Expression profile of viability and stress response genes as a result of resveratrol supplementation in vitrified and in vitro produced cattle embryos.

BACKGROUND: Resveratrol, a potent antioxidant, is known to induce the up-regulation of the internal antioxidant system. Therefore, it holds promise as a method to mitigate cryopreservation-induced injuries in bovine oocytes and embryos. This study aimed to (i) assess the enhancement in the quality of in vitro produced bovine embryos following resveratrol supplementation and (ii) monitor changes in the expression of genes associated with oxidative stress (GPX4, SOD, CPT2, NFE2L2), mitochondrial function (ATP5ME), endoplasmic reticulum function (ATF6), and embryo quality (OCT4, DNMT1, CASP3, ELOVL5). METHODS AND RESULTS: Three groups of in vitro bovine embryos were cultured with varying concentrations of resveratrol (0.01, 0.001, and 0.0001 µM), with a fourth group serving as a control. Following the vitrification process, embryos were categorized as either good or poor quality. Blastocysts were then preserved at - 80 °C for RNA isolation, followed by qRT-PCR analysis of selected genes. The low concentrations of resveratrol (0.001 µM, P < 0.05 and 0.0001 µM, P < 0.01) significantly improved the blastocyst rate compared to the control group. Moreover, the proportion of good quality vitrified embryos increased significantly (P < 0.05) in the groups treated with 0.001 and 0.0001 µM resveratrol compared to the control group. Analysis of gene expression showed a significant increase in OCT4 and DNMT1 transcripts in both good and poor-quality embryos treated with resveratrol compared to untreated embryos. Additionally, CASP3 expression was decreased in treated good embryos compared to control embryos. Furthermore, ELOVL5 and ATF6 transcripts were down-regulated in treated good embryos compared to the control group. Regarding antioxidant-related genes, GPX4, SOD, and CPT2 transcripts increased in the treated embryos, while NFE2L2 mRNA decreased in treated good embryos compared to the control group. CONCLUSIONS: Resveratrol supplementation at low concentrations effectively mitigated oxidative stress and enhanced the cryotolerance of embryos by modulating the expression of genes involved in oxidative stress response.

The effect of coenzyme Q10 on cryotolerance of in vivo-derived mouse embryos.

OBJECTIVE: Cryopreservation has some adverse effects on embryos including cell metabolism reduction, mitochondria and plasma membrane damage, excess production of 'Reactive Oxygen Species' and damage to DNA. In the present study. In this study we assessed the effect of coenzyme Q10 as an exogenous antioxidant on mouse embryos following cryopreservation. METHODS: We collected mice embryos at the morula stage from uterine horns on the third day of gestation. The morulae were divided into 9 groups (1 control, 2 vehicles and 6 experimental), then vitrified. The culture and/or vitrification media of the experimental groups were supplemented by 10 or 30 µM of CoQ10. After one week, the embryos were warmed and then cultured. After 48 hours of embryo culture, the blastocyst rate, total cell number, viability; and after 72 hours of embryo culture, we assessed the hatching rate. RESULTS: Blastocyst rate and hatching rate were significantly reduced in the groups containing 30 µM CoQ10 supplemented culture media compared to other groups (p<0.05). The hatching rate in the groups containing 10 µM CoQ10 supplemented in both culture and vitrification media was significantly higher than in the other groups (p<0.05). In groups containing 10 µM CoQ10 supplemented culture media, the viability was higher than that in the other groups (p<0.05). CONCLUSIONS: It seems that CoQ10 in a dose-dependent manner is able to improve hatching rate and viability following cryopreservation through its antioxidant and anti-apoptotic properties, and through the production of ATP.

Chlorogenic acid improves the development of porcine parthenogenetic embryos by regulating oxidative stress and ameliorating mitochondrial function.

Chlorogenic acid (CGA) is an effective phenolic antioxidant that can scavenge hydroxyl radicals and superoxide anions. Herein, the protective effects and mechanisms leading to CGA-induced porcine parthenogenetic activation (PA) in early-stage embryos were investigated. Our results showed that 50 µM CGA treatment during the in vitro culture (IVC) period significantly increased the cleavage and blastocyst formation rates and improved the blastocyst quality of porcine early-stage embryos derived from PAs. Then, genes related to zygotic genome activation (ZGA) were identified and investigated, revealing that CGA can promote ZGA in porcine PA early-stage embryos. Further analysis revealed that CGA treatment during the IVC period decreased the abundance of reactive oxygen species (ROS), increased the abundance of glutathione and enhanced the activity of catalase and superoxide dismutase in porcine PA early-stage embryos. Mitochondrial function analysis revealed that CGA increased mitochondrial membrane potential and ATP levels and upregulated the mitochondrial homeostasis-related gene NRF-1 in porcine PA early-stage embryos. In summary, our results suggest that CGA treatment during the IVC period helps porcine PA early-stage embryos by regulating oxidative stress and improving mitochondrial function.

