Undiagnosed RASopathies in infertile men.

RASopathies are syndromes caused by congenital defects in the Ras/mitogen-activated protein kinase (MAPK) pathway genes, with a population prevalence of 1 in 1,000. Patients are typically identified in childhood based on diverse characteristic features, including cryptorchidism (CR) in >50% of affected men. As CR predisposes to spermatogenic failure (SPGF; total sperm count per ejaculate 0-39 million), we hypothesized that men seeking infertility management include cases with undiagnosed RASopathies. Likely pathogenic or pathogenic (LP/P) variants in 22 RASopathy-linked genes were screened in 521 idiopathic SPGF patients (including 155 CR cases) and 323 normozoospermic controls using exome sequencing. All 844 men were recruited to the ESTonian ANDrology (ESTAND) cohort and underwent identical andrological phenotyping. RASopathy-specific variant interpretation guidelines were used for pathogenicity assessment. LP/P variants were identified in PTPN11 (two), SOS1 (three), SOS2 (one), LZTR1 (one), SPRED1 (one), NF1 (one), and MAP2K1 (one). The findings affected six of 155 cases with CR and SPGF, three of 366 men with SPGF only, and one (of 323) normozoospermic subfertile man. The subgroup "CR and SPGF" had over 13-fold enrichment of findings compared to controls (3.9% vs. 0.3%; Fisher's exact test, p = 5.5 × 10-3). All ESTAND subjects with LP/P variants in the Ras/MAPK pathway genes presented congenital genitourinary anomalies, skeletal and joint conditions, and other RASopathy-linked health concerns. Rare forms of malignancies (schwannomatosis and pancreatic and testicular cancer) were reported on four occasions. The Genetics of Male Infertility Initiative (GEMINI) cohort (1,416 SPGF cases and 317 fertile men) was used to validate the outcome. LP/P variants in PTPN11 (three), LZTR1 (three), and MRAS (one) were identified in six SPGF cases (including 4/31 GEMINI cases with CR) and one normozoospermic man. Undiagnosed RASopathies were detected in total for 17 ESTAND and GEMINI subjects, 15 SPGF patients (10 with CR), and two fertile men. Affected RASopathy genes showed high expression in spermatogenic and testicular somatic cells. In conclusion, congenital defects in the Ras/MAPK pathway genes represent a new congenital etiology of syndromic male infertility. Undiagnosed RASopathies were especially enriched among patients with a history of cryptorchidism. Given the relationship between RASopathies and other conditions, infertile men found to have this molecular diagnosis should be evaluated for known RASopathy-linked health concerns, including specific rare malignancies.

Toward clinical exomes in diagnostics and management of male infertility.

Infertility, affecting ∼10% of men, is predominantly caused by primary spermatogenic failure (SPGF). We screened likely pathogenic and pathogenic (LP/P) variants in 638 candidate genes for male infertility in 521 individuals presenting idiopathic SPGF and 323 normozoospermic men in the ESTAND cohort. Molecular diagnosis was reached for 64 men with SPGF (12%), with findings in 39 genes (6%). The yield did not differ significantly between the subgroups with azoospermia (20/185, 11%), oligozoospermia (18/181, 10%), and primary cryptorchidism with SPGF (26/155, 17%). Notably, 19 of 64 LP/P variants (30%) identified in 28 subjects represented recurrent findings in this study and/or with other male infertility cohorts. NR5A1 was the most frequently affected gene, with seven LP/P variants in six SPGF-affected men and two normozoospermic men. The link to SPGF was validated for recently proposed candidate genes ACTRT1, ASZ1, GLUD2, GREB1L, LEO1, RBM5, ROS1, and TGIF2LY. Heterozygous truncating variants in BNC1, reported in female infertility, emerged as plausible causes of severe oligozoospermia. Data suggested that several infertile men may present congenital conditions with less pronounced or pleiotropic phenotypes affecting the development and function of the reproductive system. Genes regulating the hypothalamic-pituitary-gonadal axis were affected in >30% of subjects with LP/P variants. Six individuals had more than one LP/P variant, including five with two findings from the gene panel. A 4-fold increased prevalence of cancer was observed in men with genetic infertility compared to the general male population (8% vs. 2%; p = 4.4 × 10-3). Expanding genetic testing in andrology will contribute to the multidisciplinary management of SPGF.

