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\begin{document}

\title{PCOS: a chronic disease that fails to produce adequately specialized
pro-resolving lipid mediators (SPMs).}



\author[1]{Pedro-Antonio Regidor}%
\author[2]{Xavier de la Rosa}%
\author[3]{Anna Mueller}%
\author[3]{Manuela Sailer}%
\author[4]{Fernando Gonzalez Santos}%
\author[5]{Rafael Gracia banzo}%
\author[6]{Jose Miguel Rizo}%
\affil[1]{Exeltis Germany}%
\affil[2]{Brigham and Women’s Hospital and Harvard Medical School}%
\affil[3]{Exeltis Healthcare}%
\affil[4]{Solutex Soluciones Extractivas Alimentarias}%
\affil[5]{Solutex GC SL}%
\affil[6]{Chemo Group}%


\vspace{-1em}



  
  \date{May 19, 2024}


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\selectlanguage{english}
\begin{abstract}
The present study aimed to establish lipid mediator profiles of PCOS
patients compared to healthy women to identify differences in their
resolutive and pro-inflammatory lipid parameters. Main measures:
Pro-inflammatory lipid mediators and their precursor AA; SPMs, their
precursors. The level of pro-inflammatory parameters was significantly
higher in PCOS-affected women.%
\end{abstract}%



\sloppy


Title: PCOS: a chronic disease that fails to produce adequately
specialized pro-resolving lipid mediators (SPMs).

Authors: Pedro-Antonio Regidor \textsuperscript{1,7}, Xavier de la
Rosa\textsuperscript{2}, Anna M\selectlanguage{ngerman}üller\textsuperscript{3,} Manuela
Sailer\textsuperscript{4}, Fernando Gonzalez Santos\textsuperscript{5},
Rafael Gracia Banzo\textsuperscript{6}, Jose Miguel Rizo
\textsuperscript{7}

Affiliations:

\textsuperscript{1} Exeltis Healthcare. Adalperostr. 84, 85737 Ismaning,
Germany. Phone: +49 89 4520529 -- 19,

Mobil: +491738938132, Fax: +49 89 4520529 -- 819, E-Mail:
pedro-antonio.regidor@exeltis.com

\textsuperscript{2} Center for Experimental Therapeutics and Reperfusion
Injury, Department of Anesthesiology, Perioperative and Pain Medicine,
Brigham and Women's Hospital and Harvard Medical School, Boston. 60
Fenwood Road Boston, MA 02115; USA; Tel: 617-525-5001; Fax:
617-525-5017. E-Mail:

\emph{xdelarosa@bwh.harvard.edu;}

\textsuperscript{3} Exeltis Healthcare. Adalperostr. 84, 85737 Ismaning,
Germany. Phone: +49 89 4520529 -- 19,

Mobil: +491738938145, Fax: +49 89 4520529 -- 819, E-Mail:
anna.mueller@exeltis.com

\textsuperscript{4} Exeltis Healthcare. Adalperostr. 84, 85737 Ismaning,
Germany. Phone: +49 89 4520529 -- 19,

Mobil: +491734166276, Fax: +49 89 4520529 -- 819, E-Mail:
manuela.sailer@exeltis.com

\textsuperscript{5} Solutex SA. Avenida de la Transición Espanola 24,
28108 Alcobendas. Spain. Phone: +34 91

8060477. Mobil: +34 647775178, Fax: +34 91060605, E-Mail:
fgsantos@solutexcorp.com

\textsuperscript{6} Solutex GC SL. Parque Empresarial Utebo, Avda.
Miguel Servet nº 81, 50180 Utebo, Spain. Phone:+34 976787485. E-mail:

\emph{rafael.gracia@insercolab.com}

\textsuperscript{7} OTC Chemo. Manuel Pombo Angulo 28- 4th floor, 28050
Madrid. Spain. Phone: +34 913021560