Treatment of mares with the non-steroidal anti-inflammatory drug (NSAID) phenylbutazone transiently affects in vitro maturation of equine oocytes and blastocyst development after Intracytoplasmic Sperm Injection (ICSI).

Mares enrolled in assisted reproductive technologies (ARTs) programs are often treated with non-steroidal anti-inflammatory drugs (NSAIDs), particularly phenylbutazone (Bute), due to chronic lameness. The current study was performed to determine the effect of Bute administration on the developmental competence of in vitro-matured equine oocytes subjected to Intracytoplasmic Sperm Injection (ICSI). In a Preliminary Study, immature cumulus-oocyte complexes (COCs) recovered by post-mortem ovary harvested from two healthy mares (n = 2) treated for 10 days with Bute (4.4 mg/kg, PO, BID), and four non-treated healthy mares (n = 4), were matured in vitro and subjected to Piezo-driven ICSI. Lower oocyte in vitro maturation [Bute: 25% (3/12) vs. Control: 61% (28/46)] and blastocyst rates [Bute: 0% (0/12) vs. Control: 18% (5/28)] were observed in the Bute-treated when compared to the Control mares (P < 0.05). In the Main Experiment, a group of healthy mares (n = 9) received a daily dose of Bute (4.4 mg/kg, orally, SID) for 10 days. A control group of mares (n = 10) was treated with an equal volume of placebo. Mares in both groups were subjected to ultrasound-guided transvaginal oocyte aspiration (TVA) on days 3, 33, and 77 following the last dose of Bute (PT). Recovered COCs from both mare groups were matured in vitro and subjected to Piezo-driven ICSI. By day-3 PT, oocyte in vitro maturation rate was similar between mare groups [Bute: 65% (36/55) vs. Control: 67% (78/116); P > 0.05], while oocyte recovery [Bute: 53% (55/103) vs. Control: 70% (116/166)], cleavage [Bute: 31% (11/36) vs. Control: 62% (48/78)] and blastocyst rates [Bute: [0%] (0/36) vs. Control: 28% (22/78)] were significantly different (P < 0.05). By day 33 PT and 77 PT, differences on oocyte recovery, in vitro maturation, cleavage, and blastocyst rates were not observed between mare groups. In summary, the administration of Bute for 10 consecutive days (4.4 mg/kg, PO, SID, or BID) is associated with a decrease in the ability of immature equine oocytes to undergo in vitro-maturation (Preliminary Study) and develop to the blastocyst stage following ICSI (Preliminary Study and Main Experiment). This negative effect appeared to be transient, as 30- and 77-days post-treatment, no differences on in vitro maturation, cleavage or blastocyst rates were observed.

The effects of a glyphosate-based herbicide on the bovine gametes during an in vitro embryo production model.

Roundup® (R), while it is the most used herbicide globally, and its residues are ubiquitous in urban and suburban areas, its impact on vertebrates' safety remains highly debated. Here, in three in vitro experiments, we investigated the effects of a very low dose (1 ppm) of R on the fertilization capacity and embryo development in cattle. In the first experiment, frozen-thawed bull semen exposed to R for 1 h exhibited reduced motility parameters but unaffected fertilization ability. However, after in vitro fertilization, the rates of embryo formation were significantly lower compared to the untreated controls. In the second experiment, oocytes exposed to R during in vitro maturation showed reduced cleavage rates, and the embryo yield on days 7, 8, and 9 of embryo culture was significantly lower than that of the controls. In the third experiment, oocytes were matured in the presence of R and in a medium containing both R and Zinc, chosen to offer antioxidant protection to the oocytes. Day-7 blastocysts were analyzed for the expression of genes associated with oxidative stress, apoptosis, and epigenetic reprogramming. Exposure to R markedly suppressed embryo formation rates compared to the controls. The combination of R with Zinc restored the blastocyst yield, which on days 8 and 9 was comparable to that of the controls and higher than the groups exposed only to R on all days. The gene expression analysis revealed that R promotes oxidative stress development, triggers apoptosis, and induces epigenetic changes in developing embryos, while zinc presence alleviates these adverse effects of R. These findings imply that even at very low doses, R could be highly toxic, leading to functional abnormalities in both gametes, potentially affecting fertility in both genders.

