Upregulation of ICAM-1 and HLA-G Genes Expression in Cumulus Cells of Infertile Women with Poor Response to Ovarian Stimulation with a Healthy Lifestyle

Purpose: Diagnosis of women with poor ovarian response (POR) to exogenous gonadotropin stimulation is a challenge for reproductive specialists. This study aimed to investigate the expression prole for ICAM-1 and HLA-G genes in the cumulus cells of infertile women with POR based on their healthy lifestyle. Methods: Eighty women under the age of 35 were enrolled and divided into two groups: 1- POR without a healthy lifestyle (n=40) and POR with a healthy lifestyle (n=40). The ICAM-1 and HLA-G genes expression was compared by the quantitative PCR between the groups. The ICAM-1 and HLA-G protein levels were analyzed by the western blotting technique, and the methylation pattern was investigated by the methylation-specic PCR. Results: The qRT-PCR assay showed that ICAM-1 and HLA-G genes were differentially expressed between the two groups studied. ICAM-1,HLA-G genes,and proteins expressions in POR with healthy lifestyle were upregulated compared to the second group (P<0.05). ICAM-1 and HLA-G DNA methylation status in the POR with a healthy lifestyle were decreased compared to the other group (P<0.05). The oocyte quality and clinical pregnancy ratio were signicantly higher in the POR with a healthy lifestyle women than in the other ones (P<0.05). Conclusion: Theupregulation of ICAM-1 and HLA-G in POR women is probably related to lifestyle. Lifestyle may affect the methylation pattern, protein levels and alteration of gene expression proles in the cumulus cells of POR women. Additionally, lifestyle may be considered a marker for ovulation, oocyte maturation, preimplantation and clinical pregnancy process. single center lifestyle program of POR patients in Iran. We found that upregulation of ICAM1 and HLA-G in POR appeared to be related to nutrition and lifestyle. The nutrition and lifestyle may be effective on the methylation pattern, expression prole and proteins level in cumulus cells from POR. Moreover, nutrition and lifestyle may act as a biochemical marker for ovulation and the oocyte maturation process.


Introduction
The term poor response to ovarian stimulation (POR) typically refers to woman with a reduced ovarian reserve or poor ovarian response to exogenous gonadotropin stimulation [1]. In fact, the POR or the cycle without ovulation is a cycle in which the ovary is unable to release the oocyte. As a result, ovulation does not occur. Despite the efforts to optimize the de nition of POR, unfortunately there is still limited knowledgeabout the POR pathophysiology, and the etiology hinders the practical solutions to manage the condition [2]. Indeed, the diagnosis of anovulation is not easy. Contrary to popular belief, women with anovulation have more or less a normal menstrual cycle and usually for the rst time, they nd their own problem when they want to become pregnant [3]. Recently, POSEIDON criteria have been used to diagnose POR, including age, Anti Müllerian Hormone (AMH) dose, Antra Follicle Count (AFC), and oocyte number [4]. The consequential complications of POR are intrinsic ovarian resistance to gonadotropin stimulation, reduced ovarian reserve, ovulation and oocyte maturation disorder [5]. Reduced ovarian reserve is de ned as the impaired fertility ability leading to infertility. In fact, excessive use of plastics objects in the human life has been proposed as one of the causes of creating POR in women [6]. Indeed, excessive use of plastics to store hot food and drinks can cause various diseases. Polycarbonate is a type of plastic containing BisphenolA (BPA) [7]. Over time, when plastic is heated or exposed to acidic or alkaline substances, it breaks down and BPA attaches to food and drink, so that it can enter the body [8]. BPA, by mimicking sex hormones, can disrupt the endocrine system, hormones and hormonal signaling pathways [9]. BPA even in small amounts may interfere with the ovulation, ovarian response, oocyte maturation and pregnancy [10]. In fact, lifestyle plays a key role in regulating ovarian response, oocyte maturation and genes expression. Gene's expression alteration in POR patients is a condition damaging oocyte developmental, resulting in reduced rates of fertilization, embryonic development, and implantation [11]. The role of gene's expression alteration in the pathophysiology and etiology of POR is uncertain, but studies support the hypothesis that nutrition and lifestyle play a crucial role in regulating the response of intrinsic ovarian resistance to gonadotropin stimulation, reduced ovarian reserve, oocyte maturation, implantation and gene expression [12].
ICAM-1, known as CD54, plays a pivotal role during oocyte maturation, and it is a protein encoded by the ICAM1 gene. The responsibility of ICAM-1 is encoding a cell surface glycoprotein usually expressed on endothelial cells and cells of the immune system. As mentioned, ICAM-1 plays a central role in the ovulation process [13]. Indeed, the main components of this ovulation process are the disruption of the extracellular matrix (ECM) at the follicular peak and the change in follicular vessels [14]. In these events, ICAM-1 participates by secreting proteases and active inhibitors. Indeed, biochemical markers of the oocyte maturation and ovulation are highly important. Upregulation of ICAM-1 in immature oocytes as well as downregulation of ICAM-1 in mature oocytes has been observed.
Human leukocyte antigen-G (HLA-G) is regarded to play a vital role in oocyte maturation and implantation of embryos. HLA-G can be effective in controlling trophoblast invasion and maintaining a local immunosuppressive state. The expression and distribution of HLA-G is on human spermatogenic cells, primary and secondary oocytes, and preimplantation embryos. Furthermore, the HLA-G production is related to the good quality of cumulus cells (CCs) in oocytes [15].
In the present study, we aimed to assess the genes expression, proteins level and methylation status of ICAM-1 and HLA-G in the cumulus cells of infertile POR patients based on their lifestyle following ovarian stimulation with a gonadotropin releasing hormone (GnRH) antagonist protocol.

