Spatial transcriptomics of fetal membrane-Decidual interface reveals unique contributions by cell types in term and preterm births.

During pregnancy, two fetomaternal interfaces, the placenta-decidua basalis and the fetal membrane-decidua parietals, allow for fetal growth and maturation and fetal-maternal crosstalk, and protect the fetus from infectious and inflammatory signaling that could lead to adverse pregnancy outcomes. While the placenta has been studied extensively, the fetal membranes have been understudied, even though they play critical roles in pregnancy maintenance and the initiation of term or preterm parturition. Fetal membrane dysfunction has been associated with spontaneous preterm birth (PTB, < 37 weeks gestation) and preterm prelabor rupture of the membranes (PPROM), which is a disease of the fetal membranes. However, it is unknown how the individual layers of the fetal membrane decidual interface (the amnion epithelium [AEC], the amnion mesenchyme [AMC], the chorion [CTC], and the decidua [DEC]) contribute to these pregnancy outcomes. In this study, we used a single-cell transcriptomics approach to unravel the transcriptomics network at spatial levels to discern the contributions of each layer of the fetal membranes and the adjoining maternal decidua during the following conditions: scheduled caesarian section (term not in labor [TNIL]; n = 4), vaginal term in labor (TIL; n = 3), preterm labor with and without rupture of membranes (PPROM; n = 3; and PTB; n = 3). The data included 18,815 genes from 13 patients (including TIL, PTB, PPROM, and TNIL) expressed across the four layers. After quality control, there were 11,921 genes and 44 samples. The data were processed by two pipelines: one by hierarchical clustering the combined cases and the other to evaluate heterogeneity within the cases. Our visual analytical approach revealed spatially recognized differentially expressed genes that aligned with four gene clusters. Cluster 1 genes were present predominantly in DECs and Cluster 3 centered around CTC genes in all labor phenotypes. Cluster 2 genes were predominantly found in AECs in PPROM and PTB, while Cluster 4 contained AMC and CTC genes identified in term labor cases. We identified the top 10 differentially expressed genes and their connected pathways (kinase activation, NF-κB, inflammation, cytoskeletal remodeling, and hormone regulation) per cluster in each tissue layer. An in-depth understanding of the involvement of each system and cell layer may help provide targeted and tailored interventions to reduce the risk of PTB.

PGRMC2 and HLA-G regulate immune homeostasis in a microphysiological model of human maternal-fetal membrane interface.

Chorion trophoblasts (CTCs) and immune cell-enriched decidua (DECs) comprise the maternal-fetal membrane interface called the chorio-decidual interface (CDi) which constantly gets exposed to maternal stressors without leading to labor activation. This study explored how CTCs act as a barrier at CDi. The roles of human leukocyte antigen (HLA)-G and progesterone receptor membrane component 2 (PGRMC2) in mediating immune homeostasis were also investigated. The CDi was recreated in a two-chamber microfluidic device (CDi-on-chip) with an outer chamber of primary DECs and immune cell line-derived innate immune cells and an inner chamber of wild-type or PGRMC2 or HLA-G knockout immortalized CTCs. To mimic maternal insults, DECs were treated with lipopolysaccharide, poly(I:C), or oxidative stress inducer cigarette smoke extract. Expression levels of inflammation and immunity genes via targeted RNA sequencing, production of soluble mediators, and immune cell migration into CTCs were determined. In CDi-on-chip, decidua and immune cells became inflammatory in response to insults while CTCs were refractory, highlighting their barrier function. HLA-G and PGRMC2 are found to be vital to immune homeostasis at the CDi, with PGRMC2 serving as an upstream regulator of inflammation, HLA-G expression, and mesenchymal-epithelial transition, and HLA-G serving as a frontline immunomodulatory molecule, thus preventing fetal membrane compromise.

Maternal Spargel/dPGC-1 is critical for embryonic development and influences chorion gene amplification via Cyclin E activity.

