MENSTRUAL BLOOD STEM CELL TO REDUCE CARDIOVASCULAR RISK IN

WOMEN

BY
XANDER BACCHUS

DATE: JUNE 14th 2018

DISSERTATION PRESENTED IN FULFILLMENT OF THE DEGREE

DOCTOR OF NATUROPATHIC MEDICINE

BLUE MARBLE UNIVERSITY

1
 Abstract

Women were found to be 44% more likely to have cardiovascular complications than their male

counterparts (Garcia et al., 2016). The purpose of this study explores the available literature on

menstrual cells (MenSCs) as used for regenerative therapies for positive cardiovascular

outcomes. Sources revealed the significance of the menstrual cycle in the reduction of ischemia

or acute heart disorders in women (Chidgey& Boyd, 2008; Patel et al., 2008),

Hence a quantitative analysis was conducted of a data set to determine the influence of two

stages in the menstrual cycle on cardiovascular outcomes. The data set was obtained from the

CardioVascular Research Grid following inclusion by Julian, Hecksteden, Fullagar, and Meyer

(2017) from a study conducted on the effects on the physical performance of female athletes

prior to and during the menstrual cycle in 2016. The participants were female athletes between

the ages of 18-25 who charted their menstrual cycle during the follicular and luteal phases of

their menstrual cycle following physical activity. The data’s original purpose was to determine

how the menstrual cycle would influence the athlete’s physical capability; however, the

information contained in the data set was used here to evaluate the influence of the menstrual

cycle on cardiovascular flow in correlation with the levels of estrogen and progesterone in the

athlete’s body.

This research results revealed not only can potential cardiovascular events be predicted within a

woman’s menstrual cycle, but that the increase of progesterone could be a significant hormone

treatment for people with cardiac risks. The review and analysis concludes that in the menstrual

recycling chain the obvious and readily available, non-invasive and regenerative properties of the

menstrual stem cell may be a more potent alternative for acute cardiac events.

2
 Table of Contents

List of Tables ................................................................................................. 4

List of Figures................................................................................................. 5
Chapter 1: Introduction to Study.................................................................... 6
Problem Statement................................................................................ 6
Purpose of Study................................................................................. 7
Chapter 2: Literature Review........................................................................ 10
Chapter 3: Research Methods...................................................................... 18
Data Processing................................................................................... 18
Chapter 4: Results........................................................................................ 20
Chapter 5: Discussion................................................................................... 27
Increase Estrogen in Follicular Phase Heart Rate Increases............... 28
Increase Estrogen in Luteal Phase Heart Rate Declines..................... 30
Increase Progesterone in Luteal Phase Heart Rate Declines.............. 30
Chapter 6: Conclusion and Recommendations ........................................... 32
Conclusions....................................................................................... 32
Implications and Recommendations for Future Studies.................... 33
References..................................................................................................... 35

3
 List of Tables

Table 1 Descriptive Statistics....................................................................... 20

Table 2 Bivariate Relationships................................................................... 21
Table 3 Multiple Regression Models........................................................... 22
Table 4 Heart Rate (LP) Coefficients.......................................................... 23
Table 5 Heart Rate (FP) Coefficients........................................................... 25

4
 List of Figures

Figure 1 Msc Extraction and Process............................................................... 7

Figure 2 Proliferation of Menstrual Cells........................................................ 16

5
 Chapter 1: Introduction to the Study

Problem Statement
As of 2016, cardiovascular disease accounted for as many as one in three deaths among

women in the US (Garcia, Mulvagh, Merz, Buring, J.E., & Manson, 2016). Cardiovascular

disease can often go undiagnosed in women who do not have significant risk factors like obesity,

smoking, pregnancy disorders, or diabetes (Garcia et al., 2016). In given risk factors that are

equal between men and women, women were found to be 44% more likely to have

cardiovascular complications than their male counterparts (Garcia et al., 2016). In addition,

women are more likely than men to contract autoimmune disorders, like arthritis or systemic

lupus erythematosus, due to the immune and vascular response system in women which may act

as escalator instead of inhibitor to cardiovascular concerns (Garcia et al., 2016). Though the lack

of diagnosis remains one of the most significant reasons for the higher deaths among women, the

reason that many women remain undiagnosed may be due, in part, to the efficacy of the

menstrual cycle in cardiovascular cell regulation (Khoury, Alcayaga-Miranda, Illanes, &

Figueroa, 2014). Indeed, research on progenitor cells for use in regenerative therapies suggest

that the regenerative capacity of the cells expelled during a woman’s menstrual cycle may have

previously obscured the rate of ischemic or acute heart disorders in women (Chidgey & Boyd,

2008; Patel, Park, Kuzman, Benetti, & Silva et al., 2008).

6
 Purpose of the Study

Mesenchymal stem cells (MSCs), or stem cells, are a regenerative cell set extracted from

humans in the form of tissue or fluid from embryonic, adipose, and dental tissue, bone marrow,

or menstrual blood (Khoury, Alcayaga-Miranda, Illanes, & Figueroa, 2014). These stem cells

have been used in regenerative capacities for many years; however, the use of stem cells in

cardiovascular therapies has been limited in scope and to the use of bone marrow or adipose

tissue only up until 2007 (Bockeria, Bogin, Bockeria, Le, &Alekyan et al., 2013). Figure 1 below

demonstrates the use of each MSC in relation to the source and process for extraction.

Figure 1: MSC Extraction & Process

Source: Khoury, Alcayaga-Miranda, Illanes, & Figueroa (2014, p. 2)

7
 As shown, ethical concerns exist only for embryonic tissue, while bone marrow, adipose,

and dental tissue are considered too invasive for practical use in regenerative therapies. Of the

options shown, only menstrual blood is considered ethical, has immune-modulation (capacity to

bypass the immune system), is not invasive, is autologous (can be collected from one individual

or many with the same effect), and can be extracted regularly through mensual collection.

(Khoury et al., 2014). In relation to other regenerative therapies, MenSCs remain the only option

with no side effects for extraction and contain the same, or better, progenitor cells (Patel et al.,

2008). In addition, early research showed the capacity for multipotent markers, which meant that

menstrual cells could be utilized in any other organ in the human body with the same effect

(Patel et al., 2008).

