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.

C-kit signaling promotes human pre-implantation 3PN embryonic development and blastocyst formation.

BACKGROUND: Although in vitro culture system has been optimized in the past few decades, the problem of few or no high quality embryos has been still not completely solved. Accordingly, fully understanding the regulatory mechanism of pre-implantation embryonic development would be beneficial to further optimize the in vitro embryo culture system. Recent studies have found the expression of c-kit in mouse embryo and its promotion effects on mouse embryonic development. However, it is unclear the expression, the role and the related molecular regulatory mechanism of c-kit in human pre-implantation embryo development. Therefore, the present study is to determine whether c-kit is expressed in human pre-implantation embryos, and to investigate the possible regulatory mechanism of c-kit signaling in the process of embryonic development. METHODS: The present study includes human immature oocytes and three pronucleus (3PN) embryos collected from 768 women (28-32 ages) undergoing IVF, and normal 2PN embryos collected from ICR mice. Samples were distributed randomly into three different experimental groups: SCF group: G-1™ (medium for culture of embryos from the pro-nucleate stage to day 3) or G-2™ (medium for culture of embryos from day3 to blastocyst stage) + HSA (Human serum album) solution + rhSCF; SCF + imanitib (c-kit inhibitor) group: G-1™ or G-2™ + HSA solution + rhSCF + imanitib; SCF + U0126 (MEK/ERK inhibitor) group: G-1™ or G-2™ + HSA solution + rhSCF + U0126; Control group: G-1™ or G-2™ + HSA solution + PBS; The rate of good quality embryos at day 3, blastulation at day 6 and good quality blastulation at day 6 were analysis. RT-PCR, western blot and immunofluorescence staining were applied to detect the target genes and proteins in samples collected from human or mice, respectively. RESULTS: c-kit was expressed ubiquitously in all human immature oocytes, 3PN embryos and 3PN blastocysts. In the experiment of human 3PN embryos, compared with other groups, SCF group showed obviously higher rate of good quality at day 3, better rate of blastocyst formation at day 6 and higher rate of good quality blastocyst formation at day 6. Furthermore, we observed a higher ETV5 expression in SCF group than that in other groups. Similar results were also found in animal experiment. Interestingly, we also found a higher phosphorylation level of MEK/ERK signal molecule in mice embryos from SCF group than those from other groups. Moreover, inhibition of MEK/ERK signaling would remarkably impeded the mice embryonic development, which might be due to the reduced ETV5 expression. CONCLUSIONS: The present study firstly revealed that c-kit signaling might promote the human pre-implantation embryonic development and blastocyst formation by up-regulating the expression of ETV5 via MEK/ERK pathway. Our findings provide a new idea for optimizing the in vitro embryo culture condition during ART program, which is beneficial to obtain high quality embryos for infertile patients.

Increased SCF in Follicular Fluid and Granulosa Cells Positively Correlates With Oocyte Maturation, Fertilization, and Embryo Quality in Humans.

Stem cell factor (SCF), which is derived from granulosa cells (GCs), plays a key role in the process of follicular development and oocyte maturation. The present study aimed to explore whether the levels of SCF in follicular fluid (FF) and GCs can be used as a potential marker for predicting oocyte developmental potential. Follicular fluid and GC samples from 150 female patients undergoing intracytoplasmic sperm injection were collected in this study. The SCF concentrations in FFs and SCF messenger RNA (mRNA) in GCs were evaluated by using enzyme-linked immunosorbent assay and real-time polymerase chain reaction, respectively. The results showed that the levels of SCF protein and mRNA were significantly associated with oocyte maturation, normal fertilization, cleavage, and embryo quality. Moreover, the levels of SCF protein and mRNA in pregnancy group were also higher than those in the nonpregnancy group. The cutoff value of SCF in FF for predicting high-quality embryo was 1.346, with a sensitivity of 57.8% and a specificity of 72.4%, and the cutoff value of SCF in GCs for predicting high-quality embryo was 6.650, with a sensitivity of 64.4% and a specificity of 78.1%. In conclusion, our results showed a positive and statistically significant relationship between SCF level and oocyte maturation, normal fertilization, cleavage, embryo quality, and clinical pregnancy. Therefore, the levels of SCF in FF and GCs might be considered as a new marker for predicting oocyte developmental potential.

Clinical prospect of applying the chemical bile duct embolization to achieve a chemical hepatectomy in the treatment of highly selected hepatolithiasis.

The high recurrence rate of hepatolithiasis, together with the high operative risk of hepatectomy for specifically located stones shows that an effective treatment for intrahepatic stones has not been settled upon. It is commonly accepted that a diseased biliary duct mucosa is a prerequisite for the development of intrahepatic stones, and that segmental biliary obstruction is able to induce hepatic atrophy, fibrosis, and "self-cut" the obstructed hepatic segment. Therefore, we previously put forward the hypothesis that performing deliberate chemical bile duct embolization (CBDE) to induce a segmental chemical hepatectomy might be the way of treating hepatolithiasis. In this study, we review the relative experimental basis for CBDE, preliminary report on its clinical use in 2 patients, and speculate on its future application. To completely embolize a diseased biliary duct, absolute ethanol or phenol is firstly used to ablate the biliary mucosa and eradicate biliary bacteria. Subsequently, cyanoacrylate or tissue adhesive glue is used to permanently fill the duct lumen, occupying the space where the stones would have formed. Our prior laboratory investigations and preliminary clinical treatments have confirmed that this combination of embolization agents could not only achieve the desired aim of preventing stone recurrence but could also lead to complete atrophy of the targeted hepatic segment, thereby achieving a chemical hepatectomy. In the future, CBDE is likely to help in resolving the problem of calculous recurrence and thereby reduce the incidence of surgical reintervention and endoscopic stone extractions, which are so frequently needed in patients with hepatolithiasis. Also chemical hepatectomy might provide a new less-invasive hepatectomy method, especially for the more difficult resections of the caudate or right posterior lobes.

Feasibility and effectiveness of chemical bile duct embolization for chemical hepatectomy: a preliminary study.

BACKGROUND: The high operative risk of hepatectomy for specially located intrahepatic stones is still a problem to be solved. This study was undertaken to investigate the feasibility and effectiveness of chemical bile duct embolization for chemical hepatectomy. METHODS: Oxybenzene or absolute ethanol plus N-butyl-cyanoacrylate was employed for embolization. The feasibility, effectiveness and mechanism of chemical hepatectomy were preliminarily analyzed histologically or by Fas, TIMP-1, TGF-beta(1), and collagen I. RESULTS: Oxybenzene plus cyanonacrylate can preferably destroy and embolize the intrahepatic biliary duct, leading to the disappearance of hepatocytes in the periphery of embolized lobe and the achievement of effective chemical hepatectomy. The expressions of Fas, TIMP-1 and TGF-beta(1) in oxybenzene embolism group (88.90 +/- 38.10, 619.43 +/- 183.42, 185.22 +/- 70.39) and ethanol embolism group (72.39 +/- 29.51, 407.55 +/- 134.74, 163.56 +/- 51.75) were higher than those of biliary duct-ligated group (26.31 +/- 12.07, 195.31 +/- 107.67, 74.84 +/- 40.73) (P<0.05). The collagen I-positive percentage in the oxybenzene embolism group was also greater than that of the ethanol embolism group (33.97 +/- 12.51% vs. 20.67 +/- 8.09%, P<0.05). CONCLUSION: The effect of chemical hepatectomy may be achieved by chemical bile duct embolization.