The function of spargel/dPGC-1 in Drosophila oogenesis has been unequivocally established. Here, we sought to assess whether Spargel protein or RNA is essential for developmentally competent eggs. The trans-heterozygotic combination of two spargel mutant alleles allowed us to decrease Spargel expression to very low levels. Using this model, we now demonstrated the requirement for Spargel in eggshell patterning and embryonic development, which led us to establish that spargel is a maternal effect gene. Further examination of Spargel's potential mechanism of action in eggshell biogenesis revealed that low levels of Spargel in the adult ovary cause diminished Cyclin E activity, resulting in reduced chorion gene amplification levels, leading to eggshell biogenesis defects. Thus, another novel role for spargel/dPGC-1 is exposed whereby, through Cyclin E activity, this conserved transcriptional coactivator regulates the chorion gene amplification process.

Effect of subchorionic hematoma on first-trimester maternal serum free ß-hCG and PAPP-A levels.

Objective: This study aimed to investigate the effects of the presence of subchorionic hematoma (SH) in early pregnancies with threatened miscarriage (TM) on levels of first-trimester maternal serum markers, pregnancy-associated plasma protein-A (PAPP-A), and free ß-human chorionic gonadotropin (ß-hCG) levels. Methods: The data of TM cases with SH in the first trimester between 2015 and 2021 were evaluated retrospectively. The data of age and gestational age-matched TM cases without SH were also assessed to constitute a control group. Demographic characteristics, obstetric histories, ultrasonographic findings, and free ß-hCG and PAPP-A levels of the groups were compared. Results: There were 119 cases in the study group and 153 cases in the control group. The median vertical and longitudinal lengths of the SH were 31 mm and 16 mm. The median age of both groups was similar (p=0.422). The MoM value of PAPP-A was 0.088 (.93) in the study group and 0.9 (0.63) in the control group (p=0.519). Similarly, the MoM value of free ß-hCG was 1.04 (0.78) in the study group and 0.99 (0.86) in the control group (p=0.66). No significant relationship was found in the multivariate analysis between free ß-hCG MoM, PAPP-A MoM, age, gravida, and vertical and longitudinal lengths of the hematoma (p>0.05). Conclusion: The level of PAPP-A and free ß-hCG were not affected by the SH. Therefore, these markers can be used reliably in TM cases with SH for the first-trimester fetal aneuploidy screening test.

Dehydrated Amnion Chorion Membrane versus standard of care for diabetic foot ulcers: a randomised controlled trial.

OBJECTIVE: Diabetic foot ulcers (DFUs) continue to challenge wound care practitioners. This prospective, multicentre, randomised controlled trial (RCT) evaluated the effectiveness of a dehydrated Amnion Chorion Membrane (dACM) (Organogenesis Inc., US) versus standard of care (SoC) alone in complex DFUs in a challenging patient population. METHOD: Subjects with a DFU extending into dermis, subcutaneous tissue, tendon, capsule, bone or joint were enrolled in a 12-week trial. They were allocated equally to two treatment groups: dACM (plus SoC); or SoC alone. The primary endpoint was frequency of wound closure determined by a Cox analysis that adjusted for duration and wound area. Kaplan-Meier analysis was used to determine median time to complete wound closure (CWC). RESULTS: The cohort comprised 218 patients, and these were split equally between the two treatment groups with 109 patients in each. A Cox analysis showed that the estimated frequency of wound closure for the dACM plus SoC group was statistically superior to the SoC alone group at week 4 (12% versus 8%), week 6 (22% versus 11%), week 8 (31% versus 21%), week 10 (42% versus 27%) and week 12 (50% versus 35%), respectively (p=0.04). The computed hazard ratio (1.48 (confidence interval: 0.95, 2.29) showed a 48% greater probability of wound closure in favour of the dACM group. Median time to wound closure for dACM-treated ulcers was 84 days compared to 'not achieved' in the SoC-treated group (i.e., ≥50% of SoC-treated DFUs failed to heal by week 12; p=0.04). CONCLUSION: In an adequately powered DFU RCT, dACM increased the frequency, decreased the median time, and improved the probability of CWC when compared with SoC alone. dACM demonstrated beneficial effects in DFUs in a complex patient population. DECLARATION OF INTEREST: This study was funded by Organogenesis Inc., US. JC serves as a consultant and speaker for Organogenesis. RDD serves as a speaker for Organogenesis. OMA and MLS serve as consultants for Organogenesis. The authors have no other conflicts of interest to declare.

From chromosomal aberrations to mutations in individual genes - the significance of genetic studies of chorions after miscarriage in the search for causes of miscarriages.

