Embryo Culture Research Articles

The effect of hyaluronic acid-enriched transfer medium on the implantation rate in frozen-thawed embryo transfer cycles

Background: The treatment strategy for patients with a history of multiple failures in in vitro fertilization cycles remains a subject of active research. Embryo transfer in cryocycles is known to be more successful than “fresh” embryo transfer, when ovarian stimulation, follicle puncture, fertilization, and embryo culture were performed but the overall efficiency of in vitro fertilization cycles decreases with maternal age and transfer of poor quality embryos. One of the key factors influencing the failure of high quality embryo implantation is the deficiency of the adhesive matrix, which ensures the interaction between the embryo and the endometrium. The implementation of protocols that include the use of hyaluronic acid-enriched transfer media in the process of embryo transfer is a promising direction for improving implantation outcomes. Special attention of researchers is paid to determining the indications for the use of this technique. Aim: The aim of this study was to evaluate the effect of hyaluronic acid-enriched transfer media on the pregnancy rate in cryocycles. Materials and methods: This retrospective cohort comparative study, included 609 women who underwent controlled ovarian stimulation and frozen-thawed embryo transfer. Results: The main outcomes of frozen-thawed embryo transfer cycles were analyzed, taking into account the quality of the transferred embryos and the use of hyaluronic acid-enriched transfer media. The latter was not shown to be associated with an increase in biochemical and clinical pregnancy rates in women under 35 years of age with a history of repeated implantation failures or after transfer of poor quality embryos. However, in patients over 40 years of age with poor quality embryos, the use of hyaluronic acid-enriched transfer media was effective, since it increased the clinical pregnancy rate. As expected, our data showed an increase in the clinical pregnancy rate in women with high quality embryos on the day of transfer, regardless of the patient’s age. At the same time, in patients with a thin (thickness less than 7 mm) endometrium or a history of miscarriage, the use of hyaluronic acid-enriched transfer media was also associated with an increase in the clinical pregnancy rate and live birth during embryo transfer in cryocycles. Conclusions: The data obtained have shown that the use of hyaluronic acid-enriched transfer media in cryocycles may be recommended for women over 40 years of age, regardless of the quality of the transferred embryos, as well as for women of any reproductive age with a thin endometrium and a history of miscarriage when transferring good quality embryos.

Factors Affecting the Success of Ovum Pick-Up, In Vitro Production and Cryopreservation of Embryos in Cattle

Increasing global demand for animal proteins warrants improved productivity by genetic selection of superior cattle and faster dissemination of genetics. Availability of more progeny for genomic selection should maximize chances of identifying animals with desirable traits and increase selection pressure. OPU and IVP of embryos using these oocytes will substantially increase calves produced compared to conventional embryo transfer (ET). The OPU-IVP technology not only supports genetic improvement but also contributes to reducing environmental impacts of livestock production systems by improving efficiency and optimizing resources, aligning with the Sustainable Development Goals of the United Nations. However, there are several factors influencing the success of OPU-IVP. This review is focused on these factors and the impacts of in vitro culture conditions on the lipid content of embryos and potential role of L-carnitine, a lipolytic agent, on developmental competence of IVP embryos. The documented effects of L-carnitine and current knowledge regarding regulation of the Hippo signaling pathway suggest that supplementation of embryo culture media with L-carnitine will increase post-thaw survival of IVP embryos and their subsequent developmental competence by regulating lipid metabolism, production of reactive oxygen species, and Hippo signaling. Therefore, this review highlights current advancements in the field of OPU-IVP and potential areas for refining culture conditions to yield developmentally competent embryos that survive cryopreservation procedures.

