Embryonic Developmental Potential Research Articles

Effects of Eicosapentaenoic Acid during In Vitro Maturation of Porcine Oocytes: Hormone Synthesis and Embryonic Developmental Potential

Effects of Eicosapentaenoic Acid during In Vitro Maturation of Porcine Oocytes: Hormone Synthesis and Embryonic Developmental Potential

Effects of Essential Fatty Acids during In Vitro Maturation of Porcine Oocytes: Hormone Synthesis and Embryonic Developmental Potential

Omega-3 α-linolenic acid and omega-6 linoleic acid are essential fatty acids for health maintenance of human and animals because they are not synthesized in vivo. The purpose of this study was to evaluate the effect of α-linolenic acid and linoleic acid supplementation on in vitro maturation and developmental potential of porcine oocytes. Various concentrations of α-linolenic acid and linoleic acid were added into in vitro maturation medium, and we evaluated the degree of cumulus expansion, oocyte nuclear-maturation rate, blastocyst rate, blastocyst quality, and levels of prostaglandin E2, 17b-estradiol, and progesterone in the spent medium. High doses (100 μM) of α-linolenic acid and linoleic acid supplementation significantly inhibited cumulus expansion and oocyte nuclear maturation, and prostaglandin E2 synthesis also significantly decreased compared with other groups (p < 0.05). Supplementation of 50 μM α-linolenic acid and 10 μM linoleic acid showed higher quality blastocysts in terms of high cell numbers and low apoptosis when compared with other groups (p < 0.05), and synthesis ratio of 17b-estradiol / progesterone also significantly increased compared with control group (3.59 ± 0.22 vs. 2.97 ± 0.22, 3.4 ± 0.28 vs. 2.81 ± 0.19, respectively; p < 0.05). Our results indicated that supplementation with appropriate levels of α-linolenic acid and linoleic acid beneficially affects the change of hormone synthesis (in particular, an appropriate increase in the 17b-estradiol / progesterone synthesis ratio) for controlling oocyte maturation, leading to improved embryo quality. However, high doses of α-linolenic acid and linoleic acid treatment results in detrimental effects.

Improvement of embryonic development and clinical outcomes of germinal vesicle stage oocytes using a microvibration culture system

ABSTRACTIn vitro maturation (IVM) has evolved as a clinical treatment option in assisted reproductive technology. However, the poor developmental potential of germinal vesicle (GV)-stage oocytes is still suboptimal. This study’s objective was to evaluate the effect of a microvibration culture system (MVC) during IVM and/or in vitro culture (IVC) on the clinical outcomes and the embryonic development potential of human GV-stage oocytes collected from human chorionic gonadotropin (HCG)-primed IVM and fertilization-embryo transfer (IVM/F-ET) cycles of patients with polycystic ovaries (PCO). A total of 206 HCG-primed IVM/F-ET cycles were divided into four groups according to the microvibration and static culture system applied during IVM and/or IVC: Group SS (static system during both IVM and IVC); Group SV (static system during IVM alternated with microvibration system during IVC); Group VS (microvibration system during IVM alternated with static system during IVC), and Group VV (microvibration system during both IVM and IVC). The results indicate that the rates of in vitro MII oocytes per cycle, fertilization, and cleavage were not significantly different between the groups. The rate of good-quality embryos in Group SV tended to be higher than the rate in Groups SS and VS, but there was no significant difference between Group SS and Group SV. Clinical pregnancy, implantation, and live birth rates of Groups SV and VS were slightly higher than those of Group SS. However, the rate of good-quality embryos with at least six cells on day 4, the clinical pregnancy, implantation, and live births in Group VV were significantly higher than those in Group SS. These results indicate that, compared with the static culture system, the MVC system applied for both IVM and IVC seems to improve the clinical outcomes and the quality of embryos of GV oocytes derived from HCG-primed IVM/F-ET cycles in PCO patients.Abbreviations: PCO: polycystic ovaries; HCG: human chorionic gonadotropin; GV: germinal vesicle; MII: metaphase II; IVM: in vitro maturation; IVF: in vitro fertilization; IVC: in vitro culture: MVC: microvibration culture; SC: static culture; ICSI: intracytoplasmic sperm injection; IVM/F-ET: IVM and fertilization-embryo transfer; AMH: anti-Mullerian hormone; OHSS: ovarian hyperstimulation syndrome

The protective effect of vitamin C on phenylhydrazine-induced hemolytic anemia on sperm quality and in-vitro embryo development in mice.

