TITLE: N-Acetylcysteine Compared to Metformin, Improves The
Expression Profile of Growth Differentiation Factor-9 and
Receptor Tyrosine Kinase c-Kit in The Oocytes of Patients
with Polycystic Ovarian Syndrome

  ABSTRACT.BACKGROUND:
Paracrine disruption of growth factors in women with polycystic ovarian syndrome (PCOS) results in
production of low quality oocyte, especially following ovulation induction. The aim of this study was to investigate
the effects of metformin (MET), N-acetylcysteine (NAC) and their combination on the hormonal levels and expres-
sion profile of GDF-9, BMP-15 and c-kit, as hallmarks of oocyte quality, in PCOS patients.

ABSTRACT.MATERIALS AND METHODS:
This prospective randomized, double-blind, placebo controlled trial aims to study the effects
of MET, NAC and their combination (MET+NAC) on expression of GDF-9, BMP-15 and c-kit mRNA in oocytes
[10 at the germinal vesicle (GV) stage, 10 at the MI stage, and 10 at the MII stage from per group] derived following
ovulation induction in PCOS. Treatment was carried out for six weeks, starting on the third day of previous cycle until
oocyte aspiration. The expression of GDF9, BMP15 and c-kit were determined by quantitative real time polymerase
chain reaction (RT-qPCR) and western blot analysis. Data were analyzed with one-way ANOVA.

ABSTRACT.RESULTS:
The follicular fluid (FF) level of c-kit protein significantly decreased in the NAC group compared to the other
groups. Significant correlations were observed between the FF soluble c-kit protein with FF volume, androstenedione
and estradiol. The GDF-9 expression in unfertilized mature oocytes were significantly higher in the NAC group com-
pared to the other groups (P<0.001). Similar difference was not observed between the MET, NAC+MET and control
groups. The c-kit expression in unfertilized mature oocytes were significantly lower in the NAC group compared to
the other groups (P<0.001). Similar difference was not observed between the MET, NAC+MET and control groups
(Registration number: IRCT201204159476N1).

ABSTRACT.CONCLUSION:
We concluded that NAC can improve the quality of oocytes in PCOS.

BODY.INTRODUCTION:
Anovulation associated with polycystic ovary syndrome (PCOS), as a common metabolic disorder, is the major cause of female infertility (1,2). The principal feature of PCOS is the large number of follicles arresting at early growth stage. The cytoplasmic and nuclear maturity of oocytes is reduced following ovarian stimulation and may account for embryo quality in these couples (3). Exclusive oocyte secreted factors (OSFs), such as growth differentiation factor-9 (GDF-9) and bone morphogenetic factor-15 (BMP-15), belonging to for oocyte competence (3,5). The receptor tyrosine kinase c-kit is another OSFs which plays important role in oogenesis and folliculogenesis (6). Recent evidence suggest possible involvement of c-kit and its receptor, kit ligand (KL) in PCOS pathology (7). Indeed, it has been shown that the aberrant or low expression of these exclusive oocyte secreting factors (BMP-15 and GDF-9), lead to over expression of c-kit and its receptor (8-10). Therefore, drugs which can modulate the regulation of these intra-ovarian factors may play a role in the clinical management of PCOS.   To improve the quality of oocyte, various protocol tion induction along with insulin-sensitizing drugs in PCOS patients. But, the risks of poor response, ovarian hyperstimulation, production of low quality oocytes, reduced fertilization rates and poor embryo quality remains among concern to be dealt within PCOS women undergoing in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) (11). Metformin, an insulin-lowering agent, has been extensively used for treatment of anovulation and infertility in PCOS patients. ed (12). In this regard, background studies indicate that MET does not improve the overall outcomes of assisted reproductive procedure in term of the aforementioned parameters (13,14).   On the contrary, administration of N-acetylcysteine (NAC) has been shown to improve not only the number and also the quality of oocytes in these patients. This phenomenon has been mainly related to the strong antioxidant effect of NAC, which has been shown to reduce follicle atresia and improve the quality of oocyte (15). In vitro, NAC plays a key role in cell survival through the production of trophic factor and follicular preservation (16,17). In line with these reports, Sacinositol and NAC improve ovarian function of PCOS patients. Therefore, considering the fact that oocyte secretory factors are hallmarks of oocyte quality, this study aims to evaluate the effects of NAC, MET and their co-administration on the expression of GDF-9, BMP-15 and c-kit in PCOS individuals undergoing ovarian stimulation in ICSI cycle. 

