Cilostazol and its effects on human oocyte maturation in vivo: a pilot study

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Cilostazol and its Effects on Human Oocyte Maturation in Vivo: A
Pilot Study
Master of Clinical Science (MsC) Degree Dissertation
December 2014
Dr. Laura Sech
Dr. Daniel Mishell, Jr., Dr. Richard Paulson, Dr. Kristen Bendikson
University of Southern California
December 2014
2
ABSTRACT
Background: Combined hormonal contraceptive pills (COCs) are the most frequently used
method of contraception by women in the United States. However, COCs are frequently
discontinued secondary to hormonal side effects. Moreover, COCs are contraindicated in women
with certain medical problems. Development of a non-hormonal birth control pill could
overcome these problems. In recent studies, phosphodiesterase 3 inhibitors (PDE3s) have been
shown to inhibit oocyte maturation and impair fertility in macaques, mice, and pigs. Cilostazol,
a class IIIa PDE, is currently FDA approved for the treatment of intermittent claudication.
Additionally, this drug has demonstrated fertility impairment, despite the occurrence of
ovulation, with complete reversibility in both mice and pigs. To date, no in vivo studies of
Cilostazol in humans have been undertaken to determine its effects on oocyte maturation.
Methods: This research design is a pilot study that enrolled 4 healthy women who have
previously been oocyte donors. All participants were given the study drug, Cilostazol, at the
current FDA approved dose of 100mg PO BID. The study drug was administered twice daily
during the 10-14 days of ovarian stimulation with hMG until ovulation was induced with hCG.
Participants underwent oocyte and follicular fluid retrieval following cycle stimulation. The
stage of oocyte maturation (Germinal vesicle (GV), Metaphase I (MI), Metaphase II (MII)) was
evaluated at the time of ovum retrieval and at 24 hours. The oocyte maturational stages obtained
from subjects during Cilostazol treatment were compared to those documented from these same
subjects during oocyte donation from a previous stimulation cycle while not taking study drug.
Additionally, plasma as well as follicular fluid cilostazol levels were collected for analysis.
Results: There was not a significant difference in percent of oocyte maturation retrieved at
baseline vs during treatment with Cilostozol (t3 = 0.36, p = 0.74). Plasma and follicular fluid
levels of Cilostazol were obtained and a small correlation between cilostazol levels in both the
follicular fluid and plasma and oocyte maturation was demonstrated. There was a significant
difference in diastolic blood pressure (DBP) (p = 0.008) and heart rate (HR) (p = 0.027) from
baseline to measurements made during the study. These findings are likely not clinically
relevant.
Conclusions: Our findings indicate that the FDA approved dose of Cilostazol, given at 100mg
PO BID, does not impair oocyte maturation in the human. Future studies using a higher dose of
Cilostazol are indicated but this may prove difficult given potential for toxicity at higher doses.
December 2014
3
INTRODUCTION
! Combined oral contraceptive pills (COCs) are the most commonly used hormonal form
of birth control in the United States with at least 87% of women of reproductive age reporting
oral contraceptive use at some point in their lives (9). However, COCs are contraindicated in
certain groups of women as outlined by The Centers for Disease Control Medical Eligibility
Criteria (11). Given the high prevalence of oral contraceptive users who commonly discontinue
use secondary to side effects (17) or who are not eligible for use as a result of underlying health
conditions, the development of novel oral non-hormonal methods that are equally effective at
pregnancy prevention are warranted.
Phosphodiesterase inhibitors (specifically PDE3 isoforms) have been studied in animals
such as monkeys, mice, and pigs with significant findings of impaired fertility noted (1,2,7,8,10).
The mechanism of action attributed to the impaired fertility is secondary to inhibition of oocyte
maturation rendering the ovum released from the dominant follicle at the time of ovulation
incapable of being fertilized. This mechanism of action is thought to be secondary to the
increased oocyte intracellular cAMP levels that arise when phosphodiesterase fails to breakdown
cAMP to cellular AMP (3,6). Two studies have been conducted using human oocytes with
findings of impaired oocyte maturation when cultured in vitro (12,14). No in vivo treatment of
humans has been conducted using a PDE3 with subsequent assessment of oocyte maturation.
The PDE3-inhibitor Cilostazol has been studied in mice and pigs demonstrating a similar
effect to previous studies in monkeys (7,8,9) conducted using other PDE3s-inhibitors such as
ORG 9935 (1,2,11). Cilostazol is currently FDA approved for the treatment of intermittent
claudication and has been shown to have additional health benefits (4). A recent study conducted
in mice using cilostazol showed both in vitro and in vivo inhibition of oocyte maturation with no
significant increase in heart rate or decrease in blood pressure (10). Furthermore, the most
promising study to date examining pregnancy rates of mice treated with cilostazol showed 100%
efficacy at pregnancy prevention with 100% demonstrable reversibility (1,10). No adverse
events were reported in the mice treated with this drug.
As an initial step, the present study aims to address the effects of Cilostazol on human
oocyte maturation in vivo. This effect has never been studied to date in humans. Our primary
objective is to determine if women taking Cilostazol demonstrate impairment of oocyte
maturation in comparison to paired historic controls following ovarian follicle stimulation. For
the purposes of our study, oocyte maturation is assessed using the oocyte maturational stages of
prophase I, metaphase I, and metaphase II. Thus, we will be assessing the percent of oocytes
aspirated from follicles during controlled ovarian stimulation that remain in the Germinal Vesicle
stage (prophase I) and compare this to the maturation of oocytes obtained from the same women
who underwent ovarian follicle stimulation previously while not on study drug. We propose that
women undergoing treatment with the FDA approved dose of 100mg PO BID of Cilostazol will
demonstrate an impairment of oocyte maturation in comparison to paired historic controls
following ovarian follicle stimulation.
December 2014
4
METHODS
Study participants:

