A considerable issue in assisted reproductive
technology (ART) is poor ovarian response (POR)
to gonadotropin stimulation. This condition affects
approximately 9 to 24% of the
The immune system may play an important role in pathophysiology of POR. A major part of immune system is innate immunity that generates more rapid and primary responses to pathogens than the adaptive immune system (
Previous studies have discussed the functional TLRs in the reproductive system. TLRs 1-10 are expressed in the female reproductive tract (
TLRs are activated by specific ligands and can create intra-cellular signals via MYD88-dependent and independent pathways (
Moreover activation of TLRs leads to the stimulation of chemokine and cytokine expression including interleukin (IL) - 6, IL-8 and macrophage migration inhibitory factor (MIF) that activate a variety of host responses (
Follicular cells play an important role in folliculogenesis, steroidogenesis and oocyte maturation (
Therefore, regarding the involvement of the immune system in POR pathogenesis (
This case-control study was approved by Iran University of Medical Sciences and Royan Institute Ethics Committees. Forty participants (20 infertile poor ovarian responder patients and 20 normal women with male factor infertility as control) attending the Reproductive Medicine Unit in Royan Infertility clinic, Tehran, Iran, for intracytoplasmic sperm injection (ICSI) treatment were invited to participate in the study during 2012. An information sheet was offered to all women, and informed written consent was obtained.
Inclusion criteria were infertile women aged 20-35 undergoing ICSI treatment, receiving the same standard long protocol. The exclusion criteria were endometriosis, polycystic ovarian syndrome, endocrine disorder like hyperprolactinemia, history of ovarian surgery and female reproductive tract infection.
POR were defined as any patient who had abnormal ovarian reserve test [antral follicle count (AFC) <5-6 follicle or anti-Mullerian hormone (AMH) <0.5-1.1ng/ml] and a previous poor response (<3 oocyte retrieved) in control ovarian hyperstimulation (COH).
Anthropometric measurements were taken, including BMI [BMI, calculated as weight/(height)2 (kg/m2)]. Luteinizing hormone (LH), follicle stimulating hormone (FSH), anti mullerian hormone (AMH) and testosterone levels were determined. All laboratory parameters were determined in the early follicular phase of the menstrual cycle.
Patients underwent a standard long protocol using GnRH-a (Superfact, Aventis, Frankfurt, Germany) at a daily dose of 0.5 mg subcutaneous start on the day 17-19 of the natural menstrual cycle as a pre-treatment. Once pituitary desensitization was confirmed (endometrial thickness <5 mm and serum estradiol level <50 pg/ml), the GnRH-a dose was reduced by one-half and ovarian stimulation was initiated.
In all patients, ovarian stimulation started with a dose of 150-225 IU r-FSH (Gonal-F, Merck Serono, Switzerland) depending on the age of the patient. It was continued until the day of ovulatory human chorionic gonadotropin (hCG) administration according to the ovarian response. When at least two follicles were greater than 18 mm, 10,000 IU urinary hCG (Choriomon, IBSA, Lugano, Switzerland) was administered intramuscularly for ovulation induction and oocyte retrieval was performed 34-36 hours later (
Follicular fluid aspiration was carried out with transvaginal ultrasound guidance using an aspiration needle from the largest follicle (>18mm) without flushing medium and blood contamination. The follicular fluid was transferred to a sterile Petri dish, and after the oocytes were removed, the fluid was located into a 15-mL conical tube and centrifuged at 300 g for 5 minutes. Supernatant was then removed.
One milliliter of TRI reagent (Sigma, Pool, UK) was added on the cellular pellet and homogenized for total RNA extraction following a standard protocol according to manufacturer’s instructions. Obtained total RNA in both groups was treated three times with DNase I (fermentase, sanktleon-rot, Germany) to remove genomic DNA contamination from the samples. First strand cDNA synthesis was performed using oligodT primers and reverse transcription by Super-Script II (Fermentas). Negative controls were prepared without addition of the enzyme (non-reverse transcribed controls, RT controls). The RT-PCR was performed using cDNA of each patient, Platinum Blue PCR Super Mix (Invitrogen, Pairsley, UK) and the forward and reverse primers for
list of primers were used for regular PCR and real time –PCR
|Variables||Forward primer(5-3)||Reverse primer(3-5)||Annealing temperature(C)||Product size(bp)|
Sequence of TLRs, COX2 and β-actin primers used in the current investigation in RT-PCR. TLR; Toll-like receptor and COX; Cyclooxygenase.
Result of RT-PCR for TLR1, 2, 4, 5, 6, COX2 and β-actin mRNA expression in control (A) and POR (B) groups. RTC; RT control, WC; Water control and COX; Cyclooxygenase.
