Embryonic Developmental Potential Research Articles

The impact of female BMI on sperm DNA damage repair ability of oocytes and early embryonic development potential in intracytoplasmic sperm injection cycles.

Obesity adversely influences the quality of oocytes and embryos and can affect DNA repair in embryos, leading to reproductive issues. However, the effects of body mass index (BMI) on DNA repair ability in oocytes during intracytoplasmic sperm injection (ICSI) cycles have not yet been investigated. Therefore, this retrospective study aimed to analyze the influence of sperm DNA damage on embryo development and reproductive outcomes in overweight/obese and normal-weight women in ICSI cycles. A total of 1,141 patients who received the first fresh ICSI cycle treatments were recruited from July 2017 to July 2021. Based on the BMI of the women, all patients were divided into normal weight (18.5≤BMI<25 kg/m2; n=824; 72.22%) and overweight/obese (BMI≥25 kg/m2; n=317; 27.78%) groups. Furthermore, according to the sperm DNA fragmentation index (DFI), these two groups were subdivided into two subgroups: DFI<30% and DFI≥30%. In the normal-weight women group, the embryonic development and reproductive outcomes of ICSI cycles were not statistically different between the two subgroups (DFI<30% and DFI≥30%). However, in the overweight/obese women group, couples with a sperm DFI≥30% had a significantly lower fertilization rate (76% vs. 72.7%; p=0.027), cleavage rate (98.7% vs. 97.2%; p=0.006), and high-quality embryo rate (67.8% vs. 62.6%; p=0.006) than couples with a sperm DFI<30%. When injected sperm with high DFI into the oocytes of overweight/obese women, resulting in lower fertilization, cleavage, and high-quality embryo rates in ICSI cycles, and the decreased early developmental potential of embryos from overweight/obese patients may be caused by the diminished capacity of oocytes to repair sperm DNA damage.

Epifriedelanol delays the aging of porcine oocytes matured invitro

Oocyte aging directly affects the subsequent embryonic development. Epifriedelanol is the active ingredient of Aster tataricus L.F. extract, and it possesses potential anti-cancer, anti-inflammatory and antioxidant properties. In addition, epifriedelanol can slow the aging of human skin fibroblasts. To explore the effect of epifriedelanol on the aging of porcine oocytes matured in vitro, the aging model was first established, epifriedelanol was added to in vitro maturation (IVM) medium to investigate its anti-aging effects by observing oocyte maturation and embryonic development potential, and analyzing aging-related gene expression, reactive oxygen species and mitochondrial membrane potential levels. It was found that typical aging of porcine oocytes appeared from 66 h during in vitro maturation. Compared with the 44 h group, a larger perivitelline space, increased abnormality of microtubulin formation, and significantly lower blastocyst rate were observed in the 66 h and 72 h groups. Compared with the 0 μg/mL group, the first polar body extrusion, cleavage and blastocyst rates were significantly improved (P < 0.05) in 10 μg/mL group. The expression of oocyte developmental potential-related, SIRT family-related, antioxidant and anti-apoptotic-related genes was significantly up-regulated (P < 0.05), p53 and pro-apoptotic genes were significantly down-regulated (P < 0.05). In addition, the reactive oxygen species level was significantly decreased (P < 0.01), the mitochondrial membrane potential was significantly elevated (P < 0.01) in 10 μg/mL group. In conclusion, epifriedelanol delays the aging of porcine oocytes cultured in vitro by up-regulating SIRT family gene expression, enhancing the antioxidant and anti-apoptotic capacity of oocytes.

Artificial oocyte activation with Ca2+ ionophore improves reproductive outcomes in patients with fertilization failure and poor embryo development in previous ICSI cycles.

Does artificial oocyte activation (AOA) by a calcium ionophore (ionomycin) improve the previous fertilization failure or poor embryo development of intracytoplasmic sperm injection (ICSI) account for male factor infertility or other infertility causes? This retrospective study involved 114 patients receiving ICSI-AOA in Shanghai First Maternity and Infant Hospital with previous ICSI fertilization failure or poor embryo development. The previous ICSI cycles of the same patients without AOA served as the control group. The fertilization rates, cleavage rates, transferable embryo rates and blastocyst formation rates of the two groups were compared. Additionally, the clinical pregnancy, implantation rate and live birth rates were also compared to assess the efficiency and safety of AOA. Furthermore, two subgroup analyses were performed in this study based on the cause of infertility and the reason for AOA. The fertilization rate, embryonic development potential and clinical outcome were compared among groups. Among 114 ICSI-AOA cycles, the fertilization rate, top-quality embryo rate, implantation rate, clinical pregnancy per patient and live birth rate per patient were improved significantly compared with previous ICSI cycles (p<0.05 to P< 0.001), and the miscarriage rate in the AOA group was significantly lower than that of the control group (p<0.001). In the AOA subgroups based on the cause of infertility, the fertilization rates of each subgroup were significantly improved compared with previous control cycles except for the mixed factor infertility subgroup (p<0.05 to p<0.001). In the AOA subgroups based on the reason for AOA, the fertilization rates of each subgroup were significantly increased compared with those in their previous ICSI cycle without AOA (p<0.001); however, there was no significant difference in the top-quality embryo rate. No significant improvement was found in the implantation rates and the clinical pregnancy rate in each subgroup except for the poor embryo development subgroup. In the 114 AOA cycles, 35 healthy infants (21 singletons and 7 twins) were delivered without major congenital birth defects or malformations. This study showed that AOA with the calcium ionophore ionomycin can improve the reproductive outcomes of patients with previous fertilization failure and poor embryo development after ICSI.

