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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.

Huang X, Hao C, Shen X, Zhang Y, Liu X - Reprod. Biol. Endocrinol. (2013)

Bottom Line: Two additional genes which had been found to be associated with oocyte maturation or embryo quality in non-PCOS patients (GPX3 and PTX3) were also studied.The PTX3 expression level was significantly lower in cumulus cells from oocytes with two normal pronuclei than that from oocytes that formed >2 pronuclei (MPN) after fertilization.GPX3 mRNA levels were decreased in cumulus cells isolated from oocytes that developed into blastocysts with high potential development competence.

View Article: PubMed Central - HTML - PubMed

Affiliation: Reproductive Medicine Centre, Affiliated Hospital of Qingdao Medical University, Yuhuangding Hospital of Yantai, 20 Yuhuangding Road East, Yantai, Shandong, China. cuifanghao@aliyun.com.

ABSTRACT

Background: Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder in women. The developmental competence of oocytes and embryos in PCOS patients is reduced to a certain extent (comparing to non-PCOS patients, the high quality embryo rate was decreased by 16% from the data of our centre) during the in vitro fertilization (IVF) process. Cross-talk between the oocyte and cumulus cells is critical for oocyte maturation and embryo competence. In this study, we have evaluated the transcription of specific genes in cumulus cells harvested from pre-ovulatory follicles of PCOS patients before IVF, according to individual oocyte nuclear maturity and developmental competence. Seven genes (RUNX2, PSAT1, ADAMTS9, CXCL1, CXCL2, CXCL3, and ITGB5) were targeted from our previous cDNA microarray data which isolated genes related to oocyte nuclear maturation in PCOS patients. Two additional genes which had been found to be associated with oocyte maturation or embryo quality in non-PCOS patients (GPX3 and PTX3) were also studied.

Methods: The mRNA expression levels of cumulus cells were detected by qRT- PCR.

Results: Consistent with our previous cDNA microarray data, with the exception of GPX3 and PTX3, the selected 7 genes were related to oocyte nuclear maturation in PCOS patients. Noticeably, the expression level of RUNX2 was lower in cumulus cells derived from oocytes that could develop into blastocysts than the level of expression from oocytes that could not. The PTX3 expression level was significantly lower in cumulus cells from oocytes with two normal pronuclei than that from oocytes that formed >2 pronuclei (MPN) after fertilization. GPX3 mRNA levels were decreased in cumulus cells isolated from oocytes that developed into blastocysts with high potential development competence.

Conclusions: Several cumulus cell genes were associated with oocyte maturation, fertilization and embryo quality in PCOS patients. RUNX2 and GPX3 are candidate genetic markers in the monitoring of embryo quality for PCOS patients, whereas PTX3 mainly played a role in fertilization process. Together with morphological evaluation, cumulus cells genes may serve as biomarkers of oocyte and embryo selection during the IVF process for PCOS patients and may advance our understanding of PCOS.