Mangiferin improves early porcine embryonic development by reducing oxidative stress.

Mangiferin (MGN) is primarily found in the fruits, leaves, and bark of plants of the Anacardiaceae family, including mangoes. MGN exhibits various pharmacological effects, such as protection of the liver and gallbladder, anti-lipid peroxidation, and cancer prevention. This study aimed to investigate the effects of MGN supplementation during in vitro culture (IVC) on the antioxidant capacity of early porcine embryos and the underlying mechanisms involved. Porcine parthenotes in the IVC medium were exposed to different concentrations of MGN (0, 0.01, 0.1, and 1 µM). The addition of 0.1 µM MGN significantly increased the blastocyst formation rate of porcine embryos while reducing the apoptotic index and autophagy. Furthermore, the expression of antioxidation-related (SOD2, GPX1, NRF2, UCHL1), cell pluripotency (SOX2, NANOG), and mitochondria-related (TFAM, PGC1α) genes was upregulated. In contrast, the expression of apoptosis-related (CAS3, BAX) and autophagy-related (LC3B, ATG5) genes decreased after MGN supplementation. These findings suggest that MGN improves early porcine embryonic development by reducing oxidative stress-related genes.

Deoxynivalenol leads to endoplasmic reticulum stress-mediated apoptosis via the IRE1/JNK/CHOP pathways in porcine embryos.

The cytotoxic mycotoxin deoxynivalenol (DON) reportedly has adverse effects on oocyte maturation and embryonic development in pigs. Recently, the interplay between cell apoptosis and endoplasmic reticulum (ER) stress has garnered increasing attention in embryogenesis. However, the involvement of the inositol-requiring enzyme 1 (IRE1)/c-jun N-terminal kinase (JNK)/C/EBP homologous protein (CHOP) pathways of unfolded protein response (UPR) signaling in DON-induced apoptosis in porcine embryos remains unknown. In this study, we revealed that exposure to DON (0.25 µM) substantially decreased cell viability until the blastocyst stage in porcine embryos, concomitant with initiation of cell apoptosis through the IRE1/JNK/CHOP pathways in response to ER stress. Quantitative PCR confirmed that UPR signaling-related transcription factors were upregulated in DON-treated porcine blastocysts. Western blot analysis showed that IRE1/JNK/CHOP signaling was activated in DON-exposed porcine embryos, indicating that ER stress-associated apoptosis was instigated. The ER stress inhibitor tauroursodeoxycholic acid protected against DON-induced ER stress in porcine embryos, indicating that the toxic effects of DON on early developmental competence of porcine embryos can be prevented. In conclusion, DON exposure impairs the developmental ability of porcine embryos by inducing ER stress-mediated apoptosis via IRE1/JNK/CHOP signaling.

Ovarian response and embryo ploidy following oral micronized progesterone-primed ovarian stimulation versus GnRH antagonist protocol. A prospective study with repeated ovarian stimulation cycles.