Ameliorative effect of Stevia rebaudiana Bertoni on sperm parameters, in vitro fertilization, and early embryo development in a streptozotocin-induced mouse model of diabetes.

Diabetes mellitus (DM) is a common metabolic disease characterized by high blood sugar levels. It is well known that men with diabetes frequently experience reproductive disorders and sexual dysfunction. In fact, sperm quality has a significant effect on fertilization success and embryo development. The current study aimed to investigate the effect of Stevia rebaudiana hydroalcoholic extract on serum testosterone levels, sperm parameters, in vitro fertilization (IVF) success, and in vitro embryonic developmental potential to reach the blastocyst stage in a streptozotocin (STZ)-induced mouse model of diabetes. In this research, 30 male mice were distributed randomly into control, diabetic (streptozotocin 150 mg/kg) and diabetic + Stevia (400 mg/kg) groups. The results revealed a decrease in body and testis weight and elevated blood fasting blood sugar (FBS) levels in the diabetic group, compared with the control. However, Stevia treatment significantly increased body and testis weight, while serum FBS levels were decreased compared with the diabetic group. In addition, Stevia significantly increased blood testosterone levels compared with the diabetic group. Moreover, sperm parameters were improved considerably by Stevia treatment compared with the diabetic group. Furthermore, Stevia administration significantly promoted IVF success rate and in vitro development of fertilized oocytes compared with the diabetic group. In summary, our data indicated that Stevia enhanced sperm parameters, IVF success, and in vitro embryonic developmental competency in diabetic mice, probably because of its antioxidant effects. Therefore, Stevia could ameliorate sperm parameters that, in turn, increase fertilization outcomes in experimental-induced diabetes.

Rapid and non-invasive diagnostic techniques for embryonic developmental potential: a metabolomic analysis based on Raman spectroscopy to identify the pregnancy outcomes of IVF-ET.

The non-invasive and rapid assessment of the developmental potential of embryos is of great clinical importance in assisted reproductive technology (ART). In this retrospective study, we analyzed the metabolomics of 107 samples provided by volunteers and utilized Raman spectroscopy to detect the substance composition in the discarded culture medium of 53 embryos resulting in successful pregnancies and 54 embryos that did not result in pregnancy after implantation. The culture medium from D3 cleavage-stage embryos was collected after transplantation and a total of 535 (107 × 5) original Raman spectra were obtained. By combining several machine learning methods, we predicted the developmental potential of embryos, and the principal component analysis-convolutional neural network (PCA-CNN) model achieved an accuracy rate of 71.5%. Furthermore, the chemometric algorithm was used to analyze seven amino acid metabolites in the culture medium, and the data showed significant differences in tyrosine, tryptophan, and serine between the pregnancy and non-pregnancy groups. The results suggest that Raman spectroscopy, as a non-invasive and rapid molecular fingerprint detection technology, shows potential for clinical application in assisted reproduction.

P-405 Nuclear transfer overcomes poor embryo development of in vitro grown mouse oocytes