Genome sequencing of Pakistani families with male infertility identifies deleterious genotypes in SPAG6, CCDC9, TKTL1, TUBA3C, and M1AP.

BACKGROUND: There are likely to be hundreds of monogenic forms of human male infertility. Whole genome sequencing (WGS) is the most efficient way to make progress in mapping the causative genetic variants, and ultimately improve clinical management of the disease in each patient. Recruitment of consanguineous families is an effective approach to ascertain the genetic forms of many diseases. OBJECTIVES: To apply WGS to large consanguineous families with likely hereditary male infertility and identify potential genetic cases. MATERIALS AND METHODS: We recruited seven large families with clinically diagnosed male infertility from rural Pakistan, including five with a history of consanguinity. We generated WGS data on 26 individuals (3-5 per family) and analyzed the resulting data with a computational pipeline to identify potentially causal single nucleotide variants, indels, and copy number variants. RESULTS: We identified plausible genetic causes in five of the seven families, including a homozygous 10 kb deletion of exon 2 in a well-established male infertility gene (M1AP), and biallelic missense substitutions (SPAG6, CCDC9, TUBA3C) and an in-frame hemizygous deletion (TKTL1) in genes with emerging relevance. DISCUSSION AND CONCLUSION: The rate of genetic findings using the current approach (71%) was much higher than what we recently achieved using whole-exome sequencing (WES) of unrelated singleton cases (20%). Furthermore, we identified a pathogenic single-exon deletion in M1AP that would be undetectable by WES. Screening more families with WGS, especially in underrepresented populations, will further reveal the types of variants underlying male infertility and accelerate the use of genetics in the patient management.

Urinary microRNA profiling in pregnant women identifies placental microRNAs that are candidate biomarkers for monitoring placental health.

MicroRNAs are among the key modulators of placental transcriptome dynamics. This study aimed at comparative profiling of urinary (sampling at 228-230 gestational days, g.days), serum (217-230 g.days), and placental (279-286 g.days) microRNAs in three healthy pregnant women using miRNome sequencing. Placenta showed significant enrichment of microRNAs compared with serum and urine (1174, 341, and 193, respectively; P < 10-5 ). All sample types shared 153 microRNAs, representing candidate biomarkers for placental health. Urine samples contained eight of 56 transcripts from the placenta-specific chromosome 19 microRNA cluster C19MC and one of 91 transcripts (miR-432-5p) from the chromosome 14 cluster C14MC. These data suggest an active filtering at the maternal-fetal interface in passing through only selected microRNAs. Urine represents a valid source to monitor the signature of placenta-expressed microRNAs that are differentially expressed in pregnancy complications.

DDX3Y is likely the key spermatogenic factor in the AZFa region that contributes to human non-obstructive azoospermia.

Non-obstructive azoospermia, the absence of sperm in the ejaculate due to disturbed spermatogenesis, represents the most severe form of male infertility. De novo microdeletions of the Y-chromosomal AZFa region are one of few well-established genetic causes for NOA and are routinely analysed in the diagnostic workup of affected men. So far, it is unclear which of the three genes located in the AZFa chromosomal region is indispensible for germ cell maturation. Here we present four different likely pathogenic loss-of-function variants in the AZFa gene DDX3Y identified by analysing exome sequencing data of more than 1,600 infertile men. Three of the patients underwent testicular sperm extraction and revealed the typical AZFa testicular Sertoli cell-only phenotype. One of the variants was proven to be de novo. Consequently, DDX3Y represents the AZFa key spermatogenic factor and screening for variants in DDX3Y should be included in the diagnostic workflow.

Microdeletions and microduplications linked to severe congenital disorders in infertile men.

