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Hydatidiform mole (HM) is an abnormal human pregnancy characterized by excessive growth of placental trophoblasts and abnormal early embryonic development. Following a first such abnormal pregnancy, the risk for women of successive molar pregnancies significantly increases. To date variants in seven maternal-effect genes have been shown to cause recurrent HMs (RHM). NLRP7 is the major causative gene for RHM and codes for NOD-like receptor (NLR) family pyrin domain containing 7, which belongs to a family of proteins involved in inflammatory disorders. Since its identification, all NLRP7 variants have been recorded in Infevers, an online registry dedicated to autoinflammatory diseases (https://infevers.umai-montpellier.fr/web/). Here, we reviewed published and unpublished recessive NLRP7 variants associated with RHM, scored their pathogenicity according to the American College of Medical Genetics classification, and recapitulated all functional studies at the level of both the patients and the conceptions. We also provided data on further variant analyses of 32 patients and genotypes of 36 additional molar pregnancies. This comprehensive review integrates published and unpublished data on NLRP7 and aims at guiding geneticists and clinicians in variant interpretation, genetic counseling, and management of patients with this rare condition.

Supplemental Methods Figure S1: Reports clinical information's about normal pregnant women, patients with complete hydatiform moles or choriocarcinoma. Figure S2: Experimental procedure. Figure S3: Quantification of EG-VEGF PROKR1 and PROKR2 protein expression in CTL, Complete hydatiform mole (CHM) and choriocarcinoma (CC) placental section. Figure S4: EG-VEGF effect on JEG3 migration, in the absence or presence of PROKR1 or PROKR2 antagonists. Figure S5: JEG3-luc injected mice exhibited arrested gestation and disorganized vascularization. Figure S6: Antibody microarray analysis. Figure S7: Characterization of the angiogenic status of sera and placentas collected from CTL, CHM and CC patients. Figure S8: Antibody angiogenic microarray analysis of sera collected from CTL, CHM and CC patients.

Purpose: Choriocarcinoma (CC) is the most malignant gestational trophoblastic disease that often develops from complete hydatidiform moles (CHM). Neither the mechanism of CC development nor its progression is yet characterized. We recently identified endocrine gland–derived vascular endothelial growth factor (EG-VEGF) as a novel key placental growth factor that controls trophoblast proliferation and invasion. EG-VEGF acts via two receptors, PROKR1 and PROKR2. Here, we demonstrate that EG-VEGF receptors can be targeted for CC therapy.Experimental Design: Three approaches were used: (i) a clinical investigation comparing circulating EG-VEGF in control (n = 20) and in distinctive CHM (n = 38) and CC (n = 9) cohorts, (ii) an in vitro study investigating EG-VEGF effects on the CC cell line JEG3, and (iii) an in vivo study including the development of a novel CC mouse model, through a direct injection of JEG3-luciferase into the placenta of gravid SCID-mice.Results: Both placental and circulating EG-VEGF levels were increased in CHM and CC (×5) patients. EG-VEGF increased JEG3 proliferation, migration, and invasion in two-dimensional (2D) and three-dimensional (3D) culture systems. JEG3 injection in the placenta caused CC development with large metastases compared with their injection into the uterine horn. Treatment of the animal model with EG-VEGF receptor's antagonists significantly reduced tumor development and progression and preserved pregnancy. Antibody-array and immunohistological analyses further deciphered the mechanism of the antagonist's actions.Conclusions: Our work describes a novel preclinical animal model of CC and presents evidence that EG-VEGF receptors can be targeted for CC therapy. This may provide safe and less toxic therapeutic options compared with the currently used multi-agent chemotherapies. Clin Cancer Res; 23(22); 7130–40. ©2017 AACR.

PURPOSE: To investigate the frequency of a founder mutation in NLRP7, L750V, in independent cohorts of Mexican patients with recurrent hydatidiform moles (RHMs). METHODS: Mutation analysis was performed by Sanger sequencing on DNA from 44 unrelated Mexican patients with RHMs and seven molar tissues from seven additional unrelated patients. RESULTS: L750V was present in homozygous or heterozygous state in 37 (86%) patients and was transmitted on the same haplotype to patients from different states of Mexico. We also identified a second founder mutation, c.2810+2T>G in eight (18.1%) patients, and a novel premature stop-codon mutation W653*. CONCLUSION: Our data confirm the strong founder effect for L750V, which appears to be the most common mutation in NLRP7. We also report on six healthy live births to five patients with biallelic NLRP7 mutations, two from spontaneous conceptions and four from donated ovum and discuss our recommendations for DNA testing and genetic counseling. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-021-02132-1.

Recurrent hydatidiform moles (RHMs) are human pregnancies with abnormal embryonic development and hyperproliferating trophoblast. Biallelic mutations in NLRP7 and KHDC3L, members of the subcortical maternal complex (SCMC), explain the etiology of RHMs in only 60% of patients. Here we report the identification of seven functional variants in a recessive state in three SCMC members, five in NLRP7, one in NLRP5, and one in PADI6. In NLRP5, we report the first patient with RHMs and biallelic mutations. In PADI6, the patient had four molar pregnancies, two of which had fetuses with various abnormalities including placental mesenchymal dysplasia and intra-uterine growth restriction, which are features of Beckwith-Wiedemann syndrome and Silver Russell syndrome, respectively. Our findings corroborate recent studies and highlight the common oocyte origin of all these conditions and the continuous spectrum of abnormalities associated with deficiencies in the SCMC genes.

