mRNA Expression of Aquaporins in Human Luteinized Granulosa Cells from Different Diameter Follicles / 中山大学学报(医学科学版)

ObjectiveTo investigate the mRNA expression levels of various aquaporins （AQPs） in luteinized granulosa cells from follicles of different diameters. MethodsFrom March 25， 2022 to September 23， 2022 in our reproductive medicine center， 48 women undergoing in-vitro fertilization （IVF） were enrolled and divided into the antagonist group and the agonist group according to the ovarian stimulation protocol. Follicular fluid samples were collected on the day of oocyte pick-up and granulosa cells were extracted from follicles of different diameters： small （<13 mm）， medium （13~18 mm） and large （≥18 mm）. After RNA quantification， 22 cases （66 samples） were included for analysis and mRNA expression levels of AQPs were compared among the three follicle groups. ResultsThe mRNA expression of aquaporin 2 （AQP2） in luteinized granulosa cells increased with the increase of follicle diameter （linear trend P = 0.004） and the difference was statistically significant between two groups of large and small follicles （P = 0.017）. Statistical difference was found in the antagonist group （P = 0.049 6）， but not in the agonist group （P = 0.108）. ConclusionThe mRNA level of AQP2 in luteinized granulosa cells increases with the increase of follicle diameter and its expression is related to the ovarian stimulation protocol， suggesting that AQP2 may play a role in follicle growth and follicular fluid formation， and its mRNA expression level may be regulated by follicle stimulating hormone （FSH） and luteinizing hormone （LH）.

Comparison of M-CSF and its receptor mRNA expression in luteinized granulosa cells after using recombinant human follicle stimulating hormone / 中国临床药理学与治疗学

AIM:To explore the effect of MCSF and its receptor on the response of ovarian stimulation by comparing the expression of macrophage colony stimulating factor (M-CSF) and its receptor mRNA in luteinized granulosa cells in patients after using combinant human follicle stimulating hormone.METHODS:Ninety-six patients with polycystic ovary syndrome (PCOS) and 157 patients with non-PCOS underwent in vitro fertilization were divided into four groups,i.e.the PCOS fast and slow reaction group,and the non PCOS fast and slow reaction group,according to their response to recombinant human follicle stimulating hormone (r-FSH).Luteinized granulosa cells were then collected after mature follicular puncture.SYBR Green quantitative RT-PCR method was used to detect the expression of M-CSF,M-CSFR and GAPDH in the mRNA gene of the granulosa cells samples.The relative quantity of these genes were determined by comparing the threshold value (CT value of the target gene subtract CT value of housekeeping gene).The difference of gene expression between two groups was compared by t test,and Spearman correlation analysis was used to describe the data relationships.RESULTS:No significant difference was observed in the use of r-FSH among the different groups (P ＞ 0.05).Neither was there any significant difference in mRNA quantity of M-CSF or M-CSFR between the entire PCOS and non PCOS patients (P ＞ 0.05).After grouping,no significant difference was observed between any two groups in the expression of M-CSF (P ＞ 0.05).The expression of M-CSFR in PCOS slow response group was significantly lower than that of PCOS fast response group (P =0.006).Meanwhile,the Spearman analysis showed that the correlation between the quantification of M-CSFR mRNA and the days of r-FSH in PCOS group was statistically significant (P =0.023);the correlation coefficient was 0.511.CONCLUSION:The slow response to ovarian stimulation in PCOS patients is possibly related to the reduction of granulocyte MCSFR expression.The M-CSF and its receptor may be involved in the ovarian stimulation response process.

Interrelationship of aging and mitochondrial DNA deletion in luteinized granulosa cells / 대한산부인과학회지

OBJECTIVE: Many clinical trials have proven the close relationship between the loss of human mitochondrial DNA and aging process. The purpose of this study was to evaluate the different types of mitochondrial DNA deletion and its frequency in luteinized granulosa cells in different aged groups of women undergoing in vitro fertilization (IVF). METHODS: The ovum pick up was done in 51 women who participated in Konyang University IVF program, and mitochondrial DNAs extracted from luteinized granulosa cells, were screened to search for different types of deletion and its frequency. The deleted mitochondrial DNA were analyzed by polymerase chain reaction method. DNA sequencing was performed to reveal exact deletion point. RESULTS: Three different types of deletions (4,977 bp, 7,150 bp, and 5,777 bp) were confirmed. To find the difference between the aged groups, we have divided women into groups younger than 32 years, between 32 to 37 years, and older than 37 years. The deletion of 4,977 bp was 60.9% (14/23) in younger than 32 years, 46.2% (6/13) in 32 to 37 years, 46.7% (7/15) in older than 37 years. There was no statistical significance between aged groups and the incidence of the deletion. The deletion of 7150 bp was 34.8% (8/23), in younger than 32 years, 30.8% (4/13) in 32 to 37 years, 40% (6/15) in older than 37 years. We investigated relationship between the frequency of deletion and the aging, but there was no statistical significance. In case of 5,777 bp, the deletion was 43.5% (10/23) in younger than 32 years, 30.8% (4/13) in 32 to 37 years, 53.3% (8/15) in older than 37 years. It showed no statistical significance as well as other types. CONCLUSION: In this study we have found three different types of deletion of mitochondrial DNA obtained from luteinized granulosa cells in women with infertility. There were no significant differnces of each type of deletion in 3 different aged groups of infantile women. The limitation of this study is that the analyze were done qualitatively. If we could provide the quantitative analyze it could be applied clinically.

