Preimplantation Period Research Articles

Comparative study of hematopoietic reconstitution in patients of the Arnaldo Milian Hospital from Santa Clara

A mathematical model was applied to the hematopoietic reconstitution study of a group of thirteen patients with Hodgkin and Non-Hodgkin Lymphoma after having undergone Autologous Transplantation of Hematopoietic Progenitors in the Hematology Service of the Surgical Clinical University Hospital "Arnaldo Malian". Based on the model, the recovery process dynamics were computationally simulated, and the hematopoietic recovery times of each patient collected in the clinical data were compared with the times predicted by the simulations. Despite the simplicity of the mathematical model, it is surprising how well the reconstitution curves coincide with the clinical data collected, in such a manner that the model is able to predict with acceptable accuracy when hematopoietic recovery occurs. Model forecasts can be used to estimate the average duration of the pre-implant period, in which severe aplasia and immunodeficiency condition the occurrence of hemorrhagic and infectious complications that are often severe. In addition, it is an important tool for planning next patient´s transplant by the team, thereby reducing the time spent in planning the logistics required between one transplant and the next, which, in turn, will increase the number of patients benefited by this procedure.

Maternal age affects pronuclear and chromatin dynamics, morula compaction and cell polarity, and blastulation of human embryos.

Does maternal ageing impact early and late morphokinetic and cellular processes of human blastocyst formation? Maternal ageing significantly affects pronuclear size and intra- and extra-nuclear dynamics during fertilization, dysregulates cell polarity during compaction, and reduces blastocoel expansion. In ART, advanced maternal age (AMA) affects oocyte yield, fertilization, and overall developmental competence. However, with the exception of chromosome segregation errors occurring during oocyte meiosis, the molecular and biochemical mechanisms responsible for AMA-related subfertility and reduced embryo developmental competence remain unclear. In particular, studies reporting morphokinetics and cellular alterations during the fertilization and pre-implantation period in women of AMA remain limited. A total of 2058 fertilized oocytes were stratified by maternal age according to the Society for Assisted Reproductive Technology classification (<35, 35-37, 38-40, 41-42, and >42 years) and retrospectively analysed. AMA effects were assessed in relation to: embryo morphokinetics and morphological alterations; and the presence and distribution of cell polarity markers-Yes-associated protein (YAP) and protein kinase C-ζ (PKC-ζ)-involved in blastocyst morphogenesis. A total of 1050 cycles from 1050 patients met the inclusion criteria and were analysed. Microinjected oocytes were assessed using a time-lapse culture system. Immature oocytes at oocyte retrieval and mature oocytes not suitable for time-lapse monitoring, owing to an excess of residual corona cells or inadequate orientation for correct observation, were not analysed. Phenomena relevant to meiotic resumption, pronuclear dynamics, cytoplasmic/cortical modifications, cleavage patterns and embryo quality were annotated and compared among groups. Furthermore, 20 human embryos donated for research by consenting couples were used for immunofluorescence. Static microscopic observation revealed that blastocyst formation and expansion were impaired in the 41-42 and >42-year groups (P < 0.0001). The morphological grades of the inner cell mass and trophectoderm were poorer in the >42-year group than those in the <35-year group (P = 0.0022 and P < 0.0001, respectively). Time-lapse microscopic observation revealed a reduction in nucleolus precursor body alignment in female pronuclei in the 41-42 and >42-year groups (P = 0.0010). Female pronuclear area decreased and asynchronous pronuclear breakdown increased in the >42-year group (P = 0.0027 and P < 0.0122, respectively). Developmental speed at cleavage stage, incidence of irregularity of first cleavage, type and duration of blastomere movement, and number of multinucleated cells were comparable among age groups. Delayed embryonic compaction and an increased number of extruded blastomeres were observed in the >42-year group (P = 0.0002 and P = 0.0047, respectively). Blastulation and blastocyst expansion were also delayed in the 41-42 and >42-year groups (P < 0.0001 for both). YAP positivity rate in the outer cells of morulae and embryo PKC-ζ immunoflourescence decreased in the >42-year group (P < 0.0001 for both). At the cellular level, the investigation was limited to cell polarity markers. Cell components of other developmental pathways should be studied in relation to AMA. The study indicates that maternal ageing affects the key functions of embryo morphogenesis, irrespective of the well-established influence on the fidelity of oocyte meiosis. This study was supported by the participating institutions. The authors have no conflicts of interest to declare. N/A.

