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.

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.

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.

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.

Monogenic causes of non-obstructive azoospermia: challenges, established knowledge, limitations and perspectives.

It is estimated that one in 100 men have azoospermia, the complete lack of sperm in the ejaculate. Currently, ~ 20% of azoospermia cases remain idiopathic. Non-obstructive azoospermia (NOA) is mostly explained by congenital factors leading to spermatogenic failure, such as chromosome abnormalities. The knowledge of the monogenic causes of NOA is very limited. High genetic heterogeneity due to the complexity of spermatogenesis and testicular function, lack of non-consanguineous familial cases and confirmatory studies challenge the field. The reported monogenic defects cause syndromic NOA phenotypes presenting also additional congenital problems and isolated NOA cases, explained by spermatogenic defects. The established and recently reported NOA genes (n = 38) represent essential guardians of meiosis, transcriptional and endocrine regulators of reproduction. Despite the list being short, 92% of these loci are predicted to functionally interact with each other (STRING analysis: average 5.21 connections/gene, enrichment P < 10-16). Notably, ~ 50% of NOA genes have also been implicated in primary ovarian insufficiency, amenorrhea and female genital anomalies, referring to overlapping mechanisms. Considering the knowledge from respective female phenotypes and animal models, exploring the scenarios of di/oligogenic and de novo mutations represent perspective directions in the genetic research of NOA. Knowing the exact genetic cause in each patient improves the management of infertility and other health risks (e.g., cancer), and facilitates the counseling of family members about their reproductive health. Uncovering the loci and biological processes implicated in NOA will also broaden the understanding of etiologies behind spermatogenic failure and promote the development of novel non-invasive treatments for male infertility.

C-allele of rs4769613 Near FLT1 Represents a High-Confidence Placental Risk Factor for Preeclampsia.

The variant rs4769613 T/C within the enhancer element near FLT1, an acknowledged gene in preeclampsia, was previously identified as a risk factor for preeclampsia in the genome-wide association study (GWAS) targeting placental genotypes. We aimed to test the robustness of this association in 2 Estonian cohorts. Both placental sample sets HAPPY PREGNANCY (Development of novel non-invasive biomarkers for fertility and healthy pregnancy; preeclampsia, n=44 versus nonpreeclampsia, n=1724) and REPROMETA (REPROgrammed fetal and/or maternal METAbolism; 52/277) exhibited suggestive association between rs4769613[C] variant and preeclampsia (logistic regression adjusted for gestational age and fetal sex, nominal P<0.05). Meta-analysis across 2 samples (96/2001) replicated the genome-wide association study outcome (Bonferroni corrected P=4×10-3; odds ratio, 1.75 [95% CI, 1.23-2.49]). No association was detected with gestational diabetes mellitus, preterm birth, and newborn parameters. Also, neither maternal nor paternal rs4769613 genotypes predisposed to preeclampsia. The exact role of placental rs4769613 genotype in the preeclampsia pathogenesis is to be clarified as no effect was detected on maternal baseline serum sFlt-1 (soluble fms-related receptor tyrosine kinase 1) levels. However, when placental FLT1 gene expression and maternal serum sFlt-1 measurements were stratified by placental rs4769613 genotypes, significantly higher transcript and biomarker levels were detected in preeclampsia versus nonpreeclampsia cases in the CC- and CT- (Student t test, P≤0.02), but not in the TT-genotype subgroup. We suggest that rs4769613 represents a conditional expression Quantitative Trait Locus, whereby only the enhancer with the C-allele reacts to promote the FLT1 expression in unfavorable placental conditions. The study highlighted that the placental FLT1 rs4769613 C-allele is a preeclampsia-specific risk factor. It may contribute to early identification of high-risk women, for example, when genotyped in the cffDNA available in maternal blood plasma.

Single-Tube Multimarker Assay for Estimating the Risk to Develop Preeclampsia.