Data on the clinical validity of DNA copy number variants (CNVs) in spermatogenic failure (SPGF) is limited. This study analyzed the genome-wide CNV profile in 215 men with idiopathic SPGF and 62 normozoospermic fertile men, recruited at the Andrology Clinic, Tartu University Hospital, Estonia. A two-fold higher representation of > 1 Mb CNVs was observed in men with SPGF (13%, n = 28) compared to controls (6.5%, n = 4). Seven patients with SPGF were identified as carriers of microdeletions (1q21.1; 2.4 Mb) or microduplications (3p26.3, 1.1 Mb; 7p22.3-p22.2, 1.56 Mb; 10q11.22, 1.42 Mb, three cases; Xp22.33; 2.3 Mb) linked to severe congenital conditions. Large autosomal CNV carriers had oligozoospermia, reduced or low-normal bitesticular volume (22-28 ml). The 7p22.3-p22.2 microduplication carrier presented mild intellectual disability, neuropsychiatric problems, and short stature. The Xp22.33 duplication at the PAR1/non-PAR boundary, previously linked to uterine agenesis, was detected in a patient with non-obstructive azoospermia. A novel recurrent intragenic deletion in testis-specific LRRC69 was significantly overrepresented in patients with SPGF compared to the general population (3.3% vs. 0.85%; χ2 test, OR = 3.9 [95% CI 1.8-8.4], P = 0.0001). Assessment of clinically valid CNVs in patients with SPGF will improve their management and counselling for general and reproductive health, including risk of miscarriage and congenital disorders in future offspring.

Diverse monogenic subforms of human spermatogenic failure.

Non-obstructive azoospermia (NOA) is the most severe form of male infertility and typically incurable. Defining the genetic basis of NOA has proven challenging, and the most advanced classification of NOA subforms is not based on genetics, but simple description of testis histology. In this study, we exome-sequenced over 1000 clinically diagnosed NOA cases and identified a plausible recessive Mendelian cause in 20%. We find further support for 21 genes in a 2-stage burden test with 2072 cases and 11,587 fertile controls. The disrupted genes are primarily on the autosomes, enriched for undescribed human "knockouts", and, for the most part, have yet to be linked to a Mendelian trait. Integration with single-cell RNA sequencing data shows that azoospermia genes can be grouped into molecular subforms with synchronized expression patterns, and analogs of these subforms exist in mice. This analysis framework identifies groups of genes with known roles in spermatogenesis but also reveals unrecognized subforms, such as a set of genes expressed across mitotic divisions of differentiating spermatogonia. Our findings highlight NOA as an understudied Mendelian disorder and provide a conceptual structure for organizing the complex genetics of male infertility, which may provide a rational basis for disease classification.

Screening of Gestational Diabetes and Its Risk Factors: Pregnancy Outcome of Women with Gestational Diabetes Risk Factors According to Glycose Tolerance Test Results.

Background: Gestational diabetes mellitus (GDM) can cause maternal and neonatal health problems, and its prevalence is increasing worldwide. We assessed the screening of GDM during a 7-year period and compared the outcome of pregnancies at high risk for GDM. Methods: We analyzed non-selected pregnant women (n = 5021) receiving antenatal care in Tartu University Hospital, Estonia in 2012-2018. Pregnant women were classified based on the absence or presence of GDM risk factors as low risk (n = 2302) or high risk for GDM (n = 2719), respectively. The latter were divided into subgroups after the oral glycose tolerance test (OGTT): GDM (n = 423), normal result (n = 1357) and not tested (n = 939). Results: The proportion of women with GDM risk factors increased from 43.5% in 2012 to 57.8% in 2018, and the diagnosis of GDM more than doubled (5.2% vs. 13.7%). Pregnancies predisposed to GDM but with normal OGTT results were accompanied by an excessive gestational weight gain and increased odds to deliver a LGA baby (AOR 2.3 (CI 1.8-3.0)). Conclusions: An increasing number of pregnancies presenting GDM risk factors are diagnosed with GDM. Pregnant women with GDM risk factors are, despite normal OGTT, at risk of increased weight gain and LGA newborns.

Novel Early Pregnancy Multimarker Screening Test for Preeclampsia Risk Prediction.

