides the energy to carry out the menstrual cycle2. This nutritional imbalance has become known in athletic amenorrhea as low energy availability21.

Long term energy deficits often result in a low percentage of body fat. Rose Frisch was a leader in the field proposing the necessity of a minimal body fat percentage and energy intake for restoration and maintenance of menses. A 1991 review by Frisch suggested that a higher percentage of body fat (around 26-28%) was essential for regular ovulation and reproductive health22. Her review demonstrated that a moderate weight loss of only 10-15% in women in the normal weight-for-height category could result in amenorrhea. The Frisch hypothesis also found that total caloric intake was lower by 500 calories per day in amenorrheic runners compared with eumenorrheic runners who had similar BMI’s22. This hypothesis suggested that metabolic fuels, such as food and internal fuel reserves, need to adequately match energy expenditure of not only athletes, but all women, and that a caloric, or fuel, deficiency is the main factor in chronic anovulation23. While the suggested body fat levels of Frisch have since been debunked, the current paradigm still includes the energy imbalance she suggested even though it has morphed to energy availability24.

Stress is another known factor that may contribute to secondary amenorrhea. Excessive exercise is one means of causing stress on the body, and a similar feedback mechanism occurs with other types of stress such as psychological stress1,25,26. These stress-inducing states cause the same alterations as undernutrition or exercise and fall into the category of hypothalamic amenorrhea. This stress and/or lower body weight sends feedback to the hypothalamus in the form of altered hormone levels due to the hypothalamic-pituitary-adrenal axis being activated, 9 and cortisol inhibits the hypothalamic-pituitary-ovarian axis1. This is a homeostatic mechanism where hormones from various organs control the centers in the brain, and is why it is named hypothalamic amenorrhea.

Hypothalamic Amenorrhea

Hypothalamic amenorrhea is one of the most common types of secondary amenorrhea4.

It is often referred to as functional hypothalamic amenorrhea (FHA) or hypothalamic pituitary amenorrhea (HPA). The exact mechanism of the root cause is unknown, and mitigation of the behavior causing the issue tends to restore reproductive function including the menstrual cycle, hence the term functional27. FHA is responsible for about 20-35% of secondary amenorrhea cases as reported by The American Society of Reproductive Medicine4. It has also been shown that the longer the duration of insult, the longer it takes to return to normal menses28.

The menstrual cycle is a complex process that involves many different hormonal signals which can change cycle length or even stop it completely. Many pathways of information in the endocrine system start in the hypothalamus with a “releasing factor”, which stimulates the pituitary to send additional signals that continue to the effector organ- an endocrine gland29.

The effector organ then releases the hormones that regulate the body. The complex signaling pathways are considered to work in loops where hormones released from the effector organs also feedback to the brain to influence the hypothalamus and pituitary gland. In addition to this loop of feedback for the reproductive system, the hypothalamus and pituitary also orchestrate similar loops for the adrenal, thyroid and liver as effector organs controlling the release of cortisol, thyroxine, and IGF-1 respectively, and each of these organ pathways are often referred to as axes. 10 There is a tight link between the hypothalamic pituitary axis and GnRH drive. When any of the factors that affect GnRH secretion from the hypothalamus are significantly altered, it will influence the release of FSH and LH from the pituitary. This deficiency in GnRH secretion results in a diminished cascade of hormones to the ovaries, and eventually affects ovulation30. Pulse frequency of these reproductive hormones is progressively reduced in athletic amenorrheic women. For example, studies have shown LH pulse frequency to be reduced by 50% in this population31. It is well-known that factors that stimulate the hypothalamic-pituitary-adrenal axis will increase cortisol, and cortisol will inhibit the hypothalamic-pituitary-ovarian axis as well as the thyroid and liver (IGF-1) axes32. The complex orchestration and signaling in the brain controls the peripheral organs28,2.

Work by Loucks et al has been instrumental in demonstrating brain to effector organ signaling. A study of runners, cyclists, and triathletes as early as 1989 showed that LH pulse variation was greater with a lower number of pulses in athletic women who reported amenorrhea. Athletic amenorrheic women also had an increased LH response to externally administered GnRH32. This alteration was purported to be caused by the pituitary becoming hypersensitive when signals from the hypothalamus are consistently low, and reacting intensely when there is a signal32. This line of research has continued to define the female athlete triad as we know it today.

In a landmark study in 1998, Dr. Loucks was the first to show that low energy availability, defined as dietary intake minus exercise energy expenditure, was the cause of altered LH pulsatility and not the stress caused by exercise. This study defined the stress of exercise independent from energy availability by providing the recommended number of 11 calories needed for adequate energy availability or restricting energy intake while having normally cycling women perform exercise. They then compared these pulsatilities to those demonstrated in women with similar energy availabilities, but who did not exercise. In the exercising women with low energy availability, LH pulse frequency was reduced by 10% and amplitude was increased by 36%. In women with adequate energy, the stress of exercise alone did not alter pulse frequency or amplitude significantly. The results from this study changed how we look at the disruption of LH in exercising amenorrheic women, proving that the changes are due to low energy availability and not exercise alone21.

A later study (2003) performed by Loucks and Therma took habitually sedentary women and had them perform exercise to expend 15 kcal/kg of lean body mass (LBM). For the first trial, the women consumed a diet of 45 kcal/kg LBM/day, and after a two-month washout, were assigned to groups for 30, 20, or 10 kcal/kg LBM/day with the same exercise protocol in a second trial. In the groups assigned 20 and 10 kcal/kg LBM/day, LH pulse amplitude increased and frequency decreased. This was the original study demonstrating that habitually consuming less than 30 kcal/kg LBM/day is the culprit in alterations in LH secretion33.

These modified signals from the brain lead to a decrease in production of estrogens by the ovaries4. Estrogen is important for its role in maintaining pregnancy as it works with progesterone to maintain endometrial cyclicity4,34. When estrogen is low, it inhibits GnRH release further, causing an increase in severity of the menstrual disorder, thus spectrum of insult4. Loucks et al demonstrated this nicely in the 1998 LH pulsatility study of normally cycling sedentary and athletic women and athletic amenorrheic women. The study cited basal estradiol

(a type of estrogen) levels to be 186 + 16 pmol/L in cycling sedentary women compared to 114 12 + 22 pmol/L in athletic amenorrheic women32. This showed the level of ovarian suppression in amenorrheic women compared to regularly cycling women.

Estrogen is responsible for influencing other hormones in the menstrual cycle, such as

FSH. Hypoestrogenism is a known characteristic of hypothalamic amenorrhea due to low energy availability35. De Souza et al studied exercising women, and found that the day of peak estrogen excretion was significantly later in women with luteal phase defects, and those with amenorrhea had significant suppression of estrogen as well36. Overall, exercising women had significantly lower progesterone excretion. This imbalance of hormones affects the menstrual cycle and causes the issues of amenorrhea, low bone density, and suppressed metabolism.

Activation of the adrenal axis also causes a decrease in thyroid stimulating hormone

(TSH). Without TSH stimulating the thyroid, triiodothyronine (T3 or TT3) levels decrease. This can lead to hypothyroidism, which may also cause infertility due to changes in GnRH, prolactin, sex steroid levels, and sex-hormone binding proteins37,38. T3 is significantly lower in exercising women, but especially lower in exercising amenorrheic women39. One study showed that an intervention in two groups of diet restriction or diet restriction plus exercise lowered T3 levels in both, but more so in the exercise group with a greater energy deficit40. The thyroid axis is also influenced by low energy availability.

Along with low GnRH, the stress caused by decreased energy availability causes increased corticotropin-releasing hormone (CRH) levels. This increases adrenocorticotropic hormone (ACTH) levels, which act on the adrenal gland to increase cortisol secretion27. Cortisol is a glucocorticoid steroid hormone that plays a wide role in bodily processes. Normally, cortisol is secreted in a pulsatile fashion with a well-defined rhythm41. 13 It is well-known that various kinds of stress elevate cortisol levels42,43. In amenorrheic athletes, cortisol levels are higher than in eumenorrheic counterparts26,44. In a study by Ding et al, most women with amenorrhea had cortisol levels that not only exceeded average levels, but were outside (higher than) the ranges of any cortisol assay, showing the large effect stress has on these hormones41. The body perceives low EA as stress with hormonal responses back to the brain.

There are many other organ-related hormonal factors that act centrally at the hypothalamic level. Starvation causes a negative feedback loop from the stomach to increase the release of the hormone ghrelin. Ghrelin’s main role is to send hunger cues to the brain to stimulate feeding. It is considered the primary peripheral signal for energy homeostasis as it indicates an energy deficit39. Basal ghrelin levels have been shown to be responsive to decreases in body weight, fat mass, and resting metabolic rate that were caused by an energy deficit45. In amenorrheic women, ghrelin levels are high, signaling the need for more energy.

DeSouza et al found ghrelin levels to be 100% higher in exercising amenorrheic women, compared to controls who cycled normally39. Ghrelin is a compensatory mechanism that may reflect a prolonged chronic state of energy deficiency and signal the brain to increase food intake46. Athletes with constant low energy availability may generate this starvation signal from the stomach to signal the body’s need for fuel.

Body fat, or adipose tissue, also acts as an endocrine organ to release leptin. When leptin levels are decreased due to lower levels of body fat, they act in the negative feedback loop4,29. Scientists have hypothesized that leptin could be regulated by energy availability, meaning it would be altered in FHA47. When energy intake is restricted in amenorrheic women, 14 leptin levels are reduced. This leads to a down-regulation of the hypothalamic-pituitary axis48.

Low-dose leptin replacement has also been shown to prevent decreased thyroid axis function in energy-restricted diets29. Thong et al found a positive correlation between percent body fat and plasma leptin levels. This led them to hypothesize that leptin may be a peripheral signal of energy availability47. Köpp et al also found that leptin levels can predict lifetime occurrence of amenorrhea, proving leptin is another important hormone in energy homeostasis and reproductive functioning48. These studies are in line with findings by early research by Matkovic et al showing leptin’s role as a stimulus to triggering the reproductive cycle24. Low body fat stores mean lower leptin, and this hormone is another regulating signal based on energy reserves.

Furthermore, to facilitate energy conservation, other metabolic shifts including suppression of liver insulin-like growth factor (IGF-1) as well as elevated peptide YY (PYY) levels occur. Insulin-like growth factor binding protein (IGFBP-1) is acutely regulated by exercise and may be a marker of metabolic stress. It has been demonstrated in a study by Waters et al that this protein rises throughout exercise49. IGFBP-1’s influence on IGF-1 can be another signal of reduced fuel availability50. Similarly, elevated PYY levels are also present in this energy-deficient population. PYY is a peptide secreted by endocrine cells in the small intestine. It’s secretion increases after food consumption to provide a feeling of satiety, and it also regulates bone metabolism as it activates receptors that inhibit bone formation due to decreased osteoblast activity51. Russell et al demonstrated PYY levels of 111 + 52 in amenorrheic athletes and 61 + 29 pg/ml in eumenorrheic athletes. This suggests that in amenorrheic women, PYY levels may be

15 higher than normal51. This is not only an issue for the menstrual cycle, but also contributes to low bone density discussed later.

To demonstrate the integration of multiple factors influencing the menstrual cycle,

Williams et al developed an animal model of menstrual dysfunction. Previously unstressed monkeys were placed in three groups: low levels of psychosocial stress, moderate exercise + caloric restriction, and a group that had all three factors. They found that 7 out of 10 members of the group who endured all three stressors had disrupted menstrual cycles longer than 45 days in length where the typical average is 30 days, with some monkeys having cycles longer than 100 days in length52. In these monkeys, there was predominately an increase in the follicular phase length where the ovaries prepare a mature follicle and release it or ovulate.

While it is not a human study, this primate study showed that stress caused by many factors induces amenorrhea in 70% of subjects.

Risk Associated with Amenorrhea

Fertility

Many potential risks are associated with prolonged amenorrhea including compromised bone health, fertility, and altered resting metabolic rates2. Fertility issues are one of the acute risks of FHA often due to anovulation or altered luteal phases53. The long-term effects of prior amenorrhea on fertility in athletes have not been well studied.

There is more research on infertility in other amenorrheic women such as women with eating disorders (ED). These issues are comparable to HPA as they often include decreased energy availability and ceasing of menses. The prevalence of infertility in the ED population has been measured in different ways such as EDs in infertile women, or infertility in ED patients. 16 The results of these studies are confounding, but may still provide background for adulthood fertility for amenorrheic athletes.

Questionnaire work also contributes to the literature for the influence of eating disorders on fertility. A study by Easter et al collected survey information from pregnant women querying on if they had experienced any EDs in their lifetime, time to conception, and fertility treatments54. Their findings showed that women who reported a history of anorexia nervosa (AN) as well as AN and bulimia nervosa (BN) together were more likely to see a doctor due to a lifetime of fertility issues compared to those not reporting ED histories54. Women with a history of AN + BN also took 6 months longer to conceive and needed treatment to do so, showing issue with fertility in women with ED histories. However, it should be noted that the sample group was small and the classifications for ED were based on self-report data, not a clinical diagnosis54.

Eating disorder questionnaires employed in fertility clinics have also led to interesting results. Cousins and Freizinger showed women experiencing infertility reported significantly higher symptoms of eating disorders than those without55. Specifically, these women experiencing infertility had greater scores for measures on drive for thinness based on the

Eating Disorder Index Referral Form55. A similar investigation by Freizinger et al published 5 years later determined the prevalence of ED in a fertility clinic and found that 20.7% of participants met criteria for past or current ED, showing fertility issues in this population56.

Appearance of a higher rate of ED in fertility clinics suggests a potential relationship.

Anorexia studies led to a similar suggestion. Shomento et al followed women for four years after being hospitalized for AN. These women were all able to conceive within a year of 17 trying after 92% of normal weight for height was reached to resume menses, leading the researchers to conclude that a history of AN does not affect fertility57. Results from this population are conflicting, but do show some evidence that disordered eating behaviors leading to low energy availability may affect fertility.

Bone Density

Peak bone mass is the amount of bone tissue that is present at the end of bone maturation. Most rapid growth and development happens during adolescence as accumulation of bone is most powerful then58. The accumulation of bone is thought to stop at an average age of thirty years old, but can continue for several more years in some individuals58. This means that there is still room for bone density growth in early adulthood, including college-aged women.

Estrogen is a major hormonal factor in the changes in bone health. It works with calcium and vitamin D to build and maintain strong bones. Estrogen deficiency affects bone density by accelerated bone turnover with not enough formation to match resorption59. Mechanically, estrogen inhibits osteoclastic activity. Therefore, when circulating estrogen is low, it allows higher osteoclastic activity without upregulating osteoblast activity, preventing bone resorption and yielding a net bone loss60. Bone physiologists have suggested that estrogen deficiency is stronger than calcium deficiency as a stimulus for resorption. Since estrogen deficiency is present in about 55% of amenorrheic women, compromised bone health is an intuitive outcome59.

The imbalance in osteoclast/osteoblast activity in remodeling is often termed uncoupling. Bone modeling is when the skeleton develops and grows by the independent action 18 of osteoblasts and osteoclasts61. Bone remodeling is when osteoclasts and osteoblasts work in succession in the same bone61. First, in the resorption stage, osteoclasts break down old bone62.

Then, in the formation stage, osteoblasts lay down new bone until all bone that was absorbed is completely replaced62. In a low energy available state, resorption is disproportionate to formation, causing bone loss59. After peak bone mass is reached, remodeling is stable for about two decades. After this, age-related bone loss begins61. Lawrence-Riggs suggested that the pro- inflammatory cytokines in amenorrheic women increase bone resorption as they increase the number of pre-osteoclasts in bone marrow60. A small change in one cytokine in the microenvironment of any bone can alter the concentration of others, causing a cascade of issues that damage bone health60.

There are several methods of measuring bone density including: dual-energy x-ray absorptiometry (DXA), computed tomography (CT), peripheral quantitative computed tomography (pQCT), and quantitative ultrasound (QUS)63. The results from these measurements score an individual within normative data for age, gender, and ethnicity. These numbers are given as z-scores, numbers of standard deviations from the mean. Generally, a z- score falling below -1 is considered low bone density for age, according to the International

Society for Clinical Densitometry6,64,65.

Several studies have researched the relationship of bone development in sport, and have found that weight-bearing exercise has a positive effect on increasing bone density66,58.

However, in amenorrheic women who have hormonal imbalances, exercise may not provide the same benefit to bone. Barbara Drinkwater is one investigator who provided much of the seminal literature in this area of study. A study by Drinkwater et al compared bone mass at the 19 lumbar spine and the radius of fourteen amenorrheic athletes to that of fourteen eumenorrheic athletes67. Their findings showed that bone density at the lumbar spine was significantly lower in amenorrheic athletes67. They reported that the lumbar bone mass of the amenorrheic athletes was comparable to that of a 51.2 year old woman. It was suggested that this could be due to the significantly lower levels of estrogen in the amenorrheic athletes67.

The prevalence of low bone density in amenorrheic women has been estimated by reviews and may by up to 40% in exercising women2,68. Bone loss may also be greater in women who experience amenorrhea for longer periods of time. Davies et al showed that vertebral bone density was inversely related to the duration of a subjects amenorrhea65. The relationship of amenorrhea and bone density is well-demonstrated.

Given that osteoporosis is sometimes called a “silent disease” with the beginnings in adolescence, studies on amenorrhea during the growth years should be given special consideration. Amenorrheic athletes have been shown to have slower bone mineral accretion compared with eumenorrheic counterparts, specifically at the lumbar spine69. A study by Soyka et al used adolescents with AN to report the impact of chronic undernutrition on bone accrual.