BODY.MATERIALS AND METHODS:
Antibodies directed against c-kit and β-actin was
obtained from Cell Signaling Technology (Beverly,
MA, USA). BMP-15 antibody was obtained from Abcam
Technology (Cambridge, MA, USA) and GDF-9
antibody from Santa Cruz Biotechnology (CA, USA).
Other reagents used in this study were obtained from
Sigma-Aldrich (St. Louis, MO, USA). Cell culture media
and sera were obtained from Gibco BRL (Carlsbad,
CA, USA).

BODY.MATERIALS AND METHODS.STUDY DESIGN:
This study was performed in continuation of our prospective
randomized, double-blind, placebo controlled
trial, in the IVF Unit of Infertility Research Center of
the Academic Center for Education, Culture and Research
(ACECR), Qom/Iran. 80 infertile PCOS women
at the age of 25-35 years, in the interval between
July 2012 and February 2013, who planned to undergo
ICSI were included in this study (19). Individuals were
diagnosed as PCOS according to the Rotterdam consensus
workshop (20). Based on this consensus, each
individual needed to have two out of three criteria: i.
Biochemical or clinical hyperandrogenism, ii. Chronic
oligo or anovulation and iii. Polycystic ovaries at ultrasound
examination. Ethical consideration and further
information on this clinical trial are provided in previous
studies (19). This study was approved by the Ethics
Committee (EC/91/1041) of Royan Institute, Tehran,
Iran. The patients provided an informed consent and
committed to avoid any changes in their normal physical
activity, diet or starting a new medical regimen
throughout the study.

BODY.MATERIALS AND METHODS.TREATMENT DESIGN AND OVULATION INDUCTION:
The female partner of ICSI candidates were examined
and randomly divided into 4 groups (n=20): i.
Placebo (PLA) receiving oral rehydration solution
(ORS, Poursina, Iran), ii. MET receiving MET (Glucophage,
Merck, West Drayton, UK, 500 mg), iii.
NAC receiving NAC (Holzkrichen, Germany, batch
no. 6N5483, 600 mg) and iv. MET+NAC group receiving
the combination of MET and NAC at the
aforementioned doses. Treatment was carried out
three times daily for a period of six weeks. The dose
and duration of NAC treatment was chosen according
to recent studies (21-23).  Gonadotropin-releasing hormone (GnRH) agonist
protocol (18) was used to induce ovulation. The female
partner of ICSI candidates randomized to four groups
received PLA, MET, NAC or MET+NAC from the third
day of last menstrual period (LMP) of previous cycle
until the day of oocyte aspiration. Oral contraceptive
pills (OCPs) were also included in the regimen for 21
days starting simultaneously with placebo, MET, NAC
or MET+NAC on the day 3 of menstrual cycle prior
to the treatment cycle. For ovarian down-regulation,
daily injections of Bucerelin acetate (1 mg, Suprefact,
Aventis, Germany) were administered from the day 19
of the preceding, menstrual cycle until day 2 of the next
cycle. On the second day on the next cycle if the endometrial
thickness was less than 4 mm, the dose of
Burcerelin acetate was reduced to 0.5 mg.  Ovulation induction was induced from the day two of
the cycle with average daily injections of 2 ampoules
of recombinant follicle stimulating hormone (rFSH,
Gonal-f, Merck Serono S.A., Geneva, Switzerland).
Vaginal ultrasound (Honda Electronics HS 4000-Japan)
was also used to monitor the cycles. 10,000 IU
human chorionic gonadotropin (hCG, Pregnyl, Organon,
Netherlands) was administered to induce ovulation.
36 hours after the administration of hCG, when at
least three follicles had reached the diameters of 16-18
mm, transvaginal oocyte aspiration was performed under
ultrasound guidance and general anesthesia. This
protocol of induction ovulation was used for all the individuals
in the 4 group. During the treatment the participants were asked to report any probable side effects
such as abdominal discomfort, diarrhea and nausea.
Due to these side effects, 20 couples (5 per group) were
excluded from the study (Fig .1).

