Assessment of the calcium releasing machinery in oocytes that failed to fertilize after conventional ICSI and assisted oocyte activation.

RESEARCH QUESTION: Can oocyte-related activation deficiencies be evaluated in oocytes that failed to fertilize after intracytoplasmic sperm injection (ICSI) combined with assisted oocyte activation (AOA)? DESIGN: Evaluation of the spindle-chromosome complexes and intracellular distribution of inositol trisphosphate type 1 receptors (IP3R1) in in-vitro matured (IVM) and failed-to-fertilize oocytes from patients undergoing AOA. Assessment of the oocyte-related Ca2+ releasing capacity in response to Ca2+ ionophores and sperm microinjection in oocytes that failed to fertilize after ICSI or ICSI-AOA. RESULTS: IVM oocytes from patients undergoing conventional ICSI (control) and ICSI-AOA (study group) revealed a similar normalcy of spindle-chromosome complexes and distribution patterns of IP3R1. Failed-to-fertilize oocytes from both groups showed significant differences in proportion of normal or abnormal spindle-chromosome complex conformations. However, migration of IP3R1 was identified in a higher proportion of failed-to-fertilize oocytes after ICSI-AOA than after conventional ICSI. It was further observed that oocytes which failed to fertilize, either after ICSI or ICSI-AOA, mostly retain their capacity to respond to stimuli such as exposure to Ca2+ ionophores or to sperm microinjection. CONCLUSIONS: Evaluation of spindle-chromosome normalcy and distribution of IP3R1 does not help identify the presence of Ca2+ releasing deficiencies in these oocytes. However, oocyte Ca2+ analysis adds value in identifying Ca2+ releasing incapacity of oocytes that failed to fertilize after ICSI or ICSI-AOA. Some patients experiencing fertilization failure after ICSI-AOA present with a suspected activation deficiency downstream of the Ca2+ machinery, which cannot be overcome by ICSI-AOA based on the use of Ca2+ ionophores.

Patients with a high proportion of immature and meiotically resistant oocytes experience defective nuclear oocyte maturation patterns and impaired pregnancy outcomes.

Patients presenting with abnormally high numbers of immature oocytes at retrieval are more likely to exhibit maturation resistant oocytes. However, the clinical relevance of such events remains unknown. We investigated nuclear maturation competence of immature oocytes from patients showing >40% of collected immature oocytes (Study group) and Controls, in which a normal number of mature oocytes (≥60%) was retrieved. Following in-vitro culture, oocytes were classified as maturation resistant or in-vitro matured (IVM). Treatment outcomes were evaluated in Study and Control groups based on presence of maturation resistant oocytes. Overall, similarly high spindle and chromosome abnormality rates were observed in maturation resistant oocytes from both Study and Control groups. IVM oocytes from the Study group revealed significantly higher percentages of misaligned chromosomes compared with Controls (P < 0.05). Remarkably, Study group patients with at least one maturation resistant oocyte showed significantly reduced cumulative pregnancy and live birth rates compared with Control group maturation resistant patients (P < 0.05). When further investigating the aetiology, a maturation resistant mouse model revealed defective Ca2+ signalling of maturation resistant oocytes at germinal vesicular breakdown and parthenogenetic activation. In conclusion, appropriate treatment strategies, including clinical utilization of IVM oocytes from Study group patients, warrant further investigation.

Culture conditions affect Ca2+ release in artificially activated mouse and human oocytes.

Inconsistent fertilisation and pregnancy rates have been reported by different laboratories after application of ionomycin as a clinical method of assisted oocyte activation (AOA) to overcome fertilisation failure. Using both mouse and human oocytes, in the present study we investigated the effects of ionomycin and Ca2+ concentrations on the pattern of Ca2+ release and embryonic developmental potential. In the mouse, application of 5µM ionomycin in potassium simplex optimisation medium (KSOM) or 10µM ionomycin in Ca2+-free KSOM significantly reduced the Ca2+ flux and resulted in failure of blastocyst formation compared with 10µM ionomycin in KSOM. Increasing the Ca2+ concentration up to three- or sixfold did not benefit mouse embryonic developmental potential. Similarly, 10µM ionomycin-induced rise in Ca2+ in human oocytes increased with increasing total calcium concentrations in the commercial medium. Remarkably, we observed significantly reduced mouse embryo development when performing AOA over a period of 10min in Quinn's AdvantageTM Fertilisation medium (Cooper Surgical) and IVFTM medium (Vitrolife) compared with Sydney IVF COOK cleavage medium (Cook Ireland), using the same sequential culture system from the post-activation stage to blastocyst formation stage in different AOA groups. In conclusion, concentrations of both ionomycin and Ca2+ in culture media used during AOA can have significant effects on Ca2+ release and further embryonic developmental potential.

