Homozygous variants in CDC23 cause female infertility characterized by oocyte maturation defects.

Oocyte maturation defects are major phenotypes resulting in female infertility. Although many genetic factors have been found to be responsible for these phenotypes, the underlying pathogenic genes and variants remain to be identified. The anaphase promoting complex or cyclosome (APC/C) is known to be essential in the metaphase-to-anaphase transition. In this study, we identified two homozygous missense variants (c.986A > G, p.Y329C and c.988C > T, p.R330C) in CDC23 that are responsible for female infertility characterized by oocyte maturation defects in three infertile individuals. CDC23 (cell division cycle 23) is one of the core subunits of the APC/C. In vitro experiments showed that the variant c.986A > G (p.Y329C) led to a decrease in CDC23 protein level and the variant c.988C > T (p.R330C) changed the localization of CDC23 in HeLa cells and mouse oocytes. In vivo studies showed that Cdc23Y329C/Y329C mice successfully mimicked the patients' phenotype by causing low expression of CDC23 and APC4 and the accumulation of securin and cyclin B1 in oocytes. AZ3146 treatment was able to rescue the phenotype. Taken together, our findings reveal the important roles of CDC23 in human oocyte maturation and provide a new genetic marker for female infertility.

Follistatin-like 3, an activin A binding protein, is involved in early pregnancy loss.

BACKGROUND: Early pregnancy loss (EPL), a common and severe complication in pregnancy, has a long-term personal and social impact. It was previously reported that follistatin-like 3(FSTL3), an activin A binding protein, contributes to the invasion and migration of trophoblast. Simultaneously, activin A induces the release of FSTL3 and the elevated activin A is found to be associated with pregnancy loss in women. This study aimed to identify the roles of FSTL3 in the establishment and maintenance of pregnancy, and to determine whether FSTL3 is involved in the pathophysiology of EPL. METHODS: Endometrial Ishikawa cells and JAR cells were cultured and FSTL3 siRNA was used to silence FSTL3. The trophoblast spheroids mimicking embryos were used in an embryonic adhesion system. The system aimed to investigate the role of FSTL3 silence on embryonic adhesion onto endometrial cell in vitro. The ICR mice model in vivo was used to investigate whether the FSTL3 works in embryonic implantation. The western blotting was used to determine the expression of FSTL3 and activin A. RESULTS: In the in vitro study, silence of FSTL3 in JAR cells significantly reduced the number of trophoblast spheroids adhered onto Ishikawa cells compared with the scramble siRNA. For the in vivo study, the number of embryos implanted in the uterine horn injected with FATL3 siRNA mixture was significantly less than that in control group. In the case control study, both the expression of FSTL3 and activin A in EPL women were significantly higher than that in controls. CONCLUSIONS: FSTL3 plays a biological role in the establishment and maintenance of normal pregnancy. Moreover, FSTL3 may be involved in the early pregnancy loss via neutralizing the elevated activin A.

Declined placental PLAC1 expression is involved in preeclampsia.

BACKGROUND: This study aimed to clarify the change of the expression of placenta-specific 1 (PLAC1) in the placenta of preeclamptic women and to explore the regulatory effects on thophoblast by PLAC1. METHODS: Nineteen women with preeclampsia and 19 with normal pregnancies were recruited, and then we determined the expression of PLAC1 by immunohistochemistry (IHC) and Western blotting. To observe the effect of hypoxia on the expression of PLAC1, trophoblasts were cultured at the normoxia or hypoxia condition. Small interference of ribonucleic acid (siRNA) was used to silence PLAC1. The proliferation, migration and invasion of trophoblasts were evaluated with cell counting kit-8 and transwell analysis, and the apoptosis of trophoblast was evaluated by flow cytometry with FITC and PI staining. RESULTS: Placental PLAC1 expression was significantly decreased in severe preeclampsia compared with control (P < .001). The expression of PLAC1 in trophoblasts was significantly decreased after treated with low oxygen concentration (P = .018). PLAC1 siRNA significantly inhibited the proliferation (P < .001), the migration (P < .001) and invasion (P < .001) of trophoblasts, but increased the apoptosis (P = .004 for Swan-71; P = .031 for Jar). CONCLUSIONS: The expression of PLAC1 was declined in preeclampsia and this inhibited the function of trophoblast, suggesting PLAC1 may play a role in the development of preeclampsia.