Their findings showed that young females with AN had reduced bone mineral accrual over the year they were followed, compared to healthy counterparts70. Barrack et al also performed a study comparing the bone mineral accrual between adolescent runners and other non- endurance running sport participants. Their findings suggest that runners exhibited suppressed bone mineral accrual71. These studies demonstrate the risk of slower accretion and risk of not reaching peak bone mass in amenorrheic athletes.

20 Women with HPA/FHA, have been found to have lower bone density than other classifications of amenorrhea, and even lower bone density than eumenorrheic non-active women2,5. A review by De Souza and colleagues suggests a low BMD prevalence of 0-15% in exercising women (if z-score < -2) and 0-40% with BMD scores between -1.0 and -2.02. This prevalence is of concern as low BMD can lead to injury.

Fortunately, bone loss in the amenorrheic years may be reversible once the menstrual cycle is restored. Another study by Drinkwater et al measured athletes bone density while they were amenorrheic, then again after had regained cycles for 15.5 months72. They found an increase in bone density of 6.3% in the spine of the previously amenorrheic women, concluding that the resumption of menses can help restore bone mass72.

Low bone mass can increase risk for bone-related injury, such as stress fractures. Risk of injury in amenorrheic athletes has been well-explored. Rauh et al followed high school athletes for one year. They reported that athletes who have experienced oligo- or amenorrhea had a three-fold greater risk for injury related to bone73. Cromer et al also found stress fractures to be

2-4 times more common in female athletes with amenorrhea than normally menstruating athletes2. These injuries can unfortunately be how these athletes discover their low bone density and energy availability deficit.

Resting Metabolic Rate

Resting metabolic rate (RMR) is also compromised concomitant to amenorrhea. RMR is a measurement of whole-body metabolism reflecting the activities at the hypothalamic- pituitary-thyroid axis. It is an indicator of how many calories a person burns at rest and is also known as resting energy expenditure (REE). 21 An additional metabolic adaptation to conserve energy in the face of low energy availability induced amenorrhea is suppression of RMR. In a study performed by Koehler et al, they found RMR to be 8% lower than predicted in exercising women with amenorrhea74. A similar study showed RMR in amenorrheic runners compared to eumenorrheic runners to be

200 kilocalories lower75. The relationship between the ovarian and thyroid axis or at least a concomitant effect is confirmed with these studies.

A decreased resting metabolic rate may be a problem for long-term health as it can affect weight loss in the future76. A study by Brownell et al altered the diets of male rats, restricting calories and refeeding in a cycle to see how it affected weight regain77. Their findings showed that the cycling of decreased energy intake caused higher “food efficiency” leading to greater weight gain after having low energy availability77. Another study by Steen et al looked at the RMR of adolescent wrestlers who took part in weight cycling. They found that wrestlers who “cut weight” for matches had lower RMRs compared to wrestlers who did not cut.

It is possible that amenorrheic athletes may experience similar effects during their post- competitive years. Theoretically, if a decrease in resting metabolic rate persists, maintaining a healthy weight in later years may become more difficult. However, Weinsier et al found that though RMR was reduced in energy-restricted states, once adequate energy was provided again, RMR increased back to a normal, expected level78. At this time, there is a paucity of research on the influence of long-term energy deficiency on RMR once energy intake becomes sufficient.

22 Measurement Tools

Dual energy x-ray absorptiometry (DXA) is a method often chosen to measure body composition and bone density for many reasons. DXA is a non-invasive technique that has minimal operator variability and provides results for the whole body. The DXA machine also provides a much lower dose of radiation when compared to other bone assessment tools. A review by Toombs and colleagues suggests that the DXA is a convenient and minimal-risk tool for BMD and body composition79.

Since low bone density puts athletes at risk for bone injuries, estimating bone strength is another method to quantify the effects of amenorrhea on an athlete’s skeletal health.

Peripheral quantitative computed tomography (pQCT) provides a three-dimensional image that allows predictions of the mechanical properties of bone80. The pQCT also gives a picture of the cross-sectional area of bone and estimates the cross-sectional moment of inertia as an estimate of bone strength . The pQCT scan also allows visualization of the geometric bone properties and amount of cortical and trabecular bone82,83. Using a pQCT measurement, which better estimates bone strength, along with a DXA scan allows comparison and stronger estimates of bone health and fracture risk81.

RMR can be measured using various methods. The most accurate measurement uses a whole-body calorimeter, and is consider a direct method as it measures the heat given off by the body. There are very few direct calorimeters in the world. Thus, the most widely used method to measure RMR is through indirect calorimetry. This method measures the rate of oxygen consumption with or without carbon dioxide production by a subject and translates oxygen use into calories needed if one were to be at rest for a full day42. One brand of indirect 23 calorimetry analysis is the ReeVue Indirect Calorimeter84. A study by Henes et al tested the validity of the Reevue machine and found no significant difference between its measurements compared to gold-standard indirect calorimeters85.

Predicted Prevalence of Amenorrhea in Athletes

Menstrual disturbances have been reported to be present in 1-31% of high school and collegiate athletes who play a variety of sports36. More specifically, HPA is present in 6-43% of runners, which is a sport known to expend high calories and require a lean body type36. De

Souza et al estimated that 50% of exercising women experienced abnormal menstrual cycles compared to 4.2% of sedentary women36. In more detail, their study showed that 33.7% of the exercising women displayed amenorrhea. Given that amenorrhea leads to compromised bone health and higher risk for injury, this means that one in three female athletes may be at a higher risk for bone injury.

Diagnosis of Amenorrhea

Defining the evaluation of an athlete with amenorrhea depends on the managing physician. Klein and Poth discussed many factors for diagnosis of amenorrhea86. First, they suggest reviewing the physical characteristics such as breast development, presence of acne, hirsutism, and genital exam. They also suggest labs like pregnancy test and hormones including

LH, FSH, prolactin, and thyroid stimulating hormone (TSH). In other cases, where anatomical issues may be present but not visible from the outside, a pelvic ultrasound may be necessary86.

The practice guidelines published by the Endocrine Society also suggests obtaining diet history, eating disorder history, exercise history, and psychological tests assessing personality traits such as perfectionism along with the blood tests28. Clearly, diagnosing amenorrhea as well as 24 classifying the specific type can be difficult, and may require several rounds of testing that could still lead to inconclusive results.

There are other reasons for amenorrhea thus HPA may often be seen as a diagnosis of exclusion6. Other common diagnoses for amenorrhea which need to be excluded are polycystic ovary syndrome (PCOS), pituitary tumor, and Kallman syndrome. PCOS is a hormonal disorder, like HPA, that causes women to have infrequent or prolonged menses, which is why it can sometimes be confused for amenorrhea. One differentiating quality of PCOS is the presence of excess androgen hormone and small follicles on the ovaries, affecting the release of eggs87. The prevalence of PCOS is as high as 15-20%, but this number varies based on the criteria used for diagnosis88. Pituitary tumors, or a prolactinomas, produce high levels of prolactin, causing hyperprolactinemia. The prevalence of prolactinomas are 30 in every 100,000 women89. Finally,

Kallman syndrome is a form of hypogonadotrophic hypogonadism, meaning there is a lack of hormones present. This syndrome is characterized by delayed or absent puberty90. The National

Institute of Health predicts the prevalence of Kallman’s to be of 1 in every 120,000 females90.

These more serious diagnoses need to be excluded through an appropriate physician assessment.

Research on reproductive hormones is costly, and normative values coincide with the timing of the menstrual cycle. Research in female athletes to examine menstrual status will often rely on self-report questionnaires as the primary tool for gathering information.

Questionnaires have been found to be an easy tool for obtaining information from a large group of individuals. Online questionnaires also provide immediate response and allow for minimal participant error. 25 Whether HPA is formally diagnosed or self-reported, treatment is important to helping an athlete avoid injury. Unfortunately, all too often athletes fail to realize that amenorrhea is signaling an underlying issue and holds a higher risk for injury. Since dysfunction at the hypothalamic pituitary axis is multi-factorial and associated with stress, weight loss, excessive exercise, or any combination of the three, the treatment should be multi-faceted.

Treatment for Amenorrhea

The most successful treatment routines will likely rely on accurate assessment of habits.

Loucks et al concluded from the athletic and menstrual histories collected from amenorrheic and normally cycling athletic women that alterations in the hypothalamic-pituitary-ovarian axis endure as long as exercise habits with inadequate energy to support them continue32. There is not a definite treatment method for FHA other than trying to restore normal menses through optimal energy availability28. FHA is typically reversible with increased energy intake, decreased exercise, or both28. These strategies are often used to increase weight and allow for normal hormonal functioning.

There are case studies in the literature demonstrating these recovery strategies. A case by Hind in 2008 followed an athlete with amenorrhea for 6 years with 3 meetings per year. At baseline, the athlete (aged 21.7 years old) had been amenorrheic for 3 years, and measured a lumbar BMD z-score of -2.2, and a body fat percentage from the DXA of 5.9%. A recovery plan was put in place that consisted of cognitive behavioral theory, weight gain, improved dietary intake, and reduced training load. At the age of 25, the athlete was considered oligomenorrheic and at the age of 26, eumenorrheic. After six years, this athlete regained menstrual function,

BMD z-score improved to -0.6, and body fat to 23%. Within four months of regularly 26 menstruating, this athlete was pregnant. This case concluded that fertility and peak bone mass may not be fully jeopardized in former amenorrheic athletes91.

In some amenorrhea diagnoses, such as PCOS, the opposite treatments are prescribed.

As weight gain is typically seen preceding the clinical features of PCOS, weight loss may be prescribed by increased activity or decreased caloric intake88. The symptoms of PCOS are often measured with medicines such as spironolactone for hirsutism and metformin for insulin resistance. Other treatment methods may be used to regulate cycles and include birth control methods or hormone therapies28,1. It is important to realize that amenorrhea has different causes and, thus different treatments.

What We Do Not Know

Although the prevalence, causes, and impacts on bone health due to functional hypothalamic amenorrhea have been researched in-depth, the impact on fertility later in adulthood has not been adequately explored. More specifically, there have been no studies to date that have looked strictly at prior varsity collegiate athlete’s reproductive status, bone health, and resting energy expenditure. Nicola Rinaldi et al described how seemingly healthy eating and exercising can still lead to loss of the menstrual cycle. Her book entitled No Period,

Now What contains case stories from women who have had issues with fertility because of their history with amenorrhea, stating that 69% of 240 cases needed to use fertility treatments to get pregnant the first time. Her work also examined second pregnancies, where 62% conceived naturally92. Dr. Rinaldi’s work is part of the impetus for the current study.

Though it has been studied that bone density is likely able to be restored, more research is beneficial to find the prevalence of lingering low bone mass in previously amenorrheic 27 women. There is also little research on whether resting metabolic rate is restored to an optimal level in these women. None of these factors associated with amenorrhea have been measured together in one sample of participants.

28 Chapter 3

Methodology

Research Design

This current study was conceptualized as a large scale multi-institutional fertility questionnaire to be sent to the past female athletes from participating institutions. This pilot study was smaller in scope to pre-test the questionnaire with Ohio State alumnae. In addition to piloting the questionnaire, local participants were invited for a laboratory visit to collect objective data. The pilot study was approved by the Institutional Review Board (2018H0285).

Questionnaire participants agreed to participate at the beginning of the digital questionnaire, and lab visit volunteer participants signed a separate hardcopy consent/HIPAA form at the beginning of the lab visit.

This descriptive, cross-sectional, retrospective study was a pilot study at Ohio State

University with the potential to expand it nationwide in the future. This questionnaire was sent to participants through Alumni Services and was accessible in Qualtrics® and queried on sport and activity history, menstrual history, and fertility. At the end of the questionnaire, participants who were local to Columbus, OH were asked if they would like to participate in a local lab visit. The local lab visit provided further evaluations including iDXA for bone mineral density and body composition, and a resting metabolic rate measurement using REEVUE indirect calorimetry.

Recruitment and Description of Human Subjects

The sampling approach included prior female athletes from The Ohio State University provided from the database via Alumni Services. Alumni Services required that any research 29 distributed widely to alumni has to be sent through an email from their office. The email invitation (Appendix A) contained a link to the anonymous questionnaire where subjects provided historical information about activity, menstrual status, pregnancy, and fertility issues.

The last section of the questionnaire invited subjects for a lab visit for further testing of bone density, body composition, and resting metabolic rate.

Exclusion Criteria

Female subjects participating in the local lab visit would have been excluded from that subset if they were found to be pregnant (according to a urine hCG dipstick) at the time if the

DXA scans. No one was disqualified from this study.

Study Questionnaire

The questionnaire was developed by experts in the fields of sports nutrition and fertility.

The main focus was to determine the prevalence of amenorrhea in prior female athletes and describe the impact on later fertility. More specifically, the questionnaire queried subjects on the following information:

• Demographics of the participant

• Age of sports participation, type of sport played, hours per week participating in sport,

and any extra physical activity performed

• Menstrual history, including irregularities and duration, diagnoses, and any treatments

provided by a physician

• Fertility and pregnancy history

• Use of birth control

30 Face Validity

Before the survey was sent out to the target audience, it was tested for face validity.

The survey was distributed to one prior collegiate athlete from each decade, starting in the 20s and up through the 50s, who is now a nutrition professional. Feedback from these athletes was taken into consideration and the survey was adjusted as needed. This field of fertility in prior athletes has not been well-established, thus there is not a widely-accepted and validated tool to gather this information.

Laboratory Visit

Questionnaire participants willing to come for a laboratory visit were coordinated for the lab. Participants were scheduled in the morning before eleven to help prevent eating and exercising prior. Upon arrival, they were consented for the lab visit and filled out the iDXA intake form. Participants provided a small urine sample to ensure they were not pregnant, and the urine was examined using the refractometer to document hydration status.

Anthropometrics and Bone Density

Trained researchers collected height and weight on a standardized Health-O-Meter digital scale according to standard protocols. Once height and weight were recorded, participants were scanned on the DXA for multi-compartment body composition. This study used a GE Lunar iDXA system paired with the enCORE™ software (version 15, service pack 2)93.

The protocol included: total body scan for total body bone density and body composition, precise mode lumbar spine scan, precise mode hip scan of the non-dominant hip, and standard radial scan of the non-dominant forearm. According to the Duke radiation dose calculator based on a Hologic QDR 4500 DXA system (not GE Lunar iDXA), the subjects would receive 31 about 28 mrem of radiation, but the estimated radiation for the GE iDXA system is considerably less at about 1.4 mrem. As pasted from the Duke website94:

Scan Duke estimation94 GE Lunar iDXA

Total body DXA 3 mrem 0.096 mrem

Spine DXA 10 mrem 0.677 mrem

Hip DXA 10 mrem 0.615 mrem

Wrist DXA 5 mrem 0.006 mrem

Total 28 mrem 1.394 mrem

Table 1. Comparison of Duke radiation estimator to GE Lunar iDXA actual radiation

Resting Metabolic Rate

A REEVUE indirect calorimetry machine was used to obtain a resting metabolic rate. This machine measures oxygen extracted from the ambient air to estimate resting energy expenditure. The results of the RMR are most accurate early in the day and prior to eating, exercise, or stimulant intakes such as caffeine. Subjects were instructed to arrive fasted of food and drinks other than water for at least 4 hours. They were also instructed to not have exercised the morning before the visit. RMRs were estimated with the subject lying supine on the iDXA or clinic table.

Lean Versus Non-Lean Sports

After the lab visits, participants were categorized into lean versus non-lean sport groups to find the prevalence of each in our study. Lean sports are often defined as those in which leanness directly affects performance and success95. These were classified in line with previous

32 studies done by colleagues where lean sports were considered: swimming, synchronized swimming, cross country, rowing/crew, track, and gymnastics95,96.

Statistical Analysis

Data were collated and cleaned from the questionnaire export, iDXA export, and manual entry of the RMR as well as measured height and weight into an Excel spreadsheet. Data were then imported into IBM Statistical Package for the Social Sciences (SPSS) (version 25) for analysis97. Frequency and means analyses were used to describe the demographics of each sample (questionnaire participants and lab visit participants).

The data set for the full questionnaire respondent group was examined for frequency of sports participated in, age, menstrual and fertility issues. Subjects were classified as amenorrheic if they reported an occurrence of 3 or more months without a menstrual cycle for high school or college. The questionnaire asked if the amenorrhea was due to birth control, and those particular subjects were excluded in comparisons between eumenorrheic and amenorrheic groups. Eumenorrheic participants did not report amenorrhea during high school or college. Frequencies were tallied and T-test was used to compare the simple groups.

Participants who had tried to get pregnant were sorted by menstrual status, and the Chi-Square test was used to compare the frequency of fertility issues.

In addition to the above evaluation, a smaller dataset for the local lab visit was examined for the average and range of bone density by skeletal site, RMR, and body composition. T-tests were used to find BMD issues associated with the history of amenorrhea.

Lab participants were grouped according to history for amenorrhea (“yes” or “no”) and group means for Z-scores for all skeletal sites, estimated RMR, measured RMR, percent of Harris- 33 Benedict-estimated RMR, age, and percent body fat were compared using an independent sample two-tailed t-test. The assumption of underlying equality of variances was examined using Levene’s statistic. If Levene’s statistic was p<0.20, variances were considered unequal, and the non-parametric Mann Whitney U statistic was calculated. Statistical significance for all comparisons was defined where p<0.05.

34 Chapter 4

Results

A total of 298 participants consented and completed the full survey. It is estimated that the questionnaire invitation was sent to 1936 athlete alumnae, thus an approximate response rate of 15%. The participant details are contained in Table 1. Average age of participants was 38

+ 11.75 years, with a range of 21 to 60 years. The average self-reported weight of subjects was

155.5 + 29.85 pounds with self-reported height 66.5 + 2.92 inches. Ethnicity of subjects is shown in Table 2. The majority of participants (91.9%) identified most closely with white. Those who reported other as their ethnicity were bi- or multi-racial according to the write-in text boxes.

