Volume 16, Issue 12 (December 2018)                   IJRM 2018, 16(12): 775-782 | Back to browse issues page


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Hendarto H, Widyanugraha M Y A W, Widjiati W. Curcumin improves growth factors expression of bovine cumulus-oocyte complexes cultured in peritoneal fluid of women with endometriosis. IJRM 2018; 16 (12) :775-782
URL: http://ijrm.ir/article-1-1333-en.html
1- Department of Obstetrics and Gynecology, Faculty of Medicine, Airlangga University/Dr Soetomo Hospital Surabaya, Indonesia , hndhendy@yahoo.com
2- Department of Obstetrics and Gynecology, Faculty of Medicine, Airlangga University/Dr Soetomo Hospital Surabaya, Indonesia
3- Laboratory of Embriology, Faculty of Veterinary Medicine, Airlangga University, Surabaya, Indonesia.
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Endometriosis is one of the most common gynae- cological diseases characterized by the growth of endometrial-like tissue outside the uterus that sub- sequently induces a chronic inflammatory reaction (1, 2). Numerous symptoms are associated with endometriosis, including dysmenorrhea, pelvic pain, dyspareunia, and infertility, as well as reduced quality of life (2–4). The relationship between endometriosis and infertility remains unclear. One of the postulations of decreased fertility in women with endometriosis is the reduction of oocyte quality caused by abnormal folliculogenesis related to peritoneal fluid (PF) inflammation and oxidative stress (5–8).
Folliculogenesis is a dynamic process marked by proliferation and differentiation of granulosa cells and maturation of oocyte. The regulation of ovarian folliculogenesis, determined by a number of growth factors and classic endocrine mech- anism, provides an optimal environment to pro- duce fertilizable oocyte (9, 10). There are two important growth factors that contribute to the regulation of ovarian folliculogenesis including the interactions between oocyte and granulosa cells. Those growth factors are Growth Differentiation Factor 9 (GDF-9) produced by oocyte, which is useful for granulosa cell proliferation and differ- entiation, and Kit Ligand (KitL) secreted by gran- ulosa cells, which induces oocyte maturation (11– 14).
In women with endometriosis, ovaries are natu- rally bathed in PF that is rich in inflammatory medi- ators. This may cause abnormal folliculogenesis and subsequently result in infertility. The PF of women with endometriosis contains a variety of inflamma-
tory mediators, including Tumor Necrosis Factor 𝛼
(TNF𝛼), Interleukin-6 (IL-6), and Interleukin-8 (IL-8)
(15–17).
Curcumin, which is derived from Curcuma longa (turmeric), has a strong potential anti-inflammatory activity. It has been widely used both traditionally

and scientifically to treat various conditions includ- ing inflammatory diseases such as rheumatoid arthritis, chronic anterior uveitis, and ulcerative col- itis through various intracellular and extracellular molecular pathways (18–20). Treatment with cur- cumin can reduce implant size and cell proliferation in endometriosis model rat (21), but the effects   of curcumin on  bovine  ovarian  growth  factors  in endometriosis and infertility remain controver- sial.
The objective of our study was to evaluate whether curcumin could improve growth factors expression  in  bovine  cumulus-oocyte complexes
(COC)s by analyzing GDF-9, KitL, and TNF𝛼 expres-
sions  in  culture  medium  with  PF  from  infertile
women with endometriosis.
 
 

1.Materials and Methods

In this laboratory experimental study, two growth factors expression of folliculogenesis reg- ulation on bovine COCs cultured in three different types of medium was evaluated. Group 1 (con- trol group) Tissue Culture Medium 199 (TCM199) only, Group 2 (endometriosis group) TCM199 plus PF from infertile women with endometriosis, and Group 3 (endometriosis+curcumin) TCM199 plus PF from infertile women with endometriosis added with curcumin. A total of 21 bovine COCs were cultured in the medium; each group contained 7 COCs.
 
 

1.1.Endometriosis peritoneal   fluid (PF)

 
PF samples were collected from 21 women aged between 20 and 40 years with endometriosis undergoing laparoscopy for infertility evaluation at Dr Sutomo Hospital from January to July 2015. The diagnosis of endometriosis was made by visual inspection and peritoneal biopsy according to the American Society for Reproductive Medicine criteria (22, 23). PF has been collected by aspira- tion from posterior cul-de-sac during the laparo- scopic procedure. The samples were placed in a tube and centrifuged at 600 g for 10 min. The supernatants were stored at –80ºC until analy- sis.
 
 

1.1.    Curcumin

 
Curcumin has been obtained from Merck Schuchardt OHG (85662 Hohenbrunn, Germany). Curcumin 0.2 ml/medium (curcumin 20 μ/ml dissolved into Carboxymethyl Cellulose Sodium/ Na-CMC) was added and homogenized in 10 ml
of TCM199 medium and PF from infertile women
with endometriosis (30 𝜇l). BSA (3%) was added until the pH reached to 7.4–7.8. The solution was
then  filtered  through  a  0.22-𝜇m  microfilter, and
100 mL of solution was placed in a Petri dish for
culture.
 