BackgroundPhenylhydrazine (PHZ) induced anemia and was shown to have harmful effects on the male reproductive system. ObjectiveTo investigate the protective effect of vitamin C (Vit C) on sperm parameters quality, in vitro fertilization potential and embryonic development in a mouse model of hemolytic anemia induced by PHZ.Materials and MethodsThirty-two NMRI adult male mice (n = 8/each) were randomly classified into four groups. Group I (control) received normal saline, Group II (PHZ) received 8 mg/100 gr body weight PHZ as initial dose, continued by 6 mg/100 gr intraperitoneally every 48 hr, Group III (Vit C) received Vit C (10 mg/kg, daily, intraperitoneally), and group IV (PHZ + Vit C) received PHZ and Vit C. After 35 days, sperm quality parameters, the percentage of sperm with DNA damage and in vitro fertilization outcomes up to blastocyst stage were evaluated.ResultsA significant (p 0.001) reduction in all of the sperm parameters (count, motility, viability and normal morphology) were observed in group II (PHZ) compared with group I (control). In group IV (PHZ Vit C), these parameters and sperm DNA damage (p 0.001) improved significantly when compared with PHZ-treated mice. Furthermore, PHZ caused a significant (p 0.001) decrease in the fertilization rate and the percentage of pre-implantation embryos' (two cell embryo and blastocyst) formation in comparison to group I (control), and Vit C supplementation in mice of group IV improved significantly the fertilization rate (p = 0.002), but it could not improve the percentage of two cell embryos and blastocyst production.ConclusionThe data from this study indicated that Vit C decreased the adverse effects of PHZ on the quality of sperm parameters and in vitro fertilization rate, but it is insufficient to restore the in-vitro embryonic development and fertility potential.

Using time-lapse technology to explore vacuolization in embryos on Day 3 and Day 4.

To investigate the occurrence and development state of embryo vacuoles between the 8-cell and morula stages, and to explore how vacuoles affected the development of embryos. A retrospective study of a cohort of 422 patients undergoing conventional in vitro fertilization or intracytoplasmic sperm injection. With the help of time-lapse imaging, the development processes and outcomes of good quality embryos with or without vacuoles were analyzed. Vacuole positive embryos had significantly lower blastulation rate and good quality blastulation rate than vacuole negative embryos, p < 0.05. Compared to vacuole negative embryos, the number of best and good quality blastocysts was significantly reduced, while the number of fair and discarded ones was significantly increased, p < 0.05. The average starting time of vacuolization was 73.7 ± 9.3h after insemination. The proportion of blastomeres affected by vacuoles was associated with embryonic developmental potential. Vacuolization on Day 3 and Day 4 was frequently observed and was detrimental to embryo development. The proportion of blastomeres affected by vacuoles may be an indicator of embryo developmental potential.

Mitochondrial Dynamics Is Critical for the Full Pluripotency and Embryonic Developmental Potential of Pluripotent Stem Cells

While the pluripotency of stem cells is known to determine the fate of embryonic development, the mechanisms underlying the acquisition and maintenance of full pluripotency largely remain elusive. Here, we show that the balance between mitochondrial fission and fusion is critical for the full pluripotency of stem cells. By analyzing induced pluripotent stem cells with differential developmental potential, we found that excess mitochondrial fission is associated with an impaired embryonic developmental potential. We further uncover that the disruption of mitochondrial dynamics impairs the differentiation and embryonic development of pluripotent stem cells; most notably, pluripotent stem cells that display excess mitochondrial fission fail to produce live-born offspring by tetraploid complementation. Mechanistically, excess mitochondrial fission increases cytosolic Ca2+ entry and CaMKII activity, leading to ubiquitin-mediated proteasomal degradation of β-Catenin protein. Our results reveal a previously unappreciated fundamental role for mitochondrial dynamics in determining the full pluripotency and embryonic developmental potential of pluripotent stem cells.