Mean Std Dev Min Max Age 38.5 11.75 21 60

Self-Reported 66.51 2.92 60 76 Height (in) Self-Reported 155.55 29.852 100 280 Weight (lbs) Table 2. Survey Participant Descriptive Data

Race Count Percent White 274 91.9% Black or African 14 4.7% American Asian 3 1.0% Hispanic 3 1.0% Other 4 1.3% Table 3. Ethnicity of Participants

35 Amenorrhea and Fertility

The first menstrual status analysis included the larger questionnaire group. Amenorrhea was classified as 3 or more months without a bleed that was not due to birth control. Seventy- eight participants reported a history of amenorrhea. The prevalence in this sample was 26.7%, showing more than one-quarter of participants experienced amenorrhea. Some respondents

(n=32) were not included in the amenorrheic history group due to reporting use of birth control.

Of those 78 reporting a history of amenorrhea, 44 reported trying to conceive. Thirty- nine of these participants were successful, and 5 (11.36%) were not. Of the eumenorrheic participants, 114 reported trying to conceive, and 11 (9.65%) were not successful. The Chi

Square between groups was ᵡ2=0.103 with p=0.749. There was no difference in the frequency of fertility issues between eumenorrheic and amenorrheic subjects in the alumnae from the larger questionnaire.

Lab Visit

A total of 44 participants signed up for the local lab visit for iDXA and ReeVue measurements. The average age of the lab visit subjects was 33.7 + 10.94 years with height 66.4

+ 2.37 inches, 160.5 + 33.30 pounds, and 30.6% + 8.69% body fat (Table 3). The lab visit group was slightly younger than the overall questionnaire participants. Lab participant’s hydration was measured as urine specific gravity (USG) is known to affect DXA results98. USG of participants was 1.020 + 0.010.

36

Mean Std Dev Min Max Age 33.7 10.94 21 60

Height (in) 66.4 2.37 60.5 70.6

Weight (lbs) 160.5 33.30 100.1 257.7

Region 30.6% 8.69% 15.4% 47.3% Percent Fat*

USG for* 1.020 0.010 1.000 1.040

Table 4. Lab Participant Descriptive Data

Lab visit participants were grouped according to menstrual status identical to the questionnaire participants. Fifteen of the 44 total lab visit participants (34.1%) were considered amenorrheic. Data for lab measures are reported for the 44 subjects then compared by menstrual status.

Bone Density

Bone density scores were also compared between participants with eumenorrheic and amenorrheic histories. None of the differences in Z-scores for the various skeletal sites for bone density were found to be significantly different. The details of these comparisons are outlined in Table 4. Total body z-score showed a mean difference of -0.54 + 0.332 between amenorrheic and eumenorrheic women (p = 0.11). Differences in femoral neck, L1-L4 spine, and radius UD z- scores were -0.109 + 0.304 (0.73), -0.38 + 0.502 (0.46), and -0.51 + 0.472 (0.28) respectively.

The p-value of the total body differences (p=0.109) may be considered a trend by some, thus a

37 power analysis was performed99. A power calculation using the means and pooled standard deviation demonstrated a power of 54.1% with 76 subjects required for a power value of 80%.

Amenorrhea Eumenorrhea Mean difference + t-score p-value std error Total Body Z- 1.46 + 1.04 2.00 + 1.01 -0.54 + 0.332 -1.64 0.109 Score Neck Z-Score 0.50 + 1.08 0.61 + 0.85 -0.11 + 0.304 -0.35 0.725

L1-L4 Z-Score* 0.68 + 1.80 1.06 + 0.96 -0.38 + 0.502 -0.76 0.457

Radius UD Z- -0.71 + 1.49 -0.19 + 1.43 -0.52 + 0.472 -1.10 0.279 Score Table 5. Bone Density Measures *equal variances not assumed using Levene’s Test for Equality of Variances, p-value reported for Mann Whitney U test

The prevalence of low BMD for age in the lab visit cohort using z-scores for each skeletal site is shown in Table 5 which includes all lab participants, not separated by menstrual status.

The highest prevalence of low BMD was shown in the radius. The only site for any participant that scored below -2 was also at the radius with the lowest z-score being -2.4 (n=2). When the low bone mass for age subjects are divided by menstrual group there do not appear to be any differences in frequency of low bone mass, and the frequencies are displayed in Table 6.

38 Site Low BMD

Total Body Z-Score 0

Neck Z-Score 3

L1-L4 Z-Score 2

Radius UD Z-Score 15

Table 6. Frequency of Low BMD for age

Amenorrheic Eumenorrheic

n=15 n=26

Total Hip 1 0

Femoral Neck 2 1

Lumbar Spine 1 0

Distal radius 6 8

Table 7. Frequency of Low Bone Mass by Skeletal Z-score for Amenorrhea and Eumenorrheic Participants (3 subjects excluded due to OCP amenorrhea)

Resting Metabolic Rate

The RMR of participants is shown in Table 7. RMR results using the ReeVue measurement were found to be 1565 + 248 kcals/day with a minimum of 1109 and maximum of 2160. This was compared to the estimated RMR of subjects, using the Harris-Benedict

39 prediction equation which was 1507 + 149 kcals/day with a minimum of 1262 and a maximum of 1945. The average percent difference in actual RMR to estimated RMR was 103.7% higher than prediction-estimated RMR.

Mean Std Dev Min Max RMR (kcal) 1565 248 1109 2160 Estimated RMR 1507 149 1262 1945 (kcal) Difference 58 99 -153 215 Table 8. Resting Metabolic Rate Values *rounded to the nearest whole number

Amenorrhea Eumenorrhea Mean difference + t-score p-value std error RMR* 1589 + 309 1547 + 224 41.34 + 91.107 0.50 0.654 Percent of 1.05 + 0.129 1.02 + 0.10 0.03 + 0.038 0.66 0.518 Predicted RMR Table 9. Differences in Resting Metabolic Rate Based on Menstrual History *rounded to the nearest whole number

The mean difference in RMR of those with a history of amenorrhea to those without was compared using a T-test. The RMR in prior amenorrheic athletes was an insignificant difference of 41.34 + 91.107 kcals higher than eumenorrheic history athletes (p = 0.654). Table

8 demonstrates the RMR details between groups. Similarly, when the menstrual history groups

40 were compared for the percentage of actual to equation-estimated RMR values, the 3% difference was not significantly significant.

Sport Frequency

The study did not limit the lab visit to any particular group of people, but it was apparent throughout the study that we may have experienced some recruiting bias. Varsity sport frequency was reported for the larger sample. The sports with the largest numbers for participation were swimming (14%), track and field (13.4%), and rowing/crew (10.7%). The remainder of the counts for sport participation are listed in Table 8.

The questionnaire sport count was compared to the counts for sports played by lab visit participants. The sports with largest numbers in this group were swimming (20.5%), rowing/crew (15.9%), and cross country (13.6%). These results are also reported in Table 9 for ease of comparison.

41 Survey Lab Visit Sport Frequency Percent Frequency Percent Cross Country* 26 8.7% 6 13.6% Track and Field* 40 13.4% 5 11.4% Soccer 15 5% 2 4.5% Field Hockey 13 4.3% 3 6.8% Lacrosse 14 4.7% 3 6.8% Swimming* 42 14% 9 20.5% Tennis 11 3.7% Volleyball 9 3% Softball 20 6.7% 2 4.5% Basketball 7 2.3% Gymnastics* 23 7.7% 1 2.3% Rowing/Crew* 32 10.7% 7 15.9% Pistol and Rifle 1 0.3% Synchronized 26 8.7% 5 11.4% Swimming* Fencing 7 2.3% Spirit 1 0.3% Other 22 7.4% 4 9.1% Table 10. College Sport Frequency *Denotes those sports considered lean-sports

Lean athletes who completed the survey and/or lab visit are denoted with * in Table 8.

For the survey, a total of 189 (63.4%) athletes competed in lean sports. For the lab visit, a total of 33 (75%) of the participants were involved in lean sports in college.

42 Discussion

The prevalence of amenorrhea in this study was similar to other investigations. The current study found that 26.7% of women reported amenorrhea at some point in their high school or college athletic career. Previous studies predicted about one-third of athletic women experience this level of menstrual disorder, which is in line with the current findings36.

The number of women with an amenorrheic history could potentially be higher in this study as those who reported amenorrhea and hormonal birth control use were not included in the analysis of amenorrheic and eumenorrheic histories. Some women may have stopped their cycles via birth control, but still had an energy imbalance that would not have allowed for cycles even without contraception. Additionally, we did not ask “eumenorrheic” participants who did not report amenorrhea if the reason they had a normal cycle was due to birth control. In either of these cases, we may have excluded or erroneously categorized the participants.

Alternatively, if the study invitation appeared to only be useful for alumni athletes who experienced amenorrhea, the study may have under-recruited eumenorrheic alumni, thus underestimating eumenorrheic subjects.

The primary objective of this study was to compare the fertility issues in past athletes with a history of amenorrhea to those with a history of eumenorrhea. Comparison between amenorrheic and eumenorrheic alumni athletes showed that there was not a significant difference in prevalence of fertility issues between the groups. These findings are comparable to those found in Hind et al who also suggested that fertility may not be completely compromised in athletes with a history of amenorrhea91. However, severity of menstrual

43 dysfunction was also not assessed, and may have impacted those who reported or measured as currently having the risks factors we studied associated with amenorrhea.

There are other factors that could be present in the participants that are influencing fertility both in the amenorrheic or eumenorrheic group. Although not assessed, participants may have had other health concerns such as endometriosis, fibroid tumors, or ovarian cysts.

This survey included an open comment box where participants could provide additional information in which some of the mentioned health concerns were present and may have affected the prevalence of fertility issues in either group.

These findings are similar to those in the ED literature. Though we did not look into participants who reported still experiencing amenorrhea and if they were the population experiencing fertility issues, it would not be surprising to find that as the case. It could be explained that those women who had experienced amenorrhea regained cycles and were subsequently successful with conception, similar to the findings in work by Shomento et al.

There were no significant differences in the current bone mass values for amenorrheic compared with eumenorrheic participants. Most participants were in the healthy range for bone density at all sites, suggesting no identifiable higher risk for osteoporosis later in life for amenorrheic athletes in this study. These findings compare to those by Hind et al showing that once menses are resumed, bone mass begins to increase again. However, Keen & Drinkwater reported that former oligo- and amenorrheic athletes exhibited significantly lower bone mass at the lumbar spine than athletes who had always had regular menstrual cycles100. It should be noted that athletes with a history of amenorrhea were the only group who presented low bone mass for total hip and lumbar spine. 44 The lowest z-scores shown in the iDXA data were at the distal radius. This is not surprising as this is an area of measurement that gets less impact than the spine and femoral neck. Impact loading is likely the cause for increased bone density thus fewer inadequacies at the more loaded sites. The prevalence of low BMD and osteoporosis for each site were not compared for those with a history of amenorrhea to those without, though this should be examined in the future. Severity of menstrual issues was also not examined in relation to bone density and it is possible that length of time with menstrual dysfunction could influence outcomes.

Resting metabolic rates were also not significantly different between menstrual groups.

Somewhat surprisingly, RMRs were slightly higher in the group reporting amenorrhea. There are likely a number of factors that could be analyzed or considered to explore these differences such as length of amenorrhea, sports played, and history of body weight. RMR may have also been altered or increased if the participant had inadvertently eaten, exercised, or consumed a beverage other than water before the visit. While the lab visit instructions clearly outlined the protocol, it is difficult to ensure the guidelines were followed. The lab staff did ask participants if they had refrained from eating, drinking, and exercising, and it was apparent that a few subjects did not follow the protocol.

This was one of the first studies to investigate RMR in prior athletes who experienced amenorrhea. Therefore, it is difficult to conclude whether there are long-term effects of amenorrhea on RMR. It is still important to educate athletes as they are about to enter their post-competitive years, especially those who are still experiencing amenorrhea, on the effects that a decreased RMR may have on changes in weight. 45 Given that the lab visit participants seemed to be dominated by swimmers and crew athletes, the research team was also compelled to examine the frequency of sport for the questionnaire and lab visit groups. Interestingly enough, those who participated in sports where thinness is often required and/or body weight is a factor in performance were those who came to the lab visit. This could be due to experiences in their collegiate careers where they needed to be thin to perform. It would also be interesting to know if these athletes feel a continued anxiety that led them to volunteer for the lab visit. Regardless, about 75% of the lab visit volunteers were from lean sports.

It was also interesting to see that the majority (63.4%) of survey participants were also those involved in lean sports in college. This is possibly because the leanness needed for their sport may have led to a higher prevalence of amenorrhea in these groups, causing them to be more apt to complete the survey and assist in the research for future athletes. Although the prevalence of amenorrhea in each reported sport in this study was not assessed, it would not be surprising to find a higher prevalence in lean sports.

Limitations

A limitation of the survey was that the response rate was only 15%. This survey was not tested for reliability or validity and the length of the survey could have caused respondent fatigue leading to misreported data. The survey also relied on self-report data that required participants to think back many years into their menstrual history, which may lead to over or under reporting. The statistical analysis assumes that participant memory was accurate and the athletes with differing menstrual histories were similarly attracted to fill out the questionnaire.

46 There was a small number of participants who volunteered for the lab visit and when that was limited to those who were eumenorrheic (n=26) and those who were amenorrheic

(n=15) there were a total of 41 participants. Further examination of the total body bone density trend for a lower Z-score in the amenorrheic subjects demonstrated that the study would have needed seventy-six subjects to attain a power value of 80%. Thus, it is possible the indifferences between the amenorrheic vs eumenorrheic subjects wer based on a lack of power for the study.

A limitation of the RMR measurement was that the ReeVue machine only measures oxygen consumption. Since this method does not also measure carbon dioxide expiration, it may have altered measurements. However, this was the machine available at the facility for use and is believed to be comparable to other measurements85.

An additional limitation was the relatively young age of participants in both the survey and the lab visit. Having participants of a younger age could be the reason why the number of respondents who had tried for conception was less than half. The respondents who had not tried for conception may have issues with fertility, but have not experienced them yet.

The imbalance between lean and non-lean sports was unanticipated, and it may have been of benefit or interest to recruit according to sport played to allow for a wider comparison.

Additionally, the cross-sectional nature of the study precludes drawing conclusions about the changes that may have happened over time for the lab participants. It may also be meaningful to look at the frequency and duration of amenorrhea to provide a marker of severity as it may have influenced infertility and bone density.

47 For the nation-wide study, the questionnaire will need to be simplified so that it is shorter and does not cause respondent fatigue. The larger study will also look into severity of menstrual disturbances as well as any additional reasons for fertility issues. Along with this, it will examine the prevalence of amenorrhea in lean sport populations.

Conclusions

There were no significant differences between past amenorrheic athletes from eumenorrheic athletes in this cohort for fertility issues, bone density measurements, or RMRs to demonstrate that a history of amenorrhea puts one at higher risk for other adverse health outcomes later in life. This study did not address any of these risks in the student athletes while their amenorrhea was occurring, which is when many of these issues may present most acutely.

Implications for Practice

While this study failed to show a higher risk for lingering bone or metabolic damage for amenorrheic athletes, it is better to error on the side of caution until more research is completed. The prevalence of amenorrhea in our study is of concern and should be addressed through further education of athletes at all ages and levels to prevent any associated risks.

Though our findings may be considered positive as there is no higher prevalence of any issues with fertility, bone density, or RMR later in life, these risks should still be of focus at the time of insult. Many young women are not aware of the other risks that accompany amenorrhea and need to be informed. The field of fertility following athletic amenorrhea still requires more research. A larger sample size may show different results while also looking at the severity of menstrual issues.

48 Athletes should also be educated on the transition after college to their post- competitive years. Specifically, athletes who currently are or have experienced amenorrhea need to be told the importance of restoring and maintaining their menses. Many athletes think that after they are done playing a competitive sport, they need to drastically decrease their energy intake. Although a decrease in energy intake may be acceptable, lowering intake too much may keep them in a low energy availability state. Restoring menses post-competitive years is also important as there is still time for these athletes to accrue bone if they had low bone mass for age while in a low EA state. These athletes also need to be informed that some form of weight-bearing exercise along with adequate nutritional intake is needed to build and maintain their bone mass.

Finally, it is important for athletes leaving collegiate athletics to be educated on healthy weight control strategies. As mentioned before, amenorrheic athletes should try to restore their menstrual cycle post-sport if they were unable to during, but it is also important to educate all athletes on how the decrease in activity may make it difficult for them to maintain their current weight. Providing resources for these athletes may be beneficial in keeping them in a healthy weight range.

49 Chapter 5

Bone Mass and Resting Metabolic Rate in Prior Female Athletes with a History of Amenorrhea

Introduction

The female menstrual cycle is a complex relationship between hormonal signals, energy availability, and reproductive organs. When there is low energy availability present to support normal functions, the body downregulates functions that are not necessary for immediate survival, such as the menstrual cycle. Low energy availability can be due to inadequate nutritional intake, excess exercise, or a combination of both.

With increased participation in collegiate athletics since the inception of Title IX, the number of female athletes has increased. This increase in participation likely leads to an increased number of women with inadequate fueling (Title IX). This issue can lead to a cascade of problems due to the shift in hormones such as decreased bone density and suppressed resting metabolic rate (RMR)2,4,6,.

Menstrual function is described as the continuum of eumenorrhea, or normal menstrual cycles lasting 21-35 days in length, to amenorrhea, or the absence of menses for 3 or more months at a time8. There are several causes of amenorrhea such as pregnancy, contraceptives, stress, and energy imbalance101. Energy deficits are often caused by inadequate nutritional intake to support activity or excessive exercise that exceeds the number of calories consumed.