BODY.MATERIALS AND METHODS.PREPARATION OF OOCYTES, FOLLICULAR FLUID AND BLOOD SAMPLES:
Based on our pervious study, oocytes and follicular
fluid (FF) from multiple follicles, from each subject
were pooled as explained (18). Following oocyte retrieval,
their cumulus cells were removed by exposure
to 20 IU/ml hyaluronidase (ART-4007A, SAGE BioPharma,
USA) in HEPES-based medium for 30 seconds
followed by mechanical pipetting in HEPES-buffered
HTF containing 5 mg/ml human serum albumin (ART-
3001, SAGE BioPharma, USA).  The nuclear status of each oocyte was determined
under the stereo microscope (Olympus Co., Japan) and
classified into three categories: i. Unfertilized mature
oocyte [metaphase II (MII)] following ICSI, ii. Germinal
vesicle (GV) stage, iii. Without first polar body
called metaphase I (MI). For gene expression analysis,
in each experimental group, 10 GV, 10 MI, and 10 MII
oocytes were separately pooled and washed in phosphate-
buffered saline (PBS) and transferred into RNasefree
microcentrifuge tubes. 50 μl of RNAlater, RNA Stabilization
Reagent (Qiagen, USA) were added to each
tube and all samples were stored in a -80°C freezer until
analysis. Only MII oocytes were used for ICSI.  The FF, from the first aspirated with no visible blood
contamination was collect and immediately centrifuged
at 3000 rpm for 10 minutes, and the supernatants
were stored at -70°C for further analysis. Fasting
blood sample were also collected from each participant
once prior to the start of treatment (day 2 of pervious
cycle) and once on the day of ovum pick up of ICSI
cycle. The samples were immediately centrifuged for
10 minutes at 3000 rpm (Hettich, EBA20, UK) and the
resulting serum were stored at -70°C for evaluation.  The levels of luteinizing hormone (LH, mIU/ml), FSH
(mIU/ml), total testosterone (TT, ng/ml), Progesterone
(ng/ml), estradiol (E2, pg/ml) and androstenedione (ng/
ml) in the FF and serum were measured in all samples
using the ELISA enzyme immunoassay (Demeditec Diagnostics
GmbH, Germany) according to the manufacturer's
protocol. The FF soluble protein level of c-Kit (pg/ml)
was measured with the ELISA Kit (Abnova Corporation,
Taiwan) by sandwich enzyme immunoassay technique,
according to the manufacturer's protocol.  Fig.1Flowchart of participants in this study.MET; Metformin, NAC; N-acetylcysteine, and PLA; Placebo.

BODY.MATERIALS AND METHODS.GENE EXPRESSION ANALYSIS:
Total RNA from the oocytes of each group were isolated
using the EZ-10 total RNA mini-prep Kit (Bio
Basic Inc., Canada), according to the manufacturer's
protocol. All samples were stored at -80oC till
analysis. Complementary DNA (cDNA) was synthesized,
using random hexamers [using the RevertAid
First Strand cDNA synthesis Kit (Thermo scientific,
USA)]. To determine the relative expression of target
genes, quantitative real time polymerase chain reaction
(RT-qPCR) was carried out using SYBR-Green/
ROX qPCR master mix assay (Thermo scientific,
USA) by gene-specific primers (Table 1). Relative
gene expression was calculated as the abundance ratio
of each target gene relative to β-actin. The ABI
step one plus (ABI, USA) instrument was used for
real time PCR experiments and the ΔΔCt method for
data calculation.   Table 1  Identity and sequence details of polymerase chain reaction (PCR) primers used to analyses mRNA expression in oocytes Sequence primer (5´-3´) Gene F: CCAATAGAAGTCACCTCR: GCGATCCAGGTTAAATAGCA GDF-9 F: CAGTCCTCTATTGCCCTTCTR: AATGGTGCGGTTCTCTCTA  BMP-15 F: ACGAATGAGAATAAGCAGAATGAAR: GAGAGGACAGCGGACCAG  c-Kit F: GGACTTCGAGCAAGAGATGGR: AGCACTGTGTTGGCGTACAG β-actin   Total proteins from each pool of oocytes were extracted
using RIPA lysis and extraction buffer Kit (Cat No:
89900, Thermo Scientific, USA), according to the manufacturer's
instruction. Concentration of proteins was determined
according to Bradford's method using bovine
serum albumin (BSA) as a reference standard (Bradford,
1976). Total proteins were electrophoresed in 12.5% sodium
dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) gel, transferred to polyvinylidene fluoride
membranes, and probed with specific antibodies. Membranes
were developed using enhanced chemiluminescence
reagents (Amersham Bioscience, USA) and the
intensity of immunoreactive polypeptides was analyzed
subsequent to visualization of the bands developed on a
photographic film. Protein bands on photographic film
were quantified by densitometry scanning after background
subtraction. Integrated densities of bands were
measured by Image J software.

