Subsequent Embryo Research Articles

Lipid metabolites, interleukin-6 and oxidative stress markers in follicular fluid and their association with serum concentrations in mares.

The application of trans-vaginal ovum pick up (OPU) and intracytoplasmic sperm injection (ICSI) is well established for commercial in vitro embryo production in horses. These assisted reproductive techniques are especially applied during the non-breeding season of the mare. However, little is known about how the health of the oocyte donor may affect the biochemical composition of the follicular fluid (FF) in small and medium-sized follicles routinely aspirated during OPU. This study aimed to investigate associations between systemic and FF concentrations of interleukin-6 (IL-6), total cholesterol, triglycerides, non-esterified fatty acids (NEFA), reactive oxygen metabolites (d-ROMs), biological antioxidant potential (BAP), and oxidative stress index (OSI) during the non-breeding season in mares. At the slaughterhouse, serum and FF of small (5-10mm in diameter), medium (> 10-20mm in diameter), and large (> 20-30mm in diameter) follicles were sampled from 12 healthy mares. There was a strong positive association (P < 0.01) between the concentration of IL-6 in serum and those measured in small (r = 0.846), medium (r = 0.999), and large (r = 0.996) follicles. Serum concentrations of NEFA were positively correlated (P < 0.05) with those measured in small (r = 0.726), medium (r = 0.720), and large (r = 0.974) follicles. Values of total cholesterol and OSI in serum and medium follicles were significantly associated (r = 0.736 and r = 0.696, respectively). The serum concentrations of all lipid metabolites were markedly higher than those measured in FF of small- and medium-sized follicles. Values of IL-6 and OSI did not change significantly between serum and all follicle classes (P ≥ 0.05). To conclude, changes in the blood composition associated with inflammation, oxidative stress, and disturbed lipid metabolism of mares may lead to an inadequate oocyte microenvironment, which could affect oocyte quality and the success rate of OPU/ICSI programs. Further research should indicate whether these changes may ultimately affect in vitro oocyte developmental capacity and subsequent embryo quality.

The bisphenol S contamination level observed in human follicular fluid affects the development of porcine oocytes.

Bisphenol S (BPS), the main replacement for bisphenol A (BPA), is thought to be toxic, but limited information is available on the effects of Bisphenol S on ovarian follicles. In our study, we demonstrated the presence of Bisphenol S in the follicular fluid of women at a concentration of 22.4nM. The effect of such concentrations of Bisphenol S on oocyte maturation and subsequent embryo development is still unknown. Therefore, we focused on the effect of Bisphenol S on in vitro oocyte maturation, fertilization, and embryo development. As a model, we used porcine oocytes, which show many physiological similarities to human oocytes. Oocytes were exposed to Bisphenol S concentrations similar to those detected in female patients in the ART clinic. We found a decreased ability of oocytes to successfully complete meiotic maturation. Mature oocytes showed an increased frequency of meiotic spindle abnormalities and chromosome misalignment. Alarming associations of oocyte Bisphenol S exposure with the occurrence of aneuploidy and changes in the distribution of mitochondria and mitochondrial proteins were demonstrated for the first time. However, the number and quality of blastocysts derived from oocytes that successfully completed meiotic maturation under the influence of Bisphenol S was not affected.

Does smooth endoplasmic reticulum aggregation in oocytes impact the chromosome aneuploidy of the subsequent embryos? A propensity score matching study