STUDY QUESTION: Is there any difference in ovarian response and embryo ploidy following progesterone-primed ovarian stimulation (PPOS) using micronized progesterone or GnRH antagonist protocol? SUMMARY ANSWER: Pituitary downregulation with micronized progesterone as PPOS results in higher number of oocytes retrieved and a comparable number of euploid blastocysts to a GnRH antagonist protocol. WHAT IS KNOWN ALREADY: Although the GnRH antagonist is considered by most the gold standard protocol for controlling the LH surge during ovarian stimulation (OS) for IVF/ICSI, PPOS protocols are being increasingly used in freeze-all protocols. Still, despite the promising results of PPOS protocols, an early randomized trial reported potentially lower live births in recipients of oocytes resulting following downregulation with medroxyprogesterone acetate as compared with a GnRH antagonist protocol. The scope of the current prospective study was to investigate whether PPOS with micronized progesterone results in an equivalent yield of euploid blastocysts to a GnRH antagonist protocol. STUDY DESIGN, SIZE, DURATION: In this prospective study, performed between September 2019 to January 2022, 44 women underwent two consecutive OS protocols within a period of 6 months in a GnRH antagonist protocol or in a PPOS protocol with oral micronized progesterone. PARTICIPANTS/MATERIALS, SETTING, METHODS: Overall, 44 women underwent two OS cycles with an identical fixed dose of rFSH (225 or 300 IU) in both cycles. Downregulation in the first cycles was performed with the use of a flexible GnRH antagonist protocol (0.25 mg per day as soon as one follicle of 14 mm) and consecutively, after a washout period of 1 month, control of LH surge was performed with 200 mg of oral micronized progesterone from stimulation Day 1. After the completion of both cycles, all generated blastocysts underwent genetic analysis for aneuploidy screening (preimplantation genetic testing for aneuplody, PGT-A). MAIN RESULTS AND THE ROLE OF CHANCE: Comparisons between protocols did not reveal differences between the duration of OS. The hormonal profile on the day of trigger revealed statistically significant differences between protocols in all the tested hormones except for FSH: with significantly higher serum E2 levels, more elevated LH levels and higher progesterone levels in PPOS cycles as compared with antagonist cycles, respectively. Compared with the GnRH antagonist protocol, the PPOS protocol resulted in a significantly higher number of oocytes (12.7 ± 8.09 versus 10.3 ± 5.84; difference between means [DBM] -2.4 [95% CI -4.1 to -0.73]), metaphase II (9.1 ± 6.12 versus 7.3 ± 4.15; DBM -1.8 [95% CI -3.1 to -0.43]), and 2 pronuclei (7.1 ± 4.99 versus 5.7 ± 3.35; DBM -1.5 [95% CI -2.6.1 to -0.32]), respectively. Nevertheless, no differences were observed regarding the mean number of blastocysts between the PPOS and GnRH antagonist protocols (2.9 ± 2.11 versus 2.8 ± 2.12; DBM -0.07 [95% CI -0.67 to 0.53]) and the mean number of biopsied blastocysts (2.9 ± 2.16 versus 2.9 ± 2.15; DBM -0.07 [95% CI -0.70 to 0.56]), respectively. Concerning the euploidy rates per biopsied embryo, a 29% [95% CI 21.8-38.1%] and a 35% [95% CI 26.6-43.9%] were noticed in the PPOS and antagonist groups, respectively. Finally, no difference was observed for the primary outcome, with a mean number of euploid embryos of 0.86 ± 0.90 versus 1.00 ± 1.12 for the comparison of PPOS versus GnRh antagonist. LIMITATIONS, REASONS FOR CAUTION: The study was powered to detect differences in the mean number of euploid embryos and not in terms of pregnancy outcomes. Additionally, per protocol, there was no randomization, the first cycle was always a GnRH antagonist cycle and the second a PPOS with 1 month of washout period in between. WIDER IMPLICATIONS OF THE FINDINGS: In case of a freeze-all protocol, clinicians may safely consider oral micronized progesterone to control the LH surge and patients could benefit from the advantages of a medication of oral administration, with a potentially higher number of oocytes retrieved at a lower cost, without any compromise in embryo ploidy rates. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by an unrestricted grant from Theramex. N.P.P. has received Research grants from Merck Serono, Organon, Ferring Pharmaceutical, Roche, Theramex, IBSA, Gedeon Richter, and Besins Healthcare; honoraria for lectures from: Merck Serono, Organon, Ferring Pharmaceuticals, Besins International, Roche Diagnostics, IBSA, Theramex, and Gedeon Richter; consulting fees from Merck Serono, Organon, Besins Healthcare, and IBSA. M.d.M.V., F.M., and I.R. declared no conflicts of interest. TRIAL REGISTRATION NUMBER: The study was registered at Clinical Trials Gov. (NCT04108039).

In vitro blastocyst implantation and trophoblast migration are disrupted by the UV filter benzophenone-3 (BP3).

Benzophenone-3 (BP3) is a common ingredient in personal care products (PCPs) due to its well-established effectiveness in absorbing UV radiation. Sunscreen products are among the most widely used PCPs-containing BP3 applied to the skin, resulting in significant human exposure to BP3 primarily through a dermal application. In the present work, we have tested the action of three environmentally relevant concentrations of BP3 (2, 20 and 200 µg/L) on an in vitro model of implantation of murine blastocysts and on migration ability of the human trophoblast cell line Swan 71. We showed that BP3 caused a significant reduction of blastocyst expansion and a delayed hatching in a non-monotonic way. Besides, embryos displayed a delayed attachment in the three BP3 groups, resulting in a smaller implantation area on the 6th day of culture: BP3(2) (0.32 ± 0.07 mm2); BP3(20) (0.30 ± 0.08 mm2) and BP3(200) (0.25 ± 0.06 mm2) in comparison to the control (0.42 ± 0.07 mm2). We also found a reduced migration capacity of the human first-trimester trophoblast cell line Swan 71 in a scratch assay when exposed to BP3: the lowest dose displayed a higher uncovered area (UA) at 6h when compared to the control, whereas a higher UA of the wound was observed for the three BP3 concentrations at 18 and 24 h of exposure. The changes in UA provoked by BP3 restored to normal values in the presence of flutamide, an androgen receptor (AR) inhibitor. These results indicate that a direct impairment on early embryo implantation and a defective migration of extravillous trophoblast cells through the androgen receptor pathway can be postulated as mechanisms of BP3-action on early gestation with potential impact on fetal growth.