Preeclampsia (PE) is a common pregnancy-linked disease, causing preterm births, complicated deliveries, and health consequences for mothers and offspring. We have previously developed 6PLEX, a multiplex assay that measures PE-related maternal serum biomarkers ADAM12, sENG, leptin, PlGF, sFlt-1, and PTX3 in a single test tube. This study investigated the potential of 6PLEX to develop novel PE prediction models for early pregnancy. We analyzed 132 serum samples drawn at 70-275 gestational days (g days) from 53 pregnant women (PE, n = 22; controls, n = 31). PE prediction models were developed using a machine learning strategy based on the stepwise selection of the most significant models and incorporating parameters with optimal resampling. Alternative models included also placental FLT1 rs4769613 T/C genotypes, a high-confidence risk factor for PE. The best performing PE prediction model using samples collected at 70-98 g days comprised of PTX3, sFlt-1, and ADAM12, the subject's parity and gestational age at sampling (AUC 0.94 [95%CI 0.84-0.99]). All cases, that developed PE several months later (onset 257.4 ± 15.2 g days), were correctly identified. The model's specificity was 80% [95%CI 65-100] and the overall accuracy was 88% [95%CI 73-95]. Incorporating additionally the placental FLT1 rs4769613 T/C genotype data increased the prediction accuracy to 93.5% [AUC = 0.97 (95%CI 0.89-1.00)]. However, 6PLEX measurements of samples collected at 100-182 g days were insufficiently informative to develop reliable PE prediction models for mid-pregnancy (accuracy <75%). In summary, the developed model opens new horizons for first-trimester PE screening, combining the easily standardizable 6PLEX assay with routinely collected antenatal care data and resulting in high sensitivity and specificity.

Maternal Pyelonephritis as a Potential Cause of Perinatal Periventricular Venous Infarction in Term-Born Children.

Introduction: The study was designed to assess the prevalence of pregnancy and delivery associated risk factors in children suffering from neonatal or presumed periventricular venous infarction. Methods: Antenatal records and pregnancy outcome data were retrospectively assessed in children with presumed periventricular venous infarction (n = 43, born ≥36 gestational weeks) or neonatal periventricular venous infarction (n = 86, born <36 gestational weeks) and compared to a matched control group (n = 2168, ≥36 gestational weeks) from a prospective study. Results: Children with presumed periventricular venous infarction had significantly more maternal bacterial infections compared to the control group (47% vs 20%, respectively, P < .001), whereas no difference was found compared to the neonatal periventricular venous infarction group (49%, P = .80). Mothers with bacterial infection in the presumed periventricular venous infarction group had significantly more often pyelonephritis compared to the control group (50% vs 3.4%, respectively, P < .001). Conclusions: Our data show an increased risk for developing periventricular venous infarction in the case of maternal bacterial infections, especially between gestational weeks 21 and 31.

DDB1- and CUL4-associated factor 12-like protein 1 (Dcaf12l1) is not essential for male fertility in mice.

Male infertility is a common condition affecting at least 7% of men worldwide and is often genetic in origin. Using whole exome sequencing, we recently discovered three hemizygous, likely damaging variants in DDB1- and CUL4-associated factor 12-like protein 1 (DCAF12L1) in men with azoospermia. DCAF12L1 is located on the X-chromosome and as identified by single cell sequencing studies, its expression is enriched in human testes and specifically in Sertoli cells and spermatogonia. However, very little is known about the role of DCAF12L1 in spermatogenesis, thus we generated a knockout mouse model to further explore the role of DCAF12L1 in male fertility. Knockout mice were generated using CRISPR/Cas9 technology to remove the entire coding region of Dcaf12l1 and were assessed for fertility over a broad range of ages (2-8 months of age). Despite outstanding genetic evidence in men, loss of DCAF12L1 had no discernible impact on male fertility in mice, as highlighted by breeding trials, histological assessment of the testis and epididymis, daily sperm production and evaluation of sperm motility using computer assisted methods. This disparity is likely due to the parallel evolution, and subsequent divergence, of DCAF12 family members in mice and men or the presence of compounding environmental factors in men.