Influence of selective conditions in vitro on variability of created flax forms

Fiber flax is a strategic crop. Flax products are widely used in various sectors of the economy, including the production of fabrics, paints, medical cotton wool, gunpowder, etc. The occurrence of diseases on flax crops entails a decrease in crop yield. The quality of flax fiber obtained from the affected flax plant deteriorates, and infected seeds reduce germination and biological potential. The most harmful diseases of fiber flax at present are fusarium wilt, rust, anthracnose, pasmo, and bacteriosis. The goal of the research conducted in 2021–2023 on the basis of the laboratory of breeding and biotechnology of the Federal State Budgetary Institution “Federal Scientific Center for Bast Crops” (Tver Region) was to create new flax genotypes resistant to anthracnose (Colletotrichum lini) using biotechnological methods. As a result of research, new forms, more resistant to the pathogen, were created in vitro from flax genotypes susceptible to anthracnose. It was revealed that on a selective background, plants with varying degrees of resistance were formed on the basis of immature embryos. Both resistant and moderately susceptible forms were noted susceptible forms formed on the basis of the same genotype. It was noted that the majority of regenerants obtained during in vitro selection from genotypes susceptible to anthracnose, under the newly created selective conditions, were characterized by resistance and average susceptibility to the cultural filtrate. It has been suggested that the formation of regenerated plants, different from the original forms, against a selective background in the culture of immature fiber flax embryos, is caused by the occurrence of a spontaneous mutation process, which, apparently, does not always depend on the creation of selective conditions. The studies revealed differences in the used strains of the anthracnose pathogen according to morphological characteristics. It was established that the intensity of sporulation and growth of the strains used did not depend on their virulence.

IVF cycle safety when a positive passive air sampling occurs under laminar flow hood in absence of a detectable contamination in the embryo culture

Microbiological contamination in the embryo culture media might affect embryo early development and clinical outcomes during IVF procedures. Infections in the genital tract represent the most common causes of culture contamination, but also environmental air quality might have a detrimental effect on reproductive outcomes of infertile couples undergoing IVF procedures. Monitoring microbiological contamination in an embryology laboratory is mandatory and daily tests are performed under laminar vertical flow hood. In this study, we investigated the IVF outcome of procedures carried out during 5 years of laboratory activity when a positive passive air sampling occurs under laminar flow hood in the absence of clear contamination in the embryo culture. We performed 570 air samplings, and we isolated at least 1 CFU of microorganisms in the TSA settle plate in 13 cases (2.28%). No infections were suspected in the culture media given the absence of detectable microorganisms under the microscope or a turbidity/color change of culture media visible to the naked eye (0% contamination rate). There were no statistically significant differences in biochemical pregnancy, live birth rate, and abortion between the “contaminated” Group P and the “negative” Group N. Surprisingly, we found a better outcome in terms of clinical pregnancy rate in Group P as compared to Group N, a finding likely due to the accidental lower age of Group P (p = 0.0133). Data showed that, in the absence of a detectable contamination in the embryo culture media, IVF cycles are safe when an air positive sample occurs in Grade A environment.

One carbon metabolism supplementation in maturation medium but not embryo culture medium improves the yield of blastocysts from bovine oocytes

Optimizing oocyte maturation and embryo culture media could enhance in vitro embryo production. The purpose of the present study was to investigate the role of supplementing one carbon metabolism (OCM) substrates and its cofactors (Cystine, Zinc, Betaine, B2, B3, B6, B12 and 5-methyltetrahydrofolate) in maturation and/or embryo culture media on the rate of blastocyst formation and pregnancy outcomes following the transfer of the resulting blastocysts in bovines. In the first experiment, 2537 bovine oocytes were recovered from slaughterhouse ovaries and then matured either in conventional maturation medium (IVM) or IVM supplemented with OCM substrates (Sup-IVM). After in vitro fertilization, the putative zygotes from each treatment (IVM or Sup-IVM) were cultured in the media either without (IVM/IVC or Sup-IVM/IVC) or with (IVM/Sup-IVC or Sup-IVM/Sup-IVC) OCM supplementation. The blastocyst rate, assessed on day 8, was significantly increased in Sup-IVM/IVC group (34.90 ± 2.52) as compared to IVM/IVC (17.06 ± 1.69; P = 0.0001) and Sup-IVM/Sup-IVC (20.29 ± 2.75; P = 0.004) and non-significantly as compared to IVM/Sup-IVC (24.86 ± 5.37). In the second experiment, non-matured bovine oocytes were collected by transvaginal ovum pick up after FSH stimulation, randomly allocated into IVM/IVC (n = 275) and Sup-IVM/IVC (n = 260) and the blastocysts achieved at day 7 were transferred in recipient cattle. The blastocyst rate was significantly higher in Sup-IVM/IVC group (38.85%) as compared to the IVM/IVC group (23.64%; P < 0.0001). After single embryo transfer, the supplemented blastocysts were at least as competent as non-supplemented ones with a non-significantly higher (20% vs. 14%) pregnancy rate and the advantage of several good quality blastocysts available for future use. In conclusion, optimizing the maturation medium with OCM substrates and its cofactors could enhance the formation of viable blastocysts with the potential to increase the cumulative birth rate in cattle.