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Related in: MedlinePlus

Distribution tree of COCs involved in the study. A total of 212 COCs were retrieved from the 15 PCOS patients. Except for 6 COCs which are atretic or with fractured zones pellucidae were discarded, there have 3 GV stage COCs, 30 MI stage COCs, and 173 MII stage COCs. 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 “CC2PN” and “CCMPN” 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 six subgroups randomly. The other 73 embryos were cultured to the blastocyst stage. Cumulus cells from oocytes yielding blastulas after 5/6 days in vitro culture were classified as the “CCB+” group (n = 37), and cumulus cells from oocytes that stopped developing at the embryo stage after Day 6 were divided into “CCB-” groups (n = 36). COC, cumulus-oocyte-complex; CCGV, cumulus cells from GV oocyte; CCMI, cumulus cells from MI oocyte; CCMII, cumulus cells from MII oocyte; CC0PN, cumulus cells from oocyte which formed no pronuclei (0PN) after fertilization; CC2PN, cumulus cells from oocyte which formed two pronuclei (2PN) after fertilization; CCMPN, cumulus cells from oocyte which formed multi pronuclei (MPN) after fertilization; CCWLGE, cumulus cells from oocyte yielding a weak or low grade embryo at Day3; CCTQE, cumulus cells from oocyte yielding a top quality embryo at Day 3; CCB-, cumulus cells from oocyte which stopped development at the embryo stage at Day 5/6; CCB+, cumulus cells from oocyte yielding a blastocyst after 5/6 days of in vitro culture.
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Figure 1: Distribution tree of COCs involved in the study. A total of 212 COCs were retrieved from the 15 PCOS patients. Except for 6 COCs which are atretic or with fractured zones pellucidae were discarded, there have 3 GV stage COCs, 30 MI stage COCs, and 173 MII stage COCs. 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 “CC2PN” and “CCMPN” 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 six subgroups randomly. The other 73 embryos were cultured to the blastocyst stage. Cumulus cells from oocytes yielding blastulas after 5/6 days in vitro culture were classified as the “CCB+” group (n = 37), and cumulus cells from oocytes that stopped developing at the embryo stage after Day 6 were divided into “CCB-” groups (n = 36). COC, cumulus-oocyte-complex; CCGV, cumulus cells from GV oocyte; CCMI, cumulus cells from MI oocyte; CCMII, cumulus cells from MII oocyte; CC0PN, cumulus cells from oocyte which formed no pronuclei (0PN) after fertilization; CC2PN, cumulus cells from oocyte which formed two pronuclei (2PN) after fertilization; CCMPN, cumulus cells from oocyte which formed multi pronuclei (MPN) after fertilization; CCWLGE, cumulus cells from oocyte yielding a weak or low grade embryo at Day3; CCTQE, cumulus cells from oocyte yielding a top quality embryo at Day 3; CCB-, cumulus cells from oocyte which stopped development at the embryo stage at Day 5/6; CCB+, cumulus cells from oocyte yielding a blastocyst after 5/6 days of in vitro culture.

Mentions: According to different stages of oocytes or embryos, the corresponding cumulus cells were divided into different groups, viz.: CCMI/GV, CCMII, CC2PN, CCMPN, CCB+, CCB-. 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 CCMI/GV groups (n = 33). In each CCMI/GV subgroup, there were ten CCMI and one CCGV. 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 “CC2PN” and “CCMPN” 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 CC2PN 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 “CCB+” group (n = 37), and cumulus cells from oocytes that arrested at the cleavage stage after Day 6 were divided into “CCB-” groups (n = 36). The number of CCs and biological replicates in the different groups are shown in Table 1.


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.

Huang X, Hao C, Shen X, Zhang Y, Liu X - Reprod. Biol. Endocrinol. (2013)

Distribution tree of COCs involved in the study. A total of 212 COCs were retrieved from the 15 PCOS patients. Except for 6 COCs which are atretic or with fractured zones pellucidae were discarded, there have 3 GV stage COCs, 30 MI stage COCs, and 173 MII stage COCs. 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 “CC2PN” and “CCMPN” 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 six subgroups randomly. The other 73 embryos were cultured to the blastocyst stage. Cumulus cells from oocytes yielding blastulas after 5/6 days in vitro culture were classified as the “CCB+” group (n = 37), and cumulus cells from oocytes that stopped developing at the embryo stage after Day 6 were divided into “CCB-” groups (n = 36). COC, cumulus-oocyte-complex; CCGV, cumulus cells from GV oocyte; CCMI, cumulus cells from MI oocyte; CCMII, cumulus cells from MII oocyte; CC0PN, cumulus cells from oocyte which formed no pronuclei (0PN) after fertilization; CC2PN, cumulus cells from oocyte which formed two pronuclei (2PN) after fertilization; CCMPN, cumulus cells from oocyte which formed multi pronuclei (MPN) after fertilization; CCWLGE, cumulus cells from oocyte yielding a weak or low grade embryo at Day3; CCTQE, cumulus cells from oocyte yielding a top quality embryo at Day 3; CCB-, cumulus cells from oocyte which stopped development at the embryo stage at Day 5/6; CCB+, cumulus cells from oocyte yielding a blastocyst after 5/6 days of in vitro culture.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222840&req=5