BODY.MATERIALS AND METHODS.STATISTICS:
The normality of continuous variables was confirmed
using the Kolmogrov-Smirnov test and data were reported
as means ± SEM. Data analysis were performed using
one-way ANOVA and Tukey's test for post-hoc. Means
were considered significantly different at P<0.05. Pearson's
correlation test defined the relation between variables.
All data were analyzed with the statistical software
SPSS (version16.0 for windows, Chicago, IL, USA).

BODY.RESULTS:
Patients characteristics including age, body mass index
(BMI), the level of LH, FSH, E2 and TT were not significantly
different among the groups PCOS prior to treatment.

BODY.RESULTS.FOLLICULAR FLUID ANALYSIS:

FF volume and FF level of androstenedione, E2 and
progesterone were similar in all groups (P>0.05, Table 2),
but the level of soluble c-kit protein in the FF significantly
decreased in the NAC group compared to other groups
(P<0.01). Our results also showed a significant correlation
between the soluble c-kit protein in the FF of all the
population with the FF volume (r=0.508, P=0.02), androstenedione
(r=0.682, P=0.01), and E2 (r=0.638, P=0.01)
(Fig .2).

BODY.RESULTS.EVALUATION OF OOCYTE AND EMBRYO QUALITY:
The number of immature oocytes (MI+GV) and abnormal
mature oocytes significantly decreased in the NAC
group (P<0.01) compared to the other groups. Similar
reduction was also observed in MET and MET+NAC
groups but the reduction was not significant compared
to the placebo group (P>0.05). The fertilization rate of
metaphase II oocytes were similar in all groups (P>0.05).
The number of good embryo (grade I) on day 3 showed a
significant increase in the NAC group (P<0.02) compared
to placebo group. This improvement was also observed in
the MET and MET+NAC groups when compared to the
placebo (P>0.05), but remained insignificant (Fig .3). The
percentage of top grad embryos was not different between
the three NAC with MET and MET+NAC groups.   Table 2  Comparison of the biochemical parameters of follicular fluid in PCOS patients PlA NAC+MET MET NAC Parameter Follicular fluid Volume (ml) 5.4 ± 1.13 a 5.7 ± 1.1 a 4.96 ± 1.1 a 4.9 ± 1.1 a Estradiol (pg/ml) 466.6 ± 34.8 a 496.6 ± 44.8 a 470.2 ± 48.4 a 436 ± 40.16 a Progesterone (ng/ml) 3983.7 ± 353.9 a 3916.9 ± 359.5 a 3501.2 ± 326.9 a 3255.3 ± 414.8 a Androstenedione (ng/ml) 426.2 ± 30.3 a 435.2 ± 46.7 a 487.6 ± 42.9 a 548.3 ± 42.36 a Soluble c-Kit (pg/ml) 317.8 ± 27.5 b 380.8 ± 30.3 a 429.8 ± 28.7 a 455.2 ± 28.75 a Data are shown as mean ± SEM. Analysis was performed by ANOVA and Tukey’s test for multiple comparisons. Means without a common letter are significantly different (P<0.05). PCOS; Polycystic ovarian syndrome, MET; metformin, and NAC; N-acetylcysteine, and PLA; Placebo.   Fig.2Correlation between parameters of follicular fluid (FF) in all the population. A. Soluble c-Kit with volume of FF, B. Estradiol (E2), and C. Androstenedione.
R2; Determination of coefficient.  Fig.3Distribution of oocytes retrieved, quality of oocytes and embryos in polycystic ovary syndrome patients undergoing treatment of N-acetylcysteine
(NAC), metformin (MET), NAC+MET and Placebo (control).Data are the mean ± SEM. Statistical analyses were performed by ANOVA followed by Tukey's test for multiple comparisons. Means without a common
letter are significantly different (P<0.05).

