Mark A. Garthwaite

Human uterine and ovarian expression of growth hormone–releasing hormone messenger RNA in benign and malignant gynecologic conditions

OBJECTIVE To determine uterine and ovarian expression of growth hormone-releasing hormone (GHRH) messenger RNA (mRNA) in benign and pathologic gynecologic states. DESIGN Case-control study. SETTING Tertiary-care academic department. PATIENT(S) Women undergoing hysterectomy for benign or malignant gynecologic conditions. INTERVENTION(S) Ovarian and uterine tissue was obtained for measurement of GHRH mRNA levels by reverse transcription polymerase chain reaction. MAIN OUTCOME MEASURE(S) Levels of GHRH mRNA in normal tissues were compared with those in tissues with pathologic abnormalities. RESULT(S) Growth hormone-releasing hormone mRNA was detectable in the ovary, endometrium, myometrium, fallopian tubes, and placenta. Levels of GHRH mRNA were significantly increased in secretory endometrium compared with proliferative endometrium. Hormone replacement therapy did not affect endometrial GHRH mRNA levels. Uterine myomas expressed similar levels of GHRH mRNA as normal myometrium. No changes in endometrial GHRH mRNA were detected in endometrial cancers compared with normal endometrium or myometrium obtained from the same patient; however, these levels were higher than those in noncancerous myometrial tissue obtained from other patients with benign gynecologic disease. In ovarian tissue, no differences in GHRH mRNA were found between premenopausal and postmenopausal women. Ovarian GHRH mRNA was significantly decreased in endometriotic cysts, whereas significantly greater GHRH expression occurred in ovarian cancer compared with normal ovarian tissue. CONCLUSION(S) Endometrial and ovarian GHRH gene transcription are altered in selective physiologic and pathologic states and are influenced by such factors as ovarian hormones. Because it is a growth factor, GHRH may promote endometrial proliferation and may be involved in the pathogenesis of ovarian and endometrial cancer and endometriosis.

Embryonic stem cells as models of trophoblast differentiation: progress, opportunities, and limitations

While the determination of the trophoblast lineage and the facilitation of placental morphogenesis by trophoblast interactions with other cells of the placenta are crucial components for the establishment of pregnancy, these processes are not tractable at the time of human implantation. Embryonic stem cells (ESCs) provide an embryonic surrogate to derive insights into these processes. In this review, we will summarize current paradigms which promote trophoblast differentiation from ESCs, and potential opportunities for their use to further define signals directing morphogenesis of the placenta following implantation of the embryo into the endometrium.

Characterization of decidual leukocyte populations in cynomolgus and vervet monkeys.

The objective of this study was the phenotypic and functional evaluation of decidual immune cells in the cynomolgus and vervet monkeys. Early pregnancy (days 36-42) deciduas were obtained by fetectomy for histological evaluation and decidual mononuclear leukocyte (MNL) isolation. While peripheral NK (pNK) cells in these species do not express CD56, CD56(+) NK cells were abundant in decidual samples. The majority of decidual NK (dNK) cells (>80%) had high light-scatter characteristics and were CD56(bright)CD16(+) cells with no or very low levels of natural cytotoxicity receptors (NKp46, NKp30) and NKG2A, while a minor population were small CD56(dim)CD16(-) lymphocytes also expressing less NKp46, NKp30 and NKG2A than pNK cells. All dNK cells were found to be perforin(+); however, their cytotoxic potential was low and cynomolgus dNK cells showed strongly reduced cytotoxicity against target cells compared with pNK cells. Macrophages and T cells together comprised approximately 25-30% of decidual MNL. Decidual T cells contained a higher proportion of the minor T cell subtypes (gammadeltaT cells, CD56(+) T cells) compared with peripheral blood. A subset of DC-SIGN(+) macrophages, with a distribution adjacent to areas of placental attachment in contrast to the widespread setting of general CD68(+) cells, was identified in both species. Together, these results demonstrate that the maternal-fetal interface in both cynomolgus and vervet monkeys is very rich in immune cells that have similar phenotypes to those seen in humans, indicating that both species are excellent models to study the contributions of distinct immune cell populations to pregnancy support.