9507, Mobil: + 34 618721034. E-Mail: josemiguel.rizo@chemogroup.com

\textsuperscript{1} responsible for correspondence

Funding: Insud Pharma

Conflict of Interest: Pedro-Antonio Regidor is an employee of Exeltis
Healthcare, Anna Müller and Manuela Sailer are employees of Exeltis
Germany, Fernando Gonzalez Santos and Rafael Gracia Banzo are employees
of Solutex Spain, Jose Miguel Rizo is an employee of Chemo OTC Spain.
Xavier de la Rosa declares no conflict of interest

Ethical approval: Ethical approval was obtained from the investigational
center. The overall approval for the study was given on Jul 17, 2020, by
the Ethical Committee of the Bayerischen Landesärztekammer; Nr. 20056.

Data availability: Not applicable.

Author Contribution

Pedro-Antonio Regidor: Responsible for the concept of resolution of
inflammation in PCOS

Anna Müller: Responsible for literature research and writing of the
manuscript

Manuela Sailer: Responsible for graphic design

Fernando Gonzalez Santos: Responsible for the development of SPMs use in
humans and therefore for essential scientific data

Jose Miguel Rizo: responsible for data acquisition

Rafael Gracia Banzo: Responsible for laboratory analyses

Xavier de la Rosa: Responsible for coordination of the manuscript

Abstract:

Introduction: Polycystic Ovary Syndrome (PCOS) is an endocrinologic
disorder that affects 5-15 \% of women of their reproductive age and is
a frequent cause of infertility. Major symptoms include
hyperandrogenism, ovulatory dysfunction, a characteristic
multi-follicular morphology of the ovary, an elevated ratio of LH/FSH,
and often obesity and/or insulin resistance. PCOS also represents a
state of chronic low-grade inflammation that is closely interlinked with
the metabolic features. Inflammatory processes consist of the acute
inflammatory response and resolution processes initiated concomitantly.
''Classical'' pro-inflammatory lipid mediators like prostaglandins (PG),
leukotrienes (LT), or thromboxanes (TX) are derived from arachidonic
acid (AA) and are crucial for the initial response. Resolution processes
are driven by four families of so-called specialized pro-resolving
mediators (SPMs): resolvins, maresins, lipoxins, and protectins. SPM
biosynthesis starts from the essential polyunsaturated fatty acids DHA,
DPA, or EPA via certain hydroxylated intermediates (18-HEPE, 17-HDHA,
14-HDHA). The present study aimed to establish lipid mediator profiles
of PCOS patients compared to healthy women to identify differences in
their resolutive and pro-inflammatory lipid parameters.

Material and Methods: Blood samples were taken (20 ml), separated into
plasma and serum, and analyzed by HPLC/MS-QQQ. Fifteen female patients
(18-45 years) were diagnosed with PCOS according to Rotterdam criteria,
and five healthy women, as comparator group, were recruited for the
study. The main outcome measures were: Pro-inflammatory lipid mediators
(PG, LT, TX) and their precursor AA; SPMs (Resolvins, Maresins,
Protectins, Lipoxins), their precursors EPA, DHA, DPA, and their active
biosynthesis pathway intermediates (18-HEPE, 17-HDHA, 14-HDHA). Ratio
{[}(sum of pro-inflammatory molecules)/sum of SPMs{]}.

Results: The level of pro-inflammatory parameters in serum was
significantly higher in PCOS-affected women. The ratio {[}(sum of
pro-inflammatory molecules) / (sum of SPMs plus hydroxylated
intermediates){]} reflecting the inflammatory state was significantly
lower in the group of healthy women.

Conclusion: There is a strong pro-inflammatory state in PCOS patients.
Further research will clarify whether supplementation with SPMs or their
precursors may improve this state.

Keywords: PCOS, obesity, inflammation, specialized pro-resolving
mediators (SPMs)

Key Clinical Message: SPMs, which derive from 18-HEPE, 17-HDHA, and
14-HDHA, and are biosynthesized from their respective precursor
omega-3-fatty acids EPA and DHA, have a possible influence on the
resolution of inflammation associated with polycystic ovary syndrome.