BackgroundThe appearance of smooth endoplasmic reticulum aggregation (SERa) is one of the most common dysmorphic phenotypes of oocytes, however, the impact of SERa occurrence on in vitro fertilization (IVF) outcomes is controversial. This study aimed to investigate the impact of SERa in oocytes on the aneuploidy of the subsequent embryos in IVF.MethodsIn this retrospective cohort study, a total of 114 intracytoplasmic sperm injection (ICSI) cycles with the appearance of SERa undergoing preimplantation genetic testing for aneuploidy (PGT-A) were enrolled, and among them there were 323 SERa(+) oocytes and 1253 sibling unaffected oocytes. The 907 PGT-A cycles without SERa during the same period were enrolled as controls. A propensity score matching of 1:1 ratio between these two groups resulted in 113 matched cycles. The outcome parameters between the SERa(+) cycles/oocytes and the controls were compared. IVF laboratory outcomes, PGT-A outcomes, and clinical and neonatal outcomes were the main outcomes.ResultsIncreased abnormal fertilization rate and reduced blastocyst formation rate can be observed in both SERa(+) cycles and oocytes, some other parameters on developmental potential, such as available embryo rate at Day 3 and available blastocyst rate, were also impaired in the case of SERa occurrences. Among the 910 blastocysts for PGT-A, the percentage of euploid embryos was similar between the matched cohorts, while an unpredicted increase of the proportions of euploid in the SERa(+) oocytes, compared to the SERa(-) oocytes. Moreover, there was no significance in terms of clinical and neonatal outcomes, such as implantation rate, biochemical pregnancy rate, clinical pregnancy rate, miscarriage rate, and live birth rate, regardless of the presence of SERa in cycles and oocytes.ConclusionsThe appearance of SERa within mature oocytes has no significant impact on the aneuploidy of subsequent blastocysts. It is recommended to utilize these oocytes, especially for those with few oocytes or advanced maternal age, which is likely to increase the cumulative pregnancy rate. This study may offer evidence to assist embryologists to make clinical decisions concerning SERa(+) oocytes more consciously and rationally.

Dendrobine enhances bovine oocyte maturation and subsequent embryonic development and quality

Strategies for improving the quality of oocytes have important theoretical and practical significance for increasing the efficiency of livestock breeding. In this respect, the accumulation of reactive oxygen species (ROS) is a major factor affecting the development of oocytes and embryos. This study investigated the effects of Dendrobium nobile extract (DNE) on the in vitro maturation of bovine oocytes and embryonic development after IVF. DNE is an extract from Dendrobium rhizomes that contains alkaloids with anti-inflammatory, anti-cancer and anti-ageing functions. Various concentrations of DNE (0, 5, 10, 20 and 50 μmol/L) were added during oocyte maturation in vitro, and we found that 10 μmol/L of DNE remarkably increased the oocyte maturation rate, the subsequent blastocyst formation rate and embryo quality. Further, we found that DNE treatment decreased the frequency of spindle/chromosome defects and ROS and increased the oocyte glutathione and mitochondrial membrane potential in oocytes. Moreover, DNE upregulated the expression of oxidative stress-related genes (Sirt1, Sirt2, Sirt3 and Sod1) in oocytes and apoptosis-related genes (Caspase-3, Caspase-4, Bax, Bcl-xl and Survivin) in blastocysts. These results suggest that DNE supplementation can promote oocyte maturation and subsequent embryonic development by regulating redox reactions and inhibiting embryonic apoptosis.

Vitrification of porcine immature oocytes and zygotes results in different levels of DNA damage which reflects developmental competence to the blastocyst stage.

The present study investigated the effects of vitrification of porcine oocytes either at the immature Germinal Vesicle (GV) stage before in vitro maturation (GV-stage oocytes) or at the pronuclear stage after in vitro maturation and fertilization (zygotes) on DNA integrity in relevance with their subsequent embryo development. Vitrification at the GV stage but not at the pronuclear stage significantly increased the abundance of double-strand breaks (DSBs) in the DNA measured by the relative fluorescence after γH2AX immunostaining. Treatment of GV-stage oocytes with cryoprotectant agents alone had no effect on DSB levels. When oocytes were vitrified at the GV stage and subjected to in vitro maturation and fertilization (Day 0) and embryo culture, significantly increased DSB levels were detected in subsequent cleavage-stage embryos which were associated with low cell numbers on Day 2, the upregulation of the RAD51 gene at the 4-8 cell stage (measured by RT-qPCR) and reduced developmental ability to the blastocyst stage when compared with the non-vitrified control. However, total cell numbers and percentages of apoptotic cells (measured by TUNEL) in resultant blastocysts were not different from those of the non-vitrified control. On the other hand, vitrification of zygotes had no effect on DSB levels and the expression of DNA-repair genes in resultant embryos, and their development did not differ from that of the non-vitrified control. These results indicate that during vitrification GV-stage oocytes are more susceptible to DNA damages than zygotes, which affects their subsequent development to the blastocyst stage.