Figure 1: Distribution tree of COCs involved in the study. A total of 212 COCs were retrieved from the 15 PCOS patients. Except for 6 COCs which are atretic or with fractured zones pellucidae were discarded, there have 3 GV stage COCs, 30 MI stage COCs, and 173 MII stage COCs. 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 “CC2PN” and “CCMPN” 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 six subgroups randomly. The other 73 embryos were cultured to the blastocyst stage. Cumulus cells from oocytes yielding blastulas after 5/6 days in vitro culture were classified as the “CCB+” group (n = 37), and cumulus cells from oocytes that stopped developing at the embryo stage after Day 6 were divided into “CCB-” groups (n = 36). COC, cumulus-oocyte-complex; CCGV, cumulus cells from GV oocyte; CCMI, cumulus cells from MI oocyte; CCMII, cumulus cells from MII oocyte; CC0PN, cumulus cells from oocyte which formed no pronuclei (0PN) after fertilization; CC2PN, cumulus cells from oocyte which formed two pronuclei (2PN) after fertilization; CCMPN, cumulus cells from oocyte which formed multi pronuclei (MPN) after fertilization; CCWLGE, cumulus cells from oocyte yielding a weak or low grade embryo at Day3; CCTQE, cumulus cells from oocyte yielding a top quality embryo at Day 3; CCB-, cumulus cells from oocyte which stopped development at the embryo stage at Day 5/6; CCB+, cumulus cells from oocyte yielding a blastocyst after 5/6 days of in vitro culture.
Mentions: According to different stages of oocytes or embryos, the corresponding cumulus cells were divided into different groups, viz.: CCMI/GV, CCMII, CC2PN, CCMPN, CCB+, CCB-. 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 CCMI/GV groups (n = 33). In each CCMI/GV subgroup, there were ten CCMI and one CCGV. 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 “CC2PN” and “CCMPN” 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 CC2PN 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 “CCB+” group (n = 37), and cumulus cells from oocytes that arrested at the cleavage stage after Day 6 were divided into “CCB-” groups (n = 36). The number of CCs and biological replicates in the different groups are shown in Table 1.

Bottom Line: Two additional genes which had been found to be associated with oocyte maturation or embryo quality in non-PCOS patients (GPX3 and PTX3) were also studied.The PTX3 expression level was significantly lower in cumulus cells from oocytes with two normal pronuclei than that from oocytes that formed >2 pronuclei (MPN) after fertilization.GPX3 mRNA levels were decreased in cumulus cells isolated from oocytes that developed into blastocysts with high potential development competence.

View Article: PubMed Central - HTML - PubMed

Affiliation: Reproductive Medicine Centre, Affiliated Hospital of Qingdao Medical University, Yuhuangding Hospital of Yantai, 20 Yuhuangding Road East, Yantai, Shandong, China. cuifanghao@aliyun.com.

ABSTRACT

Background: Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder in women. The developmental competence of oocytes and embryos in PCOS patients is reduced to a certain extent (comparing to non-PCOS patients, the high quality embryo rate was decreased by 16% from the data of our centre) during the in vitro fertilization (IVF) process. Cross-talk between the oocyte and cumulus cells is critical for oocyte maturation and embryo competence. In this study, we have evaluated the transcription of specific genes in cumulus cells harvested from pre-ovulatory follicles of PCOS patients before IVF, according to individual oocyte nuclear maturity and developmental competence. Seven genes (RUNX2, PSAT1, ADAMTS9, CXCL1, CXCL2, CXCL3, and ITGB5) were targeted from our previous cDNA microarray data which isolated genes related to oocyte nuclear maturation in PCOS patients. Two additional genes which had been found to be associated with oocyte maturation or embryo quality in non-PCOS patients (GPX3 and PTX3) were also studied.

Methods: The mRNA expression levels of cumulus cells were detected by qRT- PCR.

Results: Consistent with our previous cDNA microarray data, with the exception of GPX3 and PTX3, the selected 7 genes were related to oocyte nuclear maturation in PCOS patients. Noticeably, the expression level of RUNX2 was lower in cumulus cells derived from oocytes that could develop into blastocysts than the level of expression from oocytes that could not. The PTX3 expression level was significantly lower in cumulus cells from oocytes with two normal pronuclei than that from oocytes that formed >2 pronuclei (MPN) after fertilization. GPX3 mRNA levels were decreased in cumulus cells isolated from oocytes that developed into blastocysts with high potential development competence.

Conclusions: Several cumulus cell genes were associated with oocyte maturation, fertilization and embryo quality in PCOS patients. RUNX2 and GPX3 are candidate genetic markers in the monitoring of embryo quality for PCOS patients, whereas PTX3 mainly played a role in fertilization process. Together with morphological evaluation, cumulus cells genes may serve as biomarkers of oocyte and embryo selection during the IVF process for PCOS patients and may advance our understanding of PCOS.

Show MeSH
Related in: MedlinePlus