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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.

Objective: What are the cost per live birth and the incremental cost of preventing a miscarriage with preimplantation genetic testing for aneuploidy (PGT-A) by polar body biopsy and array-based comprehensive genome hybridisation (aCGH) versus regular IVF/ICSI without PGT-A for infertility treatment in women 36–40 years of age?. Design: Decision tree model. Population: A randomised clinical trial on PGT-A (ESTEEM study). Methods: Two treatment strategies were compared: one cycle of IVF/ICSI with or without PGT-A. Costs and effects were analysed with this model for four different cost scenarios: high-, higher medium, lower medium and low-cost. Base case, sensitivity, threshold, and probabilistic sensitivity analyses were used to examine the cost-effectiveness implications of PGT-A. Results: PGT-A increased the cost per live birth by approximately 15% in the high-cost scenario to approximately 285% in the low-cost scenario. Threshold analysis revealed that PGT-A would need to be associated with an absolute increase in pregnancy rate by 6% to >39% or, alternatively, would need to be US$2,969 (high-cost scenario) to US$4,888 (low-cost scenario) cheaper. The incremental cost to prevent one miscarriage by PGT-A using the base case assumptions was calculated to be US$34,427 (high-cost scenario) to US$51,146 (low-cost scenario). A probabilistic sensitivity analysis showed cost-effectiveness for PGT-A from 1.9% (high-cost scenario) to 0.0% (low-cost scenario) of calculated samples. Conclusions: While avoiding unnecessary embryo transfers and miscarriages are important goals, patients and doctors need to be aware of the high-cost implications of applying PGT-A using aCGH on polar bodies. Tweetable abstract: PGT-A by polar body biopsy and comprehensive genome hybridisation increases cost per live birth and requires high financial spending per miscarriage averted.

Oocyte apoptosis can be used as an indicator of oocyte quality and development competency. Phospholipase C (PLC) is a critical enzyme that participates in phosphoinositide metabolic regulation and performs many functions, including the regulation of reproduction. In this study, we aimed to explore whether PLC participates in the regulation of apoptosis in porcine oocytes and investigated its possible mechanism. In porcine oocytes, 0.5 μM U73122 (the PLC inhibitor) was considered to be the best concentration to facilitate maturation, and 0.5 μM m-3M3FBS (the PLC activator) was regarded as the most appropriate concentration to inhibit maturation. The percentage of cleavage and blastocysts treated with 0.5 μM U73122 was lower than that of the control group. Furthermore, the percentage of cleavage and blastocysts treated with 0.5 μM m-3M3FBS was higher than that of the control group. The relative PLC messenger RNA (mRNA) expression tested by a quantitative real-time polymerase chain reaction was found to be inhibited by 0.5 μM U73122 or activated by 0.5 μM m-3M3FBS. The relative mRNA abundance of BAK, BAX, CASP3, CASP8, and TP53 and protein abundance of Bak, cleaved caspase-3, caspase-8, and P53 was activated by U73122 or inhibited by m-3M3FBS, while the relative mRNA and protein level of BCL6 showed the opposite trend. The intracellular Ca2+ concentration increased and the expression of PLCB1 protein also increased in porcine oocytes when they were cultured with 0.5 μM m-3M3FBS for 44 hours. The abundance of proteins PKCβ and CAMKIIα and the expression of several downstream genes (CDC42, NFATc1, NFATc2, NFκB, and NLK) were activated by m-3M3FBS or inhibited by U73122. Our findings indicate that PLC inhibits apoptosis and maturation in porcine oocytes. The intracellular Ca2+ concentration, two Ca2+ -sensitive proteins, and several downstream genes were positively regulated by PLC.

Allicin, a chemical component of garlic, has strong antioxidant activity and is thought to exert antiaging effects in vitro. We investigated whether allicin treatment would protect porcine oocytes and embryos from postovulatory aging mediated by apoptosis and autophagy. The rates of oocyte survival and polar body extrusion in samples treated with 1 µM allicin (1 AL) were significantly higher than in untreated samples (0 AL). In addition, 1 AL prevented defects in spindle formation and chromosome alignment, as well as decreases in the expression of maturation markers, during in vitro aging. In this study, we considered allicin to be a regulator of autophagy rather than an antioxidant or antiapoptotic agent. At the embryo level, although the cleavage rate after parthenogenetic activation was similar in all groups, the blastocyst formation rate was higher in the 1 AL group than in the 0 AL group. Our findings demonstrate that allicin effectively prevents the deterioration of porcine oocytes during aging in vitro, and could therefore be used to improve the quality of aged oocytes used in in vitro experiments.

Cytoskeleton which includes microtubule and actin filaments plays important roles during mammalian oocyte maturation. In the present study, we showed that protein kinase C mu (PKC mu) was one potential key molecule which affected cytoskeleton dynamics in mouse oocytes. Our results showed that PKC mu expressed and localized at the poles of the spindle during oocyte maturation, and PKC mu expression reduced in the oocytes from 6-month-old mice or 24 hr in vitro culture. We knocked down the expression of PKC mu in oocytes using morpholino injection to explore the relationship between PKC mu and subcellular structure defects. The loss of PKC mu reduced oocyte maturation competence, showing with decreased polar body extrusion rate and increased rate of symmetric division. Further analysis indicated that PKC mu decrease caused the spindle organization defects, and this could be confirmed by the decreased tubulin acetylation level. Moreover, we found that PKC mu affected the phosphorylation level of cofilin for actin assembly, which further affected cytoplasmic actin distribution and spindle positioning. In summary, our data indicated that PKC mu is one key factor for oocyte maturation through its roles on the spindle organization and actin filament distribution.

