RUNX2, GPX3 and PTX3 gene expression profiling in cumulus cells are reflective oocyte/embryo competence and potentially reliable predictors of embryo developmental competence in PCOS patients

This project was approved by the Institutional Ethical Review Board of Affiliated Hospital of Qingdao Medical University (Yuhuangding Hospital of Yantai). PCOS patients referred to our center for in vitro fertilization (IVF) were included in this study after written informed consent. All of the PCOS patients were characterized by the presence of two or more of the following features: chronic oligo-ovulation or anovulation, androgen excess and polycystic ovaries. We excluded patients with Cushing’s syndrome, congenital adrenal hyperplasia, and androgen-secreting tumors. The inclusion criteria of the recruited PCOS patients in this study were as follows: age ranging between 30 and 38 years, BMI ranging between 20 and 26 kg/m², number of obtained oocytes ranging between 13 and 19 per cycle, number of obtained blastocysts ranging between 4 and 9 per cycle, basal serum LH/FSH more than 2.0, serum androgen more than 0.5 ng/ml, and normal spermiogram of the partner according to the WHO criteria. Ovarian stimulation and oocyte retrieval protocols were performed as our previously described in all PCOS patients [25].

The method of retrieving cumulus cells and the process of oocyte insemination and culture were performed as described previously [25]. In brief, after COC retrieval, a proportion of the CCs surrounding a single oocyte were removed using a sharp needle, lysed in 80 ml of RLT buffer (RNeasy Mini Kit, Qiagen) supplemented with 1% (v/v) 2-β-mercaptoethanol (M-3148; Sigma, Lyon, France), and stored at −80°C (CCs from one oocyte per vial) until RNA extraction. Oocytes were further inseminated and embryos were cultured in sequential media of SAGE (CooperSurgical, Leisegang Medical, Berlin) individually in 20 ul droplets covered by mineral oil. Embryos were transferred or vitrified on Day 3 and the other embryos were cultured to blastula stage on Day 5–6.

The morphological characteristics of the oocytes and embryos were individually recorded. Oocytes were denudated to assess the maturation stage 3 h after insemination. The oocytes were first classified into two categories based on nuclear status: (i) Immature MI oocytes exhibiting no polar bodies (PB) or immature oocytes at the germinal vesicle stage (GV), and (ii) mature MII oocytes that extruded a clearly visible PB. Fertilization was observed at 16–18 h after insemination and zygotes were classified into two groups: (i) oocyte exhibiting normal fertilization (2 pronuclei, 2PN) and (ii) oocyte exhibiting abnormal fertilization (more than 2 pronuclei, MPN). Oocytes with no pronuclei (0PN) were discarded. On Day 2 (44–46 h post-IVF), individually cultured embryos were evaluated according to the number of blastomeres, the fragmentation rate and the presence of multinucleated blastomeres. Embryos with one or several multinucleated blastomeres and un-cleaved embryos were excluded. On Day 3, embryos containing at least 7 cells, <10% fragmentation and no multinucleated blastomeres were classified as ‘top quality embryo (TQE)’. Others were classified as ‘weak or low grade embryo (WLGE)’ [24]. Two or three embryos were transplanted into the uterus of patients on Day 3, whereas the others were frozen or cultured to the blastocyst stage. The blastocysts were scored on the expansion of the blastocoel cavity, the cohesiveness of the inner cell mass, and trophectodermal cells [26]. The best blastocyst quality on Day 5, i.e. blastocyst score 1 = BL 3–5, AA or AB (inner cell mass/ trophectoderm , ICM/TE) with ≤10% fragmentation. The good blastocyst quality on Day 5, blastocyst score 1 or 2 with score 2 = BL 1,2 or BL 3–5 with BA or BB (ICM/TE score) with ≤20% fragmentation.