Abstract Study question Is the nuclear transfer technology able to overcome the compromised embryonic development of in vitro cultured secondary follicles from young B6D2 mice? Summary answer Nuclear transfer technology was able to restore embryonic development to levels similar of in vivo grown oocytes. What is known already In vitro follicle culture has been proposed as a strategy for fertility preservation in cancer patients. Studies in human follicle culture remain scarce, due to the low availability of human tissue. Mouse models have been extensively studied to improve follicle maturation and investigate the potential of in vitro grown (IVG) gametes. Despite significant improvements reported over the years, including increased maturation rates, advanced embryonic development and generation of fertile offspring, the quality of IVG oocytes remains inferior compared to their in vivo grown counterparts. Study design, size, duration The experimental study was conducted between October 2020 and January 2023, after approval by the Animal Ethics Committee of Ghent University Hospital (ECD no 19/60). In total, 108x 16-days-old B6D2 females were used for secondary follicle isolation and in vitro culture, 81x 8-12-week-old females for collection of in vivo grown oocytes, 4x 9-14-week-old B6D2 males for sperm freezing, 5x 10-24-week-old CD1 vasectomised males and 10x 8-12-weeks-old CD1 females as surrogate mothers for embryo transfer. Participants/materials, setting, methods Follicles isolated from 16-days-old B6D2 mice were cultured for 9 days, followed by maturation for 16-18hrs. Mature oocytes were assessed on diameter, spindle morphology, calcium releasing ability, mitochondrial membrane and embryonic developmental potential. In vivo grown oocytes from stimulated mice were used as controls and cytoplasmic donors for nuclear transfer. Spindle (ST) and Pronuclear transfer (PNT) were applied to overcome poor development of IVG embryos, by transferring the spindle/pronuclei of IVG oocytes/zygotes to enucleated controls. Main results and the role of chance In total, 1509 secondary follicles were cultured and 73.6% survived to Day 9. From the 719 cumulus oocyte complexes, 364 (50.6%) oocytes matured. Oocyte diameters were significantly smaller between IVG (67.4μm) and controls (73.1μm) (p &amp;lt; 0.001). Spindle staining revealed a similar number of normal spindles between the two groups (71.4% and 82.6% respectively, p = 0.138). Calcium release was significantly lower in IVG oocytes (1.6) vs controls (5.7) (p &amp;lt; 0.001), as well as mitochondrial membrane potential ((0.9) vs (2.2), p &amp;lt; 0.001). These data implicate a cytoplasmic inferiority in IVG oocytes. Following parthenogenetic activation, 2-cell (59.4%) and blastocyst rate (36.3%) in IVG group was significantly lower than control (89.4% and 88.2% respectively, p &amp;lt; 0.001). For this reason, we performed ST, using controls as cytoplasmic recipients for IVG spindles. The 2-cell rate significantly increased to 100% (p &amp;lt; 0.001) in ST oocytes, reaching 100% blastocyst formation. Next, to create bi-paternal embryos, we performed IVF. From the IVG group, 2-cell (40%) and blastocyst (0%) rates were significantly lower than control (94.4% and 83.3%, p &amp;lt; 0.001). Embryo development was restored following PNT (98% 2-cell and 86% blastocyst rate, p &amp;lt; 0.001). Genetic analysis of PNT embryos revealed that 5/6 were chromosomally normal, similar to controls (8/10). Embryo transfer of PNT and control embryos is ongoing. Limitations, reasons for caution Both ST and PNT are promising technologies to overcome poor embryonic development of IVG oocytes, but the benefit for human IVG oocytes remains to be investigated, along with the safety of the technique. Wider implications of the findings Our results demonstrate that ST and PNT have the potential to restore embryonic developmental competence of IVG oocytes, without increasing chromosomal abnormalities. This technology could be investigated in the future, as a mean to overcome poor quality of IVG oocytes in human. Trial registration number Not applicable

P-123 Early irregular division of human embryos changes in style between the first and second divisions

Abstract Study question Is there a difference in irregular division kinetics between first and second division of human embryos? Summary answer The incidence of rapid cleavage was reduced in the second division, but the developmental potential of the blastomeres by this dynamics was unexpectedly low. What is known already The styles of irregular division of early human embryos are known: direct cleavage (DC), in which one cell becomes three (or more) cells without a two-cell phase, and rapid cleavage (RaC), in which one (or both) cells dividing rapidly after two-cell phase, both of which reduces the embryonic developmental potential. However, there are no reports that have examined in detail whether these division styles occur similarly in the first and second divisions, and how these dynamics affect the potential. Study design, size, duration This is a retrospective study of 635 embryos collected in 2020 at our clinic and cultured for at least 5 days. The embryos were time-lapse monitored by EmbryoScope (Vitrolife, Sweden) to observe the first and second divisions, and the blastomeres of each embryo were classified according to their style of division. Participants/materials, setting, methods It was observed whether there was a DC (division into 3 cells without a 2-cell phase) or RaC (One or two blastomeres divide within 5 hours after the 2-cell phase) at the first division. First dividing normal embryos were observed for the presence of a DC or RaC at the second division. Blastomeres by DC or RaC were observed for subsequent development and whether they participated in blastocysts or not was recorded. Main results and the role of chance Among the subject embryos, 63 embryos had DC and 199 embryos had RaC in the first division, and their blastomeres were classified into the DC1 and RaC1 groups, respectively. Of the 373 first division normal embryos, 39 had DC and 23 had RaC (there were 4 embryos with both DC and RaC) in the second division, and their blastomeres were classified into DC2 and RaC2 groups. The blastomeres blastocyst participation rate was 4.4% in the DC1 group and 19.5% in the RaC1 group, respectively, the former was significantly lower (P &amp;lt; 0.01), however, in the DC2 and RaC2 groups, 23.3% and 4.2%, respectively, the latter was significantly lower (P &amp;lt; 0.01). The incidence of DC was similar in the first (9.9%) and second (10.5%) divisions, but the incidence of RaC was 31.3% in the first division and 6.2% in the second division, the latter was significantly lower (P &amp;lt; 0.01). The development rate of good blastocysts (≥4BC in Gardner grade) was 4.8% in embryos with DC1 and 19.5% in embryos with RaC1, with the former significantly lower (P &amp;lt; 0.01), 37.0% in embryos with DC2, and 26.3% in embryos with RaC2, with no significant difference. Limitations, reasons for caution As of 2020, the clinical use of PGT-A is not approved in principle in Japan, and the subject embryos have not been chromosomally analyzed. Wider implications of the findings RaC would lead to inadequate DNA proliferation, but the incidence of this and the prognosis of the blastomeres were different in the first and second divisions. In human embryos, the mechanisms that control blastomeres from dividing may change as development progresses, but further studies are needed to elucidate this. Trial registration number not applicable