The combined effect of BMI and age on ART outcomes.

For a woman with infertility and overweight/obesity, can infertility treatment be postponed to first promote weight loss? Advice regarding a delay in IVF treatment to optimize female weight should consider female age, particularly in women over 38 years for whom only substantial weight loss in a short period of time (3 months) seems to provide any benefit. Body weight excess and advanced age are both common findings in infertile patients, creating the dilemma of whether to promote weight loss first or proceed to fertility treatment immediately. Despite their known impact on fertility, studies assessing the combined effect of female age and BMI on cumulative live birth rates (CLBRs) are still scarce and conflicting. We performed a multicentre retrospective cohort study including 14 213 patients undergoing their first IVF/ICSI cycle with autologous oocytes and subsequent embryo transfers, between January 2013 and February 2018 in 18 centres of a multinational private fertility clinic. BMI was subdivided into the following subgroups: underweight (<18.5 kg/m2), normal weight (18.5-24.9 kg/m2), overweight (25.0-29.9 kg/m2), and obesity (≥30.0 kg/m2). The primary outcome was CLBR. The secondary outcome was time to pregnancy. To assess the influence of female age and BMI on CLBR, two multivariable regression models were developed with BMI being added in the models as either an ordinal categorical variable (Model 1) or a continuous variable (Model 2) using the best-fitting fractional polynomials. CLBR was estimated over 1-year periods (Model 1) and shorter timeframes of 3 months (Model 2). We then compared the predicted CLBRs according to BMI and age. When compared to normal weight, CLBRs were lower in women who were overweight (adjusted odds ratio (aOR) 0.86, 95% CI 0.77-0.96) and obese (aOR 0.74, 95% CI 0.62-0.87). A reduction of BMI within 1 year, from obesity to overweight or overweight to normal weight would be potentially beneficial up to 35 years old, while only a substantial reduction (i.e. from obesity to normal BMI) would be potentially beneficial in women aged 36-38 years. Above 38 years of age, even considerable weight loss did not compensate for the effect of age over a 1-year span but may be beneficial in shorter time frames. In a timeframe of 3 months, there is a potential benefit in CLBR if there is a loss of 1 kg/m2 in BMI for women up to 33.25 years and 2 kg/m2 in women aged 33.50-35.50 years. Older women would require more challenging weight loss to achieve clinical benefit, specifically 3 kg/m2 in women aged 35.75-37.25 years old, 4 kg/m2 in women aged 37.50-39.00 years old, and 5 kg/m2 or more in women over 39.25 years old. This study is limited by its retrospective design and lower number of women in the extreme BMI categories. The actual effect of individual weight loss on patient outcomes was also not evaluated, as this was a retrospective interpatient comparison to estimate the combined effect of weight loss and ageing in a fixed period on CLBR. Our findings suggest that there is potential benefit in weight loss strategies within 1 year prior to ART, particularly in women under 35 years with BMI ≥25 kg/m2. For those over 35 years of age, weight loss should be considerable or occur in a shorter timeframe to avoid the negative effect of advancing female age on CLBR. A tailored approach for weight loss, according to age, might be the best course of action. No specific funding was obtained for this study. All authors have no conflicts to declare. N/A.

