Human chorionic gonadotropin (hCG) is classically regarded as signal for maternal recognition of pregnancy affecting the corpus luteum to prevent luteolysis and stimulating the ovary to produce progesterone. Intact hCG is the predominant form of hCG produced by the trophoblast during pregnancy and is a hetero-dimer of hCGα and hCGβ subunits. It is often erroneously referred to as hCGβ but β subunit of hCG is other thing (freeβ) 13. Either high or diminished concentrations of intact hCG have been associated with maternal or embryo-placenta abnormalities. Its measurements becomes a useful method to detect higher risk of miscarriage, ectopic pregnancy, predict pre-eclampsia, intrauterine fetal growth restriction, fetal hydrops or identify trisomy 14. Recently an electro-chemiluminescence (ECL) method has been able to detect intact hCG secreted by embryos in spent culture media of day 1, day 3 and day 5 15. The embryonic hCG secretion was found to be associated with morphological grading in blastocyst stage and embryos having strong implantation potential both on day 3 and day 5 suggesting that morphological scoring system combined with embryonic hCG concentration may be a reasonable way for selecting competent human embryos in clinical IVF reproductive medicine. The hCG molecule has several isoforms attributed to varying degrees of glycosylation and stages of metabolic degradation some of which have specific functions different from that of classically recognized. Studies on the qualitative hCG output of developing embryos in culture as analyzed by hCG isoforms expressed in the secretome shows that hCGβ (intact hCG) is detectable in media from 2 pronuclear (2PN) stage embryos through to the blastocyst stage and absent in 1PN and arrested embryos 16. Prior to hatching, hyper-glycosylated hCG (hCGh) is observed selectively in 3PN embryos, but after hatching becomes along with intact hCG the dominant hCG molecule. Therefore hCG isoforms have potential roles as biomarkers of embryo viability and indicative of potential implantation success. This is of matter particularly considering the number of paracrine actions played by hCG around the time of implantation 171819. In fact there is growing in vitro and in vivo evidence that the hCG initially produced by the implanting embryo regulates the endometrial receptivity over the role played by the ovarian steroid hormones 182021222324. In the baboon, as long as adequate ovarian steroids priming of the endometrium is provided, the intrauterine addition of recombinant-hCG (rhCG) exerts an independent and additional modulation of the endometrial function 1721. Infusion of interleukin 1B (IL1B) at the time of implantation into the non-pregnant baboon treated with rhCG synergizes with hCG and mimics the early endometrial events associated with the presence of an embryo 22. Modulation of endometrial cell receptivity via rhCG is not limited to IL1 receptor ’agonists and antagonists but also extends to other IL1 family members which, again, regulate embryo attachment, endometrial remodeling, angiogenesis and immune responses around the implanting blastocyst 192324. Overall these data suggest that important biological functions are regulated in endometrium by signals from the implanting primate embryo to enhance endometrial receptivity and support embryo implantation 17182021222324. In vitro studies, using urinary-hCG (uhCG) and rhCG, show additional effects on embryo attachment 19 and rapid biosynthesis of prostaglandins by epithelial-glandular cells, respectively 2526. In humans, the intrauterine injection of hCG (500 UI) during the secretive phase of the cycle resulted to be associated with increased secretion of cytokines (PRL, LIF, VEGF, MMP-9) important for endometrial decidualization and vascularization 20. Actions on stromal cells are probably secondary to eicosanoids produced by endometrial epithelial-glandular cells as first response to hCG 27. Finally, hCG not only acts on the endometrium but probably also on the entire fetus-placental unit establishing the way for the initial embryo-maternal dialogue and placenta development as shown by other studies on trophoblast differentiation, tissue remodeling and trophoblast invasion 202829.