The timeline for advancements in the field of menstrual blood banking and usage is

marked by the increase in research articles in 2008, 2012, and 2015. Between 2008 and 2012,

scientists were determining plasticity of menstrual blood and predicting uses in regenerative

medicine (Patel et al., 2008; Lin et al., 2011), and by 2015, they were evaluating the benefit of

multi-lineage differentiation (Alcayaga-Miranda et al., 2015) and calculating the advantages over

that of cord or tissue due to the visible proliferation of cells found within menstrual blood

(Gajbhiye, 2016). Despite these discoveries, research on the use of menstrual cells in the

treatment of diseases not involving the reproductive organs has been limited. And, despite the

consistent advancements over the last 14 years, there remains a significant gap in the field of

cardiovascular heath involving regenerative outcomes. To follow is a discussion on the rise of

menstrual blood as one of the most promising regenerative therapies available for cardiovascular

diseases, including early failures by decades-long studies like that of the Women’s Health

8
Initiative, and a quantitative analysis of cardiovascular reductions from menstrual tissue as a

regenerative therapy as found in studies between 2008 and 2016.

9
 Chapter 2: Literature Review

Much of the available research in the field has been based on a cardiovascular dynamics

study conducted in 1974 which reported that “an increased cardiac output might play a part in the

development of systemic hypertension in some women receiving oral contraceptives and could

also add risk to women with pre-existing heart disease” (p. 567). This study remains the

foundation for risk factors in relation to cardiovascular disease and concerns when prescribing

women oral contraceptives (Shufelt& Merz, 2009). The results may have complicated the

progression of future studies which focused on the role of both estrogen and progestin in

cardiovascular health outcomes. The results of the Littler, Bueno, and Banks study inspired a

decade’s long observational study by the Women’s Health Initiative which proposed that a

combination of hormone therapies using estrogen and progestin in women following menopause

would reduce their risk, or potentially even begin to reverse, the onset of cardiovascular disease

(Rossouw, Anderson, Prentice, LaCroix, &Kooperberg et al., 2002). The study recruited 16,608

women ages 50-79 between the years 1993-1998 and administered placebo and a hormone

therapy using estrogen and progestin to a control and intervention group based on the concept

that “invasive breast cancer exceeded the stopping boundary for adverse effects and the global

index statistic supported risks exceeding benefits” (p. 321). Despite a promising start, the study

ended with complications and a higher percentage risk factor in which the determination was

made that “the risk-benefit profile found in this trial is not consistent with the requirements for a

viable intervention for primary prevention of chronic diseases, and the results indicate that this

regimen should not be initiated or continued for primary prevention of CHD” (Rossouw et al.,

2002, p. 321).

10
 However, the work upon which the Women’s Health Initiative study was built remained

tantalizing to scientists and medical review sources began promoting the benefits of estrogen in

combating cardiovascular diseases, noting that “as long as women are having regular menstrual

cycles, they enjoy a significant, although not absolute, level of protection” (Agatston, 2008, par.

1). In addition, “when a woman’s estrogen production plummets in her late forties to early fifties,

she begins to lose her hormonal advantage” (Agatston, 2008, par. 1). Though the science behind

statements like these remained fuzzy, at best, the concept was confirmed again and again in cases

where ischemic and acute cardiac episodes seemed to only occur in women following a

menstrual cycle. Further, the concept began to receive accolades following a study by Hida,

Nishiyama, Moyoshi, Kira, and Segawa et al. (2008) which found that “potent cardiac precursor-

like cells can be harvested from human menstrual blood [and] represents a new, noninvasive, and

potent source of cardiac stem cell therapeutic material” (p. 1695).

In addition, though this finding was significant to the field, it wasn’t until years later that

researchers turned their focus from rising and falling hormones during menstruation to the

occurrence of menstruation itself as the primary indicator of reduced cardiovascular occurrences.

For the purpose of this discussion, the menstrual cycle contains four phases: (1) menstrual phase,

(2) follicular phase, (3) ovulation phase – considered the end of the follicular phase, and (4) the

luteal phase. A woman’s cycle generally lasts around 28 days and the phase transitions may

blend together depending on the speed at which the woman’s hormones, mainly estrogen and

progesterone, rise and fall. Estrogen will generally be at its highest during the follicular phase,

while progesterone will generally be at its highest during the luteal phase. Available literature

breaks the menstrual cycle down into halves, wherein the follicular phase is contained within the

first half and the luteal phase is contained within the second half.

11
 Prior to the use as a regenerative treatment option, menstrual blood-driven stem cells

(MenSCs) were first considered an outcome in extrauterine growths as a means to regenerate the

endometrium and reverse disorders in the uterine lining like endometriosis and infertility

(Taylor, 2004). As research in the field continued, MenSCs were found to be more practical and

have far more uses in regenerative therapies than bone marrow or embryonic cells due to their

“ability to directly differentiate into cardiomyocytes, as well as to secrete angiogenic and trophic

factors, which assist in regeneration and possibly activation of endogenous cardiac stem cells”

(Bockeria et al., 2013, p. 2). This meant that the cells found in menstrual blood were more

equipped for regenerative uses, due to the speed at which the cells could determine the area of

concern and begin the process of regeneration or replacement of damaged tissues and cells. In

addition, MenSCs “appear to be immune privileged and immune modulatory [making them] poor

stimulators of allogeneic immunity and in many cases, [can] actively inhibit ongoing immune

responses” (Bockeria et al., 2013, p. 2). In other words, like embryonic cells MenSCs, could

bypass antigens without triggering a foreign-body response in the human’s immune system,

which commonly occurred in bone marrow transplants due to the inherent regenerative

properties of the cells (Chidgey & Boyd, 2008).

Further, though early research indicated that progenitor cells were the key to successful

stem and regenerative therapies, it wasn’t until 2004 that scientists discovered that progenitor

cells existed in menstrual blood and that menstrual blood could potentially be used for any

regenerative therapies currently requiring embryonic cells or bone marrow (Patel, Park, Kuzman,

Benetti, & Silva et al., 2008). One of the first significant discoveries in the field found that the

progenitor cells responsible for the regenerative properties within MenSCs may also be the

12
inciting factor in the onset of endometriosis (Yang & Huang, 2014), which led to the potential

regenerative relationship between MenSCs and other cell types.