O-097 Predicting live birth rate using combined Day3-spent embryo culture medium metabolomics and cumulus cell gene expression based algorithm to optimize embryo selection in unexplained infertility

Abstract Study question Can combination of metabolomics of spent embryo culture medium (sECM) and cumulus cell (CC) genes judge live birth rate in unexplained infertility (UI)? Summary answer A combination of day3 metabolic profiling of sECM with CC gene expression yields an algorithm to predict live birth rate in UI. What is known already Optimizing embryo selection is rate-limiting for pregnancy success. The metabolomic profile of sECM can aid in advanced prediction of embryonic developmental potential. Recent transcriptomics studies proposed prokinectin 2 (PROK2), and pregnancy up-regulated nonubiquitous CaM kinase (PNCK) as two novel gene/s responsible for perifollicular vascularization and embryonic development respectively; two important events regulating bidirectional CC-oocyte signalling and implantation ability. Given that UI is contributory to impaired oocyte quality, we delineated if combined metabolic signature of CC gene expression encompassing metabolic, developmental competence, and extracellular matrix facet/s and sECM could address live birth rate in women with UI following single embryo transfer. Study design, size, duration A prospective cohort study was conducted at Institute of Reproductive Medicine between January 2022 and October 2022. 48 women (age: 30-39 years) diagnosed with UI undergoing oocyte-retrieval offered consented participation and were sub-stratified according to pregnancy outcome. Cleavage-stage embryos from intra cytoplasmic sperm injection (ICSI) cycles were scored according to combination of gene expression pattern of CC-samples and integral value of metabolite obtained from nuclear magnetic resonance (NMR) spectroscopy of sECM (range 0-3, 4-6, 7-9). Participants/materials, setting, methods Expression profiles of metabolic (LDHA, PFKP, PKM2), extracellular matrix (HAS2, PTX3, TNFAIP6, VCAN), and developmental competence (PNCK, PROK2) related genes were evaluated by real time-quantitative polymerase-chain-reaction (qRT-PCR) from retrieved CC. Day-3 sECM (30 μL) was collected for NMR from 43 single transferred embryos. Univariate and multivariate analysis was performed after NMR data acquisition. Live birth rate was evaluated by area-under-the-curve (AUC) values using combined score panel. Main results and the role of chance The groups were similar for patient age, BMI, AMH, infertility diagnosis, and stimulation protocol. A total of 43 cycles were included in the analysis [embryo transfer cancelled (n = 2), insufficient cumulus mass (n = 3)]. Clinical pregnancy was confirmed in 16 patients (37.21%). Mean mRNA levels for all genes were similar in CC associated with oocyte that fertilized normally (2PN) compared with those that either failed to fertilize or were abnormal (i.e. 1PN, 3PN). Similar results were observed in terms of speed of embryonic cleavage (≥7 cell on Day 3 vs. ≤6 cell on Day 3). Expression of VCAN, (OR: 1.102, 95% CI: 1.010-1.202, p &amp;lt; 0.02), PNCK (OR: 0.931, 95% CI: 0.916-0.947, p &amp;lt; 0.001) and PROK2 (OR: 1.273, 95% CI: 1.011-1.231, p &amp;lt; 0.001) was significantly higher (p &amp;lt; 0.01) in CC that achieved live birth compared with those that failed to result a successful pregnancy. Multivariate and univariate analysis revealed distinct metabolomic signatures between pregnant and non- pregnant group/s. Lactate, pyruvate and proline were the most significant (p &amp;lt; 0.01) altered metabolite in sECM of non-pregnant group when compared to their pregnant counterpart. The combination demonstrated a “good” (VCAN, pyruvate: AUC: 0.87), “fair” (PROK2, proline: AUC: 0.79) and “modest” (PNCK, glucose: AUC: 0.71) in terms of live-birth rate. Limitations, reasons for caution The study population is heterogeneous in terms of duration of infertility and previous infertility treatment outcome, limited by small sample size and subjective nature of embryo scoring. The presumable involvement of unknown factors in developmental competence of UI and impact of frozen cycle/s should also be addressed. Wider implications of the findings The study provides reliable accuracy measures to support relationship between live birth rate and combined score panel in patients with UI. It is envisioned integration of CC gene expression/s and metabolomics of sECM if combined with patient characteristics may tailor therapy by artificial intelligence to model the outcome in UI. Trial registration number Not applicable

Near-infrared fluorophore IR-61 delays postovulatory aging of mouse oocytes through suppressing oxidative stress mediated by mitochondrial protection.