Hydatidiform mole (HM) is an aberrant human pregnancy characterized by excessive trophoblastic proliferation and abnormal embryonic development. HM has two morphological types, complete (CHM) and partial (PHM), and non-recurrent ones have three genotypic types, androgenetic monospermic, androgenetic dispermic, and triploid dispermic. Most available studies on risk factors predisposing to different types of HM and their malignant transformation mainly suffer from the lack of comprehensive genotypic analysis of large cohorts of molar tissues combined with accurate postmolar hCG follow-up. Moreover, 10–20% of patients with one HM have at least one non-molar miscarriage, which is higher than the frequency of two pregnancy losses in the general population (2–5%), suggesting a common genetic susceptibility to HM and miscarriages. However, the underlying causes of the miscarriages in these patients are unknown. Here, we comprehensively analyzed 204 HM, mostly from patients referred to the Quebec Registry of Trophoblastic Diseases and for which postmolar hCG monitoring is available, and 30 of their non-molar miscarriages. We revisited the risk of maternal age and neoplastic transformation across the different HM genotypic categories and investigated the presence of chromosomal abnormalities in their non-molar miscarriages. We confirm that androgenetic CHM is more prone to gestational trophoblastic neoplasia (GTN) than triploid dispermic PHM, and androgenetic dispermic CHM is more prone to high-risk GTN and choriocarcinoma (CC) than androgenetic monospermic CHM. We also confirm the association between increased maternal age and androgenetic CHM and their malignancies. Most importantly, we demonstrate for the first time that patients with an HM and miscarriages are at higher risk for aneuploid miscarriages [83.3%, 95% confidence interval (CI): 0.653–0.944] than women with sporadic (51.5%, 95% CI: 50.3–52.7%, p value = 0.0003828) or recurrent miscarriages (43.8%, 95% CI: 40.7–47.0%, p value = 0.00002). Our data suggest common genetic female germline defects predisposing to HM and aneuploid non-molar miscarriages in some patients.

Recurrent miscarriage (RM) is defined by the occurrence of at least two pregnancy losses prior to 22 weeks of gestation and affects up to 5% of couples trying to conceive.1–3 RM has a significant emotional impact on couples and the repetitive nature intensifies the grief experienced. A recessive missense in cyclin B3 ( CCNB3 ) has recently been shown in two sisters with RM and triploidy of maternal origin.4 Here, we report a novel recessive CCNB3 mutation, c.4091+1G>A, p.Val1321Glyfs*4, in a patient with 16 RM and show that one of her miscarriages is triploid digynic resulted from the failure of meiosis I. RM is clinically and genetically highly heterogeneous. After comprehensive clinical and laboratory testing, in 50% of couples, no abnormalities are identified, and such cases are categorised as RM of unexplained clinical aetiology. To date, little is known about their genetic causes, and known genes explain only a minority of cases. One of the many factors that have hampered our understanding of the genetics of recurrent miscarriages is their complexity, genetic heterogeneity and the difficulties in homogenising these entities to simplify their studies. In many cases of recurrent miscarriages of unknown clinical aetiology, it is impossible to know whether the defect originates from the male or the female, and whether it is in a dominant or a recessive state. Also, it is impossible to know if the defect is transmitted from the parents to the miscarried conception or if it occurred de novo in the miscarriage. While the germline origin of male causes of miscarriages could be sometimes diagnosed based on semen analysis, diagnosing the origin of female causes of miscarriages is more challenging; in many cases, it is impossible to distinguish germline from uterine or systemic defects. Consequently, despite the use of next-generation sequencing, which has greatly facilitated …

Study question What causes non-molar miscarriages in women with one hydatidiform mole (HM)? Summary answer We found a higher rate of aneuploidies in the non-molar miscarriages of women with HM than in those from women with sporadic or recurrent miscarriages. What is known already Women with hydatidiform moles have higher rates of miscarriages and women with recurrent miscarriages have higher rates of moles than women from the general population. Study design, size, duration We retrieved archived formalin-fixed paraffin embedded tissues from non-molar miscarriages of patients with one HM and analyzed them for the presence of aneuploidies using single nucleotide polymorphism (SNP)-microarray. We next determined the meiotic origin of the aneuploidies by genotyping the aneuploid non-molar miscarriages along with the parental genomes using microsatellite markers. Participants/materials, setting, methods All participants and some of their partners provided written consent to participate in our study, agreed to a blood draw for genotyping analysis, and agreed for us to retrieve their molar and non-molar tissues from various histopathology laboratories for research purposes. Main results and the role of chance We demonstrate for the first time that patients with an HM and miscarriages are at higher risk for aneuploid miscarriages [83.3%, 95% confidence interval (CI): 0.653–0.944] than women with sporadic (51.5%, 95% CI: 50.3–52.7%, p value = 0.0003828) or recurrent miscarriages (43.8%, 95% CI: 40.7–47.0%, p value = 0.00002). Genotyping the aneuploid miscarriages and the parental genomes demonstrated that most of the aneuploidies originated from errors in maternal meiosis I or II. Limitations, reasons for caution We were able to retrieve only 30 non-molar miscarriages from women with one HM for analysis. Expanding such analysis to a larger and independent cohort of miscarriages from such patients will be important to validate our observations. Wider implications of the findings: Our data suggest common genetic female germline defects predisposing to HM and aneuploid non-molar miscarriages in some patients. Trial registration number Not applicable

The inflammatory gene NLRP7 is the major gene responsible for recurrent complete hydatidiform moles (CHM), an abnormal pregnancy that can develop into gestational choriocarcinoma (CC). However, the role of NLRP7 in the development and immune tolerance of CC has not been investigated. Three approaches were employed to define the role of NLRP7 in CC development: (i) a clinical study that analyzed human placenta and sera collected from women with normal pregnancies, CHM or CC, (ii) an in vitro study that investigated the impact of NLRP7 knockdown on tumor growth and organization, and (iii) an in vivo study that used two CC mouse models, including an orthotopic model. NLRP7 and circulating inflammatory cytokines were upregulated in tumor cells and in CHM and CC. In tumor cells, NLRP7 functions in an inflammasome-independent manner and promoted their proliferation and 3D organization. Gravid mice placentas injected with CC cells invalidated for NLRP7, exhibited higher maternal immune response, developed smaller tumors, and displayed less metastases. Our data characterized the critical role of NLRP7 in CC and provided evidence of its contribution to the development of an immunosuppressive maternal microenvironment that not only downregulates the maternal immune response but also fosters the growth and progression of CC.

Recurrent hydatidiform moles (RHMs) are human pregnancies with abnormal embryonic development and hyperproliferating trophoblast. Biallelic mutations in NLRP7 and KHDC3L, members of the subcortical maternal complex (SCMC), explain the etiology of RHMs in only 60% of patients. Here we report the identification of seven functional variants in a recessive state in three SCMC members, five in NLRP7, one in NLRP5, and one in PADI6. In NLRP5, we report the first patient with RHMs and biallelic mutations. In PADI6, the patient had four molar pregnancies, two of which had fetuses with various abnormalities including placental mesenchymal dysplasia and intra-uterine growth restriction, which are features of Beckwith-Wiedemann syndrome and Silver Russell syndrome, respectively. Our findings corroborate recent studies and highlight the common oocyte origin of all these conditions and the continuous spectrum of abnormalities associated with deficiencies in the SCMC genes.