Cell-free fat extract improves embryo development and clinical outcomes in older women with previous in-vitro fertilization failure

BackgroundPreimplantation embryos in vivo are exposed to various growth factors in the female reproductive tract that are absent in in vitro embryo culture media. Cell-free fat extract exerts antioxidant, anti-ageing, and ovarian function-promoting effects. However, its effects on embryo quality are yet to be investigated.MethodsWe assessed the effect of cell-free fat extract supplementation on embryo culture using a naturally ageing mouse model. We assessed the model’s efficacy in influencing embryo development and pregnancy rates in older women with in vitro fertilization failure. In addition, we performed immunofluorescence staining, multiplex immunoassay, whole-genome amplification and DNA sequencing, time-lapse embryo monitoring, and in vitro experiments.ResultsCell-free fat extract-supplemented media has a suitable osmolarity and pH and contains high levels of bioactive growth factors. Cell-free fat extract promoted embryo development and implantation in aged mice, probably by increasing embryo growth rate, inhibiting cell apoptosis, and promoting blastocyst adhesion. Clinical results showed that the cell-free fat extract group had significantly higher rates of the day 3 available and high-quality embryos than the control group, and the rate of usable embryos tended to be higher in the cell-free fat extract group. Furthermore, implantation and clinical pregnancy rates improved in the cell-free fat extract group than in the control group.ConclusionsOur study implies that cell-free fat extract supplementation can promote embryo development and clinical outcomes and may serve as a rescue strategy for older women with in vitro fertilization failure.

The Impact of Cytokine Additives in Embryo Culture Media on Intracytoplasmic Sperm Injection Outcomes and Reproductive Success

The Impact of Cytokine Additives in Embryo Culture Media on Intracytoplasmic Sperm Injection Outcomes and Reproductive Success

Human fallopian tube organoids provide a favourable environment for sperm motility.

Does a human fallopian tube (HFT) organoid model offer a favourable apical environment for human sperm survival and motility? After differentiation, the apical compartment of a new HFT organoid model provides a favourable environment for sperm motility, which is better than commercial media. HFTs are the site of major events that are crucial for achieving an ongoing pregnancy, such as gamete survival and competence, fertilization steps, and preimplantation embryo development. In order to better understand the tubal physiology and tubal factors involved in these reproductive functions, and to improve still suboptimal in vitro conditions for gamete preparation and embryo culture during IVF, we sought to develop an HFT organoid model from isolated adult stem cells to allow spermatozoa co-culture in the apical compartment. Over a 2-year period, fallopian tube tissues were collected for organoid culture purposes from 10 'donor' patients undergoing bilateral salpingectomy by laparoscopy for definitive sterilization. After tissue digestion, isolated cells from the isthmus and ampulla regions were separately seeded in 3D Matrigel and cultured with conventional growth factors for organoid culture and specific factors for differentiation of the female genital tract. HFT organoids were characterized by light microscopy, scanning and transmission electron microscopy, immunofluorescence, and transcriptome analysis. Following simultaneous organoid culture on specific inserts, spermatozoa from five donors were placed either in control media or in the apical compartment of colon or HFT organoids (isthmus and ampulla separately) for 96 h. Vitality and motility and kinematic parameters were assessed at 0, 48, and 96 h on 200 spermatozoa in each condition and in duplicate and compared using the Wilcoxon test. Specific fallopian tube differentiation of our model was confirmed by immunofluorescence, transcriptome analysis, and electron microscopy observations that exhibited ciliated and secretory cells. We succeeded in releasing spermatozoa in the apical compartment of HFT organoids and in recovering them for sperm analysis. Sperm vitality values were similar in HFT organoids and in commercial sperm media. We demonstrated a superiority of the HFT organoid apical compartment for sperm motility compared with other controls (colon organoids, organoid culture media, and conventional commercial sperm fertilization media). At 48 h of incubation, progressive sperm motility was higher in the apical compartment of HFT organoids (ampulla 31% ± 17, isthmus 29% ± 15) than in commercial fertilization media (15% ± 15) (P < 0.05) and compared with all other conditions. At 96 h, progressive sperm motility was almost nil (<1%) in all conditions except for spermatozoa in HFT organoids (P < 0.05): 12% ± 15 and 13% ± 17 in ampulla and isthmus organoids, respectively. Computer-assisted sperm analysis (CASA) analysis also showed that the organoids were able to maintain significantly higher levels of kinematic parameters (curvilinear velocity, average path velocity, straight linear velocity, and amplitude of lateral movement of the head) and therefore more efficient mobility compared with commercial IVF media. N/A. This was an in vitro study in which conditions of organoid culture could not exactly mimic the in vivo environment of the extracellular matrix and vascularization of fallopian tubes. This work opens up perspectives for better understanding of HFT physiology. For the first time, it highlights the possibility of developing HFT organoids for reproductive purposes. In the future, it could help us to improve gamete fertilizing abilities and embryo culture conditions during human ARTs. This study was funded by a grant from the Occitanie region, and by financial allocations from the DEFE and IRSD research teams. The authors have no conflicts of interest to report.

