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ORIGINAL ARTICLE
Year : 2017  |  Volume : 1  |  Issue : 2  |  Page : 63-68

Expression of cellular FLICE-like inhibitory protein in granulosa cells in In Vitro fertilization patients with advanced endometriosis


Human Assisted Reproduction Center, Shanxi Women and Children's Hospital, Taiyuan 030013, China

Date of Web Publication17-Oct-2017

Correspondence Address:
Xue-Qing Wu
Human Assisted Reproduction Center, Shanxi Women and Children's Hospital, Taiyuan 030013
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2096-2924.216861

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  Abstract 


Background: This study evaluated the expression of cellular FLICE-like inhibitory protein (cFLIP) in granulosa cells (GCs) obtained from in vitro fertilization-embryo transfer patients with advanced endometriosis.
Methods: A total of 267 patients with advanced endometriosis were enrolled in this study. They were divided into clinical pregnancy group (n = 114) and nonpregnancy group (n = 153). The expressions of cFLIP in mRNA and protein level were measured by real-time polymerase chain reaction (RT-PCR) and Western blotting. The related factors on the clinical pregnancy were analyzed using logistic regression analysis. Coefficients of correlation were calculated using the nonparametric rho-Spearman test.
Results: The number of oocytes retrieved, fertilization rate, and cleavage rate were significantly and independently related with clinical pregnancy (P < 0.05). RT-PCR and Western blotting analysis showed that the expressions of cFLP in mRNA and protein level were significantly higher in the clinical pregnancy group than in nonpregnancy group (P < 0.05). cFLIP had a significantly positive correlation with the number of oocytes retrieved (P < 0.05) and no correlation with fertilization rate and cleavage rate (P > 0.05).
Conclusion: Higher expression of cFLIP increased the pregnancy rate in women with advanced endometriosis.

Keywords: Antral Follicle Count; Cellular FLICE-like Inhibitory Protein; Clinical Pregnancy; Endometriosis; In Vitro Fertilization-Embryo Transfer


How to cite this article:
Zhang JP, Yan MQ, Wang YQ, Yang F, Liu J, Wu XQ. Expression of cellular FLICE-like inhibitory protein in granulosa cells in In Vitro fertilization patients with advanced endometriosis. Reprod Dev Med 2017;1:63-8

How to cite this URL:
Zhang JP, Yan MQ, Wang YQ, Yang F, Liu J, Wu XQ. Expression of cellular FLICE-like inhibitory protein in granulosa cells in In Vitro fertilization patients with advanced endometriosis. Reprod Dev Med [serial online] 2017 [cited 2022 Jun 30];1:63-8. Available from: https://www.repdevmed.org/text.asp?2017/1/2/63/216861




  Introduction Top


As an intracellular anti-apoptotic protein,[1] abnormal anti-apoptotic protein cellular FLICE-like inhibitory protein (cFLIP) expression has previously been shown in various diseases such as multiple sclerosis, diabetes mellitus, rheumatoid arthritis, Alzheimer's disease, cardiovascular diseases, and various cancers.[2],[3],[4],[5],[6],[7],[8] cFLIP expression during apoptosis of ovarian granulosa cells (GCs) was firstly detected in rats and pigs in 2002.[9],[10] Subsequently, the role of cFLIP during the apoptosis of ovarian GCs and follicular atresia in humans was reported. Goto et al.[11] found that cFLIP mRNA was highly expressed in follicular GCs prepared from healthy follicles in humans. It was found that cFLIP was an essential pro-survival factor for GCs which prevented GC apoptosis by inhibiting procaspase-8 activation.[12],[13] There are no reports which show the relationship between cFLIP expression and clinical pregnancy in endometriosis patients undergoingin vitro fertilization-embryo transfer (IVF-ET). Therefore, the aim of the present study is to evaluate the expression of cFLIP in mRNA and protein level in GCs of IVF patients with advanced endometriosis and explore the correlation between cFLIP expression and clinical pregnancy.


  Methods Top


Ethical approval

The study was approved by the Institutional Ethics Committee of Shanxi Women and Children Health Hospital. Written informed consent was obtained from all the participants.

Patients

This cohort study included 267 infertile women aged between 24 and 38 years. All patients were diagnosed with stage III/IV endometriosis through laparoscopic surgery using the revised American Fertility Society (AFS) classification of endometriosis before they received in vitro fertilization-embryo tranfer (IVF-ET)/intracytoplasmic sperm injection (ICSI) treatment.