Global analyses and potential effects of extracellular vesicles on the establishment of conceptus implantation during the peri-implantation period.

Intrauterine extracellular vesicles (EVs) are involved in establishing proper conceptus-endometrial communication, which is essential for conceptus implantation and subsequent successful placentation. Despite several studies on intrauterine EVs, the composition and quantitative changes in conceptus and endometrial EVs, as well as the effects of intrauterine EVs on endometrial epithelial cells (EECs) during the peri-implantation period, have not been well characterized. To elucidate global changes in proteins in EVs extracted from uterine flushings (UFs) during the pre-implantation (P17), just-implantation (P20), and post-implantation (P22) periods, the datasets of the proteome iTRAQ analysis were compared among P17, P20, and P22 EVs. These analyses revealed that the composition and function of proteins in the EVs changed dramatically during peri-implantation in cattle. Notably, intrauterine P17 EVs affected the high expression of "Developmental Biology" and "morphogenesis of an endothelium" compared with those in P20 and P22 EVs. Furthermore, P20 EVs had the functions of the high expression of "mitochondrial calcium ion homeostasis" and "Viral mRNA Translation" compared with those in P17 EVs. Transcripts extracted from EECs treated with P17, P20, or P22 EVs were subjected to RNA-seq analysis. These analyses identified 60 transcripts in EECs commonly induced by intrauterine EVs recovered from P17, P20, and P22, a large number of which were associated with "type I interferon signaling pathway". Collectively, these findings reveal the presence and multiple functions of EVs that are potentially implicated in facilitating conceptus implantation into the uterine epithelium during the peri-implantation period.

Glutathione peroxidase 1 and 2 during preimplantation period of pregnancy in mouse.

This study describes the presence of glutathione peroxidase (GPx) 1 and 2 in oocytes, preimplantation embryos, and mouse female genital organs. GPx1 and 2 are antioxidant enzymes, which play important roles in protection of cells against oxidation, which may be formed during cell physiological processes. After superovulation of female mice, oocytes and preimplantation embryos (O/PE) were isolated for immunofluorescent analysis, female genital organs were removed for immunohistochemical and Western blot analyses. Using immunofluorescence, GPx1 was detected in all OP/E, where it formed clusters near nuclei, or under the cytoplasmic membrane. GPx2 was not detected in any O/PE. Using immunohistochemistry, GPx1 was observed in corpus luteum and oocytes, and in oviductal and uterine epithelium. GPx2 was detected in corpus luteum, but not in oocytes. Oviductal and uterine epithelium was mostly negative. Using Western blot, two GPx1 bands of different molecular weights were detected in uterus. One GPx2 band was observed in all investigated organs. These results show that both enzymes may be important during preimplantation period of pregnancy in genital organs, GPx1 also in O/PE. GPx2 may play roles in embryo after the implantation, when gastrointestinal tract is formed (Fig. 10, Ref. 75).

Effects of Cardiac Contractility Modulation Electrodes on Tricuspid Regurgitation in Patients with Heart Failure with Reduced Ejection Fraction: A Pilot Study.

Cardiac contractility modulation (CCM) is an innovative therapy for heart failure with reduced ejection fraction delivered by a cardiac implantable device (Optimizer Smart®). One of the most prominent periprocedural complications common to all cardiac implantable devices (CIDs) is tricuspid regurgitation (TR) due to the placement of the right ventricular endocardial leads. To date, no published studies have assessed the changes in the TR degree in patients with heart failure with reduced ejection fraction (HFrEF) who received an implantable cardioverter-defibrillator (ICD) after the implantation of cardiac contractility modulation therapy devices. This study aimed to evaluate the effect of the implantation of the trans-tricuspid leads required to deliver CCM therapy on the severity of TR in patients with HFrEF who previously underwent ICD implantation. We enrolled 30 HFrEF patients who underwent CCM therapy between November 2020 and October 2021. For all the patients, echocardiographic evaluations of TR were performed according to current guidelines 24 h before and six months after the Optimizer Smart® implant was applied. At the 6-month follow-up, the grade of TR remained unchanged compared to the preimplant grade. The value of the vena contracta (VC) of TR was 0.40 ± 0.19 cm in the preimplant period and 0.45 ± 0.21 cm at the 6-month follow-up (p = 0.33). Similarly, the TR proximal isovelocity surface area (PISA) radius value was unchanged at follow-up (0.54 ± 0.22 cm vs. 0.62 ± 0.20 cm; p = 0.18). No statistically significant difference existed between the preimplant VC and PISA radius values, irrespective of the device type. The implantation of right ventricular electrodes for the delivery of CCM therapy did not worsen tricuspid regurgitation in patients with HFrEF and ICD.