BACKGROUND: Preeclampsia (PE) affects 2%-8% of all pregnancies worldwide. The predictive value of the currently used maternal serum fms-like tyrosine kinase-1/ placental growth factor (sFlt-1/PlGF) test is < 40% for PE onset within 4 weeks. We aimed to develop an innovative multiplex assay to improve PE prediction. METHODS: The 6PLEX assay combining the measurements of ADAM12, sENG, leptin, PlGF, sFlt-1, and PTX3 was developed for the Luminex® xMAP platform. Assay performance was evaluated using 61 serum samples drawn from 53 pregnant women between 180 and 275 gestational days: diagnosed PE cases, n = 4; cases with PE onset within 4-62 days after sampling, n = 25; controls, n = 32. The B·R·A·H·M·S Kryptor sFlt-1/PlGF test (Thermo Fisher Scientific, Hennigsdorf, Germany) was applied as an external reference. Alternative PE prediction formulae combining 6PLEX measurements with clinical parameters were developed. RESULTS: There was a high correlation in sFlt-1/PlGF estimated for individual sera between the 6PLEX and B·R·A·H·M·S Kryptor immunoassays (Spearman's r = 0.93, P < 0.0001). The predictive power of the 6PLEX combined with gestational age and maternal weight at sampling reached AUC 0.99 (95% CI 0.97-1.00) with sensitivity 100.0% and specificity 96.9%. In all models, sFlt-1/PlGF derived from the B·R·A·H·M·S immunoassays exhibited the lowest AUC value (<0.87) and sensitivity (<80%) with broad confidence intervals (13%-92%). The estimated prognostic yield of the 6PLEX compared to the B·R·A·H·M·S assay was significantly higher (96.5% vs 73.7%; P = 0.0005). CONCLUSIONS: The developed single-tube multimarker assay for PE risk estimation in combination with clinical symptoms reached high prognostic yield (96.5%) and exhibited superior performance compared to the sFlt-1/PlGF test.

The decline of FANCM immunohistochemical expression in prostate cancer stroma correlates with the grade group.

Prostate adenocarcinoma (PCa) stromal markers have recently gained attention as complementary diagnostic tools. The DNA reparation complex protein FANCM has been shown to express in the normal prostate stroma and FANCM gene alterations to be associated with PCa susceptibility; this has led to the hypothesis that an insufficient level of FANCM expression may provide additional information for the evaluation of PCa. The study cohort comprised 60 radical prostatectomy specimens. The controls involved 11 autopsies (CTRL) and non-cancerous tissue (NCT) areas from the prostatectomy specimen. The samples were stained with the FANCM antibody. The quantification of the stromal staining index (SSI) was made using ImageJ and QuPath. Overall, 655 regions of interest (ROI) were analyzed. FANCM expression appeared equally intense and stroma specific in both CTRL and NCT, indicating the absence of underlying baseline alterations. Within the age span of the cohort 47-89 years, no significant effect of the age of the patients on the FANCM expression was seen. FANCM demonstrated Gleason grade (G) dependent decline in PCa, being statistically significant in controls versus G1 and G2 versus G3. In other adjacent International Society of Urological Pathology (ISUP) groups, it remained insignificant, still being meaningful between high and low-grade cancers.

Bi-allelic Recessive Loss-of-Function Variants in FANCM Cause Non-obstructive Azoospermia.