OBJECTIVE: To determine the frequency of chromosomal aberrations in chorions after a miscarriage. The second was to examine selected euploid chorions using a next-generation sequencing (NGS) panel designed to assess 43 genes associated with pregnancy loss. MATERIALS AND METHODS: The 1244 chorions were tested by targeted quantitative fluorescent PCR (QF-PCR, 827 chorions) and microarray-based comparative genomic hybridization (aCGH, 417 chorions). Then, 9 euploid chorions were examined using a designed NGS panel. RESULTS: Trisomies were the most common chromosomal aberration identified in the spontaneous miscarriage samples. The second chromosomal abnormality in the aCGH group and the third most common in the QF-PCR group was monosomy X. Structural aberrations were the third most common aberration in the samples screened by aCGH (7.7% of chorions). In 19% of 647 couples who submitted chorions for analysis after pregnancy loss, the chromosomal abnormality in the chorion originated from a woman with a balanced chromosomal rearrangement. This discovery was statistically significant compared to patients with normal chorions. Using the designed NGS panel, we identified a potentially pathogenic de novo variant in the F5 gene in two euploid chorions. Additionally, among the patients who experienced miscarriages and were screened using the NGS panel, we identified variants in the MDM, ACE, and NLRP2 genes that could be associated with a predisposition to pregnancy loss. CONCLUSION: Numerical aberrations are the most common cause of miscarriages, but structural chromosomal aberrations also account for a significant proportion of abnormal results. Our findings indicate that couples with structural chromosomal abnormalities in material post-miscarriage are at increased risk of carrying balanced chromosomal abnormalities. Moreover, NGS-based analyses can uncover previously unidentified causes of miscarriages in the chorionic villi.

Improving the Prognosis of Periodontally Involved Teeth at the Time of Extraction of Adjacent Teeth With an Amnion-Chorion Barrier and Bioactive Dentin Graft.

With increased awareness, both in the dental literature and by the general public, of peri-implant disease, a growing trend in dentistry is to save teeth with a "questionable" periodontal prognosis. This prospective study involving such patients was designed to evaluate the effects of combining a bioactive barrier and graft, not on the socket but to augment adjacent periodontal conditions on teeth with severe periodontal bone loss at the time of extraction of an adjacent tooth. Fifteen patients were selected; teeth were extracted, ground, prepared with a pH 11 cleanser, partially demineralized, and made into a graft. This mixture was used to augment socket volume and perform periodontal regenerative surgery. The graft was covered with a bioactive amnion-chorion barrier membrane. Bioactive membranes can stimulate host cells in the surrounding gingival and periosteal tissues to accelerate site closure and healing, simultaneously exerting positive effects on the underlying bone and graft material not observed to the same extent with other membranes. This can improve healing and site regeneration as shown clinically and radiographically in this report. Use of these bioactive barrier membrane and dentin graft materials may have additive effects and provide stimulus for conversion to host bone after site healing. The combination of an amnion-chorion membrane with autologous dentin graft appears to maximize the benefits of the individual materials, improving guided tissue regeneration results and the prognoses of periodontally involved teeth.

Human amniotic membrane modulates collagen production and deposition in vitro.

Pathological fibrosis is a significant complication of surgical procedures resulting from the accumulation of excess collagen at the site of repair which can compromise the tissue architecture and severely impede the function of the affected tissue. Few prophylactic treatments exist to counteract this process; however, the use of amniotic membrane allografts has demonstrated promising clinical outcomes. This study aimed to identify the underlying mechanism of action by utilizing relevant models that accurately represent the pathophysiology of the disease state. This study employed a pro-fibrotic in vitro system using TGFß1 stimulation and macromolecular crowding techniques to evaluate the mechanism by which amniotic membrane allografts regulate collagen biosynthesis and deposition. Following treatment with dehydrated human amnion chorion membrane (DHACM), subsequent RNA sequencing and functional enrichment with Reactome pathway analysis indicated that amniotic membranes are indeed capable of regulating genes associated with the composition and function of the extracellular matrix. Furthermore, macromolecular crowding was used in vitro to expand the evaluation to include both the effects of DHACM and a lyophilized human amnion/chorion membrane (LHACM). DHACM and LHACM regulate the TGFß pathway and myofibroblast differentiation. Additionally, both DHACM and LHACM modulate the production, secretion, and deposition of collagen type I, a primary target for pathological fibrosis. These observations support the hypothesis that amniotic membranes may interrupt pathological fibrosis by regulating collagen biosynthesis and associated pathways.

