Reduced PATL2 Impairs the Proliferation of Ovarian Granulosa Cells by Decreasing ADM2 Expression in Patients with PCOS.

It is recognized that PCOS patients are often accompanied with aberrant follicular development, which is an important factor leading to infertility in patients. However, the relevant regulatory mechanisms of abnormal follicular development are not well understood. In the present study, by collecting human ovarian granulosa cells (GCs) from PCOS patients who underwent in vitro fertilization (IVF), we found that the proliferation ability of GCs in PCOS patients was significantly reduced. Surprisingly, PATL2 and adrenomedullin 2 (ADM2) were obviously decreased in the GCs of PCOS patients. To further explore the potential roles of PATL2 and ADM2 on GC, we transfected PATL2 siRNA into KGN cells to knock down the expression of PATL2. The results showed that the growth of GCs remarkably repressed after knocking down the PATL2, and ADM2 expression was also weakened. Subsequently, to study the relationship between PATL2 and ADM2, we constructed PATL2 mutant plasmid lacking the PAT construct and transfected it into KGN cells. The cells showed the normal PATL2 expression, but attenuated ADM2 expression and impaired proliferative ability of GCs. Finally, the rat PCOS model experiments further confirmed our findings in KGN cells. In conclusion, our study suggests that PATL2 promoted the proliferation of ovarian GCs by stabilizing the expression of ADM2 through "PAT" structure, which is beneficial to follicular development, whereas, in the ovary with polycystic lesions, reduction of PATL2 could result in the decreased expression of ADM2, subsequently weakened the proliferation ability of GCs and finally led to the occurrence of aberrant follicles.

Aberrant BMP15/HIF-1α/SCF signaling pathway in human granulosa cells is involved in the PCOS related abnormal follicular development.

AIMS: To investigate the regulatory mechanism of SCF expression in human GCs of PCOS related follicles. MATERIALS AND METHODS: SCF, BMP15 and HIF-1α were evaluated in human serums, follicular fluids (FFs) and GCs, which were collected from 69 PCOS patients and 74 normal ovulatory patients. KGN cell line was used in this study. RESULTS: Our results showed that the rate of MII oocyte and 2PN fertilization was lower in PCOS group, though PCOS patients retrieved much more oocytes. The level of BMP15 in FF and the level of SCF in serum and FF were also lower in PCOS patients. We found a weakened expression of HIF-1α and SCF in GCs from PCOS patients when compared with the non-PCOS patients. The expression of HIF-1α and SCF was significantly increased in KGN cells after treating cells with rhBMP15, however, this promotion effects of BMP15 on HIF-1α and SCF expression were obviously abolished by co-treatment with BMP-I receptor inhibitor (DM). Moreover, knock down of HIF-1α expression in KGN cells significantly reduced the expression of SCF in human GCs, in spite of activating BMP15 signaling pathway. CONCLUSIONS: The present study suggest that BMP15 could induce SCF expression by up-regulating HIF-1α expression in human GCs, the aberrance of this signaling pathway might be involved in the PCOS related abnormal follicular development.

A novel heterozygous variant in PANX1 is associated with oocyte death and female infertility.

PURPOSE: Oocyte death is a severe clinical phenotype that causes female infertility and recurrent in vitro fertilization and intracytoplasmic sperm injection failure. We aimed to identify pathogenic variants in a female infertility patient with oocyte death phenotype. METHODS: Sanger sequencing was performed to screen PANX1 variants in the affected patient. Western blot analysis was used to check the effect of the variant on PANX1 glycosylation pattern in vitro. RESULTS: We identified a novel PANX1 variant (NM_015368.4 c.86G > A, (p. Arg29Gln)) associated with the phenotype of oocyte death in a non-consanguineous family. This variant displayed an autosomal dominant inheritance pattern with reduced penetrance. Western blot analysis confirmed that the missense mutation of PANX1 (c.86G > A) altered the glycosylation pattern in HeLa cells. Moreover, the mutation effects on the function of PANX1 were weaker than recently reported variants. CONCLUSION: Our findings expand the inheritance pattern of PANX1 variants to an autosomal dominant mode with reduced penetrance and enrich the variational spectrum of PANX1. These results help us to better understand the genetic basis of female infertility with oocyte death.

