«Molecular basis of the embryo-maternal crosstalk in the female reproductive tract Inaugural-Dissertation zur Erlangung des Doktorgrades der ...»
Molecular basis of the embryo-maternal crosstalk in the
female reproductive tract
Erlangung des Doktorgrades der
der Heinrich-Heine-Universität Düsseldorf
Dunja Maria Baston-Büst
Aus der Frauenklinik, Universitäres interdisziplinäres Kinderwunschzentrum Düsseldorf,
der Heinrich-Heine Universität Düsseldorf
Gedruckt mit der Genehmigung der
Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf Referent: Prof. Dr. J.-S. Krüssel Koreferent: Prof. Dr. P. Proksch
Tag der mündlichen Prüfung:
A. Table of contents Table of contents
1.1 The female reproductive tract
1.2 Menstrual cycle and hormonal regulation of fallopian tube and endometrium.............. 13
1.3 Ovulation, fertilization, embryo passage, embryo-maternal dialogue and implantation
1.4 Aim of the studies
2.1 Interleukin-1 system in the human fallopian tube - No spatial but a temporal regulation of mRNA and protein expression (Molecular and cellular Endocrinolgy 303 (2009), 7Cathepsin S system at the feto-maternal interface (Reproduction 139 (2010), 741-748)
2.3 CXCL1 expression in human decidua in vitro is mediated via the MAPK signalling cascade (submitted to Journal of Clinical Endocrinology and Metabolism – under review)
2.4 Syndecan-1 knock-down in decidualized human endometrial stromal cells leads to significant changes in cytokine and angiogenic factor expression patterns (submitted to Reproductive Biology and Endocrinology - under review)
2.5 In-vitro culture does not alter the expression of vascular endothelial growth factor and its receptors in single murine preimplantation embryos (accepted for publication in Gynecologic and obstetric investigation)
2.6 Expression of the vascular growth factor receptor Neuropilin-1 in the human endometrium (Journal of Reproductive Immunology 79 (2009) 129-136).................. 148
2.7 Expression of vascular endothelial growth factor receptor Neuropilin-1 at the human fetal-maternal interface (in resubmission to European Journal of Obstetrics, Gynecology and Reproductive Biology)
4. Scientific Curriculum Vitae
KdS1 knock-down Syndecan-1 (St-T1 cells stably transfected with shRNA for Syndecan-1) l, ml, µl liter, milli-, microliter LB luria bertani broth (lysogeny broth) M, mM, µM molarity, milli-, micro-molarity MCS multiple cloning site (polylinker)
Vol. volume v/v volume per volume W watt (unit of performance) WHO world health organization w/v weight per volume Zeo zeocin (formula C55H83N19O21S2Cu)
1. Introduction Reproduction is an important feature of life. Until today, the molecular basis of embryonic preimplantation development on its passage through the oviduct into the uterus and subsequent implantation into a receptive maternal endometrium to successfully establish a pregnancy is not well elucidated. International scientific societies as the European Society of Human Reproduction and Embryology (ESHRE), the American Society of Reproductive Medicine (ASRM) and the Society of Gynaecologic Investigation (SGI) deal with human fertility and embryology supporting experimental and practical research and deciding clinical practice guidelines in the field of the human reproductive tract and the treatment of infertility applying assisted reproductive technology (ART).
General aspects regarding basic processes of the female cycle, fertility and early embryonic development will be explained further in the following paragraphs.
1.1 The female reproductive tract In human, the sexual development starts at the 6th week of gestation. The proximal oviduct, the uterus and the vagina are of paramesonephric origin (ductus muellerius). The ductus mesonephricus (ductus wolfius) also takes part in the development of the vagina. The cranial parts of the ductus muellerius differentiate into the fallopian tubes. The ovaries originate from epithelial coelom and primordial germ cells. Within the 5th and 6th week of gestation, primordial germ cells migrate into the indifferent gonadal segment. These cells initiate mitosis and form gametocytes (about 6×106 gametocytes starting in the 12th week of gestation).
