Murine uterine gland branching is necessary for gland function in implantation.

Uterine glands are branched, tubular structures whose secretions are essential for pregnancy success. It is known that pre-implantation glandular expression of leukemia inhibitory factor (LIF) is crucial for embryo implantation; however, the contribution of uterine gland structure to gland secretions, such as LIF, is not known. Here, we use mice deficient in estrogen receptor 1 (ESR1) signaling to uncover the role of ESR1 signaling in gland branching and the role of a branched structure in LIF secretion and embryo implantation. We observed that deletion of ESR1 in neonatal uterine epithelium, stroma, and muscle using the progesterone receptor PgrCre causes a block in uterine gland development at the gland bud stage. Embryonic epithelial deletion of ESR1 using a Müllerian duct Cre line, Pax2Cre, displays gland bud elongation but a failure in gland branching. Reduction of ESR1 in adult uterine epithelium using the lactoferrin-Cre (LtfCre) displays normally branched uterine glands. Unbranched glands from Pax2Cre Esr1flox/flox uteri fail to express glandular pre-implantation Lif, preventing implantation chamber formation and embryo alignment along the uterine mesometrial-antimesometrial axis. In contrast, branched glands from LtfCre Esr1flox/flox uteri display reduced expression of ESR1 and glandular Lif resulting in delayed implantation chamber formation and embryo-uterine axes alignment but mice deliver a normal number of pups. Finally, pre-pubertal unbranched glands in control mice express Lif in the luminal epithelium but fail to express Lif in the glandular epithelium, even in the presence of estrogen. These data strongly suggest that branched glands are necessary for pre-implantation glandular Lif expression for implantation success. Our study is the first to identify a relationship between the branched structure and secretory function of uterine glands and provides a framework for understanding how uterine gland structure-function contributes to pregnancy success.

Activation of sperm Toll-like receptor 2 induces hyperactivation to enhance the penetration to mucus and uterine glands: a trigger for the uterine inflammatory cascade in cattle.

It is known that sperm and seminal plasma (SP) affect uterine immunity. In cattle, artificial insemination enables breeding by depositing frozen and largely diluted sperm with a negligible amount of SP into the uterus. Thus, the present study focused on the impact of frozen-thawed sperm on bovine uterine immunity. We have previously shown that in the bovine uterus, sperm swim smoothly over the luminal epithelium and some sperm interact with uterine glands to induce a weak inflammatory response mainly via the endometrial Toll-like receptor 2 (TLR2) signaling. However, the process by which sperm is encountered in the uterine glands is not completely clear. The present study intended to evaluate the role of sperm-TLR2 in sperm-uterine mucus penetration for reaching the glandular epithelium to induce the uterine immune response. To activate and block sperm-TLR2, they were treated with TLR2 agonist and antagonist, respectively. TLR2 activation enhanced sperm hyperactivation and improved its capacity to penetrate the artificial viscoelastic fluid and estrous-uterine-mucus. In contrast, TLR2-blocked sperm showed completely opposite effects. It is noteworthy, that the TLR2-activated sperm that penetrated the uterine mucus exhibited increased motile activity with hyperactivation. In the sperm-endometrial ex-vivo model, a greater amount of TLR2-activated sperm entered the uterine glands with an immune response, which was seen as the upregulation of mRNA expression for TNFA, IL1B, IL8, PGES, and TLR2 similar to those in control sperm. On the other hand, a lesser amount of TLR2-blocked sperm entered the uterine glands and weakened the sperm-induced increase only in PGES, suggesting that penetration of a certain number of sperm in the uterine gland is necessary enough to trigger the inflammatory response. Altogether, the present findings indicate that activation of sperm-TLR2 promotes their hyperactivation and mucus penetration with greater motility, allowing them to enter into the uterine glands more. This further suggests that the hyperactivated sperm contributes to triggering the pro-inflammatory cascade partly via TLR2 in the uterus.

Three-Dimensional Visualization of Mouse Endometrial Remodeling After Superovulation.

Uterine status determines pregnancy success. Although it is well known that superovulation operations can disrupt uterine function, our understanding of the morphological changes in the uterine endometrium at the three-dimensional (3D) level is limited. Here, combining the tissue clearing with 3D deep imaging, we reveal an increase in epithelial density and angiogenesis after ovarian stimulation, which is accompanied by a circulating surge in P4 levels. Using an ovariectomized mouse model, we further detected the separate regulatory effects of P4 and E2 on the uterine endometrium, with P4 promoting endothelial cell growth and E2 inducing epithelial proliferation. Additionally, we observed that the effects of E2 can be partially neutralized by P4, and vice versa. By analyzing the 3D uterine imaging, we discovered an interesting phenomenon in which the growing blood vessels closely surround the remodeling uterine epithelium, indicating a close relationship between angiogenesis and epithelial growth. These findings provide new insight into the uterine epithelial changes and angiogenesis at the 3D level, and explain a potential reason for endometrial changes due to the low implantation rate in patients undergoing clinic super-ovulation.

Implantation and extravillous trophoblast invasion: From rare archival specimens to modern biobanking.