Though this has not been explicitly stated in the literature, there may be a connection

between the menstrual cycle in women and a reduced rate of cardiovascular disease. A study in

2001 was the first to claim that the menstrual cycle regulated blood flow to the heart due to a rise

in ischemic episodes in perimenopausal women (Kawano, Motoyama, Ohgushi, Kugiyama, &

Ogawa et al., 2001). The authors found that not only were cases of ischemia following

menstruation more likely, but that the risk for blockage was exponentially reduced during

ovulation and prior to menstruation. The authors link this finding to the rise and fall of estrogen,

being higher during ovulation; however, research by Hamelin, Methot, Arsenault, Pilote, and

Poirier et al. (2003) later explained that the rise and fall of estrogen was not connected to the rate

at which blood was passed to and from the heart. Instead, as seen in every participant involved in

the study, the occurrence of any acute coronary event was exclusively linked to the first portion

of the menstrual cycle, or immediately following the participant’s menstruation (Hamelin et al.,

2003).

Advancements, and thus the promise of regenerative therapies by companies on a global

scale, flourished between 2004 and 2007, as both scientists and private corporations realized the

value of a resource that could be extracted with a near-exponential frequency. The

commercialization of menstrual blood as a regenerative therapy began with earnest in 2007

where companies, including a popular company known as C’elle, under the larger and publicly

traded corporation Cryo-Cell International, promised to extract and store personalized stem cells

from menstrual blood sent in by consumers to enable exact match treatment options similar to

that of bone marrow or embryonic stem cells at some point in the future (Cryo-Cell, 2016). At

13
the same time, a company known as Medistem began the manufacture of endometrial cells using

menstrual blood provided by a system of donors (Bockeria et al., 2013). The company

manufactured the endometrial cells into a proprietary formula called Endometrial Regenerative

Cells and entered into clinical trials as early as 2009 to test the use of endometrial cells on failing

heart systems (Bockeria et al., 2013). By 2012, Cryo-Cell ended C’elle and promoted banking

menstrual stem cells as one of several services including cord and tissue banking under one

banner. Indeed, this more comprehensive shift may have been due to C’elle’s poor market

performance or the damaging criticism that the company was relying on technology and science

that didn’t yet exist (Roberts, 2007). Similar companies, like Stemedica, StemCells, and Vet-

Stem also act as commercial banks for menstrual, cord, and/or stem cells; although Cryo-Cell

remains the highest valued commercial banking entity on the market as of 2017 (Owler, 2017).

Medistem, on the other hand, moved into a second phase of clinical testing in 2013, and

promised the creation of a universal donor system through which more than 20,000 people could

be cured through the donation of just one menstrual cycle extraction (Bockeria et al., 2013).

Bockeria et al. (2013) explain that the process of implanting endometrial cells was also far easier

than any other previous regenerative therapy, noting that “the use of a novel ‘off the shelf’ cell

together with a minimally invasive 30-minute delivery method provided a potentially paradigm-

shifting approach to cardiac regenerative therapy” (p. 1). However, research in cardiac cell

therapy and the application of regenerative therapies remain in the early stages and few studies

have passed the initial study stage or beyond the second phase in clinical trials. As Bockeria et

al. (2013) informs, “cardiac cell therapy is currently limited by efficacy and lack of scalable

delivery methods” (p. 5) and a means and framework to scale the delivery system remains

unfulfilled.

14
 Sources like Francis, Joel, and Mathew (2016) believe that the reason additional studies

have not been pushed through to clinical trials may be due to the stigma involved in the concept

of menstrual blood as a regenerative therapy. The authors note that this is a serious concern and

“generating more studies and proving its benefits in the curative aspects of major diseases could

result in the menstrual blood stem cells giving a promising future to many who are sick” (p. 5).

In addition, research by Lin, Ziang, Zhang, Allickson, and Xiang (2011) concluded that the study

of menstrual cells remains complicated by the lack of research on the outcome of cell population

in multiple circumstances. The authors note that “the population may involve many other cells

that have not been identified. Reports to date have not indicated whether a single cell or cells

with the same genotype possess these remarkable features of differentiation” (Lin et al., 2011, p.

378). By this, the authors are referring to the autologous aspect of menstrual cells, in which cells

can repeatedly be extracted from one human and provide the same results; while the inclusion of

multiple subjects from multiple races and ages may yield different results or indicate features

within the menstrual cells which are not capable of total regeneration.

Moreover, even studies with promising results have yielded reason for concern. In a

study of endometrial cells, it was found that cells performed in relation to regeneration

assumptions; however, a certain number of cells also grew with aberrant morphology and were

not capable of regeneration in the nature required (Rossignoli, Caselli, Grisendi, Piccinno, &

Burns et al., 2016). In relation to this conclusion, studies expanded the donor criteria to women

in specific age groups to determine whether the fertile state of the woman would yield higher

results in regeneration and a reduced rate of aberrant morphology. One study, finding that “the

proliferative activity of the cells obtained from the younger women (aged between 30 and 40

years) was more than that of the cells obtained from the older women (aged between 40 and 50

15
years)” (Mehrabani, Nazarabadi, Kasaraeian, Tamadon, &Dianatpour et al., 2016, p. 137) which

confirms the theory that cells from younger women, within the peak range of fertility, would

yield more promising results. In addition, the authors found that menstrual cells retrieved at peak

fertility yielded no “mutations or other visible abnormalities at the chromosomal level”

(Mehrabani et al., 2016, p. 138).

A study that was able to isolate cell function and generate a positive rate of regeneration

under several conditions found that there were still areas of cell expression that were lacking

(Alcayaga-Miranda, Cuenca, Luz-Crawford, Aguila-Diaz, & Fernandez et al., 2015).

