The focus of my life in science, has been to improve reproductive health for women, with an emphasis on the endometrium, the most dynamic tissue in the human body: its remarkable cyclical remodelling is essential for establishment of pregnancy. The most notable events in a woman’s endometrial cycle are menstruation and endometrial repair, regeneration of the endometrium during the proliferative phase, attainment of receptivity by the mid-secretory phase of the cycle and the embryo-maternal interactions that initiate peri-implantation events within the microenvironment of the uterine cavity. I have contributed to understanding the molecular and cellular changes underpinning these events, and how disturbance of them leads to menstrual disorders, infertility and endometrial diseases including abnormal uterine bleeding, endometriosis and endometrial cancer. My team have contributed to changes in clinical IVF practice, to a new diagnostic for endometrial receptivity in infertile women, and to enhancing endometrial repair. I have shared my world with many amazing younger scientists: it has indeed been a privileged journey.
Christine A White and Lois A Salamonsen
Within the last decade, the development of DNA microarray technology has enabled the simultaneous measurement of thousands of gene transcripts in a biological sample. Conducting a microarray study is a multi-step process; starting with a well-defined biological question, moving through experimental design, target RNA preparation, microarray hybridisation, image acquisition and data analysis – finishing with a biological interpretation requiring further study. Advances continue to be made in microarray quality and methods of statistical analysis, improving the reliability and therefore appeal of microarray analysis for a wide range of biological questions. The purpose of this review is to provide both an introduction to microarray methodology, as well as a practical guide to the use of microarrays for gene expression analysis, using endometrial biology as an example of the applications of this technology. While recommendations are based on previous experience in our laboratory, this review also summarises the methods currently considered to be best practice in the field.
Naomi B Morison, Tu'uhevaha J Kaitu'u-Lino, Ian S Fraser and Lois A Salamonsen
Many women using progestogen (P)-only contraceptives experience uterine bleeding problems. In clinical trials, a single low dose of mifepristone, given to Implanon users at the beginning of a bleeding episode reduced the number of bleeding days by ∼50% compared with controls. In this study, a single dose of mifepristone was administered to etonogestrel (ENG)-exposed pseudo-pregnant mice, 5 days after artificial decidualization was induced when the endometrium showed signs of bleeding. Control mice received vehicle alone. Mice were culled 12-, 18-, 24- and 48-h post-treatment. In the continued presence of ENG, a single dose of mifepristone stimulated tissue breakdown followed by very rapid repair: most treated tissues were fully restored to the pre-decidualized state by 48 h post-treatment. During repair, proliferating cells (Ki67 immunostained) were localized to a band of cells around the basal area in breaking down tissues and to the repairing luminal epithelium and glands. Progesterone receptor-positive cells were largely localized to the basal area of the breaking down tissue in treated mice compared with decidual cells in controls. Oestrogen receptor-positive cells were observed in the repairing luminal epithelium and glands compared with the decidua and the basal region in control tissues. It is concluded that mifepristone treatment stimulates rapid restoration of luminal epithelial integrity: such action may be a key event in reducing the number of bleeding days observed in women using Implanon who were treated with a single dose of mifepristone.
Katie L Meehan, Adam Rainczuk, Lois A Salamonsen and Andrew N Stephens
Over the past decade, high-throughput proteomics technologies have evolved considerably and have become increasingly more commonly applied to the investigation of female reproductive diseases. Proteomic approaches facilitate the identification of new disease biomarkers by comparing the abundance of hundreds of proteins simultaneously to find those specific to a particular clinical condition. Some of the best studied areas of female reproductive biology applying proteomics include gynaecological cancers, endometriosis and endometrial infertility. This review will discuss the progress that has been made in these areas and will highlight some of the emerging technologies that promise to contribute to better understanding of the female reproductive disease.
Rebecca L Jones, Chelsea Stoikos, Jock K Findlay and Lois A Salamonsen
Transforming growth factor β (TGFβ) superfamily members are closely associated with tissue remodelling events and reproductive processes. This review summarises the current state of knowledge regarding the expression and actions of TGFβ superfamily members in the uterus, during the menstrual cycle and establishment of pregnancy. TGFβs and activin β subunits are abundantly expressed in the endometrium, where roles in preparation events for implantation have been delineated, particularly in promoting decidualisation of endometrial stroma. These growth factors are also expressed by epithelial glands and secreted into uterine fluid, where interactions with preimplantation embryos are anticipated. Knockout models and embryo culture experiments implicate activins, TGFβs, nodal and bone morphogenetic proteins (BMPs) in promoting pre- and post-implantation embryo development. TGFβ superfamily members may therefore be important in the maternal support of embryo development. Following implantation, invasion of the decidua by fetal trophoblasts is tightly modulated. Activin promotes, whilst TGFβ and macrophage inhibitory cytokine-1 (MIC-1) inhibit, trophoblast migration in vitro, suggesting the relative balance of TGFβ superfamily members participate in modulating the extent of decidual invasion. Activins and TGFβs have similar opposing actions in regulating placental hormone production. Inhibins and activins are produced by the placenta throughout pregnancy, and have explored as a potential markers in maternal serum for pregnancy and placental pathologies, including miscarriage, Down’s syndrome and pre-eclampsia. Finally, additional roles in immunomodulation at the materno-fetal interface, and in endometrial inflammatory events associated with menstruation and repair, are discussed.