Reichert's membrane - a continuing enigma for developmental biologists.

Reichert's membrane (RM) is a basement membrane of gigantic proportions that surrounds the mammalian embryo following implantation. It is part of the parietal yolk sac, which originates from the wall of the preimplantation blastocyst. RM persists from implantation to birth in rodents and analogous structures occur in other mammals, including primates. RM fulfils a vital role in early postimplantation rodent development, by mechanically buffering the elongating, pre-gastrulation embryo against vigorous uterine contractions, which occur at that stage of pregnancy. It persists and enlarges throughout the remainder of gestation, to match the increasing volume of the embryo/fetus and its extraembryonic membranes, although its function at these later stages is unknown. By contrast, in whole embryo culture, RM fails to expand sufficiently to enable normal embryonic development, so that routine preparation for culture includes opening and removal of RM. Modifying the culture conditions can improve embryonic outcomes when RM is intact, but does not normalise development. Possible reasons for the dichotomy between the in vivo and in vitro significance of RM include: the absence of uterine contractions in vitro, which may serve to induce RM expansion in vivo; the absence of a decidual influence, which plays a vital role in maintaining embryos beyond implantation; failure of RM to grow in vitro, owing to insufficient proliferation or survival of the parietal endoderm, which synthesises RM; insufficient cellular recruitment into the parietal endoderm from the visceral endoderm; failure of RM to lose its physical integrity in vitro, thereby limiting its expansion. Distinguishing between these possible mechanisms will require new experimental research on RM.

Study of the feasibility of polar body transfer combined with preimplantation genetic testing for blocking the intergenerational transmission of mitochondrial genetic diseases