Infertility affects around 7% of men worldwide. Idiopathic non-obstructive azoospermia (NOA) is defined as the absence of spermatozoa in the ejaculate due to failed spermatogenesis. There is a high probability that NOA is caused by rare genetic defects. In this study, whole-exome sequencing (WES) was applied to two Estonian brothers diagnosed with NOA and Sertoli cell-only syndrome (SCOS). Compound heterozygous loss-of-function (LoF) variants in FANCM (Fanconi anemia complementation group M) were detected as the most likely cause for their condition. A rare maternally inherited frameshift variant p.Gln498Thrfs∗7 (rs761250416) and a previously undescribed splicing variant (c.4387-10A>G) derived from the father introduce a premature STOP codon leading to a truncated protein. FANCM exhibits enhanced testicular expression. In control subjects, immunohistochemical staining localized FANCM to the Sertoli and spermatogenic cells of seminiferous tubules with increasing intensity through germ cell development. This is consistent with its role in maintaining genomic stability in meiosis and mitosis. In the individual with SCOS carrying bi-allelic FANCM LoF variants, none or only faint expression was detected in the Sertoli cells. As further evidence, we detected two additional NOA-affected case subjects with independent FANCM homozygous nonsense variants, one from Estonia (p.Gln1701∗; rs147021911) and another from Portugal (p.Arg1931∗; rs144567652). The study convincingly demonstrates that bi-allelic recessive LoF variants in FANCM cause azoospermia. FANCM pathogenic variants have also been linked with doubled risk of familial breast and ovarian cancer, providing an example mechanism for the association between infertility and cancer risk, supported by published data on Fancm mutant mouse models.

FSHB -211 G>T is a major genetic modulator of reproductive physiology and health in childbearing age women.