Extravillous trophoblast invasion serves to attach the placenta to the uterus and to enable access to nutrients for the embryo throughout pregnancy - secretions of the uterine glands in the first trimester, maternal blood in the second and third trimester. For assessing extravillous trophoblast invasion, histology (in combination with immunohistochemistry) still plays a major role in placental research. This is especially true for the re-assessment of rare archival specimens from early human implantation sites or placenta in utero with the background of recent knowledge which may help to strengthen current hypotheses. This review summarizes the recently expanded picture of extravillous trophoblast invasion, gives an overview about fundamental archival specimens in placental research, presents new images of archival specimens, gives insights into the latest developments in the field of biobanking and provides insight into the current situation on sample usage in the absence of biobanks. Modern techniques allow expanding our hitherto believed concept of extravillous trophoblast invasion, which is not restricted to spiral arteries: Extravillous trophoblasts also invade into uterine glands and uterine veins and thereby connect all these luminal structures with the intervillous space. All biomedical research dramatically depends on the quality of the assessed biological samples. Hence, researchers should be aware that the time between collection of a sample from a body and the beginning of analysis (pre-analytical phase) may have more impact on the outcome of a study than previously assumed.

The trophoblast plug during early pregnancy: a deeper insight.

During the first trimester of pregnancy, foetal endovascular trophoblasts invade into maternal spiral arteries, accumulate and form plugs in the lumen of the vessels. These plugs only allow blood plasma to seep through. Hence, during the first trimester of pregnancy, a first flow of fluids through the placental intervillous space is established, resulting in a physiological oxygen gradient between mother and foetus. The trophoblast plugs block spiral arteries until the beginning of the second trimester (11-14 weeks). In parallel, uterine glands are invaded and opened by endoglandular trophoblasts towards the intervillous space of the placenta, without showing the formation of plugs (Moser et al. in Hum Reprod 25:1127-1136, 2010, Hum Reprod Oxf Engl 30:2747-2757, 2015). This enables histiotrophic nutrition of the embryo prior to onset of maternal blood flow into the placenta. Failure of these endovascular and endoglandular invasion processes may lead to miscarriage or pregnancy disorders such as intrauterine growth restriction (IUGR). After dissolution of the plugs, the onset of maternal blood flow allows maternal blood cells to enter the intervillous space and oxygen concentrations rise up. In this study, we demonstrate for the first time serial cross sections through a trophoblast plug in a first trimester placental bed specimen. Invaded and plugged arteries as well as invaded uterine glands in week 11 of gestation are visualized with specific immunohistochemical double staining techniques. We show that spiral artery plugs appear throughout the placental invasion zone and illustrate erythrocytes stowed due to trophoblast plugs. In addition, we give evidence of the presence of MMP-1 in plugs of invaded spiral arteries. The results reveal a better understanding and a closer insight into the morphological appearance of trophoblast plugs and the consequences for placental and uterine blood flow.

Evidence from the very beginning: endoglandular trophoblasts penetrate and replace uterine glands in situ and in vitro.

STUDY QUESTION: How is histiotrophic nutrition of the embryo secured during the first trimester of pregnancy? SUMMARY ANSWER: Rather than specifically focusing on invasion into spiral arteries, extravillous trophoblasts also invade into uterine glands (endoglandular trophoblast) from the very beginning and open them toward the intervillous space. WHAT IS KNOWN ALREADY: Extravillous trophoblasts can be found in close contact and within the lumen of uterine glands, sometimes replacing glandular epithelial cells. STUDY DESIGN, SIZE, DURATION: As well as extensive screening of specimens from first trimester placentation sites in situ we used a previously established three-dimensional co-culture in vitro model system of first trimester villous explants with non-invaded decidua parietalis. PARTICIPANTS/MATERIALS, SETTING, METHODS: First trimester placentas were obtained from elective terminations of pregnancies (n = 48) at 5-11 weeks of gestational age. A subset was processed for confrontation co-culture (n = 31). Invaded decidua basalis was obtained from 20 placentas. All tissues were sectioned, subsequently immunostained and immunodoublestained with antibodies against keratin 7 (KRT7), major histocompatibility complex, class I, G (HLA-G), matrix metallopeptidase 9 (MMP9), von Willebrand factor (VWF) and the appropriate Immunoglobulin G (IgG) negative controls. Replacement of endothelial/epithelial cells by extravillous trophoblasts was quantified semi-quantitatively. Additionally, hematoxylin and eosin-stained archival specimens from early implantation sites were assessed. MAIN RESULTS AND THE ROLE OF CHANCE: The earliest available specimen was from around Day 10 after conception; already at this stage trophoblasts had penetrated into uterine glands and had started to replace the epithelium of the glands. Endoglandular trophoblasts replaced uterine glands in vitro and in situ and could be found in the lumen of invaded glands. Quantitative analysis revealed significantly more replacement of epithelial cells in glands (63.8 ± 22.1%) compared with endothelial cells in vessels (26.4 ± 8.8%). Accumulated detached glandular epithelial cells could be repeatedly observed in the lumen of invaded glands. Additionally, in areas of trophoblast invasion the glandular epithelium seemed to be completely disintegrated compared with glandular epithelium in the non-invaded parts of the decidua. Whole tissue specimens were used in vitro and in situ instead of cell lines; these systems mostly maintain the context of the in vivo situation. LIMITATIONS, REASONS FOR CAUTION: This is a descriptive study supported by in vitro experiments. However, a histological section will always only be a snapshot and quantification from histological sections has its limitations. WIDER IMPLICATIONS OF THE FINDINGS: This study further strengthens the hypothesis of histiotrophic nutrition of the embryo prior to the establishment of the maternal blood flow toward the placenta. Invasion of uterine glands by endoglandular trophoblasts may have more impact on the outcome of early pregnancy than assumed up to now.