Postovulatory aging can trigger deterioration of oocyte quality and subsequent embryonic development, and thus reduce the success rates of assisted reproductive technology (ART). The molecular mechanisms underlying postovulatory aging, and preventative strategies, remain to be explored. The near-infrared fluorophore IR-61, a novel heptamethine cyanine dye, has the potential for mitochondrial targeting and cell protection. In this study, we found that IR-61 accumulated in oocyte mitochondria and reduced the postovulatory aging-induced decline in mitochondrial function, including mitochondrial distribution, membrane potential, mtDNA number, ATP levels, and mitochondrial ultrastructure. In addition, IR-61 rescued postovulatory aging-caused oocyte fragmentation, defects in spindle structure, and embryonic developmental potential. RNA sequencing analysis indicated that the postovulatory aging-induced oxidative stress pathway might be inhibited by IR-61. We then confirmed that IR-61 decreased the levels of reactive oxygen species and MitoSOX, and increased GSH content in aged oocytes. Collectively, the results indicate that IR-61 may prevent postovulatory aging by rescuing oocyte quality, promoting successful rate in ART procedure.

A long-acting recombinant FSH supports high-quality mouse follicle development and oocyte maturation in vitro by coordinating somatic and germ cell transcriptomes.

Strategies to maximize individual fertility chances are constant requirements of ART. In vitro folliculogenesis may represent a valid option to create a large source of immature ovarian follicles in ART. Efforts are being made to set up mammalian follicle culture protocols with suitable FSH stimuli. In this study, a new type of recombinant FSH (KN015) with a prolonged half-life is proposed as an alternative to canonical FSH. KN015 supports the in vitro development of mouse follicles from primary to preovulatory stage with higher efficiency than canonical FSH and enhanced post-fertilization development rates of the ovulated oocytes. The use of KN015 also allows us to compare the dynamic transcriptome changes in oocytes and granulosa cells at different stages, in vivo and in vitro. In particular, KN015 facilitates mRNA accumulation in growing mouse oocytes and prevents spontaneous luteinization of granulosa cells in vitro. Novel analyses of transcriptome changes in this study reveal that the in vivo oocytes were more efficient than in vitro oocytes in terms of maternal mRNA clearing during meiotic maturation. KN015 promotes the degradation of maternal mRNA during in vitro oocyte maturation, improves cytoplasmic maturation and, therefore, enhances embryonic developmental potential. These findings establish new transcriptome data for oocyte and granulosa cells at the key stages of follicle development, and should help to widen the use of KN015 as a valid and commercially available hormonal support enabling optimized in vitro development of follicles and oocytes.

Diminished ovarian reserve may not be associated with a poorer fresh cycle outcome in women < 38 years

BackgroundPrevious studies have discussed the pregnancy outcomes of diminished ovarian reserve (DOR) patients. However, data on embryonic development potential, neonatal outcomes, and maternal complications of DOR patients still remained unknown. This is the first study to investigate the risk of DOR on pregnancy and perinatal outcomes among women < 38 years.MethodsRetrospective cohort study was conducted. Patients (< 38 years of age) undergoing their first oocyte retrieval cycle were included. Patients were divided into DOR group and non-DOR group. Pregnancy outcomes of fresh cycle and cumulative live birth rate and perinatal outcomes after one oocyte retrieved cycle were compared between DOR and non-DOR group.Result(s)From January 2016 to September 2020, there were 8,179 patients involved: 443 patients in the DOR group and 7,736 patients in the non-DOR group. The incidences of live birth and clinical pregnancy did not differ significantly between patients with or without DOR after fresh cycle transfer, but the cumulative live birth rate was significantly lower in DOR group. Among women who had singleton live births, after binary logistic regression, the rates of maternal complications and neonatal outcomes were comparable in the two groups.Conclusion(s)DOR patients (< 38 years of age) showed similar pregnancy outcomes in the first fresh embryo transfer cycle but a lower chance of live birth after a whole oocyte retrieval cycle to non-DOR patients and DOR is not associated with adverse perinatal outcomes.

Effects of high-altitude hypoxia on embryonic developmental potential in women undergoing IVF/ICSI procedures.