Following institutional review board approval, participants from the University of
Southern California Fertility clinic were recruited. Participant recruitment from this population
was necessary because data regarding oocyte maturational stages up to 24 hours following
retrieval in the absence of study drug were available for all study participants. Additionally,
having already acted as oocyte donors, participants understood the risks inherent to oocyte
donation and were well informed regarding hormonal side effects during cycle stimulation.
Women were recruited via telephone contact if they met primary eligibility criteria. Criteria for
inclusion were: previous oocyte donors to the USC IVF clinic within past 3 years, age 18-33,
willingness and ability to commit to the time requirements of the study, willingness to donate
oocytes for research purposes, willingness to discontinue current hormonal contraception, and
otherwise healthy subjects. Criteria for exclusion were: any contraindications to combined
hormonal contraceptive use (CDC MEC class 3 or 4), pregnancy, history of cardiac arrhythmias,
history of heart failure, history of bleeding disorder, concomitant use of anti-platelet therapy such
as aspirin, current use of drugs that inhibit cytochrome P450 CYP 3A4 (erythromycin, diltiazem,
ketoconazole, itraconazole) or CYP 2C19 (omeprazole) as they may lead to increased serum
levels of cilostazol, bleeding peptic ulcer disease, history of intracranial bleeding, or any
hypersensitivity reactions to cilostazol. Thirty women were eligible for study enrollment from
this population, and 4 women were ultimately enrolled. Reasons to decline study participation
included currently undergoing cycle stimulation for oocyte donation, inconvenience, or lack of
sufficient compensation.
Oocyte stimulation:

After screening and informed consent, study participants underwent a baseline
transvaginal ultrasound. Initial blood pressure and heart rate was monitored on the day of study
onset and throughout the treatment phase visits. The protocols for ovarian suppression followed
by hMG stimulation mimicked the previous stimulation cycles for each of the four women. The
frequency of transvaginal ultrasound to assess follicular growth and serum estradiol levels were
unchanged from each participants previous donation cycle. Women enrolled in the study began
taking 100mg Cilostazol q 12 hours starting on day one of hMG administration (as established
in macaques (7)). They continued taking the medication until the day of oocyte retrieval.
During the treatment phase, plasma Cilostazol levels were collected once following cycle day 4
(when drug reaches steady state), and on the morning of oocyte retrieval.
Oocyte retrieval:
! When ovarian follicles reached an appropriate size as determined by Reproductive
Endocrinology and Infertility attending staff, participants received an injection of hCG 10,000
IU. 34 to 36 hours following administration, participants were scheduled for an oocyte retrieval
at the USC IVF outpatient surgical center. The oocytes, once retrieved, were then examined
under a light microscope by an experienced embryologist, and were graded at retrieval as well as
December 2014
5
at 24 hours to determine percent germinal vesicle (GV), percent germinal vesicle breakdown
(MI), and percent of oocytes demonstrating extrusion of the first polar body (MII). The
maturational stage of oocytes obtained from cilostazol-treated subjects were then compared to
those proportions documented from the same subjects during oocyte donation from a prior cycle
at the same infertility center within the past three years.
Plasma and follicular fluid Cilostazol levels:

Quantification of both plasma and follicular fluid Cilostazol levels were established using
methodology in previously published protocols (18).
STATISTICAL ANALYSIS

Comparison of blood pressure and heart rate between baseline and study measurements
were performed using longitudinal general linear models to account for repeated measures within
participants. Effect sizes (Cohen’s D for adjusted means) were calculated to gauge the size of the
effect.
Percent egg maturation between historic controls and Cilostozol were made using paired
t-tests. Pearson correlations were performed to test the correlations between percent egg
maturation and cilostazol levels in both the plasma and follicular fluid.
Given that there are no previously published studies regarding oocyte maturation in
human subjects, a power calculation was not performed.
RESULTS

The participant characteristics for the 4 enrolled subjects are shown in table 1. No
participants discontinued despite all reporting a headache for the first 2 days following initiation
of study drug. All headaches resolved by day 4 and were treated appropriately with
acetaminophen.
December 2014
6
Table 1. Participant Characteristics
There was not a significant difference in percent of egg maturation retrieved at baseline vs during
treatment with Cilostozol (t3 = 0.36, p = 0.74). Examination of the data for individual
participants shows that one participant increased, one decreased, and two were almost identical.
(Figure 1). Nearly all oocytes demonstrated progression to MII by 24 hours following retrieval
for both groups (data not shown).
Figure 1. Percent oocyte maturation at the time of oocyte retrieval. The blue bars represent the percent of mature
oocytes (MII) at the time of retrieval during the historical control cycle. The green bars represent the percent of
mature oocytes (MII) during the Cilostazol cycle.
The plasma and follicular fluid levels of Cilostazol for all participants is shown in table 2.
December 2014
7
Participant Day 4 Plasma
Cilostazol (ng/mL)
Retrieval Plasma
Cilostazol (ng/mL)
Follicular Fluid
Cilostazol (ng/mL)
1 249 274 374
2 344 1080 745
3 256 695 726
4 640 738 585
Table 2
The correlations of percent retrieval was 0.30 with plasma Cilostozol levels at day 4, -0.82 with
plasma Cilostazol levels at retrieval, and -0.89 with follicular levels (Figure 2).
Figure 2: Pearson correlations reveal a negative correlation between follicular fluid and retrieval plasma Cilostazol
levels and oocyte maturation. There is a weakly positive correlation between day 4 plasma Cilostazol levels and
oocyte maturation.
December 2014
8
There was a significant difference in diastolic blood pressure (DBP) (p = 0.008) and heart rate
(HR) (p = 0.027) from baseline to measurements made during the study. DBP was lower during
the study (M+SE = 69.0+1.5) than at baseline (74.0+3.1), a very large effect d = 2.2. HR during
the study was higher during the study (81.3+3.0) than at baseline (70.5+7.1), also a very large
effect d = 2.1. There was not a significant difference in systolic blood pressure during the study
(116.6+4.1) than at baseline (117.8+3.3), a small difference d = 0.3. These changes in heart rate
and diastolic blood pressure, although statistically significant, may not be clinically relevant as
they still fall within the normal human range.
DISCUSSION

It does not appear that cilostazol at the current FDA approved dose of 100mg PO q 12
hours has any effect on human egg maturation in vivo. There did appear, however, to be an
association between plasma levels of cilostazol on the day of oocyte retrieval as well as follicular
fluid levels and percent oocyte maturation. That is, the higher the concentration of cilostazol in
both the plasma and follicular fluid, the lower the percentage of mature oocytes. This association
is difficult to interpret, however, with only 4 study participants. This study is extremely
important as it is the first human in vivo trial using an FDA approved PDE3 inhibitor to assess
oocyte maturation. Research in this area is novel in that it attempts to address the need of non-
hormonal contraceptive methods.