Restoration of Developmental Competence in Low-Quality Porcine Cumulus–Oocyte Complexes through the Supplementation of Sonic Hedgehog Protein during In Vitro Maturation

The sonic hedgehog (SHH) pathway is an important signaling pathway for mammalian ovarian folliculogenesis and oocyte maturation. A previous study demonstrated that low-quality porcine cumulus-oocyte complexes (COCs) have low developmental competence, with lower SHH signaling protein expression before and after in vitro maturation (IVM) than high-quality COCs. However, there is no reported evidence on the restorative effects of SHH protein supplementation during the IVM of low-quality porcine COCs. Therefore, this study investigated the effects of SHH protein supplementation on the IVM of low-quality porcine COCs, as assessed by brilliant cresyl blue (BCB) staining. To examine this, we designed four groups: (i) BCB- (low-quality), (ii) BCB- + SHH, (iii) BCB+ (high-quality), and (iv) BCB+ + SHH. While the supplementation of SHH protein with high-quality COCs had no effect, supplementation with low-quality COCs significantly improved cumulus cell expansion, metaphase II rate, and subsequent embryo development following parthenogenetic activation. Our results provide the first evidence that the low developmental competence of low-quality porcine COCs can be improved by supplementation with the SHH protein. These results indicate that an active SHH signaling pathway is required for the acquisition of developmental competence in porcine COCs.

Should the Treatment of Patients with Repeated Embryo Implantation Failure Be Adapted as a Function of the Endometrial Cytokine Profile? A Single-Center Experience.

Repeated embryo implantation failures (RIF) is a source of distress and frustration for patients and clinicians alike. Today's approaches for treating RIF are largely empirical and have limited effectiveness. The main causes of RIF are poor endometrial receptivity and poor-quality embryos. Recent studies have suggested the involvement of immune dysregulation due to an imbalance between T-helper (Th) 1 and Th2 cytokines; this opens up perspectives for treating women with RIF and increasing the implantation rate. We conducted an interventional, longitudinal, prospective cohort study of the impact of correcting the cytokine imbalance on the clinical pregnancy rate in women with RIF. Seventy-seven women with RIF underwent an endometrial biopsy during the implantation window. The cytokine profile was evaluated by studying the activation and maturation of uterine natural killer (uNK) cells, the IL-15/Fn-14 mRNA ratio (a biomarker of uNK activation/maturation), and the IL-18/TWEAK mRNA ratio (a marker of angiogenesis and the Th1/Th2 balance). Personalized treatment was initiated for women with an abnormal endometrial cytokine profile (hyper-activation or hypo-activation). We documented the clinical pregnancy rate after subsequent embryo transfers. In total, 72.7% (56/77) of patients had an abnormal endometrial cytokine profile (hyper-activation in 68.8% (n = 53) and hypo-activation in 3.9% (n = 3). After treatment (or not) as a function of the endometrial profile, the overall clinical pregnancy rate was 30.2%. Our results indicated a potential positive effect of appropriate treatment on the ongoing pregnancy rate in women with RIF, despite the small number of cases analyzed. The results must now be validated in randomized studies with larger numbers of well-characterized patients. By applying a previously published decision tree, this treatment approach could be implemented in clinics worldwide.

Deep learning analysis of endometrial histology as a promising tool to predict the chance of pregnancy after frozen embryo transfers.