Cumulus-oocyte interactions and programmed cell death in bovine embryos produced in vitro

This study investigated the interactions between cumulus cells (CCs) and oocytes and programmed cell death in bovine cumulus-oocyte complexes (COCs) with different morphological characteristics. DNA fragmentation was assessed in CCs at 0 and 24 h of maturation, as well as parthenogenetic developmental competence on the 9th day post-activation, blastocyst quality and BCL-2 and BAX transcript levels in matured CCs. Most immature oocytes in the COC-A group (full cumulus and several compact layers) were in the initial germinal vesicle (iGV) stage, exhibiting minimal or no DNA damage. In contrast, after follicle removal, the COCB (partial cumulus and one or two cell layers) and C (expanded cumulus) groups presented in more advanced GV stages and exhibited DNA fragmentation. After maturation, significant increases in fragmented nuclei were noted in COCC and COCB groups. Embryos resulting from the COC-A developed more rapidly and had increased competence compared to embryos resulting from groups COCB and COCC. The COCB group exhibited the highest BAX protein levels and a reduced BCL-2/BAX protein ratio. The results show a negative correlation between nuclear fragmentation and embryonic development potential in COCs with different morphologies. In addition, a low BCL-2/BAX protein ratio might be associated with an increase in nuclear fragmentation in CCs.

Spent embryo culture medium metabolites are related to the in vitro attachment ability of blastocysts

The metabolomic profile of an embryo culture medium can aid in the advanced prediction of embryonic developmental potential and genetic integrity. But it is not known if this technology can be used to determine the in vitro potential of inner cell mass (ICM) in adherence and proliferation. Here, we investigated the developmental potential of mouse 2-cell embryos carrying cisplatin-induced DNA lesions (IDL), beyond blastocyst stage using ICM outgrowth assay. The genetic integrity of ICM cells was determined by comet assay. The metabolic signatures of spent medium were recorded 84 hours post injection of hCG (hpi-hCG), and after 96 hours of extended in vitro culture (Ex 96) by NMR spectroscopy. We observed that blastocysts that lack the ability to adhere in vitro had an increased requirement of pyruvate (p < 0.01), lactate (p < 0.01), and were accompanied by a significant reduction of pyruvate-alanine ratio in the culture medium. We propose that the aforementioned metabolites from 84 hpi-hCG spent medium be further explored using appropriate experimental models, to prove their potential as biomarkers in the prediction of implantation ability of in vitro derived human embryos in clinical settings.

Simulated physiological oocyte maturation has side effects on bovine oocytes and embryos.

Oocyte maturation is a complex process involving nuclear and cytoplasmic modulations, during which oocytes acquire their ability to become fertilized and support embryonic development. The oocyte is apparently "primed" for maturation during its development in the dominant follicle. As bovine oocytes immediately resume meiosis when cultured, it was hypothesized that delaying resumption of meiosis with cyclic nucleotide modulators before in vitro maturation (IVM) would allow the oocytes to acquire improved developmental competence. We tested the Simulated Physiological Oocyte Maturation (SPOM) system that uses forskolin and 3-isobutyl-1-methylxanthine for 2h prior to IVM against two different systems of conventional IVM (Con-IVM). We evaluated the ultrastructure of matured oocytes and blastocysts and also assessed the expression of 96 genes related to embryo quality in the blastocysts. In summary, the SPOM system resulted in lower blastocyst rates than both Con-IVM systems (30 ± 9.1 vs. 35 ± 8.7; 29 ± 2.6 vs. 38 ± 2.8). Mature SPOM oocytes had significantly increased volume and number of vesicles, reduced volume and surface density of large smooth endoplasmic reticulum clusters, and lower number of mitochondria than Con-IVM oocytes. SPOM blastocysts showed only subtle differences with parallel undulations of adjacent trophectoderm plasma membranes and peripherally localized ribosomes in cells of the inner cell mass compared with Con-IVM blastocysts. SPOM blastocysts, however, displayed significant downregulation of genes related to embryonic developmental potential when compared to Con-IVM blastocysts. Our results show that the use of the current version of the SPOM system may have adverse effects on oocytes and blastocysts calling for optimized protocols for improving oocyte competence.

