Assessing The Effectiveness of Growth Hormone Releasing Protein-6 in Improving Human Oocyte Maturation and Meiotic Progression in In Vitro Maturation Culture Media.

In vitro maturation (IVM) is a patient-friendly and cost effective technique in human assisted reproductive tech nology (ART) that involves the maturation of immature oocytes obtained from small antral follicles. In recent years, IVM has gained popularity in the field of reproductive medicine for its application to carefully selected patients, such as those at risk of ovarian hyper stimulation syndrome (OHSS), polycystic ovary syndrome (PCOS), or patients seeking fertility preservation before cancer treatments ( 1 ). Additionally, the clinical applications of IVM have expanded to include patients experiencing repeated failures in assisted reproduction due to resistant ovary syndrome and/or poor ovarian response ( 2 ) with only germinal vesicle (GV) oocytes in their stimulation cycles. However, the complex process of IVM and its gener ally lower success rates compared to conventional in vitro fertilization (IVF) remain significant challenges in reproductive science ( 3 - 5 ). Various factors influence IVM out comes, with one of the most crucial being the optimization of the IVM culture system ( 6 ). Popular media used for the IVM procedure include tissue culture medium 199 (TCM-199), human tubal fluid (HTF), Ham’s-F10 medium, glucose-free medium (P1), Chang’s medium, and blastocyst medium. Different constituents in the IVM medium can impact oocyte maturation and subsequent embryo development. Over the years of study, various supplements such as proteins, hormones, carbohydrates, and antioxidants have been added to the culture media to enhance the efficiency of IVM and approximate the ovarian niche ( 6 ). In this study, we examined the effects of Ghrelin hormone agonist at different doses in human IVM culture media to investigate oocyte maturation rates. Ghrelin is a 28-amino acid peptide known as a gastric hormone with multiple physiological functions, including the regulation of food intake, energy balance, and adiposity ( 7 - 9 ). The ghrelin gene encodes a 117-amino acid pre-pro-ghrelin peptide, which undergoes processing for mat uration and activation ( 9 , 10 ). Ghrelin has two systemic forms: acyl-ghrelin, the active form, and des-acyl-ghrelin ( 11 , 12 ). This acyl modification is catalyzed by ghrelin O-acyl-transferase (GOAT) ( 11 ) , which is an essential ghrelin-processing enzyme. Previous studies have indicated that ghrelin regulates various reproductive functions ( 12 ). Ghrelin binds to Ghrelin receptors GHSR-1a and GHSR-1b, with only GHSR-1a being functionally active ( 13 ). According to over a decade of studies, GHSR is distributed in the pituitary gland, hypothalamus, stomach, heart, small intestine, lungs, blood vessels, adipose tissue, immune system, multiple central nervous systems, reproductive cells/tissues and solid tissues such as human breast tumors ( 11 , 14 ). The binding of Ghrelin to GHSR in the brain activates multiple signaling pathways, including the Phospholipase C (PLC) signaling pathway, which leads to increased inositol triphosphate (IP3) levels and the activation of protein kinase C (PKC), resulting in the release of stored calcium in cells. These processes mediate the growth, development, and metabolism of the organism. The result of all of these processes is mediating the growth, development, and metabolism of the organism. Real-time polymerase chain reaction (PCR) results confirmed that the expression of Ghrelin mRNA varied depending on the developmental stage reproductive cells and embryo, with minimal expression in GV oocytes and the highest expression level in metaphase 2 oocytes (MII). The mRNA expression decreased at the cleavage stages and gradually increased further to the blastocyst stage. Studies have shown that the levels of GHSR-1a mRNA