BODY.RESULTS.EXPRESSION PROFILE OF GDF-9, BMP-15 AND C-KIT IN
OOCYTES:
The level of BMP-15 protein in the mature unfertilized
oocytes and GV oocytes did not differ significantly among
the groups (P>0.05, Figes.4, 5A, D, E). The expression level
of GDF-9 in the GV oocytes significantly increased in
all groups compared to the placebo (P<0.001) (Fig.4B, D,
F), while for unfertilized mature oocytes, GDF-9 mRNA
and protein levels only significantly increased in the NAC
group (P<0.001, Fig.5B, D, F). The expression of c-kit in
the GV oocytes significantly decreased in the NAC and
MET groups compared to the placebo group (P<0.001),
but not in NAC+MET group (Fig.4C, D, G). The c-kit
expression in the unfertilized mature oocytes significantly
decreased in the NAC group compared to the MET and
other treatment groups (P<0.001), but no significant difference
was found in the MET and NAC+MET groups
when compared to the placebo group (P>0.05, Fig.5C, D,
G). It is important to note that the results for MI oocytes
were similar to GV oocyte; therefore, in this article only
the results of GV were presented. This observation is in
accordance with pervious literature (24).  Fig.4Effects of NAC and MET on BMP-15, GDF-9 and c-kit mRNA and protein expression in immature oocytes (IMO, GV oocytes) of PCOS patients. Results
of reverse transcriptase real-time polymerase chain reaction (PCR) for mRNAs of A. BMP-15, B. GDF-9, C. c-kit, in GV oocytes, D. Immunoblots of BMP-15,
GDF-9 and c-kit from oocyte cell lysates. Densities of E. BMP-15, F. GDF-9 and G. c-kit protein bands in the experimental groups are shown. Means without
a common letter are significantly different (P<0.05).NAC; N-acetylcysteine, MET; Metformin, GV; Germinal vesicle, and PCOS; Polycystic ovarian syndrome.  Fig.5Effects of NAC and MET on BMP-15, GDF-9 and c-kit mRNA and protein expression in unfertilized mature oocytes (UMO, MII oocytes) of PCOS
patients. Results of reverse transcriptase real-time polymerase chain reaction (PCR) for mRNAs of A. BMP-15, B. GDF-9, C. c-kit in MII oocytes, D. Immunoblots
of BMP-15, GDF-9 and c-kit from oocyte cell lysates. Densities of E. BMP-15, F. GDF-9, and G. c-kit protein bands in the experimental groups are
shown. Means without a common letter are significantly different (P<0.05).NAC; N-acetylcysteine, MET; Metformin, and PCOS; Polycystic ovarian syndrome.