To assess the feasibility of first polar body transfer (PB1T) combined with preimplantation mitochondrial genetic testing for blocking the transmission of a pathogenic mitochondrial DNA 8993T>G mutation. A Chinese family affected with Leigh syndrome which had attended the Reproductive Medicine Centre of the First Affiliated Hospital of Anhui Medical University in September 2021 was selected as the study subject. Controlled ovarian hyperstimulation was carried out for the proband after completing the detection of the mitochondrial DNA 8993T>G mutation load among the pedigree members. Mature MII oocytes were inseminated by intracytoplasmic sperm injection (ICSI), cultured in vitro for 5 to 6 days to the blastocyst stage, and trophoblastocytes were obtained by microbiopsy. Mitochondrial DNA testing (PGT-MT) and chromosomal aneuploidy (PGT-A) analyses were carried out after whole-genome amplification, and the embryos with zero mutation load were selected for transfer. Amniotic fluid and umbilical cord blood samples were collected during middle pregnancy and after birth respectively for mitochondrial DNA testing to verify the reliability of embryo screening. As an attempt, PB1 with good morphology of MII oocytes was selected for transfer into the enucleated oocytoplasm from healthy donors, followed by ICSI fertilization, blastocyst culture and PGT of embryos using the same procedure. This study has been approved by the Ethics Committee of the First Affiliated Hospital of Anhui Medical University (No. 2021zhyx-B12). An antagonist protocol was used for ovarian stimulation, and a total of 19 oocytes were obtained, of which 14 MII were fertilized by ICSI, and 2 had developed into blastocysts. PGT-MT was carried out on biopsied trophoblastocytes, in which the mitochondrial DNA 8993T>G mutation load was not detected in one embryo, the other was 100% mutated, and the mutation loads of the remaining unfertilized eggs and developmentally arrested embryos ranged from 0% ~ 100%, presenting a clear biased distribution. With fully informed consent, one PGT-MT zero mutation load blastocyst was transferred and clinical pregnancy was achieved. Mitochondrial DNA and chromosomal testing of amniotic fluid cells during middle pregnancy had revealed no abnormalities. The proband had delivered a healthy boy through Caesarean section at 39+5 weeks of gestation, and no mutation was detected in the cord blood sample. Five well-formed PBs from 14 eggs were selected for PB1 transfer, followed by ICSI and culture, and two of the reconstituted embryos had formed blastocysts, with none of the above mutations detected in the biopsied samples. The PGT-MT technology can help families affected with mitochondrial diseases to have healthy offspring. PB1 transfer in combination with ICSI and PGT-MT holds the promise of turning waste into treasure and providing an alternative means of fertility for such families.

Study on the embryotoxic effects and potential mechanisms of Aconitum diterpenoid alkaloids in rat whole embryo culture through morphological and transcriptomic analysis

Ethnopharmacological relevanceThe lateral root of Aconitum carmichaelii Debeaux, or Fuzi, is recognized in Asia for its anti-inflammatory, analgesic, and cardiotonic effects. Its main active compounds are diester diterpenoid alkaloids (DDAs) such as aconitine (AC), mesoacitine (MA), and hypoaconitine (HA), which are also toxic and have a narrow therapeutic window, limiting their clinical use. Although Aconitum DDAs are known for cardiotoxic and neurotoxic effects, their impact on embryonic development remains unclear. Aim of the studyThe embryotoxicity of three representative Aconitum DDAs (AC, MA, and HA) and their metabolites were systematically assessed, and the mechanisms underlying AC-induced embryotoxicity was explored. Materials and methodsThe embryotoxicity of these DDAs was assessed by indicators such as morphological scores in a whole embryo culture (WEC) system. Immunofluorescence analysis was conducted to detect DNA damage and apoptosis in embryos, and transcriptomic analysis and western blotting were performed to explore the underlying mechanisms. ResultsDDAs, particularly AC, induced dose-dependent developmental retardation and malformation in rat embryos. Notably, the embryotoxicity of AC metabolites such as benzoyltrypine (BAC) and aconine, was significantly reduced. AC treatment caused substantial DNA damage and apoptosis in embryos. Transcriptomic analysis indicate that AC treatment may impair DNA replication and histone synthesis by activating the p53/p21/CDK2/NPAT pathway, ultimately affecting embryonic development. ConclusionAmong these Aconitum DDAs, AC exhibited the strongest embryotoxicity, mainly through DNA damage and regulation of histone genes via the p53/p21/CDK2/NPAT pathway.

3-methyl-4-nitrophenol disturbs the maternal-to-zygotic transition of early embryos by damaging mitochondrial function and histone modification.

