Mechanisms governing protective pregnancy-induced adaptions of the pelvic floor muscles in the rat pre-clinical model

Background: The intrinsic properties of pelvic soft tissues in women who do and do not sustain birth injuries are likely divergent, however little is known about this. Rat pelvic floor muscles undergo protective pregnancy-induced structural adaptations, sarcomerogenesis and increase in intramuscular collagen content, that protect against birth injury. Objectives: We aimed to test the following hypotheses: 1) increased mechanical load of gravid uterus drives antepartum adaptations; 2) load-induced changes are sufficient to protect pelvic muscles from birth injury. Study Design: Independent effects of load uncoupled from hormonal milieu of pregnancy were tested in 3- to 4-month-old Sprague-Dawley rats randomly divided into four groups, N=5-10/group: (1) load-/pregnancy hormones- (controls); (2) load+/pregnancy hormones-; (3) reduced load/pregnancy hormones+; (4) load+/pregnancy hormones+. Mechanical load simulating a gravid uterus was simulated by weighing uterine horns with beads similar to fetal rat size and weight. Reduced load was achieved by unilateral pregnancy after unilateral uterine horn ligation. To assess acute and chronic phases required for sarcomerogenesis, rats were sacrificed at 4 hours or 21 days post bead loading. Coccygeus, iliocaudalis, pubocaudalis and non-pelvic tibialis anterior were harvested for myofiber and sarcomere length measurements. Intramuscular collagen content was assessed using hydroxyproline assay. Additional 20 load+/pregnancy hormones- rats underwent vaginal distention to determine whether load-induced changes are sufficient to protect from mechanical muscle injury in response to parturition-associated strains of various magnitude. Data, compared using two-way repeated measures analysis of variance/pairwise comparisons, are presented as mean +/- standard error of mean. Results: Acute increase in load resulted in significant pelvic floor muscle stretch, accompanied by acute increase in sarcomere length compared to non-loaded control muscles (coccygeus: 2.69+/-0.03 vs 2.30+/-0.06 micrometers, P<0.001; pubocaudalis: 2.71+/-0.04 vs 2.25+/-0.03 micrometers, P<0.0001; iliocaudalis: 2.80+/-0.06 vs 2.35+/-0.04 micrometers, P<0.0001). After 21 days of sustained load, sarcomeres returned to operational length in all pelvic muscles (P>0.05). However, the myofibers remained significantly longer in load+/pregnancy hormones- compared to load-/pregnancy hormones- in coccygeus (13.33+/-0.94 vs 9.97+/-0.26 millimeters, P<0.0001) and pubocaudalis (21.20+/-0.52 vs 19.52+/-0.34 millimeters, P<0.04) and not different from load+/pregnancy hormones+ (12.82+/-0.30 and 22.53+/-0.32millimeters, respectively, P>0.1), indicating that sustained load induced sarcomerogenesis in these muscles. Intramuscular collagen content in load+/pregnancy hormones- group was significantly greater relative to controls in coccygeus (6.55+/-0.85 vs 3.11+/-0.47 micrograms/milligram, P<0.001) and pubocaudalis (5.93+/-0.79 vs 3.46+/-0.52 micrograms/milligram, P<0.05) and not different from load+/pregnancy hormones+ (7.45+/-0.65 and 6.05+/-0.62 micrograms/milligram, respectively, P>0.5). Iliocaudalis required both mechanical and endocrine cues for sarcomerogenesis. Tibialis anterior was not affected by mechanical or endocrine alterations. Despite equivalent extent of adaptations, load-induced changes were only partially protective against sarcomere hyperelongation. Conclusions: Load induces plasticity of the intrinsic pelvic floor muscle components that renders protection against mechanical birth injury. The protective effect, which varies between individual muscles and strain magnitudes, is further augmented by the presence of pregnancy hormones. Maximizing impact of mechanical load on pelvic floor muscles during pregnancy, such as with specialized pelvic floor muscle stretching regimens, is a potentially actionable target for augmenting pregnancy-induced adaptations to decrease birth injury in women who may otherwise have incomplete antepartum muscle adaptations.