In this study we examined the effects of long-term adaptation to hypoxia on embryonic developmental potential of oocytes collected from women who underwent IVF/ICSI procedures. We selected young infertile women who lived in a low-altitude normoxic environment (n = 80, altitude < 500m) or high-altitude hypoxic environment (n = 100, altitude > 2500m) for a lengthy period of time and who planned to undergo IVF/ICSI procedures. We then determined the baseline reproductive hormone levels, gonadotropin (Gn) dose and Gn treatment duration during controlled ovarian hyperstimulation (COH), number of oocytes retrieved, number of mature oocytes, oocyte maturation rate, fertilization rate, normal fertilization rate, day (D3) embryo-formation rate, blastocyst formation rate, good-quality formation rate, D5 blastocyst formation rate, and D6 blastocyst formation rate between the two groups. Compared with the low-altitude normoxic group, the various reproductive hormone markers of women in the high-altitude hypoxia group were lower, with LH and T levels significantly reduced (P < 0.05) at 72.29 and 72.44% of the normoxic group, respectively (normoxic group vs. hypoxic group, 5.24 ± 1.61 vs. 3.79 ± 1.21; 0.61 ± 0.18 vs. 0.42 ± 0.15; P < 0.05). During ovarian hyperstimulation, a greater Gn dose and longer Gn treatment duration were required for the hypoxic group to complete COH (normoxic group vs. hypoxic group, 2152.08IU ± 52.76 vs. 2622.09IU ± 123.28; 9.96days ± 1.27 vs. 11.54days ± 1.34, respectively; P < 0.05). The fertilization, cleavage, and D3 embryo-formation rates tended to be higher in the normoxic group than in the hypoxic group (P > 0.05); while the normal fertilization rate tended to lower than in the hypoxic group (P > 0.05). When we conducted an analysis of blastocyst formation rates at different timepoints, we ascertained that the blastocyst formation rate, usable blastocyst rate, and good-quality blastocyst rate of the hypoxic group were all lower than in the normoxic group, with the difference in usable blastocyst rate the most highly significant (normoxic group vs. hypoxic group, 75.31 ± 5.53 vs. 56.04 ± 6.10%, respectively; P < 0.05). In addition, the D5 and D6 blastocyst-formation rates in the normoxic group were slightly higher than in the hypoxic group, revealing that not only were fewer blastocysts formed in the hypoxic group but that there was also a delay in blastocyst formation. In young women undergoing IVF/ICSI treatment, long-term hypoxic adaptation required augmented Gn dose and Gn treatment duration during COH, and blastocyst developmental potential was also attenuated.

WNT Co-Receptor LRP6 Is Critical for Zygotic Genome Activation and Embryonic Developmental Potential by Interacting with Oviductal Paracrine Ligand WNT2.

Mammalian preimplantation development depends on the interaction between embryonic autocrine and maternal paracrine signaling. Despite the robust independence of preimplantation embryos, oviductal factors are thought to be critical to pregnancy success. However, how oviductal factors regulate embryonic development and the underlying mechanism remain unknown. In the present study, focusing on WNT signaling, which has been reported to be essential for developmental reprogramming after fertilization, we analyzed the receptor-ligand repertoire of preimplantation embryonic WNT signaling, and identified that the WNT co-receptor LRP6 is necessary for early cleavage and has a prolonged effect on preimplantation development. LRP6 inhibition significantly impeded zygotic genome activation and disrupted relevant epigenetic reprogramming. Focusing on the potential oviductal WNT ligands, we found WNT2 as the candidate interacting with embryonic LRP6. More importantly, we found that WNT2 supplementation in culture medium significantly promoted zygotic genome activation (ZGA) and improved blastocyst formation and quality following in vitro fertilization (IVF). In addition, WNT2 supplementation significantly improved implantation rate and pregnancy outcomes following embryo transfer. Collectively, our findings not only provide novel insight into how maternal factors regulate preimplantation development through maternal-embryonic communication, but they also propose a promising strategy for improving current IVF systems.

Developmental competence of chimeric porcine embryos through the aggregation of parthenogenetic embryos and somatic cell nuclear transfer embryos

The efficiency of somatic cell nuclear transfer (NT) in pigs is low and requires enhancement. We identified the most efficient method for zona pellucida (ZP) removal and blastomere aggregation in pigs and investigated whether the aggregation of NT and parthenogenetic activation (PA) of blastomeres could reduce embryonic apoptosis and improve the quality of NT-derived embryos by investigating. Embryonic developmental competence after ZP removal using acid Tyrode's solution or protease (pronase E). The embryonic developmental potential of NT-derived blastomeres was also investigated using well-of-the-well or phytohemagglutinin-L. We analyzed apoptosis in aggregate-derived blastocysts. The aggregation rate of protease-treated embryos was lower than that of Tyrode’s solution-treated embryos (69.2% vs. 88.3%). No significant difference was observed between phytohemagglutinin-L and well-of-the-well (35.7%–38.5%). However, 2P1N showed a higher number of blastocysts compared to 3N (73.8% vs. 24.3%) and an increased blastocyst diameter compared to the control and 1P2N (274 μm vs. 230–234 μm). In blastomeres aggregated using phytohemagglutinin-L, the apoptotic cell ratio was significantly higher in 1P2N and 3N than in 3P (5.91%–6.46% vs. 2.94%, respectively). Our results indicate that aggregation of one NT embryo with two PA embryos improved the rate of blastocysts with increased blastocyst diameter.

Glutathione and selenium nanoparticles have a synergistic protective effect during cryopreservation of bull semen.