STUDY QUESTION: Are the genetic variants FSHB -211 G>T (rs10835638), FSHR c.2039 A>G (Asn680Ser, rs6166) and FSHR -29 G>A (rs1394205) associated with serum FSH, LH and anti-Müllerian hormone (AMH) levels in reproductive age women, their menstrual cycle parameters and risk of infertility? SUMMARY ANSWER: Only the FSHB -211 G>T variant was a major genetic determinant of serum gonadotropin levels in both, eumenorrheic healthy women and female infertility patients, and the T-allele carrier status was enriched among idiopathic infertility cases. WHAT IS KNOWN ALREADY: There are accumulating data on common genetic variants modulating reproductive parameters and fertility potential. FSHB -211 G>T represents the strongest acknowledged genetic factor contributing to male circulating gonadotropins levels. Respective data in women are limited and the two previously published studies have reached conflicting results. In addition, previous studies have consistently associated FSHR c.2039 A>G (but not FSHR -29 G>A) with female serum FSH level. STUDY DESIGN, SIZE, DURATION: The study aimed to test robust and clinically meaningful genetic effects (if present) of the FSHB -211 G>T, FSHR c.2039 A>G and FSHR -29 G>A variants on female basal FSH, LH and AMH levels, and linked reproductive parameters. Genetic association testing was performed in two independent and clinically different study groups (i) eumenorrheic healthy women without known fertility problems (n = 169; 27.6 ± 6.1 years) and (ii) female partners of infertile couples (n = 186; 32.4 ± 4.7 years). The study groups were compared for allelic and genotypic distributions of the analysed variants. PARTICIPANTS/MATERIALS, SETTING, METHODS: All participants were recruited during the HAPPY PREGNANCY study (2013-2015) at the Women's Clinic, Tartu University Hospital, Estonia. Serum FSH, LH and AMH were measured in the follicular phase (Days 2-6) of the menstrual cycle. All three single nucleotide polymorphisms (SNPs) were genotyped by PCR and Taqman allelic discrimination assay. The effect of the analysed variants on hormonal measurements and menstrual cycle data was assessed using linear regression under additive and recessive models adjusted by age, BMI and smoking status. Results of the two subgroups were combined in a meta-analysis applying the fixed effects model. Restricted maximum likelihood analysis was applied to estimate the proportion of total phenotypic variance of analysed reproductive parameters, explainable by the tested genetic variants. In case-control analysis, genetic association with infertility status was tested using Fisher's exact test and logistic regression adjusted by age, BMI and smoking status. MAIN RESULTS AND THE ROLE OF CHANCE: In both study groups, T-allele of the FSHB -211 G>T was associated with significantly higher serum levels of FSH and LH. Results of the meta-analysis (additive genetic model) remained significant after Bonferroni correction for multiple testing: FSH, T-allele effect 0.80 IU/L, P = 1.2 × 10-3; LH, 1.58 IU/L, P = 1.8×10-8. A more pronounced effect of T-allele of the FSHB -211 G>T on circulating LH was identified as a driving factor to increased LH/FSH ratio (meta-analysis, P = 4.7 × 10-3). In healthy women, the FSHB -211 G>T variant was estimated to explain 3.5 and 7.1% of the total variance of the measured serum FSH and LH levels, respectively. The corresponding numbers for the infertility patients were 1.6 and 10.5%. Women with idiopathic infertility compared to controls exhibited a doubled T-allele frequency (23.6 versus 12.4%; P = 8.9 × 10-3) and a >3-fold excess of TT homozygotes (5.6 versus 1.8%; P = 3.5 × 10-2). The only association of the FSHR c.2039 A>G was detected with serum FSH levels in eumenorrheic healthy women, explaining 3.9% of the total parameter variance (G-allele effect 0.56 IU/L, P = 4.6 × 10-3). In the study group of healthy reproductive age women, the highest serum FSH levels were detected among the FSHB -211 T-allele carriers with the FSHR c.2039 GG-genotype (median 7.7 IU/L). In contrast, the lowest hormone concentrations were measured for the women carrying the combination of the FSHB -211 GG- and the FSHR c.2039 AA-homozygosity (median 5.8 IU/L, P = 9.6 × 10-3). None of the analysed reproductive parameters was associated with the FSHR -29 G>A variant. In our study groups, the tested polymorphisms did not reach significant associations with serum AMH measurements, menstrual cycle length or age at menarche. LIMITATIONS, REASONS FOR CAUTION: Small sample size and the design involving two clinical groups with different reproductive histories may have limited the capacity to replicate the associations with the age at menarche and length of menstrual cycle, initially reported in large genome-wide association studies. Small sample size may have also affected the accuracy in estimating the contribution of the tested variants to the total phenotypic variance of measured gonadotropin concentrations. The group of eumenorrheic healthy women had its limitations as a control to estimate the true effect of analysed genetic variants on individual's fertility potential as the recruitment strategy had been targeted mostly towards younger women, who may not yet have planned to conceive a child by this age. WIDER IMPLICATIONS OF THE FINDINGS: We propose that like in men, also in women the FSHB -211 G>T represents a key genetic modulator of circulating gonadotropin, leading to various possible downstream effects on reproductive physiology. This claim is strongly supported by the reports of genome-wide association studies on various female reproductive traits and diseases. In perspective, FSHB -211 G>T may have a diagnostic value in fertility clinics to detect female patients with genetically inherited elevated basal FSH and LH levels. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by Estonian Science Foundation Grant (ETF9030 for M.L.); Institutional Research Grant (IUT34-12 for M.L.) and European Union through the European Regional Development Fund (project HAPPY PREGNANCY, 3.2.0701.12-0047; for M.L. and K.R.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the article. We have no competing interests to declare. TRAIL REGISTRATION NUMBER: Not applicable.

Stanniocalcin-1 Hormone in Nonpreeclamptic and Preeclamptic Pregnancy: Clinical, Life-Style, and Genetic Modulators.