Identification of the Inner Cell Mass and the Trophectoderm Responses after an In Vitro Exposure to Glucose and Insulin during the Preimplantation Period in the Rabbit Embryo.

The prevalence of metabolic diseases is increasing, leading to more women entering pregnancy with alterations in the glucose-insulin axis. The aim of this work was to investigate the effect of a hyperglycemic and/or hyperinsulinemic environment on the development of the preimplantation embryo. In rabbit embryos developed in vitro in the presence of high insulin (HI), high glucose (HG), or both (HGI), we determined the transcriptomes of the inner cell mass (ICM) and the trophectoderm (TE). HI induced 10 differentially expressed genes (DEG) in ICM and 1 in TE. HG ICM exhibited 41 DEGs involved in oxidative phosphorylation (OXPHOS) and cell number regulation. In HG ICM, proliferation was decreased (p < 0.01) and apoptosis increased (p < 0.001). HG TE displayed 132 DEG linked to mTOR signaling and regulation of cell number. In HG TE, proliferation was increased (p < 0.001) and apoptosis decreased (p < 0.001). HGI ICM presented 39 DEG involved in OXPHOS and no differences in proliferation and apoptosis. HGI TE showed 16 DEG linked to OXPHOS and cell number regulation and exhibited increased proliferation (p < 0.001). Exposure to HG and HGI during preimplantation development results in common and specific ICM and TE responses that could compromise the development of the future individual and placenta.

ANEUPLOIDY SCREENING AFTER PREIMPLANTATION GENETIC TESTING: A NATIONAL SURVEY OF PHYSICIAN KNOWLEDGE AND PRACTICE

Preimplantation genetic testing (PGT) can assist in selecting embryos for implantation, however genetic testing in the preimplantation period does not preclude the need to offer prenatal genetic screening and invasive diagnostic testing. The American College of Obstetricians and Gynecologists recommends all patients be offered prenatal genetic screening and diagnostic testing, including screening for aneuploidy, regardless of PGT status. With the recent rise in the utilization of in vitro fertilization and PGT, general obstetric practitioners may be less familiar with current guidelines for aneuploidy screening in this growing patient population.

278 Awardee Talk: Progesterone Regulates the Abundance of Mrnas and Proteins Involved in Polyamine Metabolism in the Sheep Endometrium

Abstract Agmatine and polyamines (putrescine, spermidine, and spermine), metabolites of L-arginine, are important for development of mammalian conceptuses. The objective of this study was to determine if exogenous progesterone administered during the pre-implantation period, which accelerates conceptus development, alters the expression of mRNAs and proteins involved in the metabolism of agmatine and polyamines in the sheep uterus during pregnancy. Mature Suffolk ewes were bred to fertile rams and assigned randomly to be treated with daily i.m. injections of either 1ml corn oil (CO;n=28) or 25mg progesterone in corn oil (P4;n=20) from Day 1.5 through Day 8 after breeding (Day 0). Ewes were hysterectomized on Day 9, 12, or 125 of gestation and endometrial samples were collected. The expression of candidate mRNAs was quantified using qPCR and proteins localized using immunohistochemistry. On Day 9 of pregnancy, the expression of mRNAs for AMD1 (P &amp;lt; 0.01), ASL (P &amp;lt; 0.01), and SLC12A8 (P &amp;lt; 0.05) was greater in endometria of P4-treated compared to CO-treated ewes. The expression for MAT2B mRNA was greater (P &amp;lt; 0.001) for P4-treated compared to CO-treated ewes on both Days 9 and 12. On Day 125 of gestation, endometria from P4-treated ewes tended to have greater expression of SAT1 (P &amp;lt; 0.10) mRNA compared to CO-treated ewes. In contrast, the expression of AMD1(P &amp;lt; 0.01), MAT2B (P &amp;lt; 0.05), OAT (P &amp;lt; 0.05), SMOX (P &amp;lt; 0.05), and SLC12A8 (P &amp;lt; 0.05) mRNAs was lower in endometria from P4-treated compared to CO-treated ewes on Day 125. Exogenous P4 appeared to increase the relative abundance of SAT1 and SMOX proteins in the uterine luminal and glandular epithelia on Day 125 of pregnancy. These results suggest that early administration of progesterone may alter the biosynthesis of agmatine and polyamines in the sheep uterus during the pre-implantation period of pregnancy with potential ‘programming’ effects manifested in late pregnancy.