Tributyltin oxide (TBTO) has been widely used as marine antifouling composition, preservative, biocide, and a stabilizer in plastic industry. Previous studies have indicated that TBTO can cause immunotoxicity as an environmental pollutant. However, little is known about its reproductive toxicity, especially on female oocyte maturation and the underlying mechanisms. In this study, mouse oocytes were cultured with different concentrations of TBTO in vitro, and several crucial events during meiotic maturation were evaluated. We found that the first polar body extrusion rate was significantly reduced, which reflected the disruption of meiotic maturation. The rate of abnormal spindle organization increased significantly, accompanied with a higher rate of chromosome misalignment. In addition, TBTO treatment increased reactive oxygen species generation markedly, which also accelerated the early-stage apoptosis. Moreover, heterogeneous mitochondrial distribution, mitochondrial dysfunction, and higher rate of aneuploidy were detected, which consequently disrupted in vitro fertilization. In conclusion, our results indicated that TBTO exposure could impair mouse oocyte maturation by affecting spindle organization, chromosome alignment, mitochondria functions, oxidative stress, and apoptosis.

Timely degradation of protein regulators of the cell cycle is essential for the completion of cell division. This degradation is promoted by the E3 anaphase-promoting complex/cyclosome (APC/C) and mediated by the E2 ubiquitin-conjugating enzymes (Ube2s). Unlike the ample information gathered regarding the meiotic E3 APC/C, the E2s participating in this cell division have never been studied. We identified Ube2C, -S, and -D3 as the E2 enzymes that regulate APC/C activity during meiosis of mouse oocytes. Their depletion reduces the levels of the first meiotic cytokinesis by 50%, and their overexpression doubles and accelerates its completion (50% as compared with 4% at 11 h). We also demonstrated that these E2s take part in ensuring appropriate spindle formation. It is noteworthy that high levels of Ube2C bring about the resumption of the first meiotic division, regardless of the formation of the spindle, overriding the spindle assembly checkpoint. Thus, alongside their canonical function in protein degradation, Ube2C and -S also control the extrusion of the first polar body. Overall, our study characterizes new regulators and unveils the novel roles they play during the meiotic division. These findings shed light on faithful chromosome segregation in oocytes and may contribute to better understanding of aneuploidy and its consequent genetic malformations.

Generation of a haploid female germ cell, the egg, consists of two rounds of asymmetric cell division (meiosis I and meiosis II), yielding two diminutive and nonviable polar bodies and a large haploid egg. Animal eggs are also unique in the lack of centrioles and therefore form meiotic spindles without the pre-existence of the two dominant microtubule organizing centers (centrosomes) found in mitosis. Meiotic spindle assembly is further complicated by the unique requirement of sister chromatid mono-oriented in meiosis I. Nonetheless, the eggs appear to adopt many of the same proteins and mechanisms described in mitosis, with necessary modifications to accommodate their special needs. Unraveling these special modifications will not only help understanding animal reproduction, but should also enhance our understanding of cell division in general.

In starfish oocytes, microtubules (MTs) form a spindle, which plays an important role in contributing to the selective loss of chromosomes and centrosomes to the polar bodies (PBs) during meiosis. When Taxol was locally injected near the germinal vesicle (GV) or the mitotic apparatus during meiosis I, PB formation was inhibited as mentioned below. In the oocytes, which were injected with Taxol after spindle formation, the spindle became large, and then the volume of the first PB also increased more than that of the control. In contrast, in the oocytes injected with Taxol before the spindle formation, chromosome capture and alignment were inhibited. These oocytes did not form PB, but only a bulge at the cell cortex was occasionally observed. Moreover, in the oocytes injected with Taxol before GV breakdown, the chromosomes did not gather in one place, and then two asters were observed at distant positions from the cell cortex. These results suggested that MTs lost not only the ability to obtain the bipolar attachment of chromosomes by Taxol injection but also the aster closer to the cell cortex lost its interaction with the cell cortex of the animal pole. © 2012 Wiley Periodicals, Inc.

Recent work shows that cytokinesis and other cellular morphogenesis events are tuned by an interplay among biochemical signals, cell shape, and cellular mechanics. In cytokinesis, this includes cross-talk between the cortical cytoskeleton and the mitotic spindle in coordination with cell cycle control, resulting in characteristic changes in cellular morphology and mechanics through metaphase and cytokinesis. The changes in cellular mechanics affect not just overall cell shape, but also mitotic spindle morphology and function. This review will address how these principles apply to oocytes undergoing the asymmetric cell divisions of meiosis I and II. The biochemical signals that regulate cell cycle timing during meiotic maturation and egg activation are crucial for temporal control of meiosis. Spatial control of the meiotic divisions is also important, ensuring that the chromosomes are segregated evenly and that meiotic division is clearly asymmetric, yielding two daughter cells - oocyte and polar body - with enormous volume differences. In contrast to mitotic cells, the oocyte does not undergo overt changes in cell shape with its progression through meiosis, but instead maintains a relatively round morphology with the exception of very localized changes at the time of polar body emission. Placement of the metaphase-I and -II spindles at the oocyte periphery is clearly important for normal polar body emission, although this is likely not the only control element. Here, consideration is given to how cellular mechanics could contribute to successful mammalian female meiosis, ultimately affecting egg quality and competence to form a healthy embryo.