Involvement of follistatin-like 3 in preeclampsia.

INTRODUCTION: Preeclampsia is a main cause of maternal and perinatal mortality and morbidity. The expression of follistatin-like 3 (FSTL3) is enhanced in maternal serum and placenta of preeclamptic women. However, whether FSTL3 is involved in the pathophysiologic of preeclampsia has not been clarified yet. METHOD: Trophoblast cell lines Swan71 and JAR cells were cultured and siRNA was used to silence FSTL3. The expression of FSTL3 was determined by Western blotting. The matrigel-coated transwell and wound healing assays were used to assess invasion and migration, cell proliferation and apoptosis were detected by CCK-8 and flow cytometric analysis, respectively. Oil red O staining was used to detect the lipid storage in trophoblast. RESULTS: Hypoxia culture significantly enhanced the expression of FSTL3 by trophoblast. Down-regulation of FSTL3 significantly suppressed the proliferation, migration, invasion and lipid storage but increased apoptosis of trophoblast. DISCUSSION: Aberrant expression of FSTL3 in preeclampsia led to the dysfunction of trophoblast, indicating its involvement in the pathogenesis of preeclampsia.

Alteration in methylation level at differential methylated regions of MEST and DLK1 in fetus of preeclampsia.

OBJECTIVES: Offspring born to preeclamptic women are at high risk for metabolic diseases in later life, but the mechanisms are not known. The purposes of the current investigation were to clarify the changes in DNA methylation at MEST and DLK1 DMRs in fetus of preeclampsia and to explore the possible mechanisms behind the high risk of adult diseases in the offspring of preeclampsia. METHODS: Fetal lymphocytes were isolated from umbilical cord blood of 78 women with preeclampsia and 95 women with normal pregnancy. Genomic DNA was extracted and then DNA methylation levels of MEST and DLK1 DMRs were determined by MassARRAY quantitative methylation analysis. RESULTS: The methylation levels were detected in 20 CpG sites of MEST DMR and 16 sites of DLK1 DMR. Methylation changes were significantly different at CPG1, 3, 4, 7.8, 15, 18.19, and 20 of MEST between preeclampsia and normal pregnancy (P = 0.014, 0.001, <0.001, <0.001, = 0.001,  = 0.005, and = 0.003, respectively). Significant differences were also observed at CPG 3 and 9 of DLK1 (P = 0.002 and 0.027, respectively). However, overall methylation at these DMRs were not affected. CONCLUSION: We conclude methylation changes at some CpG sites of MEST and DLK DMRs in preeclamptic group. This may be among the mechanisms behind the high risk of adult diseases in the later life of offspring born to preeclamptic pregnancies. ABBREVIATIONS: DMR: Differentially Methylated Region; MEST: Mesoderm Specific Transcript.

Involvement of viperin in prevention of intrauterine transmission of hepatitis B virus.

The aim of this study was to explore the role of viperin in the prevention of intrauterine infection of hepatitis B virus (HBV). Placental samples were collected from seven HBV-positive pregnant women with their infants infected via intrauterine transmission (infected group), 30 HBV-positive women with non-infected infants (non-infected group), and 30 HBV-negative women (controls). The expression of viperin in placenta was analyzed with immunohistochemistry and Western blotting. The expression of viperin of placental trophoblast cell line Swan71 was determined after exposed to HBV. Viperin was localized to syncytiotrophoblast, and there was a significant difference in placental viperin levels among control, non-infected group and infected group (F = 12.824, p < 0.001). The expression of viperin was significantly higher in non-infected group than in control (p = 0.019) and in infected group (p < 0.001), and viperin expression was significantly lower in infected group than in control (p = 0.001). The exposure to HBV significantly increased the expression of viperin in Swan 71 (p < 0.001). Exposure to HBV up-regulates viperin expression in vivo and in vitro in placental trophoblast, and lack of this up-regulation is associated with intrauterine transmission of HBV.