Background Gestational trophoblastic diseases (GTDs) encompass a group of clinically heterogeneous tumors of trophoblastic cell origin. In some populations and studies, single NLRP7 heterozygous mutations and some non-synonymous variants (NSVs) have been shown to confer genetic susceptibility to sporadic GTDs. Our study aims to investigate whether single rare heterozygous NSVs in NLRP7 predispose patients to GTDs in Chinese. Methods We performed Sanger sequencing of all NLRP7 exons and exon-intron boundaries in a cohort of Chinese patients with non-recurrent sporadic GTDs and normal childbearing women. Using in-silico analysis and several prediction programs, we investigated the effects of identified NSVs on the function of the protein. Then we examine the function in inflammatory pathway to assessing the functional consequences of the identified rare NSVs in in-vitro studies.Results We found that the number of patients with rare single heterozygous NSVs that are predicted to have functional effects on the protein is significantly higher in the first cohort of 292 patients than in the 300 controls (p=0.028). Analysis of the validation cohort of 130 patients confirmed the significant higher number of patients with rare NSVs than controls (p=0.0014). We show that most of the rare variants that are identified only in the patients are predicted to have functional effects on the protein by various algorithms and in-vitro experiments. Conclusion Our results demonstrate that Chinese patients with GTDs have a higher burden of single heterozygous NLRP7 variants which were predicted to have effects on the protein than controls. We suggest that some of these single NSVs may contribute to the genetic susceptibility for GTDs in China.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Androgenetic complete hydatidiform moles are human pregnancies with no embryos and affect 1 in every 1,400 pregnancies. They have mostly androgenetic monospermic genomes with all the chromosomes originating from a haploid sperm and no maternal chromosomes. Androgenetic complete hydatidiform moles were described in 1977, but how they occur has remained an open question. We identified bi-allelic deleterious mutations in MEI1, TOP6BL/C11orf80, and REC114, with roles in meiotic double-strand breaks formation in women with recurrent androgenetic complete hydatidiform moles. We investigated the occurrence of androgenesis in Mei1-deficient female mice and discovered that 8% of their oocytes lose all their chromosomes by extruding them with the spindles into the first polar body. We demonstrate that Mei1−/− oocytes are capable of fertilization and 5% produce androgenetic zygotes. Thus, we uncover a meiotic abnormality in mammals and a mechanism for the genesis of androgenetic zygotes that is the extrusion of all maternal chromosomes and their spindles into the first polar body. © 2018 American Society of Human Genetics

Introduction Hydatidiform mole (HM) is an aberrant human pregnancy with abnormal trophoblastic development, migration/invasion of the extravillous trophoblast in the decidua. These abnormalities are established in a hypoxic environment during the first trimester of gestation. Methods Using text mining, we identified 72 unique genes that are linked to HM (HM-linked genes) that we studied by bioinformatic analysis in publicly available transcriptomes of primary chorionic villous cells (cytotrophoblast, syncytiotrophoblast, extravillous trophoblast, and arterial and venous endothelial) isolated from normal placentas or established trophoblastic cell lines cultured under different oxygen concentrations. Results We show that the majority of HM-linked genes (75%) are involved in normal trophoblastic differentiation, arranged in clusters, and some of them are implicated in chorionic villous invasion or regulated by oxygen concentrations. Discussion Our analysis integrates the various aspects of the pathophysiology of HM and highlights the importance of trophoblastic differentiation in this pathology.

A hydatidiform mole (HM) is an abnormal human pregnancy characterised by absence of, or abnormal, embryonic development, excessive trophoblastic proliferation and hydropic degeneration of placental villi. The common types of moles are sporadic, not recurrent, and affect 1 in 1000 pregnancies in western countries. HM may recur in the same patient, which is referred to as recurrent HM (RHM), and indicates that the patient is genetically susceptible to HM. Through the examination of rare familial cases of RHM, two maternal-effect genes, NLRP7 and KHDC3L, responsible for this condition have been identified. Pathogenic variants in these genes appear to impair imprinting establishment during oogenesis. Herein, we review current knowledge on the genetics and epigenetics of RHM, and highlight the benefits of testing patients for pathogenic variants in the known genes. Key Concepts RHM from patients with recessive pathogenic variants in NLRP7 or KHDC3L are diploid biparental and originate from a different mechanism than sporadic androgenetic or triploid HM. At the epigenetic level, RHM from patients with recessive pathogenic variants in NLRP7 or KHDC3L lack maternal methylation marks on differentially methylated regions (DMR) and mimic diploid androgenetic CHM, which lack a maternal genome. RHM from patients with recessive pathogenic variants in NLRP7 have defective oocytes and benefit from ovum donation. Diploid biparental HM from patients with pathogenic variants in NLRP7 have an imbalance between embryonic tissue differentiation and trophoblastic proliferation. The same imbalance is also observed in sporadic androgenetic and triploid HM. NLRP7 is part of the innate immune system and its pathogenic variants downregulate inflammation. Keywords: hydatidiform mole; recurrent hydatidiform mole; NLRP7; KHDC3L; reproductive loss; imprinting; epigenetics; oocyte; trophoblastic proliferation; androgenetic monospermic HM; triploid dispermic HM; ovum donation

Hydatidiform mole (HM) is an abnormal human pregnancy characterized by excessive trophoblastic proliferation and abnormal embryonic development. There are two types of HM based on microscopic morphological evaluation, complete HM (CHM) and partial HM (PHM). These can be further subdivided based on the parental contribution to the molar genomes. Such characterization of HM, by morphology and genotype analyses, is crucial for patient management and for the fundamental understanding of this intriguing pathology. It is well documented that morphological analysis of HM is subject to wide interobserver variability and is not sufficient on its own to accurately classify HM into CHM and PHM and distinguish them from hydropic non-molar abortions. Genotyping analysis is mostly performed on DNA and tissues from formalin-fixed paraffin-embedded (FFPE) products of conception, which have less than optimal quality and may consequently lead to wrong conclusions. In this article, detailed protocols for multiplex genotyping and flow cytometry analyses of FFPE molar tissues are provided, along with the interpretation of the results of these methods, their troubleshooting, and integration with the morphological evaluation, p57KIP2 immunohistochemistry, and fluorescence in situ hybridization (FISH) to reach a correct and robust diagnosis. Here, the authors share the methods and lessons learned in the past 10 years from the analysis of approximately 400 products of conception.