Association between a deep learning-based scoring system with morphokinetics and morphological alterations in human embryos

Research questionWhat is the association between the deep learning-based scoring system, iDAScore, and biological events during the pre-implantation period? DesignRetrospective observational study of patients (n = 925) who underwent oocyte retrieval in a clomiphene citrate-based minimal stimulation cycle and obtained expanded blastocysts between October 2019 and December 2020. The association between iDAScore with morphokinetics and morphological alteration during fertilization, cleavage stage, compaction and blastocyst stage was analysed. ResultsThe duration of the cytoplasmic halo was significantly prolonged in low-scoring blastocysts (P < 0.0001). The timing of female and male pronuclei breakdown was significantly delayed in low-scoring blastocysts compared with high-scoring blastocysts (P < 0.0001 in both). Embryos with either trichotomous, multi-chotomous, rapid or reverse cleavage or asymmetric division had a lower score than embryos with normal cleavage (P < 0.0001–0.0098). The cell number and amount of blastomere fragmentation on days 2 and 3 were significantly associated with iDAScore (P < 0.0001–0.0008). Delayed compaction, blastulation and blastocyst expansion were observed in low-scoring embryos (P < 0.0001 in all). The incidence of blastomere exclusion and extrusion during embryonic compaction was significantly higher in low-scoring embryos than in high-scoring embryos (P ≤ 0.0001 in both). Blastocyst morphology was significantly associated with iDAScore (P < 0.0001). Multiple linear regression analysis revealed that, during the transformation to blastocyst stage, morphokinetic and morphological events were strongly associated with iDAScore (P < 0.0001–0.0116). ConclusionsiDAScore was significantly correlated with morphokinetics and morphological alterations of pre-implantation embryos, especially during the late pre-implantation period. Our findings contribute to research on deep learning model-based embryo selection, which may provide patients with a compelling explanation of blastocyst selection.

P-167 Male infertility and polycystic ovary syndrome (PCOS) affect absolute and relative embryo morphokinetics observed by time-lapse imaging