CONTEXT AND OBJECTIVES: The study represents the first comprehensive analysis of Stanniocalcin-1 (STC1) hormone in human pregnancy, assessing clinical, lifestyle, and genetic determinants of circulating STC1 at term. DESIGN, SETTING, AND PARTICIPANTS: Participants included women with (n = 50) and without (n = 316) preeclampsia (PE) at delivery, recruited in the REPROgrammed fetal and/or maternal METAbolism (REPROMETA) study (2006-2011, Estonia). Genetic association analysis combined PE cases (n = 597) and controls (n = 623) from the REPROMETA and Finnish Genetics of Preeclampsia Consortium (2008-2011) studies. MAIN OUTCOME MEASURE(S): Maternal postpartum plasma STC1 was measured by ELISA (n = 366) and placental STC1 gene expression by TaqMan quantitative RT-PCR (n = 120). Genotyping was performed using Sequenom MassArray. RESULTS: Significantly higher STC1 plasma level was measured for the PE (median, 1952 pg/mL; 1030-4284 pg/mL) compared with non-PE group (median, 1562 pg/mL; 423-3781 pg/mL; P = 3.7 × 10-4, Mann-Whitney U test). Statistical significance was enhanced after adjustment for cofactors (linear regression, P = 1.8 × 10-6). STC1 measurements were negatively correlated with maternal smoking. Prepregnancy body mass index had a positive correlation with STC1 only among PE patients (r = 0.45; P = .001). The strongest genetic association with hormone concentrations was detected for STC1 single nucleotide polymorphisms rs3758089 (C allele: minor allele frequency, 5%; linear regression: ß = 249.2 pg/mL; P = .014) and rs12678447 (G allele: minor allele frequency, 7%; ß = 147.0 pg/mL; P = .082). rs12678447 placental genotypes were significantly associated with STC1 gene expression (P = .014). The REPROMETA/Finnish Genetics of Preeclampsia Consortium meta-analysis suggested an increased risk to develop late-onset PE for the rs12678447 G allele carriers (P = .05; odds ratio = 1.38 [0.98-1.93]). CONCLUSIONS: Increased STC1 hormone represents a hallmark of late-onset PE. STC1 gene variants modulate placental gene expression and maternal hormone levels.

Extensive load of somatic CNVs in the human placenta.

Placenta is a temporary, but indispensable organ in mammalian pregnancy. From its basic nature, it exhibits highly invasive tumour-like properties facilitating effective implantation through trophoblast cell proliferation and migration, and a critical role in pregnancy success. We hypothesized that similarly to cancer, somatic genomic rearrangements are promoted in the support of placental function. Here we present the first profiling of copy number variations (CNVs) in human placental genomes, showing an extensive load of somatic CNVs, especially duplications and suggesting that this phenomenon may be critical for normal gestation. Placental somatic CNVs were significantly enriched in genes involved in cell adhesion, immunity, embryonic development and cell cycle. Overrepresentation of imprinted genes in somatic duplications suggests that amplified gene copies may represent an alternative mechanism to support parent-of-origin specific gene expression. Placentas from pregnancy complications exhibited significantly altered CNV profile compared to normal gestations, indicative to the clinical implications of the study.

Structural genomic variation as risk factor for idiopathic recurrent miscarriage.

Recurrent miscarriage (RM) is a multifactorial disorder with acknowledged genetic heritability that affects ∼3% of couples aiming at childbirth. As copy number variants (CNVs) have been shown to contribute to reproductive disease susceptibility, we aimed to describe genome-wide profile of CNVs and identify common rearrangements modulating risk to RM. Genome-wide screening of Estonian RM patients and fertile controls identified excessive cumulative burden of CNVs (5.4 and 6.1 Mb per genome) in two RM cases possibly increasing their individual disease risk. Functional profiling of all rearranged genes within RM study group revealed significant enrichment of loci related to innate immunity and immunoregulatory pathways essential for immune tolerance at fetomaternal interface. As a major finding, we report a multicopy duplication (61.6 kb) at 5p13.3 conferring increased maternal risk to RM in Estonia and Denmark (meta-analysis, n = 309/205, odds ratio = 4.82, P = 0.012). Comparison to Estonian population-based cohort (total, n = 1000) confirmed the risk for Estonian female cases (P = 7.9 × 10(-4) ). Datasets of four cohorts from the Database of Genomic Variants (total, n = 5,846 subjects) exhibited similar low duplication prevalence worldwide (0.7%-1.2%) compared to RM cases of this study (6.6%-7.5%). The CNV disrupts PDZD2 and GOLPH3 genes predominantly expressed in placenta and it may represent a novel risk factor for pregnancy complications.