According to different stages of oocytes or embryos, the corresponding cumulus cells were divided into different groups, viz.: CC_MI/GV, CC_MII, CC_2PN, CC_MPN, CC_B+, CC_B-. Each group had ≥3 replicates. Each subgroup, containing 9–12 cumulus cells, represented a biological replicate. The distribution of the collected COCs is depicted in Figure 1. In our experiment, a total of 212 COCs were retrieved from the 15 PCOS patients. Among these COCs, only 3 GV stage COCs were retrieved and were combined into the CC_MI/GV groups (n = 33). In each CC_MI/GV subgroup, there were ten CC_MI and one CC_GV. On Day 1 (16–18 hours after insemination), 130 CCs separated from oocytes with normal fertilization (2PN) and 36 CCs separated from mature oocytes with abnormal fertilization (MPN) were classed into a “CC_2PN” and “CC_MPN” group, respectively. On Day 3, 54 of 127 embryos that developed from oocytes with normal fertilization (2PN) were transplanted or frozen and their corresponding CCs were classified into 6 subgroups of CC_2PN while the other 73 embryos were cultured to the blastocyst stage. In each subgroup, there are 9 cumulus cells randomly from 3 or 4 PCOS patients. Cumulus cells from oocytes yielding blastulas (top or good blastocyst quality)after 5–6 days in culture were classified as the “CC_B+” group (n = 37), and cumulus cells from oocytes that arrested at the cleavage stage after Day 6 were divided into “CC_B-” groups (n = 36). The number of CCs and biological replicates in the different groups are shown in Table 1.

The cumulus cells of each subgroup were pooled together to extract total RNA and were analysed individually. Total RNA isolation was performed using the Qiagen RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. This RNA isolation kit significantly reduces contamination from both genomic DNA and proteins. The RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Kista, Sweden). Only samples with RNA integrity number (RIN) >6.0 in the cumulus cells subgroups were included. A good correlation between RIN values >5.5 and the outcome of a real-time PCR experiment has been shown and RIN values ≥ 5.5 are considered as high quality [27]. The first-strand complementary synthesis reaction was performed using the PrimeScript RT reagent Kit (Perfect Real time; TaKaRa Biotechnology, Dalian, China).

qRT-PCR was performed on 9 genes (RUNX2, PSAT1, ADAMTS9, CXCL1, CXCL2, CXCL3, ITGB5, GPX3 and PTX3). Primers for all the target genes were designed using the Universal Probe Library software (Roche Diagnostics, Roche Applied Science) and were chosen to be intron spanning. Amplification reactions were conducted using SYBR Premix Ex Taq (Perfect Real Time) (TaKaRa Biotechnology, Dalian, China) and an ABI PRISM 7300 system. The gene-specific qRT-PCR primers used are listed in Table 2. To normalize the expression level, the housekeeping gene GAPDH was used because its expression was stable in all CCs groups. The PCR thermal cycling conditions were 95°C for 10 min for polymerase activation and the initial denaturation step, followed by 40 cycles with denaturation at 95°C for 15 s, annealing at 60°C for 60 s and extension at 72°C for 30 s. A melting curve analysis was recorded at the end of the amplification to evaluate the absence of contaminants or primer dimers. Each set of qRT-PCR reactions was repeated three times, and the fold change in the expression of each gene was analysed using the DDCt method [28].

Student’s t-test of independent data was used for statistical analysis (SPSS Inc., Chicago, IL). The differences among groups were considered significant when the p-value was <0.05.

In the CC_MII (n = 173) compared to CC_MI/GV (n = 33) groups (Figure 2), mean transcript levels of three genes were significantly higher, with 2.18-, 3.37- and 2.26- fold increases observed for RUNX2 (2.14 ± 0.63 versus 0.98 ± 0.33), PSAT1 (4.38 ± 0.92 versus 1.3 ± 0.3), and ADAMTS9 (1.65 ± 0.3 versus 0.73 ± 0.23), respectively, whereas the expression levels of four genes were significantly decreased in the CC_MII samples, with 4.05-, 4.21-, 6- and 3.27- fold decreases observed for CXCL1 (0.19 ± 0.09 versus 0.77 ± 0.26), CXCL2 (0.19 ± 0.02 versus 0.8 ± 0.18), CXCL3 (0.16 ± 0.02 versus 0.96 ± 0.09) and ITGB5 (0.26 ± 0.03 versus 0.85 ± 0.13), respectively. The transcripts levels of the other two genes (GPX3 and PTX3) exhibited no significant differences between the CC_MII and CC_MI/GV groups.