3-methyl-4-nitrophenol (PNMC), a chemical prevalent in various industries for drug, dye, and leather production, also serves as a primary byproduct of organophosphate insecticides. Despite its global recognition as an endocrine disruptor with documented reproductive toxicity, its detrimental impact on preimplantation embryonic development has yet to be thoroughly investigated. In this study, through the in vitro culture of mice embryos, it was initially observed that even low concentrations of PNMC exposure led to a significant reduction in blastocyst formation and a sharp decline in the ratio of inner cell mass within the blastocysts. SMART-seq2 transcriptome sequencing further confirmed that PNMC treatment disrupted global gene expression in 2-cell embryos, with differentially expressed genes enriched in multiple signaling pathways, including those related to autophagy, apoptosis, fertilization, embryonic development, transcription, and mRNA processing. Integration of transcriptome data with open databases revealed that both zygotic genome activation genes and maternal factors experienced significant transcript-level disruptions. Moreover, the study demonstrated that these gene expression changes were closely associated with mitochondrial dysfunction, evidenced by diminished mitochondrial membrane potential, reduced ATP production, aberrant expression of mitochondria-related genes, increased ROS accumulation, and heightened DNA damage in PNMC-treated embryos. Additionally, PNMC exposure induced defects in histone modification, as shown by altered levels of H3K9me3 and H3K27me3, H3K9ac and H3K27ac. Lastly, the findings indicated that PNMC triggered apoptosis in embryos, validated by elevated BAX and CASPASE3 expression, alongside positive TUNEL staining. In summary, PNMC exposure impairs the maternal-to-zygotic transition, likely through mitochondrial dysfunction and histone modification, culminating in developmental arrest and apoptosis in mouse preimplantation embryos.

Speed breeding advancements in safflower (Carthamus tinctorius L.): a simplified and efficient approach for accelerating breeding programs.

This study investigated the potential of extended irradiation combined with immature embryo culture techniques to accelerate generation advancements in safflower (Carthamus tinctorius L.) breeding programs. We developed an efficient speed breeding method by applying light-emitting diodes (LEDs) that emit specific wavelengths, alongside the in vitro germination of immature embryos under controlled environmental conditions. The experimental design for light treatments followed a 2 × 4 completely randomized factorial design with four replications, incorporating two safflower varieties, Remzibey-05 and Dinçer, and four LED treatments (white, full-spectrum, red + blue + white, and control). A lighting regimen of 22 h of light and 2 h of darkness was applied for all the LED treatments, whereas the control received 18 h of light and 6 h of darkness. Additionally, the immature embryo culture experiment used a 2 × 2 × 4 factorial arrangement, assessing two safflower cultivars, two media types, and four embryo developmental stages, with three replications. The parameters evaluated included plant height, branch number, seed number per plant, seed number per head, time to flower initiation, time to 50% flowering, time to harvest, and germination percentage of in vitro cultured immature embryos at various developmental stages. The harvest time among the light treatments ranged from 50.62 to 73.12days, with the shortest time achieved under the red + blue + white LED combination and the longest under the control treatment. The plant height, number of seeds per plant, and number of seeds per head were highest under the full-spectrum LED, control and red + blue + white LED combinations, respectively. Immature embryos rescued at 10dayspost-pollination presented a 57% germination rate, with an increasing trend in germination as the number of dayspost-pollination increased. The germination rates did not significantly differ across varieties or hormone treatments. This study demonstrated the potential to achieve six generations per year by combining prolonged illumination with targeted LED lighting and immature embryo culture techniques. These findings provide valuable insights for optimizing safflower growth and development and advancing speed breeding in controlled environments.

Modification of Gene Expression Involved in Alkaloid Production in Opium Poppy by VIGS Combined With Pretreatment of Macerozyme Enzyme.

Papaver somniferum L., a medicinal plant renowned for its pharmaceutical alkaloids, has captivated scientific interest due to its rich secondary metabolite profile. This study explores a novel approach to manipulating alkaloid biosynthesis pathways by integrating virus-induced gene silencing (VIGS) with macerozyme enzyme pretreatment. Targeting key genes in the benzylisoquinoline alkaloid (BIA) pathway (CODM, T6ODM, COR, DIOX2), the research aimed to elucidate the transformative potential of enzymatic preconditioning in somatic embryo cultures. To address the cell wall barrier, a known limitation in genetic manipulation, macerozyme pretreatment was employed, significantly enhancing gene silencing efficacy. Quantitative reverse transcription PCR analyses revealed significant alterations in gene expression profiles with macerozyme pretreatment, whereas no changes were observed in its absence. The T6ODM + DIOX combination was the most effective, reducing CODM, T6ODM, and DIOX2 expression by 72%, 65%, and 60%, respectively. Conversely, T6ODM expression increased by up to 107% in the CODM treatment. Notably, COR expression displayed dual regulatory dynamics, with suppression (47% decrease in T6ODM + DIOX) and enhancement (49% increase in CODM+DIOX) observed under different conditions. These findings underscore the complex interplay of gene regulation in the morphine biosynthesis pathway. This study highlights the critical role of macerozyme enzymatic pretreatment in overcoming cell wall barriers, enabling effective VIGS applications in somatic suspension cultures. The combination of VIGS and enzymatic pretreatment provides a robust platform for targeted metabolic engineering, offering insights into the regulation of morphine biosynthesis and paving the way for advancements in pharmaceutical alkaloid production and functional genomics in medicinal plants.