These primary oocytes arrest between meiotic pro- and metaphase until the age of puberty 1.
During fetal development, the uterus changes in shape and growth due to caudal growth of the uterovaginal primordium, rostral extension of the fused portion of the paramesonephric
Some layering of the mucosa is evident at this time, and by 20th week of gestation smooth muscle cells develop in the muscular layer (myometrium), growing during the second half of gestation and resulting in the pear-like shaped uterus as seen in adults (Figure 1) 4. Before the 20th week of gestation, the uterine cavity of the human fetus is lined by a single layer of columnar epithelium without glands. At mid-gestation, the epithelial lining of the cavity forms pouches of cells that grow into the underlying mesenchyme, which become glands by the 7th month of pregnancy. At this time, the endometrium responds to the high levels of steroid hormones from the placenta. By the 9th month of gestation, the fetal endometrium is hypertrophic, with an oedematous stroma, blood vessels and actively secreting, coiled glands. In the newborn, the endometrium has features of proliferative and secretory 4,5 endometrium. During infancy and childhood the uterus grows consistent with overall body growth. The glands elongate and reach the basal portion of the endometrium. After puberty, uterine (endometrial and myometrial) growth is stimulated by circulating estradiol (E2) and progesterone (P4), with the corpus becoming larger than the cervix, as in the adult (Figure 1).
Figure 1: The adolescent female reproductive tract consisting of ovaries, fallopian tubes, uterus and vagina.
In the adult, each oviduct is about 13 cm long and is divided into four regions:
1. intramural region (passing through the uterine wall),
2. isthmus (proximal 1/3 of the tube, extending laterally from the intramural region to the ampulla),
3. ampulla (distal 2/3 of the oviduct, contiguous with the isthmus),
4. infundibulum (trumpet-shaped end of the tube, open to the peritoneal cavity by the abdominal osteum and containing multiple folds, the “fimbriae”) 6.
The oviduct is composed of three distinct histologic layers: an inner mucosa, middle muscularis, and the outer serosa. The mucosa forms an elaborate system of highly branched, interconnected, longitudinal folds in the ampulla, resembling a labyrinth, with less branching and folding along the tube towards the isthmus 6. A single columnar epithelium lines the mucosa and is comprised of ciliated cells and non-ciliated secretory cells. The lamina propria provides the support for the mucosa and is comprised of a thin vascular layer of loose connective tissue. The muscularis is comprised of an inner circular layer and an outer, longitudinal layer of smooth muscle and connective tissue. The serosa is the outer layer 6.
The uterus is divided into three anatomical parts:
1. corpus (body - containing the cavum uteri),
2. fundus (top portion),
3. isthmus (which is contiguous with the cervix).
The uterine corpus is composed of three histological layers: the inner endometrium, the muscular myometrium and the outer perimetrium or serosa adjacent to the peritoneum.
The endometrium lining the corpus is most profoundly affected by changes in circulating steroid hormones E2 and P4.
The endometrium has a complex cellular composition, including simple columnar epithelium (glandular and luminal) of secretory and ciliated cells, stromal fibroblasts, vascular endothelium and smooth muscle, and immune cells 7. The layer of columnar epithelium, “luminal epithelium” is the interface between the uterine cavity and the endometrial mucosa.
Glandular epithelium lines the tubular or branched glands that can extend as deep as the endometrial-myometrial junction. The stromal compartment (lamina propria) is comprised of highly cellular connective tissue with an extracellular matrix that contains few connective tissue fibers and resembles embryonic mesenchyme. The endometrial-myometrial junction is indistinct, without an intervening submucosal membrane. Parts of the basal endometrium extend in the proximate regions of adjacent myometrium 8.
The endometrium is comprised of four histologically defined zones 9. These compartments
1. zone I, comprised of luminal epithelia and sub-adjacent, densely packed stroma;
2. zone II, the upper endometrium in which the straight region of the glands course and are widely separated by stroma;
3. zone III, mid-regions of the glands, widely separated by stroma;
4. zone IV, the deepest portion of the glands in a fibrous stroma, adjacent to the endometrial-myometrial junction.