Protocol of ovulation induction and oocyte retrieval

All patients received a long protocol. Patients received the gonadotropin-releasing hormone agonist triptorelin acetate (0.05–0.1 mg/d, Ferring International Center, SA, Switzerland) through subcutaneous injection during the previous midluteal phase for 14 days. Controlled ovarian hyperstimulation (COH) commenced after pituitary downregulation (estradiol [E2] <50 ng/L, luteinizing hormone, and follicle-stimulating hormone [FSH] <5 IU/L) was achieved. The starting dose of recombinant FSH (75 IU/d or 450 IU/d, Gonal-F, Merck Serono S.p.A., Italy) and/or human menopausal gonadotropins (75 IU/ampoule, menotropins, Detergent Pharmaceutical Group, China) ranged from 150 to 450 IU/d which was determined in accordance with the body constitution. Ovarian response was monitored by regular serum E2 measurements and transvaginal ultrasonography. When at least the diameter of one follicle was ≥18 mm, two follicles reached 17 mm or three follicles reached 16 mm, recombinant human chorionic gonadotropin (rhCG) solution (250 μg, Merck Serono S.p.A., Italy) was administered through injection. Oocyte retrieval was performed using transvaginal ultrasound-guided puncture of follicles 34–36 h after hCG administration under intravenous or local anesthesia.

Granulosa cell (GCs) isolation

Human GCs were obtained from follicular fluid during oocyte retrieval. The follicular fluid was prepared by initial centrifugation followed by layering on 40% Percoll (Sigma). The middle ring-like layer of three layers was collected and washed by centrifugation for 10 min at 400 g. The pellet was then resuspended in phosphate-buffered saline (PBS), and red blood cell lysate was added (volume 1:3) at room temperature for 10 min. Finally, it was centrifuged at 2,862 g for 1 min and washed three times with PBS at 2,862 g. The GCs were then collected.

Real-time polymerase chain reaction (RT-PCR)

Total RNA was extracted with E.Z.N.A ® Tissue RNA Kit (OMEGA) according to the manufacturer's instructions. One microgram of total RNA was carried out by reverse transcription using QuantiTect ® Reverse Transcription Kit (QIAGEN). RT-PCR was conducted using CFX Connect™ Real-time System (Bio-Rad Laboratories, Inc.) and accompanying software (CFX Manager Software, Bio-Rad Laboratories, Inc., Hercules, CA, USA). Forward primer was 5'-TTCTGGGAGAGGCACTGC-3' and reverse primer was 5'-GTGGTGTCAGAGGGVVAG-3. The productive size was 208 bp. The PCR was initiated by incubation at 94°C for 5 min, and then was denatured at 94°C for 30 s, annealed at 58/51.9°C for 30 s, and extended at 72°C for 40 cycles. Melting curves were evaluated from 60°C to 95°C. The comparative Ct method was used to analyze the data. All analyses were performed in triplicate, and the ratio of target gene expression was normalized with housekeeping gene GAPDH. For each sample, the value of 2−ΔCt was calculated, and data were graphically indicated as relative expression.

Western blotting

Total protein was extracted from human GCs. Twenty-five micrograms of protein was separated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and then transferred electrophoretically to a 0.2 μm polyvinylidene difluoride membrane. The membrane was blocked with 5% nonfat milk in Tris-buffered saline and Tween-20 for 2 h at room temperature. Then, the membranes were incubated separately with primary antibodies: rabbit anti-human cFLIP antibody (Abcam) overnight at 4°C, and β-actin antibody (protein) was used as the internal control. Finally, the membranes were incubated for 2 h at room temperature with secondary antibody: goat anti-rabbit antibody (protein). The immune complexes were detected by chemiluminescence. ImageJ 1.46 software (National Institute of Health, Bethesda, MD, USA) was used to determine the intensity of bands and performed the quantitative analyses of each gray numerical value. The relative expression level was calculated by the ration of target protein/β-actin.