Introduction

Polycystic Ovary Syndrome (PCOS) is a disease that causes irregular
bleeding, chronic anovulation, androgen excess, and a typical ovarian
ultrasound feature {[}1{]}. It affects between 5 and 10 \% of women in
their reproductive age, thus representing one of the most frequent
causes of infertility {[}2{]}. The reasons for the development of PCOS
have not been resolved yet. Genetic predisposition, together with the
gestational environment and lifestyle factors, seem to be critical
contributors {[}3{]}. Apart from the cardinal diagnostic criteria
including hyperandrogenism, ovulatory dysfunction, and/or the morphology
of polycystic ovaries as defined by the so-called ''Rotterdam-Criteria''
{[}4{]}, other characteristics are related to the disease. PCOS is often
accompanied by obesity {[}5{]}, and 30-40\% of women with PCOS show a
reduced glucose tolerance, often accompanied by insulin resistance
{[}6{]}. 80\% of obese women and 30-40\% of lean individuals with PCOS
suffer from hyperinsulinemia {[}6,7{]}. It has been found that
hyperinsulinemia is a crucial factor in the clinical pathogenesis of
PCOS and seems to be independent of weight {[}8{]}. Excess insulin may
lead to enhanced androgen synthesis by direct stimulation of the
androgen production on the one hand and by reducing the serum levels of
sex hormone-binding globulin (SHBG) on the other side, thereby
contributing to the androgen excess characteristic for PCOS {[}8{]}. In
addition, obesity has a substantial impact on the severity of PCOS
symptoms {[}9{]}. Apart from reinforcing insulin resistance, adipocytes
show an altered hormone metabolism that contributes to the
endocrinologic disorder {[}10{]}.

Furthermore, excess adipose tissue is a source of chronic low-grade
inflammatory processes, and PCOS is considered an inflammatory disease
{[}11{]}. Inflammatory response has been defined as an ensemble of
initiation and active resolution processes. Within this perception, the
resolution of inflammation is dependent on a class of lipid mediator
molecules called specialized pro-resolving mediators (SPM) {[}12{]}.
These molecules are derived from polyunsaturated fatty acids EPA
(Eicosapentaenoic Acid) and DHA (Docosahexaenoic Acid) and are
synthesized via specific intermediate molecules by cells of the immune
system. This publication will focus on the role of SPMs in chronic
inflammatory diseases like PCOS and the potential benefit of
supplementation with their precursor molecules.

Obesity, insulin resistance, and inflammation

A state of chronic systemic inflammation is characteristic of obesity.
It can be determined by measuring increased serum levels of inflammatory
cytokines and altered frequencies and functions of peripheral blood
lymphocytes {[}13, 14, 15{]}. These changes are manifested at the tissue
level of the adipose-, liver- and other tissue beds {[}14, 15{]}. They
might be responsible for comorbidities often related to obesity, such as
atherosclerosis, diabetes, and steatohepatitis {[}16, 17, 18, 19, 20{]}.
This kind of inflammation is often attributed to irregularities in
innate immunity. However, innate and adaptive immune systems are closely
interlinked, and consequently, obesity-related inflammation is
associated with both processes {[}21{]}. For example, in obese
individuals, systemic levels of free fatty acids are elevated {[}10{]}.
These molecules are primary ligands of Toll-like receptors, which are
critical regulators of the innate immune response {[}22, 23{]}. In this
way, the systems, which regulate obesity and inflammation, are linked
directly.

A further relationship between inflammation and the metabolic system is
visible on the cellular level since adipocytes and macrophages are
closely related. Their evolution might be traced back to a conventional
primordial precursor cell {[}24{]}.

It has also been demonstrated that insulin resistance and inflammatory
processes are closely linked and may stimulate each other {[}25{]}. Both
subclinical inflammation and insulin resistance are important markers
for the development of cardiovascular disease {[}26{]}. For women with
PCOS, whose cardiovascular risks are elevated, a connection between
inflammation and their hormonal- metabolic features was shown {[}27{]}.