 

1.2.    Bovine cumulus-oocyte complex

 
COCs aspirated from antral follicles with a diam- eter of 3–8 mm were obtained from bovine ovaries in a slaughterhouse. The ovaries were  washed and stored in 0.89% NaCl with penicillin-G (1000 IU/ml) and streptomycin sulfate (0.2 ug/l) at a temperature of 30–35ºC. Before follicle aspiration, the diameter of the follicles was measured with   a caliper. The COCs were aspirated using an 18-  G needle connected to a 5-ml syringe containing
1 ml of phosphate  buffered  saline  (PBS)  with  3% bovine serum albumin (BSA) and 50 ug/ml gentamycin.
The COCs were washed three times successively in PBS medium and one time in TCM199, placed   in TCM199 medium with 50 mIU/ml FSH and 50 mIU/ml LH, divided into three groups by placing them  into  medium  groups  1,  2, and  3, and then
incubated at a temperature of 38ºC in 5% CO2
for 24 hr. Subsequently, the COCs of each group were fixated in a glass flask and subjected to immunohistochemical staining for GDF-9 (Bioss Antibodies  Inc  USA,  catalog  no.  Bs-4720R), KitL
(Abcam  USA,  catalog  no.  ab52603),  and  TNF𝛼
(Bioss Antibodies Inc USA, catalog no. Bs-2081R)
expressions.
The three expressions were semi-quantitatively assessed according to the modified Remmele method which is the result of multiplication between the percentage score of immunoreactive cells (positive cells) with the color intensity score generated on the cell (24).
 

1.1.Ethical consideration

 
The study has been approved by the Ethical Committee for Health Research at Dr Sutomo Hos- pital Surabaya (428/Panke. KKE/X/2014). Written informed consent was obtained from participants before starting the study.
 

1.2.Statistical analysis

 
Data analysis was performed using SPSS statis- tical software (Statistical Package for the Social Sciences, version 17.0, SPSS Inc, Chicago, Illinois, USA). Normality of variable was tested with the Kolmogorov–Smirnov  test.  One-way  analysis   of
variance (ANOVA) test was used to detect  signif-
icant differences of all variables. p < 0.05 was accepted as statistically significant.
 
 

2.Results

2.1.GDF-9 expression

 
The GDF-9 expression in bovine COC was deter- mined by the dark color of immunoreactive cells on the immunohistochemical staining result, and  it was different among the three groups (Figures 1A, B, C). The mean expression of GDF-9 in bovine endometriosis+curcumin (2.67 ± 0.98) increased COC  cultured  in  PF  from  infertile  women  with
compared to those cultured without curcumin (0.50
± 0.67) but reduced compared to the control (5.83
± 1.58). (p ≤ 0.001) (Figure 2).
 

1.1.Kit ligand expression

 
The KitL expression in bovine COC was deter- mined by the dark color of immunoreactive cells on the immunohistochemical staining result, and  it was different among the three groups (Figures 1D, E, F). The semi-quantitative results of KitL expression in bovine COC were similar to those of GDF-9 expression. The mean expression of KitL in bovine COC cultured in PF from infertile women
with endometriosis (0.33 ± 0.49) reduced compared to the control (3.92 ± 2.02) and those in the endometriosis + curcumin group (2.67 ± 1.23) (p
≤  0.001).  There was also a significant   difference
between KitL expression in bovine COC cultured

in PF from infertile women with endometriosis + curcumin and control group (p = 0.03) (Figure 2).

1.2.TNF𝛼 expression

The semi-quantitative results of TNF𝛼 expression in bovine COC cultured in control,  endometriosis,
and endometriosis+curcumin groups were 0.00 ± 0.00; 8.67 ± 3.72; and 2.17 ± 1.69, respectively (25).  TNF𝛼   expression  in  bovine  COC  cultured
in PF from infertile women with endometriosis increased  compared  to  those  in  control  group,
whereas TNF𝛼 expression in bovine COC cultured in
PF from infertile women with endometriosis added
with curcumin reduced compared to those cultured without  curcumin;  however,   the  level  increased
compared to the control (p ≤ 0.001) (Figure 2).
In this study, the relationship of three variables
was also examined using regression analysis and
revealed a significant association among TNF𝛼, GDF-9, and KitL expression cultured in PF from
curcumin             (p             <             0.05). infertile  women  with  endometriosis  added with



Figure 1: The immunoreactive cells (black arrow) of GDF-9 (A,B,C) and KitL (D,E,F) on immunohistochemical staining of bovine COC
in the following medium: 1. control, 2. endometriosis, and 3. endometriosis + curcumin (p < 0.05). GDF9 = Growth Differentiation
Factor-9; KitL = Kit Ligand; COC = cumulus oocyte complex. Magnification 400x; Olympus BX-50. Pentax optio 230 Digital Camera
2.0 megapixel.




Figure 2: The expression of GDF-9, KitL, and TNF􀀀 on bovine COC in the following medium: 1. control, 2. endometriosis, and 3.
endometriosis + curcumin (p < 0.05).