The activated DNA double-strand break repair pathway in cumulus cells from aging patients may be used as a convincing predictor of poor outcomes after in vitro fertilization-embryo transfer treatment.

Women with advanced maternal age exhibit low anti-Müllerian hormone (AMH) levels and an altered follicular environment, which is associated with poor oocyte quality and embryonic developmental potential. However, the underlying mechanism is poorly understood. The present study aimed to assesswhether aging patients exhibit an activated DNA double-strandbreak (DSB) repair pathway in cumulus cells and thus, an association with poor outcomes after in vitro fertilization-embryo transfer (IVF-ET) treatment. Cumulus cells from young (≤29 y) and aging (≥37 y) human female patients were collected after oocyte retrieval. Our results indicated that aging patients showed a higher rate of γ-H2AX-positive cells than in young patients (24.33±4.55 vs.12.40±2.31, P<0.05). We also found that the mRNA expression levels of BRCA1, ATM, MRE11 and RAD51 were significantly elevated in aging cumulus cells. Accordingly, significantly increased protein levels of phospho-H2AX, BRCA1, ATM, MRE11 and RAD51 could be observed in aging cumulus cells. Moreover, aging cumulus cells showed a more frequent occurrence of early apoptosis than young cumulus cells. This study found that increases in DSBs and the activation of the repair pathway are potential indicators that may be used to predictoutcomes after IVF-ET treatment.

Meiotic spindle position and second polar body orientation enable the prediction of embryonic developmental potential and embryo euploidy

Meiotic spindle position and second polar body orientation enable the prediction of embryonic developmental potential and embryo euploidy

Non-invasive metabolomic profiling of culture media of ICSI- and IVF-derived early developmental cattle embryos via Raman spectroscopy

The aim of the present study was to compare differences in composition between in vitro cultured early developmental embryos resulting from either in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Non-invasive metabolomic profiling of culture media was conducted with laser tweezer Raman spectroscopy (LTRS), providing molecular information that was used to aid the diagnosis or treatment of embryos that were adversely affected by ICSI treatment, ultimately improving the ICSI embryonic developmental potential. Cattle embryos were generated via ICSI and IVF with development to the 2-, 4-, 8-, 16-,32-cell, and blastocyst stages with individual in vitro culturing occurring for 4 h. The culture media for embryos in different developmental stages were separately analyzed using LTRS. The resulting composition of culture media used for culturing IVF- and ICSI-derived embryos was mainly altered in contents of carbohydrates, lipids, DNA, and proteins. Bands at 1004 cm−1 (phenylalanine) and 1529 cm−1 (-C = C-carotenoid) had specific patterns related to the metabolicactivity of embryos; using LTRS, and these may be considered as biomarkers for embryonic development. Furthermore, the vibrations of lipids at different stages increased more with assessment of ICSI culture media than in IVF media. Discriminant function analysis can be utilized for the classification of culture media used for culture of ICSI- and IVF-derived embryos. In conclusion, LTRS can be used for development of an independent assay to assess embryo status during both ICSI and IVF procedures, which provides novel insights into differences in structure and components of single cells.

Zygotic Fluorescence Recovery After Photo-bleaching Analysis for Chromatin Looseness That Allows Full-term Development.

Live imaging is a powerful tool that allows for the analysis of molecular events during ontogenesis. Recently, chromatin looseness or openness has been shown to be involved in the cellular differentiation potential of pluripotent embryonic stem cells. It was previously reported that compared with embryonic stem cells, zygotes harbor an extremely loosened chromatin structure, suggesting its association with their totipotency. However, until now, it has not been addressed whether this extremely loosened/open chromatin structure is important for embryonic developmental potential. In the present study, to examine this hypothesis, an experimental system in which zygotes that were analyzed by fluorescence recovery after photo-bleaching can develop to term without any significant damage was developed. Importantly, this experimental system needs only a thermos-plate heater in addition to a confocal laser scanning microscope. The findings of this study suggest that fluorescence recovery after photo-bleaching analysis (FRAP) analysis can be used to investigate whether the molecular events in zygotic chromatin are important for full-term development.

Resveratrol promotes the embryonic development of vitrified mouse oocytes after in vitro fertilization.