Progesterone inactivation in decidual stromal cells: A mechanism for inflammation-induced parturition.

The process of human parturition involves inflammation at the interface where fetal chorion trophoblast cells interact with maternal decidual stromal (DS) cells and maternal immune cells in the decidua (endometrium of pregnancy). This study tested the hypothesis that inflammation at the chorion-decidua interface (CDI) induces labor by negating the capacity for progesterone (P4) to block labor and that this is mediated by inactivation of P4 in DS cells by aldo-keto reductase family 1 member C1 (AKR1C1). In human, Rhesus macaque, and mouse CDI, AKR1C1 expression increased in association with term and preterm labor. In a human DS cell line and in explant cultures of term human fetal membranes containing the CDI, the prolabor inflammatory cytokine, interleukin-1ß (IL-1ß), and media conditioned by LPS-stimulated macrophages increased AKR1C1 expression and coordinately reduced nuclear P4 levels and P4 responsiveness. Loss of P4 responsiveness was overcome by inhibition of AKR1C1 activity, inhibition of AKR1C1 expression, and bypassing AKR1C1 activity with a P4 analog that is not metabolized by AKR1C1. Increased P4 activity in response to AKR1C1 inhibition was prevented by the P4 receptor antagonist RU486. Pharmacologic inhibition of AKR1C1 activity prevented parturition in a mouse model of inflammation-induced preterm parturition. The data suggest that inflammatory stimuli at the CDI drive labor by inducing AKR1C1-mediated P4 inactivation in DS cells and that inhibiting and/or bypassing of AKR1C1-mediated P4 inactivation is a plausible therapeutic strategy to mitigate the risk of inflammation-associated preterm birth.

Orthologs of NOX5 and EC-SOD/SOD3: dNox and dSod3 Impact Egg Hardening Process and Egg Laying in Reproductive Function of Drosophila melanogaster.

The occurrence of ovarian dysfunction is often due to the imbalance between the formation of reactive oxygen species (ROS) and the ineffectiveness of the antioxidative defense mechanisms. Primary sources of ROS are respiratory electron transfer and the activity of NADPH oxidases (NOX) while superoxide dismutases (SOD) are the main key regulators that control the levels of ROS and reactive nitrogen species intra- and extracellularly. Because of their central role SODs are the subject of research on human ovarian dysfunction but sample acquisition is low. The high degree of cellular and molecular similarity between Drosophila melanogaster ovaries and human ovaries provides this model organism with the best conditions for analyzing the role of ROS during ovarian function. In this study we clarify the localization of the ROS-producing enzyme dNox within the ovaries of Drosophila melanogaster and by a tissue-specific knockdown we show that dNox-derived ROS are involved in the chorion hardening process. Furthermore, we analyze the dSod3 localization and show that reduced activity of dSod3 impacts egg-laying behavior but not the chorion hardening process.

The relationship between thyroid autoantibodies and X chromosome monosomy in the chorionic tissue of patients with missed miscarriage.

OBJECTIVE: The aim of this study was to investigate the relationship between thyroid autoantibodies (TGAb and TPOAb) and X chromosome monosomy in the chorionic tissue of patients with missed early miscarriage. METHODS: The baseline data, thyroid function, thyroid antibody and the chromosomes from the chorionic tissue of 228 patients with missed early miscarriage were examined. RESULTS: (1) Among the 228 patients, 121 had a normal chromosome number, and 107 had an abnormal chromosome number. The majority of them were autosomal trisomy, of which trisomy 16 (40.19%) was predominant. Sex chromosome monosomy (28.04%) was secondary. (2) Among the 228 patients, 208 patients in this study had normal thyroid function (including 134 cases of negative thyroid antibodies and 74 cases of positive thyroid antibodies alone); 6 patients had abnormal thyroid function (including 2 cases of clinical hyperthyroidism, 3 cases of subclinical hypothyroidism, 1 case of hypothyroxinemia); and 14 patients had normal TSH and elevated T4 alone.(3) After exclusion of patients with thyroid function abnormalities, there were no significant differences in baseline data between the normal chromosome group and the abnormal chromosome group (P > 0.05). However, there was a significant difference in TGAb and TPOAb between the normal chromosome and abnormal chromosome group with 45, X karyotype, with a higher proportion of TGAb and/or TPOAb positivity in the 45, X karyotype group (P < 0.05). Additionally, compared to TGAb and/or TPOAb-positive patients, the risk of X chromosome monosomy was significantly reduced in TGAb and TPOAb-negative patients (P < 0.05). Moreover, both TGAb and TPOAb titer values in the X chromosome monosomy group were higher than those in the chromosomally normal group (P < 0.05). CONCLUSION: There is a correlation between TGAb, TPOAb and X chromosome monosomy in the chorionic tissue of patients with missed early miscarriage, although the mechanism remains to be further investigated.

