Monopronucleated (1PN) and tripronuclear (3PN) zygotes formation during assisted reproduction in POSEIDON group 4 patients: Emphasizing on polar bodies.

AIM: Abnormal fertilization (1PN/3PN) and its accompanying polar body (PB) conditions have been less discussed in poor ovarian responders. By observing the PBs, we analyzed the mechanisms of abnormal fertilization and aimed to explore the role of intracytoplasmic sperm injection/in vitro fertilization (ICSI/IVF) in POSEIDON group 4 patients. METHODS: An observational study. All fresh IVF/ICSI cycles from January 2018 to December 2019 were evaluated. The inclusion criteria were POSEIDON group 4. Fertilization and PB conditions were assessed 16-18 h post-insemination. Primary observation endpoints including normal fertilization, abnormal fertilization, and total fertilization failure rate. RESULTS: A total of 351 cycles involving 180 patients met the inclusion criteria. Of these, 15 cycles reported no retrieved oocytes. Finally, 336 cycles (IVF, n = 267; ICSI, n = 69) were included. A total of 1005 oocytes and 939 embryos were assessed. The mean female age was 40.8 years, and the mean AMH level was 0.6 ng/mL. The normal fertilization rate was 69.7%. The zygote distribution was 18.7% 0PN, 3.9% 1PN, 66.9% 2PN, 9.5% 3PN, and 1.0% ≥4PN. For 1PN zygotes, 59% were denoted as 1PN2PB. The mean 3PN rate was 8.9%. CONCLUSIONS: In POSEIDON group 4, most of the monopronucleated zygotes were 1PN2PB. Digyny (3PN1PB), due to failure to extrude the second PB, was the major cause of triploidy in which ICSI could not circumvent. The distribution of abnormally fertilized zygotes was similar in IVF and ICSI. To investigate the mechanisms of abnormal fertilization and assess whether ICSI is necessary, analysis of PB will provide important clues.

Cobra venom proteome and glycome determined from individual snakes of Naja atra reveal medically important dynamic range and systematic geographic variation.

Recent progress in snake venomics has shed much light on the intra-species variation among the toxins from different geographical regions and has provided important information for better snakebite management. Most previous reports on snake venomics were based on venoms pooled from different snakes. In this study, we present the proteomic and glycomic profiles of venoms from individual Naja atra snakes. The results reveal wide dynamic range of three-finger toxins. Systematic classification based on cardiotoxin (CTX-) profiles of A2/A4 and A6, respectively, allowed the identification of two putative subspecies of Taiwan cobra from the eastern and western regions. We also identified four major N-glycan moieties on cobra snake venom metalloproteinase on the bi-antennary glycan core. ELISA showed that these glycoproteins (<3%) could elicit much higher antibody response in antiserum when compared to other high-abundance cobra venom toxins such as small molecular weight CTXs (~60%). By removing these high-molecular weight glycoproteins from the immunogen, we demonstrated better protection than that achieved with conventional crude venom immunization in mice challenged by crude venom. We conclude that both intra-species and inter-individual variations of proteomic and glycomic profiles of snake venomics should be considered to provide better antivenomic approach for snakebite management. BIOLOGICAL SIGNIFICANCE: Based on the proteomic and glycomic profiles of venoms obtained from individual snakes, we demonstrated a surprisingly wide dynamic range and geographical variation of three-finger toxins in cobra venomics. This provides a reasonable explanation for the variable neutralization effects of antivenom treatment on victims suffering from cobra snakebite and suggests a simple and economic method to produce potent antivenom with better efficacy. Since two major venomic profiles with distinct dynamic ranges were observed for Taiwan cobra venoms isolated from the eastern and western regions, the current venomic profile should be used as a quality control for future production of antivenom in clinical applications.

Global disulfide bond profiling for crude snake venom using dimethyl labeling coupled with mass spectrometry and RADAR algorithm.