Embryo transfer and clinical pregnancy

Day 3 embryo transfer (ET) was performed using the prodimed embryo transfer catheter under ultrasound guidance. Three embryos were transferred in patients aged ≥35 years and two embryos were transferred in patients aged <35 years. Luteal-phase support was performed by daily progesterone intramuscular injection (Xianju, Zhejiang, China) after oocyte retrieval. All patients underwent IVF or ICSI cycles. Embryos were graded on days 2 and 3 using a score of 1–4 (with 1 is the best), which was based on cell symmetry, fragmentation, and blastomere number.[14] Pregnancies were confirmed by a rising concentration of serum β-hCG tested 14 days after ET. Clinical pregnancy was defined as the presence of an intrauterine gestational sac using transvaginal ultrasonography examination 30–35 days after ET.

Statistical analysis

The statistical analyses were performed by SPSS software version 18.0 (SPSS Inc., Chicago, IL, USA). Values were expressed as mean ± standard error of mean (SEM) or percentages as required. Statistically significant differences were determined using t-test or Chi-square test as appropriate. The related factors on the clinical pregnancy were analyzed using logistic regression analysis and the values were expressed as mean ± standard error (SE). Coefficients of correlation were calculated using the nonparametric rho-Spearman test. P< 0.05 was considered statistically significant except that P< 0.10 was considered statistically significant in logistic regression analysis.


  Results Top


Patients' basal characteristics and COH parameters

A total of 267 patients with endometriosis-related infertility underwent IVF. The patients were divided into clinical pregnancy group (n = 114) and nonpregnancy group (n = 153). Patients' basal characteristics and COH parameters were summarized in [Table 1]. Patients of two groups were comparable in terms of age, body mass index (BMI), duration of infertility, day 3 FSH level, and antral follicle count (AFC) (P > 0.05). Similarly, there were no differences in gonadotropin initial doses, duration of stimulation, and serum progesterone level on the day of hCG administration between the two groups (P > 0.05). Statistically significantly lower total doses of gonadotropin and higher serum E2 levels on the day of hCG administration were observed in clinical pregnancy group than those of nonpregnancy group (P< 0.05).
Table 1: Patients' basal characteristics and parameters of COH in clinical pregnancy group versus control group

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Patients' embryological data

The embryological data were compared between 114 clinical pregnancy and 153 nonpregnancy patients. The number of oocytes retrieved, metaphase II (MII) oocytes, fertilization rate, the number of two-pronucleated oocytes, cleavage rate, and good-quality embryo rate in the clinical pregnancy group were significantly higher compared with those of nonpregnancy group (P< 0.05) [Table 2].
Table 2: Embryological data in clinical pregnancy group versus nonpregnancy group

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Expression of cFLIP in human GCs

The amplification curves and the melting peak of cFLIP gene were shown in [Figure 1]a and [Figure 1]b. Compared with women without pregnancy, women with clinical pregnancy had a significantly higher expression of cFLIP mRNA (0.27 ± 0.31 vs. 0.16 ± 0.18, respectively, P < 0.05) [Figure 1]c. cFLIP protein expression was also detected by Western blotting in the two groups [Figure 2]a. Compared with women without pregnancy, women with clinical pregnancy had a significantly higher expression of protein level (0.55 ± 0.03 vs. 0.32 ± 0.02, respectively, P < 0.05) [Figure 2]b.
Figure 1: cFLIP mRNA expression in clinical pregnancy and nonpregnancy groups. (a) Amplification curves of cFLIP gene. (b) Melting peak of cFLIP gene. (c) cFLIP mRNA levels in clinical pregnancy and nonpregnancy groups (data expressed as mean ± SEM, *P < 0.05). cFLIP: Cellular FLICE-like inhibitory protein; SEM: Standard error of the mean.

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Figure 2: cFLIP protein expression in clinical pregnancy and nonpregnancy groups. (a) cFLIP protein expression detected by Western blotting. (b) Gray analysis showing cFLIP protein expression in clinical pregnancy and nonpregnancy groups (data expressed as mean ± SEM, *P < 0.05). cFLIP: Cellular FLICE-like inhibitory protein; SEM: Standard error of the mean.