Endometrial histology on hematoxylin and eosin (H&E)-stained preparations provides information associated with receptivity. However, traditional histological examination by Noyes' dating method is of limited value as it is prone to subjectivity and is not well correlated with fertility status or pregnancy outcome. This study aims to mitigate the weaknesses of Noyes' dating by analyzing endometrial histology through deep learning (DL) algorithm to predict the chance of pregnancy. Endometrial biopsies were taken during the window of receptivity from healthy volunteers in natural menstrual cycles (group A) and infertile patients undergoing mock artificial cycles (group B). H&E staining was performed followed by whole slide image scanning for DL analysis. In a proof-of-concept trial to differentiate group A (n=24) vs. B (n=37), a DL-based binary classifier was trained, cross-validated, and achieved 100% for accuracy. Patients in group B underwent subsequent frozen-thawed embryo transfers (FETs) and were further categorized into "pregnant (n=15)" or "non-pregnant (n=18)" sub-groups based on the outcomes. In the following trial to predict pregnancy outcome in group B, the DL-based binary classifier yielded 77.8% for accuracy. Its performance was further validated by an accuracy of 75% in a "held-out" test set where patients had euploid embryo transfers. Furthermore, the DL model identified histo-characteristics including stromal edema, glandular secretion, and endometrial vascularity as important features related to pregnancy prediction. DL-based endometrial histology analysis demonstrated its feasibility and robustness in pregnancy prediction for patients undergoing FETs, indicating its value as a prognostic tool in fertility treatment.

Laparoscopy versus open surgery for the surgical management of tubo-ovarian abscess (TOA). Is there a beneficial impact of early endoscopic intervention in terms of fertility rates?

To compare success rates and complications in women undergoing laparoscopic versus open surgical management of tubo-ovarian abscess. We further examined whether early laparoscopic intervention has any impact on pregnancy rates in a subgroup of infertile patients following frozen-thawed embryo transfer. Hospital records of 48 patients diagnosed with TOA between January 2015 and December 2020, who underwent surgical intervention or received only medical treatment were analyzed. All patients were hospitalized, and parenteral antibiotics were commenced on admission initially. Laparoscopic or open surgery was performed within 48 hours course of intravenous antibiotherapy (early intervention) or later according to the clinical findings and antibiotherapy response. Of 48 patients with TOA, 18 (37.5%) underwent laparoscopic and 30 (62.5%) underwent open surgical intervention. The median postoperative hospital stay was shorter (4.5 days vs 7.5 days, respectively; p = 0.035), and postoperative opioid analgesic requirement was lesser in the laparoscopy group compared to open surgery group (22% vs 53%, respectively; p = 0.034). Intra- and post-operative complication rates were similar between the groups. Of these 48 patients, seven were diagnosed to have TOA following oocyte retrieval, and four of these conceived with frozen thawed embryo transfer all of whom underwent laparoscopic surgery within 48 hours of diagnosis. Minimal invasive surgery should be preferred even in the presence of severely adhesive and inflammatory TOA in order to improve postoperative outcomes. Moreover, early laparoscopic intervention may be considered in infertile patients with an aim to optimize pregnancy rates in a subsequent frozen-thawed embryo transfer.

GnRHa trigger—the story of the ugly duckling

The objective of this invited review is to describe the introduction and development of the GnRH agonist (GnRHa) trigger protocol in modern invitro fertilization, focusing on ovarian hyperstimulation syndrome (OHSS) prevention and, equally important on the role of GnRHa trigger as an opener of the luteal phase "black box." The GnRHa trigger and freezing of all embryos is the ultimate weapon against OHSS in the OHSS-risk patient. In the non OHSS-risk patient, GnRHa trigger followed by a modified luteal phase support with lutein hormone activity and subsequent fresh embryo transfer results in excellent reproductive outcomes. Thus, the GnRHa trigger has paved the way for a virtually OHSS-free clinic, and equally important is the fact that the early lessons learned from studying the GnRHa trigger opened the "black box" of the luteal phase enabling improved reproductive outcomes in both fresh and frozen embryo transfer cycles.