In silico-designed vitrification protocols: an approach to improve survival of in vitro produced-bovine embryos.

In brief: In silico predictions validated in this study demonstrate the potential for designing shorter equilibration protocols that improve post-warming re-expansion and hatching rates of D7 and D8 in vitro-produced bovine embryos. Our results benefit the livestock industry by providing a refined and reproducible approach to cryopreserving bovine embryos, which, in addition, could be useful for other mammalian species. Abstract: The cryopreservation of in vitro-produced (IVP) embryos is vital in the cattle industry for genetic selection and crossbreeding programs. Despite its importance, there is no standardized protocol yielding pregnancy rates comparable to fresh embryos. Current approaches often neglect the osmotic tolerance responses to cryoprotectants based on temperature and time. Hereby, we propose improved vitrification methods using shorter dehydration-based protocols. Blastocysts cultured for 7 (D7) or 8 days (D8) were exposed to standard equilibration solution (ES) at 25ºC and 38.5ºC. Optimized exposure times for each temperature and their impact on post-warming re-expansion, hatching rates, cell counts, and apoptosis rate were determined. In silico predictions aligned with in vitro observations, showing original volume recovery within 8 min 30 s at 25ºC or 3 min 40 s at 38.5ºC (D7 blastocysts) and 4 min 25 s at 25ºC and 3 min 15 s at 38.5ºC (D8 blastocysts) after exposure to ES. Vitrification at 38.5ºC resulted in D7 blastocysts re-expansion and hatching rates (93.1% and 38.1%, respectively) comparable to fresh embryos (100.0% and 32.4%, respectively), outperforming the 25ºC protocol (86.2% and 24.4%, respectively; P < 0.05). No differences were observed between D7 and D8 blastocysts using the 38.5ºC protocol. Total cell number was maintained for D7 and D8 blastocysts vitrified at 38.5ºC but decreased at 25ºC (P < 0.05). Apoptosis rates increased post-warming (P < 0.05), except for D8 blastocysts vitrified at 38.5ºC, resembling fresh controls. In conclusion, based on biophysical permeability data, new ES incubation times of 3 min 40 s for D7 blastocysts and 3 min 15 s for D8 blastocysts at 38.5ºC were validated for optimizing vitrification/warming methods for bovine IVP blastocysts.

Impact of media supplements FGF2, LIF and IGF1 on the genome activity of porcine embryos produced in vitro.

In this article, we focused on the impact of precisely chemically modified FLI maturation medium enriched with fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), insulin-like growth factor 1 (IGF1), and polyvinyl alcohol (PVA) and its potential to improve the efficiency of in vitro production of porcine embryos. We hypothesized that enhancing the composition of the maturation medium could result in an elevated production of embryos in vitro and can affect EGA. FLI medium resulted in a significantly higher rate of oocyte blastocyst maturation and formation compared to the control DMEM medium. In addition, immunocytochemical labelling confirmed the detection of UBF in 4-cell FLI parthenogenic embryos, suggesting similarities with natural embryo development. Through RNAseq analysis, upregulated genes present in 4-cell FLI embryos were found to play key roles in important biological processes such as cell proliferation, cell differentiation, and transcriptional regulation. Based on our findings, we demonstrated the positive influence of FLI medium in the evaluation of in vitro embryo production, EGA detection, transcriptomic and proteomic profile, which was confirmed by the positive activation of the embryonal genome in the 4-cell stage of parthenogenetically activated embryos.

Reassessing the impact of letrozole co-administration in controlled ovarian hyperstimulation: findings from a single-center repeated measures study.