Vitrification of oocytes is closely associated with the lower embryonic developmental potential, which involves the cryopreservation injury occurred during vitrification. It indicates that vitrification may need to be further optimized. Therefore, we studied the effects of resveratrol, an antioxidant, on the developmental potential of vitrified mouse oocytes after in vitro fertilization. After adding a series of concentrations of resveratrol (0, 1, 10, 25, and 50μM) into vitrification, warming, and post-warming mediums, we found that 25 and 50μM resveratrol increased the blastocyst formation rate of vitrified oocytes. We further showed that 25μM resveratrol increased the mean cell numbers of blastocyst from vitrified oocytes. 25 μM resveratrol reduced oxidative stress of vitrified oocytes through decreasing the levels of reactive oxygen species (ROS) and increasing the levels of glutathione (GSH), and 25μM resveratrol alleviated the abnormal mitochondrial distribution pattern of oocytes after vitrification. In conclusion, our study implied that resveratrol could diminish the cryopreservation injuries during the vitrification of mouse oocytes and further confirmed that resveratrol may be an effective antioxidant to optimize vitrification.

Strontium fails to induce Ca2+ release and activation in human oocytes despite the presence of functional TRPV3 channels.

STUDY QUESTIONAre the transient receptor potential cation channels vanilloid 3 (TRPV3) present and able to mediate strontium (Sr2+) induced artificial activation in human oocytes?SUMMARY ANSWERSr2+ did not induce Ca2+ rises or provoke activation in human oocytes, however, mRNA for the TRPV3 channel was present in metaphase II (MII) human oocytes after IVM and TRPV3 agonists induced Ca2+ rises and oocyte activation, demonstrating the channels were functional.WHAT IS KNOWN ALREADYSelective activation of TRPV3 by agonists induces Ca2+ entry and promotes mouse oocyte activation, and the absence of TRPV3 channels in mouse oocytes prevents Sr2+ mediated artificial activation. Sr2+ is sometimes used to overcome fertilization failure after ICSI in the clinic, but its efficiency is still controversial and the mechanism(s) of how it mediates the Ca2+ flux has not been studied yet in human.STUDY DESIGN, SIZE, DURATIONThe protein distribution (n = 10) and mRNA expression level (n = 19) of the TRPV3 channels was investigated in human MII oocytes after IVM. The Sr2+ (10 mM) and TRPV3 agonists (200 μM 2-aminoethoxydiphenyl borate [2-APB] and 200 μM carvacrol)-induced Ca2+ response was analyzed in human (n = 15, n = 16 and n = 16, respectively) and mouse oocytes (n = 15, n = 19 and n = 26, respectively). The subsequent embryonic developmental potential following the parthenogenetic activation using these three agents was recorded in human (n = 10, n = 9 and n = 9, respectively) and mouse (n = 20 per agent) oocytes, by determining pronucleus, or 2-cell and blastocyst formation rates.PARTICIPANTS/MATERIALS, SETTING, METHODSMII oocytes from B6D2F1 mice (6–10 weeks old) as well as human IVM oocytes and IVO oocytes (from patients aged 25–38 years old) with aggregates of smooth endoplasmic reticulum clusters were used. The expression of TRPV3 channels was determined by immunofluorescence staining with confocal microscopy and RT-PCR, and the temporal evolution of intracellular Ca2+ concentration was measured by time-lapse imaging after exposure to Sr2+ and TRPV3 agonists (2-APB and carvacrol). Artificial activation efficiency was assessed using these agents.MAIN RESULTS AND THE ROLE OF CHANCESr2+ did not promote Ca2+ oscillations or provoke activation in human oocytes. Transcripts of TRPV3 channels were present in IVM MII human oocytes. TRPV3 protein was expressed and distributed throughout the ooplasm of human oocytes, rather than particularly concentrated in plasma membrane as observed in mouse MII oocytes. Both agonists of TRPV3 (2-APB and carvacrol), promoted a single Ca2+ transient and activated a comparable percentage of more than half of the exposed human oocytes (P > 0.05). The agonist 2-APB was also efficient in activating mouse oocytes, however, significantly fewer mouse oocytes responded to carvacrol than 2-APB in both the Ca2+ analysis and activation test (P < 0.001).LIMITATIONS REASONS FOR CAUTIONThe availability of fresh IVO matured oocytes in human was limited. Data from TRPV3 knockout model are not included.WIDER IMPLICATIONS OF THE FINDINGSThe benefit of clinical application using Sr2+ to overcome fertilization failure after ICSI requires further validation.STUDY FUNDING/COMPETING INTEREST(S)This study was supported by FWO-Vlaanderen, China Scholarship Council and Special Research Fund from Ghent University (Bijzonder Onderzoeksfonds, BOF). No competing interests are declared.