A systematic analysis of the suitability of preimplantation genetic diagnosis for mitochondrial diseases in a heteroplasmic mitochondrial mouse model.

STUDY QUESTION: What is the reliability of preimplantation genetic diagnosis (PGD) based on polar body (PB), blastomere or trophectoderm (TE) analysis in a heteroplasmic mitochondrial mouse model? SUMMARY ANSWER: The reliability of PGD to determine the level of mitochondrial DNA (mtDNA) heteroplasmy is questionable based on either the first or second PB analysis; however, PGD based on blastomere or TE analysis seems more reliable. WHAT IS KNOWN ALREADY: PGD has been suggested as a technique to determine the level of mtDNA heteroplasmy in oocytes and embryos to avoid the transmission of heritable mtDNA disorders. A strong correlation between first PBs and oocytes and between second PBs and zygotes was reported in mice but is controversial in humans. So far, the levels of mtDNA heteroplasmy in first PBs, second PBs and their corresponding oocytes, zygotes and blastomeres, TE and blastocysts have not been analysed within the same embryo. STUDY DESIGN, SIZE AND DURATION: We explored the suitability of PGD by comparing the level of mtDNA heteroplasmy between first PBs and metaphase II (MII) oocytes (n = 33), between first PBs, second PBs and zygotes (n = 30), and between first PBs, second PBs and their corresponding blastomeres of 2- (n = 10), 4- (n = 10) and 8-cell embryos (n = 11). Levels of mtDNA heteroplasmy in second PBs (n = 20), single blastomeres from 8-cell embryos (n = 20), TE (n = 20) and blastocysts (n = 20) were also compared. PARTICIPANTS/MATERIALS, SETTING, METHODS: Heteroplasmic mice (BALB/cOlaHsd), containing mtDNA mixtures of BALB/cByJ and NZB/OlaHsd, were used in this study. The first PBs were biopsied from in vivo matured MII oocytes. The ooplasm was then subjected to ICSI. After fertilization, second PBs were biopsied and zygotes were cultured to recover individual blastomeres from 2-, 4- and 8-cell embryos. Similarly, second PBs were biopsied from in vivo fertilized zygotes and single blastomeres were biopsied from 8-cell stage embryos. The remaining embryo was cultured until the blastocyst stage to isolate TE cells. Polymerase chain reaction followed by restriction fragment length polymorphism was performed to measure the level of mtDNA heteroplasmy in individual samples. MAIN RESULTS AND THE ROLE OF CHANCE: Modest correlations and wide prediction interval [PI at 95% confidence interval (CI)] were observed in the level of mtDNA heteroplasmy between first PBs and their corresponding MII oocytes (r(2) = 0.56; PI = 45.96%) and zygotes (r(2) = 0.69; PI = 37.07%). The modest correlations and wide PI were observed between second PBs and their corresponding zygotes (r(2) = 0.65; PI = 39.69%), single blastomeres (r(2) = 0.42; PI = 48.04%), TE (r(2) = 0.26; PI = 54.79%) and whole blastocysts (r(2) = 0.40; PI = 57.48%). A strong correlation with a narrow PI was observed among individual blastomeres of 2-, 4- and 8-cell stage embryos (r(2) = 0.92; PI = 11.73%, r(2) = 0.86; PI = 18.85% and r(2) = 0.85; PI = 21.42%, respectively), and also between TE and whole blastocysts (r(2) = 0.90; PI = 23.58%). Moreover, single blastomeres from 8-cell stage embryos showed a close correlation and an intermediate PI with corresponding TE cells (r(2) = 0.81; PI = 28.15%) and blastocysts (r(2) = 0.76; PI = 36.43%). LIMITATIONS, REASONS FOR CAUTION: These results in a heteroplasmic mitochondrial mouse model should be further verified in patients with mtDNA disorders to explore the reliability of PGD. WIDER IMPLICATIONS OF THE FINDINGS: To avoid the transmission of heritable mtDNA disorders, PGD techniques should accurately determine the level of heteroplasmy in biopsied cells faithfully representing the heteroplasmic load in oocytes and preimplantation embryos. Unlike previous PGD studies in mice, our results accord with PGD results for mitochondrial disorders in humans, and question the reliability of PGD using different stages of embryonic development. TRIAL REGISTRATION NUMBER: Not applicable.

Structural basis of the subcortical maternal complex and its implications in reproductive disorders.