When there is a significant energy imbalance, it causes a shift in hormones that signal the hypothalamus to decrease gonadotropin releasing hormone (GnRH), which then affects the release of a cascade of hormones including decreased estrogen, altering the menstrual cycle102,1. When amenorrhea is present due to changes in these hormones, it is termed 50 hypothalamic amenorrhea103,27. This type of amenorrhea is often seen in collegiate female athletes as their activity level is high and often not met with adequate calories46.

The risks associated with these alterations in hormones include, but are not limited to, decreased bone density and suppressed RMR2. Although weight-bearing exercise is known to have a positive effect on bone density, when there is inadequate energy available exercise may not provide benefit to bone as bone building is not prioritized58. Decreased estrogen availability also affects bone density in amenorrheic athletes104. When estrogen levels are low, it stimulates osteoclast activity and without upregulating osteoblast activity, yielding a net bone loss60. De

Souza and colleagues have shown bone density to have a prevalence of 0-15% in exercising women, showing it is a problem in this population2.

RMR is often suppressed in amenorrhea as well. This is done to decrease the amount of fuel needed for the day74. Studies have shown that RMR may be 8% lower than predicted in exercising women with amenorrhea74. Less is known if the metabolic suppression continues into adulthood.

Though these measurements have been studied in women with acute amenorrhea, they have not been well explored in an older population. It is important to know if these risks continue into post-competitive years. The purpose of the current study was to investigate if there is any evidence of long-term bone or metabolic damage in athletic women who have experienced amenorrhea.

Objective

Compare the prevalence of low bone density and low resting metabolic rate in prior athletes with a history of amenorrhea to those without a history of amenorrhea. 51 Methods

Research design and Recruitment

This descriptive, cross-sectional, retrospective study is a pilot study at a single institution. The questionnaire was developed by experts in the fields of sports nutrition and fertility and sent to prior varsity athletes via email invitation through Alumni Services. At the end of the questionnaire, participants who wished to come for further measurements were asked if they would like to participate in a lab visit including iDXA for bone mineral density and body composition and a resting metabolic rate measurement using REEVUE indirect calorimetry.

Inclusion Criteria

All prior female varsity athletes from Ohio State University according to the database of

Alumni Services were invited to participate in the questionnaire. All questionnaire respondents were invited to participate in the local lab visit.

Exclusion Criteria

Female subjects would have been excluded from that subset if they were found to be pregnant (according to a urine dipstick) at the time if the DXA scans.

Study Questionnaire

The questionnaire, created in Qualtrics, queried subjects on the following information:

• Demographics of the participant

• Age of sports participation, type of sport played, hours per week participating in sport,

and any extra physical activity performed

52 • Menstrual history, including irregularities and their duration, diagnoses, and any

treatments provided by a physician

• Fertility and pregnancy history

• Use of birth control

Alumnae who volunteered for the laboratory visit were coordinated and instructed for the

lab visit via email. They were instructed to wear non-metal clothing, be normally hydrated,

and refrain from eating, drinking, or exercising the morning of the visit.

Laboratory Visit

The first step in the laboratory visit was to review and sign the informed consent and iDXA intake form. Participants then provided a urine sample which was tested for pregnancy as well as hydration using hCG dipsticks and refractometer. Height and weight were recorded using standard methods on a calibrated stadiometer scale.

Bone Density

After anthropometrics were recorded and urine was tested, subjects were scanned for total body, non-dominant hip, lumbar spine, and non-dominant radius. These measurements were taken on the GE Lunar iDXA machine using enCORE™ software (version 15 service pack

2)93. Finally, subjects either remained supine on the iDXA table for the RMR measurement or walked a short distance across the hall where they laid down and the measurement was taken following ReeVue protocols.

Resting Metabolic Rate

A REEVUE indirect calorimetry machine was used to obtain a resting metabolic rate. This machine measures oxygen extracted from the ambient air to estimate the number of calories 53 needed at rest, or resting energy expenditure. The results of the RMR are most accurate early in the day and prior to eating or exercise or stimulant intakes such as caffeine. Subjects were instructed to come fasted of food and drinks other than water for at least 4 hours. They were also instructed to not have exercised before the visit.

Statistical Analysis

Data were collated from the questionnaire to find history of amenorrhea. iDXA results were exported and RMR, and measured height and weight were manually entered into an Excel spreadsheet. Data were imported into IBM Statistical Package for the Social Sciences (SPSS)

(version 25) for analysis97. Frequency and means analyses were used to describe the demographics of each sample (questionnaire participants and lab visit participants.

The data was examined for the average and range of bone density by skeletal site, RMR, and body composition. Simple T-test comparisons were produced to look for evidence of bone mass issues associated with the history of amenorrhea. Frequencies for BMD scores were also tallied.

Participants were grouped according to history for amenorrhea (yes or no) and group means for Z-scores for all skeletal sites, estimated RMR, measured RMR, percent of estimated

RMR, age, and percent body fat were compared using an independent sample two-tailed t-test after assessment for equality of variances using Levene’s statistic. If Levene’s statistic was p<0.20, variances were considered unequal, and the non-parametric Mann Whitney U statistic was calculated. Statistical significance for all correlations and comparisons was defined where p<0.05

54 Results

A total of 44 participants signed up for the local lab visit for iDXA and ReeVue measurements. The average age of the lab visit subjects was 33 + 10.9 years with height 66.4 +

2.37 inches, 160.5 + 33.30 pounds, and 30.6% + 8.69% body fat. Descriptive statistics are displayed in Table 1. Hydration of subjects who participated in the lab visit was measured via a urine refractometer. Participant’s hydration measured as urine specific gravity (USG) is known to affect DXA results98. USG of subjects was 1.02 + 0.01. Prevalence of amenorrhea in the lab visit was 34.1% with 15 reporting history and 29 without.

Mean Std Dev Min Max Age 33.7 10.94 21 60

Height (in) 66.4 2.37 60.5 70.6

Weight (lbs) 160.5 33.30 100.07 257.68

Region 30.6% 8.69% 15.4% 47.3% Percent Fat*

USG* 1.020 0.010 1.000 1.040

Table 11. Lab Participant Descriptive Data

55 Resting Metabolic Rate

The RMR of participants is shown in Table 2. RMR results using the ReeVue measurement were found to be 1565 + 248 kcals/day with a minimum of 1109 and maximum of 2160. This was compared to the estimated RMR of subjects, using the Harris-Benedict prediction equation which was 1507 + 149 kcals/day with a minimum of 1262 and a maximum of 1945. The average percent difference in actual RMR to estimated RMR was 103.7% higher than prediction-estimated RMR.

Mean Std Dev Min Max RMR (kcal) 1565 248 1109 2160 Estimated RMR 1507 149 1262 1945 (kcal) Difference 58 99 -153 215 Table 12. RMR Values *rounded to the nearest whole number

The mean difference in RMR of those with a history of amenorrhea to those without was compared using a T-test. The RMR in prior amenorrheic athletes was an insignificant difference of 41.34 + 91.107 kcals higher than eumenorrheic history athletes (p = 0.654). Table

3 demonstrates the RMR details between groups. Similarly, when the menstrual history groups were compared for the percentage of actual to equation-estimated RMR values, the 3% difference was not significantly significant.

56

Amenorrhea Eumenorrhea Mean difference + t-score p-value std error RMR* 1589 + 309 1547 + 224 41.34 + 91.107 0.50 0.654 Percent of 1.05 + 0.129 1.02 + 0.10 0.03 + 0.038 0.66 0.518 Predicted RMR Table 13. Differences in RMR Based on Menstrual History *rounded to the nearest whole number

Bone Density

Bone density scores were also compared between participants with eumenorrheic and amenorrheic histories. None of the differences in Z-scores for the various skeletal sites for bone density were found to be significantly different. The details of these comparisons are outlined in Table 4. Total body z-score showed a mean difference of -0.54 + 0.332 between amenorrheic and eumenorrheic women (p = 0.11). Differences in femoral neck, L1-L4 spine, and radius UD z- scores were -0.109 + 0.304 (0.73), -0.38 + 0.502 (0.46), and -0.51 + 0.472 (0.28) respectively.

The p-value of the total body differences (p=0.109) may be considered a trend by some, thus a power analysis was performed99. A power calculation using the means and pooled standard deviation demonstrated a power of 54.1% with 76 subjects required for a power value of 80%.

57

Amenorrhea Eumenorrhea Mean difference + t-score p-value std error Total Body Z- 1.46 + 1.04 2.00 + 1.01 -0.54 + 0.332 -1.64 0.11 Score Neck Z-Score 0.50 + 1.08 0.61 + 0.85 -0.11 + 0.304 -0.35 0.73

L1-L4 Z-Score* 0.68 + 1.80 1.06 + 0.96 -0.38 + 0.502 -0.76 0.46

Radius UD Z- -0.71 + 1.49 -0.19 + 1.43 -0.52 + 0.472 -1.10 0.28 Score Table 14. Bone Density Measures *equal variances not assumed using Levene’s Test for Equality of Variances

The prevalence of low BMD and osteoporosis in this lab visit cohort using z-scores for each skeletal site is shown in Table 5. The highest prevalence of low BMD was shown in the radius. The only site for any participant that scored below -2 was also at the radius with the lowest z-score being -2.4 (n=2). There do not appear to be any differences in frequency of low bone mass.

58 Amenorrheic Eumenorrheic

n=15 n=26

Total Hip 1 0

Femoral Neck 2 1

Lumbar Spine 1 0

Distal radius 6 8

Table 15. Frequency of Low Bone Mass by Skeletal Z-score for Amenorrhea and Eumenorrhea Participants (3 subjects excluded due to OCP amenorrhea)

Discussion

The prevalence of amenorrhea in this study was similar to other investigations. The current study found that 34.1% of women experienced amenorrhea at some point in their high school or college athletic career. The number of women with an amenorrheic history could potentially be higher in this study as those who reported amenorrhea and hormonal birth control use were not included in the analysis of amenorrheic and eumenorrheic histories. Some women may have stopped their cycles via birth control, but still had an energy imbalance that would not have allowed for cycles even without contraception.

Bone density measurements between menstrual history groups were not found to be significantly different. Most participants were in the healthy range (greater than -1 Z-score) for their bone density at all sites, failing to demonstrate a higher risk for bone healthy issues later in life for amenorrheic athletes.

59 The lowest z-scores shown in the iDXA data were at the distal radius. This is not surprising as this is an area of measurement that gets less impact than the spine and femoral neck. Impact loading is likely the cause for increased bone density thus fewer inadequacies at the more loaded sites. The prevalence of low BMD and osteoporosis for each site were not compared for those with a history of amenorrhea to those without, though this should be examined in the future. Severity of menstrual issues was also not examined in relation to bone density.

Resting metabolic rates were also not significantly different between menstrual groups.

Somewhat surprisingly, RMRs were slightly higher in the group reporting amenorrhea. There are likely a number of factors that could be analyzed or considered to explore these differences such as length of amenorrhea, sports played, and history of body weight. RMR may have also been altered or increased if the participant had inadvertently eaten, exercised, or consumed a beverage other than water before the visit. While the lab visit instructions clearly outlined the protocol, it is difficult to ensure the guidelines were followed. Lab staff did ask participants if they had refrained from eating, drinking and exercising, and it was apparent that a few subjects did not follow the protocol.

Limitations

All studies have limitations that are important to examine. A limitation of menstrual status history was that it relied on self-report data that required participants to think back many years into their menstrual history. This could mean that instances of amenorrhea were over or under reported. The statistical analysis assumes that participant memory was accurate.

Additionally, the cross-sectional nature of the study precludes drawing conclusions about the 60 changes that may have happened over time for the lab participants. It may also be meaningful to look at the frequency of amenorrhea to provide a marker of severity.

Conclusions

There were no significant differences between past amenorrheic athletes from eumenorrheic athletes for bone density measurements or RMRs to demonstrate that a history of amenorrhea puts one at higher risk for other adverse health outcomes later in life. This study did not address any of these risks acutely in the student athletes while their amenorrhea was occurring.

Implications for Practice

While this study failed to show a higher risk for lingering bone or metabolic damage for amenorrheic athletes, it is better to error on the side of caution until more research is completed. The prevalence of a history amenorrhea of those who participated is of concern.

Though our findings may be considered positive as there is no higher prevalence of any issues with fertility, bone density, or RMR later in life, these risks should still be of focus at the time of insult. Many young women are not aware of the other risks that accompany amenorrhea and need to be informed.

Athletes entering their post-competitive years should be educated on the importance of maintaining a healthy weight and restoring menses if they experience amenorrhea. The importance of weight-bearing exercise along with adequate nutritional intake for bone and menstrual health is of importance. Athletes may decrease energy intake too much due to a decrease in activity, or may stop activity and not optimize bone accrual. Education and resources should be provided for these athletes. 61 References

1. Fourman LT, Fazeli PK. Neuroendocrine Causes of Amenorrhea—An Update. J Clin Endocrinol Metab. 2015;100(3):812-824. doi:10.1210/jc.2014-3344

2. De Souza MJ, Koltun KJ, Etter CV, Southmayd EA. Current Status of the Female Athlete Triad: Update and Future Directions. Curr Osteoporos Rep. 2017;15(6):577-587. doi:10.1007/s11914-017-0412-x

3. Title IX and the Rise of Female Athletes in America. Women’s Sports Foundation. https://www.womenssportsfoundation.org/education/title-ix-and-the-rise-of-female- athletes-in-america/. Published September 2, 2016. Accessed October 22, 2018.

4. Meczekalski B, Katulski K, Czyzyk A, Podfigurna-Stopa A, Maciejewska-Jeske M. Functional hypothalamic amenorrhea and its influence on women’s health. J Endocrinol Invest. 2014;37(11):1049-1056. doi:10.1007/s40618-014-0169-3

5. Devleta B, Adem B, Senada S. Hypergonadotropic amenorrhea and bone density: new approach to an old problem. J Bone Miner Metab. 2004;22(4). doi:10.1007/s00774-004- 0495-1

6. Mountjoy M, Sundgot-Borgen J, Burke L, et al. The IOC consensus statement: beyond the Female Athlete Triad—Relative Energy Deficiency in Sport (RED-S). Br J Sports Med. 2014;48(7):491-497. doi:10.1136/bjsports-2014-093502

7. eumenorrhea. https://medicine.academic.ru/122180/eumenorrhea. Accessed October 21, 2018.

8. Menstrual cycle: What’s normal, what’s not. Mayo Clinic. http://www.mayoclinic.org/healthy-lifestyle/womens-health/in-depth/menstrual-cycle/art- 20047186. Accessed October 21, 2018.

9. Draper CF, Duisters K, Weger B, et al. Menstrual cycle rhythmicity: metabolic patterns in healthy women. Sci Rep. 2018;8. doi:10.1038/s41598-018-32647-0

62 10. Bopp B, Shoupe D. Luteal phase defects. J Reprod Med. 1993;38(5):348-356.

11. pmhdev. Oligomenorrhea - National Library of Medicine. PubMed Health. https://www.ncbi.nlm.nih.gov/pubmedhealth/PMHT0030345/. Accessed October 21, 2018.

12. Rebar R. Evaluation of Amenorrhea, Anovulation, and Abnormal Bleeding. In: De Groot LJ, Chrousos G, Dungan K, et al., eds. Endotext. South Dartmouth (MA): MDText.com, Inc.; 2000. http://www.ncbi.nlm.nih.gov/books/NBK279144/. Accessed August 29, 2018.

13. Polycystic Ovary Syndrome, Oligomenorrhea, and Risk of Ovarian Cancer Histotypes: Evidence from the Ovarian Cancer Association Consortium | Cancer Epidemiology, Biomarkers & Prevention. http://cebp.aacrjournals.org/content/27/2/174.long. Accessed October 22, 2018.

14. The Practice Committee for the American Society for Reproductive Medicine. Current evaluation of amenorrhea. Fertil Steril. 2008;90(5):S219-S225. doi:10.1016/j.fertnstert.2008.08.038

15. What causes amenorrhea? | NICHD - Eunice Kennedy Shriver National Institute of Child Health and Human Development. https://www.nichd.nih.gov/health/topics/amenorrhea/conditioninfo/causes. Accessed October 21, 2018.

16. McNeilly AS. Lactation and Fertility. J Mammary Gland Biol Neoplasia. 1997;2(3):291-298. doi:10.1023/A:1026340606252

17. What Are the Side Effects & Disadvantages of Depo-Provera? https://www.plannedparenthood.org/learn/birth-control/birth-control-shot/what-are-the- disadvantages-of-the-birth-control-shot. Accessed October 23, 2018.

18. Clark M, Sowers M, Levy B, Nichols S. Bone mineral density loss and recovery during 48 months in first-time users of depot medroxyprogesterone acetate. Fertil Steril. 2006;86(5):1466-1474. doi:10.1016/j.fertnstert.2006.05.024

63 19. Cromer BA, Blair JM, Mahan JD, Zibners L, Naumovski Z. A prospective comparison of bone density in adolescent girls receiving depot medroxyprogesterone acetate (Depo-Provera), levonorgestrel (Norplant), or oral contraceptives. J Pediatr. 1996;129(5):671-676. doi:10.1016/S0022-3476(96)70148-8

20. Hillard P. Menstrual suppression: current perspectives. Int J Womens Health. June 2014:631. doi:10.2147/IJWH.S46680

21. Loucks AB, Verdun M, Heath EM, (With the Technical Assistance of T. Law, Sr. and J. R. Thuma). Low energy availability, not stress of exercise, alters LH pulsatility in exercising women. J Appl Physiol. 1998;84(1):37-46. doi:10.1152/jappl.1998.84.1.37

22. Frisch RE. Body weight, body fat, and ovulation. Trends Endocrinol Metab. 1991;2(5):191- 197. doi:10.1016/1043-2760(91)90018-I

23. Bringer J, Lefebvre P, Boulet F, Clouet S, Renard E. Deficiency of energy balance and ovulatory disorders. Hum Reprod. 1997;12(suppl 1):97-109. doi:10.1093/humrep/12.suppl_1.97

24. Matkovic V, Ilich JZ, Skugor M, et al. Leptin Is Inversely Related to Age at Menarche in Human Females. 1997;82(10):7.