PURPOSE: To explore whether letrozole improved outcomes in subsequent controlled ovarian hyperstimulation (COH) cycles. METHODS: This was a retrospective repeated measures cohort study examining COH cycles. Patients were included if they underwent two cycles for unexplained infertility, male factor infertility, or planned oocyte/embryo cryopreservation. The first cycles for all patients implemented a non-letrozole, conventional gonadotropin protocol. Second cycles for the study group included letrozole (2.5-7.5 mg for 5 days) with no medication change to second cycles amongst controls. Our primary objective was to compare oocyte yield. Cohorts were then subdivided by pursuit of oocyte (OC) or embryo (IVF) cryopreservation. Secondary outcome amongst the OC subgroup was oocyte maturation index (metaphase II (MII)/total oocytes). Secondary outcomes amongst the IVF subgroup were normal fertilization rate (2-pronuclear zygotes (2PN)/oocytes exposed to sperm), blastocyst formation rate (blastocysts/2PNs), and embryo ploidy (%euploid and aneuploid). RESULTS: Fifty-four cycles (n = 27) were included in letrozole and 108 cycles (n = 54) were included in control. Oocyte yield was higher in second cycles (p < 0.008) in the letrozole group but similar in second cycles (p = 0.26) amongst controls. Addition of letrozole did not impact MII index (p = 0.90); however, MII index improved in second cycles amongst controls (p < 0.001). Both groups had similar rates of normal fertilization (letrozole: p = 0.52; control: p = 0.61), blast formation (letrozole: p = 0.61; control: p = 0.84), euploid (letrozole: p = 0.29; control: p = 0.47), and aneuploid embryos (letrozole: p = 0.17; control: p = 0.78) between cycles. CONCLUSIONS: Despite improved oocyte yield, letrozole did not yield any difference in oocyte maturation or embryo outcomes.

Differential Gene Regulation of the Human Blastocyst Trophectoderm and Inner Cell Mass by Progesterone.

Knowledge of action of progesterone (P4) on the human preimplantation embryo is lacking. The objective of this study was to determine expression of a mitochondrial P4 receptor (PR-M) in the trophectoderm (TE) and the inner cell mass (ICM) of the human blastocyst and to determine P4-induced gene expression during growth from the cleavage to the blastocyst stage. Previously cryopreserved cleavage stage embryos were treated with P4 (10-6 M) or vehicle until blastocyst development. Cells from the TE and the ICM of dissected euploid embryos underwent RNA-seq analysis, while other embryos were used for analysis of nuclear PR (nPR) and PR-M expression.PR-M expression was confirmed in the TE, the ICM, and a human embryonic stem cell line (HESC). Conversely, nPR expression was absent in the TE and the ICM with low expression in the HESC line. RNA-seq analysis revealed P4 effects greater in the TE with 183 significant pathway changes compared to 27 in the ICM. The TE response included significant upregulation of genes associated with DNA replication, cell cycle phase transition and others, exemplified by a 7.6-fold increase in the cell proliferation gene, F-Box Associated Domain Containing. The majority of ICM pathways were downregulated including chromosome separation, centromere complex assembly and chromatin remodeling at centromere. This study confirms that human blastocysts express PR-M in both the TE and the ICM, but lack expression of nPR. P4-induced gene regulation differs greatly in the two cell fractions with the predominant effect of cell proliferation in the TE and not the ICM.

Oocyte Vitrification Reduces its Capability to Repair Sperm DNA Fragmentation and Impairs Embryonic Development.

Oocytes play a crucial role in repairing sperm DNA damage, which can affect the next generation; however, certain factors can impair this ability. This study examined whether oocyte vitrification, a widely used method for fertility preservation, negatively affects repair ability. Male DBA/2 mice (n = 28) were injected with 101.60 µmol/100 g body weight of tert-Butyl hydroperoxide (tBHP) for 14 days to induce sperm DNA damage. Histological changes, sperm functions, and DNA fragmentation were assessed using the TUNEL assay. Cumulus-oocyte-complexes (COCs) of superovulated female DBA/2 mice (n = 28) were vitrified using the Cryotop method. Fresh and vitrified oocytes were then fertilized by tBHP-treated and untreated sperms, and subsequent embryonic development was monitored. Additionally, the expression of Mre11a, Rad51, Brca1, and Xrcc4 was assessed in resulting zygotes and blastocysts using real-time PCR. The sperm tBHP treatment reduced differentiated spermatogenic cells in the testicular tissue, sperm concentration, and motility, while increasing DNA fragmentation (P < 0.05). The fertilization rate was decreased in the tBHP-treated sperm-vitrified oocyte group (P < 0.05), and the two-cell rate diminished in tBHP-treated sperm-fresh and vitrified oocyte groups (P < 0.05). The four-cell to blastocyst rate decreased in the untreated sperm-vitrified oocyte and the tBHP-treated sperm-fresh and vitrified oocyte groups (P < 0.05), and the tBHP-treated sperm-vitrified oocyte groups had the lowest blastocyst rate. In zygotes, Brca1 was upregulated in the tBHP-treated sperm-vitrified oocyte group (P < 0.05). Also, in blastocysts, Rad51, Brca1, and Xrcc4 were significantly upregulated in the untreated sperm-vitrified oocytes group (P < 0.05). Damages to the oocyte due to vitrification can disrupt the repair of sperm DNA fragmentation and consequently impair the embryo development.

