Sex differences in innate and adaptive immunity impact fetal, placental, and maternal health†.

The differences between males and females begin shortly after birth, continue throughout prenatal development, and eventually extend into childhood and adult life. Male embryos and fetuses prioritize proliferation and growth, often at the expense of the fetoplacental energy reserves. This singular focus on growth over adaptability leaves male fetuses and neonates vulnerable to adverse outcomes during pregnancy and birth and can have lasting impacts throughout life. Beyond this prioritization of growth, male placentas and fetuses also respond to infection and inflammation differently than female counterparts. Pregnancies carrying female fetuses have a more regulatory immune response, whereas pregnancies carrying male fetuses have a stronger inflammatory response. These differences can be seen as early as the innate immune response with differences in cytokine and chemokine signaling. The sexual dimorphism in immunity then continues into the adaptive immune response with differences in T-cell biology and antibody production and transfer. As it appears that these sex-specific differences are amplified in pathologic pregnancies, it stands to reason that differences in the placental, fetal, and maternal immune responses in pregnancy contribute to increased male perinatal morbidity and mortality. In this review, we will describe the genetic and hormonal contributions to the sexual dimorphism of fetal and placental immunity. We will also discuss current research efforts to describe the sex-specific differences of the maternal-fetal interface and how it impacts fetal and maternal health.

Maternal physiology and blastocyst morphology are correlated with an inherent difference in peri-implantation human embryo development.

OBJECTIVE: To determine what patient and embryo characteristics are correlated with the developmental potential of the peri-implantation embryo. DESIGN: Retrospective study. SETTING: Research laboratory. PATIENTS: Six hundred fifty-one cryopreserved human blastocysts donated for research with informed patient consent. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Blastocyst attachment to fibronectin-coated plates, trophectoderm outgrowth area, epiblast cell number, total cell number, human chorionic gonadotropin secretion. RESULTS: Patients' body mass index, age, follicle-stimulating hormone: luteinizing hormone ratio on menstrual cycle day 3, antral follicle count on menstrual cycle day 3, antimüllerian hormone level on menstrual cycle day 3, and blastocyst morphological grade were correlated with peri-implantation development outcomes. After controlling for good-quality morphological grades, blastocysts from patients of advanced maternal age developed fewer epiblast cells than blastocysts from younger patients. CONCLUSIONS: Extended embryo culture during the peri-implantation period mirrors several disparities in fertility treatment outcome that we see clinically, including those from patients with advanced maternal age, high body mass index, and low ovarian reserve and from embryos with lower-quality morphological grades. This model system may be useful by providing an alternative or more sensitive endpoint assessment in studying patient, clinical, or laboratory factors that may influence preimplantation embryo developmental potential.

Absence of SARS-CoV-2 (COVID-19 virus) within the IVF laboratory using strict patient screening and safety criteria.

RESEARCH QUESTION: Is there a risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral exposure and potential cross-contamination from follicular fluid, culture media and vitrification solution within the IVF laboratory using strict patient screening and safety measures? DESIGN: This was a prospective clinical study. All women undergoing transvaginal oocyte retrieval were required to have a negative SARS-CoV-2 RNA test 3-5 days prior to the procedure. Male partners were not tested. All cases used intracytoplasmic sperm injection (ICSI). The first tube of follicular fluid aspirated during oocyte retrieval, drops of media following removal of the embryos on day 5, and vitrification solution after blastocyst cryopreservation were analysed for SARS-CoV-2 RNA. RESULTS: In total, medium from 61 patients, vitrification solution from 200 patients and follicular fluid from 300 patients was analysed. All samples were negative for SARS-CoV-2 viral RNA. CONCLUSIONS: With stringent safety protocols in place, including testing of women and symptom-based screening of men, the presence of SARS-CoV-2 RNA was not detected in follicular fluid, medium or vitrification solution. This work demonstrates the possibility of implementing a rapid laboratory screening assay for SARS-CoV-2 and has implications for safe laboratory operations, including cryostorage recommendations.

Dynamics of trophoblast differentiation in peri-implantation-stage human embryos.