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.

Effect of Alpha Lipoic Acid on in vitro Maturation of Porcine Oocytes and Subsequent Embryonic Development after Parthenogenetic Activation

Alpha lipoic acid (ALA) is a biological membranes compound. As the antioxidant, it decreases the oxidized forms of other antioxidant substances such as vitamin C, vitamin E, and glutathione (GSH). To examine the effect of ALA on the in vitro maturation (IVM) of porcine oocytes, we investigated intracellular GSH and reactive oxygen species (ROS) levels, and subsequent embryonic development after parthenogenetic activation (PA). Intracellular GSH levels in oocytes treated with 50uM ALA increased significantly (P < 0.05) and exhibited a significant (P < 0.05) decrease in intracellular ROS levels compared with the control group. Oocytes matured with 50 uM of ALA during IVM displayed significantly higher cleavage rates (67.8% vs. 83.4%, respectively), and higher blastocyst formation rates and total cell number of blastocysts after PA (31.6%, 58.49 vs. 46.8%, 68.58, respectively) than the control group. In conclusion, these results suggest that treatment with ALA during IVM improves the cytoplasmic maturation of porcine oocytes by increasing the intracellular GSH levels, thereby decreasing the intracellular ROS levels and subsequent embryonic developmental potential of PA.

157 Sodium Fluoride Exposure Exerts Toxic Effects on Porcine Oocyte Maturation

Excessive long-term fluoride intake is associated with several health problems, including infertility. However, limited information is available on the toxic effects of fluoride exposure on the female reproductive system, especially oocyte maturation. In this study, we investigated the toxic effect of sodium fluoride (NaF) exposure on porcine oocyte maturation and its possible underlying mechanisms. The level of reactive oxygen species, glutathione (GSH), mitochondrial membrane potential, apoptosis, and DNA damage and cathepsin B in the blastocysts were observed by staining with 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA), 4-chloromethyl-6.8-difluoro-7-hydroxycoumarin (CMF2HC, Invitrogen, Carlsbad, CA, USA), 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1), annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit, anti-γH2A.X (Ser139) and Magic Red cathepsin B assay kit, respectively. Data obtained from the experimental groups were compared using Student’s t-test. Our results showed that treatment with 60, 100, and 150 μg mL−1 NaF significantly decreased the degree of cumulus cell expansion (CEI: 2.64 ± 0.27, 1.32 ± 0.52, and 0.57 ± 0.15 v. 3.58 ± 0.19; P &lt; 0.05) and the rate of maturation (74.33% ± 4.32%, 57.83% ± 7.14%, 44.00% ± 5.93% v. 84.50% ± 3.62%; P &lt; 0.05) in a dose-dependent manner during in vitro maturation for 44 h. Cell cycle analysis showed that NaF exposure blocked meiotic resumption, disturbed spindle dynamics, disrupted chromosome separation, and increased aneuploidy in porcine oocytes. Moreover, NaF exposure disturbed mitochondrial function, triggered DNA damage response, and induced early apoptosis in porcine oocytes. Exposure to NaF also induced oxidative stress, decreased GSH level, and increased cathepsin B activity in and impaired the further development potential of porcine oocytes, as indicated by a decrease in blastocyst formation rate, increase in apoptosis, and inhibition of cell proliferation. Together, these results indicate that NaF exposure impairs the maturation capacity of porcine oocytes by inhibiting cumulus cell expansion, disturbing cytoskeletal dynamics, and blocking nuclear and cytoplasmic maturation, thus decreasing the quality and affecting the subsequent embryonic development potential of porcine oocytes.