Abstract Study question This study aimed to test if morphokinetics are altered in embryos from patients with male infertility, polycystic ovary syndrome (PCOS) or recurrent pregnancy loss (RPL). Summary answer Morphokinetics differed significantly between embryos from patients with male infertility or PCOS compared to the control groups, possibly suggesting a negative impact on development potential. What is known already Time-lapse imaging technology allows continuous observation of embryonic morphology and developmental kinetics during the preimplantation period. The ESHRE Working group on Time-lapse technology recently summarised common absolute morphokinetic time points and intervals [DOI: 10.1093/hropen/hoaa008]. Additionally, four relative morphokinetic expressions were introduced by Cetinkaya et al.: Cleavage Synchronicity 2-8-, 4-8-, 2-4-cell stage (CS2-8, CS4-8, CS2-4) and DNA Replication time ratio (DR) [DOI: 10.1007/s10815-014-0341-x]. A study by Freis et al. observed worse CS2-8 and CS4-8 ratios in embryos from patients with endometriosis [DOI: 10.1177/1933719117741373]. Other studies have found that male infertility and PCOS may affect morphokinetic time points. Study design, size, duration In this retrospective observational study n = 1433 two-pronuclei oocytes from n = 433 patients between 18 and 45 years undergoing IVF/ICSI-treatment between 09/2016 and 12/2019 were examined. The use of a time-lapse incubator was obligatory. Exclusion criteria were the presence of endometriosis, history of chemotherapy, preimplantation genetic testing (PGT) cycles and genetic abnormalities. Every patient was included only once. Control groups were uterine, tubal factor and idiopathic infertility. Participants/materials, setting, methods Male infertility (n = 928 oocytes; 288 patients) versus control (n = 400; 115), PCOS (n = 48; 14) versus control (n = 400; 115) and RPL (n = 79; 21) versus control (n = 321; 94) were compared. Patient and treatment characteristics were compared using Mann-Whitney-U and Fisher’s exact test. Times normalised to fading of pronuclei (tPNf), intervals and ratios were analysed using a Generalised Linear Mixed Model (GLMM). Main results and the role of chance The male infertility group was significantly different (p &amp;lt; 0.05) from the control group regarding male age (p = 0.009), fertilisation rate (p = 0.008), number of embryos per patient (p = 0.033), treatment method (p &amp;lt; 0.001) and use of Calcium-Ionophore (p = 0.049) or SpermMobile (p = 0.003). DR was significantly higher in the male infertility group compared to the control group (p = 0.031). The start of blastulation (tSB-tPNf; p = 0.008) and start of expansion of the blastocyst (tEB-tPNf; p = 0.002) were significantly delayed. The PCOS group was significantly different concerning female age (p = 0.023) and AMH blood level (p &amp;lt; 0.001). CS2-8 was significantly lower (p = 0.003) and CS2-4 significantly higher (p = 0.001) in the PCOS group compared to the control group. The time intervals from three- to four-cell (s2; p = 0.013), from five- to eight-cell (s3; p = 0.032) and from four- to eight-cell stage (ECC3; p = 0.043) were significantly longer in the PCOS group. There was no significant difference in patient or treatment related characteristics between the RPL group and the control group. Also, none of the morphokinetic parameters analysed in this study was significantly different between the groups. Limitations, reasons for caution Small group sizes decreased further at later developmental time points. In general, time-lapse data show a high intra- and inter-observer variability. A GLMM was performed to take statistical dependencies between embryos of one patient into account. Its Gaussian assumption was partially violated by the true distribution of the data. Wider implications of the findings These data support the hypothesis that patients’ co-morbidities may affect embryo morphokinetics. The impact on clinical outcome should be examined further. Accordingly, our working group conducted another study about the predictive value of morphokinetics on blastocyst quality and implantation (DRKS-ID: DRKS00022983). The long-term goal is to improve embryo assessment. Trial registration number not applicable

Actions of WNT family member 5A to regulate characteristics of development of the bovine preimplantation embryo†.

WNT signaling is important for regulation of embryonic development. The most abundant WNT gene expressed in the bovine endometrium during the preimplantation period is WNT5A. One objective was to determine whether WNT5A regulates competence of the bovine preimplantation embryo to become a blastocyst and alters the number of cells in the inner cell mass and trophectoderm. A second objective was to delineate features of the cell-signaling mechanisms involved in WNT5A actions. WNT5A caused a concentration-dependent increase in the proportion of embryos developing to the blastocyst stage and in the number of inner cell mass cells in the resultant blastocysts. A concentration of 200ng/mL was most effective, and a higher concentration of 400ng/mL was not stimulatory. Bovine serum albumin in culture reduced the magnitude of effects of WNT5A on development to the blastocyst stage. WNT5A affected expression of 173 genes at the morula stage; all were upregulated by WNT5A. Many of the upregulated genes were associated with cell signaling. Actions of WNT5A on development to the blastocyst stage were suppressed by a Rho-associated coiled-coil kinase (ROCK) signaling inhibitor, suggesting that WNT5A acts through Ras homology gene family member A (RhoA)/ROCK signaling. Other experiments indicated that actions of WNT5A are independent of the canonical β-catenin signaling pathway and RAC1/c-Jun N-terminal kinase (JNK) signaling. This is the first report outlining the actions of WNT5A to alter the development of the mammalian embryo. These findings provide insights into how embryokines regulate maternal-embryonic communication.