The hCG molecule responsible for early modulation of uterine receptivity, embryo attachment and then embryo/decidua invasion might not be the same molecule. In fact in the first 3 or 4 weeks of pregnancy the predominant form of hCG is the hCGh but it is soon replaced with native hCG during a few weeks after implantation when it declines from more than 80% to 50% of total hCG forms. This shift in the glycosylation pattern results from production initially by cytotrophoblast, specifically extravillous endovascular trophoblast cells, gradually shifting to that by syncytotrophoblast 14. hCGh and not intact hCG has an autocrine rather endocrine function and directly modulates cell growth and cytotrophoblast invasion in early pregnancy. Recent data, obtained by using highly sensitive urine hCG tests, show that on the day of implantation pregnancies destined to fail as spontaneous abortions or biochemical pregnancies are marked by low production of hCGh 30. This suggests that failure to produce appropriately glycosylated-hCG most probably translates into invasion process abnormalities. hCGh is made exclusively by cytotrophoblast (extra villous trophoblast cells) and is the primary component of blastocyst invasion. This is in line with the finding that invading trophoblast from hydatiform mole and chorioncarcinoma secretes very high amounts of mostly hCGh 31. In addition, these tumors over express hCG receptors compared to normal trophoblast suggesting that hCG has a role in trophoblast transformation, growth, and invasion in gestational trophoblastic neoplasms 3233. It is noteworthy, that grade of trophoblast invasion, amount of hCG receptors- expression and hCGh are all dependent on the percentage of paternal-ploidy inherited at fertilization. The invasive characteristics are made possible by concomitant HCG-dependent actions on maternal immunotolerance and endometrial apoptosis by regulating Fas-Fas ligand system 34.

Variant molecular forms of hCG exist each one having different plasma life, receptor binding affinity and bioactivity. We refer to the extensive review studies on this topic 1314. Briefly, the cell type (cytotrophoblast, synciotrophoblast, pituitary, tumor cells) determines the sort of oligosaccharide processing on α and β subunits but it is the α-β combination which modulates the extension of this processing. The set of enzymes contained in the secretory cell determines the nature of the processing and composition of the oligosaccharides added to both subunits. The metabolic sequence, cleavage of signal peptides, assembly of the native hCG, sequential posttranslational glycosylation and formation of the disulfide bonds happens at the same time that the hCG molecules are translocated from the place of synthesis up to the cell surface 14. Fine tuning knowledge of how the carbohydrate units affect in vivo bioactivity in the clinical setting is, however, still missing.

In vitro studies on the angiogenic and immunological actions played by hCG confirm how variability present in glycosylation rates among the various hCG molecules used (urinary-, recombinant- or hyper-glycosylated-hCG), is important 19. Recombinant hCG (rhCG) has a direct angiogenic function on uterine endothelial cells via a G protein coupled receptors to PLC/IP3 –DAG/Ca2+/ protein kinase C 35. Studies on endothelial cells recruitment and vessel formation in co-cultures of umbilical vein endothelial cells (HUVECs) and aortic smooth muscle cells (AoSMCs) in presence of rhCG showed involvement of different signal transduction systems for different actions (PKA and PKC pathways) 36. Besides a direct effect on endothelial cells proliferation, an indirect angiogenic action is possible through a hCG-mediated release of prostaglandins from epithelial and glandular endometrial cells and subsequent prostaglandins-stimulation of important angiopoietins (VEGF, BFGF, PLGF) by stromal cells 3738. In experiments where hCGh rather than rhCG has been used, different receptor coupling and internal pathway were found 39. The hCGh molecule has large carbohydrate side chains structure similar to that found in transforming growth factor β (TGF β) and because of this, it does compete with the TGFβ for binding to appropriate receptors 30. In experimental model, using endothelial and mural cells of the aortic ring assay, it has been shown that the potent angiostimulation effect played by hCGh was independent of the classic signaling pathway and an alternative TGFβ-receptor II was identified as the only receptor for the hCGh 39. Similar observations are valid for the hCGh-mediated actions on uterine natural killer cell proliferation (representing 70% of the endometrial leukocytes), uterine macrophages activation and interleukin 8 production by blood-circulating monocytes: in all these cases the HCG signal was not mediated by classical pathways but via the mannose receptor, a member of the c-type lectin receptor family that binds glycoproteins with N-linked carbohydrate side chains 4041. These last data suggest that the embryo may transmit the information about its presence to maternal immune systems also using hCGh which mimics some products released in bacterial infection. Such an immune-endocrine network involving hCG and peripheral blood immune cells might be compromised by infections and explain recurrent pregnancy losses following Chlamydia Trachomatis infection 40.

