Pre-attachment Period Research Articles

The Relationship Between Continuity of Care and Enhancement of Clinical Outcomes Among Patients with Chronic Conditions.

Continuity of care is one of the main principles of family medicine, described as a relationship with a single provider that extends beyond a single illness episode. This retrospective study, conducted at King Saud University Family Medicine Center in Riyadh, Saudi Arabia, aimed to investigate the impact of having a regular primary care provider on clinical outcomes and preventive service delivery for patients with diabetes and/or hypertension. The study, spanning 2017 to 2019, included 400 patients diagnosed with diabetes and/or hypertension for at least six months before the 6-month pre-attachment period to regular family medicine physicians in 2018. Data before and after attachment for at least six months were compared using electronic health records. The mean age of the patients was 60.9, with a predominant female representation (66.8%) and 90.7% Saudis. Results indicated a significant improvement in glycated hemoglobin (HbA1c) levels (p = 0.005) and systolic blood pressure (p = 0.014) post-attachment. Preventive service delivery saw notable enhancements, with increased colon cancer screening (p = 0.03), breast cancer screening (p < 0.001), and retinal screening (p < 0.001) post-attachment. This study's findings underscore the importance of continuity of care in chronic disease management and provide valuable and promising insights into the Saudi healthcare context, aligning with the Saudi Ministry of Health's vision for universal access to regular primary care providers.

Bioinformatic analysis of endometrial miRNA expression profile at day 26–28 of pregnancy in the mare

The establishment of the fetomaternal interface depends on precisely regulated communication between the conceptus and the uterine environment. Recent evidence suggests that microRNAs (miRNAs) may play an important role in embryo-maternal dialogue. This study aimed to determine the expression profile of endometrial miRNAs during days 26–28 of equine pregnancy. Additionally, the study aimed to predict target genes for differentially expressed miRNAs (DEmiRs) and their potential role in embryo attachment, adhesion, and implantation. Using next-generation sequencing, we identified 81 DEmiRs between equine endometrium during the pre-attachment period of pregnancy (day 26–28) and endometrium during the mid-luteal phase of the estrous cycle (day 10–12). The identified DEmiRs appear to have a significant role in regulating the expression of genes that influence cell fate and properties, as well as endometrial receptivity formation. These miRNAs include eca-miR-21, eca-miR-126-3p, eca-miR-145, eca-miR-451, eca-miR-491-5p, members of the miR-200 family, and the miRNA-17-92 cluster. The target genes predicted for the identified DEmiRs are associated with ion channel activity and sphingolipid metabolism. Furthermore, it was noted that the expression of mucin 1 and leukemia inhibitory factor, genes potentially regulated by the identified DEmiRs, was up-regulated at day 26–28 of pregnancy. This suggests that miRNAs may play a role in regulating specific genes to create a favorable uterine environment that is necessary for proper attachment, adhesion, and implantation of the embryo in mares.

Early pregnancy-induced transcripts in peripheral blood immune cells in Bos indicus heifers

Immune cells play a central role in early pregnancy establishment in cattle. We aimed to: (1) discover novel early-pregnancy-induced genes in peripheral blood mononuclear cells (PBMC); and (2) characterize the temporal pattern of early-pregnancy-induced transcription of select genes in PBMC and peripheral blood polymorphonuclear cells (PMN). Beef heifers were artificially inseminated on D0 and pregnancies were diagnosed on D28. On D10, 14, 16, 18, and 20, blood was collected for isolation of PBMC and PMN from heifers that were retrospectively classified as pregnant (P) or non-pregnant (NP). PBMC samples from D18 were submitted to RNAseq and 220 genes were differentially expressed between pregnant (P) and non-pregnant (NP) heifers. The temporal abundance of 20 transcripts was compared between P and NP, both in PBMC and PMN. In PBMC, pregnancy stimulated transcription of IFI6, RSAD2, IFI44, IFITM2, CLEC3B, OAS2, TNFSF13B, DMKN and LGALS3BP as early as D18. Expression of IFI44, RSAD2, OAS2, LGALS3BP, IFI6 and C1R in PMN was stimulated in the P group from D18. The novel early-pregnancy induced genes discovered in beef heifers will allow both the understanding of the role of immune cells during the pre-attachment period and the development of technologies to detect early pregnancies in beef cattle.