Since obesity, insulin resistance, and inflammation, key features of
PCOS, are correlated, it is worthwhile looking at the possible pathways
of inflammation that accompany PCOS, considering the modern perception
of inflammatory processes.

The present study was therefore conceived to describe the physiological
status of the innate immune response and its resolution potential by
blood profiling of eicosanoid parameters and pro-resolving mediators in
the plasma and sera of patients suffering from a PCOS as this
information is lacking to date and comparing the results with a healthy
group to describe the intensity of the pro-inflammatory lipid mediator's
exacerbation in the peripheral blood in these patients.

Material and methods

Human plasma and serum of PCOS patients

Fifteen PCOS patients were evaluated in this study. A control group of
five healthy patients was used for comparison. The healthy patients were
probands with no evident clinical acute disease or known pathological
anamnesis in the medical history. The BMI of both groups was similar.

All patients were recruited at the Lubos Klinik in Munich, Germany.

Samples were obtained at 8 am under fasting conditions.

Table 1 shows the demographic data of the patients.

The whole study was performed following a protocol designed and
conducted following the ethical principles that have their origin in the
Declaration of Helsinki and are consistent with GCP and existing
regulatory requirements. Institutional review board approval was
obtained from the study site.

Ethical approval

Ethical approval was obtained for the investigational center. The
overall approval for the study was given on Jul 17, 2020, by the Ethical
Committee of the Bayerischen Landesärztekammer; Nr. 20056.

Clinical trial registration: DRKS-ID: DRKS00022337. Date of
registration: Jun 29, 2020.

Blood sample analyses.

Blood samples of plasma and serum were obtained for each patient. Each
of these samples was considered a mono-replicate.

After standard preliminary treatment, samples were stored at -80 degrees
Celsius until they were processed in the laboratory. They were all
analyzed separately.

Lipid mediator extraction and profiling (LC-MS/MS)

Lipid mediators were extracted from human plasma and serum samples
following the SPE (Solid Phase Extraction) method described below.
Internal labeled standards d\textsubscript{8}-5-HETE,
d\textsubscript{5}-RvD2, d\textsubscript{5}-LXA\textsubscript{4},
d\textsubscript{4}-LTB\textsubscript{4},
d\textsubscript{4}-PGE\textsubscript{2} (500 pg each, Cayman Chemical
Company) in 4 mL of methanol (Methanol Optima LC/MS Grade, Fisher
Chemical) were added to each sample (plasma or serum, 1 mL) previously
thawed on ice. These labeled standards were used for the amount
determined and the calculations of the recovery of the lipid mediators.
Next, the samples were placed at -80ºC for 30 minutes to allow the
precipitation of proteins. The probes were centrifugated in the
following working step (2000 g, 10 min, 4ºC). The supernatants were
obtained from each sample, and SPE was carried out according to
optimized and reported methods {[}28, 29{]}. Samples were rapidly
acidified to pH=3.5 with 9 mL of acidic water (HCl) before loading onto
SPE columns (100mg, 10 mL, Biotage) and pH neutralized with 4 mL of
MiliQ water, followed by 4 mL of n-hexane wash step. After, compounds
were eluted with 9 mL of methyl format. Extracts from the SPE were
brought to dryness under a gentle stream of nitrogen and immediately
resuspended in methanol/water (50:50 vol/vol) (MeOH/Water Optima LC/MS
Grade, Fisher Chemical, both) before injection into an LC-MS/MS system.