Kien C Luu, Gui Ying Nie, Anne Hampton, Guo-Qiang Fu, Yi-Xun Liu and Lois A Salamonsen
The endometrium is hostile to embryo implantation except during the ‘window of receptivity’. A change in endometrial gene expression is required for the development of receptivity. Calbindin-d9k (CaBP-d9k) and calbindin-d28k (CaBP-d28k) are proteins possessing EF-hand motifs which have high affinity for Ca2+ ions. Previously, it has been demonstrated that, in mouse endometrium, the expression of both calbindins is highly regulated during implantation and that both proteins play critical but functionally redundant roles at implantation. This study was the first to determine the expression of these two calbindins in the human and rhesus monkey endometrium. Initial RT-PCR analysis demonstrated that CaBP-d28k but not CaBP-d9k mRNA expression is detectable in the endometrium of both species. Western blot analysis confirmed the presence of immuno-reactive CaBP-d28k protein in the primate endometrium. Furthermore, the endometrial expression pattern of CaBP-d28k mRNA and protein was examined by Northern blot analysis and immunohistochemistry respectively in both species across the menstrual cycle and during early pregnancy. Semi-quantitative statistical analysis of the immunohistochemistry results revealed that, in the human, CaBP-d28k protein expression was maximal in luminal and glandular epithelium during the mid-secretory phase, coinciding with the time when the endometrium is receptive to embryo implantation. Expression in rhesus monkey showed a similar trend. These results suggest that, in the primate endometrium, only CaBP-d28k is expressed and that the specific regulation of this calbindin is potentially important for the establishment of uterine receptivity.
Naomi B Morison, Jin Zhang, Tu’uhevaha J Kaitu’u-Lino, Ian S Fraser and Lois A Salamonsen
Breakthrough bleeding (BTB), a major side effect of long-acting progestogen (p)-only contraceptives in women, is the main reason for discontinuation of their use. To understand the mechanisms of BTB, a mouse model of endometrial breakdown and repair was adapted to evaluate the effects of long-term progestogens on the endometrium. Appropriately prepared mice received either an etonogestrel (ENG)- or levonorgestrel (LNG)-releasing subdermal implant. Forty eight hours after decidualization was induced in one uterine horn the majority of tissues were highly decidualized, designated 0 day (0d). Uteri were collected subsequently at 5-day intervals (to 45d) and both decidualized and non-decidualized horns were analysed for morphological changes, leukocyte infiltration and matrix metalloproteinase expression (MMP). In decidualized horns, large blood vessels (BV) developed and disturbance of tissue integrity was observed at 5d with substantial stromal breakdown by 10d, progressing until 25d when re-epithelialization was initiated. By 45d, the tissue was restored to its pre-decidualized state but with considerable tortuosity of the luminal epithelium. Tissue remodelling was not apparent in the non-decidualized horns before 35d, when hyperproliferation of the luminal epithelium resulted in tortuosity. Changes in morphology were similar with the two progestogens, but occurred more rapidly with LNG. Apart from macrophages, few leukocytes were present in non-decidualized horns but large infiltrates of neutrophils and uterine natural killer cells (uNK) were associated with tissue breakdown in decidualized tissue, many of these cells were MMP9-positive. MMP7 was primarily associated with tissue repair. Therefore, this model mimics some of the changes observed in endometria of women using p-only contraceptives and provides an opportunity for functional studies.
Rebecca L Jones, Tu’uhevaha J Kaitu’u-Lino, Guiying Nie, L Gabriel Sanchez-Partida, Jock K Findlay and Lois A Salamonsen
Maternal–fetal communications are critical for the establishment of pregnancy. Embryonic growth and differentiation factors produced by the oviduct and uterus play essential roles during the pre- and early post-implantation phases. Although several studies indicate roles for activin in embryonic development, gene-knockout studies have failed to identify a critical role in mammalian embryogenesis. We hypothesized that activin is produced by maternal tissues during the establishment of pregnancy, and thus maternally derived activin could compensate for the absence of embryonic activin in null homozygotes during critical developmental stages. We investigated the expression of inhibin α, activin βA, and βB subunits in the mouse oviduct and uterus during the estrous cycle and early pregnancy, and in the early conceptus. Inhibin α subunit was weakly expressed, while activin βA and βB subunits were strongly expressed in oviduct and uterus at estrous, and dramatically upregulated in the uterus on each day of pregnancy between days 3.5 and 8.5 post coitum. Prior to implantation, activin βA and βB subunits were immunolocalized to oviductal and uterine epithelial cells; following implantation they were expressed in the stroma, in a wave preceding decidualization. Later in pregnancy, activin βA and βB subunits were present in decidua basalis, trophoblast giant cells, and labyrinth zone of the developing placenta. Expression of activin βA subunit was also detected in blastocysts and early post-implantation embryos. These data are consistent with a role for maternally derived activins in the support of the pre-implantation embryo, and during gastrulation and embryogenesis.