Menstrual flow as a non-invasive source of endometrial organoids

Assessment of the endometrium often necessitates a biopsy, which currently involves an invasive, transcervical procedure. Here, we present an alternative technique based on deriving organoids from menstrual flow. We demonstrate that organoids can be derived from gland fragments recovered from menstrual flow. To confirm they faithfully reflect the in vivo state we compared organoids derived from paired scratch biopsies and ensuing menstrual flow from patients undergoing in vitro fertilisation (IVF). We demonstrate that the two sets of organoids share the same transcriptome signature, derivation efficiency and proliferation rate. Furthermore, they respond similarly to sex steroids and early-pregnancy hormones, with changes in morphology, receptor expression, and production of ‘uterine milk’ proteins that mimic those during the late-secretory phase and early pregnancy. This technique has wide-ranging impact for non-invasive investigation and personalised approaches to treatment of common gynaecological conditions, such as endometriosis, and reproductive disorders, including failed implantation after IVF and recurrent miscarriage.

Giant breast fibroadenoma in pregnancy

Overview Objective: To present unusual case of rapidly growing breast fibroadenoma during pregnancy. Case report: Breast fibroadenomas are hormone-dependent tumors which can grow rapidly during pregnancy. We present a case of giant fibroadenoma, first detected before pregnancy, which grew rapidly under the influence of pregnancy hormones and was excised in the third trimester. Conclusion: Fibroadenomas are benign breast lesions in young women. They are hormone-dependent tumors, which can grow rapidly especially during pregnancy and/ or lactation and imitate malignancy. In each case of a newly created breast lesion, it is necessary to proceed responsibly to the clinical examination, which is a base for further decision concerning the necessity of taking a bio­psy for histological verification. Keywords: breast fibroadenoma – pregnancy – lactation – phyllodes tumor

Pregnancy Changes the Response of the Vomeronasal and Olfactory Systems to Pups in Mice

Motherhood entails changes in behavior with increased motivation for pups, induced in part by pregnancy hormones acting upon the brain. This work explores whether this alters sensory processing of pup-derived chemosignals. To do so, we analyse the expression of immediate early genes (IEGs) in the vomeronasal organ (VNO; Egr1) and centers of the olfactory and vomeronasal brain pathways (cFos) in virgin and late-pregnant females exposed to pups, as compared to buttons (socially neutral control). In pup-exposed females, we quantified diverse behaviors including pup retrieval, sniffing, pup-directed attack, nest building and time in nest or on nest, as well as time off nest. Pups induce Egr1 expression in the VNO of females, irrespective of their physiological condition, thus suggesting the existence of VNO-detected pup chemosignals. A similar situation is found in the accessory olfactory bulb (AOB) and posteromedial part of the medial bed nucleus of the stria terminalis (BSTMPM). By contrast, in the medial amygdala and posteromedial cortical amygdala (PMCo), responses to pups-vs-buttons are different in virgin and late-pregnant females, thus suggesting altered sensory processing during late pregnancy. The olfactory system also shows changes in sensory processing with pregnancy. In the main olfactory bulbs, as well as the anterior and posterior piriform cortex, buttons activate cFos expression in virgins more than in pregnant females. By contrast, in the anterior and especially posterior piriform cortex, pregnant females show more activation by pups than buttons. Correlation between IEGs expression and behavior suggests the existence of two vomeronasal subsystems: one associated to pup care (with PMCo as its main center) and another related to pup-directed aggression observed in some pregnant females (with the BSTMPM as the main nucleus). Our data also suggest a coactivation of the olfactory and vomeronasal systems during interaction with pups in pregnant females.

Characterization of Organoid Cultures to Study the Effects of Pregnancy Hormones on the Epigenome and Transcriptional Output of Mammary Epithelial Cells

The use of mouse derived mammary organoids can provide a unique strategy to study mammary gland development across a normal life cycle, as well as offering insights into how malignancies form and progress. Substantial cellular and epigenomic changes are triggered in response to pregnancy hormones, a reaction that engages molecular and cellular changes that transform the mammary epithelial cells into “milk producing machines”. Such epigenomic alterations remain stable in post-involution mammary epithelial cells and control the reactivation of gene transcription in response to re-exposure to pregnancy hormones. Thus, a system that tightly controls exposure to pregnancy hormones, epigenomic alterations, and activation of transcription will allow for a better understanding of such molecular switches. Here, we describe the characterization of ex vivo cultures to mimic the response of mammary organoid cultures to pregnancy hormones and to understand gene regulation and epigenomic reprogramming on consecutive hormone exposure. Our findings suggest that this system yields similar epigenetic modifications to those reported in vivo, thus representing a suitable model to closely track epigenomic rearrangement and define unknown players of pregnancy-induced development.