Patient selection
In the present study, eighty women under the age of 35 and BMI 18-25 Kg /m2, who participated in an intracytoplasmic sperm injection (ICSI) program, were selected. The patients who were diagnosed by the Poseidon group1 subgroup 1b that included; [patients <35 years old with adequate ovarian reserve parameters (AFC>5; AMH>1.2 ng/ml) and with an unexpected poor or suboptimal ovarian response (subgroup 1b: 4-9 oocytes retrieved] (4). The patients were divided into two groups: without a healthy lifestyle (n=40, infertile women with poor response (POR) to ovarian stimulation history), and with a healthy lifestyle (n=40, infertile women with poor response (POR) to ovarian stimulation history). The healthy lifestyle group included women who did not use plastic containers for hot food, hot drink and foodstuffs store. However, the group without a healthy lifestyle included women who used excessive plastic containers to store hot food, hot drink and foodstuffs. Information on a healthy lifestyle was obtained using a validated food frequency questionnaire. The main exclusion criteria were women affected by endometriosis, and women with a history of uterus and ovaries operation. Furthermore, in this study, at the request of the patients, the treatment IVF/ICSIcycle was canceled, while they had a low number of follicles or lacked growth of follicles. The Ethics Committee at Royan Institute, Iran approved this prospective case-control study (No. IR.ACECR.ROYAN.REC.1394.150). All participants gave informed consent prior to inclusion in the study. We ensured the con dentiality of the patients' identities by data anonymization during analysis. This research did not affect the treatment of patients.

Antagonist stimulation protocol
In this study, Controlled of ovarian stimulation (COS) was controlled from the third day of the cycle. The patients received regular, daily subcutaneous (SC) injections of recombinant follicle stimulating hormone (rFSH, Gonal-F, Serono, Switzerland). The rst dose of rFSH for each patient was started according to sonographic monitoring, and AFC, estradiol (E2) level, and AMH for each patient were evaluated. In the stage of growing follicles >12 mm, the patients received SC injections of a GnRH antagonist, cetrorelix (Cetrotide®, Merck Serono, Germany). The protocol consisted of daily Cetrotide® SC injections until the criteria for human chorionic gonadotropin (hCG) administration were met. When more than 3 follicles reached diameters of at least 18 mm and E2 levels of 1000-4000 pg/mL When at least three follicles reached diameters of ≥18 mm as well as E2 levels of 1000-4000 pg/mL, each patient received an intramuscular (IM) injection of 10000 IU of hCG (Pregnyl®, Organon, Netherlands) or SC injection of 250 μgOvidrel (Merck Serono, Germany).