LSM1-mediated Major Satellite RNA decay is required for nonequilibrium histone H3.3 incorporation into parental pronuclei

Epigenetic reprogramming of the parental genome is essential for zygotic genome activation and subsequent embryo development in mammals. Asymmetric incorporation of histone H3 variants into the parental genome has been observed previously, but the underlying mechanism remains elusive. In this study, we discover that RNA-binding protein LSM1-mediated major satellite RNA decay plays a central role in the preferential incorporation of histone variant H3.3 into the male pronucleus. Knockdown of Lsm1 disrupts nonequilibrium pronucleus histone incorporation and asymmetric H3K9me3 modification. Subsequently, we find that LSM1 mainly targets major satellite repeat RNA (MajSat RNA) for decay and that accumulated MajSat RNA in Lsm1-depleted oocytes leads to abnormal incorporation of H3.1 into the male pronucleus. Knockdown of MajSat RNA reverses the anomalous histone incorporation and modifications in Lsm1-knockdown zygotes. Our study therefore reveals that accurate histone variant incorporation and incidental modifications in parental pronuclei are specified by LSM1-dependent pericentromeric RNA decay.

Nicotinamide mononucleotide restores oxidative stress-related apoptosis of oocyte exposed to benzyl butyl phthalate in mice.

Benzyl butyl phthalate (BBP) is a chemical softener and plasticizer commonly used in toys, food packaging, wallpaper, detergents and shampoos. The estrogenic actions of BBP have detrimental effects on humans and animals. In this study, the specific influence of BBP on mouse oocyte maturation was investigated using in vivo and in vitro models. The experiment first verified that BBP exposure significantly affected the rate of oocyte exclusion of the first polar body, although it did not affect germinal vesicle breakdown (GVBD) through in vitro oocyte culture system. Results of in vitro fertilization show that BBP exposure affects blastocyst rate. Subsequently, the results obtained by immunofluorescence staining technology showed that oocyte spindle organization, chromosomal arrangement and the distribution of cortical actin were disrupted by BBP exposure, and led to the failure of oocyte meiotic maturation and the subsequent early embryo development. Singe-cell transcriptome analysis found that BBP exposure altered the expression levels of 588 genes, most associated with mitochondria-related oxidative stress. Further analysis demonstrated that the detrimental effects of BBP involved the disruption of mitochondrial function and oxidative stress-induced early apoptosis. Nicotinamide mononucleotide (NMN) supplementation reduced the adverse effects of BBP. Collectively, these findings revealed a mechanism of BBP-induced toxicity on female reproduction and showed that NMN provides an effective treatment for BBP actions.

Modelling of the Decidualization of Mouse Endometrial Stromal Cells with Subsequent Embryo Implantation In Vitro

Modelling of the Decidualization of Mouse Endometrial Stromal Cells with Subsequent Embryo Implantation In Vitro

Elective single versus double blastocyst-stage embryo transfer in women aged 36 years or older: a retrospective cohort study

We evaluated if elective single-blastocyst transfer (eSBT) could be adopted in women aged 36 or older. In this retrospective cohort, women aged ≥36 years received IVF ovarian stimulation cycles and had ≥ two blastocysts. A total of 240 women underwent eSBT and 189 double-blastocyst transfer (DBT) in the first transfer cycle. The subsequent frozen-thawed embryo transfer cycles were a combination of single- and double- blastocyst transfers. Analysis was stratified for patients in age groups 36–37, 38–39 and ≥40, considering the quality of the blastocyst transferred. The cumulative live birth rates (cLBR) were 74.2% (178/240) versus 63.0% (119/189) after eSBT versus DBT, respectively (aOR: 1.09 (0.68, 1.75)). Time to live birth did not vary significantly between the two groups (HR: 0.85 (0.68, 1.08)). The total number of children born was 194 after eSBT (162 singletons and 16 pairs of twins) versus 154 (84 singletons and 35 twins) after DBT. The odds ratios for preterm birth (0.37 (0.21–0.64)), and low birth weight (0.31 (0.16, 0.60)) were all lower in eSBT. In women aged ≥36 years, cLBR following single- versus double- blastocyst transfer was comparable while the odds of multiple live births and adverse perinatal outcomes were reduced.