25. Osofsky HJ, Fisher S. Psychological Correlates of the Development of Amenorrhea in a Stress Situation: Psychosom Med. 1967;29(1):15-23. doi:10.1097/00006842-196701000-00002

26. Marcus MD, Loucks TL, Berga SL. Psychological correlates of functional hypothalamic amenorrhea. Fertil Steril. 2001;76(2):310-316. doi:10.1016/S0015-0282(01)01921-5

27. Functional Hypothalamic Amenorrhea: An Endocrine Society Clinical Practice Guideline. Presented at the: 2017.

28. Gordon CM, Ackerman KE, Berga SL, et al. Functional Hypothalamic Amenorrhea: An Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab. 2017;102(5):1413- 1439. doi:10.1210/jc.2017-00131 64 29. Moreno-Aliaga MJ, Lorente-Cebrián S, Martínez JA. Regulation of adipokine secretion by n-3 fatty acids. Proc Nutr Soc. 2010;69(03):324-332. doi:10.1017/S0029665110001801

30. Angeline N. Beltsos. Hypothalamic Amenorrhea - Fertility Centers of Illinois | Fertility Centers of Illinois. https://fcionline.com/our-center/for-physicians/articles-on- reproductive-medicine/hypothalamic-amenorrhea-by-angeline-n-beltsos-m-d/. Accessed October 9, 2018.

31. LAUGHLIN GA, DOMINGUEZ CE, YEN SSC. Nutritional and Endocrine-Metabolic Aberrations in Women with Functional Hypothalamic Amenorrhea. 2018;83(1):8.

32. Loucks AB, Mortola JF, Girton L, Yen SSC. Alterations in the Hypothalamic-Pituitary-Ovarian and the Hypothalamic-Pituitary-Adrenal Axes in Athletic Women*. J Clin Endocrinol Metab. 1989;68(2):402-411. doi:10.1210/jcem-68-2-402

33. Loucks AB, Thuma JR. Luteinizing Hormone Pulsatility Is Disrupted at a Threshold of Energy Availability in Regularly Menstruating Women. J Clin Endocrinol Metab. 2003;88(1):297-311. doi:10.1210/jc.2002-020369

34. Understanding the role of estrogen in the development of benign prostatic hyperplasia - ScienceDirect. https://www.sciencedirect.com/science/article/pii/S1110570418300055. Accessed October 22, 2018.

35. De Souza MJ, West SL, Jamal SA, Hawker GA, Gundberg CM, Williams NI. The presence of both an energy deficiency and estrogen deficiency exacerbate alterations of bone metabolism in exercising women. Bone. 2008;43(1):140-148. doi:10.1016/j.bone.2008.03.013

36. De Souza MJ, Toombs RJ, Scheid JL, O’Donnell E, West SL, Williams NI. High prevalence of subtle and severe menstrual disturbances in exercising women: confirmation using daily hormone measures. Hum Reprod. 2010;25(2):491-503. doi:10.1093/humrep/dep411

37. Sowiński J, Sawicka-Gutaj N, Gutaj P, Ruchała M. The role of free triiodothyronine in pathogenesis of infertility in levothyroxine-treated women with thyroid autoimmunity – a

65 preliminary observational study. Gynecol Endocrinol. 2015;31(2):116-118. doi:10.3109/09513590.2014.964200

38. Dittrich R, Beckmann MW, Oppelt PG, et al. Thyroid hormone receptors and reproduction. J Reprod Immunol. 2011;90(1):58-66. doi:10.1016/j.jri.2011.02.009

39. De Souza MJ, Leidy HJ, O’Donnell E, Lasley B, Williams NI. Fasting Ghrelin Levels in Physically Active Women: Relationship with Menstrual Disturbances and Metabolic Hormones. J Clin Endocrinol Metab. 2004;89(7):3536-3542. doi:10.1210/jc.2003-032007

40. Phinney SD, LaGrange BM, O’Connell M, Danforth E. Effects of aerobic exercise on energy expenditure and nitrogen balance during very low calorie dieting. Metabolism. 1988;37(8):758-765. doi:10.1016/0026-0495(88)90011-X

41. Ding J-H. High Serum Cortisol Levels in Exercise-Associated Amenorrhea. Ann Intern Med. 1988;108(4):530. doi:10.7326/0003-4819-108-4-530

42. Herrera AY, Nielsen SE, Mather M. Stress-induced increases in progesterone and cortisol in naturally cycling women. Neurobiol Stress. 2016;3:96-104. doi:10.1016/j.ynstr.2016.02.006

43. Dueck CA, Manore MM, Matt KS. Role of Energy Balance in Athletic Menstrual Dysfunction. Int J Sport Nutr. 1996;6(2):165-190. doi:10.1123/ijsn.6.2.165

44. Dalsky GP. Effect of exercise on bone: permissive influence of estrogen and calcium. Med Sci Sports Exerc. 1990;22(3):281.

45. Leidy HJ, Gardner JK, Frye BR, et al. Circulating ghrelin is sensitive to changes in body weight during a diet and exercise program in normal-weight young women. J Clin Endocrinol Metab. 2004;89(6):2659-2664. doi:10.1210/jc.2003-031471

46. De Souza MJ, Lee DK, VanHeest JL, Scheid JL, West SL, Williams NI. Severity of energy- related menstrual disturbances increases in proportion to indices of energy conservation in exercising women. Fertil Steril. 2007;88(4):971-975. doi:10.1016/j.fertnstert.2006.11.171

66 47. Thong FSL, McLean C, Graham TE. Plasma leptin in female athletes: relationship with body fat, reproductive, nutritional, and endocrine factors. J Appl Physiol. 2000;88(6):2037-2044. doi:10.1152/jappl.2000.88.6.2037

48. Köpp W, Blum WF, von Prittwitz S, et al. Low leptin levels predict amenorrhea in underweight and eating disordered females. Mol Psychiatry. 1997;2(4):335-340. doi:10.1038/sj.mp.4000287

49. Waters DL, Qualls CR, Dorin R, Veldhuis JD, Baumgartner RN. Increased Pulsatility, Process Irregularity, and Nocturnal Trough Concentrations of Growth Hormone in Amenorrheic Compared to Eumenorrheic Athletes. 2018;86(3):7.

50. Narita K, Nagao K, Bannai M, et al. Dietary Deficiency of Essential Amino Acids Rapidly Induces Cessation of the Rat Estrous Cycle. PLoS ONE. 2011;6(11). doi:10.1371/journal.pone.0028136

51. Russell M, Stark J, Nayak S, et al. Peptide YY in adolescent athletes with amenorrhea, eumenorrheic athletes and non-athletic controls. Bone. 2009;45(1):104-109. doi:10.1016/j.bone.2009.03.668

52. Williams NI, Berga SL, Cameron JL. Synergism between psychosocial and metabolic stressors: impact on reproductive function in cynomolgus monkeys. Am J Physiol-Endocrinol Metab. 2007;293(1):E270-E276. doi:10.1152/ajpendo.00108.2007

53. Gordon CM. Clinical practice. Functional hypothalamic amenorrhea. N Engl J Med. 2010;363(4):365-371. doi:10.1056/NEJMcp0912024

54. Easter A, Treasure J, Micali N. Fertility and prenatal attitudes towards pregnancy in women with eating disorders: results from the Avon Longitudinal Study of Parents and Children: Fertility in women with eating disorders. BJOG Int J Obstet Gynaecol. 2011;118(12):1491- 1498. doi:10.1111/j.1471-0528.2011.03077.x

55. Cousins A, Freizinger M, Duffy ME, Gregas M, Wolfe BE. Self‐Report of Eating Disorder Symptoms Among Women With and Without Infertility. J Obstet Gynecol Neonatal Nurs. 2015;44(3):380-388. doi:10.1111/1552-6909.12573 67 56. Freizinger M, Franko DL, Dacey M, Okun B, Domar AD. The prevalence of eating disorders in infertile women. Fertil Steril. 2010;93(1):72-78. doi:10.1016/j.fertnstert.2008.09.055

57. S.H. Shomento, R.E Kreipe. Menstruation and Fertility Following Anorexia Nervosa. Adolesc Pediatr Gynecol. 1994;7(3):142-146.

58. Recker RR, Davies KM, Hinders M, Kimmel DB. Bone Gain in Young Adult Women. :6.

59. Miller KK, Klibanski A. Amenorrheic Bone Loss. J Clin Endocrinol Metab. 1999;84(6):1775- 1783. doi:10.1210/jcem.84.6.5688

60. Riggs BL. The mechanisms of estrogen regulation of bone resorption. J Clin Invest. 2000;106(10):1203-1204. doi:10.1172/JCI11468

61. Langdahl B, Ferrari S, Dempster DW. Bone modeling and remodeling: potential as therapeutic targets for the treatment of osteoporosis. Ther Adv Musculoskelet Dis. 2016;8(6):225-235. doi:10.1177/1759720X16670154

62. Hadjidakis DJ, Androulakis II. Bone remodeling. Ann N Y Acad Sci. 2006;1092:385-396. doi:10.1196/annals.1365.035

63. Glüer C-C, Blake G, Lu Y, Blunt1 BA, Jergas1 M, Genant1 HK. Accurate assessment of precision errors: How to measure the reproducibility of bone densitometry techniques. Osteoporos Int. 1995;5(4):262-270. doi:10.1007/BF01774016

64. Nattiv A, Loucks AB, Manore MM, Sanborn CF, Sundgot-Borgen J, Warren MP. The Female Athlete Triad. Med Sci Sports Exerc. 2007;39(10):1867. doi:10.1249/mss.0b013e318149f111

65. Lewiecki EM, Gordon CM, Baim S, et al. International Society for Clinical Densitometry 2007 Adult and Pediatric Official Positions. Bone. 2008;43(6):1115-1121. doi:10.1016/j.bone.2008.08.106

68 66. Bassey EJ, Ramsdale SJ. Increase in femoral bone density in young women following high- impact exercise. Osteoporos Int. 1994;4(2):72-75. doi:10.1007/BF01623226

67. Drinkwater BL, Nilson K, Chesnut CH, Bremner WJ, Shainholtz S, Southworth MB. Bone Mineral Content of Amenorrheic and Eumenorrheic Athletes. N Engl J Med. 1984;311(5):277-281. doi:10.1056/NEJM198408023110501

68. Gibbs JC, Williams NI, De Souza MJ. Prevalence of individual and combined components of the female athlete triad. Med Sci Sports Exerc. 2013;45(5):985-996. doi:10.1249/MSS.0b013e31827e1bdc

69. Bennell KL, Malcolm SA, Wark JD, Brukner PD. Skeletal effects of menstrual disturbances in athletes. Scand J Med Sci Sports. 1997;7(5):261-273. doi:10.1111/j.1600- 0838.1997.tb00151.x

70. Soyka LA, Misra M, Frenchman A, et al. Abnormal Bone Mineral Accrual in Adolescent Girls with Anorexia Nervosa. J Clin Endocrinol Metab. 2002;87(9):4177-4185. doi:10.1210/jc.2001-011889

71. Barrack MT, Rauh MJ, Nichols JF. Cross-sectional evidence of suppressed bone mineral accrual among female adolescent runners. J Bone Miner Res. 2010;25(8):1850-1857. doi:10.1002/jbmr.63

72. Drinkwater BL, Nilson K. Bone Mineral Density After Resumption of Menses in Amenorrheic Athletes. :3.

73. Rauh MJ, Nichols JF, Barrack MT. Relationships Among Injury and Disordered Eating, Menstrual Dysfunction, and Low Bone Mineral Density in High School Athletes: A Prospective Study. J Athl Train. 2010;45(3):243-252. doi:10.4085/1062-6050-45.3.243

74. Koehler K, Williams NI, Mallinson RJ, Southmayd EA, Allaway HCM, De Souza MJ. Low resting metabolic rate in exercise-associated amenorrhea is not due to a reduced proportion of highly active metabolic tissue compartments. Am J Physiol-Endocrinol Metab. 2016;311(2):E480-E487. doi:10.1152/ajpendo.00110.2016

69 75. Myerson M, Gutin B, Warren M, et al. Resting metabolic rate and energy balance in amenorrheic and eumenorrheic runners. Med Sci Sports Exerc. 1991;23(1):15-22.

76. Horswill CA. Weight Loss and Weight Cycling in Amateur Wrestlers: Implications for Performance and Resting Metabolic Rate. Int J Sport Nutr. 1993;3(3):245-260. doi:10.1123/ijsn.3.3.245

77. Brownell KD, Greenwood MR, Stellar E, Shrager EE. The effects of repeated cycles of weight loss and regain in rats. Physiol Behav. 1986;38(4):459-464.

78. Weinsier RL, Nagy TR, Hunter GR, Darnell BE, Hensrud DD, Weiss HL. Do adaptive changes in metabolic rate favor weight regain in weight-reduced individuals? An examination of the set-point theory. Am J Clin Nutr. 2000;72(5):1088-1094. doi:10.1093/ajcn/72.5.1088

79. Toombs RJ, Ducher G, Shepherd JA, De Souza MJ. The Impact of Recent Technological Advances on the Trueness and Precision of DXA to Assess Body Composition. Obesity. 2012;20(1):30-39. doi:10.1038/oby.2011.211

80. Horton JA, Murray GM, Spadaro JA, Margulies BS, Allen MJ, Damron TA. Precision and Accuracy of DXA and pQCT for Densitometry of the Rat Femur. J Clin Densitom. 2003;6(4):381-390. doi:10.1385/JCD:6:4:381

81. Leung KS, Siu WS, Qin L. pQCT bone strength index may serve as a better predictor than bone mineral density for long bone breaking strength. J Bone Miner Metab. 2003;21(5):316- 322. doi:10.1007/s00774-003-0427-5

82. Ashe MC, Khan KM, Kontulainen SA, et al. Accuracy of pQCT for evaluating the aged human radius: an ashing, histomorphometry and failure load investigation. Osteoporos Int. 2006;17(8):1241-1251. doi:10.1007/s00198-006-0110-5

83. Ashe MC, Liu-Ambrose T, Khan KM, White N, McKay HA. Optimizing Results From pQCT. J Clin Densitom. 2005;8(3):335-340. doi:10.1385/JCD:8:3:335

70 84. ReeVue Medical RMR Metabolic Rate Analysis System | KORR. KORR Medical Technologies. https://korr.com/products/medical-metabolic-rate-analysis-system/. Accessed April 4, 2019.

85. Henes ST, Johnson A, Toner M, et al. Assessing Resting Metabolic Rate in Overweight and Obese Adolescents With a Portable Indirect Calorimeter: A Pilot Study for Validation and Reliability. :7.

86. KLEIN DA, POTH MA. Amenorrhea: An Approach to Diagnosis and Management. 2013;87(11):8.

87. Polycystic ovary syndrome (PCOS) - Symptoms and causes. Mayo Clinic. https://www.mayoclinic.org/diseases-conditions/pcos/symptoms-causes/syc-20353439. Accessed January 30, 2019.

88. Sirmans S, Pate K. Epidemiology, diagnosis, and management of polycystic ovary syndrome. Clin Epidemiol. December 2013:1. doi:10.2147/CLEP.S37559

89. Yatavelli RKR, Bhusal K. Prolactinoma. In: StatPearls. Treasure Island (FL): StatPearls Publishing; 2019. http://www.ncbi.nlm.nih.gov/books/NBK459347/. Accessed March 13, 2019.

90. Reference GH. Kallmann syndrome. Genetics Home Reference. https://ghr.nlm.nih.gov/condition/kallmann-syndrome. Accessed January 30, 2019.

91. Hind K. Recovery of bone mineral density and fertility in a former amenorrheic athlete. :4.

92. Nicola J. Rinaldi, Stephanie G. Buckler, Lisa Sanfilippo Waddell. No Period, Now What? Waltham, MA: Antica Press LLC; 2016.

93. GE Healthcare. iDXA for Bone Health - DXA for Bone Health - Bone Health - Categories. Lunar iDXA for Bone Health. http://www3.gehealthcare.com.au/en- au/products/categories/bone_health/dxa/lunar_idxa_for_bone_health. Published 2018. Accessed March 7, 2019. 71 94. Robert E Reiman. Radiation Risk Statements. IRB Protocol Radiation Risk Statements. https://vmw-oesoapps.duhs.duke.edu/radsafety/consents/irbcf_asp/default.asp. Published 2004. Accessed March 7, 2019.

95. Beals KA, Hill AK. The Prevalence of Disordered Eating, Menstrual Dysfunction, and Low Bone Mineral Density among US Collegiate Athletes. Int J Sport Nutr Exerc Metab. 2006;16(1):1-23. doi:10.1123/ijsnem.16.1.1

96. Watson S, Buell J. Body Composition, Resting Metabolic Rate and Dietary Habits of Lean and Non-lean Female Athletes. :43.