Snake venom consists of toxin proteins with multiple disulfide linkages to generate unique structures and biological functions. Determination of these cysteine connections usually requires the purification of each protein followed by structural analysis. In this study, dimethyl labeling coupled with LC-MS/MS and RADAR algorithm was developed to identify the disulfide bonds in crude snake venom. Without any protein separation, the disulfide linkages of several cytotoxins and PLA2 could be solved, including more than 20 disulfide bonds. The results show that this method is capable of analyzing protein mixture. In addition, the approach was also used to compare native cytotoxin 3 (CTX III) and its scrambled isomer, another category of protein mixture, for unknown disulfide bonds. Two disulfide-linked peptides were observed in the native CTX III, and 10 in its scrambled form, X-CTX III. This is the first study that reports a platform for the global cysteine connection analysis on a protein mixture. The proposed method is simple and automatic, offering an efficient tool for structural and functional studies of venom proteins.

Endocytotic routes of cobra cardiotoxins depend on spatial distribution of positively charged and hydrophobic domains to target distinct types of sulfated glycoconjugates on cell surface.

Cobra cardiotoxins (CTX) are a family of three-fingered basic polypeptides known to interact with diverse targets such as heparan sulfates, sulfatides, and integrins on cell surfaces. After CTX bind to the membrane surface, they are internalized to intracellular space and exert their cytotoxicity via an unknown mechanism. By the combined in vitro kinetic binding, three-dimensional x-ray structure determination, and cell biology studies on the naturally abundant CTX homologues from the Taiwanese cobra, we showed that slight variations on the spatial distribution of positively charged or hydrophobic domains among CTX A2, A3, and A4 could lead to significant changes in their endocytotic pathways and action mechanisms via distinct sulfated glycoconjugate-mediated processes. The intracellular locations of these structurally similar CTX after internalization are shown to vary between the mitochondria and lysosomes via either dynamin2-dependent or -independent processes with distinct membrane cholesterol sensitivity. Evidence is presented to suggest that the shifting between the sulfated glycoconjugates as distinct targets of CTX A2, A3, and A4 might play roles in the co-evolutionary arms race between venomous snake toxins to cope with different membrane repair mechanisms at the cellular levels. The sensitivity of endocytotic routes to the spatial distribution of positively charged or hydrophobic domains may provide an explanation for the diverse endocytosis pathways of other cell-penetrating basic polypeptides.

YueF overexpression inhibits cell proliferation partly through p21 upregulation in renal cell carcinoma.

YueF is a novel putative tumor suppressor gene that can inhibit proliferation and induce apoptosis in hepatoma cells, but its role in renal cell carcinoma (RCC) remains unclear. Here, we examined the expression of the YueF gene in RCC tissues and the effect of YueF on cell proliferation in RCC 786-0 cells. The results showed that YueF was expressed at high levels in normal kidney tissues and cell lines but was reduced or absent in RCC tissues and 786-0 cells. Lentivirus-mediated YueF overexpression in RCC 786-0 cells caused cell-cycle arrest in the G1 phase and dramatically reduced proliferation in culture. YueF overexpression resulted in increased protein levels of p53 and p21(WAF1/Cip1), whereas the protein levels of cyclin D1 and pRb were decreased. The proliferation defects caused by YueF overexpression could be partially rescued by the expression of p21 siRNA. These findings suggest a critical role for p21 in the YueF-induced growth inhibition of 786-0 cells and provide novel insights into the mechanism underlying the tumor-suppressive action of YueF.

L-arginine-glycine amidinotransferase, betaine-homocysteine S-methyltransferase, and neuropolypeptide H3 are diminished in renal clear cell carcinoma of humans.

OBJECTIVE: To identify renal clear cell carcinoma-associated marker proteins. METHODS: Twelve patients with renal cell carcinoma (RCC) were collected and processed in the Department of Urology, Renmin Hospital, Wuhan University, China, between January 2008 and September 2009. Two-dimensional polyacrylamide gel electrophoresis and matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) were employed to investigate differentially expressed protein spots between RCC tissues and adjacent normal tissues, then reverse transcription polymerase chain reaction and western blot were employed to confirm the proteomic results. RESULTS: One protein spot was upregulated, 13 were downregulated, and 22 were absent in RCC tissues. Four of the absent proteins were L-arginine-glycine amidinotransferase (AGAT), Betaine-homocysteine S-methyltransferase (BHMT), Ketohexokinase (KHK), and Neuropolypeptide h3 (NPh3). The reverse transcriptase-polymerase chain reaction analysis demonstrated mRNA expression of AGAT, BHMT, and Nph3 was significantly decreased in 12 RCC tissues. In addition, Western blot analysis showed AGAT protein was absent in 11/12, BHMT in 9/12, and Nph3 in 5/12 RCC tissues. CONCLUSION: Absence of AGAT, BHMT, and Nph3 is common events in clear cell RCC; hence, it may be involved in the development of RCC; therefore, they have the potential to be tumor markers for diagnosis, treatment, and prognosis of RCC patients.