decrease from GV to MII, increase at the 2-cell stage, and then remain stable until the blastocyst stage, indicating high GHSR-1a mRNA expression at the GV stage ( 15 ). Various studies have explored the effect of Ghrelin on the maturation of germinal vesicles in the IVM system of different animal species. Ghrelin has been found to shorten the duration of IVM and hinder early embryonic development in sheep ( 16 ). In another study ghrelin exerts a specific and direct role on the oocyte, and accelerating its maturational process on Bovine ( 12 ). And also it had been reported in Ovine, ghrelin can promote oocytes maturation in vitro ( 17 ). However, some studies have reported inhibitory effects of Ghrelin on microtubule organization and no improvement in meiotic maturation in porcine models ( 18 ). Growth hormone releasing protein-6 (GHRP-6) is a synthetic hexa-peptide which specifically stimulates secretion of growth hormone (GH) by pituitary somatotrophs ( 19 ). It had been demonstrated, GHRP-6 as a synthetic activator of GHS-R1a can mimic the activity of full length Ghrelin in chicken in vitro ( 20 ). The knowledge of Ghrelin’s role in oocyte maturation and pre-implantation development is limited ( 12 , 21 - 23 ), and further investigation is needed due to inconsistent results and lack of information in human studies. In this study, the first examination of GHRP-6 in human oocyte IVM, immature oocytes derived from intra-cytoplasmic sperm injection (ICSI) cycles were used. These oocytes, which often discarded and considered useless, have the potential for maturation and early embryo development in vitro . They can achieve maturation and early embryo development in vitro ( 24 ) leading to possible pregnancy outcomes ( 25 ). If these oocytes could be rescued to maturation for clinical purposes, the patients may benefit from it. In this study, we utilized GV stage oocytes retrieved from ICSI cycles to determine the effects of Ghrelin hormone agonist (GHRP-6) ( 24 ) on human oocyte nucleic and cytoplasmic maturation and to identify the optimal concentration in vitro .

The total number of 108 couples participated in this sec tion of study. A number of 2.21 ± 1.06 ( 1 - 5 ) GV-stage oo cyte was donated from each patient ( Table 2 ). This num ber normalized by inclusion and exclusion criteria. Primer sequences used in real-time polymerase chain reaction ( 29 ) Baseline characteristics of the study population Data are presented as n (%) or mean ± SD (range). The t test was applied for statistical analysis. There was no statistically significant difference in any parameter between the GHRP-6 and control groups, and Sham group. GHRP-6; Growth hormone releasing protein-6, HTF; Human tubal fluid, GV; Germinal vesicle, MI; Metaphase I, MII; Metaphase II, and SD; Standard deviation. A total of 240 GV-stage oocytes were cultured in six different concentrations of GHRP-6, along with two control groups, for 48 hours. After 24 hours, 116 GV oocytes reached the MII stage (48.3%), while 51 (21.2%) arrested at the MI stage. Maturation of 73 oocytes (30.4%) was blocked at the GV stage ( Table 3 ). A significant difference was observed among the three types of oocytes between the 75 ng/ml, sham, and control groups compared to other groups (P=0.000). The number of MII oocytes after 24 hours was significantly higher at 75 ng/ml than in other experimental groups and the sham group (P<0.05). The rate of GV-arrested oocytes at 75 ng/ml was 3.3%, significantly lower than both the sham and control groups, which had rates of 30% (P=0.012). The rate of MI-stage arrested oocytes at 75 ng/ml was 26.7%, while the control and sham groups had rates of 3.3 and 20.0%, respective ly, which was not statistically significant (P=0.111 and P=0.750). Evaluation of 6 different dose of GHRP-6 and single step culture and HTF after 24 hours GV; Germinal vesicle, MI; Metaphase I, MII; Metaphase