Cellular interactions during implantation in domestic ruminants

Implantation is a critical step in the progress of pregnancy, during which the conceptus acquires a fixed position within the uterine lumen, and leads to the establishment of the placental structures. This process implies some cellular modifications of both the uterine epithelium and the trophoblast to ensure cell adhesion between the two tissues. In ruminants, the implantation process is characterized by three main steps: a long pre-attachment period lasting 2-3 weeks during which the conceptus elongates considerably, an apposition stage when cellular contacts are established between the trophoblast and the uterine epithelium, and an adhesion stage which ends the process and gives rise to the cellular structure of an epithelio-chorial placenta. Trophoblast apposition begins in the vicinity of the embryo by day 15, 18 and 19 in sheep, goats and cows, respectively. The trophoblast cells surrounding the embryo show morphological and functional changes. These modifications are local within the conceptus since non-implanted areas of trophoblast still display the morphological and functional features that characterized this tissue during the pre-attachment period. As the implantation process spreads towards the extremity of the elongated conceptus, these cellular transformations progressively affect the whole trophoblast. Apposition is completed by a close adhesion between the interdigitating uterine microvilli and the trophoblastic plasma membrane. By this stage, trophoblastic binucleate cells migrate through the trophoblast monolayer and fuse with individual uterine cells to form a syncytial tissue. During this process placental lactogen hormones and pregnancy serum proteins (PSP) produced by the binucleate cells are transported to the endometrial tissues and then to the maternal blood circulation.

Effects of cell-attachment and extracellular matrix on bone formation in vivo in collagen-hydroxyapatite scaffolds.

Cell-based tissue engineering can be used to replace missing or damaged bone, but the optimal methods for delivering therapeutic cells to a bony defect have not yet been established. Using transgenic reporter cells as a donor source, two different collagen-hydroxyapatite (HA) scaffolds, and a critical-size calvarial defect model, we investigated the effect of a cell-attachment period prior to implantation, with or without an extracellular matrix-based seeding suspension, on cell engraftment and osteogenesis. When quantitatively compared, the in-house scaffold implanted immediately had a higher mean radiopacity than in-house scaffolds incubated overnight. Both scaffold types implanted immediately had significantly higher area fractions of donor cells, while the in-house collagen-HA scaffolds implanted immediately had higher area fractions of the mineralization label compared with groups incubated overnight. When the cell loading was compared in vitro for each delivery method using the in-house scaffold, immediate loading led to higher numbers of delivered cells. Immediate loading may be preferable in order to ensure robust bone formation in vivo. The use of a secondary ECM carrier improved the distribution of donor cells only when a pre-attachment period was applied. These results have improved our understanding of cell delivery to bony defects in the context of in vivo outcomes.

Presence of Transcription Factor OCT4 Limits Interferon-tau Expression during the Pre-attachment Period in Sheep.