Non-Invasive Staging of In Vitro Mice Embryos by Means of Volatolomics

Current methods for embryo selection are limited. This study assessed a novel method for the prediction of embryo developmental potential based on the analysis of volatile organic compounds (VOCs) emitted by embryo samples. The study included mice embryos monitored during the pre-implantation period. Four developmental stages of the embryos were tested, covering the period from 1 to 4 days after fecundation. In each stage, the VOCs released by the embryos were collected and examined by employing two different volatolomic techniques, gas-chromatography coupled to mass-spectrometry (GC-MS) and a nanoarray of chemical gas sensors. The GC-MS study revealed that the VOC patterns emanating from embryo samples had statistically different values at different stages of embryo development. The sensor nanoarray was capable of classifying the developmental stages of the embryos. The proposed volatolomics analysis approach for embryos presents a promising potential for predicting their developmental stage. In combination with conventional morphokinetic parameters, it could be effective as a predictive model for detecting metabolic shifts that affect embryo quality before preimplantation.

The people behind the papers - Manoj Madhavan and Ripla Arora.

The 3D architecture of the uterus plays a key role in determine pregnancy outcome. A new paper in Development reveals the dynamic changes in luminal folding pattern that occur during the preimplantation period in mice. To hear more about the story, we caught up with first author Manoj Madhavan and corresponding author Ripla Arora, Assistant Professor at Michigan State University.

Prognostic Value of Cardiopulmonary Exercise Test Parameters in Ventricular Assist Device Therapy.

Cardiopulmonary exercise test (CPET) parameters are established prognosticators in heart failure. However, the prognostic value of preimplantation and postimplantation CPET parameters in left ventricular assist device (LVAD) therapy is unclear and it is evaluated in this study. Adult patients who were implanted with an LVAD and underwent CPET during the preimplantation or postimplantation period were retrospectively analyzed. Five CPET parameters were calculated: vO2 max, oxygen uptake efficiency slope (OUES), VE/vCO2 Slope, VE/vCO2 min, and VE/vCO2 max. The relationship between CPET parameters and postimplantation outcomes was evaluated with multivariable analysis. Pre and postimplantation CPET cohorts included 191 and 122 patients, respectively. Among preimplantation CPET parameters: vO2 max and OUES were associated with 1, 3, and 5 year mortality, VE/vCO2 min was associated with 3 and 5 year mortality, whereas VE/vCO2 Slope was associated with 5 year mortality. From postimplantation CPET parameters: vO2 max was an independent predictor of 3 and 5 year mortality, whereas VE/vCO2 max was an independent predictor of 3 year mortality following LVAD implantation. Preimplantation CPET parameters have a prognostic value for long-term survival following LVAD implantation, whereas their association with early postimplantation outcomes appears to be weaker. Postimplantation vO2 max and VE/vCO2 max values are associated with survival on device support and may provide a second chance for prognostication in patients without preimplantation CPET data.

Perfusate Metabolomics Content and Expression of Tubular Transporters During Human Kidney Graft Preservation by Hypothermic Machine Perfusion.

Ischemia-related injury during the preimplantation period impacts kidney graft outcome. Evaluating these lesions by a noninvasive approach before transplantation could help us to understand graft injury mechanisms and identify potential biomarkers predictive of graft outcomes. This study aims to determine the metabolomic content of graft perfusion fluids and its dependence on preservation time and to explore whether tubular transporters are possibly involved in metabolomics variations. Kidneys were stored on hypothermic perfusion machines. We evaluated the metabolomic profiles of perfusion fluids (n = 35) using liquid chromatography coupled with tandem mass spectrometry and studied the transcriptional expression of tubular transporters on preimplantation biopsies (n = 26), both collected at the end of graft perfusion. We used univariate and multivariate analyses to assess the impact of perfusion time on these parameters and their relationship with graft outcome. Seventy-two metabolites were found in preservation fluids at the end of perfusion, of which 40% were already present in the native conservation solution. We observed an increase of 23 metabolites with a longer perfusion time and a decrease of 8. The predictive model for time-dependent variation of metabolomics content showed good performance (R 2 = 76%, Q 2 = 54%, accuracy = 41%, and permutation test significant). Perfusion time did not affect the mRNA expression of transporters. We found no correlation between metabolomics and transporters expression. Neither the metabolomics content nor transporter expression was predictive of graft outcome. Our results call for further studies, focusing on both intra- and extratissue metabolome, to investigate whether transporter alterations can explain the variations observed in the preimplantation period.