Figure 2: Proliferation of Menstrual Cells

Source: Alcayaga-Miranda, Cuenca, Luz-Crawford, Auila-Diaz, & Fernandez et al. (2015, p. 7)

Figure 2, above, demonstrates the cell growth of MenSCs compared against other MSCs

like embryonic cells. As shown, MenSCs grew at exponential rates in the first image and

“showed significantly higher proliferation kinetics” (Alcayaga-Miranda et al., 2015, p. 7). The

second image shows the time elapsed and the difference in proliferation capacity when

compared. The authors explain that, “here, we show for the first time, that MenSCs possess

strong feeder properties as evidenced by a high expansion, maintenance of stemness, and

16
differentiation potential. Their support of other cultures was superior to that of any of the MSCs

[tested against] as well as a higher number of early progenitor colonies was observed”

(Alcayaga-Miranda et al., 2015, p. 12). The authors conclude that MenSCs “possess increased

proliferative, migration, and pro-angiogenic capacity in comparison to MSC populations

obtained from bone marrow tissues” (p. 12). In short, previous explorations of MenSCs may

have involved only analogous donors and therefore been unable to determine whether the

mutations or potential issues in regeneration were linked to one specific donor or, even more

consequentially, one specific age group. Therefore, “MenSCs can be obtained repetitively and

expanded to the larger quantities required for their therapeutic application” The authors

acknowledge that even if MenSCs did not display the same regenerative capacity as other MSCs,

the fact that they can be collected routinely and in near-exponential portions, makes MenSCs the

priority regenerative source for therapies like that for cardiovascular disease.

17
 Chapter 3: Research Methods

A quantitative analysis was conducted of a data set to determine the influence of two

stages in the menstrual cycle on cardiovascular outcomes. The data set was obtained from the

CardioVascular Research Grid following inclusion by Julian, Hecksteden, Fullagar, and Meyer

(2017) from a study conducted on the effects on the physical performance of female athletes

prior to and during the menstrual cycle in 2016. The participants were female athletes between

the ages of 18-25 who charted their menstrual cycle during the follicular and luteal phases of

their menstrual cycle following physical activity. The data’s original purpose was to determine

how the menstrual cycle would influence the athlete’s physical capability; however, the

information contained in the data set can also be used to evaluate the influence of the menstrual

cycle on cardiovascular flow in correlation with the levels of estrogen and progesterone in the

athlete’s body.

Data Processing

The data set was cleaned for missing values and clarified variable names. Participants

without a documented heart rate, estrogen, and progesterone value for both luteal and follicular

phase were removed. The dependent variable is the average heart rate during the follicular and

luteal phases and will be tested against the independent variables of estrogen (in follicular and

luteal phases) and progesterone (in follicular and luteal phases)to determine the significance of

fluctuations in the menstrual cycle against changes to the heart rate at luteal and follicular phase

averages. In addition, heart rate, estrogen, and progesterone were measured at normal states and

high states during both the luteal and follicular phases, providing an average range for all

variables and allowing for the influence of progesterone in the luteal phase to be compared

18
against heart rate from the follicular phase, for instance. Additional variables including age,

height, and weight will be used for classification and analysis of correlations. Following these

considerations, the data set was then analyzed using IBM SPSS ver. 21 for statistical significance

using bivariate relationships, multiple regression, and coefficient model analyses.

19
 Chapter 4: Results

An assessment of the descriptive statistics, shown in Table 1, below, indicates that the

participants, aged between 18 and 25, were between 155 cm and 170 cm tall, and weighed

between 70-86 kg. Heart rate during follicular and luteal phases was between 89-99 and 100-114,

respectively. Estrogen during follicular and luteal phases was between 14.26-69.11 and 90.66-

120.01, respectively; and progesterone during follicular and luteal phases was between 1.08-2.67

and 3.99-9.02. Standard deviation is relatively low for many of the variables, signaling a close

grouping of data points; however, estrogen (FP) and estrogen (LP) had high standard deviations

which signals a wide spread between the data points. Wide disparity between data points would

generally indicate that participants had wildly fluctuating estrogen measurements at both

follicular and luteal phases, but even so deviations would be indicative of the general health and

hormone ranges between participants.

Table 1: Descriptive Statistics

Minimum Maximum Mean Std. Deviation

Age 18 25 21.54 2.23
Height 155 170 165.85 3.58
Weight 70 86 78.38 4.19
Heart Rate (FP) 89 99 95.15 2.92
Heart Rate (LP) 100 114 105.15 4.72
Estrogen (FP) 14.26 69.11 40.56 19.23
Estrogen (LP) 90.66 120.01 110.39 7.43
Progesterone (FP) 1.08 2.67 1.56 0.45
Progesterone (LP) 3.99 9.02 7.15 1.25

For the purpose of comparative analysis during the discussion to follow, age height and

weight have been tallied. Of the participants, 10 were between the ages of 18-20, 10 were

between the ages of 21-23, and 6 were between the ages of 24-25. Height in centimeters ranged

with 7 participants between 155-163, 9 between 164-167, and 10 between 169-170. Finally, 6

20
participants weighed between 70-75 kg, 13 participants weighed between 76-80 kg, and 7

weighed between 81-86 kg. In a quick correlation model, height was the most varied indicator to

weight; although none of the results were statistically significant. Based on this, age, weight, and

height are not correlated in any statistically significant way and may only be used at the

discretion of the researcher for the classification of results within the discussion.

Following this, correlations were found by testing for bivariate relationships, as shown in

Table 2 below.