Sodium fluoride exposure exerts toxic effects on porcine oocyte maturation

Excessive long-term fluoride intake is associated with several health problems, including infertility. However, limited information is available on the toxic effects of fluoride exposure on the female reproductive system, especially oocyte maturation. In this study, we investigated the toxic effect of sodium fluoride (NaF) exposure on porcine oocyte maturation and its possible underlying mechanisms. Our results showed that NaF exposure during porcine oocyte maturation inhibited cumulus cell expansion and impaired polar body extrusion. Cell cycle analysis showed that NaF exposure blocked meiotic resumption, disturbed spindle dynamics, disrupted chromosome separation, and increased aneuploidy in porcine oocytes. Moreover, NaF exposure disturbed mitochondrial function, triggered DNA damage response, and induced early apoptosis in porcine oocytes. NaF exposure also induced oxidative stress, decreased GSH level, and increased cathepsin B activity in and impaired the further development potential of porcine oocytes, as indicated by a decrease in blastocyst formation rate, increase in apoptosis, and inhibition of cell proliferation. Together, these results indicate that NaF exposure impairs the maturation capacity of porcine oocytes by inhibiting cumulus cell expansion, disturbing cytoskeletal dynamics, and blocking nuclear and cytoplasmic maturation, thus decreasing the quality and affecting the subsequent embryonic development potential of porcine oocytes.

Abnormal cleavage patterns in embryos are associated with aneuploidy and poor morphology scores

Morphokinetic parameters as measured through time-lapse imaging may predict embryonic developmental potential. While there are several studies on the timing of early cell divisions, there are limited data on abnormal cleavage (AC) events and their relationship to embryo quality. An AC is the division from a mother blastomere cell to three daughter cells. This is thought to be secondary to abnormal mitotic spindle assembly, leading to an abnormal segregation of chromosomes. Previous studies report that embryos exhibiting ACs have decreased blastulation and worse implantation rates. In our study, we sought to determine if ACs are associated with aneuploidy. We also evaluated the relationship between ACs and developmental potential as well as day 3 and day 5 morphologic scoring. Our primary objective was to determine if ACs are related to ploidy status. Secondary objectives were to determine if ACs are correlated with blastulation rates and with embryo morphologic scores at the cleavage and blastocyst stages. Previously cryopreserved human embryos donated for research were thawed and cultured in the Miri Time-lapse incubator for 6 days. A staff embryologist recorded standard time-lapse parameters and day 3 and 5 morphology assessments. Trophectoderm biopsies were performed on embryos that grew to blastocysts on day 5 or 6. Otherwise whole embryos were sent for preimplantation genetic screening (PGS). Next generation sequencing was used to determine embryo ploidy status. Morphologic grading of embryos was performed per Society of Assisted Reproductive Technology guidelines for cleavage stage embryos. We used methods for blastocyst morphologic grading described by Gardner, et al with minor modifications. Statistics were performed using Chi-square, Fisher’s Exact, ANOVA, and logistic regression. 50 embryos were included our analysis. 32/50 (64%) of thawed embryos grew to the blastocyst stage. 35/50 (70%) of embryos were euploid. ACs were visualized in 15/50 (30%) embryos. 8/15 (53.3%) of these embryos experienced an AC after the 1st cytokinesis. 7/15 (46.7%) of embryos had ACs after the 2nd cytokinesis. 4/15 (26.7%) embryos had more than 1 AC event. Aneuploid embryos had a significantly higher percentage of AC events (73.3% vs 22.9%, p<0.001). AC events did not reliably predict which embryos grew to the blastocyst stage or those that arrested. Embryos that had at least 1 AC took an average of 10.7 hours longer to develop from a morula to a fully expanded blastocyst compared to those without ACs (β=10.7, p=0.01). Cleavage stage morphology scores did not correlate with ACs. In blastocysts with ACs, there was a higher proportion of poor quality embryos compared with good/fair embryos (66.7% vs 33.3%, p=0.03). Based on our findings, ACs were more likely to occur in aneuploid embryos. While they did not predict blastocyst formation, they were associated with delayed blastulation and with poor blastocyst morphology scores. When selecting embryos for transfer, particularly those not undergoing PGS, those with ACs may have a worse prognosis for achieving a successful pregnancy.