In the present study, the synergistic protective effect of co-supplementation of glutathione (GSH) with selenium nanoparticles (SeNPs) on the cryopreservation efficiency of bull semen was analyzed. After collection, the ejaculates of Holstein bulls were subsequently diluted with a Tris extender buffer supplemented with different concentrations of SeNPs (0, 1, 2, and 4 μg/ml), followed by semen equilibration at 4°C and assessment of sperm viability and motility. Subsequently, the ejaculates of Holstein bulls were pooled, split into four equal groups, and diluted with a Tris extender buffer supplemented with basic extender (negative control group, NC group), 2 μg/ml SeNPs (SeNPs group), 4 mM GSH (GSH group), and 4 mM GSH plus 2 μg/ml SeNPs (GSH + SeNPs group). After cryopreservation, motility, viability, mitochondrial activity, plasma membrane integrity, acrosome integrity, concentration of malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT), and ability of frozen-thawed sperm cells to support in vitro embryonic development were evaluated. No side effect of SeNPs concentrations applied in the current study on the motility and viability of equilibrated bull spermatozoa was found. Meanwhile, supplementation of SeNPs significantly promoted the motility and viability of equilibrated bull spermatozoa. Furthermore, the co-supplementation of GSH with SeNPs effectively protected bull spermatozoa from cryoinjury as expressed by promoting semen motility, viability, mitochondrial activity, plasma membrane integrity, and acrosome integrity. Finally, the enhanced antioxidant capacity and embryonic development potential in the frozen-thawed bull spermatozoa cryopreserved by co-supplementation of GSH with SeNPs further confirmed the synergistic protective effect of co-supplementation of GSH with SeNPs on the cryopreservation of bull semen.

Effect of Paternal Body Mass Index on In Vitro Fertilization and Neonatal Outcomes among Oligozoospermia and Asthenospermia Patients.

Male overweight and obesity could affect sperm quality and reproductive health. However, the impact of body mass index (BMI) on assisted reproductive technology (ART) outcomes in oligospermia and/or asthenospermia patients is yet lacking. This study aims to assess the impact of paternal BMI on ART and neonatal outcomes among oligozoospermia and/or asthenospermia patients undergoing in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI). In this study, 2,075 couples undergoing their first fresh embryo transfer between January 2015 and June 2022 were recruited. Following the World Health Organization's (WHO's) categories, couples were stratified into three cohorts based on paternal BMI: normal weight (18.5-24.9 kg/m²), overweight (25.0-29.9 kg/m²), and obese (≥30.0 kg/m²). Modified Poisson regression models were used to assess the associations of paternal BMI with fertilization, in vitro embryonic development, and pregnancy outcomes. Logistic regression models were performed to investigate the associations of paternal BMI with pregnancy loss and neonatal outcomes. Furthermore, stratified analyses were performed based on fertilization methods, male infertility cause, and maternal BMI. Higher paternal BMI is associated with a lower likelihood of achieving normal fertilized (p-trend=0.002), Day 3 transferable (p-trend=0.007), and high-quality embryos (p-trend=0.046) in IVF cycles, rather than in ICSI cycles. Paternal BMI of oligospermia or asthenospermia was negatively correlated with day 3 transferable (p-trend=0.013 and 0.030) and high-quality embryos (p-trend=0.024 and 0.027). Moreover, for neonatal outcomes, paternal BMI was positively associated with macrosomia (p-trend=0.019), large for gestational age (LGA) (p-trend=0.031), and very LGA (p-trend=0.045). Our data suggested that higher paternal BMI was associated with fetal overgrowth, reduced fertilization, and embryonic development potential. Among males with oligospermia and/or asthenospermia, the impact of overweight and obesity on the choice of fertilization method and the long-term effects on their offspring need to be further investigated.

Nicotinamide riboside supplementation ameliorated post-ovulatory oocyte quality decline.

Oocyte quality and its NAD+ level decrease with time during in vitro culture. This study shows that nicotinamide riboside (NR) supplementation improves early embryonic development potential in post-ovulatory oocytes by decreasing the reactive oxygen species (ROS) levels and reducing DNA damage and apoptosis which could potentially increase the success rate of assisted reproductive technology (ART). The quality of post-ovulatory oocytes deteriorates over time, impacting the outcome of early embryonic development during human ART. We and other groups have found that NAD+, a prominent redox cofactor and enzyme substrate, decreases in both aging ovaries and oocytes. In this study, we found that the NAD+ levels decreased in the post-ovulatory mouse oocytes during in vitro culture and this decrease was partly prevented by NR supplementation. NR treatmenty restored MII oocyte quality and enhanced the early embryonic development potential of post-ovulatory oocytes via alleviating mitochondrial dysfunction and maintaining normal spindle/chromosome structure. Also, treatment with NR decreased ROS levels and reduced DNA damage and apoptosis in post-ovulatory oocytes. Taken together, our findings indicated that NR supplementation increases the oocyte quality and early embryonic development potential in post-ovulatory oocytes which could potentially increase the success rate of ART.