Genetic characterization of cpti and nptii genes in the hybrids of transgenic and non-transgenic apple

To investigate the inheritance and expression of exogenous cowpea trypsin inhibitor (CpTI) and neomycin phosphotransferase II (nptII) genes in the progenies of transgenic apples (Malus domestica Borkh.), hybrid (F1) fruits and seeds were obtained using transgenic Gala and non-transgenic Fuji for cross and reciprocal cross experiments. Transgenic apple hybrid F1 plants were obtained using the established apple embryo culture system. The law of segregation of nptII in the hybrid progeny was studied through successive selections under high-level kanamycin pressure. The genetic linkage between CpTI and nptII was tested by PCR analysis. The effects of exogenous genes on transgenic apple fruits were measured by nptII enzyme activity, fruit weight, fruit shape index, fruit hardness, soluble solids content, acid content, and other related indicators in the transgenic Gala fruit. The kanamycin resistance test in F1 plantlets 1:1 Mendelian ratio of kanamycin resistance to non-resistance . Thus, the nptII marker gene, a dominant heterozygous gene, passed onto the progenies through sexual hybridization. PCR analysis also showed that CpTI was closely linked to nptII . The activity of nptII enzyme was detected in most transgenic apples, but the intensity of nptII enzyme activity in different fruits was different.. Transgenic Gala fruit and the control fruit were oblate or nearly round, with the fruit shape index ranging between 0.83 and 0.90. Acid content of a transgenic fruit was not significantly different compared to the control. Fruit hardness and soluble solids content were significantly lower compared to the control. The results provide a reference for further studies on the genetic characteristics of transgenic apples and the utilization of transgenic products. Bangladesh J. Bot. 53(4): 1043-1053, 2024 (December)

Efficiency of methods of cultivation of feline embryos depending on the terms of their obtaining after ovarioectomy

Efficiency of methods of cultivation of feline embryos depending on the terms of their obtaining after ovarioectomy

Comparison of Non-Invasive and Minimally Invasive Preimplantation Genetic Testing for Aneuploidy Using Samples Derived from the Same Embryo Culture.

Background/Objectives: To assess the ploidy status of embryos via preimplantation genetic testing for aneuploidy (PGT-A), a biopsy of trophectoderm (TE) cells can be performed. However, this approach is considered invasive, and therefore the aim of this study was to identify the optimal sample type and sampling day for non-invasive or minimally invasive PGT-A (ni/miPGT-A) in terms of data quality and concordance rates with TE biopsies derived from the same embryos. Methods: This study was performed using 239 embryo cultures. After optimization using 96 embryos, non-invasive spent culture media (SCM) and a minimally invasive combination of blastocoel fluid and SCM (BF+SCM), along with the corresponding TE samples, were collected from 143 embryos cultured for 5 days (n = 70) or 6 days (n = 73), and all were subjected to ni/miPGT-A with whole-genome amplification followed by next-generation sequencing. Results: The amplification failure rate was lower for SCM samples than for BF+SCM (SCM: 0.7%, 1/143 vs. BF+SCM: 7.7%, 11/143; p = 0.005). The rate of ploidy concordance with TE was significantly higher for SCM samples than for BF+SCM samples (SCM: 83.7%, 118/141 vs. BF+SCM: 58%, 76/131; p < 0.001). Among SCM samples, concordance rates were higher for samples derived from embryos cultured for 6 days (87.5%, 63/72) than for 5 days (79.7%, 55/69). In the embryos cultured for 6 days, discordant cases included five (6.9%) SCM samples with falsely negative (euploid) results that were deemed to be mosaic according to TE and four (5.6%) samples falsely found to be aneuploid. Conclusions: SCM samples derived from embryos cultured for 6 days can be applied in niPGT-A with subsequent verification of aneuploid samples using TE biopsy.