Actionable secondary findings following exome sequencing of 836 non-obstructive azoospermia cases and their value in patient management.

STUDY QUESTION: What is the load, distribution and added clinical value of secondary findings (SFs) identified in exome sequencing (ES) of patients with non-obstructive azoospermia (NOA)? SUMMARY ANSWER: One in 28 NOA cases carried an identifiable, medically actionable SF. WHAT IS KNOWN ALREADY: In addition to molecular diagnostics, ES allows assessment of clinically actionable disease-related gene variants that are not connected to the patient's primary diagnosis, but the knowledge of which may allow the prevention, delay or amelioration of late-onset monogenic conditions. Data on SFs in specific clinical patient groups, including reproductive failure, are currently limited. STUDY DESIGN, SIZE, DURATION: The study group was a retrospective cohort of patients with NOA recruited in 10 clinics across six countries and formed in the framework of the international GEMINI (The GEnetics of Male INfertility Initiative) study. PARTICIPANTS/MATERIALS, SETTING, METHODS: ES data of 836 patients with NOA were exploited to analyze SFs in 85 genes recommended by the American College of Medical Genetics and Genomics (ACMG), Geisinger's MyCode, and Clinical Genome Resource. The identified 6374 exonic variants were annotated with ANNOVAR and filtered for allele frequency, retaining 1381 rare or novel missense and loss-of-function variants. After automatic assessment of pathogenicity with ClinVar and InterVar, 87 variants were manually curated. The final list of confident disease-causing SFs was communicated to the corresponding GEMINI centers. When patient consent had been given, available family health history and non-andrological medical data were retrospectively assessed. MAIN RESULTS AND THE ROLE OF CHANCE: We found a 3.6% total frequency of SFs, 3.3% from the 59 ACMG SF v2.0 genes. One in 70 patients carried SFs in genes linked to familial cancer syndromes, whereas 1 in 60 cases was predisposed to congenital heart disease or other cardiovascular conditions. Retrospective assessment confirmed clinico-molecular diagnoses in several cases. Notably, 37% (11/30) of patients with SFs carried variants in genes linked to male infertility in mice, suggesting that some SFs may have a co-contributing role in spermatogenic impairment. Further studies are needed to determine whether these observations represent chance findings or the profile of SFs in NOA patients is indeed different from the general population. LIMITATIONS, REASONS FOR CAUTION: One limitation of our cohort was the low proportion of non-Caucasian ethnicities (9%). Additionally, as comprehensive clinical data were not available retrospectively for all men with SFs, we were not able to confirm a clinico-molecular diagnosis and assess the penetrance of the specific variants. WIDER IMPLICATIONS OF THE FINDINGS: For the first time, this study analyzed medically actionable SFs in men with spermatogenic failure. With the evolving process to incorporate ES into routine andrology practice for molecular diagnostic purposes, additional assessment of SFs can inform about future significant health concerns for infertility patients. Timely detection of SFs and respective genetic counseling will broaden options for disease prevention and early treatment, as well as inform choices and opportunities regarding family planning. A notable fraction of SFs was detected in genes implicated in maintaining genome integrity, essential in both mitosis and meiosis. Thus, potential genetic pleiotropy may exist between certain adult-onset monogenic diseases and NOA. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Estonian Research Council grants IUT34-12 and PRG1021 (M.L. and M.P.); National Institutes of Health of the United States of America grant R01HD078641 (D.F.C., K.I.A. and P.N.S.); National Institutes of Health of the United States of America grant P50HD096723 (D.F.C. and P.N.S.); National Health and Medical Research Council of Australia grant APP1120356 (M.K.O'B., D.F.C. and K.I.A.); Fundação para a Ciência e a Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Inovação grant POCI-01-0145-FEDER-007274 (A.M.L., F.C. and J.G.) and FCT: IF/01262/2014 (A.M.L.). J.G. was partially funded by FCT/Ministério da Ciência, Tecnologia e Ensino Superior (MCTES), through the Centre for Toxicogenomics and Human Health-ToxOmics (grants UID/BIM/00009/2016 and UIDB/00009/2020). M.L.E. is a consultant for, and holds stock in, Roman, Sandstone, Dadi, Hannah, Underdog and has received funding from NIH/NICHD. Co-authors L.K., K.L., L.N., K.I.A., P.N.S., J.G., F.C., D.M.-M., K.A., K.A.J., M.K.O'B., A.M.L., D.F.C., M.P. and M.L. declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Value of soluble fms-like tyrosine kinase-1/placental growth factor test in third trimester of pregnancy for predicting preeclampsia in asymptomatic women.