Endometrial immune cell ratios and implantation success in patients with recurrent implantation failure

The objective of this study was to compare the endometrial immune cells quantities and ratios during the mid-luteal phase between women with recurrent implantation failure (RIF) with successful and unsuccessful embryo implantation. For this purpose, endometrial biopsies from 116 women aged between 29 and 46 with history of RIF undergoing Assisted Reproductive Technology (ART) without endometrial pathologies were immunohistochemically stained for CD3 + T-cells, CD4 + T-helpers, CD8 + T-killers, CD14 + monocytes, CD68 + macrophages, CD56 + NK cells and CD79α+ B-cells. Endometrial immune cells quantities and ratios were compared based on the embryo implantation outcome in the subsequent embryo transfer cycle. Spearman correlation analysis and Mann-Whitney U test were used to analyse the obtained data. Patients who experienced successful implantation at the subsequent cycle had significantly lower percentage of CD3 + T cells, and higher ratios of CD4 + /CD8 + , CD4 + /CD3 + and CD68 + /CD3 + than the patients who experienced another failure in implantation. In addition, the ratios of CD3 + /CD14 + , CD79α+ /CD14 + and CD56 + /CD14 + were significantly lower in the successful implantation group than that in the unsuccessful one. A cut off value of CD68 + /CD3 + ratio higher than 0.85 (AUC 0.67, 95% CI 0.56–0.79), CD4 + /CD3 + ratio higher than 0.19 (AUC 0.67, 95% CI 0.56–0.79) and CD4 + /CD8 + ratio higher than 0.43 (AUC 0.62, 95% CI 0.50–0.73) could be predictive factors for successful implantation in RIF patients. Knowledge on the immune cell composition could assist in the evaluation of the endometrial receptivity in RIF patients.

Isorhamnetin improves in vitro maturation of oxidative stress-exposed porcine oocytes and subsequent embryo development.

This study investigated the effect of the flavonoid-based compound isorhamnetin (ISO) on maturation and developmental competence in oxidative stress-exposed porcine oocytes in vitro. Treatment with 2 μM ISO (2 ISO) increases the developmental rate of oxidative stress-exposed porcine oocytes during in vitro maturation (IVM). The glutathione level and mRNA expression of antioxidant-related genes (NFE2L2 and SOD2) were increased in the 2 ISO-treated group, whereas the reactive oxygen species level was decreased. Treatment with 2 ISO increased mRNA expression of a cumulus cell expansion-related gene (SHAS2) and improved chromosomal alignment. mRNA expression of maternal genes (CCNB1, MOS, BMP15 and GDF9) and mitogen activated protein kinase (MAPK) activity were increased in the 2 ISO-treated group. The total cell number per blastocyst and percentage of apoptotic cells were increased and decreased in the 2 ISO-treated group, respectively. Treatment with 2 ISO increased mRNA expression of development-related genes (SOX2, NANOG, and POU5F1) and anti-apoptotic genes (BCL2L1 and BIRC5) and decreased that of pro-apoptotic genes (CASP3 and FAS). These results demonstrate that 2 ISO improves the quality of porcine oocytes by protecting them against oxidative stress during IVM and enhances subsequent embryo development in vitro. Therefore, we propose that ISO is a useful supplement for IVM of porcine oocytes.

Effect of post-activation culture conditions on the development of parthenogenetic embryos in cattle