The subcortical maternal complex (SCMC) plays a crucial role in early embryonic development. Malfunction of SCMC leads to reproductive diseases in women. However, the molecular function and assembly basis for SCMC remain elusive. Here we reconstituted mouse SCMC and solved the structure at atomic resolution using single-particle cryo-electron microscopy. The core complex of SCMC was formed by MATER, TLE6 and FLOPED, and MATER embraced TLE6 and FLOPED via its NACHT and LRR domains. Two core complexes further dimerize through interactions between two LRR domains of MATERs in vitro. FILIA integrates into SCMC by interacting with the carboxyl-terminal region of FLOPED. Zygotes from mice with Floped C-terminus truncation showed delayed development and resembled the phenotype of zygotes from Filia knockout mice. More importantly, the assembly of mouse SCMC was affected by corresponding clinical variants associated with female reproductive diseases and corresponded with a prediction based on the mouse SCMC structure. Our study paves the way for further investigations on SCMC functions during mammalian preimplantation embryonic development and reveals underlying causes of female reproductive diseases related to SCMC mutations, providing a new strategy for the diagnosis of female reproductive disorders.

Effect of two assisted oocyte activation protocols used to overcome fertilization failure on the activation potential and calcium releasing pattern.

OBJECTIVE: To assess the effect of two assisted oocyte activation (AOA) protocols with the use of two calcium (Ca(2+)) ionophores, ionomycin and A23187 (calcimycin), on the intracellular Ca(2+) level in mouse and human oocytes and the fertilization rates. DESIGN: Comparison of two Ca(2+) ionophores, ionomycin and A23187, regarding their capacity to increase the intracellular Ca(2+) level and to support subsequent oocyte activation and development. SETTING: University hospital research laboratory. PATIENT(S)/ANIMAL(S): Patients undergoing intracytoplasmic sperm injection (ICSI) treatment and B6D2F1 mice. INTERVENTION(S): Assisted oocyte activation and microinjection of mouse and human oocytes with sperm. MAIN OUTCOME MEASURE(S): Measurement of the fertilizing and Ca(2+)-releasing ability of human sperm. RESULT(S): Ionomycin was more potent than A23187 in provoking Ca(2+) increases in both mouse and human oocytes with significantly higher amplitude and area under the receiver operating characteristic curve. The oocyte activation rate was significantly higher when mouse oocytes were activated with the use of the ionomycin- rather than the A23187-based AOA protocol. Furthermore, oocyte activation rate was higher when human in vitro matured oocytes were activated with the ionomycin-based AOA protocol, but the difference did not reach statistical significance. CONCLUSION(S): In both mouse and human oocytes, the AOA protocol that used ionomycin was more efficient than the one that used A23187. Bearing in mind that mammalian fertilization is successful when the total dose of Ca(2+) released reaches a minimal threshold, the use of ionomycin for human AOA might be justified instead of the use of A23187.

Cellular Heterogeneity in the Level of mtDNA Heteroplasmy in Mouse Embryonic Stem Cells.

Variation in the level of mtDNA heteroplasmy in adult tissues is commonly seen in patients with a mixture of wild-type and mutant mtDNA. A mixture of different mtDNA variants may influence such variation and cause mtDNA segregation bias. We analyzed cellular heterogeneity in embryonic stem cells (ESCs) derived from a polymorphic mouse model containing NZB and BALB mtDNA genotypes. In ESCs, inter-colony heterogeneity varied up to 61%, whereas intra-colony heterogeneity varied up to 100%. Three out of five cell lines displayed nearly homoplasmic BALB and NZB mtDNA haplotypes in differentiated single cells. The proportion of NZB mtDNA genotype increased with progressive passaging (0.39%; p = 0.002). These results demonstrate the bimodal segregation of mtDNA haplotypes, indicating the occurrence of tissues with variable levels of heteroplasmies in individuals with mtDNA mutations. Furthermore, proliferation of one mtDNA genotype over another may pose the risk of accumulating mutant mtDNAs during subsequent cell divisions.

Assessment of nuclear transfer techniques to prevent the transmission of heritable mitochondrial disorders without compromising embryonic development competence in mice.

To evaluate and compare mitochondrial DNA (mtDNA) carry-over and embryonic development potential between different nuclear transfer techniques we performed germinal vesicle nuclear transfer (GV NT), metaphase-II spindle-chromosome-complex (MII-SCC) transfer and pronuclear transfer (PNT) in mice. No detectable mtDNA carry-over was seen in most of the reconstructed oocytes and embryos. No significant differences were seen in mtDNA carry-over rate between GV NT (n=20), MII-SCC transfer (0.29 ± 0.63; n=21) and PNT (0.29 ± 0.75; n=25). Blastocyst formation was not compromised after either PNT (88%; n=18) or MII-SCC transfer (86%; n=27). Further analysis of blastomeres from cleaving embryos (n=8) demonstrated undetectable mtDNA carry-over in all but one blastomere. We show that NT in the germ line is potent to prevent transmission of heritable mtDNA disorders with the applicability for patients attempting reproduction.