Single-cell RNA sequencing of cells from cultured human blastocysts has enabled us to define the transcriptomic landscape of placental trophoblast (TB) that surrounds the epiblast and associated embryonic tissues during the enigmatic day 8 (D8) to D12 peri-implantation period before the villous placenta forms. We analyzed the transcriptomes of 3 early placental cell types, cytoTB (CTB), syncytioTB (STB), and migratoryTB (MTB), picked manually from cultured embryos dissociated with trypsin and were able to follow sublineages that emerged from proliferating CTB at the periphery of the conceptus. A unique form of CTB with some features of STB was detectable at D8, while mature STB was at its zenith at D10. A form of MTB with a mixed MTB/CTB phenotype arose around D10. By D12, STB generation was in decline, CTB had entered a new phase of proliferation, and mature MTB cells had begun to move from the main body of the conceptus. Notably, the MTB transcriptome at D12 indicated enrichment of transcripts associated with IFN signaling, migration, and invasion and up-regulation of HLA-C, HLA-E, and HLA-G. The STB, which is distinct from the STB of later villous STB, had a phenotype consistent with intense protein export and placental hormone production, as well as migration and invasion. The studies show that TB associated with human embryos is in rapid developmental flux during peri-implantation period when it must invade, signal robustly to the mother to ensure that the pregnancy continues, and make first contact with the maternal immune system.

BRCA1 regulates HMGA2 levels in the Swan71 trophoblast cell line.

During early placental development, tumor suppressors and oncogenes work synergistically to regulate cell proliferation and differentiation in a restrained manner compared with the uncontrollable growth in cancer. One example of this partnership is the regulation of the oncofetal protein HMGA2 by BRCA1. BRCA1 forms a repressor complex with ZNF350 and CtIP to bind to the promoter of HMGA2, preventing transcription. Chromatin immunoprecipitation determined BRCA1 forms this repressor complex in human trophoblast cells, suggesting a role in the placenta. Furthermore, miR-182 has been shown to target BRCA1 mRNA in ovarian cancer cells, blocking the formation of the BRCA1 repressor complex and allowing increased transcription of HMGA2. miR-182 was one of the first miRNAs described as elevated in the serum and placentas of preeclamptic women. Therefore, we hypothesized that BRCA1 is essential for normal trophoblast cell development. We used CRISPR-Cas9 genome editing and miR-182 overexpression to decrease BRCA1 protein in the Swan71 cell line. HMGA2 was significantly increased in the BRCA1 KO and miR-182 overexpressing cells compared to controls. We also determined that BRCA1 repressor complex binding to HMGA2 was significantly reduced in BRCA1 KO and miR-182 overexpressing cells compared with controls, leading us to conclude that increased HMGA2 was because of decreased binding of the BRCA1 repressor complex. Finally, we found that the caspase activity was significantly higher in BRCA1 KO and miR-182 overexpressing cells suggesting an increased amount of apoptosis. These data suggest that BRCA1 is an important regulator of the oncofetal protein HMGA2 and promotes cell survival in human placental cells.

LIN28B regulates androgen receptor in human trophoblast cells through Let-7c.

LIN28B is an RNA-binding protein necessary for maintaining pluripotency in stem cells and plays an important role in trophoblast cell differentiation. LIN28B action on target gene function often involves the Let-7 miRNA family. Previous work in cancer cells revealed that LIN28 through Let-7 miRNA regulates expression of androgen receptor (AR). Considering the similarities between cancer and trophoblast cells, we hypothesize that LIN28B also is necessary for the presence of AR in human trophoblast cells. The human first-trimester trophoblast cell line, ACH-3P was used to evaluate the regulation of AR by LIN28B, and a LIN28B knockdown cell line was constructed using lentiviral-based vectors. LIN28B knockdown in ACH-3P cells resulted in significantly decreased levels of AR and increased levels of Let-7 miRNAs. Moreover, treatment of ACH-3P cells with Let-7c mimic, but not Let-7e or Let-7f, resulted in a significant reduction in LIN28B and AR. Finally, forskolin-induced syncytialization and Let-7c treatment both resulted in increased expression of syncytiotrophoblast marker ERVW-1 and a significant decrease in AR in ACH-3P. These data reveal that LIN28B regulates AR levels in trophoblast cells likely through its inhibitory actions on let-7c, which may be necessary for trophoblast cell differentiation into the syncytiotrophoblast.

Shifting perspectives from "oncogenic" to oncofetal proteins; how these factors drive placental development.