Reproductive physiology in young men is cumulatively affected by FSH-action modulating genetic variants: FSHR -29G/A and c.2039 A/G, FSHB -211G/T.

Follicle-Stimulating Hormone Receptor (FSHR) -29G/A polymorphism (rs1394205) was reported to modulate gene expression and reproductive parameters in women, but data in men is limited. We aimed to bring evidence to the effect of FSHR -29G/A variants in men. In Baltic young male cohort (n = 982; Estonians, Latvians, Lithuanians; aged 20.2 ± 2.0 years), the FSHR -29 A-allele was significantly associated with higher serum FSH (linear regression: effect 0.27 IU/L; P = 0.0019, resistant to Bonferroni correction for multiple testing) and showed a non-significant trend for association with higher LH (0.19 IU/L) and total testosterone (0.93 nmol/L), but reduced Inhibin B (-7.84 pg/mL) and total testes volume (effect -1.00 mL). Next, we extended the study and tested the effect of FSHR gene haplotypes determined by the allelic combination of FSHR -29G/A and a well-studied variant c.2039 A/G (Asn680Ser, exon 10). Among the FSHR -29A/2039G haplotype carriers (A-Ser; haplotype-based linear regression), this genetic effect was enhanced for FSH (effect 0.40 IU/L), Inhibin B (-16.57 pg/mL) and total testes volume (-2.34 mL). Finally, we estimated the total contribution of three known FSH-action modulating SNPs (FSHB -211G/T; FSHR -29G/A, c.2039 A/G) to phenotypic variance in reproductive parameters among young men. The major FSH-action modulating SNPs explained together 2.3%, 1.4%, 1.0 and 1.1% of the measured variance in serum FSH, Inhibin B, testosterone and total testes volume, respectively. In contrast to the young male cohort, neither FSHR -29G/A nor FSHR haplotypes appeared to systematically modulate the reproductive physiology of oligozoospermic idiopathic infertile patients (n = 641, Estonians; aged 31.5 ± 6.0 years). In summary, this is the first study showing the significant effect of FSHR -29G/A on male serum FSH level. To account for the genetic effect of known common polymorphisms modulating FSH-action, we suggest haplotype-based analysis of FSHR SNPs (FSHR -29G/A, c.2039 A/G) in combination with FSHB -211G/T testing.

A modest but significant effect of CGB5 gene promoter polymorphisms in modulating the risk of recurrent miscarriage.

OBJECTIVE: To confirm the effect of single nucleotide polymorphisms (SNPs) in chorionic gonadotropin beta (CGB) genes in modulating the susceptibility to recurrent miscarriage (RM) in Danes and in a meta-analysis across Danes and the discovery samples from Estonia and Finland. DESIGN: Case-control association study, restriction fragment length polymorphism genotyping, resequencing. SETTING: Fertility clinics at the Rigshospitalet, Copenhagen, and Aalborg Hospital, Aalborg, Denmark. PATIENT(S): Four hundred fifty Danish women and men from couples with RM and 119 women with children and no miscarriages in new study. A total of 634 women and men from RM couples and 314 female controls in a combined study of Estonians, Finns, and Danes. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Distribution of CGB5 and CGB8 allele and haplotype frequencies in patients and controls. RESULT(S): For the majority of studied SNPs, the allelic and haplotypic distribution differed statistically between the Danish and the previous Estonian-Finnish sample. In Danes, two CGB5 promoter SNPs (c5-155; c5-142) exhibited a nonsignificant trend for higher allele frequency in fertile women compared with RM patients. The meta-analysis of results from three populations confirmed a modest but significant effect on carriage of c5-155C (odds ratio = 0.64; 95% confidence interval [CI] 0.44-0.94) and c5-142A (odds ratio = 0.66; 95% CI, 0.45-0.94) variants in reducing the risk of RM. None of the investigated genetic variants in the CGB8 gene was associated with RM. CONCLUSION(S): Carriage of particular variants in the promoter of the CGB5 gene seems to protect against RM. No common genetic variants in CGB5 and CGB8 were associated with increased RM susceptibility in the studied North European populations.