Interferon-tau (IFNT) is thought to be the conceptus protein that signals maternal recognition of pregnancy in ruminants. We and others have observed that OCT4 expression persists in the trophectoderm of ruminants; thus, both CDX2 and OCT4 coexist during the early stages of conceptus development. The aim of this study was to examine the effect of CDX2 and OCT4 on IFNT gene transcription when evaluated with other transcription factors. Human choriocarcinoma JEG-3 cells were cotransfected with an ovine IFNT (-654-bp)-luciferase reporter (-654-IFNT-Luc) construct and several transcription factor expression plasmids. Cotransfection of the reporter construct with Cdx2, Ets2 and Jun increased transcription of -654-IFNT-Luc by about 12-fold compared with transfection of the construct alone. When cells were initially transfected with Oct4 (0 h) followed by transfection with Cdx2, Ets2 and/or Jun 24 h later, the expression of -654-IFNT-Luc was reduced to control levels. OCT4 also inhibited the stimulatory activity of CDX2 alone, but not when CDX2 was combined with JUN and/or ETS2. Thus, when combined with the other transcription factors, OCT4 exhibited little inhibitory activity towards CDX2. An inhibitor of the transcriptional coactivator CREB binding protein (CREBBP), 12S E1A, reduced CDX2/ETS2/JUN stimulated -654-IFNT-Luc expression by about 40%, indicating that the formation of an appropriate transcription factor complex is required for maximum expression. In conclusion, the presence of OCT4 may initially minimize IFNT expression; however, as elongation proceeds, the increasing expression of CDX2 and formation of the transcription complex leads to greatly increased IFNT expression, resulting in pregnancy establishment in ruminants.

Persistence of an immunoreactive MUC1 protein at the feto - maternal interface throughout pregnancy in the mare

A polyclonal human mucin-1 (MUC1) antibody was used to stain immunohistochemically for the presence of MUC1 on the endometrium and fetal membranes in mares between 20 and 309 days of gestation. Western blot analysis demonstrated the presence of a protein equivalent in size to a human MUC1 isoform, MUC1/Y, in equine endometrium, allantochorion and amnion. At all stages of gestation examined immunoreactivity to the MUC1 antibody was detected on the apical surface of the lumenal epithelium of the endometrium and the epithelium lining the mouths and apical regions of the endometrial glands. Furthermore, it persisted unchanged on the surface of the lumenal epithelium lying beneath the highly-invasive chorionic girdle component of the trophoblast before, during and after development of the endometrial cups. The MUC1 immunoreactive protein was also present on the trophoblast and other components of the fetal membranes during the post-fixation, pre-attachment period of gestation (20-40 days) and it persisted on the apical surface of the non-invasive trophoblast of the allantochorion before, during and after attachment, microvillous interdigitation and development of the microcotyledonary epitheliochorial placenta. Hence, the delayed placentation response in mares appears to occur independently of the persistence of an immunoreactive MUC1 protein at the feto-maternal interface.

An Overview of Molecular and Cellular Mechanisms Associated with Porcine Pregnancy Success or Failure

Prenatal mortality remains one of the major constraints for the commercial pig industry in North America. Twenty to thirty per cent of the conceptuses are lost early in gestation and an additional 10-15% is lost by mid-to-late gestation. Research over the last two decades has provided critical insights into how uterine capacity, placental efficiency, genetics, environment, nutrition and immune mechanisms impact successful conceptus growth; however, the exact cause and effect relationship in the context of foetal loss has yet to be determined. Similar to other mammalian species such as the human, mouse, rat, and primates, immune cell enrichment occurs at the porcine maternal-foetal interface during the window of conceptus attachment. However, unlike other species, immune cells are solely recruited by conceptus-derived signals. As pigs have epitheliochorial placentae where maternal and foetal tissue layers are separate, it provides an ideal model to study immune cell interactions with foetal trophoblasts. Our research is focused on the immune-angiogenesis axis during porcine pregnancy. It is well established that immune cells are recruited to the maternal-foetal interface, but their pregnancy specific functions and how the local milieu affects angiogenesis and inflammation at the site of foetal arrest remain unknown. Through a better understanding of how immune cells modulate crosstalk between the conceptus and the mother, it might be possible to therapeutically target immune cells and/or their products to reduce foetal loss. In this review, we provide evidence from the literature and from our own work into the immunological factors associated with porcine foetal loss.