BODY.DISCUSSION:
A typical characteristic of PCOS patient commonly observed
during induction stimulation for ART cycles is increased
number of low quality oocytes which is mainly
related to state of endocrine disorder in these individuals
(25). Considering the indispensable role of OSFs in oocyte
development and maturation, many researches have shown
impaired expression of OSFs particularly GDF-9, BMP-
15 and c-kit, may account for low quality oocyte in PCOS
undergoing ovarian stimulation (26). This may explain, at
least a part of the folliculogenesis disorders found in these
patients (27-29). Background literature in this filed is very
discrepant. Some authors have reported reduce expression
of GDF-9 with no significant alteration in the expression
of BMP-15 (8), while others have shown no alteration in
expression of these two factors both at RNA and protein
level (9) in oocyte of PCOS individuals. The exact reason
of such discrepancy is not well understood.  In continue to our previous study, we demonstrated that
unlike MTE and NAC+MET groups, the administration
of NAC compared to placebo group, improves the maturation
and quality of oocytes and also embryo development
in PCOS patients undergoing ICSI (18). Therefore, in this
we aimed to evaluate whether NAC could alter BMP-15,
GDF-9 and c-kit levels, as the main OSFs in the oocytes
of PCOS patients in comparison to MET and MET+NAC.  Compelling evidence suggest that GDF-9 and BMP-15
members of the TGFβ superfamily are exclusively expressed
in the oocyte and their expression increases as follicle development
progresses (30). During postnatal ovarian development,
c-kit mRNA and protein are localized in the oocytes
(6), and in this regard Brankin et al. (31) has shown a relation
between KL/c-kit interaction with antrum formation,
steroidogenesis and oocyte quality. Furthermore, genetic and
descriptive studies have implicated the involvement of c-kit
receptor and its ligand, KL, in oocyte growth (32).  Low GDF-9 levels is associated with abnormally increased
KL level in PCOS, which could lead to abnormal
ovarian features such as enlarged oocytes and increased
follicle numbers (7, 32). In PCOS patients, the GDF-9
mRNA level within the oocytes is lower than in oocyte
derived from normal individuals (8), and it is believed
that there is a negative association between GDF-9 expression
and KL/c-kit expression. Tuck (7) believes that
excess androgens may act to further reduce the inhibitory
effect of GDF-9, thus resulting in an abnormal increase in
the KL/c-kit protein level in PCOS individual. Considering
the inverse relationship between c-kit and GDF-9 in
PCOS (7, 8, 32), therefore, improving the expression of
GDF-9 is expected to cause a reduction in c-kit levels.  Our results displayed a significant increase in the expression
of GDF-9 in the unfertilized mature oocytes of
PCOS patients after administration of NAC compared
to MET, indicating that NAC, as an anti-oxidant/antiapoptotic
agent, could enhance the expression of GDF-9
through inhibiting the activity of NF-kB and AP-1 transcription
factors, therefore affecting the activity of MAPkinase
signaling and related genes expression (33), which
may be able to alleviate PCOS follicular disorders and
prevent follicular developmental detention and atresia.  Our study, in agreement with aforementioned studies, also
showed a significant decrease in the expression of c-kit in
the oocytes of PCOS patients and also the soluble c-kit protein
in the FF following administration of NAC compared to
control. In addition, evidence has indicated the relationship
between KL/c-kit system with MAPK pathway and/or PI3K/
Akt pathway, which are both necessary for follicle development
(34). It is likely that NAC decreases the expression
of c-kit through interference in MAPK pathway, all of this
could be the underlying reason in the role of NAC in preventing
follicular developmental detention and atresia and
alleviation of follicular disorders in PCOS patients.  Although the FF content may be an invaluable hallmark
for PCOS diagnosis, but the NAC ability to modulate
these intra-ovarian factors of the oocyte may have interesting
pharmacological perspectives for clinical management
of PCOS patients. According to literature (7, 8, 32),
there is an inverse relationship between c-kit and GDF-9.
Therefore, improves expression of GDF-9 by NAC treatment,
is expected to follow by a reduction in c-Kit and
indeed MET appears to mask this effect of NAC, how this
masking effect is performed, remains to be elucidated.  In this regard it has been shown that with increased follicular
size and E2 production, the amount of soluble c-kit
protein in human FF also increase (35), which is consistent
with the correlation observed in this study between
soluble c-kit with the FF volume, E2, and androstenedione
concentrations.  It has been demonstrated that the excess secretion of
anti-mullerian hormone (AMH) in the FF of PCOS patients
may directly inhibit the production of OSFs such
as GDF-9 and BMP-15, which can explain the low levels
of OSFs in PCOS oocytes (36). Our findings showed
a reduction in the AMH level in the FF of NAC treated
group (18). Although this reduction was not statistically
significant but may be considered as an underlying reason
for the increased levels of GDF-9 in the NAC treated patients.
Moreover, in agreement with other findings (8, 9),
our study revealed no significant difference in the level of
BMP-15 mRNA among the studies groups.

BODY.CONCLUSION:
Considering the fact that NAC improves oocyte maturation
and embryo quality, and decreases the rate of immature
oocytes in women with PCOS while being a safe and welltolerated
agent, we suggest the administration of NAC as
an alternative to other insulin-sensitizing agents like MET.
Therefore, the present study argues that NAC possibly improves
the oocyte quality of PCOS patients compared to
MET through modulating the c-kit and GDF-9 expression,
indicating that NAC supplement may be a therapeutic alternative
to the insulin-sensitizing agents in PCOS management.

Question: Compared to (MET+NAC), metformin (MET), what was the result of N-acetylcysteine (NAC) on GDF-9 expression?
Evidence: The follicular fluid (FF) level of c-kit protein significantly decreased in the NAC group compared to the other
groups. 
Answer: significantly increased