Biogenesis of Extracellular Vesicles (EVs) and the Potential Use of Embryo-Derived EVs in Medically Assisted Reproduction.

Extracellular vesicles (EVs) are lipid bilayer-bound particles released from cells that cannot replicate on their own, play a crucial role in intercellular communication, and are implicated in various physiological and pathological processes. Within the domain of embryo culture media research, extensive studies have been conducted to evaluate embryo viability by analyzing spent culture medium. Advanced methodologies such as metabolomic profiling, proteomic and genomic analyses, transcriptomic profiling, non-coding RNA assessments, and oxidative status measurements have been employed to further understand the molecular characteristics of embryos and improve selection criteria for successful implantation. In the field of EVs, only a limited number of studies have been conducted on embryo-conditioned medium, indicating a significant gap in knowledge regarding the potential role of EVs in embryo development and implantation. Therefore, this review aims to evaluate current research findings on EVs enriched from animal and human embryo spent medium. By unraveling the potential link between embryo-derived EVs and embryo selection in clinical settings, such research might enhance embryo-selection methods in assisted reproductive technologies, eventually increasing the success rates of fertility treatments and advancing our understanding of mechanisms underlying successful embryo development and implantation in humans.

Molecular Markers in Embryo Non-Development: Analysis of Gene Expressions (Ki-67, hTERT, HIF-1α) in Spent Embryo Culture Medium.

An embryo culture medium is a specialized set of ambient conditions, technological equipment, and nutrients that embryos require to grow properly. We aimed to investigate the Ki-67, hTERT, and HIF-1α gene expression differences between developing and non-developing embryos in spent embryo culture medium. Ki-67, hTERT, and HIF-1α gene expressions were determined from the spent embryo culture medium containing developing and non-developing embryos of 20 normoresponder patients admitted to the Bahçeci Umut IVF Center. An increase in hTERT gene expression (p < 0.05) and a decrease in HIF-1α gene expression (p < 0.001) were observed in mediums of developing compared to the non-developing embryos. No difference was observed in Ki-67 gene expression (p > 0.05). While there was a correlation between Ki-67 and HIF-1α genes in the non-growing group (r < 0.01); no correlation was observed in the developing group (r > 0.05). Both normoresponder groups will be similar in terms of proliferation rate. The low HIF-1α expression that observed high telomerase activity in embryo development maintains continuity and avoids mechanisms that result in cell death. A molecular study of the embryo development in patients with similar characteristics may help to understand the pathogenesis of the disease and establish a diagnosis and specific treatment.

Controlled Dynamic Microfluidic Culture of Murine, Bovine, and Human Embryos Improves Development: Proof-of-Concept Studies.

Classical preimplantation embryo culture is performed in static fluid environments. Whether a dynamic fluid environment, like the fallopian tube, is beneficial for embryo development remains to be determined across mammalian species. Objectives of these proof-of-concept studies were to determine if controllable dynamic microfluidic culture would enhance preimplantation murine, bovine, and human embryo development compared to static culture. This prospective randomized controlled trial tested static versus controlled dynamic culture of preimplantation mouse (n = 397), bovine (n = 242), and human (n = 512) zygotes to blastocyst stages with outcome measures of embryo cleavage, cellular fragmentation, apoptosis, and blastocyst conversion rates. Dynamic culture of mouse and bovine zygotes with microfluidics significantly improved embryo development. Mouse placental imprinted gene expression was significantly different between embryos derived in vivo, by static culture, and by dynamic culture. Using human sibling zygotes, this dynamic microfluidic culture system increased the number of blastomeres per cleavage-stage embryo, reduced cellular fragmentation or apoptosis, improved blastocyst conversion rates, and enhanced blastocyst developmental stages. In conclusion, species-specific longitudinal studies demonstrated that dynamic microfluidic culture significantly improved embryo development, independent of culture media composition, temperature, and gaseous environment. These cellular indicators represent improved embryo development that can translate into higher pregnancy rates in transgenics, domestic livestock and endangered species and treating human infertility.