Association of gene expression in ovarian granulosa cells with embryonic development potential among patients with polycystic ovary syndrome

To assess the association of gene expression with development potential of early embryos derived from patients with polycystic ovary syndrome (PCOS). Three pairs of infertile patients with respectively matched age, body mass index, ovarian reserve and treatment with gonadotrophin-releasing hormone (GnRH) antagonists were selected. Patients with fewer embryos were assigned as the case group (n = 3), whilst the remainders were assigned as the control group (n = 3). Ovarian granulosa cells from patients were collected for the extraction of total RNA and subjected to RNA sequencing. The results were subjected to differential gene expression and functional enrichment analyses. Compared with the control group, 76 genes were up-regulated and 110 genes were down-regulated in the case group. The level of estradiol (E2) was significantly higher in the control group on the trigger day with the injection of human chorionic gonadotrophin (HCG). Compared with the control group, the KRT7 gene was most significantly up-regulated, whilst the CCNYL2 gene was most significantly down-regulated in the case group. Gene ontology (GO) entries enrichment has found those associated with chromosome segregation, cell cycle regulation, and fatty acid metabolism to be significantly enriched. The genes participating in the regulation of cell assembly, differentiation, negative regulation of cell cycle, negative regulation of development, extracellular regulated protein kinases (ERK), ERK1 and ERK2 signaling pathways to be significantly down-regulated. KEGG enrichment analysis of cell signaling pathways revealed that steroid hormone biosynthesis-related genes were enriched. Among patients treated with GnRH antagonists, the significant difference in the number of oocytes fertilized in vitro and the number of available embryos are associated with the difference in the expression of genes of ovarian granulosa cells.

Variations in mitochondrial DNA coding and D-loop region are associated with early embryonic development defects in infertile women.

Mitochondrial DNA (mtDNA) plays a critical role in oocyte maturation, fertilization, and early embryonic development. Defects in mtDNA may determine the alteration of the mitochondrial function, affecting cellular oxidative phosphorylation and ATP supply, leading to impaired oocyte maturation, abnormal fertilization, and low embryonic developmental potential, ultimately leading to female infertility. This case-control study was established to investigate the correlation between mtDNA variations and early embryonic development defects. Peripheral blood was collected for next-generation sequencing from women who suffered the repeated failures of in vitro fertilization (IVF) and/or intracytoplasmic sperm injection (ICSI) cycles due to early embryonic development defects as well as in-house healthycontrols, and the sequencing results were statistically analyzed for all subjects. This study found that infertile women with early embryonic development defects carried more mtDNA variants, especially in the D-loop region, ATP6 gene, and CYTB gene. By univariate logistic regression analysis, 16 mtDNA variants were associated with an increased risk of early embryonic development defects (OR > 1, p < 0.05). Furthermore, we identified 16 potentially pathogenic mtDNA variants only in infertile cases. The data proved that mtDNA variations were associated with early embryonic development defects in infertile Chinese women.

Developmental potential of different embryos on day 3: a retrospective study

To investigate how different quality of day 3 (D3) embryos affect blastocyst formation and clinical outcomes. This retrospective study analysed 699 patients undergoing assisted reproductive technology (ART) between January 2017 and February 2021. A total of 2517 D3 embryos were transferred to blastocyst medium for extended culture. D3 embryos were divided into five groups. Grade A, 6–10 cells, symmetrical blastomeres and <20% fragmentation; grade B, 6–10 cells, uneven blastomeres and ≥20% fragmentation; grade C, >10 cells, symmetrical blastomeres and <20% fragmentation; grade D, >10 cells, uneven blastomeres and ≥20% fragmentation; grade E, <6 cells. Status of day 5 (D5) and day 6 (D6) blastocysts and the clinical outcomes (blastocyst transfer) of each D3 embryo were recorded. The grade C group showed a higher D5 blastocyst formation rate and a high-quality blastocyst rate than other groups (p<.05). However, the clinical pregnancy rates in the grade A group were higher than other groups (p<.05). Embryos with low speed of development (grade E group) showed considerable clinical outcomes that were still worth investigating. D3 embryos with less fragmentation and ≥6 symmetrical blastomeres revealed a higher developmental potential, while embryos with 6–10 blastomeres showed the ideal clinical outcomes. Impact Statement What is already known on this subject? Accurate embryo evaluation can effectively reflect the developmental potential of different embryos. The number of blastomeres, proportion of fragmentation, and blastomere symmetry are three important and popular morphologic parameters used for evaluating day 3 (D3) embryos. However, in existing reports, combining these three parameters for embryo evaluation often results in different results. This is because different researchers have chosen different criteria for these three parameters. What do the results of this study add? In this retrospective study, we summarised the medical records of our reproductive centre in the past three years, redefined the evaluation method of the D3 embryos, and analysed the corresponding developmental potential and clinical outcomes. We conclude that although the embryonic development potential of grade C embryos (>10 cells, symmetrical blastomeres and/or <20% fragmentation) is relatively good, the results of grade A embryos (6–10 cells, symmetrical blastomeres and/or <20% fragmentation) are better in terms of clinical outcomes. What are the implications of these findings for clinical practice and/or further research? We believe this is meaningful for embryologists to choose embryos for transfer and predict the clinical outcome of IVF cycles.