II, HSA; Human serum albumin, HTF; Human tubular fluid, and SD; Standard deviation. The total number of oocyte cultured in each group (T). The maturation rate was only statistically significant in 75 ng/ml after 24 hours (P<0.05). On the second day of culture, a significant difference was observed among the three types of oocytes between the 75 ng/ml GHRP-6 group and the other experimen tal groups (P=0.048). However, this difference was not significant when compared to the sham (P=0.172) and control groups (P=0.313). The maturation rate of oocytes treated with GHRP-6 reached 80%, while the control and sham groups had maturation rates of 66.7% ( Table 4 ). Degenerated oocytes were only observed in cultures treated with 200 ng/ml and 300 ng/ml of GHRP-6, but this was not statistically significant. Evaluation of 6 different dose of GHRP-6 and single step culture and HTF after 48 hours GHRP-6; Growth hormone releasing protein-6, GV; Germinal vesicle, MI; Metaphase I, MII; Metaphase II, HSA; Human serum albumin, HTF; Human tubular fluid, and SD; Standard deviation. Oocytes nuclear maturation rate resume in 48 hours in all groups, but this improvement was not statistically significant in any of them (P>0.05). 10% of oocytes degenerated in 200 and 300 ng/ml, but this rate was not statistically significant (P>0.05). These findings suggest that GHRP-6 treatment significantly promotes meiosis and the extraction of the first PB in a shorter timeframe. A total number of 162 GV-stage oocyte from 81 cycles, randomly distributed into 75 ng/ml GHRP-6, sham and control group for 24 hours (54 oocyte in each group for triplicate run). All women was under 35 [30.51 ± 3.12 ( 22 , 35 )] ( Table 5 ). In present study, to assess the effects of GHRP-6 on cytoplasmic maturation and two related gene mRNA expres sion quality, RT-PCR was performed. The results showed that, CENP-E and LINGO2 expression level compared with housekeeping gene ( GAPDH ) were down-regulated insignificantly in 75 ng/ml GHRP-6-treated oocytes. CENP-E is related to meiosis, while LINGO2 prepares the oocyte for future embryonic development. Baseline characteristics of the study population Data are presented as n (%) or mean ± SD (range). The t test was applied for statistical analysis. There was no statistically significant difference in any parameter between the 75 ng/ml of GHRP-6, sham and control group. GHRP-6; Growth hormone releasing protein-6, GV; Germinal vesicle, MI; Metaphase I, MII; Metaphase II, HSA; Human serum albumin, HTF; Human tubular fluid, and SD; Standard deviation. According to this results, mRNA expression levels of CENP-E (P=0.706) and LINGO2 (P=0.663) in oocytes treated with GHRP-6 were not significantly down-regulated compared with control group, and sham group (LINGO2: P=0.585; CENP-E: P=0.949) and both of them have approximately the same effects on cytoplas mic maturation of oocytes ( Fig .1 ). Also, this data led to the hypothesis that exposure of GHRP-6 to the human GVs resulted in a low expression level of CENP-E and LINGO2. Comperison between mRNA expression of LINGO2 and CENP-E genes in 75 ng/ml GHRP-6 (test group) ,single-step culture medium (con trol) and, human tubular fluid (HTF+10% human serum albumin) media culture as sham group by using 2–ΔΔCT method. Despit inequality among experimental, control and sham group, there was no significant difference statistically. Electrophoresis gel analysis of conventional PCR prod ucts for CENP-E and LINGO2 showed that LINGO2 am plification was present in all three groups, being most significant in the control group and mildly significant in the sham and 75 ng/ml GHRP-6 groups. CENP-E amplification was only sufficient for visualization in the control group ( Fig .S1 , See Supplementary Online Information at www.ijfs.ir ). The sequences obtained from Finch TV soft ware were aligned with UCSC reference genes for primer verification.