Table 2: Bivariate Relationships

Heart Heart Estrogen Estrogen Progeste Progeste

Rate Rate (FP) (LP) rone (FP) rone
(FP) (LP) (LP)

Heart Rate Pearson’s r 1.00 .508 .602 -.515 .015 -.479

(FP)
Significance 1.00 .008 .001 .007 .941 .013

Heart Rate Pearson’s r .508 1.00 .386 -.482 -.033 -.575

(LP)
Significance .008 1.00 .052 .013 .873 .002

Estrogen Pearson’s r .602 .386 1.00 -.716 .208 -.536

(FP)
Significance .001 .052 1.00 .000 .307 .005

Estrogen Pearson’s r -.515 -.482 -.716 1.00 .012 .474

(LP)
Significance .007 .013 .000 1.00 .953 .015

Progesterone Pearson’s r .015 -.033 .208 .012 1.00 .306

(FP)
Significance .941 .873 .307 .953 1.00 .129

Progesterone Pearson’s r -.479 -.575 -.536 .474 .306 1.00

(LP)
Significance .013 .002 .005 .015 .129 1.00

21
 Significant correlations, highlighted above, are shown at the 0.01 level using the 2-tailed

test. Of the fifteen marked significant correlations, there are three of greatest significance

between the dependent and independent variables, including: (1) estrogen (FP) and heart rate

(LP) at 0.001, (2) estrogen (LP) and heart rate (FP) at .007, and (3) progesterone (LP) and heart

rate (LP) at 0.002. There are also significant interactions seen between the independent variables;

however, these relationships are not within the scope of this discussion. As shown, the

correlations indicate that estrogen (FP) has a positive correlation to heart rate (FP), estrogen (LP)

has a negative correlation to heart rate (FP), and progesterone (LP) has a negative correlation to

heart rate (LP). Therefore, three base conclusions can be made: (1) when estrogen increases in

the follicular phase, so does heart rate, (2) when estrogen increases in the luteal phase, the heart

rate will decline, and (3) when progesterone increases in the luteal phase, the heart rate will

decline. Next, a model summary has been conducted using linear regression to determine the

percentage of significance between the dependent and independent variables.

Table 3, below, is split into two separately conducted models to determine the

relationship between the independent variables and each of the dependent variables using

multiple regression. First, each of the independent variables were tested against heart rate (FP),

and then tested against heart rate (LP), as indicated.

Table 3: Multiple Regression Models

Model R R Square R Square Change Durbin-Watson

Heart Rate (FP) 1 0.602 0.362 0.362

2 0.614 0.377 0.014

3 0.620 0.384 0.008

4 0.637 0.405 0.021 1.287

22
Heart Rate (LP) 1 0.386 0.149 0.149

2 0.486 0.236 0.087

3 0.488 0.238 0.002

4 0.649 0.421 0.183 2.051

(1) estrogen (FP)

(2) estrogen (FP), estrogen (LP),
(3) estrogen (FP), estrogen (LP), and progesterone (FP)
(4) estrogen (FP), estrogen (LP), progesterone (FP), and progesterone (LP)

Each model is entered at each new stage against the previously used variables. For heart

rate (FP), model 1 results in an R square of 0.362, meaning that the variable of estrogen (FP)

accounts for 36.2% of the heart rate (FP). Each model to follow accounts for more and more of

the heart rate (FP) variable, to a maximum of 40.5%. In addition, the R square change field

indicates that average heart rates (FP) from the last model were 4.3% different from the first

model. For heart rate (LP), model 1 results in an R square of 0.386, wherein estrogen (FP)

account for 38.6% of the heart rate (LP). The maximum accounted for between the dependent

and independent variables is 64.9%, with an R square difference of 26.3%. The Durbin-Watson

of 1.287 and 2.051 are both rated as expected and mean that all of the independent variables are

at least minimally significant to the dependent variables. Based on these models, the independent

variables as a group are more significant to the heart rate (LP) than the heart rate (FP); and

further, the difference indicated by the change in R square shows a 22% difference between the

influence of the variables on heart rate (LP) over the heart rate (FP).Finally, coefficients were

tested using the same models as above as shown in Table 4 and Table 5, to follow.

Table 4: Heart Rate (LP) Coefficients

Model B Std Coeff. Beta T test Significance alpha <0.05

23
1 Estrogen (FP) 0.094 0.386 2.047 0.052

2 Estrogen (FP) 0.020 0.082 0.316 0.755

Estrogen (LP) -0.269 -0.423 -1.622 0.118

3 Estrogen (FP) 0.025 0.105 0.373 0.713

Estrogen (LP) -0.258 -0.407 -1.484 0.152

Progesterone (FP) -0.515 -0.050 -0.255 0.801

4 Estrogen (FP) -0.060 -0.245 -0.861 0.399

Estrogen (LP) -0.243 -0.382 -0.861 0.399

Progesterone (FP) 2.091 0.203 1.011 0.324

Progesterone (LP) -2.202 -5.87 -2.574 0.018

(1) estrogen (FP)

(2) estrogen (FP), estrogen (LP),
(3) estrogen (FP), estrogen (LP), and progesterone (FP)
(4) estrogen (FP), estrogen (LP), progesterone (FP), and progesterone (LP)

As shown in Table 4, above, the value for model 4 in unstandardized B (shown as B in

the table) indicates that, as progesterone (LP) increases, the heart rate (LP) will decrease by a

value of -2.202. For the same model, standard coefficients beta indicates that an increase in

progesterone (FP) is more important to one standard deviation of change in the dependent

variable than any other independent variable. In addition, model 4 demonstrates a rise and fall in

progression once each new independent variable is added in the successive model, as seen in the

standard coefficients beta column. For instance, model 4 begins with estrogen (FP) at -0.245,

decreases to -0.382 with estrogen (LP), increases to .203 with progesterone (FP), and decreases

significantly once more to -5.87 with progesterone (LP). These rapid transitions indicate that

each new independent variable has a variable influence on the importance of one standard of

deviation. Given the first and second variable additions, the drastic escalation to 0.203 with

24
progesterone (FP) indicates that estrogen was less and less significant to one standard deviation

until the addition of progesterone (FP), which increased the significance of the model rapidly.

The incredible drop off to -5.87 then indicates that progesterone (LP) is the least significant to

one standard deviation. Ultimately, this showcases the influence of the models and the impact of

each successive independent variable on the rate of change in the dependent variable. The t test

also indicates that progesterone (FP), at 1.011, is the most significant of the independent

variables. Finally, the significance column indicates that progesterone (LP), at 0.018, is the most

statistically significant.