Clinical Significance of Apoptotic Rates of Human Luteinized Granulosa Cells in In Vitro Fertilization and Embryo Transfer / 대한산부인과학회잡지

OBJECTIVE: To investigate the clinical significance of apoptotic rates of human luteinized granulosa cells in in vitro fertilization and embryo transfer (IVF-ET) program. MATERIALS AND METHODS: From 62 infertile patients undergoing controlled ovarian hyperstimulation (COH) for IVF-ET, luteinized granulosa cells were isolated during oocyte retrieval. The apoptotic rates of luteinized granulosa cells were measured by immunocytochemical staining with TUNEL method, and compared with the various clinical parameters. RESULTS: The apoptotic rates were not significantly different among the groups with the various etiologic factors of infertility. The presence of endometriosis, as well as the extent of endometriosis, did not show any differences in the apoptotic rates. The apoptotic rates were negatively correlated with the embryo quality and serum estradiol levels on hCG day. The apoptotic rates of 2.92% or below in the luteinized granulosa cells could predict a successful pregnancy with the sensitivity of 38.9% and the specificity of 93.2%. CONCLUSION: The apoptotic rates of human luteinized granulosa cells may be used to predict the outcome in IVF-ET program.

HCG attenuates ATP induced apoptosis in cultured human luteinized granulosa cells (hLGCs) / 대한산부인과학회잡지

OBJECTIVE: To determine the effects of hCG on extracellular ATP induced apoptosis in cultured human luteinized granulosa cells (hLGCs) METHODS: The addition of various concentrations of ATP (0, 0.1, 0.25, 0.5, 0.75 mM) and 5 IU hCG to luteinized granulosa cells obtained during in vitro fertilization ovum pickup procedures. After culture for 24 hours, purinoceptor activity and functional changes in mitochondria were measured by patch clamp, flow cytometry, and confocal microscopy. RESULTS: Calcium imaging with fura-2 revealed that ATP elevated [Ca2+]i by mobilizing intracellularly stored Ca2+. A patch clamp study showed that ATP exerted its effect by initially binding to the P2Y type purinoceptor, as evidenced by the ATP-evoked outward Ca2+-activated K+ current. Probing mitochondria with JC-1, a mitochondrial transmembrane potential-sensitive dye, revealed that ATP induced mitochondrial depolarization in a concentration-dependent manner. A quantitative flow cytometric analysis with Annexin V showed that apoptotic cells were increased in number in proportion to the concentration of ATP, having 18.57% of apoptotic cell populations in the presence of 0.75 mM ATP compared to 7.88% in the control. Moreover, treatments with human chorionic gonadotropin (hCG) at 5 IU reversed both the ATP-induced mitochondrial depolarization and apoptosis (18.57 vs 6.32%). CONCLUSION: Taken together, these results indicate, first, extracellular ATP recognized by the P2Y type purinoceptor on h-LGCs increases the intracellular Ca2+. Second, the increased intracellular Ca2+ triggers the apoptotic cascade by acting at least, in part, on mitochondria. Third, hCG reverses the ATP-induced apoptosis, raising a possible clinical implication of hCG in the treatment of degeneration of granulosa cells such as follicular atresia.

Effects of valproate on steroid production and correlative steroidogenesis enzyme mRNA in human luteinized granulose cells / 中国药理学通报

AIM Valproate (VPA) is widely used to treat epilepsy. Long-term treatment of women with VPA has been reported to be associated with a PCOS-like syndrome. The aim of the present study was to investigate the effects of clinically relevant concentrations of valproate on steroidogenesis and steroidogenesis acute regulatory protein (StAR),cholesterol side-chain cleavage enzyme (P450scc)and cytochrome P450 aromatase (P450arom) mRNA expression in human luteinized granulosa cells in vitro. METHODS Human luteinized granulosa cells were isolated from oocyte retrieval of in vitro fertilization procedure and were cultured with DMEM medium and treated with various concentrations of valproate (0, 100, 250 mg?L -1), the culture media was collected after 2 days for progesterone and estradiol measurements by standard radiommunoassay, StAR, P450scc and P450arom mRNA in granulose cells were detected by fluorescent real-time RT-PCR (TaqMan assay). RESULTS Valproate caused significant increase of progesterone (P