The human LH/hCG receptor is encoded by a single gene located in the short arm of chromosome 2. The gene is 80kb and consists of 10 introns and 11 exons. While the extra cellular domain arises from splicing of exons 1-10, the entire serpentine and C-terminal domains are encoded in exon 11 42. Transcription of the LH/hCG receptor gene gives rise to multiple mRNA species and the relative abundance of each six transcripts differs between gonadal and endometrial tissue. Mammalian cells transfected with the cDNAs for the rat and human LH/hCG receptor display three distinct species with molecular masses of 65-75 kDa, 85-95 kDa and 165-200 kDa 42. The 85-95 kDa band is the mature LH/hCG receptor present at the cell surface while the 65-75 kDa form of the LH/hCG receptor is a precursor of the cell surface receptor. This second form is located intracellularly because it is insensitive to surface proteolysis. Although the precursor of the LH/hCG receptor has a conformation that permits hormone binding this conformation is not the same as that of the mature form of the LH/hCG receptor 42. In endometrium at least three different protein-bands migrating at 90 kDa, 68 kDa and even lower (35 kDa) molecular weight have been reported 4344and their functional properties have been the subject of debate in original studies. Presence and function of extra-gonadal LH/hCG receptors in a number of different human tissues (including the endometrium) have been largely described by Rao et coll 3233434546. These investigators would have provided in vitro experimental data suggesting for a direct coupling of the endometrial hCG receptors to the adenylyl cyclase enzyme with a cAMP dependent eicosanoids production. On the other side, the existence of full receptor expression in human endometrium has been questioned by others and identical functional results were obtained also by incubating endometrial cells with other hormones such as TSH, FSH and alpha subunit 4748. Because no known receptor could transduce the effects of all these hormones the existence of a unique uterine receptor has been speculated. Accordingly, it has been found that compared to gonadal receptors, the endometrial LH/hCG receptor genes express mRNA for the highly conserved transmembrane portion of the hCG receptor but not for the extra cellular ligand-binding portion that recognizes and specifically binds LH/hCG 4749.

To ascertain whether human uterine receptor is identical to the cloned gonadal receptor, a further study utilizing reverse transcription, selective pre-amplification of full-length hCG/LH receptor mRNA + several shorter fragments of the receptor gene, and nested PCR with internal primers (followed by DNA sequencing) has been attempted 50. The results of this study, once for all, could substantiate the view that human endometrium expresses the classical hCG/LH receptor. This study also showed that alternative splicing is a regulatory mechanism by which the endometrial tissue controls the expression of full-length and functional receptors. Down regulation of full-length hCG/LH receptor mRNA appears to take place during the late secretory phase and early pregnancy decidua whereas up to the mid-secretory phase full-length receptor mRNA is expressed in all samples. Pattern of receptor mRNA expression reported in this study for human endometrium supports the concept of a possible direct role for ovulatory LH as well as embryonic hCG during the early secretory changes of endometrium and pre-implanting embryo arrival into the uterine cavity.

We have characterized in vivo the HCG receptors, G proteins and the adenylyl cyclase enzyme in membranes from human endometrium 274451. Using polyclonal LH/hCG receptors antibodies raised against a synthetic N-terminus amino acid sequence of 15-38 (these antibodies recognize also the truncated receptor protein) we firstly found that in human endometrial cell membranes a single band migrating at 68 kDa was present. Secondly despite the abundance of G proteins and adenylyl cyclase enzyme this system was not, in endometrium, coupled to the LH/hCG receptors when saturating concentrations of uhCG were added. On the contrary, the endometrial adenylyl cyclase enzyme was well responsive in vivo to PGE2. In internal control luteal membranes multiple LH/hCG receptor-bands migrating at higher molecular weight were found and the addition of the ligand (uhCG) did, as expected, stimulate the adenylyl cyclase enzyme activity. We also noted that after few days of progesterone the Gs protein expression was significantly increased and the adenylyl cyclase resulted to be maximally responsive to its classical stimulators. We concluded that the endometrial LH/hCG receptors are not active throughout the classical signal pathway described for gonadal tissue. Considering however that many independent in vitro studies all reported a rapid synthesis of prostaglandins after endometrial cells exposure to the hCG hormone we argued that the endometrial LH/hCG receptor must somehow be functional even though probably by means of alternative pathways.