To evaluate whether target genes were involved in the fertilization process, the transcript levels of the target genes were detected in CC_2PN (n = 130) and CC_MPN (n = 36) groups (Figure 3). The PTX3 expression level exhibits significant differences between CCs isolated from oocytes which formed two normal pronuclei (2PN) and that from oocytes which formed more than two pronuclei (MPN) after fertilization. The mean expression level of PTX3 was 2- fold lower in the CC_2PN group than in the CC_MPN group (0.5 ± 0.05 versus 1 ± 0.17). The expression levels of the other 8 target genes exhibited no significantly changes.

The transcripts levels of the target genes were then evaluated according to the outcome of the normal fertilized oocytes after 5–6 days of culture. Among the 9 target genes, two genes (RUNX2 and GPX3) exhibited differential expression between the CC_B+ (n = 37) and CC_B- (n = 36) groups (Figure 4), whereas the other 7 genes exhibited no significantly changes. The mean transcripts levels of RUNX2 was decreased by 2.14- fold in the CC_B+ group compared with the CC_B- group (0.44 ± 0.03 versus 0.94 ± 0.05), whereas the mean expression level of GPX3 was decreased by 3.18- fold in the CC_B+ group compared with the CC_B- group (0.33 ± 0.13 versus 1.05 ± 0.22).

Among the selected nine genes, the expression levels of six genes (PSAT1, ADAMTS9, CXCL1, CXCL2, CXCL3, ITGB5) exhibited significant differences in CCs from oocytes at different nuclear maturation stages. No change was observed between the CC-_2PN and CC-_MPN groups or between the CC-_B+ and CC-_B- groups. The results implied that these genes may play roles during the oocyte nuclear maturation progress. In our previous report [25], the genes were identified as being involved in different biological processes as follows: amino acid biosynthesis (PSAT1), development (ADAMTS9), the inflammatory response (CXCL1, CXCL2, CXCL3) and cell-matrix adhesion (ITGB5).

The relative mRNA abundance of PSAT1and ADAMTS9 in cumulus cells from oocytes at the MII stage was higher than that from oocytes at MI/GV stages. PSAT1, phosphoserine aminotransferase, is an enzyme implicated in serine biosynthesis and has been linked with cell proliferation in vitro [31]. PSAT1 plays an important role in the G2/M phase to modify the cell cycle and stimulate cell proliferation [32]. ADAMTS9, a gene involved in extracellular matrix binding, is widely expressed during mouse embryo development [33]. Furthermore, the extracellular matrix of CCs plays a major role in folliculogenesis and is crucial for ovulation, oviduct passage, and fertilization [34].

Comparing the CC-_MII group with the CC-_MI/GV group, the expression levels of four genes (CXCL1, CXCL2, CXCL3, ITGB5) were decreased. To our knowledge, these four genes, which are related with the inflammatory response (CXCL1, CXCL2, CXCL3) or cell–matrix adhesion (ITGB5), have been isolated from the CCs of PCOS patients for the first time by cDNA microarray in our previous research [25]. Though the role of chemokines during oocyte maturation is unclear, previous researches have demonstrated that the inflammatory response, which is induced by other chemokines (such as IL-8), increases the rates of meiotic arrest and the failure of germinal vesicle breakdown [35]. Indeed, chemokines have been implicated in a number of reproductive events, including ovulation, menstruation, embryo implantation, and parturition and disease conditions (i.e., endometriosis and cancer) [36, 37]. ITGB5 (integrin beta 5) has also been implicated in cell migration and angiogenesis during embryo implantation due to its apical expression on glandular and luminal epithelial cells [38]. Previous research has demonstrated that the expression of ITGB5, which regulated by chemokines (such as CX3CL1 and CCL14), could increase trophoblast adherence and migration at the maternal-fetal interface [39]. In the present research, we observed the same trend (down-regulation) in mRNA expression of four genes (CXCL1, CXCL2, CXCL3, ITGB5) during the oocyte maturation process. Although it remains to be clarified whether the expression of ITGB5 is regulated by chemokines (CXCL1, CXCL2, CXCL3), our findings suggest that the expression of these genes in cumulus cells is negatively related to oocyte maturation and may be involved in embryo implantation.