The absence of ovarian niches results in suboptimal human oocyte maturation in vitro ( 6 ). Several studies have reported morphological and structural differences in IVM oocytes ( 30 ). Developing an IVM culture system with a higher success rate by incorporating effective ingredi ents is of clinical significance. Despite numerous animal studies on the effects of Ghrelin hormone on follicle cell growth and maturation ( 12 , 16 - 18 , 20 , 21 ) its role in hu man IVM remains unexplored. In this study, we demonstrated the influence of the Ghrelin agonist GHRP-6 on the growth and maturation of immature human oocytes using six different doses in IVM culture media. We identified the most effective dose for oocyte maturation and compared GHRP-6 treatment with two conventional media: blastocyst media and HTF10% as control and sham groups, respectively. According to Du et al. report ( 15 ), GV-stage oocytes exhibit the highest expression of the ghrelin receptor GHSR-1a among all meiotic stages, suggesting strong re sponsiveness to ghrelin at this stage. Additionally, GHSR 1a mRNA levels decrease from the GV-stage oocyte to the metaphase MII-stage oocyte, increase at the two-cell stage, and remain stable until the blastocyst stage in sheep. We demonstrated that GHRP-6 promotes nuclear matura tion in GV-stage oocytes, as evidenced by the appearance of the first PB at lower doses during the first day of culture. This finding underscores the presence of GHSR-1a in GV-stage oocytes and suggests that low concentrations of ghrelin positively influence naked immature oocytes, while higher concentrations exert detrimental effects. After 48 hours of culture, lower doses of GHRP-6 effec tively prevented meiotic arrest in GV-stage oocytes compared to HTF 10% and blastocyst media. These findings align with the Du et al. ( 15 ) observations, which reported high responsiveness of GV-stage oocytes to GHRP-6 and its ability to promote meiotic progression. However, they contrast with Chouzouris et al. ( 31 ) results, where 800 pg/ mL of ghrelin inhibited maturation of bovine GVs. According to the aforementioned study by Wang et al. ( 16 ), in sheep COCs, 200 ng/mL Ghrelin having signifi cant effect on IVM a day after culture. In present study, 200 ng/ml of GHRP-6, not only had negative effect on meiosis resumption, (by 23% maturation rate in day one), but also oocyte vitality. By decreasing the concentration of GHRP-6 in the culture media, we found that, GHRP-6 is more effective in 75 ng/ml, which exhibit the maximum nuclear maturation after 24 hours. These contrasts between results suggest that, the effectiveness of Ghrelin on IVM is higher with denuded GVs or it could be different among species. In both Wang et al. ( 16 ) and Chouzouris et al. ( 31 ) IVM study and also Sirini et al. ( 32 ) , tissue culture media sup plemented with various concentrations of Ghrelin hormone were examined on COCs maturation. Tissue cul ture media is a common media for animal IVM ( 3 ). In this study, we supplemented a basal culture medium for human IVF/IVM (HTF 10%) with GHRP-6 at different concentrations and compared the results with the blastocyst medium, which is another recommended medium for IVM/IVF ( 3 ). The maturation rate after 24 hours for the control and sham groups was 66.7 and 50%, respectively. These percentages, compared with 25, 50, and 75 ng/mL GHRP-6 (60, 56, and 70%, respectively), indicate that GHRP-6 enhances the performance of HTF 10% media to match the level of blastocyst medium. Further, the lower percentage of immature oocytes in the treatment group on day one (3.3%) suggests that ghrelin agonist likely work by accelerating meiotic progression. Bai et al. ( 17 ) pointed at positive effect of 500 ng/ml of Ghrelin on expression of Bcl2 and Bax and promotion of ovine COCs IVM after 24 hours. In present study, we observed same high viability rate and meiosis resumption after 48 hours in human naked oocyte IVM in 75 ng/ml, the differences in effective concentrations could be attributed to cumulus cell responses to ghrelin, which protect oocytes from the negative effects of high ghrelin concentrations, or to interspecies differences. However, when Sirini et al. ( 32 ), significantly lowered ghrelin concentrations in their investigations (20, 40, and 60 pg/mL), no differences in COCs viability were reported after 24 hours which has contrast with present study. These findings suggest that ghrelin is more effective at low concentrations in denuded GV-stage oocytes. After 48 hours, degeneration was observed in oocytes cultured with 200 and 300 ng/mL of