Table 5: Heart Rate (FP) Coefficients

Model B Std Coeff. Beta T test Significance alpha <0.05

1 Estrogen (FP) 0.071 0.602 3.693 0.001

2 Estrogen (FP) 0.057 0.479 2.031 0.054

Estrogen (LP) -0.053 -0.172 -0.729 0.473

3 Estrogen (FP) 0.061 0.520 2.061 0.051

Estrogen (LP) -0.044 -0.142 -0.575 0.571

Progesterone (FP) -0.458 -0.091 -0.519 0.609

4 Estrogen (FP) 0.047 0.401 1.390 0.179

Estrogen (LP) -0.041 -0.133 -0.537 0.597

Progesterone (FP) -0.030 -0.006 -0.029 0.977

Progesterone (LP) -0.362 -0.199 -0.860 0.399

(1) estrogen (FP)

(2) estrogen (FP), estrogen (LP),
(3) estrogen (FP), estrogen (LP), and progesterone (FP)
(4) estrogen (FP), estrogen (LP), progesterone (FP), and progesterone (LP)

25
 As shown in Table 5, above, the value for model 4 in unstandardized B indicates that, as

progesterone (LP) increases, the heart rate (FP) will decrease by a value of -0.362. For the same

model, standard coefficients beta indicates that an increase in estrogen (FP) is more important to

one standard deviation of change in the dependent variable than any other independent variable.

In addition, model 4 demonstrates a rise and fall in progression once each new independent

variable is added in the successive model, as seen in the standard coefficients beta column. For

instance, model 4 begins with estrogen (FP) at 0.401, decreases to -0.133 with estrogen (LP),

increases to -0.006 with progesterone (FP), and decreases significantly to -0.199 with

progesterone (LP). These transitions indicate that each new independent variable is more or less

important to one standard of deviation than the previous models. Based on these numbers, the

indication is made that estrogen (FP) is the most significant to one standard of deviation, then

significance drops until progesterone (FP), finally dropping off once more at the final variable.

Finally, the t test indicates that estrogen (FP), at 1.390, is the most significant of the independent

variables; while the significance values indicate that all of the variables run during this model

suggests that there may be more than one variable required to have legitimate significance.

26
 Chapter 5: Discussion

The purpose of this study was to explore the available literature on menstrual cells

(MenSCs) as used for regenerative therapies for positive cardiovascular outcomes. Sources

explored the significance of the menstrual cycle in the reduction of ischemia or acute heart

disorders in women (Chidgey& Boyd, 2008; Patel et al., 2008), and the documented link found

between the menstrual cycle’s occurrence and acute coronary events (Hamelin et al., 2003).

Notably, the research also explored the significance of the reduction of cardiovascular events

during or immediately following menstruation; an aspect that sources like Agaston (2008)

consider to be a hormonal advantage of being female. In addition, the results of the data analysis

indicated the formation of three base inclusions, (1) when estrogen increases in the follicular

phase, so does the heart rate, (2) when estrogen increases in the luteal phase, the heart rate will

decline, and (3) when progesterone increases in the luteal phase, the heart rate will decline.

First, it is notable that, across all the data sets, there was only a 4.3% difference between

the first participant and the last in relation to heart rate during the follicular phase. The

implications of this is difficult to judge; however, the assumption could be made that regardless

of the increase seen in estrogen or progesterone, there will be a corresponding but not relative

increase in the heart rate as well. The assumption, then, is that the heart rate will rise to a certain

range regardless of the rate of increase seen in the estrogen and progesterone. None of the

sources reviewed for this study reference this exactly; and further, this link may have been useful

to many of the studies and may have influenced the direction of other studies in the field.

Further, the maximum influence on the heart rate in both the follicular and luteal phases was

27
64.9% from all of the independent variables. This allows for the conclusion that around 35.1% of

changes in the heart rate between follicular and luteal phases is reliant on other factors not

collected or studied.

Further, the range for increase or decrease seen in the heart rate during both follicular and

luteal phases was nearly the same. As reported, estrogen in the follicular phase accounted for

36.2% of the heart rate increase, while estrogen in the luteal phase accounted for 38.6% of the

heart rate decrease. As an independent variable, this could imply that the rate of estrogen led to a

proportional increase followed by a proportional decrease during the rise seen in the follicular

phase and the decline seen in the luteal phase. Despite this relational percentage, the data also

indicated that there was a 22% difference between the influence of the independent variables on

the heart rate during the luteal phase versus the heart rate in the follicular phase. This means

that, despite the seemingly relational movements caused by estrogen, the movements were

actually more significant with greater range. Ultimately, this would indicate that the levels of

estrogen and progesterone were at least 22% different at follicular and luteal phases and yet, the

heart rate would end up within a particular range 95.7% of the time.

Increase Estrogen in Follicular Phase, Increase Heart Rate

Hamelin et al. (2003) explores the context of the follicular phase within documented

acute coronary events and explains that “all of the acute coronary events occurred within the first

phase of the menstrual cycle compared with no events during the second half of the menstrual

cycle” (p. 600), indicating that every coronary event documented in their study was contained

only within the follicular phase of the menstrual cycle. Further, “significantly more women, 19

28
women out of 27, had their acute coronary event within 6 days after the onset of menstruation

than later during the menstrual cycle” (Hamelin et al., 2003, p. 600). Generally, estrogen is at its

highest during the follicular phase, and based on the data explored in the Results, was seen rising

during the follicular phase with an onset increase in the participants’ heart rates. Based on this

information, the estrogen increases seen during the follicular phases are not only responsible for

the degree of increase seen in the heart rate, but also the potential for the onset of acute coronary

events. This could lead to the implication that, if a woman is going to have a heart attack, it is

going to be during the first phase of her menstrual cycle. If she has passed into the second phase

of her cycle, she is likely at significantly reduced risk for a coronary event.

Moreover, as seen in the data, the impact of estrogen during the follicular phase was

shown to create a proportional increase in the participant’s heart rate and accounted for as much

as 36.2% of the heart rate’s increase during the follicular phase. Though certainly other

assumptions could be made as to the influence of estrogen on cardiovascular events, the reality is

that more than a third of a woman’s heart rate is linked to her levels of estrogen following

menstruation. As Hamelin et al. (2003) explored, “there is an increased vulnerability during and

immediately after menses” (p. 601). However, the vulnerability is the body’s physiological

response to the tasks required during and immediately following menstruation during which the

follicular phase must undertake the massive responsibility of producing enough estrogen to build

and grow the uterine lining prior to ovulation (Khoury et al., 2014). As can be expected, the

proliferation of the endometrium creates a much higher rate of blood flow, thus increasing the

woman’s heart rate for a significant degree during the entirety of the follicular phase.