Recurrent hydatidiform moles (RHM) are aberrant human pregnancies characterized by absence of, or abnormal, embryonic development, hydropic degeneration of chorionic villi, and hyperproliferation of the trophoblast. Biallelic mutations in two maternal-effect genes, NLRP7 and KHDC3L, underlie the causation of RHM in 60% of patients. We performed exome sequencing on a patient with six pregnancy losses, two miscarriages and four HM, and found no variants that affect the functions of the known genes. We found biallelic missense variants that affect conserved amino acids in PADI6 and segregate with the disease phenotype in the family. PADI6 is another maternal-effect gene and a member of the subcortical maternal complex that has been shown to have recessive variants that affect the gene function in four unrelated women with infertility who also experienced early embryonic arrest during preimplantation development after IVF. We demonstrated that PADI6 co-localizes with NLRP7 in human oocytes and preimplantation embryos and reviewed the morphology and genotypes of four products of conception from our patient. Our data expand the involvement of PADI6 to other forms of reproductive loss and highlight the commonality between infertility, miscarriages, and molar pregnancies, in some cases.

WOS: 000440567300011, PubMed ID: 29463882, Hydatidiform mole is an aberrant human pregnancy characterized by early embryonic arrest and excessive trophoblastic proliferation. Recurrent hydatidiform moles are defined by the occurrence of at least two hydatidiform moles in the same patient. Fifty to eighty percent of patients with recurrent hydatidiform moles have biallelic pathogenic variants in NLRP7 or KHDC3L. However, in the remaining patients, the genotypic types of the moles are unknown. We characterized 80 new hydatidiform mole tissues, 57 of which were from patients with no mutations in the known genes, and we reviewed the genotypes of a total of 123 molar tissues. We also reviewed mutation analysis in 113 patients with recurrent hydatidiform moles. While all hydatidiform moles from patients with biallelic NLRP7 or KHDC3L mutations are diploid biparental, we demonstrate that those from patients without mutations are highly heterogeneous and only a small minority of them are diploid biparental (8%). The other mechanisms that were found to recur in patients without mutations are diploid androgenetic monospermic (24%) and triploid dispermic (32%); the remaining hydatidiform moles were misdiagnosed as moles due to errors in the analyses and/or their unusual mechanisms. We compared three parameters of genetic susceptibility in patients with and without mutations and show that patients without mutations are mostly from non-familial cases, have fewer reproductive losses, and more live births. Our data demonstrate that patients with recurrent hydatidiform moles and no mutations in the known genes are, in general, different from those with mutations; they have a milder genetic susceptibility and/or a multifactorial etiology underlying their recurrent hydatidiform moles. Categorizing these patients according to the genotypic types of their recurrent hydatidiform moles may facilitate the identification of novel genes for this entity., Reseau Quebecois en Reproduction; McGill Faculty of Medicine; RI-MUHC Desjardins Studentship in Child Health Research; CRRD; Canadian Institute of Health ResearchCanadian Institutes of Health Research (CIHR) [MOP-86546, POP-122897, MOP-130364], NMPN was supported by fellowships from Reseau Quebecois en Reproduction, McGill Faculty of Medicine, RI-MUHC Desjardins Studentship in Child Health Research, and CRRD. Yassemine Khawajkie was supported by a CRRD trainee fellowship. RS is supported by the Canadian Institute of Health Research (MOP-86546, POP-122897, and MOP-130364).

Purpose: Choriocarcinoma (CC) is the most malignant gestational trophoblastic disease that often develops from complete hydatidiform moles (CHM). Neither the mechanism of CC development nor its progression is yet characterized. We recently identified endocrine gland–derived vascular endothelial growth factor (EG-VEGF) as a novel key placental growth factor that controls trophoblast proliferation and invasion. EG-VEGF acts via two receptors, PROKR1 and PROKR2. Here, we demonstrate that EG-VEGF receptors can be targeted for CC therapy. Experimental Design: Three approaches were used: (i) a clinical investigation comparing circulating EG-VEGF in control (n = 20) and in distinctive CHM (n = 38) and CC (n = 9) cohorts, (ii) an in vitro study investigating EG-VEGF effects on the CC cell line JEG3, and (iii) an in vivo study including the development of a novel CC mouse model, through a direct injection of JEG3-luciferase into the placenta of gravid SCID-mice. Results: Both placental and circulating EG-VEGF levels were increased in CHM and CC (×5) patients. EG-VEGF increased JEG3 proliferation, migration, and invasion in two-dimensional (2D) and three-dimensional (3D) culture systems. JEG3 injection in the placenta caused CC development with large metastases compared with their injection into the uterine horn. Treatment of the animal model with EG-VEGF receptor's antagonists significantly reduced tumor development and progression and preserved pregnancy. Antibody-array and immunohistological analyses further deciphered the mechanism of the antagonist's actions. Conclusions: Our work describes a novel preclinical animal model of CC and presents evidence that EG-VEGF receptors can be targeted for CC therapy. This may provide safe and less toxic therapeutic options compared with the currently used multi-agent chemotherapies. Clin Cancer Res; 23(22); 7130–40. ©2017 AACR.

Background Hydatidiform mole (HM) is a human pregnancy with excessive trophoblastic proliferation and abnormal embryonic development that may be sporadic or recurrent. In the sporadic form, the HM phenotype is driven by an abnormal ratio of paternal to maternal genomes, whereas in the recurrent form, the HM phenotype is caused by maternal-recessive mutations, mostly in NLRP7 , despite the diploid biparental origin of the HM tissues. In this study, we characterised the expression of the imprinted, maternally expressed gene, CDKN1C (p57 KIP2 ), the genotype, and the histopathology of 36 products of conception (POC) from patients with two defective alleles in NLRP7 and looked for potential correlations between the nature of the mutations in the patients and the various HM features. Methods/results We found that all the 36 POCs are diploid biparental and have the same parental contribution to their genomes. However, some of them expressed variable levels of p57 KIP2 and this expression was strongly associated with the presence of embryonic tissues of inner cell mass origin and mild trophoblastic proliferation, which are features of triploid partial HMs, and were associated with missense mutations. Negative p57 KIP2 expression was associated with the absence of embryonic tissues and excessive trophoblastic proliferation, which are features of androgenetic complete HMs and were associated with protein-truncating mutations. Conclusions Our data suggest that NLRP7 , depending on the severity of its mutations, regulates the imprinted expression of p57 KIP2 and consequently the balance between tissue differentiation and proliferation during early human development. This role is novel and could not have been revealed by any other approach on somatic cells.