Bisphenol Exposure Disrupts Cytoskeletal Organization and Development of Pre-Implantation Embryos.

The endocrine disrupting activity of bisphenol compounds is well documented, but less is known regarding their impact on cell division and early embryo formation. Here, we tested the effects of acute in vitro exposure to bisphenol A (BPA) and its common substitute, bisphenol F (BPF), during critical stages of mouse pre-implantation embryo development, including the first mitotic division, cell polarization, as well as morula and blastocyst formation. Timing of initial cleavage was determined by live-cell imaging, while subsequent divisions, cytoskeletal organization and lineage marker labeling were assessed by high-resolution fluorescence microscopy. Our analysis reveals that brief culture with BPA or BPF impeded cell division and disrupted embryo development at all stages tested. Surprisingly, BPF was more detrimental to the early embryo than BPA. Notably, poor embryo development was associated with cytoskeletal disruptions of the actomyosin network, apical domain formation during cell polarization, actin ring zippering for embryo sealing and altered cell lineage marker profiles. These results underscore that bisphenols can disrupt cytoskeletal integrity and remodeling that is vital for early embryo development and raise concerns regarding the use of BPF as a 'safe' BPA substitute.

Effect of ultra-low O2 (2%) tension on human in-vitro embryo development.

The study was aimed to investigate the effect of culturing human in-vitro embryos in ultra-low O2 (2%) tension. A total of 2298 oocytes from 152 patients between June 2017 and December 2017 treated with conventional in vitro fertilization (IVF) were harvested in this study. Oocytes were randomly assigned to the low (5%) or ultra-low (2%) O2 tension groups on the retrieval day. We observed that the day 3 good quality embryos (43.32 versus 42.01%; p=.635) and available embryos (82.02 versus 83.47%; p=.490) rates were similar between 2% and 5% condition. No differences were observed in the D5 blastulation rate (62.79 versus 61.85%; p=.735) and the proportion of good quality blastocysts on Day 5 (44.51 versus 45.61%; p=.700), nor in the total blastulation rate (71.26 versus 70.29%; p=.710) between 2% and 5% condition. In the first transfer, the blastocysts had similar clinical pregnancy (68.12 versus 71.08%; p= .692) and ongoing pregnancy (59.42 versus 62.65%; p=.684) rates from 2% and 5% condition. The employ of ultra-low O2 tension did not benefit for human in-vitro embryo development.

GM-CSF perturbs cell identity in mouse pre-implantation embryos.

Growth factors became attractive candidates for medium supplementation to further improve the quality of embryo culture and to mimic in vivo nutrition. Granulocyte macrophage colony-stimulating factor (GM-CSF) is a cytokine influencing the maternal-fetal interface and supporting placental development in mouse and human. It is expressed in epithelial cells of the endometrium under the regulation of estrogens. The factor is already in clinical use and a large clinical trial showed that, if supplemented to an embryo culture medium, it leads to increased survival of embryos, especially in women with previous miscarriages. Animal and cell culture studies on isolated trophectoderm cells support an effect mainly on cellular expansion. Aim of this study was to investigate, if the supplementation of GM-CSF either in a human ART medium or in a mouse optimized medium, leads to a change in cell number and cell lineages in the early pre-implantation mouse embryo. Our data shows that mouse GM-CSF increased total cell numbers with increasing concentrations. This increase of cell number has not been found in embryos cultured in ART media with or without human GM-CSF (hGM-CSF) or in a mouse medium supplemented with different concentrations of hGM-CSF. The changes were caused by a marked difference in TE and primitive endoderm cell numbers but not due to a change in epiblast cell numbers. Additionally, results show an ectopic expression of NANOG among trophectoderm cells in both, human ART media (with and without GM-CSF) and at increasing concentrations in the mouse and the human GM-CSF supplemented media. In conclusion, we could show that GM-CSF has an effect on cell identity in mice, which might probably also occur in the human. Therefore, we would like to rare awareness that the use of supplements without proper research could bare risks for the embryo itself and probably also in the post-implantation phase.

Endogenous hydrogen sulfide promotes human preimplantation embryonic development by regulating metabolism-related gene expression.