97. IBM SPSS Statistics. IBM Corp. Released 2017. IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY: IBM Corp. Armonk, NY: IBM

98. Pietrobelli A, Formica C, Wang Z, Heymsfield SB. Dual-energy X-ray absorptiometry body composition model: review of physical concepts. Am J Physiol-Endocrinol Metab. 1996;271(6):E941-E951. doi:10.1152/ajpendo.1996.271.6.E941

99. Compare 2 Means 2-Sample, 2-Sided Equality | Power and Sample Size Calculators | HyLown. http://powerandsamplesize.com/Calculators/Compare-2-Means/2-Sample- Equality. Accessed April 12, 2019.

100. Keen AD, Drinkwater BL. Irreversible bone loss in former amenorrheic athletes. Osteoporos Int J Establ Result Coop Eur Found Osteoporos Natl Osteoporos Found USA. 1997;7(4):311-315.

101. Amenorrhea - Symptoms and causes - Mayo Clinic. https://www.mayoclinic.org/diseases-conditions/amenorrhea/symptoms-causes/syc- 20369299. Accessed October 21, 2018.

102. Elliott-Sale KJ, Tenforde AS, Parziale AL, Holtzman B, Ackerman KE. Endocrine Effects of Relative Energy Deficiency in Sport. Int J Sport Nutr Exerc Metab. 2018;28(4):335-349.

72 103. Gordon CM. Functional Hypothalamic Amenorrhea. N Engl J Med. 2010;363(4):365-371. doi:10.1056/NEJMcp0912024

104. De Souza MJ, Williams NI. Physiological aspects and clinical sequelae of energy deficiency and hypoestrogenism in exercising women. Hum Reprod Update. 2004;10(5):433-448. doi:10.1093/humupd/dmh033

73 Appendix A: Recruitment Email

From: Megan Stanley Sent: Wednesday, November 28, 2018 11:44 AM To: Zivoder, Jordan M. Subject: Test: We need your feedback

Hello ,

Hello, we are members of the research study Hypothalamic Amenorrhea and Fertility in Prior Athletes at The Ohio State University. This email was sent to you in the hopes that you will participate in our study. The goal for this research is to determine the historical prevalence of irregular menses in collegiate female athletes and how that has affected fertility later in life. As a previous athlete, we are reaching out to you to help us learn more about fertility issues to prevent these problems in the future.

To participate, we ask that you complete our survey using the provided anonymous link. The survey should only take about 20-30 minutes to complete if you have experienced menstrual issues. If you did not have any menstrual issues, this survey will take you less than 3 min. Your participation will provide important reproductive information to potentially help future athletes. Your answers will be completely anonymous, meaning that neither your name nor email will be attached to your responses unless you sign up for a lab visit. You can stop the survey at any time and skip any questions that you are not comfortable answering. However, the more information we receive, the better we will be able to understand the prevalence and impact of this issue.

At the end of the survey, you will be asked if you live near the Columbus Ohio area, and if you would like to participate in a nested study. This includes a lab visit to the Jameson Crane Sports Medicine Institute where we will test your bone density, body composition, and resting metabolic rate, all of which will be at no

74 cost to you, and may be beneficial knowledge for your health as well as our study.

If you wish to participate in the lab visit, provide your email on the last survey question, and further information will be sent to you. Providing your e-mail will link your data to your name and e-mail for study purposes. We do not look at individual responses on the questionnaire, but we do want you to realize this “identifies” your data. Note that if you wish to participate in this laboratory visit, you will need to fast the morning of the visit to ensure accurate measurements. Please only sign up for the nested study if you are willing to fast the morning of your appointment.

Thank you for your time, we greatly appreciate your participation!

To participate in the survey, please visit: BuckeyeWomen This survey link will expire in 30 days.

Megan Stanley Primary Graduate Student Ohio State University Medical Dietetics

Jackie Buell, PhD, RD, CSSD, ATC Asst. Professor- Clinical, Sports Nutrition Medical Dietetics & Health Sciences Sports Medicine Center The Ohio State University Medical Center

1480 W. Lane Avenue, Columbus, OH, 43221

If you would like to unsubscribe from surveys like these, click the link below:

75

Appendix B: Questionnaire Athlete Alum Fertility Study

Start of Block: Consent

Q1 Consent to Participate in Research This provides consent information for research participation. Your participation is voluntary, please consider the information carefully. Purpose/Procedure: This study will explore menstrual and fertility history in women who have participated in intercollegiate athletics. Should you choose to participate by clicking on the agree to participate button, we will ask you questions about your age and participation, if you had normal periods while you participated, if you have had issues getting pregnant, and if so what sorts of physicians and treatments, etc. If you did not have menstrual cycle issues in college and you have not had fertility issues, it will only take a few minutes to complete the questionnaire. If you do have a history of menstrual issues and/or fertility issues, we will ask more questions to collect details, and this might take 20-30 minutes. Please recognize that if we ask a question that you do not want to answer, you are not required to answer, and you can stop participation at any point if you are uncomfortable about the questions. Risk and Benefit: There is no physical risk involved in participating in this study. Anytime you are asking people about sensitive topics, there is psychological risk. There is no direct benefit to the study participants. The research gathered through this study will help inform future research and medical management of fertility issues in past athletes. Incentives: There are no formal incentives to participate. Confidentiality: Individual results will not be analyzed; research objectives only focus on group results for analysis. We will work to make sure that no one sees your survey responses without approval. But, because we are using the Internet for the questionnaire, there is a chance that someone could access your online responses without permission. We hope the questions are worded in such a way as to not collect personally identifiable information, but there is always the chance that this information could be used to identify you. Participants Rights: An Institutional Review Board responsible for human subject research at The Ohio State University reviewed this research project and found it to be acceptable, according to applicable state and federal regulations and University policies designed to protect the rights and welfare of participants in research. If you decide not to participate, there will be no penalty or loss of benefits to which you are otherwise entitled.”

Contacts and Questions: For questions, concerns, or complaints about the study, or you 76 feel you have been harmed as a result of study participation, you may contact Jackie Buell at 614 292 9812 or [email protected]. For questions about your rights as a participant in this study or to discuss other study-related concerns or complaints with someone who is not part of the research team, you may contact Ms. Sandra Meadows in the Office of Responsible Research Practices at 1-800-678-6251.

o I participated in intercollegiate athletics and I agree to participate (1)

o I prefer not to participate (2)

o I did not participate in intercollegiate athletics (3)

o I am a still a student at Ohio State and have not graduated yet. (4)

Skip To: End of Survey If Consent to Participate in Research This provides consent information for research participation. ... = I prefer not to participate Skip To: End of Survey If Consent to Participate in Research This provides consent information for research participation. ... = I did not participate in intercollegiate athletics Skip To: End of Survey If Consent to Participate in Research This provides consent information for research participation. ... = I am a still a student at Ohio State and have not graduated yet. End of Block: Consent

Start of Block: Demographics

Q2 In what NCAA division did you participate the longest?

o Division 1 (1)

o Division 2 (2)

o Division 3 (3)

o NAIA or some qualifier, such as D1AA (please provide which) (4) ______

77 Q3 How old are you?

▼ 21 (1) ... Older than 60 (60)

Q5 What race/ethnicity/culture do you most closely identify with?

o White (1)

o Black or African American (2)

o Asian (4)

o Hispanic (7)

o American Indian or Alaska Native (3)

o Native Hawaiian or Pacific Islander (5)

o Other (6) ______

End of Block: Demographics

Start of Block: Sport Participation

78 Q6 What sports did you play in high school? (check all that apply) High School Team (1) Club (2) Recreational (3)

Cross Country (1) ▢ ▢ ▢

Track and Field (2) ▢ ▢ ▢

Soccer (3) ▢ ▢ ▢

Field Hockey (4) ▢ ▢ ▢

Lacrosse (5) ▢ ▢ ▢

Swimming (6) ▢ ▢ ▢

Tennis (7) ▢ ▢ ▢

Volleyball (8) ▢ ▢ ▢

Softball (9) ▢ ▢ ▢

Basketball (10) ▢ ▢ ▢

Gymnastics (11) ▢ ▢ ▢

Other (12) ▢ ▢ ▢

79 Display This Question: If What sports did you play in high school? (check all that apply) = Cross Country [ High School Team ]

Q7 How many hours per week did you run high school cross country? (including practice, lifts, conditioning and competition)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Cross Country [ High School Team ]

Q8 How many weeks of the year were you in high school cross country season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Cross Country [ Club ]

Q9 How many hours per week did you participate club cross country? (including practice, lifts, conditioning, and competition)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Cross Country [ Club ]

Q10 How many weeks of the year were you in club cross country season?

▼ 5 or less (1) ... 25 or more (6)

80 Display This Question: If What sports did you play in high school? (check all that apply) = Cross Country [ Recreational ]

Q11 How many hours per week did you participate in recreational cross country?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Cross Country [ Recreational ]

Q12 How many weeks of the year were you in recreational cross country season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Track and Field [ High School Team ]

Q13 How many hours per week did you participate in high school track and field? (including practice, lifts, conditioning, and competition)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Track and Field [ High School Team ]

Q14 How many weeks of the year were you in high school track and field season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Track and Field [ Club ]

81 Q15 How many hours per week did you participate in club track and field? (including practice, lifts, conditioning, and competition)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Track and Field [ Club ]

Q16 How many weeks of the year were you in club track and field season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Track and Field [ Recreational ]

Q17 How many hours per week did you participate in recreational track and field?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Track and Field [ Recreational ]

Q18 How many weeks of the year were you in recreational track and field season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Soccer [ High School Team ]

Q19 How many hours per week did you participate in high school soccer? (including practice, lifts, conditioning, and games)

▼ 5 or less (1) ... 25 or more (6)

82

Display This Question: If What sports did you play in high school? (check all that apply) = Soccer [ High School Team ]

Q20 How many weeks of the year were you in high school soccer season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Soccer [ Club ]

Q21 How many hours per week did you participate in club soccer? (including practice, lifts, conditioning, and games)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Soccer [ Club ]

Q22 How many weeks of the year were you in club soccer season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Soccer [ Recreational ]

Q23 How many hours per week did you participate in recreational soccer?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Soccer [ Recreational ]

83 Q24 How many weeks of the year were you in recreational soccer season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Field Hockey [ High School Team ]

Q25 How many hours per week did you participate in high school field hockey? (including practice, lifts, conditioning, and games)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Field Hockey [ High School Team ]

Q26 How many weeks of the year were you in high school field hockey season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Field Hockey [ Club ]

Q27 How many hours per week did you participate in club field hockey? (including practice, lifts, conditioning, and games)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Field Hockey [ Club ]

Q28 How many weeks of the year were you in club field hockey season?

▼ 5 or less (1) ... 25 or more (6)

84

Display This Question: If What sports did you play in high school? (check all that apply) = Field Hockey [ Recreational ]

Q29 How many hours per week did you participate in recreational field hockey?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Field Hockey [ Recreational ]

Q30 How many weeks of the year were you in recreational field hockey season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Lacrosse [ High School Team ]

Q31 How many hours per week did you participate in high school lacrosse? (including practice, lifts, conditioning, and games)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Lacrosse [ High School Team ]

Q32 How many weeks of the year were you in high school lacrosse season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Lacrosse [ Club ]

85 Q33 How many hours per week did you participate in club lacrosse? (including practice, lifts, conditioning, and games)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Lacrosse [ Club ]

Q34 How many weeks of the year were you in club lacrosse season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Lacrosse [ Recreational ]

Q35 How many hours per week did you participate in recreational lacrosse?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Lacrosse [ Recreational ]

Q36 How many weeks of the year were you in recreational lacrosse season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Swimming [ High School Team ]

Q37 How many hours per week did you participate in high school swimming? (including practice, lifts, conditioning, and competition)

▼ 5 or less (1) ... 25 or more (6)

86

Display This Question: If What sports did you play in high school? (check all that apply) = Swimming [ High School Team ]

Q38 How many weeks of the year were you in high school swimming season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Swimming [ Club ]

Q39 How many hours per week did you participate in club swimming? (including practice, lifts, conditioning, and competition)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Swimming [ Club ]

Q40 How many weeks of the year were you in club swimming season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Swimming [ Recreational ]

Q41 How many hours per week did you participate in recreational swimming?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Swimming [ Recreational ]

87 Q42 How many weeks of the year were you in recreational swimming season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Tennis [ High School Team ]

Q43 How many hours per week did you participate in high school tennis? (including practice, lifts, conditioning, and competition)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Tennis [ High School Team ]

Q44 How many weeks of the year were you in high school tennis season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Tennis [ Club ]

Q45 How many hours per week did you participate in club tennis? (including practice, lifts, conditioning, and competition)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Tennis [ Club ]

Q46 How many weeks of the year were you in club tennis season?

▼ 5 or less (1) ... 25 or more (6)

88

Display This Question: If What sports did you play in high school? (check all that apply) = Tennis [ Recreational ]

Q47 How many hours per week did you participate in recreational tennis?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Tennis [ Recreational ]

Q48 How many weeks of the year were you in recreational tennis season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Volleyball [ High School Team ]

Q49 How many hours per week did you participate in high school volleyball? (including practice, lifts, conditioning, and games)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Volleyball [ High School Team ]

Q50 How many weeks of the year were you in high school volleyball season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Volleyball [ Club ]

89 Q51 How many hours per week did you participate in club volleyball? (including practice, lifts, conditioning, and games)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Volleyball [ Club ]

Q52 How many weeks of the year were you in club volleyball season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Volleyball [ Recreational ]

Q53 How many hours per week did you participate in recreational volleyball?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Volleyball [ Recreational ]

Q54 How many weeks of the year were you in recreational volleyball season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Softball [ High School Team ]

Q55 How many hours per week did you participate in high school softball? (including practice, lifts, conditioning, and games)

▼ 5 or less (1) ... 25 or more (6)

90

Display This Question: If What sports did you play in high school? (check all that apply) = Softball [ High School Team ]

Q56 How many weeks of the year were you in high school softball season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Softball [ Club ]

Q57 How many hours per week did you participate in club softball?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Softball [ Club ]

Q58 How many weeks of the year were you in club softball season? (including practice, lifts, conditioning, and games)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Softball [ Recreational ]

Q59 How many hours per week did you participate in recreational softball?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Softball [ Recreational ]

91 Q60 How many weeks of the year were you in recreational softball season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Basketball [ High School Team ]

Q61 How many hours per week did you participate in high school basketball? (including practice, lifts, conditioning, and games)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Basketball [ High School Team ]

Q62 How many weeks of the year were you in high school basketball season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Basketball [ Club ]

Q63 How many hours per week did you participate in club basketball? (including practice, lifts, conditioning, and games)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Basketball [ Club ]

Q64 How many weeks of the year were you in club basketball season?

▼ 5 or less (1) ... 25 or more (6)

92

Display This Question: If What sports did you play in high school? (check all that apply) = Basketball [ Recreational ]

Q65 How many hours per week did you participate in recreational basketball?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Basketball [ Recreational ]

Q66 How many weeks of the year were you in recreational basketball season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Gymnastics [ High School Team ]

Q67 How many hours per week did you participate in high school gymnastics? (including practice, lifts, conditioning, and competition)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Gymnastics [ High School Team ]

Q68 How many weeks of the year were you in varsity gymnastics season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Gymnastics [ Club ]

93 Q69 How many hours per week did you participate in club gymnastics? (including practice, lifts, conditioning, and competitions)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Gymnastics [ Club ]

Q70 How many weeks of the year were you in club gymnastics season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Gymnastics [ Recreational ]

Q71 How many hours per week did you participate in recreational gymnastics?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Gymnastics [ Recreational ]

Q72 How many weeks of the year were you in recreational gymnastics season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Other [ High School Team ]

Q73 How many hours per week did you participate in other high school sports? (including practice, lifts, conditioning, and competition)

▼ 5 or less (1) ... 25 or more (6)

94

Display This Question: If What sports did you play in high school? (check all that apply) = Other [ High School Team ]

Q74 How many weeks of the year were you in other varsity sports season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Other [ Club ]

Q75 How many hours per week did you participate in other club sports? (including practice, lifts, conditioning, and competition)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Other [ Club ]

Q76 How many weeks of the year were you in other club sports season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Other [ Recreational ]

Q77 How many hours per week did you participate in other recreational sports?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in high school? (check all that apply) = Other [ Recreational ]

95 Q78 How many weeks of the year were you in other recreational sports season?

▼ 5 or less (1) ... 25 or more (6)

96 Q79 What sports did you play in college?

97 Recreational/Intramural Varsity (1) Club (2) (3)

Cross Country (1) ▢ ▢ ▢

Track and Field (2) ▢ ▢ ▢

Soccer (3) ▢ ▢ ▢

Field Hockey (4) ▢ ▢ ▢

Lacrosse (5) ▢ ▢ ▢

Swimming (6) ▢ ▢ ▢

Tennis (7) ▢ ▢ ▢

Volleyball (8) ▢ ▢ ▢

Softball (9) ▢ ▢ ▢

Basketball (10) ▢ ▢ ▢

Gymnastics (11) ▢ ▢ ▢

Crew (13) ▢ ▢ ▢

Pistol & Rifle (14) ▢ ▢ ▢

98 Synchronized Swimming (15) ▢ ▢ ▢

Fencing (16) ▢ ▢ ▢

Spirit/Dance/Cheer (17) ▢ ▢ ▢

Other (12) ▢ ▢ ▢

Display This Question: If What sports did you play in college? = Cross Country [ Varsity ]

Q80 How many hours per week did you participate in varsity cross country? (including practice, lifts, conditioning, and competition)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Cross Country [ Varsity ]

Q81 How many weeks of the year were you in varsity cross country season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Cross Country [ Club ]

99 Q82 How many hours per week did you participate in club cross country? (including practice, lifts, conditioning, and competition)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Cross Country [ Club ]

Q83 How many weeks of the year were you in club cross country season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Cross Country [ Recreational/Intramural ]

Q84 How many hours per week did you participate in recreational/intramural cross country?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Cross Country [ Recreational/Intramural ]

Q85 How many weeks of the year were you in recreational/intramural cross country season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Track and Field [ Varsity ]

Q86 How many hours per week did you participate in varsity track and field? (including practice, lifts, conditioning, and competition)

▼ 5 or less (1) ... 25 or more (6)

100

Display This Question: If What sports did you play in college? = Track and Field [ Varsity ]

Q87 How many weeks of the year were you in varsity track and field seaon?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Track and Field [ Club ]

Q88 How many hours per week did you participate in club track and field? (including practice, lifts, conditioning, and competition)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Track and Field [ Club ]

Q89 How many weeks of the year were you in club track and field season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Track and Field [ Recreational/Intramural ]

Q90 How many hours per week did you participate in recreational/intramural track and field?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Track and Field [ Recreational/Intramural ]

101 Q91 How many weeks of the year were you in recreational/intramural track and field season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Soccer [ Varsity ]

Q92 How many hours per week did you participate in varsity soccer? (including practice, lifts, conditioning, and games)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Soccer [ Varsity ]

Q93 How many weeks of the year were you in varsity soccer season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Soccer [ Club ]

Q94 How many hours per week did you participate in club soccer? (including practice, lifts, conditioning, and games)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Soccer [ Club ]

Q95 How many weeks of the year were you in club soccer season?