However, concerns of systemic effects with the use of PDE3 inhibitors are valid. In vivo
studies in macaques examining pregnancy rates after treatment with ORG 9935 showed no
statistically significant decrease in pregnancy rates in those macaques treated with PDE3
inhibitor; however, there did appear to be evidence of a dose response as no animal became
pregnant who had serum ORG 9935 levels above 300nM/L (9). Despite a clear dose response,
authors of this study acknowledge that PDE3-Is have systemic effects that are also dose-limiting.
At higher doses, adverse effects such as tachycardia and hypotension were observed. In our
study, there was a statistically significant decrease in diastolic blood pressure and increase in
heart rate in study participants. However, these values were not within the range of clinical
tachycardia and are likely not clinically relevant. Additionally, because the primary outcome of
this study was not Cilostazol’s effect on blood pressure or heart rate, the protocol for determining
these values was not clinically stringent. Therefore, these values should be interpreted with
caution.
Limitations of our study include the small sample size and the fact that our study was not
randomized. Additionally, since this is the first in vivo study in humans, the dose of 100mg PO q
12 hours of Cilostazol was merely chosen because that is the current FDA approved dose. Based
upon our findings, this dose is likely inadequate to achieve the desired outcome, and further
studies looking at a dose response for Cilostazol are warranted. Furthermore, although the FDA
approved human dose for cilostazol is 100mg PO BID, we do not know if this dose is high
enough to prevent oocyte maturation. Additionally, comparing the oocyte maturation of women
while not receiving study drug to themselves while receiving study drug has potential
December 2014
9
confounders given that we are treating them at different points in time during which additional
factors affecting oocyte maturation that we could not have anticipated are affecting our results.

The further development of effective non-hormonal contraceptive methods continues to
be necessary as there are many women who have medical conditions that limit hormonal
contraceptive use. The medication cilostazol has additional systemic effects that may
significantly benefit women of reproductive age (ie women with history of VTE, hypertension,
and dyslipidemia). Despite our negative findings, continued research of Cilostazol and its effects
on oocyte maturation is warranted at likely higher doses so long as systemic toxicity is not
reached.
REFERENCES
1) Albarzanchi A.M., Sayes C.M., Ridha-Albarzanchi M.T., Fajt V .R., Dees W.L., and Kraemer
D.C., (2012), Cilostazol blocks pregnancy in naturally cycling mice, Contraception, In press.
2) Albarzanchi M.T., Alanssari S.M., Fajt V .R., Ash O., and Albarzanchi A.M., (2011), Cilostazol
blocks pregnancy in swine: animal model, Abstract presented at American Association of
Bioanalysts Conference.
3) Bornslaeger E.A., Mattei P., and Schultz R.M., (1986), Involvement of cAMP-dependent
protein kinase and protein phosphoryation in regulation of mouse oocyte maturation.
Developmental Biology, 114, 453-462.
4) Chapman T.M., and Goa K.L., (2003), Cilostazol: A review of its use in intermittent
claudication, American Journal of Cardiovascular Drugs, 3(2), 117-138.
5) Chi Y .W., Lavie C.J., Milani R.V ., and White C.J., (2008), Safety and efficacy of cilostazol in
the management of intermittent claudication, Vascular Health and Risk Management, 4(6),
1197-1203.
6) Downs S.M., Daniel S.A., Bornslaeger E.A., Hoppe P.C., Eppig J.J., (1989), Maintenance of
meiotic arrest in mouse oocytes by purines: modulation of cAMP levels and cAMP
phosphodiesterase activity, Gamete Research, 23, 323-334.
7) Jensen J.T., Schwinof K.M., Zelinshi-Wooten M.B., Conti M., DePaolo L.V ., and Stouffer,
R.L., (2002), Phosphodiesterase inhibitors selectively block the spontaneous resumption of
meiosis by macaque oocytes in vitro, Human Reproduction, 17, 2079-2084.
8) Jensen J.T., Zelinski-Wooten M.B., Schwinof K.M., Vance J.E., and Stouffer R.L., (2005), The
phosphodiesterase inhibitor ORG 9935 inhibits oocyte maturation during gonadotropin-
stimulated ovarian cycles in rhesus macaques, Contraception, 71, 68-73.
December 2014
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9) Jensen J.T., Stouffer R.L., Stanley J.E., and Zelinski, M.B., (2010), Evaluation of the
phosphodiesterase 3 inhibitor ORG 9935 as a contraceptive in female macaques: initial trials,
Contraception, 81, 165-171.
10) Lepkowski J.M., Mosher W.D., Davis K.E., Groves R.M., and Van Hoewyk J., (2010), The
2006-2010 National Survey of Family Growth: sample design and analysis of a continuous
survey, Vital Health Stat 2, 150, 1-36.
11) Li M., Yu Y ., Yan J., Yan L.Y ., Zhao Y ., Li R., Liu P., Hsueh A.J., and Qiao J., (2012), The
role of cilostazol, a phosphodiesterase 3 inhibitor, on oocyte maturation and subsequent
pregnancy in mice, PLos One, 7(1), e30649.
12) Medical Eligibility Criteria for Contraceptive Use, www.cdc.gov
13) Noguiera C., Albano C, Adriaenssens T., Cortvrindt R., Bourgain C., Devroey P., and Smitz
J., (2003), Human oocytes reversibly arrested in prophase I by phosphodiesterase type 3 inhibitor
in vitro, Biology of Reproduction, 69, 1042-1052.
14) Ryan A., Saad T., Kirwan C., Keegan D.J., and Acheson R.W., (2012), Maintenance of
perioperative antiplatelet and anticoagulation therapy for vitreoretinal surgery, Clinical and
Experimental Ophthalmology, In Press.
15) Shu Y ., Zeng H., Ren Z., Zhuang G., Liana X., Shen H., Yao S., Ke P., andWant N., (2008),
Effects of cilostamide and forskolin on the meiotic resumption and embryonic development of
immature human oocytes, 23, 504-513.
16) Tsifiri A., Chun S., Zhang R., Hsueh A.J., and Conti M., (1996), Oocyte maturation
compartmentalization and opposing changes of cAMP levels in follicular somatic and germ cells:
studies using selective phosphodiesterase inhibitors, Developmental Biology, 178, 393-402.
17) Vaughan B., Trussel J., Kost K., Singh S., and Jones R., (2008), Discontinuation and
resumption of contracepive use: Results from 2002 National Survey of Family Growth,
Contraception, 78, 271-283.
18) Fu, CJ., Tata P., Okada, K., Akiyama H., and Bramer SL., (1999), Simultaneous quantitative
determination of cilostazol and its metabolites in human plasma by high-performance liquid
chromatography, Journal of Chromatography, 728: 251-262.
December 2014