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Logistic regression analysis for affecting factor of clinical pregnancy

Total dose of gonadotropin used, serum E2 level on hCG administration, the number of oocytes retrieved, MII oocytes, fertilization rate, the number of two-pronucleated oocytes, cleavage rate, good-quality embryo rate, cFLIP mRNA, and protein level were compared between the two groups. All these variables were entered into the logistic regression model, the result showed that the number of oocytes retrieved, fertilization rate, cleavage rate, cFLIP expression in mRNA, and protein level were statistically significant [Table 3]. This result indicated that the five variables were significantly and independently related with clinical pregnancy (P< 0.10).
Table 3: Related factors of clinical pregnancy in endometriosis patients

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Correlation between cFLIP, number of oocytes retrieved, fertilization rate, and cleavage rate

Significant correlation coefficients (r) among cFLIP, number of oocytes retrieved, fertilization rate, and cleavage rate were analyzed. cFLIP had a significantly positive correlation with the number of oocytes retrieved in the mRNA level (r = 0.342, P < 0.05) and protein level (r = 0.91, P < 0.05). There was no correlation between the cFLIP expression and fertilization rate in the mRNA level (r = 0.079, P > 0.05) and protein level (r = 0.013, P > 0.05). There was no correlation between the cFLIP expression and cleavage rate in the mRNA level (r = 0.083, P > 0.05) and protein level (r = 0.003, P > 0.05).


  Discussion Top


The expression of cFLIP in GCs of advanced endometriosis has not been reported. In this study, we evaluated cFLIP expression in GCs of IVF patients with advanced endometriosis for the first time.

cFLIP is a mammalian homolog of viral FLIP that was initially discovered by bioinformatic research. It is a novel virus-encoded apoptosis regulatory molecule containing a death effector domain.[1] cFLIP has been identified as an intracellular anti-apoptotic protein. The cFLIP gene is located on chromosome 2q33-34 in a cluster of 200 kb together with caspase-8.[15] When death receptor Fas interacts with its ligand FasL, the cytoplasmic tail of Fas recruits cFLIP, procaspase 8, and several other proteins to form a membrane-bound receptor complex which is called the death-inducing signaling complex (DISC).[16] The homodimerization of procaspase-8 in DISC causes autocatalytic cleavage and generates activated caspase-8. The activated caspase-8 then triggers the downstream activation of caspase-3 and eventually leads to apoptosis.[17] As a potential competitive inhibitor that structurally resembles caspase-8, cFLIP prevents the recruitment of caspase-8 from the DISC and thereby interferes with caspase-8 recruitment and activation, and eventually suppresses apoptosis.[18],[19]

In our study, we hypothesize that higher cFLIP expression may contribute to clinical pregnancy in endometriosis patients undergoing IVF-ET. In addition, the pregnancy outcome may be due to other disorders such as AFC, initial dose of gonadotropins, and number of retrieved oocytes which may be caused by endometriosis itself or surgery. Therefore, we analyzed all the affecting factors of clinical pregnancy using logistic regression analysis. Of the ten predictor variables, five final variables including the number of oocytes retrieved, fertilization rate, cleavage rate, cFLIP mRNA, and protein level were significantly and independently related with the outcome of IVF patients with advanced endometriosis. Finally, correlation coefficients among cFLIP, number of oocytes retrieved, fertilization rate, and cleavage rate were calculated.

Our results demonstrate that women with clinical pregnancy have a significantly higher expression of cFLIP mRNA and protein level than women without pregnancy (P< 0.05). It appears that higher expression of cFLIP increased the chance of high pregnancy rate in women with advanced endometriosis. This may be explained by low expression of cFLIP in GCs which may be related to the abnormal status of apoptosis in advanced endometriosis patients.

To avoid confounding factors, logistic regression analysis and nonparametric rho-Spearman test were performed, and results showed that cFLIP had a significantly positive correlation with the number of oocytes retrieved (P< 0.05). We speculate that cFLIP may play a crucial role in determining follicular growth and/or atresia which eventually resulted in the difference of number of oocytes retrieved in the clinical pregnancy and nonpregnancy groups. This was supported indirectly by Sifer et al.[20] who investigated the relationship between induced apoptosis of human luteinized GCs and IVF outcome in 25 infertility women, and found that a high percentage of apoptotic GCs correlated with a significantly lower pregnancy rate.[20]

In summary, this is the first report describing the expression of cFLIP in GCs of women with advanced endometriosis. Since women with stages I–II endometriosis in our center generally select conservative treatment or intrauterine insemination first, enough samples could not be obtained. In the future, we will focus our efforts on the relationship between expression of cFLIP and different stages of endometriosis. We will also investigate whether the protein expression of cFLIP is directly related to apoptosis of GCs.

Financial support and sponsorship

This research was funded by Shanxi Women and Children's Hospital, China (No. 201529).

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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