▼ 5 or less (1) ... 25 or more (6)

102

Display This Question: If What sports did you play in college? = Soccer [ Recreational/Intramural ]

Q96 How many hours per week did you participate in recreational/intramural soccer?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Soccer [ Recreational/Intramural ]

Q97 How many weeks of the year were you in recreational/intramural soccer season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Field Hockey [ Varsity ]

Q98 How many hours per week did you participate in varsity field hockey? (including practice, lifts, conditioning, and games)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Field Hockey [ Varsity ]

Q99 How many weeks of the year were you in varsity field hockey season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Field Hockey [ Club ]

103 Q100 How many hours per week did you participate in club field hockey? (including practice, lifts, conditioning, and games)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Field Hockey [ Club ]

Q101 How many weeks of the year were you in club field hockey season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Field Hockey [ Recreational/Intramural ]

Q102 How many hours per week did you participarte in recreational/intramural field hockey?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Field Hockey [ Recreational/Intramural ]

Q103 How many weeks of the year were you in recreational/intramural field hockey season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Lacrosse [ Varsity ]

Q104 How many hours per week did you participate in varsity lacrosse? (including practice, lifts, conditioning, and games)

▼ 5 or less (1) ... 25 or more (6)

104

Display This Question: If What sports did you play in college? = Lacrosse [ Varsity ]

Q105 How many weeks of the year were you in varsity lacrosse season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Lacrosse [ Club ]

Q106 How many hours per week did you participate in club lacrosse? (including practice, lifts, conditioning, and games)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Lacrosse [ Club ]

Q107 How many weeks of the year were you in club lacrosse season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Lacrosse [ Recreational/Intramural ]

Q108 How many hours per week did you participate in recreational/intramural lacrosse?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Lacrosse [ Recreational/Intramural ]

105 Q109 How many weeks of the year were you in recreational/intramural lacrosse season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Swimming [ Varsity ]

Q110 How many hours per week did you participate in varsity swimming? (including practice, lifts, conditioning, and competition)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Swimming [ Varsity ]

Q111 How many weeks of the year were you in varsity swimming season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Swimming [ Club ]

Q112 How many hours per week did you participate in club swimming? (including practice, lifts, conditioning, and competition)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Swimming [ Club ]

Q113 How many weeks of the year were you in club swimming season?

▼ 5 or less (1) ... 25 or more (6)

106

Display This Question: If What sports did you play in college? = Swimming [ Recreational/Intramural ]

Q114 How many hours per week did you participate in recreational/intramural swimming?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Swimming [ Recreational/Intramural ]

Q115 How many weeks of the year were you in recreational/intramural swimming season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Tennis [ Varsity ]

Q116 How many hours per week did you participate in varsity tennis? (including practice, lifts, conditioning, and competition)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Tennis [ Varsity ]

Q117 How many weeks of the year were you in varsity tennis season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Tennis [ Club ]

107 Q118 How many hours per week did you participate in club tennis? (including practice, lifts, conditioning, and competition)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Tennis [ Club ]

Q119 How many weeks of the year were you in club tennis season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Tennis [ Recreational/Intramural ]

Q120 How many hours per week did you participate in recreational/intramural tennis?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Tennis [ Recreational/Intramural ]

Q121 How many weeks of the year were you in recreational/intramural tennis season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Volleyball [ Varsity ]

Q122 How many hours per week did you participate in varsity volleyball? (including practice, lifts, conditioning, and games)

▼ 5 or less (1) ... 25 or more (6)

108

Display This Question: If What sports did you play in college? = Volleyball [ Varsity ]

Q123 How many weeks of the year were you in varsity volleyball season? (including practice, lifts, conditioning, and games)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Volleyball [ Club ]

Q124 How many hours per week did you participate in club volleyball?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Volleyball [ Club ]

Q125 How many weeks of the year were you in club volleyball season? (including practice, lifts, conditioning, and games)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Volleyball [ Recreational/Intramural ]

Q126 How many hours per week did you participate in recreational/intramural volleyball?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Volleyball [ Recreational/Intramural ]

109 Q127 How many weeks of the year were you in recreational/intramural volleyball season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Softball [ Varsity ]

Q128 How many hours per week did you participate in varsity softball? (including practice, lifts, conditioning, and games)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Softball [ Varsity ]

Q129 How many weeks of the year were you in varsity softball season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Softball [ Club ]

Q130 How many hours per week did you participate in club softball? (including practice, lifts, conditioning, and games)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Softball [ Club ]

Q131 How many weeks of the year were you in club softball season?

▼ 5 or less (1) ... 25 or more (6)

110

Display This Question: If What sports did you play in college? = Softball [ Recreational/Intramural ]

Q132 How many hours per week did you participate in recreational/intramural softball?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Softball [ Recreational/Intramural ]

Q133 How many weeks of the year were you in recreational/intermural softball season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Basketball [ Varsity ]

Q134 How many hours per week did you participate in varsity basketball? (including practice, lifts, conditioning, and games)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Basketball [ Varsity ]

Q135 How many weeks of the year were you in varsity basketball season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Basketball [ Club ]

111 Q136 How many hours per week did you participate in club basketball? (including practice, lifts, conditioning, and games)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Basketball [ Club ]

Q137 How many weeks of the year were you in club basketball season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Basketball [ Recreational/Intramural ]

Q138 How many hours per week did you participate in recreational/intramural basketball?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Basketball [ Recreational/Intramural ]

Q139 How many weeks of the year were you in recreational/intramural basketball season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Gymnastics [ Varsity ]

Q140 How many hours per week did you participate in varsity gymnastics? (including practice, lifts, conditioning, and competition)

▼ 5 or less (1) ... 25 or more (6)

112

Display This Question: If What sports did you play in college? = Gymnastics [ Varsity ]

Q141 How many weeks of the year were you in varsity gymnastics season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Gymnastics [ Club ]

Q142 How many hours per week did you participate in club gymnastics? (including practice, lifts, conditioning, and competition)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Gymnastics [ Club ]

Q143 How many weeks of the year were you in club gymnastics season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Gymnastics [ Recreational/Intramural ]

Q144 How many hours per week did you participate in recreational/intramural gymnastics?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Gymnastics [ Recreational/Intramural ]

113 Q145 How many weeks of the year were you in recreational/intramural gymnastics season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Crew [ Varsity ]

Q146 How many hours per week did you participate in varsity crew? (including practice, lifts, conditioning, and competition)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Crew [ Varsity ]

Q147 How many weeks of the year were you in varsity crew season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Crew [ Club ]

Q148 How many hours per week did you participate in club crew? (including practice, lifts, conditioning, and competition)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Crew [ Club ]

Q149 How many weeks of the year were you in club crew season?

▼ 5 or less (1) ... 25 or more (6)

114

Display This Question: If What sports did you play in college? = Crew [ Recreational/Intramural ]

Q150 How many hours per week did you participate in recreational/intramural crew?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Crew [ Recreational/Intramural ]

Q151 How many weeks of the year were you in recreational/intramural crew season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Pistol & Rifle [ Varsity ]

Q152 How many hours per week did you participate in varsity pistol & rifle? (including practice, lifts, conditioning, and competition)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Pistol & Rifle [ Varsity ]

Q153 How many weeks of the year were you in varsity pistol & rifle?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Pistol & Rifle [ Club ]

115 Q154 How many hours per week did you participate in club pistol & rifle? (including practice, lifts, conditioning, and competition)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Pistol & Rifle [ Club ]

Q155 How many weeks of the year were you in club pistol & rifle season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Pistol & Rifle [ Recreational/Intramural ]

Q156 How many hours per week did you participate in recreational/intramural pistol & rifle?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Pistol & Rifle [ Recreational/Intramural ]

Q157 How many weeks of the year were you in recreational/intramural pistol & rifle season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Synchronized Swimming [ Varsity ]

Q158 How many hours per week did you participate in varsity synchronized swimming? (including practice, lifts, conditioning, and competition)

▼ 5 or less (1) ... 25 or more (6)

116

Display This Question: If What sports did you play in college? = Synchronized Swimming [ Varsity ]

Q159 How many weeks of the year were you in varsity synchronized swimming?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Synchronized Swimming [ Club ]

Q160 How many hours per week did you participate in club synchronized swimming? (including practice, lifts, conditioning, and competition)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Synchronized Swimming [ Club ]

Q161 How many weeks of the year were you in club synchronized swimming season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Synchronized Swimming [ Recreational/Intramural ]

Q162 How many hours per week did you participate in recreational/intramural synchronized swimming?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Synchronized Swimming [ Recreational/Intramural ]

117 Q163 How many weeks of the year were you in recreational/intramural synchronized swimming season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Fencing [ Varsity ]

Q164 How many hours per week did you participate in varsity fencing? (including practice, lifts, conditioning, and competition)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Fencing [ Varsity ]

Q165 How many weeks of the year were you in varsity fencing season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Fencing [ Club ]

Q166 How many hours per week did you participate in club fencing? (including practice, lifts, conditioning, and competition)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Fencing [ Club ]

118 Q167 How many weeks of the year were you in club fencing season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Fencing [ Recreational/Intramural ]

Q168 How many hours per week did you participate in recreational/intramural fencing?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Fencing [ Recreational/Intramural ]

Q169 How many weeks of the year were you in recreational/intrmaural fencing season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Spirit/Dance/Cheer [ Varsity ]

Q170 How many hours per week did you participate in varsity spirit/dance/cheer fencing? (including practice, lifts, conditioning, and competition)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Spirit/Dance/Cheer [ Varsity ]

Q171 How many weeks of the year were you in varsity spirit/dance/cheer season?

▼ 5 or less (1) ... 25 or more (6)

119 Display This Question: If What sports did you play in college? = Spirit/Dance/Cheer [ Club ]

Q172 How many hours per week did you participate in club spirit/dance/cheer fencing? (including practice, lifts, conditioning, and competition)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Spirit/Dance/Cheer [ Club ]

Q173 How many weeks of the year were you in club spirit/dance/cheer season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Spirit/Dance/Cheer [ Recreational/Intramural ]

Q174 How many hours per week did you participate in recreational/intramural spirit/dance/cheer fencing?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Spirit/Dance/Cheer [ Recreational/Intramural ]

Q175 How many weeks of the year were you in recreational/intramural spirit/dance/cheer season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Other [ Varsity ]

120 Q176 How many hours per week did you participate in other varsity sports? (including practice, lifts, conditioning, and competition)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Other [ Varsity ]

Q177 How many weeks of the year were you in other varsity sports season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Other [ Club ]

Q178 How many hours per week did you participate in other club sports? (including practice, lifts, conditioning, and games)

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Other [ Club ]

Q179 How many weeks of the year were you in other club sports season?

▼ 5 or less (1) ... 25 or more (6)

Display This Question: If What sports did you play in college? = Other [ Recreational/Intramural ]

Q180 How many hours per week did you participate in other recreational/intramural sports?

▼ 5 or less (1) ... 25 or more (6)

121

Display This Question: If What sports did you play in college? = Other [ Recreational/Intramural ]

Q181 How many weeks of the year were you in other recreational/intramural sports season?

▼ 5 or less (1) ... 25 or more (6)

Q182 In what year did you last participate in collegiate varsity sports?

▼ 2017 (1) ... 1968 (50)

Q183 How old were you when you were first a competitive athlete, meaning playing for a school or travel team?

▼ 5 or younger (1) ... 18 (14)

Q262 Do you still participate in any type of exercise/sport?

o Yes (5)

o No (6)

Display This Question: If Do you still participate in any type of exercise/sport? = No

Q184 What year did you stop as a competitive athlete?

▼ 21 or younger (1) ... Still competing (9)

122 Display This Question: If Do you still participate in any type of exercise/sport? = Yes

Q263 What type of exercise/sport do you participate in?

▢ Recreational (1)

▢ Intramural (2)

▢ Competitive (4)

Display This Question: If What year did you stop as a competitive athlete? = Still competing Or Do you still participate in any type of exercise/sport? = Yes

Q185 Including practice, competition, conditioning, and lifts, how many hours per week are you still participating in exercise/sport?

▼ 5 or less (1) ... 25 or more (6)

Q300 Did you have anyone help with your nutrition while you were playing collegiate sports?

o Yes (4)

o No (5)

Display This Question: If Did you have anyone help with your nutrition while you were playing collegiate sports? = Yes

123 Q301 Who helped you with your nutrition?

▢ Registered Dietitian (1)

▢ Athletic Trainer (2)

▢ Strength and Conditioning Coach (3)

▢ Doctor (4)

▢ Other (5) ______

Q186 Did you have access to a Registered Dietitian in college?

o Yes (1)

o No (2)

Display This Question: If Did you have access to a Registered Dietitian in college? = Yes Or Who helped you with your nutrition? = Registered Dietitian

Q187 Did you ever speak individually with the Registered Dietitian

o Yes (1)

o No (2)

Display This Question: If Did you ever speak individually with the Registered Dietitian = Yes

124 Q188 Approximately how many times did you meet with the Registered Dietitian?

▼ Once (1) ... More than three times (4)

Display This Question: If Approximately how many times did you meet with the Registered Dietitian? = More than three times

Q189 Approximately how often did you meet with a Registered Dietitian?

▼ Once a semester (1) ... Weekly (3)

End of Block: Sport Participation

Start of Block: Menses

Q190 At what age (in years) did you get your first period?

▼ 8 (1) ... Cannot remember (15)

Q191 How tall are you?

▼ less that 5' (1) ... 6' 4" or more (18)

Q192 How much did you weigh at the end of high school? enter numbers only (in pounds)

______

Q193 How much did you weigh at the end of college? enter numbers only (in pounds)

______125

Q194 How much do you weigh right now? enter numbers only (in pounds)

______

Q195 When was your highest non-pregnant weight?

▼ Cannot remember (1) ... Older than 60 (54)

Q196 What was your highest non-pregnant weight? enter numbers only (in pounds)

______

Q197 Please estimate how old were you at your lowest weight (for your full height)? enter numbers only

▼ Cannot remember (1) ... Older than 60 (45)

Q198 While involved in sport, were you aware that amenorrhea (no menses/cycles/periods) could influence/affect fertility?

o Yes (1)

o No (2)

126

Q199 Excluding pregnancy and breastfeeding, have you experienced 3 or more months at a time when you did not have periods?

o Yes (1)

o No (2)

Display This Question: If Excluding pregnancy and breastfeeding, have you experienced 3 or more months at a time when you d... = Yes

Q299 When you missed your periods for 3 or more months, was it because of birth control?

o Yes (1)

o No (2)

Display This Question: If Excluding pregnancy and breastfeeding, have you experienced 3 or more months at a time when you d... = Yes

Q200 Has this happened more than once?

o Yes (1)

o No (2)

Display This Question: If Excluding pregnancy and breastfeeding, have you experienced 3 or more months at a time when you d... = Yes

127 Q201 To the best of your ability, please indicate your menstrual status for each of these years

No No On birth No No No period period control, Had not period period Cannot missed for 1 or for 12 no started for 3-6 for 6-9 remember period 2 months periods periods months months (8) (1) months of more expected yet (7) (3) (4) (2) (5) (6) Freshman or sophomore year of o o o o o o o o high school (1) Junior or senior year of high o o o o o o o o school (2) Freshman year of college (3) o o o o o o o o Sophomore year of college (4) o o o o o o o o Junior year of college (5) o o o o o o o o Senior year of college (6) o o o o o o o o 5th year senior in college (7) o o o o o o o o After graduating from o o o o o o o o college (8)

128 Display This Question: If Excluding pregnancy and breastfeeding, have you experienced 3 or more months at a time when you d... = Yes

Q202 Did you see a physician about your absent periods?

o Yes (1)

o No (2)

Display This Question: If Did you see a physician about your absent periods? = Yes

Q203 What kind of doctor did you see about your missing periods? select all that apply

▢ OBGYN/gynecologist (1)

▢ Endocrinologist (2)

▢ Primary Care Physician (3)

▢ Other (4)

Display This Question: If What kind of doctor did you see about your missing periods? select all that apply = Other

Q204 What other kind of doctor did you see about your missing periods?

