Yiping Zhong

Background: Luteinizing hormone (LH) can stimulate mural granulosa cells to produce Amphiregulin (AREG), which can induce the resumption of meiosis in oocytes. Theca cells are present in the outer layer of follicles, providing communication with the pituitary axis through the established vascular system around the follicle. As LH target cells, it is unknown whether theca cells can produce AREG after LH stimulation. Methods: Primary cultured human theca cells were treated with LH (with or without the inhibitor of PKA, H89), or agonists of adenylate cyclase (forskolin or db-cAMP). The mRNA and protein levels of AREG were evaluated by RT-qPCR, immunochemistry, immunofluorescence, western blotting, and ELISA. Results: Immunohistochemistry of normal ovarian tissue obtained in the early-mid follicle phase showed that AREG expression was absent in both the theca layer and the granulosa cell layer of antral follicles. Double immunofluorescent staining revealed colocalization of AREG and CYP17A1 in human theca cells and colocalization of FSHR and AREG in human granulosa cells isolated from follicular fluid collected during IVF/ICSI after hCG trigger. LH significantly increased the mRNA and protein levels of AREG in human theca cells and the concentration of AREG in the culture medium. Forskolin and db-cAMP, activators of the cAMP/PKA signalling pathway, also significantly increased the mRNA and protein levels of AREG in human theca cells and the concentration of AREG in the culture medium. H89 antagonized the stimulating effect of LH on AREG expression in human theca cells. In addition, the concentration of AREG was lower in polycystic ovarian syndrome (PCOS) follicular fluid than in normal follicular fluid. The mRNA levels of AREG were significantly lower in PCOS granulosa cells and theca cells than in normal granulosa cells and theca cells. Conclusion: LH can stimulate the expression of AREG in human theca cells, and the adenylate cyclase/cAMP/PKA cascade may mediate this process. Expression of AREG is decreased in PCOS theca cells compared to normal theca cells, with or without LH stimulation.

Anticentromere antibodies (ACA) were associated with lower oocyte maturation rates and cleavage rates, while the mechanism was not clear. Aims of this study were to examine whether active immunization with centromere protein C could elicit the CENP-C autoantibody in mice and the impacts of the CENP-C autoantibody on oocyte meiosis. Mice were divided into two groups, one was the experimental group immunized with human centromere protein C and Freund’s adjuvant (CFA), and the other was the control group injected with CFA only. Serum and oocytes of BALB/c mice immunized with human centromere protein C (CENP-C) in complete Freund’s adjuvant (CFA) or injected with only CFA were studied for the development of the CENP-C antibody. Rates of germinal vesicle breakdown (GVBD), first polar body (Pb1) extrusion, abnormal spindle morphology, and chromosome misalignment were compared between the experimental group and the control group. The CENP-C antibody was only observed in serum and oocytes of mice immunized with the centromere protein C antigen. The first polar body (Pb1) extrusion rate was lower in the experimental group ( ). A higher percentage of spindle defects and chromosome congression failure were also detected in the experimental group (spindle defects: vs. control; chromosome misalignment: vs. control; for both). Oocyte meiosis was severely impaired by the CENP-C antibody, which may be the main mechanism of adverse reproductive outcomes for ACA-positive women who have no clinical symptoms of any autoimmune diseases.

Our purpose is to explore whether anti-dsDNA antibody, which was demonstrated to enter living cells and induced apoptosis, could adversely affect reproductive outcomes. A total of 259 women receiving the in vitro fertilization-embryo transfer (IVF) cycle were enrolled in this study, including 52 women with positive ANA and anti-dsDNA (ANA+/anti-dsDNA+ group), 86 women with positive ANA and negative anti-dsDNA (ANA+/anti-dsDNA− group), and 121 women with negative ANA and anti-dsDNA (ANA−/anti-dsDNA− group). 136 nonpregnant women among 259 patients in the IVF-ET cycle were enrolled in the hormone replacement therapy frozen-thawed embryo transfer (HRT-TET) cycle. We compared basic characters and IVF outcomes among three groups in fresh embryo transfer and frozen-thawed embryo transfer cycle, respectively. The number of retrieved oocytes, available embryos, and high-quality embryos in the ANA+/anti-dsDNA+ group was lower than those in the other two groups in the fresh embryo transfer cycle. The rates of fertilization, implantation, and clinical pregnancy in the ANA+/anti-dsDNA+ group were the lowest, while the early miscarriage rate was the highest in the ANA+/anti-dsDNA+ group both in the fresh embryo transfer cycle and in the frozen-thawed embryo transfer cycle. Our data suggested that anti-dsDNA antibody may be the essential marker for defective oocytes or embryos in infertile women with any type of ANA.

Antinuclear antibody (ANA) could cause reproductive failure. But treatment for women with antinuclear antibody was controversial. One hundred and thirty-three ANA+ women with one-time in vitro fertilization (IVF) implantation failure were divided into two groups randomly. The study group comprised 60 sero-positive for antinuclear antibody. These patients were treated with prednisone (10 mg/day) and aspirin (100 mg/day) starting 3 months before induction of ovulation in 60 IVF cycles. Seventy-three patients were included in untreated group. Fertilization rate, pregnancy rate, and implantation rate were significantly higher in treatment group, while abortion rate was markedly higher in non-treatment group by randomized controlled trial. Same conclusions were also proved between cycles of 60 ANA+ women with therapy and their former cycles without any treatment. Combined treatment of prednisone for immunosuppression and aspirin as an antithrombotic agent, starting 3 months before ovulation induction, may improve reproductive outcomes in ANA+ patients.