GHRP-6, highlighting its detrimental effects at higher concentrations. Although these data were not statistically significant, they indicate that low concentrations of GHRP-6 can promote human oocyte meiosis and viability. CENP-E is an essential meiotic kinetochore motor, and is required for meiotic progression. It re-localized to the mid-body during telophase ( 33 ). During metaphase-to anaphase transition, CENP-E plays a key role in cell cycle regulation and the spindle assembly checkpoint in mam malian cells ( 34 ). Human CENP-E has recently identified to be linked with the microcephaly primordial dwarfism syndromes associated with a smaller head, brain malfor mations and a prominent nose ( 35 ) inhibition of CENP-E could have defects on early zygote cleavage, including asymmetric cell division, cell cycle arrest and the developmental abnormalities in zebra fish, also it could pro mote developmental arrest, and the abnormal embryo during zebra fish embryogenesis ( 36 ). CENP-E function is essential for meiosis, because more than 95% of mouse GV-stage oocyte injected with anti-CENP-E antibody were arrested after 24 hours in MI-stage and not undergo any progress and showing PB1 ( 37 ). There has been no investigation about effect of Ghrelin or its agonists on expression level of CENP-E in animal or human studies ( in vitro or in vivo ), but the significant low rate of arrested oocytes in GV-stage and MI-stage in 25, 50 and 75 ng/ ml after 48 hours, shows meiosis resumption is promoted and not interfered in compare with higher doses. Also, real time PCR results showed no significance difference between expression level of CENP-E in sham, control and 75 ng/ml. These findings could support the report of Duesbery et al. ( 33 ) who state the inhibition of CENP-E in muse system could block meiosis resumption. There is just one study which addresses the expression level of CENP-E in IVM ( 29 ). According to Li et al. ( 29 ) expression level of CENP-E were significantly high inmatured oocyte in IVM system treated with 200 ng/ml of GH after 24 hours. Subsequently they reported higher quality of fertilization, cleavage and blastocyst rate from IVM matured oocyte treated by GH, these data showed, by making IVM media rich by needed component we could target genes which responsible for meiosis. In present study, which Ghrelin hormone agonist examined for the first time in human IVM, we illustrate that, despite higher apparition of PB1 in a shorter time, there is lower expression level of CENP-E in treatment group in compere with sham and control group. But as these finding as not statistically significant, further investigation needs to address the exact expression quality of CENP-E in human IVM. LINGO2 encodes a type 1 transmembrane protein ex pressed exclusively in neuronal tissues in mice ( 37 ), and It has been reported that the expression level of LINGO2 gene increased gradually as the mouse embryo devel oped ( 38 ). There is no report of LINGO2 function on oocyte. Only Li et al. ( 29 ) evaluated LINGO2 in human MII oocyte cultured with GH in IVM system and identifying it as a marker of cytoplasmic maturation. The reports points to high expression level of LINGO2 gene treated by 200 ng/ ml of GH. In present study, effect of GHRP-6 examined for the first time on LINGO2 . We observed an insignificant low expression in LINGO2 in oocytes treated with 75 ng/mL of GHRP-6 compared to two regular culture media. Since Li et al. ( 29 ) reports high developmental quality after ICSI procedure in oocytes treated by GH, it could be said cytoplasmic maturation is much more complete in treatment group. With low expression level of LINGO2 in present study compere with sham and control group, it could be said cytoplasmic maturation in oocyte treated by 75 ng/ml of GHRP-6 is impair. But as these data was not statistically significant and as we didn’t perform ICSI on in vitro matured MII oocyte treated by each group, we suggest more deliberation on influence of GHLR-6 of full length of Ghrelin hormone on expression level of LINGO2 in IVM and embryo development. This study had several limitations: i. The sample size of GV oocytes, though statistically adequate, but may not capture all biological variability, ii. The 24-hour obser vation period might be insufficient to assess cytoplasmic maturation effects, and iii. The use of an agonist (GHRP 6) might be not rule as full Ghrelin hormone. However, these limitations dose not invalid our main findings, future studies with larger sample group with full Ghrelin hormone protein may warranted these results.