Targeted LC-MS/MS Acquisition Parameters

LC-MS/MS system consisting of a Qtrap 5500 (Sciex) equipped with a
Shimadzu LC-20AD HPLC pump. A Kinetex Core-Shell LC-18 column (100 mm ×
4.6 mm × 2.6 \selectlanguage{greek}μ\selectlanguage{english}m, Phenomenex) was kept in a column oven maintained at 50
\selectlanguage{ngerman}°C. A binary eluent system of LC-MS/MS grade water (A) (Fisher Chemical)
and LC-MS/MS grade methanol (Fisher Chemical) (B), both with 0.01 \%
(v/v) of acetic acid, was used as mobile phase. LMs were eluted in a
gradient program respect to the composition of B is as follows: 0-2 min,
50 \%; 2-14.5 min, 80 \%; 14.6-25 min; 98 \%. The flow rate was 0.5
mL/min.

The QTRAP 5500 was operated in negative ionization mode, using scheduled
Multiple Reaction Monitoring (MRM) coupled with the
information-dependent acquisition (IDA) and an Enhanced Product Ion scan
(EPI). Each LM parameter (CE, target retention time (RT), and specific
Q1 and Q3 mass) was optimized according to reported methods {[}29,
30{]}. To monitor and quantify LMs of interest, quantities were taken as
areas under the peak. We used MRM with MS/MS matching signature ion
fragments for each molecule (at least six diagnostic ions;
\textless{}0.1 picograms was considered below the limit of detection)
using published criteria {[}30{]}. Examples of representative MRM
spectra are presented in figure 5. The laboratory analyses were
performed at Solutex GC SL.

Statistical analyses

Quantitative measurements were presented as mean, standard error,
minimum and maximum. When indicated, Outlier exclusion was calculated
using default parameters ROUT (Q=1\%) from GraphPad Prism version 9.0.2,
GraphPad Software, San Diego, California USA. Comparisons were
consequently made using an unpaired one-tailed \emph{t} -test.

All tests were done with a one-tailed t-test, and statistical
significance was considered at \emph{P} \textless{}0.05. We did not make
any adjustments for multiple testing; thus, the results are explorative
and descriptive.

A ratio between pro-inflammatory and pro resolutive parameters was
established to describe the physiology of both investigated axes
(pro-inflammatory and pro resolutive).

The proposed ratios have the purpose of seeking the overall
balance/unbalance of interconnected metabolic routes and the overall
status of the resolution of the immune response.

Evaluated parameters

Fatty acids (EPA, DHA, ARA, DPA); SPM monohydroxylated-containing
precursors (17-HDHA, 18-HEPE, 14-HDHA); SPM´s (Resolvins: RvE1, RvD1,
RvD2, RvD3, RvD4, RvD5; Maresins: MaR1, MaR2; Protectins: PD1, PDX;
Lipoxins: LXA\textsubscript{4}, LXB\textsubscript{4}). Eicosanoids:
Prostaglandins (PGE\textsubscript{2}, PGD\textsubscript{2},
PGF\selectlanguage{greek}\textsubscript{2α}.\selectlanguage{english}) Thromboxanes (TxB\textsubscript{2}) Leukotrienes
(LTB\textsubscript{4})

Results

In this observational study, we observe that the quantity of each
parameter was detectable in the sera but not in the same way in the
participants' plasma.

After quantitation, summation of total ARA-derived pro-inflammatory
mediators resulted in a statistically significant increase
(P\textless{}0.05) when comparing sera from PCOS- patients with healthy
subjects. These pro-inflammatory mediators include LTB4, PGD2, PGE2,
PGF2, and TXB2, and values altogether were 100 times higher compared to
those of healthy subjects (see figure 1). Measured prostanoids,
including PGD2, PGE2, and PGF2, all together were increased by 600\% in
serum from patients with PCOS compared to healthy subjects (figure 1).
Thromboxane TXB2 was also statistically significantly (P\textless{}0.05)
higher in the serum from patients diagnosed with PCOS as compared to
healthy subjects, which may reflect that these patients could suffer
from coagulopathies (figure 1).