Trophoblast cell adhesion and migration are carefully coordinated during normal placental development. We have compared the expression of three adhesion molecules, E-cadherin, β-catenin, and Lewis x, by immunohistochemistry during normal trophoblast differentiation, and in hydatidiform moles and choriocarcinomas. Both E-cadherin and β-catenin were expressed in normal placenta cytotrophoblast, and this expression decreased with trophoblast maturation. E-cadherin was mainly localized along the contact between cytotrophoblast and syncytiotrophoblast, which indicates its role in the differentiation of the syncytial layer. Lewis x disappeared progressively during differentiation of normal villous vessels, and was expressed in molar pregnancies. Interestingly, whereas choriocarcinomas were not, or poorly, stained, invasive hydatidiform moles (invHMs) strongly expressed Lewis x in vascular structures. This observation correlated well with E-cadherin and β-catenin expression and suggests that these three markers are associated with the invasive transformation. The presence of robust endothelial structures in invHMs could also explain their ability to maintain organized villous architecture (contrary to metastatic choriocarcinomas) during their invasion of extrauterine tissues such as the lung or the brain after dissemination through the blood flow. In our hands, Lewis x appeared to be a new, reliable marker that can be used to clearly distinguish invHMs from choriocarcinomas.

To date, two maternal-effect genes have been shown to have causative roles in recurrent hydatidiform moles (RHMs); NLRP7 that is mutated in 48-60% of patients with RHMs and C6orf221 (HUGO-approved nomenclature is now KHDC3L), a recently identified gene, that is mutated in 14% of patients with RHMs who are negative for NLRP7 mutations. We sequenced KHDC3L in 97 patients with RHMs and reproductive loss who are mostly negative for NLRP7 mutations. We identified three unrelated patients, each homozygous for one of the two protein-truncating mutations, a novel 4-bp deletion resulting in a frameshift, c.299_302delTCAA, p.Ile100Argfs*2, and a previously described 4-bp deletion, c.322_325delGACT, p.Asp108Ilefs*30, transmitted on a shared haplotype to three patients from different populations. We show that five HM tissues from one of these patients are diploid and biparental similar to HMs from patients with two defective NLRP7 mutations. Using immunofluorescence, we show that KHDC3L protein displays a juxta perinuclear signal and colocalizes with NLRP7 in lymphoblastoid cell lines from normal subjects. Using cell lines from patients, we demonstrate that the KHDC3L mutations do not change the subcellular localization of the protein in hematopoietic cells. Our data highlight the similarities between the two causative genes for RHMs, KHDC3L and NLRP7, in their subcellular localization, the parental contribution to the HM tissues caused by them, and the presence of several founder mutations and variants in both of them indicating positive selection and adaptation.

Objective To describe in vitro development of human embryos derived from an individual with a homozygous pathogenic variant in NLRP7 (19q13.42) and recurrent hydatidiform mole (HM), an autosomal recessive condition thought to occur secondary to an oocyte defect. Methods A patient with five consecutive HM pregnancies was genomically evaluated via next generation sequencing followed by controlled ovarian hyperstimulation, in vitro fertilization (IVF) with intracytoplasmic sperm injection, embryo culture, and preimplantation genetic screening. Findings in NLRP7 were recorded and embryo culture and biopsy data were tabulated as a function of parental origin for any identified ploidy error. Results The patient was found to have a pathogenic variant in NLRP7 (c.2810+2T>G) in a homozygous state. Fifteen oocytes were retrieved and 10 embryos were available after fertilization via intracytoplasmic sperm injection. Developmental arrest was noted for all 10 embryos after 144 hours in culture, thus no transfer was possible. These non-viable embryos were evaluated by karyomapping and all were diploid biparental; two were euploid and eight had various aneuploidies all of maternal origin. Conclusion This is the first report of early human embryo development from a patient with any NLRP7 mutation. The pathogenic variant identified here resulted in global developmental arrest at or before blastocyst stage. Standard IVF should therefore be discouraged for such patients, who instead need to consider oocyte (or embryo) donation with IVF as preferred clinical methods to treat infertility.

Recurrent hydatidiform mole (RHM) is defined by the occurrence of repeated molar pregnancies in affected women. Two genes, NLRP7 and KHDC3L, play a causal role in RHM and are responsible for 48–80% and 5% of cases, respectively. Here, we report the results of screening these two genes for mutations in one Iranian and one Indian patient with RHM. No mutations in NLRP7 were identified in the two patients. KHDC3L sequencing identified two novel protein-truncating mutations in a homozygous state, a 4-bp deletion, c.17_20delGGTT (p.Arg6Leufs*7), in the Iranian patient and a splice mutation, c.349+1G>A, that affects the invariant donor site at the junction of exon 2 and intron 2 in the Indian patient. To date, only four mutations in KHDC3L have been reported. The identification of two additional mutations provides further evidence for the important role of KHDC3L in the pathophysiology of RHM and increases the diversity of mutations described in Asian populations. Two new mutations have been identified in a gene associated with recurrent hydatidiform moles (RHMs). A RHM is a form of non-viable pregnancy in which the placenta develops into a mass of cysts. This condition occurs in higher frequencies in the Middle and Far East. Majid Fardaei of Shiraz University of Medical Science in Iran and colleagues screened Iranian and Indian women for mutations in two genes, NLRP7 and KHDC3L. These genes have been implicated as having a role in 48—80% and 5% of RHMs, respectively. In two patients, no mutations were found in NLRP7, but two new mutations were identified in KHDC3L that were not found in controls or two large gene databases. The findings confirm a causal role for KHDC3L in RHMs, which should be screened for when mutations are not detected in NLRP7.

To identify the genetic defect in a Lebanese family with two sibs diagnosed with Usher Syndrome.Exome capture and sequencing were performed on DNA from one affected member using Agilent in solution bead capture, followed by Illumina sequencing.This analysis revealed the presence of a novel homozygous 5-bp deletion, in Clarin 1 (CLRN1), a known gene responsible for Usher syndrome type III. The deletion is inherited from both parents and segregates with the disease phenotype in the family. The 5-bp deletion, c.301_305delGTCAT, p.Val101SerfsX27, is predicted to result in a frameshift and protein truncation after 27 amino acids. Sequencing all the coding regions of the CLRN1 gene in the proband did not reveal any other mutation or variant.Here we describe a novel deletion in CLRN1. Our data support previously reported intra familial variability in the clinical features of Usher syndrome type I and III.