Hydrogen sulfide (H2S) as an endogenous gaseous signaling molecule had been proved to play a vital role in gametes physiology, covering meiosis, maturation and aging. However, little is known about H2S involvement in embryonic development. The present study explored the positive effect of H2S on human early embryonic development. Results validated that the two H2S producing enzymes, CBS and CSE mRNA and proteins were identified in donated human cleavage and blastocyst-stage embryos. The l-cysteine incubation produced endogenous H2S in human blastocysts. NaHS positively affected in vitro blastulation. Single-cell RNA-seq analysis identified 228 differentially expressed genes (DEGs) after NaHS treatment versus the control. The Gene Ontology (GO) enrichment analysis of DEGs showed that genes for protein modification and metabolism were significantly enriched in the NaHS treatment group. For the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, 2-oxocarboxylic acid metabolism, glycosaminoglycan biosynthesis-keratan sulfate, steroid biosynthesis, carbon metabolism, and biosynthesis of amino acids were significantly enriched. Six DEGs, including Neural EGFL like 1 (NELL1), aconitase 1 (ACO1), phosphoglycerate mutase 1 (PGAM1), TP53 induced glycolysis regulatory phosphatase (TIGAR), UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 2 (B3GNT2), and carbohydrate Sulfotransferase 4 (CHST4) were validate by real-time RT-PCR. These findings suggest that H2S is a positive regulator of early embryonic development and may alter the transcription of embryonic genes for protein modification and metabolism in human embryos.

Enniatin B induces dosage-related apoptosis or necrosis in mouse blastocysts leading to deleterious effects on embryo development.

The current study has focused on the effects of enniatin B (ENN B, a major mycotoxin produced by Fusarium fungi) on early embryonic development. In in vitro analysis, mouse blastocysts were incubated in medium with ENN B (0-40 µM) or 0.5% DMSO (control group) for 24 hours. In an animal study, blastocysts were collected from mice which were intravenously injected with ENN B (1, 3, 5, and 7mg/kg body weight/day) for 4 days in order to analyze apoptosis and necrosis via Annexin V/PI staining assay; and proliferation using dual differential staining. Exposure to low ENN B concentration (10 µM in vitro and 3 mg/kg/day in vivo) promoted Reactive Oxygen Species (ROS) generation and apoptosis in the Inner Cell Mass (ICM), the mass of cells inside the blastocyst, impairing post-implantation development alone. On the other hand, exposure to a higher ENN B concentration (40 µM in vitro and 7 mg/kg/day in vivo) induced ROS generation and decreased in intracellular ATP which encouraged necrotic processes in both trophectoderm (TE) and ICM of blastocysts leading to impaired implantation and post-implantation development. Moreover, 5 and 7 mg/kg/day ENN B intraperitoneal injection to female mice for 4 days has caused downregulation of CXCL1, IL-1ß and IL-8 expressions and increased ROS generation in the liver of newborn mice. Over all, ENN B can induce apoptosis and/or necrosis depending on the treatment dosage in mouse blastocysts. ENN B-induced necrosis in blastocysts may exert long-term harmful effects on next-generation newborns.

Understanding the natural selection of human embryos: Blastocyst quality modulates the inflammatory response during the peri-implantation period.

PROBLEM: Decidualized cells display an active role during embryo implantation sensing blastocyst quality, allowing the implantation of normal developed blastocysts and preventing the invasion of impaired developed ones. Here, we characterized the immune microenvironment generated by decidualized cells in response to soluble factors secreted by blastocysts that shape the receptive milieu. METHOD OF STUDY: We used an in vitro model of decidualization based on the Human Endometrial Stromal Cells line (HESC) differentiated with medroxiprogesterone and dibutyryl-cAMP, then treated with human blastocysts-conditioned media (BCM) classified according to their quality. RESULTS: Decidualized cells treated with BCM from impaired developed blastocysts increased IL-1ß production. Next, we evaluated the ability of decidualized cells to modulate other mediators associated with menstruation as chemokines. Decidualized cells responded to stimulation with BCM from impaired developed blastocysts increasing CXCL12 expression and CXCL8 secretion. The modulation of these markers was associated with the recruitment and activation of neutrophils, while regulatory T cells recruitment was restrained. These changes were not observed in the presence of BCM from normal developed blastocysts. CONCLUSION: Soluble factors released by impaired developed blastocysts induce an exacerbated inflammatory response associated with neutrophils recruitment and activation, providing new clues to understand the molecular basis of the embryo-endometrial dialogue.