29
Increase Estrogen in Luteal Phase, Heart Rate Declines

Estrogen, though primarily responsible for work during the follicular phase, does have a

role to play in the luteal phase as well. As seen in the data analysis, estrogen was seen increasing

during the luteal phase and was correlational to a decline in the heart rate during the same phase.

Technically, the decline in the heart rate might be related to the function of the luteal phase itself

and have less to do with the fluctuations of estrogen. Research by Seippel and Backstrom (1998)

explored the connection between menstrual cycle emotions and noted that “in anovulatory

cycles, symptom cyclicity disappears and there exists a consensus that the cyclical changes are

provoked by factors from the corpus luteum” (p. 1988). Anovulatory cycles are those in which

the woman’s ovaries do not produce an egg, and in some cases, may cause breakthrough

bleeding prior to the onset of the actual menstrual phase (Seippel&Blackstrom, 1998). The

importance of this, then, is that the production of estrogen in the luteal phase would most likely

be linked to anovulatory cycles, or those in which there are issues with the production and

maintenance of the uterine lining and the body begins preparations for an early, or breakthrough,

menstrual phase.

Increase Progesterone in Luteal Phase, Heart Rate Declines

Progesterone is in highest supply during the luteal phase, during which the body is

preparing the uterine lining for a potential pregnancy while waiting to see whether an egg is

fertilized. As the luteal phase moves toward its end, the endometrium is at its thickest

(Seippel&Blackstrom, 1998; Hamelin et al., 2003). As seen in the data analysis, as progesterone

30
in the luteal phase increased, the heart rate would show a correlational decrease by a value of 2.2

beats per minute. During the entirety of the luteal phase, then, as progesterone increases within

the corpus luteum, the heart rate decreases to a correlational degree due to the function of the

phase in the menstrual cycle (Al-Asmakh, 2007). If the woman becomes pregnant, progesterone

will continue to rise from the corpus luteum until the placenta can take over production (Al-

Asmakh, 2007). Therefore, the rise in progesterone during the luteal phase is due to the function

of the luteal phase and the shift in blood flow which would prompt a lowered heart rate during

that phase.

31
 Chapter 6: Conclusion and Recommendation

Conclusions

The research began with a goal to identify the role of menstrual blood in regenerative

therapies for cardiovascular events. As found during the process of research and data analysis,

the menstrual cycle plays a significant and primary role in the totality of function of the

cardiovascular system. The links between estrogen and progesterone were connected to a rise

and fall of the heart rate, respectively, and rationale for why this may occur was explored and

connected to the data analysis. Of primary interest was the connection seen between the

follicular phase and the onset of increased heart rates in every participant with a degree of

change within 4.3%. This indicated that, despite the fluctuations between the physiological and

hormonal systems of every participant, every heart rate charted rose within a very specific range

every single time. This demonstrates that not only is estrogen responsible for the heart rate

increase, but that estrogen plays a significant role in overall cardiovascular health and outcomes.

The same was seen with the rise of progesterone and the decrease in the heart rate of every

participant during the luteal phase.

In short, this analysis lends to the assumption that not only can potential cardiovascular

events be predicted within a woman’s menstrual cycle, but that the increase of progesterone

could be a significant hormone treatment for people with cardiac risks. In addition to this, the

influence of menstrual stem cells could prove instrumental in reducing cardiovascular events and

may even provide positive outcomes for people with higher risks or previous acute cardiac

events. Ultimately, this research has shown the connection between the hormones estrogen and

progesterone during the menstrual cycle and has explored the current and available research on

32
the application of MenSCs as a regenerative therapy for cardiovascular events. It is believed that

this realm of research should continue and applications be made for the use of MenSCs in

cardiovascular treatment research.

Implications & Recommendations for Future Study

The rate of increase seen in the estrogen and progesterone data holds an impact on the

heart rate across both follicular and luteal phases due to the nature of the rise and the nature of

the fall. In other words, though the actualized increase and decrease seen in the heart rate is

explicitly linked to the rise and fall of estrogen and progesterone, there is some holdover of the

hormones that may explain the degree to which the heart rate rises or falls. It is addressed in the

literature as an “apparent paradox [which] may be explained by a latent interval between the

effects of decreasing estrogen levels on vulnerable plaque and the clinical occurrence of the

acute coronary event” (Hamelin et al., 2003, p. 600).This; however significant, cannot be tested

with the data and would require future studies or a greater number of participants.

In addition, incidental connections between the independent variables, while outside the

scope of this current study, do lend to the implication that there is room for future studies to

explore and continue to find relationships between hormones and cardiovascular features and

health outcomes. Further, there is believed to remain a stigma involved in the concept of using

MenSCs as a regenerative therapy (Francis et al., 2016) which may extend to the lack of research

conducted on the outcome of cell population by non-autologous samples (Lin et al., 2011) due to

the impression of impracticality involved in collecting samples from multiple sources over an

extensive period of time.

33
34
 References

Agatston, A. (2008). Women, hormones, and heart disease. Everyday Health. Retrieved May 18,

2018 from, https://www.everydayhealth.com/heart-health/women-hormones-heart-

disease.aspx

Al-Asmakh, M. (2007). Reproductive functions of progesterone. Middle East Fertility Society

Journal, 12(3), 147-152.

Alcayaga-Miranda, F., Cuenca, J., Luz-Crawford, P., Aguila-Díaz, C., Fernandez, A., Figueroa,

F. E., & Khoury, M. (2015). Characterization of menstrual stem cells: angiogenic effect,

migration and hematopoietic stem cell support in comparison with bone marrow

mesenchymal stem cells. Stem Cell Research & Therapy, 6(1), 32.

http://doi.org/10.1186/s13287-015-0013-5

Bockeria, L., Bogin, V., Bockeria, O., Le, T., Alekyan, B., Woods, E., Brown, A., Ichim, T., &

Patel, A. (2013). Endometrial regenerative cells for treatment of heart failure: A new

stem cell enters the clinic. Journal of Translational Medicine, 11(1), 1-8.

https://doi.org/10.1186/1479-5876-11-56

Chidgey, A.P., & Boyd, R.L. (2008). Immune privilege for stem cells: Not as simple as it looked.

