Epithelial Human Chorionic Gonadotropin Is Expressed and Produced in Human Secretory Endometrium During the Normal Menstrual Cycle1

Abstract

The objective of this study was to determine whether beta human chorionic gonadotropin (hCG) (CGB) subunits and alpha hCG (CGA) subunits are expressed and the hCG dimer is produced in normal human cyclic endometrium. Endometrial specimens were collected for histological dating from women undergoing treatment in our division of human reproduction. RNA from normal secretory endometrium was extracted, and CGB and CGA gene expression was assessed by semiquantitative PCR. Adequate secretory endometrial specimens were homogenized using protease inhibitors. Proteins present in the supernatant were separated electrophoretically, and molecular hCG isoforms were detected by Western blot. The supernatant hCG concentrations were measured by ELISA. We characterized hCG and leukocytes in endometrial specimens by immunohistochemistry. Uterine flushing was performed to confirm endometrial hCG secretion into the uterine fluid. A full-length CGB mRNA encompassing the exon 1 promoter region and the structure exons 2 and 3 (including the C-terminal peptide) was expressed in normal secretory endometrial specimens (similar to CGA) during the early secretory phase of the menstrual cycle, up to an optimum at the midsecretory to late secretory phases. In homogenate supernatants obtained from normal secretory endometrium, hormone concentrations of dimeric hCG were approximately 5 mU per 10 mg of tissue, compared with considerably smaller concentrations of corresponding single free CGB subunit. Single chains of CGB, CGA, and dimeric molecular hCG isoforms were found in endometrial specimens by Western blot. Glandular endometrial hCG production is demonstrated immunohistochemically, with an increase toward the late secretory phase vs. the early secretory phase of the normal secretory menstrual cycle. However, glandular hCG release is diminished or absent in the dyssynchronous or missing endometrial secretory transformation. Endogenous endometrial hCG may be important for implantation and maintenance of pregnancy.

Expression and Production of Human Chorionic Gonadotropin (hCG) in the Normal Secretory Endometrium: Evidence of CGB7 and/or CGB6 Beta hCG Subunit Gene Expression1

“…Recently, we confirmed both CGA mRNA and CGB mRNA gene expression, detected the CGB and CGA subunits and dimeric hCG protein isoforms by electrophoretic separation, measured the hCG and CGB hormone concentrations in endometrial homogenates, and immunohistochemically characterized increases in hCG production in synchrony with the transformation of the secretory endometrium [24]. We found that glandular hCG release is diminished or absent, respectively, in dyssynchronously transforming or nontransforming secretory cycle phase endometria.…”

“…A total of 581 patient endometrium samples used in our recent study [24], consisting of a variety of normal, dyssynchronous, or missing endometrial secretory transformation specimens, were also analyzed histologically according to our endometrium score parameters proposed in that study (glandular shape, glandular nucleus, stromal differentiation, leukocyte number; each scored 1-6 and summarized). After this analysis only the 145 patient samples identified as normal, synchronously transformed secretory endometria were selected and included in the present study.…”

“…After this analysis only the 145 patient samples identified as normal, synchronously transformed secretory endometria were selected and included in the present study. The samples were subdivided into patient groups of the early secretory (n ¼ 42, score [8][9][10][11][12], midsecretory (n ¼ 35, score 12-16), late secretory (n ¼ 30, score [16][17][18][19][20] and late predecidual secretory (n ¼ 38, score 20-24) endometrial phases [24].…”

“…The pellets were dissolved in DE-PC-treated water, and 30-ll RNA samples were quantified and stored as small aliquots at À808C. For the tissue homogenization and the preparation of supernatants used for Western blot detection, approximately 30 mg of prewashed fresh or À808C frozen samples was resuspended in 500 ll ice-cold lysis buffer of 50 mM TrisHCl buffer, pH 7.6, using the Complete Protease Inhibitor Kit (Roche), and were homogenized on ice as previously described [24]. The supernatants were prepared by repeated centrifugation of the homogenates at 19 000 3 g and 48C and were frozen immediately at À208C until examination.…”

“…A 2.5-ll aliquot was heated at 658C to eliminate DNAse and unfold the RNA, followed by cooling at 48C. A 2.5-ll aliquot of the cDNA reaction mixture prepared according to the manufacturer's instructions (Roche) was added as previously described [24]. The reverse transcription reaction was carried out using a Perkin Elmer thermocycler under the conditions of 258C for 10 min, 428C for 60 min, and finally 5 min at 948C to destroy the reverse transcriptase activity.…”