To understand the mechanisms leading to hydatidiform mole formation in patients with NLRP7 mutations, we used a combination of various approaches to characterize five products of conception, from two patients, shown by flow cytometry to contain non-diploid cells. We demonstrate that four of these conceptions are triploid and two of them originated from fertilization with more than one sperm. We show that three of these triploid conceptions fulfill the histopathological criteria of partial hydatidiform mole and one fulfills the histopathological criteria of spontaneous abortion. Our data demonstrate that some oocytes from one patient with NLRP7 mutations are not able to prevent polyspermic fertilization and highlight the importance of using several approaches to characterize the genetic complexity of molar tissues and reproductive wastage. Altogether, our previous and current data show the association of NLRP7 mutations with several types of hydatidiform moles and with triploid spontaneous abortions.

Hydatidiform mole (HM) is a human pregnancy with abnormal embryonic development. NLRP7 is a major autosomal recessive gene responsible for recurrent molar pregnancies and associated reproductive wastage in patients from several populations. Here, we report NLRP7 mutation analysis in 35 unrelated Chinese patients with recurrent reproductive wastage, including at least one HM. We describe three new protein-truncating mutations in NLRP7 and show the presence of three founder mutations in China and Asian populations. We determined the parental contribution to six molar tissues and show the occurrence of three diploid androgenetic moles in patients with one defective allele, while three diploid biparental moles occurred in patients with two defective alleles. We document the failure of pregnancies after assisted reproductive technologies (ARTs) in three patients with two defective alleles each and a successful pregnancy in one of two patients with one defective allele. Our data suggest that patients with a single defective allele have better reproductive outcomes than patients with two defective alleles, and some of them may benefit from ART.

Hydatidiform mole (HM) is an abnormal human pregnancy characterized by absence of, or abnormal, embryonic development and hydropic degeneration of chorionic villi. The common form of this condition occurs once in every 1000–1500 pregnancies in Western countries (1), but at higher frequencies in India. The incidence of HMs in India is 1 in every 83–500 pregnancies depending on regions and studies (2, 3). HMs are usually sporadic and not recurrent. Among women with sporadic moles, 1–6% will have a second mole (4–11), and 10–20% will have a second non-molar reproductive wastage, mostly as a spontaneous abortion (7, 11, 12). The frequency of familial recurrent HMs (RHMs) is not known, but such cases are believed to be very rare. To date, 23 different mutations in NLRP7 have been reported in women with RHMs and reproductive wastage. Four of these mutations, p.Arg693Pro, p.Glu710Aspfs7X, p.Leu750Val, p.Leu825X, have been found in unrelated families from the same population indicating the presence of founder effects for these mutations (13–16). We previously reported two mutations in Indian families, p.Arg693Pro and p.Asn913Ser, and the former was found in two unrelated families (14). In this study, we searched for mutations in NLRP7 in 10 new unrelated Indian patients with recurrent HMs and reproductive wastage. Eight of the cases originate from various states in Northwest India, Jammu and Kashmir, Punjab, Chandigarh, Haryana, and Uttar Pradesh, and two cases, MoIn105 and MoIn106, originate from West Pakistan and migrated to India about 50 years ago. Mutation analysis was performed as previously described by genomic DNA sequencing of the 11 NLRP7 exons in the two directions and revealed NLRP7 mutations in seven patients. All the seven patients had two previously identified mutations in Indian patients, c.2078G>C, p.Arg693Pro and c.2738A>G, p.Asn913Ser. Three patients were compound heterozygous for the two mutations; three were homozygous for p.Arg693Pro; and one was homozygous for p.Asn913Ser. The analysis of available parents and relatives demonstrated the segregation of the mutations in their respective families (Figure 1). In family MoIn93, a woman with a history of infertility of unexplained clinical origin was found compound heterozygous for the two mutations. To date, we have analyzed 12 unrelated Indian patients, selected based only on their medical history and not on linkage to the locus spanning NLRP7, and found NLRP7 mutations in nine of them (75%). This result was unexpected since only half of patients with recurrent HM and no family history of moles referred to our laboratory (from various populations) have mutations/variants in NLRP7. The DNA change leading to p.Arg693Pro was found on 12 chromosomes (66.6%) and the allele leading to p.Asn913Ser on six chromosomes (33.4%). Haplotypes were established based on alleles found in five homozygous patients and on the analysis of three heterozygous patients and their available parents and siblings at 30 single nucleotide polymorphisms in NLRP7. Haplotype analysis shows a common haplotype carrying each of the mutations and demonstrates the presence of a strong founder effect for these two mutations in the Indian population (Table 1). This is further supported by the fact that four of the analyzed patients are homozygous despite the absence of known consanguinity between their parents. The two mutations modify recognition sites for restriction enzymes and can be easily tested in Indian patients with recurrent HMs and reproductive wastage. A number of synonymous and non-synonymous variants were also found in the patients. c.574A>C, p.Met192Leu was found in a heterozygous state in patient 678 and in 2 out 38 controls of Indo-Pakistani origin. All the other variants are known polymorphisms frequently found in several populations. Figure 1 Pedigree structures and reproductive outcomes of seven Indian families with recurrent reproductive wastage and NLRP7 mutations. The reproductive outcomes are listed by chronological order from left to right. Gestational age is from the last menstrual ... Table 1 Haplotype analysis at all NLRP7 variants found in 8 unrelated Indian patients In family MoIn106, one male with one child and no reproductive problems was found compound heterozygous for the two mutations. This is the third reported male with two NLRP7 mutations and no reproductive problems (13). Our data demonstrate the presence of two founder mutations in the Indian population, expand further the involvement of NLRP7 mutations to a wider spectrum of reproductive wastage, and provide further indications that NLRP7 mutations have no consequences on male reproduction. The presence in several populations of founder effects for a condition that prevents the reproduction of homozygous females is by itself intriguing and may reveal in the future male and/or heterozygous females advantage.