______

Display This Question: If Did you see a physician about your absent periods? = Yes

129 Q205 Did you have any abnormal findings requiring follow-ups?

o Yes (1)

o No (2)

Display This Question: If Did you have any abnormal findings requiring follow-ups? = Yes

Q206 What kind of further testing did you have? (select all that apply)

▢ Blood draw for hormone levels (1)

▢ Ultrasound (2)

▢ CT or MRI of head (4)

▢ CT or MRI of pelvis (5)

▢ DNC (6)

▢ Other (3)

Display This Question: If What kind of further testing did you have? (select all that apply) = Blood draw for hormone levels

130 Q207 What were the hormones specifically? select all that apply

▢ Follicle Stimulating Hormone (FSH) (1)

▢ Estradiol (2)

▢ Testosterone (3)

▢ Prolactin (4)

▢ Anti-Mullerian hormone (5)

▢ Luteinizing Hormone (LH) (6)

▢ Dehydroepiandosterone (DHEAS) (7)

▢ Androstenedione (8)

▢ Thyroid-Stimulating Hormone (TSH) (9)

▢ Triiodothyronine (T3/T4) (10)

▢ Don't know or don't remember (11)

Display This Question: If What kind of further testing did you have? (select all that apply) = Ultrasound

131 Q208 What were the ultrasound findings? select all that apply

▢ Thin uterine lining (1)

▢ Small ovaries (2)

▢ Small uterus (3)

▢ Polycystic ovaries (4)

▢ Enlarged ovaries (5)

▢ Multicystic ovaries (6)

▢ Other (7) ______

▢ Don't know or don't remember (8)

Display This Question: If Did you have any abnormal findings requiring follow-ups? = Yes

Q267 Feel free to provide any additional information from the further testing that the past two questions do not cover.

______

Display This Question: If Did you have any abnormal findings requiring follow-ups? = Yes

Q209 What was your approximate weight at the time? enter numbers only (in pounds)

______

132 Display This Question: If Did you see a physician about your absent periods? = Yes

Q210 Were you given a diagnosis for your missing periods? select all that apply

▢ Hypothalamic Amenorrhea, Functional Hypothalamic Amenorrhea, Hypothalamic Pituitary Amenorrhea, Hypogonadotrophic Hypergonadism (1)

▢ Polycystic Ovary Syndrome (PCOS) (2)

▢ Oligomenorrhea (infrequent menstrual periods) (3)

▢ Luteal Phase Deficiency (LPD) (4)

▢ Female Athlete Triad (5)

▢ Kallmann's Syndrome (9)

▢ Unexplained (6)

▢ No diagnosis (7)

▢ Other diagnosis (8)

Display This Question: If Were you given a diagnosis for your missing periods? select all that apply = Other diagnosis

Q211 What was the "other" diagnosis?

______

Display This Question: If Did you see a physician about your absent periods? = Yes

133 Q212 Were you given any treatment for your missing periods?

o Yes (1)

o No (2)

Display This Question: If Were you given any treatment for your missing periods? = Yes

Q213 Were you told to do any of the following? select all that apply

▢ Gain weight (1)

▢ Lose Weight (6)

▢ Increase activity (5)

▢ Decrease activity (3)

▢ Increase caloric intake (2)

▢ Other dietary changes (4)

▢ Birth control pills prescribed (7)

▢ Hormone therapy prescribed (8)

▢ Other (9) ______

Display This Question: If Were you told to do any of the following? select all that apply = Other dietary changes

Q264 What dietary change did you make to regain your period?

______134

Display This Question: If Were you told to do any of the following? select all that apply = Decrease activity

Q214 How were you told to decrease your activity? (check all that apply)

▢ By type of exercise (1)

▢ By frequency (number of times exercising) (2)

▢ By duration (minutes performing exercise) (3)

Display This Question: If Excluding pregnancy and breastfeeding, have you experienced 3 or more months at a time when you d... = Yes

Q215 Did you experience eating issues during this time, along with your amenorrhea? (select all that apply)

▢ Disordered eating (1)

▢ Anorexia (2)

▢ Bulimia (3)

▢ Binge-eating (4)

▢ Restrictive eating (5)

▢ No disordered eating (6)

135 Display This Question: If Excluding pregnancy and breastfeeding, have you experienced 3 or more months at a time when you d... = Yes

Q216 During your amenorrheic time, did you count calories?

o Yes (1)

o No (2)

Display This Question: If During your amenorrheic time, did you count calories? = Yes

Q217 If you can remember, how many calories did you plan to eat per day? enter numbers only

______

Q218 Have you used birth control pills or other hormonal contraception?

o Yes (1)

o No (2)

Display This Question: If Have you used birth control pills or other hormonal contraception? = Yes

Q219 When did you use birth control pills or hormonal contraception? (choose all that apply)

▢ High school (1)

▢ College (2)

▢ After college (3)

136

Display This Question: If When did you use birth control pills or hormonal contraception? (choose all that apply) = High school

Q220 What is the total length of time that you were on birth control or hormonal contraception in high school?

▼ 6 months (1) ... Was not on birth control in high school (6)

Display This Question: If When did you use birth control pills or hormonal contraception? (choose all that apply) = High school

Q221 Did you get a bleed while on hormonal birth control in high school?

o Yes (1)

o No - pill/device intended to prevent bleeding (2)

o No (3)

Display This Question: If When did you use birth control pills or hormonal contraception? (choose all that apply) = High school

Q222 What type of birth control were you prescribed in high school? (choose all that apply)

▢ Birth control pills (1)

▢ Progesterone (2)

▢ IUD (3)

▢ Depo Provera (4)

▢ Implant (5)

137 Display This Question: If What type of birth control were you prescribed in high school? (choose all that apply) = Birth control pills

Q223 Would you prefer to answer which type of birth control pills you used in high school by brand or hormone?

o By brand (1)

o By hormone (2)

Display This Question: If Would you prefer to answer which type of birth control pills you used in high school by brand or... = By brand

138 Q224 What was the brand of birth control pill you were prescribed in high school?

o Alesse (1)

o Apri (2)

o Aviane (3)

o Enpresse (4)

o Levlite (5)

o Levora (6)

o Loestrin (7)

o Lutera (8)

o Mircette (9)

o Ortho Novum 777 (10)

o Ortho Tri-Cyclen (11)

o Seasonique (12)

o Sprintec (13)

o Taytulla (14)

o Yasmin (15)

o Yaz (16)

o Camila (17)

o Errin (18)

o Heather (19)

o Jolivette (20) 139 o Nor-Q.D. (21)

o Nora-Be (22)

o Ortho-Micronor (23)

o Don't know (24)

o Other (please enter name) (25) ______

Display This Question: If Would you prefer to answer which type of birth control pills you used in high school by brand or... = By hormone

140 Q225 What type of hormone was the birth control that you were prescribed in high school?

o Estrogen and progestin (1)

o Desogestrel and ethinyl estradiol (2)

o Ethinyl estradiol and levonorgestrel (3)

o Progesterone and estrogen (4)

o Ethinyl estradiol and norethindrone (5)

o Estrogen (6)

o Drospirenone and ethinyl estradiol (7)

o Progestin and ethinyl estradiol (8)

o Progestin (9)

o Don't know (10)

o Other (please enter name) (11) ______

Display This Question: If What type of birth control were you prescribed in high school? (choose all that apply) = IUD

141 Q226 What was the brand of IUD that you were prescribed in high school?

o ParaGard (1)

o Mirena (2)

o Kyleena (3)

o Liletta (4)

o Skyla (5)

Display This Question: If When did you use birth control pills or hormonal contraception? (choose all that apply) = College

Q227 What type of birth control were you prescribed in college? (choose all that apply)

▢ Birth control pills (1)

▢ Progesterone (2)

▢ IUD (3)

▢ Depo Provera (4)

▢ Implant (5)

Display This Question: If What type of birth control were you prescribed in college? (choose all that apply) = Birth control pills

142 Q228 Would you prefer to answer which type of birth control pills you used in college by brand or hormone?

o By brand (1)

o By hormone (2)

Display This Question: If Would you prefer to answer which type of birth control pills you used in college by brand or horm... = By brand

143 Q229 What was the brand of birth control you were prescribed in college?

o Alesse (1)

o Apri (2)

o Aviane (3)

o Enpresse (4)

o Levlite (5)

o Levora (6)

o Loestrin (7)

o Lutera (8)

o Mircette (9)

o Ortho Novum 777 (10)

o Ortho Tri-Cyclen (11)

o Seasonique (12)

o Sprintec (13)

o Taytulla (14)

o Yasmin (15)

o Yaz (16)

o Camila (17)

o Errin (18)

o Heather (19)

o Jolivette (20) 144 o Nor-Q.D (21)

o Nora-Be (22)

o Don't know (23)

o Other (please enter name) (24) ______

Display This Question: If Would you prefer to answer which type of birth control pills you used in college by brand or horm... = By hormone

Q230 What type of hormone was the birth control that you were prescribed in college?

o Estrogen and progestin (1)

o Desogestrel and ethinyl estradiol (2)

o Ethinyl estradiol and levonorgestrel (3)

o Progesterone and estrogen (4)

o Ethinyl estradiol and norethindrone (5)

o Estrogen (6)

o Drospirenone and ethinyl estradiol (7)

o Progestin and ethinyl estradiol (8)

o Progestin (9)

o Don't know (10)

o Other (please enter name) (11) ______

145 Display This Question: If What type of birth control were you prescribed in college? (choose all that apply) = IUD

Q231 What was the brand of IUD that you were prescribed in college?

o ParaGard (1)

o Mirena (2)

o Kyleena (3)

o Liletta (4)

o Skyla (5)

Display This Question: If When did you use birth control pills or hormonal contraception? (choose all that apply) = College

Q232 What is the total length of time that you were on birth control or hormonal contraception in college?

▼ 6 months (1) ... 5 years (6)

Display This Question: If When did you use birth control pills or hormonal contraception? (choose all that apply) = After college

146 Q233 What type of birth control were you prescribed? (choose all that apply)

▢ Birth control pills (1)

▢ Progesterone (2)

▢ IUD (3)

▢ Depo Provera (4)

▢ Implant (5)

▢ Early (before children) sterilization such as tubes tied or hysterectomy (6)

▢ Later (after children) sterilization such as tubes tied or hysterectomy (7)

Display This Question: If What type of birth control were you prescribed? (choose all that apply) = Birth control pills

Q234 Would you prefer to answer which type of birth control pills you used by brand or hormone?

o By brand (1)

o By hormone (2)

Display This Question: If Would you prefer to answer which type of birth control pills you used by brand or hormone? = By brand

147 Q235 What was the brand of birth control you were prescribed in college?

o Alesse (1)

o Apri (2)

o Aviane (3)

o Enpresse (4)

o Levlite (5)

o Levora (6)

o Loestrin (7)

o Lutera (8)

o Mircette (9)

o Ortho Novum 777 (10)

o Ortho Tri-Cyclen (11)

o Seasonique (12)

o Sprintec (13)

o Taytulla (14)

o Yasmin (15)

o Yaz (16)

o Camila (17)

o Errin (18)

o Heather (19)

o Jolivette (20) 148 o Nor-Q.D (21)

o Nora-Be (22)

o Don't know (23)

o Other (please enter name) (24) ______

Display This Question: If Would you prefer to answer which type of birth control pills you used by brand or hormone? = By hormone

Q236 What type of hormone was the birth control that you were prescribed?

o Estrogen and progestin (1)

o Desogestrel and ethinyl estradiol (2)

o Ethinyl estradiol and levonorgestrel (3)

o Progesterone and estrogen (4)

o Ethinyl estradiol and norethindrone (5)

o Estrogen (6)

o Drospirenone and ethinyl estradiol (7)

o Progestin and ethinyl estradiol (8)

o Progestin (9)

o Don't know (10)

o Other (please enter name) (11) ______

149 Display This Question: If What type of birth control were you prescribed? (choose all that apply) = IUD

Q237 What was the brand of IUD that you were prescribed?

o ParaGard (1)

o Mirena (2)

o Kyleena (3)

o Liletta (4)

o Skyla (5)

Display This Question: If When did you use birth control pills or hormonal contraception? (choose all that apply) = College

Q238 Did you get a bleed while on hormonal birth control in college?

o Yes (1)

o No - pill/device intended to prevent bleeding (2)

o No (3)

Display This Question: If When did you use birth control pills or hormonal contraception? (choose all that apply) = After college

Q240 What is the total length of time that you were on birth control or hormonal contraception after college?

▼ 6 months (1) ... 10 or more years (11)

150 Display This Question: If When did you use birth control pills or hormonal contraception? (choose all that apply) = After college

Q241 Did you get a bleed while on hormonal birth control after college?

o Yes (1)

o No - pill/device intended to prevent bleeding (2)

o No (3)

Display This Question: If Excluding pregnancy and breastfeeding, have you experienced 3 or more months at a time when you d... = Yes

Q242 Have your menses resumed since you lost your period?

o Yes, natural cycles (1)

o Yes, on hormonal birth control (3)

o No, and not on hormonal birth control (2)

o No, on hormonal birth control (4)

o No, had a hysterectomy (7)

o Not applicable, currently breastfeeding or pregnant (5)

o Other (6) ______

Display This Question: If Have your menses resumed since you lost your period? = Yes, natural cycles Or Have your menses resumed since you lost your period? = Yes, on hormonal birth control

Q243

151 Did you make any changes to energy intake, exercise/activity, or lifestyle in an attempt to regain cycles? While we realize that amenorrhea is typically an energy balance issues...in order to capture as much information as possible, please select all the changes that you made.

▢ Increasing energy intake (1)

▢ Decrease energy intake (10)

▢ Removing food restrictions (2)

▢ Gaining weight (3)

▢ Reducing high intensity exercise (4)

▢ Eliminating high intensity exercise (5)

▢ Reducing stress (6)

▢ Acupuncture (7)

▢ Supplements (which) (8) ______

▢ Other (please specify) (9) ______

Display This Question: If Did you make any changes to energy intake, exercise/activity, or lifestyle in an attempt to reg... = Increasing energy intake

Q265 About how many calories were you consuming before you increased your energy intake?

______

152 Display This Question: If Did you make any changes to energy intake, exercise/activity, or lifestyle in an attempt to reg... = Increasing energy intake

Q266 By about how many calories did you increase your energy intake?

______

End of Block: Menses

Start of Block: Tried to get pregnant

Q244 Have you ever tried to get pregnant?

o Yes (1)

o No (2)

End of Block: Tried to get pregnant

Start of Block: If tried to get pregnant Display This Question: If Have you ever tried to get pregnant? = Yes

Q245 How old were you when you first tried to get pregnant?

▼ Younger than 20 (1) ... 45 or older (27)

Display This Question: If Have you ever tried to get pregnant? = Yes

Q246 Did you get pregnant?

o Yes (1)

o No (2)

153 Display This Question: If Did you get pregnant? = Yes

Q54 How many months did it take you to get pregnant the first time?

▼ Less than 3 months (1) ... more than 12 months (5)

Display This Question: If Did you get pregnant? = Yes

Q247 How many biological children do you have?

▼ currently pregnant (10) ... miscarried (9)

Display This Question: If How many biological children do you have? = 2 Or How many biological children do you have? = 3 Or How many biological children do you have? = 4 Or How many biological children do you have? = 5 Or How many biological children do you have? = 6 Or How many biological children do you have? = 7 Or How many biological children do you have? = 8 or more

Q240 Did you have any issues with your later pregnancies?

o Yes (1)

o No (2)

154 Q199 Did you need any fertility treatments?

o Yes (1)

o No (2)

Display This Question: If Did you need any fertility treatments? = Yes

Q200 Did that result in getting pregnant?

o Yes (1)

o No (2)

o No, but got pregnant naturally (3)

Display This Question: If How many biological children do you have? = 1 Or How many biological children do you have? = 2 Or How many biological children do you have? = 3 Or How many biological children do you have? = 4 Or How many biological children do you have? = 5 Or How many biological children do you have? = 6 Or How many biological children do you have? = 7 Or How many biological children do you have? = 8 or more

Q56 Have you had any miscarriages?

o Yes (1)

o No (2)

155 Display This Question: If Have you had any miscarriages? = Yes Or How many biological children do you have? = miscarried

Q239 How many miscarriages have you had?

▼ 1 (1) ... 5 or more (5)

Display This Question: If Did you need any fertility treatments? = Yes

Q203 Why did you use fertility treatments?

▢ Amenorrhea (1)

▢ > 6 months trying naturally with no success (2)

▢ > 12 months trying naturally with no success (3)

▢ Male factor (4)

▢ Other (5) ______

▢ Unsure (6)

Q255 Did you need fertility treatment for any subsequent pregnancies?

o Yes (1)

o No (2)

156 Display This Question: If Did you need fertility treatment for any subsequent pregnancies? = Yes

Q256 For what reason did you use fertility treatments?

▢ Amenorrhea (1)

▢ > 6 months trying naturally with no success (2)

▢ > 12 months trying naturally with no success (3)

▢ Male factor (4)

▢ Other (5) ______

▢ Unsure (6)

Display This Question: If Excluding pregnancy and breastfeeding, have you experienced 3 or more months at a time when you d... = Yes

Q206 If you had a history of amenorhea before getting pregnant, did you regain normal cycles after being pregnant and breastfeeding?

o Yes (1)

o No (2)

o Still pregnant or breastfeeding (3)

Q258 Please provide any additional information regarding your menstrual history

______

End of Block: If tried to get pregnant

157 Start of Block: Local Lab Visit

Q259 Are you currently living near the Columbus, Ohio area and interested in participating in a Local Lab Visit for a free body composition and bone density assessment?

o Yes (1)

o No (2)

Display This Question: If Are you currently living near the Columbus, Ohio area and interested in participating in a Local... = Yes

Q260 By providing your name and email address for us to contact you with further information, you will be linking your identity to this questionnaire. Please ONLY provide your name and email address if you are willing to have your questionnaire linked to you AND you are interested in participating in the body composition and bone density assessment.

______

End of Block: Local Lab Visit

158