Pregnancy outcomes of monochorionic diamniotic and dichorionic diamniotic twin pregnancies conceived by assisted reproductive technology and conceived naturally: a study based on chorionic comparison.

OBJECTIVE: To evaluate monochorionic diamniotic (MCDA) and dichorionic diamniotic (DCDA) twin pregnancies conceived by assisted reproductive technology (ART) and conceived naturally. METHODS: We retrospectively analyzed the data on twin pregnancies conceived by ART from January 2015 to January 2022,and compared pregnancy outcomes of MCDA and DCDA twins conceived by ART with those of MCDA and DCDA twins conceived naturally, pregnancy outcomes between MCDA and DCDA twins conceived by ART, and pregnancy outcomes of DCT and TCT pregnancies reduced to DCDA pregnancies with those of DCDA pregnancies conceived naturally. RESULT: MCDA pregnancies conceived by ART accounted for 4.21% of the total pregnancies conceived by ART and 43.81% of the total MCDA pregnancies. DCDA pregnancies conceived by ART accounted for 95.79% of the total pregnancies conceived by ART and 93.26% of the total DCDA pregnancies. Women with MCDA pregnancies conceived by ART had a higher premature delivery rate, lower neonatal weights, a higher placenta previa rate, and a lower twin survival rate than those with MCDA pregnancies conceived naturally (all p < 0.05). Women with DCDA pregnancies conceived naturally had lower rates of preterm birth, higher neonatal weights, and higher twin survival rates than women with DCDA pregnancies conceived by ART and those with DCT and TCT pregnancies reduced to DCDA pregnancies (all p < 0.05). CONCLUSION: Our study confirms that the pregnancy outcomes of MCDA pregnancies conceived by ART are worse than those of MCDA pregnancies conceived naturally. Similarly, the pregnancy outcomes of naturally-conceived DCDA pregnancies are better than those of DCDA pregnancies conceived by ART and DCT and TCT pregnancies reduced to DCDA pregnancies.

Perinatal outcomes and growth discordance of triplet pregnancies based on chorionicity: a retrospective cohort study.

BACKGROUND: The worldwide occurrence of triplet pregnancy is estimated to be 0.093%, with a natural incidence of approximately 1 in 8000. This study aims to analyze the neonatal health status and birth weight discordance (BWD) of triplets based on chorionicity from birth until discharge. METHODS: This was a retrospective study. We reviewed a total of 136 triplet pregnancies at our tertiary hospital between January 1, 2001, and December 31, 2021. Maternal and neonatal outcomes, inter-triplet BWD, neonatal morbidity, and mortality were analyzed. RESULTS: Among all cases, the rates of intrauterine death, neonatal death, and perinatal death were 10.29, 13.07, and 24.26%, respectively. Thirty-seven of the cases resulted in fetal loss, including 13 with fetal anomalies. The maternal complications and neonatal outcomes of the 99 triplet pregnancies without fetal loss were compared across different chorionicities, including a dichorionic (DC) group (41 cases), trichorionic (TC) group (37 cases), and monochorionic (MC) group (21 cases). Neonatal hypoproteinemia (P < 0.001), hyperbilirubinemia (P < 0.019), and anemia (P < 0.003) exhibited significant differences according to chorionicity, as did the distribution of BWD (P < 0.001). More than half of the cases in the DC and TC groups had a BWD < 15%, while those in the MC group had a BWD < 50% (47.6%). TC pregnancy decreased the risk of neonatal anemia (adjusted odds ratio [AOR] = 0.084) and need for blood transfusion therapy after birth (AOR = 0.119). In contrast, a BWD > 25% increased the risk of neonatal anemia (AOR = 10.135) and need for blood transfusion after birth (AOR = 7.127). TC pregnancy, MCDA or MCTA, and BWD > 25% increased neonatal hypoproteinemia, with AORs of 4.629, 5.123, and 5.343, respectively. CONCLUSIONS: The BWD differed significantly according to chorionicity. Additionally, TC pregnancies reduced the risk of neonatal anemia and need for blood transfusion, but increased the risk of neonatal hypoproteinemia. In contrast, the BWD between the largest and smallest triplets increased the risk of neonatal anemia and the need for blood transfusion. TC pregnancy, MCDA or MCTA, and BWD > 25% increased the risks of neonatal hypoproteinemia. However, due to the limited number of triplet pregnancies, further exploration of the underlying mechanism is warranted.