Isolation of cumulus cells
Following follicular puncture, the cumulus-oocyte complexes (COCs) were collected and washed at least 3-5 times in G-IVFTM medium (Vitrolife, Sweden) to remove blood and excess cells. Immediately after washing, the COCs were moved to a CO2 incubator at 37˚C for 2 hours in G-IVFTM medium. Cumulus cells were denuded from COCs by 80 IU of hyaluronidase, (Sigma, USA) [16]. After oocyte denudation, a pellet of cumulus cells was washed with phosphate-buffered saline (PBS), and RNA stabilizer reagent buffer was added for further analyses. Cumulus cells were immediately transferred toliquid nitrogenfor snap freeze, and then they were stored at -80˚C. The cumulus cells were denuded from metaphase II gametes (MII). In the IVF laboratory, MII gametes were fertilized by the ICSI process within 10 minutes after denudation, and they were incubated until the embryo transfer was processed. Fertilization was evaluated (16-20 hours after ICSI). According to our IVF laboratory standards process, embryos were graded by embryologist specialists at the pronuclear (16-20 hours) and cleavage stages (48-72 hours) [17]. Then, the embryologist selected 1 or 2 embryos for transfer. For successful embryo transfer, they considered the embryo grades, age of the patients, and previous ART cycles. The quality of the embryos at the cleavage stage was classi ed based on these criteria: [The excellent quality of embryo should be (≥4 cells or ≥8 cellsand <10 % fragmentation), the good quality of embryo should be (≥ 4 cells or ≥8 cells and 10-20% fragmentation), and the poor quality of embryo should be (<4 cells or <8 cells and >20 % fragmentation)] [18].
RNA extraction, cDNA synthesis and qPCR Total RNA was extracted by a Trizol (TRI; Sigma-Aldrich, US) and treated with RNase-free DNaseI according to the manufacturer's instructions. RNA concentration and purity were quanti ed using a Nanodrop2000 Spectrophotometer (Thermo, USA). Reverse transcription was performed at 25ºC for 10 min, 42ºC for 1h and then 70ºC for 10 min. For this reaction, cDNA was synthesized using the Revert AidTM H-Minus First Strand cDNA synthesis (Fermentas, German) by the random Hexa nucleotides primer (0.2µg/µl) in a solution (20µl) containing 4µl 5x reaction buffer, 1µl Ribonuclease inhibitor (20U/µl), 2µl dNTP mix (10mM) and RNase-DNase free water.
Real-Time PCR quanti cation ICAM-1 and HLA-G were chosen as target genes, and 18srRNAwas utilized normalizae each sample. Primers were designed by the Primer Express 3.0 software for the real-time PCR. Table 1 presents the primer sequences. The real-time PCR was performed in a Step OnePlus™ instrument (Applied Biosystems, USA). The reaction contained 2µl cDNA, 10µl of Power SYBR Green master mix (TAKARA, Japan) and 1µl (of 5oo nM primer) of forward and reverse primers. The reactions were performed in duplicate. The thermal cycling condition for the ampli cation was 95°C for 10 min, followed by 40 cycles of 95°C for 15s and 60° for 10 min. The Ct data were determined using default threshold settings. The 2 -ΔΔCT method was used to analyze the relative expression level of target genes (Livak&Schmittgen, 2001). Melt curve analysis was performed to determine the speci city of the real-time PCR assay. A t-test was used to investigate whether the differences between ICAM-1 and HLA-G genes expression were calculated by the 2 -ΔΔCT method.

Methylation-speci c PCR (MSP)
The DNA methylation status of ICAM-1 and HLA-G genes in COCs samples was evaluated by the methylation-speci c PCR (MSP). For MSP, we had to use speci c primer pairs for both methylated and unmethylated promoter sequences. The primers were designed using the Meth primer and GeneRunner software. Each MSP reaction was performed in a total volume of 25 μL. One microliter of sodium bisul te converted DNA was added into a 24 μL reaction mixture containing 0.5 U of hot start Gold Taq  Sodium bisul te treated DNA was ampli ed in two separate MSP reactions, one with a set of primers speci c for methylated, and the one for unmethylated ICAM-1 and HLA-G promoters sequences ( for 4 min. MSP products for methylated and unmethylated ICAM-1 and HLA-G promoters were run on 2% agarose gels containing 40 mMTris-acetate/1.0 mM EDTA (pH = 8) and were visualized by ethidium bromide staining (Fig2). Methylation genes pattern visualization was performed using the enhanced chemiluminescence detection system (Amersham). The intensity of methylation genes bands on the agarose gel was quanti ed using the ImageJ software version 1.50i (US National Institutes of Health, Bethesda).