We have earlier described a syndrome characterized by microcephaly, cutis verticis gyrata, retinitis pigmentosa, cataracts, hearing loss and mental retardation (Mendelian inheritance in man (MIM) no: 605685) in two brothers from a non-consanguineous Lebanese family. In view of the rarity of the disorder and the high rate of inbreeding in the Lebanese population, we assumed an autosomal recessive trait inherited from a common ancestor. A genomewide scan was performed. The single locus on the long arm of chromosome 8 that showed homozygosity by descent comprised the gene responsible for Cohen syndrome (CS), VPS13B. We then sequenced VPS13B in the patients and found a homozygous splice site mutation. Several possible explanations for the overlap between CS and the clinical features observed in our patients are discussed. Our data highlight the potential of high-resolution homozygosity mapping in small populations with a high rate of inbreeding.

Usher syndrome (US) is clinically and genetically a heterogeneous group of disorders characterized by the association of deafness with retinitis pigmentosa. So far, eight genes responsible for US have been mapped, of which only the gene responsible for the most common form, USH1B, has been identified. The USH1B is a large gene containing 49 exons and encoding for an unconventional myosin-VIIA (MYO7A). Mutation analysis within the MYO7A gene showed a wide variety of mutations dispersed all over the gene. The present report refines the location of the MYO7A gene relative to microsatellite markers mapped to this region, thereby allowing a reliable and efficient carrier detection by linkage analysis.

Infevers (Internet Fevers; http://fmf.igh.cnrs.fr/ISSAID/infevers), a website dedicated to mutations responsible for hereditary autoinflammatory diseases, was created in 2002 and has continued to evolve. This new version includes eight genes; six were already present: MEFV, MVK, TNFRSF1A, NLRP3, NOD2, PSTPIP1, and two are new, LPIN2 and NLRP7. Currently, Infevers contains over 540 sequence variants. Several new database functions were recently instituted. The website now accepts confidential data and complex alleles. For each gene, a newly created menu offers: 1) a tabular list of the variants that can be sorted by several parameters; 2) a gene graph providing a schematic representation of the variants along the gene; 3) statistical analysis of the data according to the phenotype, alteration type, and location of the mutation in the gene; 4) the cDNA and gDNA sequences of each gene, showing the nucleotide changes along the sequence, with a color-based code highlighting the gene domains, the first ATG, and the termination codon; and 5) a "download" menu making all tables and figures available for the users, which, except for the gene graphs, are all automatically generated and updated upon submission of the variants. Finally, the entire database was curated to comply with the HUGO Gene Nomenclature Committee (HGNC) and HGVS nomenclature guidelines, and wherever necessary, an informative note was provided. Infevers has already proven useful for the scientific community with a mean number of visits per month of 200 in 2002 and 800 in 2007, and its new design will lead to a more comprehensive comparative analysis and interpretation of auto-inflammatory sequence variants.

Hydatidiform mole (HM) is a human pregnancy with no embryo but cystic degeneration of chorionic villi. The common form of this condition occurs in 1 in every 1500 pregnancies in western societies and at a higher incidence in some geographic regions and populations. Recurrent moles account for 2% of all molar cases and a few of them occur in more than one family member. By studying a familial form of recurrent moles, a recessive maternal locus responsible for this condition was mapped to 19q13.4 and causative mutations identified. The defective protein, NALP7, is part of the CATERPILLAR protein family with roles in pathogen-induced inflammation and apoptosis. The exact role of NALP7 in the pathophysiology of molar pregnancies is unknown yet. NALP7 could have a role either in oogenesis or in the endometrium during trophoblast invasion and decidualization. In this review, we outlined recent advances in the field of HMs and reviewed the literature in the light of the new data.

An imprinting disorder has been believed to underlie the etiology of familial biparental hydatidiform moles (HMs) based on the abnormal methylation or expression of imprinted genes in molar tissues. However, the extent of the epigenetic defect in these tissues and the developmental stage at which the disorder begins have been poorly defined. In this study, we assessed the extent of abnormal DNA methylation in two HMs caused by mutations in the recently identified 19q13.4 gene, NALP7. We demonstrate normal postzygotic DNA methylation patterns at major repetitive and long interspersed nuclear elements (LINEs), genes on the inactive X-chromosome, three-cancer related genes, and CpG rich regions surrounding the PEG3 differentially methylated region (DMR). Our data provide a comprehensive assessment of DNA methylation in familial molar tissues and indicate that abnormal DNA methylation in these tissues is restricted to imprinted DMRs. The known role of NALP7 in apoptosis and inflammation pinpoints previously unrecognized pathways that could directly or indirectly underlie the abnormal methylation of imprinted genes in molar tissues.

Hydatidiform mole (HM) is an aberrant human pregnancy with abnormal embryonic development and excessive prolifera- tion of the trophoblast. Recessive mutations in NLRP7 are responsible for recurrent HM (RHM). Women with recessive NLRP7 muta- tions fail to have normal pregnancies from spontaneous conceptions with the exception of three out of 131 reported patients. Because there is no treatment for RHM and maternal-effect genes are needed in the oocytes to sustain normal embryonic development until the activation of the embryonic genome, one patient with recessive NLRP7 mutations tried ovum donation and achieved a success- ful pregnancy. This study reports three additional live births from donated ova to two patients with recessive NLRP7 mutations. The occurrence of two live births from spontaneous conceptions to two other patients is also reported. The reproductive outcomes and mutations of all reported patients were reviewed and it was found that live births are associated with some missense mutations ex- pected to have mild functional consequences on the protein. The data support a previous observation that ovum donation appears the best management option for these patients to achieve normal pregnancies and provide an explanation for the rare occurrence of live births from natural spontaneous conceptions in patients with two NLRP7 mutations. © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

We previously mapped a maternal recessive locus responsible for familial hydatidiform moles (HMs) to 19q13.4. The candidate region has recently been narrowed down to 1.1 megabases. Here, we report the segregation of alleles at 18 genetic markers, including nine new ones, from the HMs candidate region in a recently reported consanguineous family. In this family, five affected women had a total of seven HMs, three miscarriages, and three normal children. Linkage and haplotype analyses exclude linkage to 19q13.4 and indicate the presence of a second recessive locus responsible for familial molar pregnancies. The heterogeneity in the phenotype of the conceptuses of patients with familial HMs is in agreement with previous observations and seems to be a common feature of this condition. This indicates that the homozygous genetic defects leading to hydatidiform moles can be modulated by other genetic or environmental factors. The identification of these factors may unravel natural ways to treat these forms of reproductive wastage and reverse the infertility of women with recurrent moles.