Quantitative reduction of peripheral CD4+ CD25+ FOXP3+ regulatory T cells in reproductive failure after artificial insemination by donor sperm.

PROBLEM: The objective of this study was to determine whether peripheral Treg-cell percentages were altered in women with reproductive failure after artificial insemination by donor sperm (AID) and which parameters can best discriminate women with AID failure and normal controls. METHOD OF STUDY: A retrospective case-control study of 20 fertile controls and 20 patients undergoing more than four treatment cycles with negative pregnancy test (Group I), 20 experiencing biochemical pregnancy loss (Group II), and 20 undergoing missed abortion or spontaneous miscarriage (Group III) was performed. The peripheral percentages of CD4(+) CD25(+) and CD4(+) CD25 (+) Foxp3(+) Treg cells within CD4(+) T-cell population were evaluated at both late follicular and luteal phases of menstrual cycle by flow cytometry. RESULTS: A significantly decreased percentage of CD4(+) CD25(+) Foxp3(+) Treg cells was detected at the late follicular phase in all AID failure groups compared with the controls. The percentage of CD4(+) CD25(+) Foxp3(+) Treg cells at the late follicular phase in the controls was higher than that at the luteal phase. Using receiver operating characteristic curve, we found that CD4(+) CD25(+) Foxp3(+) Treg-cell percentage <2% can best discriminate the AID failure and normal controls. CONCLUSION: Reduced percentage of peripheral CD4(+) CD25(+) Foxp3(+) Treg cells at the late follicular phase was associated with AID failure and can be a potential biomarker for predicting AID-induced failure.

Quantitative and functional changes in peripheral natural killer cells in women with reproductive failure after artificial insemination with donor sperm.

Considerable evidence indicates that quantitative and/or functional abnormalities of natural killer (NK) cells are associated with reproductive failure. The objective of this study was to determine if peripheral NK percentage and activity are altered in women with reproductive failure after artificial insemination by donor (AID), and which parameters can best discriminate women with AID failure and normal controls. A case-control study of 20 fertile controls, 25 patients undergoing more than four treatment cycles without success (Group I), 25 experiencing biochemical pregnancy loss (Group II), and 25 undergoing embryo growth arrest or miscarriage (Group III) was performed. The peripheral NK percentage and cytotoxicity was evaluated by flow cytometry. Our results showed that a significantly increased percentage of NK cell cytotoxicity (NKCC) was detected in all study groups compared with the controls. The percentage of CD56(+) NK cells was elevated in Groups II and III compared with the controls. The percentage of CD56(+)CD16(+) NK cells in the study groups was higher than that in the controls. By using receiver operating characteristic curve and logistic regression analysis, we found that NKCC combined with CD56(+)CD16(+) NK percentage had good predictive power. The overall predicted percentage correct was 0.89. These results suggest that enhanced NKCC and an elevated cytotoxic NK percentage may be associated with AID failure and can be biomarkers for predicting AID-induced failure.

Enhanced maternal anti-fetal immunity contributes to the severity of hypertensive disorder complicating pregnancy.

PROBLEM: The aim of this study was to evaluate how fetal monocyte activation and maternal anti-fetal antigen-specific antibody-secreting cells (ASC) affect the severity of hypertensive disorder complicating pregnancy (HDCP). METHOD OF STUDY: Forty-six healthy third-trimester pregnant women and 20 patients with gestational hypertension, 20 with mild pre-ecalmpsia and another 20 with severe pre-eclampsia were included in the study. Interleukin-6 (IL-6) release from cord blood monocytes was examined by intracellular cytokine staining and flow cytometric analysis. Moreover, the maternal anti-fetal antigen-specific ASC were detected by enzyme-linked immunospot assay. RESULTS: A significantly increased percentage of IL-6-positive monocytes were detected in the cord blood of study groups compared with the controls (P < 0.01). The percentage of IL-6-positive monocytes was increased as the disease progressed (P < 0.05). There were more anti-fetal antigen-specific ASC in the study groups than those in the controls (P < 0.001). Furthermore, the anti-fetal antigen-specific ASC showed difference in gestational hypertensive and severe pre-eclamptic groups (P < 0.05). CONCLUSION: We conclude that the fetal monocyte activation and the increase in maternal anti-fetal antigen-specific ASC were related to the incidence and severity of HDCP. These results provide both indirect and direct evidence for the occurrence of exaggerated maternal humoral immunity against the fetal antigens in HDCP.