PURION® processed human amnion chorion membrane allografts retain material and biological properties supportive of soft tissue repair.

The reparative properties of amniotic membrane allografts are well-suited for a broad spectrum of specialties. Further enhancement of their utility can be achieved by designing to the needs of each application through the development of novel processing techniques and tissue configurations. As such, this study evaluated the material characteristics and biological properties of two PURION® processed amniotic membrane products, a lyophilized human amnion, intermediate layer, and chorion membrane (LHACM) and a dehydrated human amnion, chorion membrane (DHACM). LHACM is thicker; therefore, its handling properties are ideal for deep, soft tissue deficits; whereas DHACM is more similar to a film-like overlay and may be used for shallow defects or surgical on-lays. Characterization of the similarities and differences between LHACM and DHACM was conducted through a series of in vitro and in vivo studies relevant to the healing cascade. Compositional analysis was performed through histological staining along with assessment of barrier membrane properties through equilibrium dialysis. In vitro cellular response was assessed in fibroblasts and endothelial cells using cell proliferation, migration, and metabolic assays. The in vivo cellular response was assessed in an athymic nude mouse subcutaneous implantation model. The results indicated the PURION® process preserved the native membrane structure, nonviable cells and collagen distributed in the individual layers of both products. Although, LHACM is thicker than DHACM, a similar composition of growth factors, cytokines, chemokines and proteases is retained and consequently elicit comparable in vitro and in vivo cellular responses. In culture, both treatments behaved as potent mitogens, chemoattractants and stimulants, which translated to the promotion of cellular infiltration, neocollagen deposition and angiogenesis in a murine model. PURION® processed LHACM and DHACM differ in physical properties but possess similar in vitro and in vivo activities highlighting the impact of processing method on the versatility of clinical use of amniotic membrane allografts.

PL-hMSC and CH-hMSC derived soluble factors inhibit proliferation but improve hGBM cell migration by activating TGF-ß and inhibiting Wnt signaling.

Glioblastoma multiforme (GBM) is one of the most common and aggressive brain tumors. GBM resists most chemotherapeutic agents, resulting in a high mortality rate in patients. Human mesenchymal stem cells (hMSCs), which are parts of the cancer stroma, have been shown to be involved in the development and progression of GBM. However, different sources of hMSCs might affect GBM cells differently. In the present study, we established hMSCs from placenta (PL-hMSC) and chorion (CH-hMSC) to study the effects of their released soluble factors on the proliferation, migration, invasion, gene expression, and survival of human GBM cells, U251. We found that the soluble factors derived from CH-hMSCs and PL-hMSCs suppressed the proliferation of U251 cells in a dose-dependent manner. In contrast, soluble factors derived from both hMSC sources increased U251 migration without affecting their invasive property. The soluble factors derived from these hMSCs decreased the expression levels of CyclinD1, E2Fs and MYC genes that promote GBM cell proliferation but increased the expression level of TWIST gene, which promotes EMT and GBM cell migration. The functional study suggests that both hMSCs might exert their effects, at least in part, by activating TGF-ß and suppressing Wnt/ß-catenin signaling in U251 cells. Our study provides a better understanding of the interaction between GBM cells and gestational tissue-derived hMSCs. This knowledge might be used to develop safer and more effective stem cell therapy that improves the survival and quality of life of patients with GBM by manipulating the interaction between hMSCs and GBM cells.

Monochorionic Twinning in Bioengineered Human Embryo Models.