Early human placental development strongly resembles carcinogenesis in otherwise healthy tissues. The progenitor cells of the placenta, the cytotrophoblast, rapidly proliferate to produce a sufficient number of cells to form an organ that will contribute to fetal development as early as the first trimester. The cytotrophoblast cells begin to differentiate, some towards the fused cells of the syncytiotrophoblast and some towards the highly invasive and migratory extravillous trophoblast. Invasion and migration of extravillous trophoblast cells mimics tumor metastasis. One key difference between cancer progression and placental development is the tight regulation of these oncogenes and oncogenic processes. Often, tumor suppressors and oncogenes work synergistically to regulate cell proliferation, differentiation, and invasion in a restrained manner compared to the uncontrollable growth in cancer. This review will compare and contrast the mechanisms that drive both cancer progression and placental development. Specifically, this review will focus on the molecular mechanisms that promote cell proliferation, evasion of apoptosis, cell invasion, and angiogenesis.

Development and Evaluation of the Lifestyle History Questionnaire (LHQ) for People Entering Treatment for Substance Addictions.

OBJECTIVE: We developed and investigated the psychometric properties of the Lifestyle History Questionnaire (LHQ), a self-report instrument designed to measure the extent of occupational dysfunction attributable to substance abuse. METHOD: The instrument was developed using concepts in the ecological models of occupational therapy and in the work of William L. White, who defined addiction culture in terms of the patterns of life in context. We analyzed data from two field tests using both classical test theory and item response theory. RESULTS: The final version of the instrument has 70 items, 1 unifying construct, and 8 subscales. We found it to be valid and reliable (α=.93) for measuring the extent of occupational dysfunction and specific areas of strengths and weaknesses. CONCLUSION: The LHQ is a promising new instrument, the first of its kind to measure occupational dysfunction in context for people with substance addictions.

Androgen receptor and histone lysine demethylases in ovine placenta.

Sex steroid hormones regulate developmental programming in many tissues, including programming gene expression during prenatal development. While estradiol is known to regulate placentation, little is known about the role of testosterone and androgen signaling in placental development despite the fact that testosterone rises in maternal circulation during pregnancy and in placenta-induced pregnancy disorders. We investigated the role of testosterone in placental gene expression, and focused on androgen receptor (AR). Prenatal androgenization decreased global DNA methylation in gestational day 90 placentomes, and increased placental expression of AR as well as genes involved in epigenetic regulation, angiogenesis, and growth. As AR complexes with histone lysine demethylases (KDMs) to regulate AR target genes in human cancers, we also investigated if the same mechanism is present in the ovine placenta. AR co-immunoprecipitated with KDM1A and KDM4D in sheep placentomes, and AR-KDM1A complexes were recruited to a half-site for androgen response element (ARE) in the promoter region of VEGFA. Androgenized ewes also had increased cotyledonary VEGFA. Finally, in human first trimester placental samples KDM1A and KDM4D immunolocalized to the syncytiotrophoblast, with nuclear KDM1A and KDM4D immunostaining also present in the villous stroma. In conclusion, placental androgen signaling, possibly through AR-KDM complex recruitment to AREs, regulates placental VEGFA expression. AR and KDMs are also present in first trimester human placenta. Androgens appear to be an important regulator of trophoblast differentiation and placental development, and aberrant androgen signaling may contribute to the development of placental disorders.

Induced pluripotency in chicken embryonic fibroblast results in a germ cell fate.

Germ cells (GCs) are critically important as the vehicle that passes genetic information from one generation to the next. Correct development of these cells is essential and perturbation in their development often leads to reproductive failure and disease. Despite the importance of GCs, little is known about the mechanisms underlying the acquisition and maintenance of the GC character. Using a reprogramming strategy, we demonstrate that overexpression of ectopic transcription factors in embryonic fibroblasts can lead to the generation of chicken induced primordial germ cells (ciPGCs). These ciPGCs express pluripotent markers POU5F1, SSEA1, and the GC defining proteins, CVH and DAZL, closely resembling in vivo sourced PGCs instead of embryonic stem cells. Moreover, CXCR4 expressing ciPGCs were capable of migrating to the embryonic gonad after injection into the vasculature of stage 15 embryos, indicating the acquisition of a GC fate in these cells. Direct availability of ciPGCs in vitro would facilitate the study of GC development as well as provide a potential strategy for the conservation of important genetics of agricultural and endangered birds using somatic cells.