Purpose: to conduct a comparative analysis of the effect of commercial media BO-IVC and СR1aa at the stage of the activation and subsequent culture of artificially activated oocytes on the formation and quality of parthenogenetic bovine embryos.Materials and methods. 3 groups of disemeters of 50 goals in each were formed. In the first experimental group, the disemeted was in a meticulous manner with a ram-industrialist (artificial kriproporchid), in the second experimental-with a penEexctomed ram-industrialist. In the third (control) group, a producer ram was used. In the first experimental group of a ram-industrialist (artificial kriproporchid) with attached taps were released into a group of sheep twice a day for 1.5-2 hours. In the second experimental group of a penEctomed ram, it was placed in the corral to the disemetery in the morning for 3 hours. In the third group, the lamb producer was constantly with the disemets for two weeks, then he was changed on a new ram i.e. Used the methodology used in the farm. During the experiment, they observed the behavior of animals of all groups. In the experimental groups, after the detection of disemeters in the hunt, their natural insemination of the manufacturer was carried out. Based on the results of the subsequent oster, the effectiveness of the reproduction of sheep was evaluated.Results. The cleavage rate did not differ between the experimental groups, varying from 73,0 to 76,5%. Also, there was not found a significant effect of the conditions for post-activation culture of oocytes on their development before late morula and late blastocyst stage, which was for the CR1aa/CR1aa, CR1aa/BO-IVC and BOIVC/ BO-IVC groups 28,9±1,7, 40,4±7,5 and 36,0±6.4%, respectively. Meanwhile, we found out the effect of tested culture conditions on the ability of parthenogenetic embryos to overcome the 8-16 cell block and their quality on the late stages of embryo development. The rate of embryos with less than 16 nuclei was the highest in the CR1aa/CR1aa group (56,8±2,1 %). The replacement of CR1aa medium to BO-IVC medium (BO-IVC/BO-IVC group) significantly reduced this level (p&lt;0,05). The positive effect was enhanced when CR1aa medium was used at the stage of culture in the presence of 6-DMAP and cycloheximide, and subsequent embryo development was in BO-IVC medium (CR1aa/BO-IVC group) (p&lt;0.001). Furthermore, when we used the mixed variant of culture, the total cell number in parthenogenetic morula and blastocyst stage embryos increased (p&lt;0.05).Conclusion. Thus, the BO-IVC medium at the stages of post-activation and subsequent development of artificially activated bovine oocytes is comparable to the CR1aa medium in terms of the efficiency of obtaining parthenogenetic embryos at the blastocyst stage. Nevertheless, its replacement at the post-activation stage with CR1aa medium makes it possible to improve the quality of parthenogenetic embryos.

Vascular endothelial growth factor isoforms are expressed in the uterus during estrous cycle of golden hamsters (Mesocricetus auratus).

We investigated VEGF expression in the uterus during the estrous cycle in the golden hamster (Mesocricetus auratus). Reverse transcription polymerase chain reaction of genes expressed in the uterus revealed the presence of at least three different VEGF isoforms (hamster VEGF188, VEGF164, and VEGF120). They were highly homologous to the respective mouse and human isoforms. Furthermore, VEGF164 and VEGF120 were predominantly expressed in the hamster uterus during the estrous cycle. In situ hybridization revealed that VEGF is expressed only in the luminal and glandular epithelium of the endometrium but not in the stromal cells or myometrium. The positive reaction of luminal and glandular epithelial cells on day 4 of the estrous cycle (day 1 = day of ovulation) was a little stronger than that of other days of the cycle. These findings suggest that VEGF molecules are secreted by endometrial epithelial cells and play an important role in the maintenance of blood vessels in the endometrial stroma. These results also suggest that uterine changes, such as edema, observed from day 4 to day 1 of the estrous cycle, are expected to occur primarily through the action of VEGF secreted by the uterine endometrial epithelium in preparation for subsequent embryo implantation.

Modelling of the Decidualization of Mouse Endometrial Stromal Cells with Subsequent Embryo Implantation &lt;i&gt;in vitro&lt;/i&gt;

The decidualization of stromal cells is a key step in acquiring endometrial receptivity to the embryo. This process is regulated by sex steroid hormones, and in the mouse is additionally induced by embryo attachment. There are many protocols for the induction decidualization of stroma in vitro, but most of them require verification of the compliance of the processes occurring in cell culture with those in a living organism. This work focuses on the morphology of stromal cells and the expression activity of genes responsible for the progression of decidualization in 2D culture with subsequent embryo attachment. The feasibility of using several variants of protocols for the induction of decidualization and the possibility of using stromal cells after cryopreservation were also investigated.