Data analysis
Relative gene expressions were calculated by the 2 -ΔΔCt method. The normalized ΔCt value of each sample was calculated using reference genes. In this study, categorical variables were presented as number (%) and continuous variables as mean ± SD. The independent t-test was used to assess the mean differences in demographic and clinical characteristics between the groups. Chi-square analysis was used for qualitative data. Univariate and backward multiple linear regression, including all variables, were used to evaluate the association between ICAM-1 and HLA-G genes, proteins, methylation and some demographic and clinical variables. Statistical analyses were conducted using the IBM SPSS Statistics for Windows, Version 22.0 (IBM Crop., Armonk, NY, USA). All sta tistical tests were 2-tailed, and a P<0.05 was considered statistically signi cant. Table 2 presents the participants' demographic and clinical characteristics. The mean (SD) number of oocytes was statistically signi cantly higher in the healthy lifestyle group (Group1) than in the group without a healthy lifestyle (Group 2), (3.83 ± 0.99 vs. 3.27 ± 0.64, p = 0.004). The number of the MII retrieved oocyte was statistically signi cantly larger in Group 1 than in Group 2 (2.83 ± 0.99 vs. 2.35 ± 0.66, p = 0.013). The clinical pregnancy rate (fetal heart detection by ultrasound) was signi cantly different between the two groups. Indeed, the clinical pregnancy rate was higher in the healthy lifestyle group than in Group 2 (1.80 ± 0.41 vs. 1.70 ± 0.46, 0.040). There was a statistically signi cant difference in the clinical variables of total embryo number and oocyte quality between the groups. The number of total embryo and oocyte quality was statistically signi cantly higher in Group 1 than in Group 2 (oocyte quality: 3.51 ± 1.05 vs. 2.50 ± 0.93, p-0.000; total embryo: 1.93 ± 026 vs. 1.30 ± 0.56, p = 0.000). In addition, a marginally signi cant difference was observed in the AFC level between the two groups (p = 0.051). There was no statistically signi cant difference in age, marriage duration, infertility duration, and AMH level.

Results
As Fig [3, (a,b)] shows, the relative expression of ICAM1 and HLA-G genes and proteins was signi cantly increased in Group 1 (p = 0.000) than in Group 2.
Based on the univariate analysis (Table 3, 4), oocyte number (p = 0.03), and oocyte quality, total embryo, and type of group (p = 0.000) in the groups studied (women with and without a healthy lifestyle) were signi cantly associated with ICAM1 andHLA-G gene expression and DNA methylation. Variables of oocyte number, and oocyte quality, total embryo, MII number and type of group were signi cantly associated (Table2) with ICAM1 and HLA-G proteins expression in the groups studied. After adjusting all variables, based on the multiple linear regression model results, the AMH level had negatively signi cant association with the ICAM1 and HLA-G proteins expression (p = 0.018); for each unit increase in the AMH level, the expected oocyte quality was decreased by 0.14. The multiple linear regression model indicated that the number of MII was positively associated with the ICAM1 and HLA-G proteins expression (p = 0.021); for each unit increase in AMH, the expected ICAM1 and HLA-G proteins expression was increased by 0.04. This multivariate model revealed in groups 1 and 2 was negatively associated with ICAM1 and HLA-G genes. Although the ICAM1 and HLA-G protein expression in women without a healthy lifestyle was decreased by nearly 13 times than in women with a healthy lifestyle (p = 0.000), the expected ICAM1 and HLA-G protein expression was decreased by nearly 0.23 times in women without a healthy lifestyle than in women with a healthy lifestyle (p = 0.000) ( Table 4). The rest of variables included in the univariate model (Table 3) were not signi cantly associated with oocyte quality in multiple models.