Our data agree with more recent in vitro demonstration that when endometrial epithelial cells from both the baboon and humans are stimulated by rhCG the LH/hCG receptors activate a cAMP-independent MAPK pathway leading to PGE2 synthesis 25. Recombinant hCG acts via its G protein coupled receptor phosphorylating protein kinase B (PKB), c-Raf and ERK1/2 in a PI3K dependent manner. ERK1/2 phosphorylation was independent of the signaling paradigms of Gs and Gi transactivation typical of gonadal cells indicating alternative signaling pattern in endometrium 2526. Blocking the LH/CG receptor expression by small interfering RNA (siRMA) transfection, in both endometrial epithelial and stromal cells, directly inhibits the signal transduction cascade that is activated by CG in these cells 52. The internal coupling of the endometrial LH/hCG receptors to this particular pathway (PKB, c-Raf, ERK1/2) has been confirmed also by others 18. According to this last study, the endometrial LH/hCG receptors have multiple functional proprieties in response to acute doses of hCG while prolonged low-dose pre-exposition to the ligand blunts any effect. This is what may happen in the clinical setting of ovarian stimulation cycles where injection of exogenous gonadotropins would become detrimental on the endometrial capacity to transduce incoming signals of native embryonic-hCG.

The above mentioned PKB mediated prostaglandins release by CG in endometrium is a novel finding and adds to the information already available for other internal pathways (PKA and PKC) better known as active pathways in gonads as well as animal uteri 53545556. All these studies demonstrate that, in endometrium, many hormones (LH, FSH, GnRh, Relaxin, CRF, and Oxytocin) bind to their cognate receptors and through different G proteins activate either the adenylyl cyclase or phospholipase C. These receptors couple to Gi or Gq proteins to activate PLC or Gs to activate adenylyl cyclase. Activation of PKC by Ca2+ and DAG and activation of PKA by cAMP then act independently or synergically to promote an increase in COX2 and prostaglandins. Since all the signal transduction systems present in endometrium may converge to produce large amounts of prostaglandins it is difficult to get a complete picture of the in vivo intracellular dynamics and to consider the independent action of HCG separately from that of other hormones probably actively present at the time of implantation. This is particularly true during cycles of ovarian stimulation when many different GPCRs may concomitantly be activated. The fact that a single ligand elicits changes in more than one second messenger system is not unprecedented as it is well known that on luteal cells Gs and Gi proteins are involved in dual coupling of the LH/HCG receptor to adenylyl cyclase and phospholipase C 4257. It is now generally accepted that a given GPCR can independently activate more than one subfamily of G proteins and these may not be the only mediators of GPCR signaling 4258. Activation of adenylyl cyclase, phospholipase C, PI3K and MAPK by G proteins coupled receptors has been interpreted either in terms of coupling to two different G proteins (Gs and Gq/11) or in terms of generation of GTP-bound α and βγ dimers derived from Gs 5758.

While investigating the signal transduction system possibly linked to endometrial hCG receptors we discovered an incredible amount of highly functioning adenylyl cyclase enzyme in human endometrium cell membranes as compared with luteal cell membranes 51. The enzymatic activity found in cell membranes from hormone replacement therapy (HRT) cycles was much higher than that present in cell membranes from controlled ovarian stimulation (COH) cycles. Within the HRT cycles this enzyme resulted at its maximum level of activity on day plus 3 rather than on days plus 6 or 9 of progesterone supplementation. Against our expectations this transduction signaling resulted not to be classically coupled with the endometrial hCG receptors 27. The biological significance however of so abundant availability of adenylyl cyclase enzyme in human endometrium was novel and remained to be interpreted. In our opinion, the abundance of this enzyme in human endometrium has probably to do with the well-known potent cAMP-action on the process of stromal decidualization independently of and in addition to that one played by progesterone 59.