OBJECTIVES: To estimate the value of screening maternal serum soluble fms-like tyrosine kinase/placental growth factor (sFlt-1/PlGF) ratio in asymptomatic women during 3rd trimester to predict preeclampsia (PE) development. METHODS: The investigated group comprised of 178 pregnant women. During this gestation, 24 cases had developed PE and 12 isolated gestational hypertension (GH); whereas 142 remained normotensive. Blood samples were collected between 180 and 259 gestational days (g.d.) when the participants were asymptomatic. Serums were analyzed using the BRAHMS sFlt-1 Kryptor/BRAHMS PlGF plus Kryptor PE ratio test (Thermo Fisher Scientific, Henningdorf, Germany). High-risk pregnancies for the PE development were defined as sFlt-1/PlGF>38. RESULTS: The detection rate (DR) for manifestation of PE≤30 days after sampling was 83.3% and overall DR during pregnancy 58.3%. Ten of 15 women having false positive prediction of PE suffered from GH, preterm birth and/or delivery of a small-for-gestational-age-newborn. False positive rate was significantly higher at 239-253 g.d. compared to sampling at 210-224 g.d. and 225-238 g.d. (21.9% vs. 7.8% and 5.3%; p < 0.05). CONCLUSIONS: The sFlt-1/PlGF test during 180-259 g.d. detected approximately half of subsequent PE cases. An optimal time to use the test for screening purposes was estimated 225-238 g.d. (DR 66.7%). False positive test results were more common to cases with other adverse pregnancy outcomes and samples drawn at higher gestational age.

Translational aspects of novel findings in genetics of male infertility-status quo 2021.

INTRODUCTION: Male factor infertility concerns 7-10% of men and among these 40-60% remain unexplained. SOURCES OF DATA: This review is based on recent published literature regarding the genetic causes of male infertility. AREAS OF AGREEMENT: Screening for karyotype abnormalities, biallelic pathogenic variants in the CFTR gene and Y-chromosomal microdeletions have been routine in andrology practice for >20 years, explaining ~10% of infertility cases. Rare specific conditions, such as congenital hypogonadotropic hypogonadism, disorders of sex development and defects of sperm morphology and motility, are caused by pathogenic variants in recurrently affected genes, which facilitate high diagnostic yield (40-60%) of targeted gene panel-based testing. AREAS OF CONTROVERSY: Progress in mapping monogenic causes of quantitative spermatogenic failure, the major form of male infertility, has been slower. No 'recurrently' mutated key gene has been identified and worldwide, a few hundred patients in total have been assigned a possible monogenic cause. GROWING POINTS: Given the high genetic heterogeneity, an optimal approach to screen for heterogenous genetic causes of spermatogenic failure is sequencing exomes or in perspective, genomes. Clinical guidelines developed by multidisciplinary experts are needed for smooth integration of expanded molecular diagnostics in the routine management of infertile men. AREAS TIMELY FOR DEVELOPING RESEARCH: Di-/oligogenic causes, structural and common variants implicated in multifactorial inheritance may explain the 'hidden' genetic factors. It is also critical to understand how the recently identified diverse genetic factors of infertility link to general male health concerns across lifespan and how the clinical assessment could benefit from this knowledge.

Coordinated Expressional Landscape of the Human Placental miRNome and Transcriptome.