Constitutive activation of transforming growth factor Beta receptor 1 in the mouse uterus impairs uterine morphology and function.

Despite increasing evidence pointing to the essential involvement of the transforming growth factor beta (TGFB) superfamily in reproduction, a definitive role of TGFB signaling in the uterus remains to be unveiled. In this study, we generated a gain-of-function mouse model harboring a constitutively active (CA) TGFB receptor 1 (TGFBR1), the expression of which was conditionally induced by the progesterone receptor (Pgr)-Cre recombinase. Overactivation of TGFB signaling was verified by enhanced phosphorylation of SMAD2 and increased expression of TGFB target genes in the uterus. TGFBR1 Pgr-Cre CA mice were sterile. Histological, cellular, and molecular analyses demonstrated that constitutive activation of TGFBR1 in the mouse uterus promoted formation of hypermuscled uteri. Accompanying this phenotype was the upregulation of a battery of smooth muscle genes in the uterus. Furthermore, TGFB ligands activated SMAD2/3 and stimulated the expression of a smooth muscle maker gene, alpha smooth muscle actin (ACTA2), in human uterine smooth muscle cells. Immunofluorescence microscopy identified a marked reduction of uterine glands in TGFBR1 Pgr-Cre CA mice within the endometrial compartment that contained myofibroblast-like cells. Thus, constitutive activation of TGFBR1 in the mouse uterus caused defects in uterine morphology and function, as evidenced by abnormal myometrial structure, dramatically reduced uterine glands, and impaired uterine decidualization. These results underscore the importance of a precisely controlled TGFB signaling system in establishing a uterine microenvironment conducive to normal development and function.

Uterine gland microstructure in the pregnant and the non-pregnant lesser galago (galago senegalensis) / Microestructura de la glándula uterina en la gálago menor (galago senegalensis) preñada y no preñada

The histology and the ultrastructure of the uterine glands of the lesser bushbaby (Galago senegalensis) were studied in six specimens (5 pregnant and one non pregnant) which were fixed partly with bouin's fixative and part with 2.5% glutaraldehyde in 0.1 M cacodylate buffer. An overview of the main results revealed uterine glands in the non-pregnant uterus are rudimentary and scarce within the mucosa. In early pregnancy (first trimester) the uterine glands profiles appear in clusters. In late stage pregnancy (third trimester) the uterine gland profiles appear opposite chorionic vesicles. In the later stages of gestation maternal glandular epithelium consisted mainly of simple columnar epithelium. The cells had abundant flattened cisternae of granular endoplasmic reticulum usually with an apical-basal orientation. Their nuclei had abundant euchromatin relative to the amount of heterochromatin. They also had a prominent Golgi apparatus quite characteristic of protein synthesizing cells. The basal plasmalemma was thrown into infoldings that have the effect of increasing the surface area across which nutrients could pass from the maternal circulation and are secreted by the cell as histiotrophe. Both physiologic hypertrophy and hyperplasia of the uterine glands are observed to occur with advancement of pregnancy.

La histología y ultraestructura de las glándulas uterinas de la gálago menor (Galago senegalensis) fueron estudiadas en seis ejemplares (5 preñadas y 1 no preñada). Una parte de las glándula se fijó con Bouin y otra con glutaraldehído al 2,5% en tampón cacodilato 0,1 M. Una visión general de los principales resultados reveló que las glándulas uterinas en el útero no gestante son rudimentarias y escasas dentro de la mucosa. Al principio de la preñez (primer trimestre) las glándulas uterinas aparecen en racimos. En la última etapa de la preñez (tercer trimestre) las glándulas uterinas aparecen opuestas a las vesículas coriónicas. En las últimas etapas de gestación el epitelio glandular materno consiste principalmente en epitelio cilíndrico simple. Las células tenían abundantes cisternas aplanadas en el retículo endoplásmico rugoso, por lo general con una orientación apico-basal. Sus núcleos tenían abundante eucromatina en relación con la cantidad de heterocromatina. También tenían un aparato de Golgi prominente bastante característico de células que sintetizan proteínas. El plasmalema basal fue rechazado en repliegues que psoeen el efecto de aumentar el área de superficie a través del cual los nutrientes podrían pasar desde la circulación materna y son secretadas por las célula como histiotrofo. Hipertrofia fisiológica e hiperplasia de las glándulas uterinas se observaron con el avance de la preñez.