Decidualization is first apparent in the stromal cells surrounding the terminal spiral arteries of the superficial layer around days 23 of a 28-day cycle. It marks the end of the limited period of endometrial receptivity during which embryo attachment takes place. The mutual potentiation of cAMP and progesterone (PR) in stimulated effects on cultured endometrial stromal cells has been clearly demonstrated, however, what correlation is in vivo for a convergence of cAMP and PR signaling is still unknown 5960. As the secretory phase progresses circulating LH and FSH levels fall but initial local arrival of hCG could indirectly sustain the cAMP signaling throughout a large and rapid production of eicosanoids (PGE2) which are known to act, in endometrium, by coupling with the adenylyl cyclase enzyme. Numerous experimental data show that decidual process is primarily dependent on elevated levels of the second messenger cAMP (not progesterone) which is produced in response to factors activating the adenylyl cyclase enzyme (relaxin, CRF, PGE2) and simultaneous down regulation of phosphodiesterases (converting cAMP into AMP) during the early-mid secretory phase of the cycle 596061. Upon continuous cAMP stimulation and protein kinase A activation, endometrial stromal cells become first responsive and then dependent on steroid hormones (progesterone and androgens). cAMP and progesterone interact and cooperate. After estradiol priming short time of progesterone supplementation induces a significant increase of Gs protein which constitutes part of the enzyme (adenylyl cyclase) producing cAMP. This second intracellular mediator in turn induces over-expression of progesterone receptors on stromal cells membranes and it is a well-known potent stimulator of embryonic hCG secretion 14.

Recent studies on endometrial stromal cells undergoing differentiation into specialized decidual cells in presence of culture-media products from growing and arrested blastocysts have opened a new way of interpreting implantation 626364. These studies suggest that the molecular changes occurring in endometrial stromal cells during early-mid secretory phase of the cycle are fundamental not only because correspond to the “uterine receptivity” but also because they play an active role in the ability to recognize, respond to and eliminate compromised implanting embryos –“window of natural embryo selection” 63. Accordingly, decidual cells behave as embryo quality-biosensors and in case of poor or arresting embryo-cleavage, a specific decidual phenotype is derived which translates into late implantation, missed embryo quality control and early pregnancy failure. It is the decidualization process to be critical for the development of a normally functioning placenta. This agrees with in vivo data showing that successful implantation in natural cycles develops within a short time period, 8-10 days after ovulation meanwhile the risk of early pregnancy loss increases with later implantation being 13%, 26%, 52% and 82% for implantation days 9, 10, 11 and 12, respectively 65. Up to date it is unknown whether the enigmatic signal “biosensoring” decidual cells in presence of arrested embryos may be that one represented by abnormal output of hCG molecule.

Micro array studies of human endometrial stromal cells decidualized in vitro in response to progesterone and cAMP have demonstrated that of the 588 genes screened marked up regulation was observed of cytokines, growth factors, nuclear transcription factors, members of cyclin family, and mediators of the cAMP signal transduction pathway 66. In addition unexpected mRNA up regulated by progesterone and cAMP included the insulin receptor, the FSH receptor, neurotransmitter receptors, inhibin/activins and TNF-related apoptosis inducing ligand (TRAIL). This demonstrates that decidualization consists in sequential reprogramming of functionally related families of genes involved in extra cellular matrix organization, cell adhesion, cytoskeleton organization, signal transduction, metabolism, differentiation and apoptosis 5966. Upon biochemical reprogramming the endometrial stromal cells acquire many new functions that critically govern successful trophoblast invasion and placenta formation. The increased in CG receptors expression around the spiral arteries in early pregnancy probably reflects a maternal adaptation to ensure adequate blood supply to the developing embryo 67. During early pregnancy cAMP up-regulates the secretion of hCGh from cytotrophoblast to favor invasion and down-regulates the expression of classical LH/CG receptors of decidua 145067. Notably, the extra villous trophoblast cells, which invade the decidua and the inner third of myometrium and then intravasate the terminal spiral arteries, are also particularly rich of LH/hCG receptors 3368. It remains not clear why in pregnant decidua the LH/hCG receptors undergo down regulation by cAMP 67. It is possible that during the early phases of placentation, in order to comply with an extraordinary angiostimulation request, these receptors undergo transcription and conformational changes (kind of TGF β-receptors) in order to better transduce signals of the special hCGh produced by the extra villous trophoblast cells. The highly invasive nature of the extra villous trophoblast cells (attributed to paternally derived factors) must be tightly controlled in a temporal and spatial fashion to accommodate the need of a growing embryo while safeguarding the maternal host. Despite their extremely high proliferative and invasive nature their malignant transformation is rare. These cells disseminate into the maternal vasculature and the amazing precision of invasion inside the spiral arteries relies on an intricate communication with the maternal decidua 68.