Placenta is a unique organ that serves its own function, and contributes to maternal gestational adaptation and fetal development. Coordination of its transcriptome to satisfy all the maternal-fetal needs across gestation is not fully understood. MicroRNAs are powerful transcriptome modulators capable to adjust rapidly the expression level and dynamics of large gene sets. This MiR-Seq based study presents a multi-omics investigation of the human placental miRNome and its synergy with the transcriptome. The analysis included 52 placentas representing three trimesters of normal pregnancy, and term cases of late-onset preeclampsia (LO-PE), gestational diabetes and affected fetal growth. Gestational-age dependent differential expression (FDR < 0.05) was detected for 319 of 417 tested miRNAs (76.5%). A shared list of target genes of dynamic miRNAs suggested their coordinated action. The most abundant miR-143-3p revealed as a marker for pregnancy progression. The data suggested critical, but distinct roles of placenta-specific imprinted C19MC and C14MC miRNA clusters. Paternally encoded primate-specific C19MC was highly transcribed during first trimester, potentially fine-tuning the early placental transcriptome in dosage-sensitive manner. Maternally encoded eutherian C14MC showed high expression until term, underlining its key contribution across gestation. A major shift in placental miRNome (16% miRNAs) was observed in LO-PE, but not in other term pregnancy complications. Notably, 13/38 upregulated miRNAs were transcribed from C19MC and only one from C14MC, whereas 11/28 downregulated miRNAs represented C14MC and none C19MC. miR-210-3p, miR-512-5p, miR-32-5p, miR-19a-3p, miR-590-3p, miR-379-5p were differentially expressed in LO-PE and cases of small-for-gestational-age newborns, supporting a shared etiology. Expression correlation analysis with the RNA-Seq data (16,567 genes) of the same samples clustered PE-linked miRNAs into five groups. Large notable clusters of miRNA-gene pairs showing directly and inversely correlated expression dynamics suggested potential functional relationships in both scenarios. The first genome-wide study of placental miR-eQTLs identified 66 placental SNVs associated with the expression of neighboring miRNAs, including PE-linked miRNAs miR-30a-5p, miR-210-3p, miR-490-3p and miR-518-5p. This study provided a rich catalog of miRNAs for further in-depth investigations of their individual and joint effect on placental transcriptome. Several highlighted miRNAs may serve as potential biomarkers for pregnancy monitoring and targets to prevent or treat gestational disorders.

Recurrent Pregnancy Loss and Concealed Long-QT Syndrome.

Background Recurrent pregnancy loss affects 1% to 2% of couples attempting childbirth. A large fraction of all cases remains idiopathic, which warrants research into monogenic causes of this distressing disorder. Methods and Results We investigated a nonconsanguineous Estonian family who had experienced 5 live births, intersected by 3 early pregnancy losses, and 6 fetal deaths, 3 of which occurred during the second trimester. No fetal malformations were described at the autopsies performed in 3 of 6 cases of fetal death. Parental and fetal chromosomal abnormalities (including submicroscopic) and maternal risk factors were excluded. Material for genetic testing was available from 4 miscarried cases (gestational weeks 11, 14, 17, and 18). Exome sequencing in 3 pregnancy losses and the mother identified no rare variants explicitly shared by the miscarried conceptuses. However, the mother and 2 pregnancy losses carried a heterozygous nonsynonymous variant, resulting in p.Val173Asp (rs199472695) in the ion channel gene KCNQ1. It is expressed not only in heart, where mutations cause type 1 long-QT syndrome, but also in other tissues, including uterus. The p.Val173Asp variant has been previously identified in a patient with type 1 long-QT syndrome, but not reported in the Genome Aggregation Database. With heterologous expression in CHO cells, our in vitro electrophysiologic studies indicated that the mutant slowly activating voltage-gated K+ channel (IKs) is dysfunctional. It showed reduced total activating and deactivating currents (P<0.01), with dramatically positive shift of voltage dependence of activation by ≈10 mV (P<0.05). Conclusions The current study uncovered concealed maternal type 1 long-QT syndrome as a potential novel cause behind recurrent fetal loss.

Variants in GCNA, X-linked germ-cell genome integrity gene, identified in men with primary spermatogenic failure.