We next studied whether these patients might have a disbalance in SPM
formation. Specifically, comparing the ratio of total pro-inflammatory
lipid mediators, including LTB4, PGD2, PGE2, PGF2, and TXB2 vs. total
SPMs formed were statistically significantly different between the test
groups (figure 1). We observed in serum that the ratio of complete
pro-inflammatory lipid mediators to the summation of SPMs, including
14-HDHA, 17-HDHA, and 18-HEPE, was statistically higher for patients
with a PCOS than those observed in the serum of healthy subjects (P
\textless{}0.05). This finding suggested that infections could impair
resolution mechanism(s) due to exacerbated inflammation.

We quantified the free-fatty-acid-precursors of resolving mediators. We
observed that DHA, DPA, EPA, and ARA were not statistically
significantly higher in PCOS patients' plasma and serum (figure 2) as
compared to those of healthy subjects. Interestingly, we observed that
PCOS patients presented statistically more elevated amounts of the ratio
pro-inflammatory parameters / SPMs, including the monohydroxylates in
the serum compared to the plasma. (figure3).

As shown in figure 1, the mean value of total prostanoids in the serum
was 30.000 pg/ml in healthy subjects and 60000 pg/ml in PCOS patients
(see figure 1). When comparing the differences for the thromboxane
values between the healthy subjects and the PCOS patients, statistically
significant differences could also be observed. PCOS patients expressed
significantly higher values than the healthy controls (see figure 1).

Figures 4 (plasma) and 5 (serum) show in a graphical way the heat maps
of the metalipidinomic results.

Human plasma or serum was extracted using SPE and subject to targeted
LC-MS/MS (see method above). Targeted LM and pathway markers were
profiled. Each mediator was identified using published criteria obtained
on their structure, including identification criteria of at least six
characteristic diagnostic ions present in their MS-MS spectra.
Representative screen captures of MS-MS enhanced product ion (EPI)
spectra captured from the chromatographic region of (A) LTB4 (B) PGD2
(C) PGE2 (D) PGF2 (E) TXB2 (F) RvD1 (G) PD1 (H) MaR1 and (I) MaR2.
Screen captures were taken using SCIEX OS software. Insets: Chemical
structures and prominent fragmentations (figure 6).

Table 2 depicts all the values in a tabular form.

Discussion

This study described the significant disbalance between pro-inflammatory
eicosanoid- derived lipid mediators and pro-resolutive markers in the
serum of PCOS patients. PCOS represents a chronic inflammatory condition
since classical indicators for an inflammatory response are present,
such as increased values of IL-6, C-reactive protein, fibrinogen, and
erythrocyte sedimentation rate (11, 31). The presented data supports
this concept, as the lipidome of PCOS patients is shifted towards the
pro-inflammatory axis with an increase in pro-inflammatory prostanoid
derivatives and an elevated ratio of {[}pro-inflammatory LM{]}/ {[}the
sum of SPMs and their hydroxylated precursors{]}.

Some widespread diseases like diabetes, cardiovascular disease, and
obesity are associated with chronic inflammation {[}15, 17, 32{]}. These
pathologies are strongly interlinked with diet, and the positive impact
of polyunsaturated fatty acids (PUFA) - rich diet on cardiovascular
health is broadly accepted {[}33{]}. Both EPA and DHA show an
anti-inflammatory effect, and in this context, their role as precursors
for SPM biosynthesis has been discussed {[}34, 35{]}. The crucial role
of SPMs in such chronic inflammatory states has become evident
throughout the past years, and the underlying molecular mechanisms are
increasingly elucidated {[}12, 39{]}

For the DHA-derived SPM protectin PD 1 and its hydroxylated precursors,
for example, a positive influence on the metabolism of fatty tissue was
demonstrated, suggesting a potential role in the management of obesity
{[}36{]}. For DHA-derived SPM RvD1, a molecular mechanism for its
possible cardioprotective effect has been demonstrated: it can activate
lipoxin A4/formyl peptide receptor 2 (ALX/FPR2), which serves as a
sensor for the resolution of inflammation in the context of coronary
heart disease. In animal experiments, ALX7FPR-null mice developed
obesity, diastolic dysfunction and showed reduced SPM- levels associated
with an impaired resolution of inflammation after cardiac injury
{[}37{]}.