To date, two maternal-effect genes have been shown to play causal roles in recurrent hydatidiform moles (RHMs). NLRP7, a major gene for this condition, codes for a nucleotide-binding oligomerization domain-like receptor and is mutated in 48 to 60% of patients with RHMs. KHDC3L is a recently identified gene that is mutated in 14% of NLRP7-negative patients. We screened KHDC3L for mutations in a total of 101 Chinese patients, 15 with at least two hydatidiform moles, 16 with at least two reproductive losses including one hydatidiform mole, and 70 with one hydatidiform mole and no other form of reproductive loss, but did not find any mutation. Our data favor the causal role of KHDC3L in a minority of RHM cases, demonstrate its noninvolvement in other forms of reproductive loss, and indicate the presence of other unidentified genes that cause or increase patients' susceptibility to RHMs in the Chinese population.

Gestational trophoblastic diseases (GTD) are a group of disorders that originate from the trophoblast, which is the outer layer of the blastocyst that normally gives rise to the placenta and is the first tissue to differentiate from the developing embryos after implantation. The human chorionic gonadotropin (hCG) is one of the pregnancy hormones and is secreted by trophoblastic cells. hCG is detected in maternal blood seven days after fertilization (Cole, 2009) and is an excellent marker to detect chemical, normal and abnormal pregnancies. GTD include pre-malignant stages, partial and complete hydatidiform moles, and gestational trophoblastic neoplasia (GTN), which encompass invasive mole, choriocarcinoma, placental site trophoblastic tumor and epithelioid trophoblastic tumor, and are all characterized by excessive proliferation of the trophoblast and therefore excessive production of hCG. Among GTD, hydatidiform mole is the most common. After initial diagnosis by ultrasound and serum hCG quantitation, hydatidiform moles are removed by dilatation and curettage suction and the patients are followed-up with serial hCG measurements until negativity or non-pregnant levels. Abnormal decrease of hCG after curettage indicates risk of persistent trophoblastic disease and malignant transformation into GTN (Fig. 1) that are classified and treated according to the International Federation of Gynecology and Obstetrics (FIGO) guidelines (FIGO Oncology Committee, 2002, Seckl et al., 2010). The implementation of these guidelines has contributed to a better management of GTD and changed their outcomes from nearly 100% fatality 60 years ago (Goldstein, 2012) to nearly 100% cure rates in current clinical practice (Bolze et al., 2015). However, some patients still present with advanced or chemoresistant GTN. In addition, hCG is a common tumor marker that is expressed by germ cell tumors of the testis and the ovary and by 20–40% of common epithelial carcinoma such as bladder, cervix, lung, and naso-pharynx (Iles, 2007). Many of these tumors are not 100% curable and those that express hCG have the lowest survival rate. Fig. 1 Clinical management timeline of a patient with post-molar gestational trophoblastic neoplasia. A standard presentation of a patient with a clinical suspicion of hydatidiform mole based high serum hCG and suggestive pelvic ultrasound. The patient underwent ... In this issue of EBioMedicine, Openshaw et al. (2016) looked for circulating free DNA (cfDNA) in the plasma of 18 women with post-molar GTN, two with metastatic gestational choriocarcinoma, and five with hCG-secreting tumors of unknown origin. They isolated cfDNA and used it in a multiplex assay that amplifies 15 short tandem repeat (STR) loci and the Amelogenin gender-determining marker to identify whether the cfDNA contains circulating tumor DNA (ctDNA). By comparing the genotypes of cellular DNA of the patients and their partners with those amplified from available preceding molar pregnancies and cfDNA, they show the presence of paternal alleles in cfDNA in 13 patients, 12 with GTN and one with a tumor of unknown origin. The amount of non-maternal ctDNA ranged from 9% to 55% of total cfDNA. Multiplex STR genotyping on the tumor DNA is commonly used to determine the parental contribution to molar pregnancies. It is also used on choriocarcinoma tumor DNA to determine whether these tumors have germ cell (e.g. ovarian origin) or gestational origin. The identification of paternal alleles is therefore a reliable and robust criterion to confirm the gestational origin of these tumors. This is particularly important for the patients because the management and prognosis of gestational and non-gestational hCG-secreting tumors are totally different (Alifrangis et al., 2013). The study by Openshaw and his colleagues demonstrated the presence of circulating tumor DNA (ctDNA) in many patients with GTN (60%) and will most likely open new testing opportunities aimed at determining the origin of hCG-secreting tumors by genotyping cfDNA. In post-molar GTN, the gestational origin of the tumor is usually less questionable and such test may not be needed. However, genotyping cfDNA is important in patients when tumor sampling exposes the patients to the risk of bleeding and tumor dissemination. Similarly, when the preceding pregnancy is not a hydatidiform mole or/and occurred several years ago, genotyping cfDNA offers a unique source for determining the origin of the tumor and therefore adjust the FIGO score to choose the best chemotherapy regimen. An illustration of the importance of genotyping cfDNA is the case of patient CFD-023 whose latest pregnancy was terminated 11 years ago. In the study by Openshaw et al. (2016), a significant correlation between the amount of ctDNA and serum hCG was found in patients with GTN and detectable ctDNA by genotyping. However, the exact relationship between circulating tumor DNA and serum hCG remains to be elucidated in future studies. Such studies may need to consider some confounding factors such as the gestational stage of the pregnancy, the time interval between the curettage and the diagnosis of GTN and collection of blood to search for ctDNA; if accounted for, may help reaching a better correlation. It would be also interesting to look for correlation between the amounts of ctDNA and the risk of developing post-molar GTN on blood samples drawn at the time of the curettage of the moles and during the follow-up and treatments (Fig. 1). Furthermore, to better understand the diagnostic value of ctDNA, it is important to know the cellular origin of the DNA that is shed in the maternal circulation. In pre-eclampsia and intra-uterine growth restriction, the amounts of circulating fetal DNA correlate with placental apoptosis and necrosis rather than with placental volume (Taglauer et al., 2014). In GTDs, apoptotic cells are found at higher amounts in hydatidiform moles that regress spontaneously than in moles that give rise to GTNs (Chiu et al., 2001). Therefore, even if all confounding factors are taken into consideration, it is possible that the amounts of ctDNA may not correlate positively with the amounts of serum hCG in all patients. In conclusion, the present study opens interesting new perspectives to better understand the pathophysiology of GTN and provides hope for a reliable diagnostic approach from a simple blood sample to improve the management of patients with hCG-secreting tumors of unknown origin.