Male infertility impacts millions of couples yet, the etiology of primary infertility remains largely unknown. A critical element of successful spermatogenesis is maintenance of genome integrity. Here, we present a genomic study of spermatogenic failure (SPGF). Our initial analysis (n = 176) did not reveal known gene-candidates but identified a potentially significant single-nucleotide variant (SNV) in X-linked germ-cell nuclear antigen (GCNA). Together with a larger follow-up study (n = 2049), 7 likely clinically relevant GCNA variants were identified. GCNA is critical for genome integrity in male meiosis and knockout models exhibit impaired spermatogenesis and infertility. Single-cell RNA-seq and immunohistochemistry confirm human GCNA expression from spermatogonia to elongated spermatids. Five identified SNVs were located in key functional regions, including N-terminal SUMO-interacting motif and C-terminal Spartan-like protease domain. Notably, variant p.Ala115ProfsTer7 results in an early frameshift, while Spartan-like domain missense variants p.Ser659Trp and p.Arg664Cys change conserved residues, likely affecting 3D structure. For variants within GCNA's intrinsically disordered region, we performed computational modeling for consensus motifs. Two SNVs were predicted to impact the structure of these consensus motifs. All identified variants have an extremely low minor allele frequency in the general population and 6 of 7 were not detected in > 5000 biological fathers. Considering evidence from animal models, germ-cell-specific expression, 3D modeling, and computational predictions for SNVs, we propose that identified GCNA variants disrupt structure and function of the respective protein domains, ultimately arresting germ-cell division. To our knowledge, this is the first study implicating GCNA, a key genome integrity factor, in human male infertility.

A common 1.6 mb Y-chromosomal inversion predisposes to subsequent deletions and severe spermatogenic failure in humans.

Male infertility is a prevalent condition, affecting 5-10% of men. So far, few genetic factors have been described as contributors to spermatogenic failure. Here, we report the first re-sequencing study of the Y-chromosomal Azoospermia Factor c (AZFc) region, combined with gene dosage analysis of the multicopy DAZ, BPY2, and CDYgenes and Y-haplogroup determination. In analysing 2324 Estonian men, we uncovered a novel structural variant as a high-penetrance risk factor for male infertility. The Y lineage R1a1-M458, reported at >20% frequency in several European populations, carries a fixed ~1.6 Mb r2/r3 inversion, destabilizing the AZFc region and predisposing to large recurrent microdeletions. Such complex rearrangements were significantly enriched among severe oligozoospermia cases. The carrier vs non-carrier risk for spermatogenic failure was increased 8.6-fold (p=6.0×10-4). This finding contributes to improved molecular diagnostics and clinical management of infertility. Carrier identification at young age will facilitate timely counselling and reproductive decision-making.

Current knowledge on genetic variants shaping placental transcriptome and their link to gestational and postnatal health.

Despite the indispensable role of the placenta in the successful course of pregnancy, regulation of its dynamic transcriptome is still underexplored. The purpose of this literature review was to give an overview and draw attention to the contribution of genetic variation in shaping the human placental gene expression. Studies of placental transcriptome shaped by chromosomal variants are limited and may be confounded by cellular mosaicism and somatic genomic rearrangements. Even in relatively simple cases, such as aneuploidies, the placental transcriptome appears to differ from the assumed systematically increased transcript levels of the involved chromosomes. Single nucleotide variants modulating placental gene expression referred to as expression quantitative trait loci (eQTLs) have been analyzed only in ten candidate gene and three genome-wide association studies (GWAS). The latter identified 417 confident placental eGenes, supported by at least two independent studies. Functional profiling of eGenes highlighted biological pathways important in pregnancy, such as immune response or transmembrane transport activity. A fraction of placental eQTLs (1-3%) co-localize with GWAS loci for adult disorders (metabolic, immunological, neurological), suggesting a co-contributory role of the placenta in the developmental programming of health. Some placental eQTLs have been identified as risk factors for adverse pregnancy outcomes, such as rs4769613 (C > T), located near the FLT1 gene and confidently associated with preeclampsia. More studies are needed to map genetic variants shaping gene expression in different placental cell types across three trimesters in normal and complicated gestations and to clarify to what extent these heritable factors contribute to maternal and offspring disease risks.