Uptake of Cerium Dioxide Nanoparticles and Impact on Viability, Differentiation and Functions of Primary Trophoblast Cells from Human Placenta

The human placenta is at the interface between maternal and fetal circulations, and is crucial for fetal development. The nanoparticles of cerium dioxide (CeO2 NPs) from air pollution are an unevaluated risk during pregnancy. Assessing the consequences of placenta exposure to CeO2 NPs could contribute to a better understanding of NPs’ effect on the development and functions of the placenta and pregnancy outcome. We used primary villous cytotrophoblasts purified from term human placenta, with a wide range of CeO2 NPs concentrations (0.1–101 μg/cm2) and exposure time (24–72 h), to assess trophoblast uptake, toxicity and impact on trophoblast differentiation and endocrine function. We have shown the capacity of both cytotrophoblasts and syncytiotrophoblasts to internalize CeO2 NPs. CeO2 NPs affected trophoblast metabolic activity in a dose and time dependency, induced caspase activation and a LDH release in the absence of oxidative stress. CeO2 NPs decreased the fusion capacity of cytotrophoblasts to form a syncytiotrophoblast and disturbed secretion of the pregnancy hormones hCG, hPL, PlGF, P4 and E2, in accordance with NPs concentration. This is the first study on the impact of CeO2 NPs using human primary trophoblasts that decrypts their toxicity and impact on placental formation and functions.

Brain Mechanisms Regulating Maternal Behavior in Nonhuman Mammals

Chapter 4 examines the roles of oxytocin (OT) and olfaction in the maternal behavior of nonhuman mammals. It also presents an overview of brain anatomy. In concert with pregnancy hormones, the release of OT into the brain, derived from the paraventricular hypothalamic nucleus, stimulates the onset of maternal behavior. Although OT is not required for the maintenance of maternal behavior, it does enhance maternal behavior during the postpartum period in challenging environments by decreasing anxiety and increasing maternal motivation. OT, in the absence of pregnancy hormones, may also enhance maternal responsiveness in alloparents. For many postpartum mammals, maternal motivation is under multisensory control, and olfaction is not required, although it is necessary for maternal selectivity in sheep. In contrast, for laboratory mice, olfaction is essential for maternal motivation. For virgin female rats and rabbits, olfactory input from pups inhibits maternal behavior, but this inhibition is eliminated at parturition.

Osteosarcoma of the frontal bone: a study of a rare case in a 17-year-old female

Head and neck osteosarcomas are infrequent and usually present in the third–fourth decades of life. However, they are extremely rare in the pediatric population. Primary involvement of the cranial vault, excluding the mandible and maxilla, is an exceedingly rare phenomenon; thus, the number of clinical studies published in the literature is limited. Because of the anatomy of the head, complete resection may be difficult to achieve. Furthermore, an aggressive surgical approach can cause a significant functional impairment or cosmetic defect. We report the case of a 17-year-old patient with an aggressive recurrence of multiple tumors in the left side of the skull accompanied with a severe headache and complete left-sided vision loss with no metastases at presentation. The recurrence of the tumor was preceded by pregnancy and delivery, which raised the question of whether pregnancy hormones were a provoking factor in the recurrence of the tumor or not.

Endometrial and decidual stromal precursors show a different decidualization capacity

Endometrial stromal cells (EnSCs) and decidual stromal cells (DSCs) originate from fibroblastic precursors located around the vessels of the human nonpregnant endometrium and the pregnant endometrium (decidua), respectively. Under the effect of ovarian or pregnancy hormones, these precursors differentiate (decidualize), changing their morphology and secreting factors that appear to be essential for the normal development of pregnancy. However, the different physiological context – that is, non-pregnancy vs pregnancy – of those precursors (preEnSCs, preDSCs) might affect their phenotype and functions. In the present study, we established preEnSC and preDSC lines and compared the antigen phenotype and responses to decidualization factors in these two types of stromal cell line. Analyses with flow cytometry showed that preEnSCs and preDSCs exhibited a similar antigen phenotype compatible with that of bone marrow mesenchymal stem/stromal cells. The response to decidualization in cultures with progesterone and cAMP was evaluated by analyzing changes in cell morphology by microscopy, prolactin and IL-15 secretion by enzyme immunoassay and the induction of apoptosis by flow cytometry. In all four analyses, preDSCs showed a significantly higher response than preEnSCs. The expression of progesterone receptor (PR), protein kinase A (PKA) and FOXO1 was studied with Western blotting. Both types of cells showed similar levels of PR and PKA, but the increase in PKA RI subunit expression in response to decidualization was again significantly greater in preDSCs. We conclude that preEnSCs and preDSCs are equivalent cells but differ in their ability to decidualize. Functional differences between them probably derive from factors in their different milieus.