QPCR was performed with the cDNA prepared from follicular cell pellet. QPCR reactions were carried out in triplicates using an ABI Prism 7300 Sequence Detector (Applied Biosystems, foster, USA) in a total volume of 20 μl containing 250 ng cDNA, 5 pmol gene specific primers and SYBR Green reagent (Applied Biosystems) with ROX dye as passive control for signal intensity. The thermal cycle profile followed 50 cycles at 95˚for 30 seconds, 60-63˚ (
The QPCR data were analyzed using the comparative CT method (
Obtained FF from each patient was centrifuged at 300 g for 5 minutes, then supernatant was used to determine the concentration of IL-6, IL-8 and MIF by commercially Enzyme-Linked Immunosorbent Assay (ELISA) kits for IL-6 (eBioscience, Vienna, Austria), IL-8 (eBioscience, Vienna, Austria) and MIF (glory science, TX, USA).
Briefly, this technique uses a microwell plate coated with monoclonal antibody to human IL-6 (eBioscience), human IL-8/NAP-1 (eBioscience), human MIF (glory science), biotin-conjugate anti human IL-6 monoclonal antibody (eBioscience), biotin-conjugate anti human IL-8/NAP-1 monoclonal antibody (eBioscience), biotin-conjugate anti human MIF monoclonal antibody (glory science), streptavidine- HRP and tetramethyl-benzidine as a substrate.
Color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of these cytokines in the samples is then determined by comparing the O.D. of the samples to the standard curve.
Cytokine concentration were considered zero, if the detected cytokine concentration was equal to or less than the lower limit of kits (IL-6: 0.92 pg/ml, IL-8: 2.0 pg/ml and MIF: 0.003 pg/ml).
The results were expressed as mean ± SEM. Statistical analysis was performed by using t test in SPSS 18 software. P<0.05 was considered significant.
Clinical characteristics of the patients are presented in table 2.There is a significant difference in AMH, number of mature oocyte and total rFSH dose (IU) between POR and control groups.
|P value||Control Group (N=20)||POR Group (N=20)||Variable|
|30.47 ± 4.62||30.75 ± 3.89||0.727|
|25.87 ± 2.92||25.20 ± 4.19||0.55|
|9.76 ± 6.04||7.70 ± 5.41||0.25|
|20 (100)||20 (100)|
|4.23 ± 2.24||5.02 ± 3.39||0.38|
|10.99 ± 3.24||8.55 ± 5.37||0.08|
|0.56 ± 0.92||0.64 ± 0.33||0.71|
|1.21 ± 0.31||1.31 ± 0.54||0.33|
|0.175 ± 0.03||0.63 ± 0.18||0.02|
|1.5 ± 0.44||10.1 ± 1.59||0.00|
|3321 ± 243.65||2390.3 ± 190.39||0.005|
Presented as mean ± SD and compared by t test.
BMI; Body mass index, AMH; Anti-mullerian hormone, LH; Luteinizing hormone, FHS; Follicle stimulating hormone and *; P<0.05.
Clinical characteristics of the patients. There is significant difference in AMH, No. of mature oocyte and total rFSH dose (IU) between POR and control groups. Data were analyzed by t test.
Figure 1 shows the results of RT-PCR for mRNA expression of
The quantitative expression profiles of
The quantitative analysis of IL-6, IL-8 and MIF concentrations in FF by ELISA are shown in figure 3. IL-6, IL-8 and MIF were significantly increased in POR compared with control (p<0.05).
QPCR was used to quantify the expression of TLR1, 2, 4, 5, 6 and COX2 mRNA in POR and control groups. Data are presented as mean ± SEM of normalized expression values against internal controls (β-actin mRNA) in POR and control. TLR1, 2, 4, 5, 6 and COX2 showed a significantly higher expression in POR patients compared with the normal women. Data were analyzed by t test. *; P<0.05, POR; Poor ovarian response, MIF; Migration inhibitory factor, TLR; Toll-like receptor and COX; Cyclooxygenase.
IL-6, IL-8 and MIF protein concentration obtained by ELISA in FF of POR and control groups. IL-6, IL-8 and MIF showed a significantly higher expression in POR compared with the control. Data were analyzed by t test. *; P<0.05, POR; Poor ovarian response, MIF; Migration inhibitory factor and IL; Interleukin.
We found higher expression of
Numerous factors affect ovarian response to gonadotropin stimulation. It is suggested that the main reason of POR is diminished ovarian reserve (
Our findings show that
However, in relation to the TLR overexpression in POR patients, several hypotheses are discussed; i. TLR overexpression is a consequence of the presence of their endogenous or exogenous ligands in FF. Keay et al. (
TLR activation leads to apoptosis through the Fas associated death domain (FADD) (
TLRs have important role in cytokines production and autoimmunity (
IL-6 diminishes aromatase activity within follicles which result in decreased intrafollicular E2 level, fertility and fertilizing capacity (
Our study showed significant higher MIF protein production, consistent with significant
Moreover, TLR signaling results in elevated levels of IL-10 in FF (
As previously stated, overstimulation of TLRs contribute to autoimmune response and tissue injury (
Despite the study being well-designed, the present study possesses the following limitations:
i. The number of included subjects was small and ii. It is a fact that POR group received increased volume of gonadotropins; therefore this increased dosage may have affected the immunological mechanisms.
The association between increased TLR expression in follicular cells in POR suggest that TLRs may play important roles in the pathophysiology of POR. Further studies should be performed in future to confirm these findings and to determine the extent TLRs or other components of the TLR signaling pathway contribute to POR.