Monochorionic twinning of human embryos increases the risk of complications during pregnancy. The rarity of such twinning events, combined with ethical constraints in human embryo research, makes investigating the mechanisms behind twinning practically infeasible. As a result, there is a significant knowledge gap regarding the origins and early phenotypic presentation of monochorionic twin embryos. In this study, a microthermoformed-based microwell screening platform is used to identify conditions that efficiently induce monochorionic twins in human stem cell-based blastocyst models, termed "twin blastoids". These twin blastoids contain a cystic GATA3+ trophectoderm-like epithelium encasing two distinct inner cell masses (ICMs). Morphological and morphokinetic analyses reveal that twinning occurs during the cavitation phase via splitting of the OCT4+ pluripotent core. Notably, each ICM in twin blastoids contains its own NR2F2+ polar trophectoderm-like region, ready for implantation. This is functionally tested in a microfluidic chip-based implantation assay with epithelial endometrium cells. Under defined flow regimes, twin blastoids show increased adhesion capacity compared to singleton blastoids, suggestive of increased implantation potential. In conclusion, the development of technology enabling large-scale formation of twin blastoids, coupled with high-sensitivity readout capabilities, presents an unprecedented opportunity for systematically exploring monochorionic twin formation and its impact on embryonic development.

Temporal ultrasound findings in the first trimester of a dual fetal demise occurring in a monochorionic diamniotic twin gestation.

Single fetal demise in monochorionic gestations in the 2nd and 3rd trimester is associated with adverse outcomes for the co-twin. We present a case of single demise in a monochorionic gestation in the 1st trimester with evidence of subsequent hemodynamic aberrations in the co-twin, supportive of feto-fetal hemorrhage occurring early in gestation.

Dehydrated human amnion/chorion membrane to treat venous leg ulcers: a cost-effectiveness analysis.

OBJECTIVE: To evaluate the cost-effectiveness of dehydrated human amnion/chorion membrane (DHACM) in Medicare enrolees who developed a venous leg ulcer (VLU). METHOD: This economic evaluation used a four-state Markov model to simulate the disease progression of VLUs for patients receiving advanced treatment (AT) with DHACM or no advanced treatment (NAT) over a three-year time horizon from a US Medicare perspective. DHACM treatments were assessed when following parameters for use (FPFU), whereby applications were initiated 30-45 days after the initial VLU diagnosis claim, and reapplications occurred on a weekly to biweekly basis until completion of the treatment episode. The cohort was modelled on the claims of 530,220 Medicare enrolees who developed a VLU between 2015-2019. Direct medical costs, quality-adjusted life years (QALYs), and the net monetary benefit (NMB) at a willingness-to-pay threshold of $100,000/QALY were applied. Univariate and probabilistic sensitivity analyses (PSA) were performed to test the uncertainty of model results. RESULTS: DHACM applied FPFU dominated NAT, yielding a lower per-patient cost of $170 and an increase of 0.010 QALYs over three years. The resulting NMB was $1178 per patient in favour of DHACM FPFU over the same time horizon. The rate of VLU recurrence had a notable impact on model uncertainty. In the PSA, DHACM FPFU was cost-effective in 63.01% of simulations at the $100,000/QALY threshold. CONCLUSION: In this analysis, DHACM FPFU was the dominant strategy compared to NAT, as it was cost-saving and generated a greater number of QALYs over three years from the US Medicare perspective. A companion VLU Medicare outcomes analysis revealed that patients who received AT with a cellular, acellular and matrix-like product (CAMP) compared to patients who received NAT had the best outcomes. Given the added clinical benefits to patients at lower cost, providers should recommend DHACM FPFU to patients with VLU who qualify. Decision-makers for public insurers (e.g., Medicare and Medicaid) and commercial payers should establish preferential formulary placement for reimbursement of DHACM to reduce budget impact and improve the long-term health of their patient populations dealing with these chronic wounds. DECLARATION OF INTEREST: Support for this analysis was provided by MiMedx Group, Inc., US. JLD, and RAF are employees of MiMedx Group, Inc. WHT, BH, PS, BGC and WVP were consultants to MiMedx Group, Inc. VD, AO, MRK, JAN, NW and GAM served on the MiMedx Group, Inc. Advisory Board. MRK and JAN served on a speaker's bureau. WVP declares personal fees and equity holdings from Stage Analytics, US.