Our observations indicate that the GHRP-6 at an optimal concentration can induce faster maturation in human IVM in HTF 10% media, but it does not enhance the expression levels of CENP-E and LINGO2 compared to blastocyst and sham culture media. Although statistical analysis showed no significant differences between the treatment group and the two popular culture media for human IVM, it suggests that using Ghrelin hormone at lower doses in culture media could expedite maturation in IVM. Further investigations are necessary to elucidate how Ghrelin affects genes related to meiosis in humans and the future development of embryos.

In this experimental study, the GV-stage oocytes were donated by patients who underwent ICSI treatment due to male factors, uterine or tubal factors and unexplained fertilities at the Mehr fertility center Rasht/Iran from May 2023 to January 2024. A total number of 240 oocyte, from 107 ICSI procedures, were included in evaluation the most effective concentration of GHRP-6 in IVM. These oocytes were not suitable for the ICSI procedure. All women participant was under 35 years of age [30.14 ± 3.51 ( 21 - 35 )]. There was no statistically difference in age among candidates (P=0.920). Cycle diagnosed as male factor infertility (n=81), tubal factor infertility (n=11), uterine factor infertility (n=10) and unexplained infertility (n=9) and those with an adequate number of MII oocytes after oocyte retrieval (>80%) were included in this study. Women with a history of chemotherapy, endometriosis, and PCOS were excluded. Ethics approval for the current study was given by the Ethics Committee at Islamic Azad University Science and Research Branch (IR.IAU.SRB.REC.1402.155). All procedures in this research were in accordance with the ethical guidelines of responsible institutional and national committees that involve human experimentation. Participants gave verbal and written consent for study participation. Follicles were punctured under ultrasound guidance 38-40 hours after the administration of 10,000 IU of human chorionic gonadotropin (hCG). Following oocyte retrieval, cumulus-oocyte complexes (COCs) were denuded through brief exposure to 10% hyaluronidase (Ravan Saze, Iran) and frequent pipetting to remove the corona cumulus cells for approximately 30 seconds. The meiotic status of the oocytes was then assessed under an inverted microscope. The status of the oocytes was categorized as follows: i. GV-stage: Presence of a GV in the cytoplasm. ii. MI-stage (Meiosis I): Absence of a GV in the ooplasm and presence of the first polar body (PB) in the perivitelline space. iii. MII-stage (Meiosis II): Presence of the first PB in the perivitelline space ( 26 ). Only MII-stage oocytes were fertilized using the ICSI technique for the patients. GV-stage oocytes with a discernible GV were donated and collected for this study, and each GV oocyte was used fresh. The GV-stage oocytes obtained each experimental day were randomly distributed among six different concentrations of the GHRP-6 (UK Peptide, England). A total of 240 GV-stage oocytes were collected. We used HTF+10% human serum albumin (HSA) culture media, which syn thesis media along with 10% HSA (Ravan Saze, Iran), as the sham group, and blastocyst media (single-step culture, Ravan Saze, Iran) as the control group. HTF culture media contain: sodium, potassium and calcium chloride, potas sium phosphate, magnesium sulfate, sodium bicarbonate, glucose, sodium lactate, sodium pyruvate and phenol red. Blastocyst media has these components in addition of 20 amino acid which needed for embryo development. The concentration gradients were set as 0 (sham), 25, 50, 75, 100, 200, 300 ng/ml of GHRP-6 ( 16 , 27 ) . All droplets were prepared and kept under light mineral oil (Ravan Saze, Iran) in a 37°C, 5% CO 2 incubator overnight before oocyte retrieval. We examined 30 oocytes in each group, with each oocyte cultured in a separate droplet. GV-stage oocytes were cultured for 24-48 hours. The appearance