Differential diagnosis of suspected cervical pregnancy and conservative treatment with the combination of laparoscopy-assisted uterine artery ligation and hysteroscopic endocervical resection.

OBJECTIVE: To determine the accuracy of differential diagnosis by team consultation of abortion in progression, low-lying implantation/cervicoisthmic pregnancy, and cervical pregnancy (CP) in patients referred for suspicion of abnormal implantation on the lower segment and cervix of the uterus and to determine the efficacy of endoscopic surgery with uterine artery blockade followed by cervical evacuation in the treatment of confirmed CP. DESIGN: Prospective observational study under multiple-clinic and multiple-hospital cooperation. SETTING: Tertiary clinical and academic medical center. PATIENT(S): Twenty-seven women with a tentative diagnosis of CP made at their primary gynecologists' offices from July 1999 to June 2003. INTERVENTION(S): Second-opinion ultrasound scanning with transabdominal and transvaginal approach and optional color Doppler use. For patients with confirmed CP, a new treatment modality with laparoscopy-assisted uterine artery ligation followed by hysteroscopic local endocervical resection to remove the ectopic pregnancy was employed. For patients with abortion in progression or low-lying implantation/cervicoisthmic pregnancy (non-CP) requiring termination, dilatation and curettage (D&C) was performed under transabdominal ultrasound guidance. MAIN OUTCOME MEASURE(S): Fulfillment of ultrasound-based diagnostic criteria and operative course, convalescence, and commencement of menstruation in those patients with confirmed CP. RESULT(S): Cervical pregnancy was diagnosed in six (22.2%) patients at

Human cumulus-free oocyte maturational profile and in vitro developmental potential after stimulation with recombinant versus urinary FSH.

BACKGROUND: This study compares the influence of recombinant (r)FSH and urinary (u)FSH stimulation on oocyte and embryo quality in patients undergoing ICSI. METHODS AND RESULTS: Denuded oocyte maturity in ICSI patients was graded on a scale from metaphase II (MII) to prophase for nuclear maturation of oocytes. The relationships of cumulus-free oocyte maturational profiles with in vitro outcome parameters were evaluated. In the study population with an unknown distribution of FSH receptor polymorphisms, the ovarian response to rFSH stimulation was significantly different from that of uFSH stimulation, including lower number of oocytes retrieved/oocytes in MII, higher fertilization rates and higher good quality embryo rates. In the study population with a similar distribution of FSH receptor polymorphisms, the ovarian responses to rFSH were lower numbers of oocytes in MII, higher fertilization rates and higher good quality embryo rates, but the total number of oocytes retrieved was not influenced, in comparison with ovarian stimulation with uFSH. CONCLUSIONS: rFSH stimulation appears to influence oocyte quality and subsequent embryo quality in comparison with uFSH stimulation. FSH receptor polymorphisms seem to be an intrinsic factor influencing the ovarian response to FSH stimulation.

The predictive value of using a combined Z-score and day 3 embryo morphology score in the assessment of embryo survival on day 5.

BACKGROUND: The purpose of this study was to evaluate the ability of using the Z-score alone, or, in combination with the day 3 embryo morphology score, to predict embryo viability at day 5 from a large cohort of embryos derived from patients undergoing treatment with IVF/ICSI. METHODS AND RESULTS: In this retrospective study, a total of 1894 zygotes from 346 treatment cycles (295 couples) was analysed between January 2001 and May 2002. The Z-scoring system was useful in predicting day 5 embryo survival. The mean +/- SD day 5 embryo survival rates were 78.2 +/- 1.7, 49.0 +/- 2.5, 21.4 +/- 3.2 and 11.8 +/- 5.6% for Z-1, Z-2, Z-3 and Z-4 zygotes groups respectively. Embryos derived from Z-1 scores and grade I day 3 embryo scores showed the best day 5 embryo survival and a very high implantation potential. CONCLUSIONS: These data suggest that a combined evaluation of the Z-score and day 3 embryo morphology is highly predictive of embryo outcome after IVF/ICSI. The Z-score could be of great help in the selection of embryos for cultures extended to later stages. The Z-score alone, or preferably in combination with day 3 embryo morphology, is useful in the determination of the most suitable embryos and the number of embryos for transfer, thus achieving the optimal chance of conception while reducing the risk of high order multiple pregnancy.