GnRH antagonist weakens endometrial stromal cells growth ability by decreasing c-kit receptor expression.

BACKGROUND: Several surveys have reported that patients treated with gonadotropin-releasing hormone antagonist (GnRH-ant) protocol showed a significantly lower rate of implantation and clinical pregnancy compared to GnRH agonist (GnRH-a) protocol during in vitro fertilization-fresh embryo transfer. Subsequent studies imputed this poor outcome to the negative effects of GnRH-ant on endometrial receptive. However, the mechanisms were not fully understood. METHODS: The clinical data of 2815 patients undergoing fresh embryo transfer in our center were analyzed. Human endometrial stromal cells (ESCs) from healthy women undergoing elective pregnancy termination of a normal pregnancy at 8-10 weeks gestation were treated with GnRH-analogs or imatinib (c-kit receptor inhibitor). CCK8 and Flow cytometry were used to investigated the growth ability of ESCs. Immunofluorescence staining and western blot was used to detected the target proteins. RESULTS: The clinical data showed that the endometrial thickness on HCG Day were significantly lower in GnRH-ant group. Although no difference of embryo quality in these two groups, GnRH-ant group showed remarkably decreased rate of HCG positive, embryo implantation and pregnancy. Moreover, GnRH-ant significantly reduced the proliferation and induced the apoptosis of ESCs. Furthermore, the expression and activation of c-kit receptor, which played pivotal roles during embryo implantation, were observably decreased by GnRH-ant. Inhibiting the activation of c-kit by imatinib remarkably suppressed the proliferation and promoted the apoptosis of ESCs. Additionally, the phosphorylation of AKT and expression of Cyclin D1, which were closely related with cellular growth, were distinctly lessened after treating with imatinib. CONCLUSIONS: In summary, our study showed that GnRH-ant weakened the activization of c-kit receptor by decreasing its expression, causing the impaired growth ability of ESCs. Our findings provided a new insight into the effects of GnRH-ant on endometrium.

Mild starting dosage ovarian stimulation combined with a modified prolonged GnRH-a protocol improved IVF/ICSI outcomes in normal ovarian responders.

INTRODUCTION: Controlled ovarian hyperstimulation (COH) is essential for artificial reproduction technology (ART). This study aimed to evaluate the effects of a mild starting dosage of r-FSH ovarian stimulation after the modified prolonged GnRH-a down-regulation protocol for COH on the clinical outcomes in normal ovarian responders undergoing in vitro fertilization/intracytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET). MATERIAL AND METHODS: In the retrospective study, the patients were separated into two groups according to the starting dosage of r-FSH: a mild dosage group (75 IU ≤ r-FSH < 150 IU, n = 858) and a conventional dosage group (150 IU ≤ r-FSH ≤ 225 IU, n = 535). Data were collected from clinical records. The baseline characteristics and clinical outcomes were compared between the two groups. RESULTS: Although the duration of r-FSH treatment was a little longer in the mild dosage group, the total r-FSH dosage and the cost of ovarian stimulation were significantly lower than those in the conventional dosage group. Furthermore, compared to the conventional dosage group, the number of retrieved oocytes was also lower in the mild dosage group, whereas the rates of two pronuclei (2PN) fertilized oocytes and good-quality embryos were remarkable higher. The implantation rate, clinical pregnancy rate and live birth rate were significantly higher in the mild dosage group. There was no difference in early miscarriages rate, incidence of moderate and severe ovarian hyper-stimulation syndrome (OHSS) or incidence of ectopic pregnancy between the two groups. CONCLUSIONS: The modified prolonged GnRH-a pituitary down-regulation regimen combined with mild r-FSH starting dosage improved IVF/ICSI outcomes and reduced the financial cost in normal ovarian responders.