Atresia Ani: A Congenital Defect and Its Successful Management in Non-Descript Calf

Congenital defects, abnormalities of structure or function present at birth, may be caused by genetic or environmental factors, or a combination of both; in many cases, the causes are unknown. The most common bovine environmental teratogens include toxic plants consumed by the dam and maternal-fetal viral infections during gestation. The developmental defects varies with pre-attachment period (before 14 days of gestation) and period of embryo (between 14 � 42 days of gestation) of fetal development are highly susceptible to mutation and teratogens, respectively (Bademkiran et al., 2009). Congenital malformation sometimes leads to perinatal mortality, and it may also decrease maternal productivity and reduce the value of the defective neonates. Severe defects results in abortion of the calf or a return to service of the calf and cow. Congenital anomalies have been estimated to occur in 0.2%�5% of calves. (Bademkiran et al., 2009). Atresia ani, (imperforate anus) is a congenital abnormality characterized by persistence of the anal membrane resulting in a thin membrane covering the normal anal canal or is the failure of the anal membrane to break down (Noden and Lahunta 1985). Merei et al. (2001) reported most congenital malformations was atresia ani and most frequently encountered in males calves and pigs. Various surgical techniques have been use to correct atresia ani in domestic animals (Singh, 1989). This report communicates a case of atresia ani in male calf, which was successfully treated by surgical intervention.

Dickkopf-1 Expression During Early Bovine Placentation and Its Down-regulation in Somatic Cell Nuclear Transfer (SCNT) Pregnancies

The precise role of Dickkopf-1 (Dkk-1) during early bovine trophoblast development and subsequent placentation is not fully understood. Using somatic cell nuclear transfer (SCNT) generated pregnancies as a model of poor placentation we have found that mean levels of Dkk-1 mRNA were 1.5 fold lower in SCNT fetal cotyledon tissue at Day 50 of gestation than those resulting from artificial insemination (AI) and 2 fold lower at Days 100 and 150 (P<0.004). Dkk-1 expression in cotyledon tissue was localized by in situ hybridization to fetal binucleate cells (BNCs). Examining conceptuses from blastocyst stage we show that Dkk-1 mRNA was first evident between Days 15-20 of gestation in trophoblast tissue (when BNCs first appear) prior to the initial expression of the BNC specific bovine placental lactogen (bPL) on Day 20. Dkk-1 mRNA levels were higher than bPL in trophoblast tissue throughout the pre-attachment period (Days 24-31), however, this reversed during cotyledon development with only a subset of the bPL immunoreactive BNCs also containing Dkk-1 protein, suggesting a specific role for Dkk-1 during early placentation. One function of Dkk-1 is as an antagonist of the Wnt signaling pathway and, although Wnt5A and Wnt7A mRNAs were expressed in Day 50 bovine cotyledons, their expression levels were similar between AI and SCNT. In addition, the nuclear localization of beta-catenin, which is an indicator of activation of the Wnt pathway, was also similar between AI and SCNT cotyledon tissue. Transcriptional control of Dkk-1 was not due to changes in DNA methylation levels in the promoter region as methylation levels were no different when comparing AI and SCNT tissues. The decreased expression of Dkk-1 in SCNT cotyledons that are prone to abnormal placentation suggests a role in cotyledon formation but the mechanism and regulatory control is yet to be revealed.

Transcriptome Studies of Bovine Endometrium Reveal Molecular Profiles Characteristic for Specific Stages of Estrous Cycle and Early Pregnancy