Human oocyte maturity in vivo determines the outcome of blastocyst development in vitro.

PURPOSE: To date, the impact of oocyte maturity at aspiration on the blastocyst formation in vitro has not been fully evaluated. This study was undertaken to assess the influence of oocyte maturity in patients undergoing in vitro fertilization and blastocyst transfer program. METHODS: A total of 1278 oocytes derived from 147-IVF cycles were retrospectivly analyzed. Oocyte maturity was graded on a scale from 1 to 5 based on the morphology of the ooplasm, cumulus mass, corona radiata, and membrana granulosa cells. RESULTS: Mature oocytes yielded the highest fertilization rates. Although the cleavage rates were similar in both groups, the percentage of poor morphology, day-3 embryos from the nonmature-oocyte group was significantly higher than from the mature-oocyte group (54.7% vs. 15.5%, P < 0.001). Although good morphology, day-3 embryos were collected from nonmature oocytes, the incidence of these embryos developing to the blastocyst stage was significantly less than from mature oocytes (33.3% vs. 71.2%, P < 0.001). Although blastocyst stage embryos were collected from nonmature oocytes, the incidence of these embryos developing to the top-scoring blastocysts was significantly less than from mature oocytes (58.3% vs. 89.5%, P < 0.001). CONCLUSIONS: These phenomena suggest that oocyte maturity produced in vivo determine the fertilization potential and subsequent blastocyst quality in vitro.

The maturity of human cumulus-free oocytes is positively related to blastocyst development and viability.

PURPOSE: We investigated whether the human oocyte maturity at the removal of cumulus/corona cells affects the embryo outcome in vitro. METHODS: A total of 620 oocytes, which subsequently underwent blastocyst culture, were included in this analysis. Oocytes that were in prophase or Metaphase I of meiosis at the removal of cumulus/corona cells were in Group II. Oocytes that were in Metaphase II at the removal of cumulus/corona cells were in Group I. RESULTS: Group I oocytes yielded the highest fertilization rates (96.3% vs. 77.1%, P < 0.001). The incidence of Group II oocytes developing to the blastocyst stage was significantly less than from Group I oocytes (38.1% vs. 86.1%, P < 0.001). The percentage of top-scoring blastocysts from Group I oocytes was higher than that of Group II oocytes (95.4% vs. 76.2%, P < 0.001). CONCLUSIONS: Oocyte maturity at the removal of cumulus/corona cells needs to be considered in selecting good quality blastocysts for embryo transfer.

Laparoscopic-assisted vaginal hysterectomy in women of all weights and the effects of weight on complications.

STUDY OBJECTIVE: To compare intraoperative and postoperative complication rates for laparoscopic-assisted vaginal hysterectomy (LAVH) between women classified as obese, normal weight, or very thin based on body mass index (BMI). DESIGN: Retrospective cohort study (Canadian Task Force classification II-3). SETTING: University hospital. PATIENTS: Six hundred seventy women (162 with BMI >25, 34 with BMI <18.5, 474 with BMI 18.5-25 kg/m(2)). INTERVENTION: LAVH. MEASUREMENTS AND MAIN RESULTS: For women with high BMIs, 34 procedures (21.0%) were converted to laparotomy, compared with 48 (10.1%) for women of normal body weight and 3 (8.8%) for those with low BMI (p = 0.001). Average blood loss was 299.3 +/- 87.8, 219.1 +/- 57.5, and 231.8 +/- 65.9 ml, respectively (p <0.001). Very thin women had similar intraoperative and postoperative complication rates (8.8 %) as women of normal body weight (8.6%) and obese women (11.1%). CONCLUSION: Obese women had increased likelihood of conversion to laparotomy and greater blood loss after LAVH than nonobese women. Very thin women had similar intraoperative and postoperative complication rates as women of normal body weight and obese women.