ITRAQ-based quantitative proteomic analysis of porcine uterine fluid during pre-implantation period of pregnancy

Proteins in the uterine luminal fluid are essential for embryo development and regulation of embryo-maternal interaction in porcine. However, little is known about the profile of proteins in uterine luminal fluid of porcine during the pre-implantation period. The present study, applied iTRAQ proteomics technology to identify and analyze uterine luminal fluid proteins on day 9 of estrus cycle and days 9, 12, and 15 of pregnancy. A total of 964 proteins were identified in the present study. Principal component analysis and hierarchical clustering revealed the dynamic developmental characteristics of embryo implantation, which indicated significant differences on day 12 or 15 of pregnancy. In addition, further analysis conducted in the present study identified 279 differentially abundance proteins among the three groups, five clusters were generated using SOTA clustering to examine changes in of the differentially abundant proteins. Results of the current study also found that the proteins in the cluster are involved in some important processes such as regulation of low-density lipoproteins and regulation of TGF-β secretion. Notably, it was found that regulation of TGF-β is essential for porcine embryonic morphological transformation. Furthermore, proteins that play vital roles in implantation, such as CTSC, CTSB, and ACP5 were identified through protein-protein interaction network. Therefore, these findings of the present study provide a basis for understanding embryo development mechanisms and implantation in pigs. SignificanceProteins are directly acting molecules for the functioning of organisms. It is important to study the regulation mechanism of embryo implantation from the perspective of protein function. In the current study, iTRAQ proteomics technology was employed to identify and explore uterine luminal fluid proteins on day 9 of estrus cycle and days 9, 12, and 15 of pregnancy. The findings provide novel insights into the process of porcine early embryo implantation. Furthermore, it is also helpful to clarify the mechanism of embryonic development and implantation.

Cleavage of Early Mouse Embryo with Damaged DNA.

The preimplantation period of embryogenesis is crucial during mammalian ontogenesis. During this period, the mitotic cycles are initiated, the embryonic genome is activated, and the primary differentiation of embryonic cells occurs. All cellular abnormalities occurring in this period are the primary cause of fetal developmental disorders. DNA damage is a serious cause of developmental failure. In the context of DNA damage response on the cellular level, we analyzed the course of embryogenesis and phenotypic changes during the cleavage of a preimplantation embryo. Our results document that DNA damage induced before the resumption of DNA synthesis in a zygote can significantly affect the preimplantation development of the embryo. This developmental ability is related to the level of the DNA damage. We showed that one-cell embryos can correct the first cleavage cycle despite low DNA damage and incomplete replication. It seems that the phenomenon creates a predisposition to a segregation disorder of condensed chromatin that results in the formation of micronuclei in the developmental stages following the first cleavage. We conclude that zygote tolerates a certain degree of DNA damage and considers its priority to complete the first cleavage stage and continue embryogenesis as far as possible.

Plasminogen activator activity and plasminogen activator inhibition in the uterus of ewes after the induction of oestrus synchronization or superovulation, involving eCG

During the preimplantation period, early mammalian embryos largely depend on uterine environment. Concurrently, uterine architecture undergoes substantial growth and remodeling under the influence of ovarian steroids. Plasminogen activators/plasmin proteolytic system that is implicated in tissue remodeling is affected by reproductive hormone variations. Thus, hormone treatments for reproductive function control possibly influence proteolytic enzyme activity in the reproductive tract and its study in uterine fluid and endometrium could demonstrate possible changes in uterine environment. This work examines (a) the effect of hormone treatment involving eCG in the dosage used for oestrus synchronization or for superovulation on plasminogen activator activity (PAA) and plasminogen activator inhibition (PAI) in the uterus of ewes before maternal recognition of pregnancy and (b) the possible relationships among blood ovarian steroid concentration, proteolytic enzyme activity and ovarian response/embryo yield. Chios ewes (n = 24) were treated with progestagen intravaginal sponges for 12 days and eCG (500 or 1000 IU) at sponge removal; ewes at the second oestrus after synchronization were used as controls. On day 6 of the oestrous cycle, after slaughter, ovarian response was assessed and embryos were collected; uterine horn flushings (UHF), caruncular endometrium (CE) and intercaruncular (ICE) endometrium samples were collected, for PAA and PAI photometrical assessment. Oestradiol-17β (E2) and progesterone (P4) concentration was assessed in blood serum samples collected before slaughter by a radioimmunoassay. A significant increase of PAA in the UHF was observed after treatment; PAA in UHF was significantly associated with the number of embryos or high-quality embryos, while PAA in UHF per embryo or high-quality embryo was not affected by treatment. No significant effect of treatment was noticed on PAA or PAI in the endometrium (CE, ICE). In UHF and in ICE, PAI against tissue-type PA had a significant positive relationship with PAI against urokinase-type PA. PAI against tissue-type PA in CE had significant negative relationship with E2 concentration. In conclusion, hormone treatment involving eCG in the dosage used for superovulation and, to a lesser degree, in the dosage used for oestrus synchronization affected PAA in the uterus of Chios ewes, before maternal recognition of pregnancy, but did not seem to disturb reproductive function.