The endometrium undergoes marked functional changes during estrous cycle and pregnancy. As the adjacent environment of the conceptus, it represents the maternal interface for embryo-maternal communication, which is essential to maintain pregnancy. Transcriptome studies provide the unique opportunity to assess molecular profiles changing in response to endocrine or metabolic stimuli or to embryonic pregnancy recognition signals. Here we review the current state of transcriptome profiling techniques and the results of a series of transciptome studies comparing bovine endometrium samples during the estrous cycle or endometrium samples from pregnant vs. non-pregnant animals. These studies revealed specific mRNA profiles which are characteristic for the functional status of the endometrium. Transcriptome studies of endometrial samples recovered during the pre-attachment period identified many interferon-stimulated genes, genes that are possibly involved in embryo-maternal immune modulation ( C1S, C1R, C4, SERPING1, UTMP, CD81, IFITM1, BST2), as well as genes affecting cell adhesion ( AGRN, CD81, LGALS3BP, LGALS9, GPLD1, MFGE8, and TGM2) and remodeling of the endometrium ( CLDN4, MEP1B, LGMN, MMP19, TIMP2, TGM2, MET, and EPSTI1). The results of these transcriptome studies were compared to those of similar microarray analyses in human, mouse and Rhesus monkey to identify similarities in endometrial biology between mammalian species and species-specific differences. Future studies will cover dynamic transcriptome changes between different stages of early pregnancy, the relationship between metabolic problems in dairy cows and the functionality of reproductive tissues as well as endometrium transcriptome profiles in recipients of somatic cell nuclear transfer embryos.

Dissection of culture media for embryos: the most important and less important components and characteristics

Improvements in culture media formulations have led to an increase in the ability to maintain the mammalian embryo in culture throughout the preimplantation and pre-attachment period. Amino acids and specific macromolecules have been identified as being key medium components, whereas temporal dynamics have been recognised as important media characteristics. Furthermore, other laboratory factors that directly impact embryo development and viability have been identified. Such factors include the use of a reduced oxygen tension, an appropriate incubation system and an adequate prescreening of all contact supplies. With rigourous quality systems in place, it is possible to obtain in vivo rates of embryo development in vitro using new media formulations while maintaining high levels of embryo viability. The future of embryo culture will likely be based on novel culture chips capable of providing temporal dynamics while facilitating real-time analysis of embryo physiology.

Holistic differential analysis of embryo‐induced alterations in the proteome of bovine endometrium in the preattachment period

During the peri-implantation period, molecular signaling between embryo and endometrium (layer of tissue lining the uterus lumen) is supposed to be crucial for the maintenance of pregnancy. To investigate embryo-induced alterations in the proteome of bovine endometrium in the preattachment period (day 18), we used monozygotic cattle twins (generated by embryo splitting) as a model eliminating genetic variability as a source for proteome differences. One of the twins was pregnant after the transfer of two in vitro produced blastocysts, while the corresponding twin received a sham-transfer and served as a nonpregnant control. The two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) analysis of the endometrium samples of three twin pairs (pregnant/nonpregnant) revealed four proteins with significantly higher abundance (p < 10(-9)) in each sample derived from the pregnant animals: Rho GDP dissociation inhibitor beta; 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD); soluble NADP(+)-dependent isocitrate dehydrogenase 1; and acyl-CoA-binding protein. To verify the accuracy of the 2-D DIGE quantification, the abundances of 20 alpha-HSD were quantified by a targeted cleavable isotope-coded affinity tag (ICAT) approach. The mass spectrometry-based ICAT quantification matched perfectly the results obtained by 2-D DIGE quantification, demonstrating the accuracy of our data. These results demonstrate that our model (monozygotic twins) in combination with the appropriate analytical tools is particularly suitable for the detection of the proteins involved in the embryo-maternal interactions.

Hormonal and non-hormonal agents at implantation as targets for contraception.

The processes leading to implantation and the establishment of pregnancy involve hormonal and non-hormonal agents that offer opportunities as targets for contraception. Hormonal agents include progesterone, luteolytic factors (prostaglandin F2 alpha) and embryonic signals (chorionic gonadotrophin, oestradiol-17 beta, interferon-tau) responsible for maintaining the corpus luteum. Non-hormonal agents include surface antigens (attachment and adhesion molecules), vasoactive agents, tissue-remodelling enzymes (matrix metalloproteinases) and inhibitors (TIMPs), growth factors (epidermal growth factor and insulin-like growth factor families) and cytokines (such as leukaemia inhibitory factor, colony-stimulating factor-1, interleukin-1 (IL-1) and IL-6) associated with the pre-attachment period and the apposition, adhesion and invasion of the blastocyst. This review describes some of the hormonal and non-hormonal agents present at the time of implantation that may be exploited as targets for contraception in feral species. Particular attention is paid to the mouse as an experimental model and potential target species. The considerable species differences which exist in the models of implantation and placentation and the way in which the female 'recognizes' the presence of a viable conceptus offer a means of conferring species specificity on potential contraceptive targets for feral species.