Cell

Cryo-Cell International. (2016). The menstrual blood banking process. Cryo-Cell International.

Retrieved May 17, 2018 from, https://www.cryo-cell.com/menstrual-stem-cell-banking

Francis, F., Joel, S.E., & Mathew, A. (2016). Menstrual blood banking: A concept ‘best out of

waste’ in the area of stem cell research. Journal of Biomedical and Applied Sciences, 3,

1-8. https://doi.org/10.15520/.v3i0.11

35
Gajbhiye, A. (2016). Menstrual blood banking. Panacea Journal of Medical Sciences, 6(2), 57-

58. Doi: 10.5958/2348-7682.2016.00003.8

Garcia, M., Mulvagh, S.L., Merz, N.B., Buring, J.E., & Manson, J.E. (2016). Cardiovascular

disease in women: Clinical Perspectives. Circulation research, 118, 1273-1293. doi:

10.1161/CIRCRESAHA.116.307547.

Hamelin, B.A., Methot, J., Arsenault, M., Pilote, S., Poirier, P., Plante, S., &Bogarty, P. (2003).

Influence of the menstrual cycle on the timing of acute coronary events in premenopausal

women. The American Journal of Medicine, 114, 599-602. doi:10.1016/S0002-

9343(03)00051-2

Hida, N., Nishiyama, N., Miyoshi, S., Kira, S., Segawa, K., Uyama, T., Mori, T., Miyado, K.,

Ikegami, Y., Cui, C., Kiyono, T., Kyo, S., Shimizu, T., Okano, T., Sakamoto, M., Ogawa,

S., &Umezawa, A. (2008). Novel cardiac precursor-like cells from human menstrual

blood-derived mesenchymal cells. Stem Cells Express, 1695-1704.

Julian, R., Hecksteden, A., Fullagar, H.H., & Meyer, T. (2017). The effects of menstrual cycle

phase on physical performance in female soccer players. PLOSone. Retrieved from

https://doi.org/10.1371/journal.pone.0173951.s001

Kawano, H., Motoyama, T., Ohgushi, M., Kugiyama, K., Ogawa, H., & Yasue, H. (2001). Blood

flow to the heart varies during the menstrual cycle in premenopausal women with heart

disease. Annals of Internal Medicine, 135, 977-981.

Khoury, M., Alcayaga-Miranda, F., Illanes, S.E., & Figueroa, F.E. (2014). The promising

potential of menstrual stem cells for antenatal diagnosis and cell therapy. Frontiers in

Immunology, 5(205), 1-8. doi: 10.3389/fimmu.2014.00205

36
Littler, W.W., Bojorges-Bueno, R., & Banks, J. (1974). Cardiovascular dynamics in women

during the menstrual cycle and oral contraceptive therapy. Thorax, 29, 567-570. doi:

10.1136/thx.29.5.567

Lin, J., Ziang, D., Zhang, J., Allickson, J., & Xiang, C. (2011). Plasticity of human menstrual

blood stem cells derived from the endometrium. Journal of Zhejiang University Science,

12(5), 372-380. doi: 10.1631/jzus.B1100015

Mehrabani, D., Nazarabadi, R.B., Kasraeian, M., Tamadon, A., Dianatpour, M., Vahdati, A.,

Zare, S., &Ghobadi, F. (2016). Growth kinetics, characterization, and plasticity of human

menstrual blood stem cells. Iranian Journal of Medical Science, 41(2), 132-139.

Owler. (2017). Cryo-Cell competitors, revenue, etc. Owler. Retrieved May 18, 2018 from,

https://www.owler.com/company/cryo-cell

Patel, A.N., Park, E., Kuzman, M., Benetti, F., Silva, F.J., &Allickson, J.G. (2008). Multipotent

menstrual blood stromal stem cells: Isolation, Characterization, and differentiation. Cell

Transplantation, 17, 303-311.

Roberts, M. (2007). Experts have expressed concern over the launch by a US company of a

service for women to store their own menstrual blood. BBC News. Retrieved May 17,

2018 from, http://news.bbc.co.uk/2/hi/health/7086548.stm

Rossignoli, F., Caselli, A., Grisendi, G., Piccinno, S., Burns, J.S., Murgia, A., Veronesi, E.,

Loschi, P., Masinin, C., Conte, P., Paolucci, P., Horwicz, E.M., &Dominici, M. (2016).

Isolation, Characterization, and Transduction of Endometrial Decidual Tissue

Multipotent Mesenchymal Stromal/Stem Cells from Menstrual Blood. BioMed Research

International, 13, 1-14. https://doi.org/10.1155/2013/901821.

37
Rossouw, J.E., Anderson, G.L., Prentice, R.L., LaCroix, A.Z., Kooperberg, C., Stefanick, M.L.,

Jackson, R.D., Beresford, S.A., Johnson, K.C., Kotchen, J.M., &Ockene, J. (2002). Risks

and benefits of estrogen plus progestin in healthy postmenopausal women: Principal

results from the Women’s Health Initiative randomized controlled trial. Journal of

American Medical Association, 288(3), 321-332.

Seippel, L., & Backstrom, T. (1998). Luteal-phase estradiol relates to symptom severity in

patients with premenstrual syndrome. Journal of Clinical Endocrinology and

Metabolism, 83(6), 1988-1992.

Shufelt, C.L., & Merz, N.B. (2009). Contraceptive hormone use and cardiovascular disease.

Journal of the American College of Cardiology, 53(3), 221-231. doi:

10.1016/j.jacc.2008.09.042

Taylor, H.S. (2004). Endometrial cells derived from donor stem cells in bone marrow transplant

recipients. JAMA, 292(1), 81-85. doi:10.1001/jama.292.1.81

Yang, J., & Huang, F. (2014). Stem cell and endometriosis: New knowledge may be producing

novel therapies. International Journal of Clinical and Experimental Medicine, 7(11),

3853-3858.

38