SPM biosynthesis is, off-course, based on the abundance of their
PUFA-precursors. However, in several experimental setups, the SPM
biosynthesis was disturbed by altered activities of the involved enzymes
{[}36{]}. Depending on the affected enzyme, supplementation with the
hydroxylated intermediates of the SPM biosynthetic pathways may be
efficacious in those cases, as demonstrated in a setting with leukocytes
of obese individuals that showed a profound deficiency in the
biosynthesis of RvD: incubating the leukocytes with 17-HDHA, the
precursor of RvD, restored the SPM production {[}38{]}.

Therefore, supplementation with DHA- or EPA-derived SPMs and their
corresponding hydroxylated precursor metabolites 18-HEPA, 17-HDHA, and
14-HDHA may represent a promising approach to address the pathologic
features often associated with PCOS: hyperandrogenemia, obesity, and
insulin resistance all aggravate each other and are all associated with
chronic inflammatory processes. In the present study, an association
with the lipid mediator profile was demonstrated that was significantly
shifted towards the pro-inflammatory axis compared to healthy women.
Exciting was the greatly increased level of thromboxane TXB2 in PCOS
patients compared to the test group, as its precursor, TXA2, plays an
essential role in platelet activation and aggregation. PCOS-affected
women are known to have a 2-fold increased risk for venous
thromboembolic events compared to healthy women.

Conclusion

Our study supports the former investigation of Duleva et al. {[}40{]}
and Rudnika et al. {[}41{]} that could demonstrate using classical
inflammatory markers that PCOS is an inflammatory disease.

The fact that white blood cells and cytokines, and interleukins are
elevated could be reaffirmed with our data showing a metalipidinomic
disbalance in women with PCOS.

Treatment of PCOS is mainly focused on weight loss, use of
anti-androgenic hormone preparations, treatment with insulin-sensitizing
agents or the whole repertoire of ovulation induction, and in vitro
fertilization techniques when child wish comes into focus. At the same
time, the underlying inflammatory processes are relatively neglected as
a therapeutic target {[}42{]}.

Targeted supplementation with SPMs or their precursors may be a valuable
novel therapeutic strategy worth further investigation.

Acknowledgement: We thank Julio Boza for correcting the manuscript and
creating the graphs.

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Legends:

Table 1: Demographical data of the study population.

Table 2: ARA, DHA, EPA, and DPA metabolome: healthy and PCOS lipid
mediators amount from human plasma and serum.

Figure 1: Results of the ARA-derived proinflammatory mediators of the
healthy and PCOS patients.

Figure 2: Results of the quantified the free-fatty-acid-precursors of
resolving mediators.

Figure 3: Results of the ratio pro-inflammatory parameters / SPMs
including the monohydroxylates in the serum compared to the plasma.

Figure 4: Heat map results in pg/ml of the human plasma analyses of the
healthy and PCOs patients.

Figure 5: Heat map results in pg/ml of the human serum analyses of the
healthy and PCOs patients.

Figure 6: Representative screen captures of LM analyzed. Top panels
indicate MS/MS spectra of synthetic lipid mediators used as standard.
Bottom panel exhibit biological MS/MS spectra of indicated LM. (A) PGE2,
(B) PGF2, (C) PD1, and (D) MaR2. Blue arrows on Y-axes denote the noise
threshold. Screen captures were taken using SCIEX OS software.\selectlanguage{english}
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\textbf{Hosted file}

\verb`Table 1.docx`  available at \url{https://authorea.com/users/369651/articles/711798-pcos-a-chronic-disease-that-fails-to-produce-adequately-specialized-pro-resolving-lipid-mediators-spms}

\textbf{Hosted file}

\verb`Table 2.docx`  available at \url{https://authorea.com/users/369651/articles/711798-pcos-a-chronic-disease-that-fails-to-produce-adequately-specialized-pro-resolving-lipid-mediators-spms}

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