Porcine Conceptuses Secrete an Interferon During the Preattachment Period of Early Pregnancy1

Maternal recognition of pregnancy in sheep and cattle is thought to be initiated by the conceptus secretory proteins ovine trophoblast protein-1 (oTP-1) and bovine trophoblast protein-1 (bTP-1), respectively. Recently, these proteins have been shown to be members of the interferon-alpha (IFN-alpha) family. In this study, we have examined whether pig conceptuses also produce IFN during early pregnancy. conceptuses were collected at Days 8, 10, 11, 12, 13, 15, and 17 of pregnancy and cultured in serum-free medium for 24 h. Day 11 conceptuses secreted a dominant 22,000-24,000 Mr cluster of acidic proteins (pI 5.2-5.4), that appeared to cross-react on immunoblots with antiserum against human IFN-alpha but not against oTP-1. Antiviral activity characteristic of an IFN was present in conceptus culture medium and uterine flushings from Day 11 through Day 17 of pregnancy, but was absent in flushings prior to Day 11 of pregnancy and in flushings from Day 12 nonpregnant gilts. The antiviral activity coeluted with a 22,000-24,000 Mr protein during partial purification through a gel filtration column. The activity was extremely labile, but could be restored by sequential protein denaturation, reduction, and renaturation. We conclude that production of IFN by early conceptuses is not restricted to ruminant species, and may therefore represent a more general phenomenon.

Normal murine and porcine embryos recruit NK cells to the uterus

Decidual NK cells, indistinguishable from those found in lymphoid tissues, are present in cell suspensions prepared from maternal decidua of random-bred mice between Days 6.5 and 10.5 of first gestation. The stringency of the correlation between NK cells and normal embryos during successful pregnancy is unknown. Our previous finding that active NK cells were unable to mediate lysis of fresh embryonic tissues at any stage during gestation suggests that if NK cells play a functional role in normal pregnancy it would be a noncytolytic role. Before studies on the function of uterine NK cells were undertaken, evidence that the association of NK cells with normal embryos is widespread was sought by assessing NK cell activity in cell suspensions from decidua of syngeneically mated mice, from decidua of multiparous, random-bred mice, and from the endometrium of pigs during first pregnancy. Neither parity nor maternal-fetal compatibility changed the pattern of high levels of decidual NK cell activity early in pregnancy followed by decline. Porcine NK cell activity was not detected in uterine cells isolated from cycling pigs but in pregnant animals it gradually increased during the preattachment period and reached levels greater than those in blood, postattachment (Day 28). Some of this activity was hormone dependent but sustained increases in NK cell activity required the presence of an embryo. These studies demonstrate that the association of functional NK cells with normal embryos is wide-spread during early pregnancy.

New developments in the biochemistry of early pregnancy in farm animals

The concept of the maternal recognition of pregnancy in farm animals is discussed with emphasis on the transmission of a signal from the conceptus to the mother. Work on the pig has revealed that pre-attachment blastocyst tissue acquires the ability to synthesize hormonal steroids, and their physiological role is discussed in terms of the transformation of ovarian activity, the mechanism of attachment and embryonic protein synthesis. Parallel studies on the establishment of pregnanoy in the sheep and cow are briefly reviewed. In the cow a laboratory test of pregnancy in thie third week after insemination based on progesterone levels in milk has revealed that pregnancy failure is often due to embryonic death during the preattachment period.