1.Discussion

In this study, GDF-9 and KitL expressions in bovine COC cultured with PF from infertile women with endometriosis were significantly reduced com- pared to control. It suggests that numerous inflam- matory mediators in the PF of infertile women with endometriosis enters the bovine COC and further inhibits oocytes and granulosa cells’ activ- ity. This results in abnormal folliculogenesis and subsequently infertility.
The PF of infertile women with endometriosis contains a variety of inflammatory mediators (15,
26) and the PF component plays  an  essential  role in the process of folliculogenesis, ovulation, and fertilization. Communication between oocytes and granulosa cells occurs during folliculogene- sis to produce mature and fertilizable oocytes. This process is impaired in infertile women with

endometriosis. We previously reported abnormal GDF-9 and KitL expression in endometriosis (7, 27).
GDF-9 is part of the TGF𝛽  family member dom-
inantly secreted by oocyte and plays in important
role in ovarian folliculogenesis. Study in GDF-9 deficient female mice has resulted in impaired follicular development after primary follicle growth that causes infertility (28). GDF-9 is a determining factor for follicular growth, maturation and sur- vival through inhibition of the apoptosis process in granulosa cell and  follicular  atresia  (29, 30). In this study, the addition of curcumin to the culture medium of PF from infertile women with endometriosis  resulted  in  more  improved  GDF-
9 expression than those without curcumin. It is possible that the anti-inflammatory effects of curcumin release oocyte suppression. By taking into  account  the  fact  that  the  expression  of GDF-9 was still lower than the control, it indi- cates that it is necessary to adjust the dosage   of curcumin. These findings indicate that the  dose of curcumin plays a role in GDF-9 expres- sion.
 
In our study, KitL expression in bovine COC cul- tured in PF from infertile women with endometriosis added with curcumin significantly increased from those cultured without curcumin. It indicates that curcumin has a positive effect on KitL expression. KitL is a growth factor produced by granulosa cells in the ovarian follicle. KitL binds to its receptor in the oocyte, c-Kit, and actively functions for signal pathways during folliculogenesis (31). The initiation of primordial follicle growth, and the development of oocyte and follicle are regulated by the KitL/c- Kit system (13, 31). Study in the buffalo ovaries  by Mahajan prove that KitL could be used to predict the oocyte maturation as well as oocyte competence (32).
 
The changes on two growth factors expression, GDF-9, and KitL suggest that curcumin has a repair effect on oocyte-granulosa cell interactions and the regulation of ovarian folliculogenesis. In order to evaluate that curcumin has a repair effect on oocyte-granulosa cell interactions and the reg- ulation of ovarian folliculogenesis, we assessed the effect of curcumin on bovine COC inflamma-
tion  via  TNF𝛼   expression.  An  increase  in TNF𝛼
expression  was  noted  in  the  PF  from  infertile
women with endometriosis compared to that from normal women. In addition, we found a correlation
between TNF𝛼  concentration and the degree of
that TNF𝛼 plays a role in the inflammatory process endometriosis severity. In fact, it has been reported
and angiogenesis, triggers follicular atresia, and impairs oocyte maturation (33–35). The findings of
increased TNF𝛼 expressions in bovine COC cultured
in  PF  from  infertile  women  with   endometriosis
suggest that TNF𝛼  from PF enters the COC and  is active. The addition of curcumin to the culture
reduced               TNF𝛼                 expression. media results in an improvement as indicated by the Curcumin exhibits anti-endometriosis activities by affecting MMP2 and TIMP2 (36). It works as anti- inflammatory medication by inhibiting cyclooxyge- nase2 and lipoxygenase and suppressing the activ- ity of NFkB and the level of inflammatory cytokines,
including  TNF𝛼,  IL-6,  IL-8,  and  IFN-𝛾  (18,  19, 20,
36). Based on its mechanism of action, curcumin
can suppress an inflammatory process in bovine COC cultured in PF from infertile women with endometriosis. Yet, we found that the decrease in
TNF𝛼 expression did not reach a normal level. We also found a significant correlation among TNF𝛼,
GDF9,  and  KitL  expression,  suggesting  that the
inflammatory factor of TNF𝛼 may play a role in GDF- 9 and KitL interaction. Studies evaluating the dose-
response effects of curcumin on the GDF-9-KitL interaction are still needed.
The limitation of our study included the use    of only one concentration of curcumin. However, this was the first attempt to evaluate the effects of curcumin on oocyte-granulosa cell interaction and the regulation of ovarian folliculogenesis in the presence of PF from infertile women with endometriosis.
 

1.Conclusion

Our findings suggest that in the culture of PF from infertile women with endometriosis, curcumin addition improved growth factors expression of bovine COCs through the decrease of inflamma- tion. The increase of GDF-9 and KitL expressions due to the anti-inflammatory effect of curcumin will improve oocyte–granulosa cell interaction and subsequently folliculogenesis. Further studies are warranted.
 

Acknowledgements

The authors thank Prof. Togas Tulandi from McGill University, Canada, for his assistance in the preparation of the manuscript.
 

Conflict of Interest

The authors report no conflict of interest.



 
Type of Study: Original Article |

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