Discussion
In the present study, our nding showed that ICAM-1 and HLA-G transcripts and proteins in POR with healthy lifestyle were upregulated compared to the POR without a healthy lifestyle. In addition, the ICAM-1 and HLA-G DNA methylation status in the POR with a healthy lifestyle was hypomethylated compared to the POR without a healthy lifestyle. In fact, lifestyle habit can affect the transcript and proteins pro le alterations in cumulus cells from POR patients. Furthermore, it may affect the methylation status in the cumulus cells of POR patients. Moreover, based on our data, the groups without a healthy lifestyle showed signi cantly lower numbers of good quality oocytes and clinical pregnancy rate compared to the group with a healthy lifestyle. As a result, lifestyle habit can affect the ovulation, oocyte maturation and clinical pregnancy rate in POR patients.
Oocyte quality is one of the major limiting factors for the success of the ART cycle in POR patients [19]. Oocyte maturation is achieved during folliculogenesis. Folliculogenesis is the maturation of the ovarian follicle. Folliculogenesis needs communication between the oocyte and surrounding somatic cells [20]. One of the kinds of somatic cells is cumulus cells.
Cumulus cells play a potential role in achieving oocyte development [21]. Based on a recent study, some genes are expressed in the cumulus cells that can be considered genetic markers for the association between oocyte and embryo quality. Probably, developmental signals from cumulus cells are transferred to the oocyte via gap junctions and pathways, which can affect the oocyte maturation [22]. The present study aimed to determine the novel biochemical marker to evaluate the oocyte quality and clinical pregnancy rate in the ART treatment of POR patients, and our data showed that lifestyle might be considered a biochemical marker for oocyte maturation increasing the clinical pregnancy rate. One of the consequent complications of POR is oocyte maturation disorder [23]. Nowadays, overuse of plastic objects to store hot food and hot drinks has caused numerous health problems in humans like reproduction disorders, including POR [24]. In fact, plastic objects made of polycarbonate and polycarbonates contain BPA [25]. According to the recent study, BPA may affect the infertility related gene expression that is closely associated with reproductive, but this association interaction mechanism is unclear. Owing to heat, acidic or alkaline substances, polycarbonate will break and BPA can attach to food and drink, and it can enter the body [26].
BPA can act like sex hormones, so that it can disrupt the endocrine system and hormonal signaling pathways. BPA in small amounts may interfere in the reproduction related gene expression, and it can interfere in the function of ovary, uterus and other reproductive organs [27]. Pednekar et al. [28] reported that infertile women showed signi cantly higher plasma concentrations of BPA. As a result, lifestyle habit plays a vital role in the regulating of ovarian response, oocyte maturation, reproductive process and genes expression. In this study, we evaluated the expression pro le for ICAM-1 and HLA-G genes in the cumulus cells of infertile women with poor response to ovarian stimulation (POR) based on their healthy lifestyle. The probable effect of plastic objects usage was observed on the genes, proteins, DNA methylation pattern, oocyte quality and clinical pregnancy rate in these women. According to other studies, altered gene expression of CCs may play a role in women with POR pathogenesis [29,30]. Therefore, assessment of genes expression is valuable to understand the etiology of infertile women with POR patients, since oocyte maturation dysfunction is one of the disorders in POR patients [31]. Recently, it has been shown that alternation of ICAM1 and HLA-G genes can affect the quality and quantity of oocytes [32]. Our ndings revealed that a major contributor to oocyte maturity disorder and infertility in the POR patients without a healthy lifestyle can be a reduction in ICAM1 and HLA-G transcript and proteins expression as well as hypermethylation of DNA in ICAM1 and HLA-G. According to recent studies, comparison of POR patients to healthy, normal ovulatory fertile women with a history of male infertility history has shown differential expression of ICAM-1 genes [33].
The ICAM-1 gene expression pattern indicated that it is important in oocyte maturity and grading. It appeared that dysregulation of ICAM-1 expression could be associated with defective oocyte maturation and grading [34].The studies demonstrated that ICAM-1 was a probable key effector of the oocyte maturation. Although the sICAM-1 release was very high in immature oocytes, it was decreased in mature oocytes. They regarded sICAM-1 as a biochemical marker for oocyte maturation and grading [35]. The ICAM-1 appears to play a critical role in oocyte development, while ICAM-1 levels play the role of a predictive marker in oocyte maturation and quality. Borgtti and Rizzo [36] proposed sICAM-1 as a biochemical marker for oocyte maturation and grading. Their ndings supported a signi cant correlation between ICAM-1 gene and protein expression and oocyte maturation. A recent study has shown that ICAM-1 is expressed on leukocytes, epithelial and endothelial cells [37]. Furthermore, HLA-G plays a vital role in the oocyte maturation and preimplantation of embryos. Jurisicova et al. detected HLA-G mRNA expression by preimplantation human embryos [38]. They demonstrated the presence of HLA-G mRNA and protein in the blastocysts tested [39]. Yao et al. [40] revealed the expression of the HLA-G protein by human preimplantation embryos with an increasing ratio of positive embryos with developmental stage. Kaneko [41] indicated that the low expression of HLA-G in cumulus cells was associated with lower oocyte quality, poor fertilization, and reduced embryo development [42]. Rooij van reported the low level of HLA-G in the cumulus cells of POR patients [43]. The gene expression alternation in the cumulus cells of women with POR after dehydroepiandrosterone (DHEA) supplementation can affect the oocyte maturation [44]. According to recent study, oocyte quality decreases with increasing age [45]. Our ndings indicated that age was directly associated with oocyte quality and aging. Furthermore, another study showed that younger POR patients' quality of oocyte and grading were better than older patients' [46]. Our study also highlighted the role of age as one of the components in oocyte maturation. The present study emphasized the association of lifestyle with the ICAM1 and HLA-G genes expression alteration and oocyte quality as an opinion for the deeper understanding of the etiology of infertile women with POR patients.

Conclusion
The current study was the rst single center lifestyle program of POR patients in Iran. We found that upregulation of ICAM1 and HLA-G in POR appeared to be related to nutrition and lifestyle. The nutrition and lifestyle may be effective on the methylation pattern, expression pro le and proteins level in cumulus cells from POR. Moreover, nutrition and lifestyle may act as a biochemical marker for ovulation and the oocyte maturation process.