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Creator Sech, Laura Ann (author)

Core Title Cilostazol and its effects on human oocyte maturation in vivo: a pilot study

Contributor Electronically uploaded by the author (provenance)

School Keck School of Medicine

Degree Master of Science

Degree Program Clinical, Biomedical and Translational Investigations

Publication Date 11/13/2014

Defense Date 11/13/2014

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag non-hormonal contraception,OAI-PMH Harvest,oocyte maturation

Format application/pdf (imt)

Language English

Advisor Azen, Stanley P. (committee chair), Mishell, Daniel (committee member), Natavio, Melissa (committee member)

Creator Email laurasech30@gmail.com,sech@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-516457

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Abstract (if available)

Abstract Background: Combined hormonal contraceptive pills (COCs) are the most frequently used method of contraception by women in the United States. However, COCs are frequently discontinued secondary to hormonal side effects. Moreover, COCs are contraindicated in women with certain medical problems. Development of a non-hormonal birth control pill could overcome these problems. In recent studies, phosphodiesterase 3 inhibitors (PDE3s) have been shown to inhibit oocyte maturation and impair fertility in macaques, mice, and pigs. Cilostazol, a class IIIa PDE, is currently FDA approved for the treatment of intermittent claudication. Additionally, this drug has demonstrated fertility impairment, despite the occurrence of ovulation, with complete reversibility in both mice and pigs. To date, no in vivo studies of Cilostazol in humans have been undertaken to determine its effects on oocyte maturation. ❧ Methods: This research design is a pilot study that enrolled 4 healthy women who have previously been oocyte donors. All participants were given the study drug, Cilostazol, at the current FDA approved dose of 100mg PO BID. The study drug was administered twice daily during the 10-14 days of ovarian stimulation with hMG until ovulation was induced with hCG. Participants underwent oocyte and follicular fluid retrieval following cycle stimulation. The stage of oocyte maturation (Germinal vesicle (GV), Metaphase I (MI), Metaphase II (MII)) was evaluated at the time of ovum retrieval and at 24 hours. The oocyte maturational stages obtained from subjects during Cilostazol treatment were compared to those documented from these same subjects during oocyte donation from a previous stimulation cycle while not taking study drug. Additionally, plasma as well as follicular fluid cilostazol levels were collected for analysis. ❧ Results: There was not a significant difference in percent of oocyte maturation retrieved at baseline vs during treatment with Cilostozol (t₃ = 0.36, p = 0.74). Plasma and follicular fluid levels of Cilostazol were obtained and a small correlation between Cilostazol levels in both the follicular fluid and plasma and oocyte maturation was demonstrated. There was a significant difference in diastolic blood pressure (DBP) (p = 0.008) and heart rate (HR) (p = 0.027) from baseline to measurements made during the study. These findings are likely not clinically relevant. ❧ Conclusions: Our findings indicate that the FDA approved dose of Cilostazol, given at 100mg PO BID, does not impair oocyte maturation in the human. Future studies using a higher dose of Cilostazol are indicated but this may prove difficult given potential for toxicity at higher doses.