Zones I and II comprise the functionalis layer, and zones III and IV comprise the basalis layer 2. In humans, the functionalis responds cyclically to ovarian steroid hormones, is the site of embryonic implantation, and is shed with the menstrual bleeding in the absence of implantation. Cells of the basalis including stem cells participate in regeneration of the tissue after the menstrual bleeding 3.
The onset of puberty is associated with high hypothalamic gonadotropin-releasing hormone (GnRH) pulsing responsible for the release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from anterior pituitary gland resulting in ovarian activation (Figure 2). Leptin, a protein hormone which is responsible for nutrition intake and metabolism, is associated with the initial GnRH pulsing. The GnRH pulse activity is critical for successful reproductive function until menopause.
Figure 2: Overview of GnRH pulsing and feedback loop (http://www.nature.com/nm/journal/v12/n6 /images/nm0606-612-F1.gif).
Fallopian tubes and endometrium are highly sensitive to changes in circulating ovarian and adrenal cortex derived, menstrual cycle dependent steroid hormones E2 and P4 critically determining the menstrual cycle.
1.2 Menstrual cycle and hormonal regulation of fallopian tube and endometrium The human endometrium underlies morphological and structural changes during the menstrual cycle in preparation for embryo implantation or menstrual shedding in absence of implantation, respectively 11 (Figure 3).
The E2-dominated proliferative phase of the endometrium, which lasts from the end of menstrual bleeding until ovulation, is characterized by proliferation of the lamina functionalis, endometrial glands and endometrial stromal tissue as well as angiogenesis of endometrial blood vessels. In the oviduct, remarkable changes occur in epithelial ciliogenesis and secretory cell development 12.
The P4-dominated secretory phase, which starts at ovulation with the peak of LH and ends with the following menstrual bleeding, is accompanied by high secretory activity of
endometrial glands, elongation and growth of spiral arteries and decidualization and reorganization of endometrial stroma enabling embryonic invasion during the window of implantation between day LH peak +8 until LH+10. Growth factors and cytokines are involved in paracrine and autocrine actions to affect the histologic and biochemical signatures of the endometrial functionalis. Under the influence of P4, the oviductal epithelium atrophies, regresses, and becomes quiescent, primarily through cellular apoptosis.
Regarding the establishment of pregnancy, all of these changes - endometrial reorganization via matrix metalloproteinases (MMP) and further matrix modifying enzymes, angiogenesis, influx of immunocompetent cells (e.g. specialized uterine natural killer cells (uNK)), biochemical changes, cleavage and maturation of the embryo after fertilization during its passage through the fallopian tube - need to be synchronized on molecular level in a coordinated embryo-maternal dialogue facilitating embryonic invasion and implantation.
The knowledge about the factors involved in these early processes on embryo and maternal side increased in the last decades supported by extensive DNA and protein microarray analyses 14-16.
In the absence of implantation, the endometrium undergoes apoptosis, tissue desquamation followed by menstrual bleeding, and regeneration ensured by mechanisms likely involving stem cells in the basalis region of the tissue 17.
Figure 3: Overview of the female menstrual cycle. a) hypothalamic GnRH release, b) subsequent FSH and LH levels in peripheral blood, c) leading to growth of follicles and ovulation, d) ovary (and adrenal cortex) derived
implantation The process of ovulation is characterized by biochemical and morphological changes finally leading to the release of a mature oocyte and the transformation of the follicle into the corpus luteum. Generally, an ovarian follicle consists of different, interacting layers of cells surrounding the developing primary oocyte. Granulosa cells encompass the oocyte and proliferate in response to circulating gonadotrophins. During the E2-dominated proliferation,
respectively, FSH stimulates the sythesis of LH receptors on granulosa cells. Granulosa cells respond to elevating LH levels by an increase of cyclicAMP (cAMP) and production of P4.