of the first PB was used as a marker for matured oocytes. In this section of study, nine pairs of GV-stage oocytes from nine different patients, with previous criteria consideration, were cultured for 24 hours in 75 ng/ml GHRP-6, sham group and control group. Each groups reserved 18 oocyte (54 oocyte in each run, and 164 oocyte in overall for triplicate run). Total RNA was extracted using the RNX TM –Plus reagent (Sinaclon, Iran) and quantitatively measured us ing Nanodrop® (ND‐2000) spectrophotometer. Then, the cDNA synthesis was carried out using cDNA synthesis kit (RT5201; Sinnagen, Iran) and Random Hexamer primers according to the manufacturer’s recommendations. cDNA from these 18 oocytes was obtained through Sina colon kit (Iran). Oocytes were washed in phosphate-buffered saline (PBS) then transferred into a labeled nuclease-free tube. Preparing briefly, RNA-primer mixture involved 1 μL random hexamer and 1 μL dNTP mix with addition of DEPC treated water up to 10 μL to the template tube on ice. The mixture incubated at 70˚C for 5 minutes and chilled on ice for 2 minutes and then briefly spine down. cDNA synthesis was performed ( 28 ) with 4 μL of 5× buffer M MulV (DTT) , 1 μL M-MulV reverse transcriptase , 1 μL RNase and topped up to 10 μL of DEPC- treated water. Ten μL of the cDNA synthesis mix were added to each RNA primer mixture tube, and the total reaction volume reached 21 μL. After a brief centrifuge, and incubation for 10 minutes in 25˚C, the mixture incubated for 50 minutes at 40˚C. The reaction terminated by tube incubation at 80˚C for 5 minutes and then tubes chilled on ice. GV stage oocytes were always collected fresh for culturing in experimental and control groups. cDNA synthesis performed after 24 hours of culture, and then tubes stored and kept at -4˚C. Real-time polymerase chain reaction (PCR) was executed as follows: 12.5 μL of SinaSYBR Blue HS-qPCR Mix was added to 1 μL of each reverse and forward primer ( Table 1 ), 1.5 μL of cDNA, and sterile water to a total vol ume of 25 μL. The gene amplification program included 5 minutes at 95°C, followed by 15 seconds at 95°C, 30 seconds at 60°C, and 10 seconds at 72°C, for a total of 40 extension cycles. All assays were performed in triplicate for both technical and biological replicates and normal ized to GAPDH as the reference gene. The Relative Expression Software Tool (REST, version 2009, Technical University Munich) was used to calculate the expression of each target gene ( LINGO2, CENP-E ). Conventional PCR was performed as follows: Three pairs of GV-stage oocytes from three different patients, with previous criteria consideration, were collected and cultured for 24 hours in 75 ng/ml, sham, and control groups (one pair of oocytes for each group). GV-stage oocytes were always collected fresh, and the synthesized cDNA was stored at -4°C. We added 1 μL of cDNA, 1 μL of each reverse and forward primer, 51 μL of nuclease-free water, and 32 μL of Sina Colon ready-to-use master mix (Iran) to a PCR tube. The PCR was processed for 5 minutes at 90°C, followed by 30 seconds at 94°C, 30 seconds at 56°C, and 45 seconds at 72°C, with a total of 40 extension cycles. The amplification products were visualized on agarose gel electrophoresis under short UV light. PCR products were sent to the Genomin Center (Tehran, Iran) for paired-end read sequencing analysis. Finch TV software was used for quality control analysis of the reads. The sequences were blasted in the NCBI browser for accuracy verification. The reference genome, downloaded from the Ensembl database (R and F), was replaced in the UCSC browser to check the accuracy of the blasted sequences in NCBI. Categorical data were displayed as counts and percent ages and analyzed using the chi-square test. Real-time PCR data were counted and analyzed using the method of 2-ΔΔCT ( 28 ), expressed as the mean ± SD. Statistical analyses were performed with SPSS software (IBM Corp, Armonk, NY, USA), and statistical significance was considered at P<0.05.