Complete hydatidiform mole with coexisting dichorionic diamniotic twins following testicular sperm extraction and intracytoplasmic sperm injection.

We present the first report of complete hydatidiform mole (HM) with coexisting dichorionic diamniotic twins. This pregnancy was achieved after testicular sperm extraction and intracytoplasmic sperm injection (ICSI) for azoospermia in the woman's husband. Standard in vitro fertilization may cause multisperm fertilization and increase triploid partial HM and complete HM, which arise from dispermic fertilization. In contrast, ICSI can avoid multisperm fertilization. In our case, paternal isodisomy in the molar tissue was confirmed by microsatellite analysis suggesting that it resulted from duplication of a haploid paternal genome following monospermic fertilization of an inactivated oocyte or from monospermic fertilization of an inactivated oocyte with a diploid sperm. Although the patient was eager to continue the pregnancy, the size of the HM component increased rapidly and termination of the pregnancy was required for pre-eclampsia-like symptoms at 15 weeks of gestation. After the operation, chemotherapy was initiated for persistent trophoblastic disease.

Bioactivity of human chorionic gonadotropin produced in frozen-thawed embryo transfer cycles with exogenous hormone replacement.

Human chorionic gonadotropin (HCG) is the glycoprotein hormone in pregnancy. It is known that hCG molecule has a variety of isoforms showing different potency in bioactivity. Taking advantage of rat Leydig cell assay to evaluate hCG bioactivity, we first tried to predict the prognosis of pregnancy in hormone replacement (HR)-cryopreserved embryo transfer cycles. There was no significant difference in serum estradiol (E2) level, immuno-hCG, or bioactive to immunoreactive hCG ratio (b/i) between normal pregnancies and miscarriages at 4 weeks of gestation. Linear regression did not show a significant correlation in E2 or b/i between normal pregnancies and miscarriages. The same analysis was performed on serum samples collected from spontaneously pregnant patients. In normal pregnancies b/i was significantly lower than that in miscarriages while serum E2 was significantly higher. The linear regression analysis was also performed to clarify the correlation between E2 and b/i, and no significant correlation was observed. To confirm the effect of E2 on hCG production, we treated trophoblastic cells with E2. E2 increased the immunoreactivity of hCG to a non-physiologically high concentration; however, it did not change its bioactivity, suggesting that E2 did not change hCG bioactivity by affecting trophoblasts directly. In this study, we presumed it possible to speculate the prognosis of the pregnancy in spontaneous cycles from b/i of hCG, but not in HR cycles. It is suggested that process of hCG secretion could undergo different endocrine regulations between the artificial sex steroid replacement cycles and spontaneous cycles with corpus luteum function.

Relaxin signaling in reproductive tissues.

The insulin/relaxin peptide family includes insulin, IGFs, relaxin1-3, INSL3/RLF, INSL4, INSL5/RIF2 and INSL6/RIF1, many without functional characterization. Based on analysis of transgenic phenotypes and phylogenetic profiling, we have discovered that two orphan leucine-rich repeat-containing G protein-coupled receptors, LGR7 and LGR8, are cognate receptors for relaxin whereas INSL3 is a specific ligand for LGR8. With the identification of the relaxin receptors, it is now possible to investigate specific cells and tissues that are responsive to relaxin in diverse physiological and pathological conditions as well as to develop agonists and antagonists for LGR7 and LGR8 as therapeutics to treat different labor disorders. Furthermore, future functional characterization of the specificity of these pluripoentent receptors with peptide ligands could lead to the understanding of related orphan ligands and receptors.

H3 relaxin is a specific ligand for LGR7 and activates the receptor by interacting with both the ectodomain and the exoloop 2.

Leucine-rich repeat-containing, G protein-coupled receptors (LGRs) represent a unique subgroup of G protein-coupled receptors with a large ectodomain. Recent studies demonstrated that relaxin activates two orphan LGRs, LGR7 and LGR8, whereas INSL3/Leydig insulin-like peptide specifically activates LGR8. Human relaxin 3 (H3 relaxin) was recently discovered as a novel ligand for relaxin receptors. Here, we demonstrate that H3 relaxin activates LGR7 but not LGR8. Taking advantage of the overlapping specificity of these three ligands for the two related LGRs, chimeric receptors were generated to elucidate the mechanism of ligand activation of LGR7. Chimeric receptor LGR7/8 with the ectodomain from LGR7 but the transmembrane region from LGR8 maintains responsiveness to relaxin but was less responsive to H3 relaxin based on ligand stimulation of cAMP production. The decreased ligand signaling was accompanied by decreases in the ability of H3 relaxin to compete for (33)P-relaxin binding to the chimeric receptor. However, replacement of the exoloop 2, but not exoloop 1 or 3, of LGR7 to the chimeric LGR7/8 restored ligand binding and receptor-mediated cAMP production. These results suggested that activation of LGR7 by H3 relaxin involves specific binding of the ligand to both the ectodomain and the exoloop 2, thus providing a model with which to understand the molecular basis of ligand signaling for this unique subgroup of G protein-coupled receptors.

Activation of orphan receptors by the hormone relaxin.

Relaxin is a hormone important for the growth and remodeling of reproductive and other tissues during pregnancy. Although binding sites for relaxin are widely distributed, the nature of its receptor has been elusive. Here, we demonstrate that two orphan heterotrimeric guanine nucleotide binding protein (G protein)-coupled receptors, LGR7 and LGR8, are capable of mediating the action of relaxin through an adenosine 3',5'-monophosphate (cAMP)-dependent pathway distinct from that of the structurally related insulin and insulin-like growth factor family ligand. Treatment of antepartum mice with the soluble ligand-binding region of LGR7 caused parturition delay. The wide and divergent distribution of the two relaxin receptors implicates their roles in reproductive, brain, renal, cardiovascular, and other functions.

The ectodomain of the luteinizing hormone receptor interacts with exoloop 2 to constrain the transmembrane region: studies using chimeric human and fly receptors.

Lutropin (LH) and follitropin (FSH) receptors belong to a group of leucine-rich repeat-containing, G protein-coupled receptors (LGRs) found in vertebrates and flies. We fused the ectodomain of human LH or FSH receptors to the transmembrane region of fly LGR2. The chimeric human/fly receptors, unlike their wild type counterparts, exhibited ligand-independent constitutive activity. Because ectodomains likely interact with exoloops to constrain the receptors, individual exoloops of the chimeric receptor containing the ectodomain of the LH receptor and transmembrane region of fly LGR2 was replaced with LH receptor sequences. Chimeric receptors with the ectodomain and exoloop 2, but not exoloop 1 or 3, from LH receptors showed decreases in constitutive activity, but ligand treatment stimulated cAMP production. Furthermore, substitution of key resides in the hinge region of fly LGR2 with LH receptor sequences led to constitutive receptor activation; however, concomitant substitution of the homologous exoloop 2 of the LH receptor decreased G(s) coupling. These results suggest that the hinge region of the LH receptor interacts with exoloop 2 to constrain the receptor in an inactive conformation whereas ligand binding relieves this constraint, leading to G(s) activation.