ACP5 (Uteroferrin): Phylogeny of an Ancient and Conserved Gene Expressed in the Endometrium of Mammals

ABSTRACT Type 5 acid phosphatase (ACP5; also known as tartrate-resistant acid phosphatase or uteroferrin) is a metalloprotein secreted by the endometrial glandular epithelium of pigs, mares, sheep, and water buffalo. In this paper, we describe the phylogenetic distribution of endometrial expression of ACP5 and demonstrate that endometrial expression arose early in evolution (i.e., before divergence of prototherian and therian mammals ∼166 million years ago). To determine expression of ACP5 in the pregnant endometrium, RNA was isolated from rhesus, mouse, rat, dog, sheep, cow, horse, armadillo, opossum, and duck-billed platypus. Results from RT-PCR and RNA-Seq experiments confirmed that ACP5 is expressed in all species examined. ACP5 was also demonstrated immunochemically in endometrium of rhesus, marmoset, sheep, cow, goat, and opossum. Alignment of inferred amino acid sequences shows a high conservation of ACP5 throughout speciation, with species-specific differences most extensive in the N-terminal and C-terminal regions of the protein. Analysis by Selecton indicated that most of the sites in ACP5 are undergoing purifying selection, and no sites undergoing positive selection were found. In conclusion, endometrial expression of ACP5 is a common feature in all orders of mammals and has been subjected to purifying selection. Expression of ACP5 in the uterus predates the divergence of therians and prototherians. ACP5 is an evolutionary conserved gene that likely exerts a common function important for pregnancy in mammals using a wide range of reproductive strategies.

Characterization of cynomolgus and vervet monkey placental MHC class I expression: diversity of the nonhuman primate AG locus

Nonhuman primates are important animal models for the study of the maternal immune response to implantation within the decidua. The objective of this study was to define the placental expression of major histocompatibility complex (MHC) class I molecules in the cynomolgus (Macaca fascicularis) and vervet (African green) (Chlorocebus aethiops) monkeys. Early pregnancy (d36-42) cynomolgus and vervet placentas were obtained by fetectomy and prepared for histological evaluation. A pan-MHC class I monoclonal antibody demonstrated MHC class I expression in both vervet and cynomolgus placental trophoblasts, with particularly high expression in the villous syncytium, as previously shown in the rhesus and baboon. Placental cytotrophoblasts were isolated by enzymatic dispersion and gradient centrifugation and cultured, and multicolor flow cytometry was used to phenotype cell populations. Culture of isolated villous cytotrophoblasts demonstrated that MHC class I expression was linked to syncytiotrophoblast differentiation. A monoclonal antibody against Mamu-AG, the nonclassical MHC class I homolog of HLA-G in the rhesus monkey, demonstrated intense immunostaining and cell surface expression in cynomolgus placental trophoblasts; however, staining with vervet placenta and cells was low and inconsistent. Reverse transcriptase polymerase chain reaction was used to clone MHC class I molecules expressed in cynomolgus and vervet placentas. While Mafa-AG messenger RNA (mRNA) was readily detectable in cynomolgus placental RNA and was >99% identical at the amino acid level with Mamu-AG, 7/8 Chae-AG complementary DNAs had an unusual 16 amino acid repeat in the α1 domain, and all clones had an unexpected absence of the early stop codon at the 3′-end of the mRNA diagnostic for rhesus, cynomolgus, and baboon AG mRNAs, as well as HLA-G. We conclude that while the vervet monkey has retained the placental expression of a primate-specific nonclassical MHC class I locus, diversity is also revealed in this locus expressed at the maternal–fetal interface, thought to participate in placental regulation of the maternal immune response to embryo implantation and pregnancy.

Trophoblast differentiation, invasion and hormone secretion in a three-dimensional in vitro implantation model with rhesus monkey embryos

BackgroundThe initiation of primate embryo invasion into the endometrium and the formation of the placenta from trophoblasts, fetal mesenchyme, and vascular components are essential for the establishment of a successful pregnancy. The mechanisms which direct morphogenesis of the chorionic villi, and the interactions between trophectoderm-derived trophoblasts and the fetal mesenchyme to direct these processes during placentation are not well understood due to a dearth of systems to examine and manipulate real-time primate implantation. Here we describe an in vitro three-dimensional (3-D) model to study implantation which utilized IVF-generated rhesus monkey embryos cultured in a Matrigel explant system.MethodsBlastocyst stage embryos were embedded in a 3-D microenvironment of a Matrigel carrier and co-cultured with a feeder layer of cells generating conditioned medium. Throughout the course of embryo co-culture embryo growth and secretions were monitored. Embedded embryos were then sectioned and stained for markers of trophoblast function and differentiation.ResultsSigns of implantation were observed including enlargement of the embryo mass, and invasion and proliferation of trophoblast outgrowths. Expression of chorionic gonadotropin defined by immunohistochemical staining, and secretion of chorionic gonadotropin and progesterone coincident with the appearance of trophoblast outgrowths, supported the conclusion that a trophoblast cell lineage formed from implanted embryos. Positive staining for selected markers including Ki67, MHC class I, NeuN, CD31, vonWillebrand Factor and Vimentin, suggest growth and differentiation of the embryo following embedding.ConclusionsThis 3-D in vitro system will facilitate further study of primate embryo biology, with potential to provide a platform for study of genes related to implantation defects and trophoblast differentiation.