Environmental constraints and pathologies that modulate equine placental genes and development.

Equine placental development is a long process with unique features. Implantation occurs around 40 days of gestation (dpo) with the presence of a transient invasive placenta from 25-35 to 100-120 dpo. The definitive, non-invasive placenta remains until term (330 days). This definitive placenta is diffuse and epitheliochorial, exchanging nutrients, gas and waste with the endometrium through microvilli, called microcotyledons. These are lined by an external layer of haemotrophic trophoblast. Moreover, histotrophic exchange remains active through the histotrophic trophoblast located along the areolae. Placental development is dependent on the maternal environment that can be affected by several factors (e.g. nutrition, metabolism, age, embryo technologies, pathologies) that may affect fetal development as well as long-term offspring health. The first section of the review focuses on normal placental development as well as definitive placental structure. Differences between the various regions of the placenta are also highlighted. The latter sections provide an overview of the effects of the maternal environment and reproductive pathologies, respectively, on trophoblast/placental gene expression and structure. So far, only pre-implantation and late gestation/term data are available, which demonstrate important placental plasticity in response to environmental variation, with genes involved in oxidative stress and tissue differentiation mostly involved in the pre-implantation period, whereas genes involved in feto-placental growth and nutrient transfers are mostly perturbed at term.

Effects of exogenous progesterone on the expression of mineral regulatory molecules by ovine endometrium and placentomes†.

This study aimed to determine whether the acceleration of conceptus development induced by the administration of exogenous progesterone (P4) during the preimplantation period of pregnancy alters calcium, phosphate, and vitamin D signaling at the maternal-conceptus interface. Suffolk ewes (n = 48) were mated to fertile rams and received daily intramuscular injections of either corn oil (CO) vehicle or 25mg of progesterone in CO (P4) for the first 8days of pregnancy and hysterectomized on either Day 9 (CO, n = 5; P4, n = 6), 12 (CO, n = 9; P4, n = 4) or 125 (CO, n = 14; P4, n = 10) of gestation. The expression of S100A12 (P < 0.05) and fibroblast growth factor receptor (FGFR2) (P < 0.01) messenger RNAs (mRNAs) was lower in endometria from P4-treated ewes on Day 12. The expression of ADAM10 (P < 0.05) mRNA was greater in endometria from P4-treated ewes on Day 125. The expression of ADAM10 (P < 0.01), FGFR2 (P < 0.05), solute carrier (SLC)20A1 (P < 0.05), TRPV5 (P < 0.05), and TRPV6 (P < 0.01) mRNAs was greater, but KL mRNA expression was lower (P < 0.05) in placentomes from P4-treated ewes at Day 125. There was lower endometrial and greater placentomal expression of mRNAs involved in mineral metabolism and transport in twin compared to singleton pregnancies. Further, the expression of mRNAs involved in mineral metabolism and transport was greater in P4-treated twin placentomes. KL, FGF23, vitamin D receptor (VDR), S100A9, S100A12, S100G, and CYP27B1 proteins were immunolocalized in endometria and placentomes. Exogenous P4 in early pregnancy altered the expression of regulators of calcium, phosphate, and vitamin D on Day 125 of pregnancy indicating a novel effect of P4 on mineral transport at the maternal-conceptus interface.