PTX3 is a downstream target gene of GDF9, which is a member of the transforming growth factor-β superfamily and plays an important role in promoting cumulus expansion [40]. Cumulus expansion is a critical process for normal oocyte development, ovulation, and fertilization [41, 42]. PTX3 co-localises with hyaluronic acid throughout the cumulus matrix from the periphery to the zona pellucida and interacts with tumour necrosis factor α-induced protein 6 (TNFAIP6) instead of binding directly to hyaluronan (HA) [43]. Inactivation of PTX3 by targeted mutagenesis has been reported to reduce the likelihood of fertilization, possibly through disruption of the structural integrity of the cumulus complex [44]. The relationship between PTX3 levels and oocyte competence is, however, controversial. Only Zhang and colleagues have reported that PTX3 mRNA levels exhibit significant differences between cumulus cells derived from unfertilized oocytes and from oocytes that developed to the eight-cell stage and led to the establishment of clinical pregnancies [18]. In previous studies, neither the PTX3 transcript level [17, 45] nor the PTX3 protein concentration in follicular fluid [46] exhibited significant association with oocyte quality. Our present results also show no significant difference between cumulus cells isolated from oocytes at different nuclear maturation stages (MII and MI stages) or cumulus cells derived from oocytes with different developmental competence (embryos that could develop to blastocyst stage or not). However, the PTX3 expression level exhibited significant differences between cumulus cells isolated from mature oocytes that formed two normal pronuclei or multi- pronuclei after fertilization (CC-_2PN and CC-_MPN groups). So, we speculated that PTX3 plays a key role in the fertilization process instead of oocyte maturation or embryo quality.

RUNX2 is a transcription factor that has been shown to play a crucial role in cell differentiation. The expression of RUNX2 was identified in rat ovary and was shown to be increased by the luteinizing hormone (LH) surge in preovulatory follicles and the luteal tissue [47]. Previous reports have confirmed the strong relationship between RUNX2 expression and ovulation, luteinization and steroidogenesis [48]. Consistent with Papamentzelopoulou’s findings [49], our data also proved that RUNX2 played an antagonistic role in regulating embryo development and could possibly be used as a genetic marker for projecting embryo quality. Recently, it was reported that the expression level of RUNX2 was lower in the central stroma of PCOS ovaries than in those of non-PCOS ovaries. The results may have implications for the PCOS-related defects in the inflammation-like ovulatory process [50]. In our research, it is interesting that the transcript level of RUNX2 to be higher in cumulus cells derived from oocytes at the MII stage than in those at the MI or GV stage of PCOS patients. Maybe the significant difference of RUNX2 transcript levels between CC_MII and CC_MI/GV was also related with the defects of inflammatory response in immature oocytes.

The expression level of GPX3 exhibited significant difference between the CC-_B+ and CC-_B- groups. Consistent with previous researches which proved that GPX3 was related with embryo development or pregnancy outcome [22, 51], we observed that the expression level of GPX3 was associated with embryo development and could be a projector of embryo quality in PCOS patients. GPX3 is regulated by hypoxia through HIF-1 binding sites which detected only in glutathione peroxidase [52]. Hypoxia can induce the formation of reactive oxygen species (ROS) which can cause lipid peroxidation, enzyme inactivation and cell damage, thereby, leading to apoptosis [53]. This phenomenon occurs both in cumulus cells and in the oocyte [54]. Moreover, the hypoxia conditions and the higher concentration of ROS in follicular fluid are negatively associated with embryonic development and pregnancy outcomes [55, 56]. Therefore, GPX3 may indicate that hypoxic conditions could act as a negative regulator of embryo development in PCOS patients.

The strengths of this study were the well-diagnosed PCOS women according to the Rotterdam criteria and the cumulus cells were classified strictly according to the different development stages of oocyte/embryo. A limitation of the study was that the cumulus cells in each subgroup which derived from 3–4 PCOS patients were pooled together to extract RNA due to the total RNA purified from a single cumulus is so limited. This may cause an imbalance of the samples and limits the endpoints that can be analyzed. But the imbalance of the samples could not be avoided absolutely, unless all the cumulus cells were derived from the same patient. Moreover, because PCOS is a common heterogeneous endocrinopathy in women of reproductive age, the molecular roles of target genes in the development of oocyte/embryo need be further verified in the large-scale PCOS patients.