Structural and functional analysis of rare missense mutations in human chorionic gonadotrophin ß-subunit.

Heterodimeric hCG is one of the key hormones determining early pregnancy success. We have previously identified rare missense mutations in hCGß genes with potential pathophysiological importance. The present study assessed the impact of these mutations on the structure and function of hCG by applying a combination of in silico (sequence and structure analysis, molecular dynamics) and in vitro (co-immunoprecipitation, immuno- and bioassays) approaches. The carrier status of each mutation was determined for 1086 North-Europeans [655 patients with recurrent miscarriage (RM)/431 healthy controls from Estonia, Finland and Denmark] using PCR-restriction fragment length polymorphism. The mutation CGB5 p.Val56Leu (rs72556325) was identified in a single heterozygous RM patient and caused a structural hindrance in the formation of the hCGα/ß dimer. Although the amount of the mutant hCGß assembled into secreted intact hCG was only 10% compared with the wild-type, a stronger signaling response was triggered upon binding to its receptor, thus compensating the effect of poor dimerization. The mutation CGB8 p.Pro73Arg (rs72556345) was found in five heterozygotes (three RM cases and two control individuals) and was inherited by two of seven studied live born children. The mutation caused ~50% of secreted ß-subunits to acquire an alternative conformation, but did not affect its biological activity. For the CGB8 p.Arg8Trp (rs72556341) substitution, the applied in vitro methods revealed no alterations in the assembly of intact hCG as also supported by an in silico analysis. In summary, the accumulated data indicate that only mutations with neutral or mild functional consequences might be tolerated in the major hCGß genes CGB5 and CGB8.

Pharmacogenetics of follicle-stimulating hormone action.

PURPOSE OF REVIEW: To review the current knowledge of genetic variants in the two genes affecting the individual responsiveness to follicle-stimulating hormone (FSH) action-the FSH beta-subunit (FSHB) and the FSH receptor (FSHR), as well as the pharmacogenetic ramifications of the findings. RECENT FINDINGS: Four common variants in the FSHB and the FSHR genes were shown to exhibit significant effect on FSH action: linked FSHR variants Thr307Ala and Asn680Ser determining common receptor isoforms, and gene expression affecting polymorphisms FSHR -29G/A and FSHB -211G/T. In women, the FSHR Thr307Ala/Asn680Ser polymorphisms show consistent predictive value for estimating the most optimal recombinant FSH dosage in controlled ovarian hyperstimulation (COH). The same variants exhibit a potential for the pharmacogenetic assessment of the treatment of polycystic ovarian syndrome. The FSHR -29G/A variant was also shown to contribute to ovarian response to COH. Pilot studies have suggested the FSHB -211 TT homozygous oligozoospermic men with genetically determined low concentration of FSH, as potentially the best responders to FSH treatment; furthermore, modulation of this response by FSHR polymorphisms is possible. SUMMARY: Genetic variants in FSHB and FSHR exhibit a potential for pharmacogenetic applications in selecting appropriate treatment options (timing and dosage) in male and female conditions requiring or benefiting from FSH therapy.

HYPEST study: profile of hypertensive patients in Estonia.

BACKGROUND: More than one third of adult population in Estonia has problems with elevated blood pressure (BP). The Hypertension in Estonia (HYPEST) study represents the country's first hypertension-targeted sample collection aiming to examine the epidemiological and genetic determinants for hypertension (HTN) and related cardiovascular diseases (CVD) in Estonian population. The HYPEST subjects (n = 1,966) were recruited across Estonia between 2004-2007 including clinically diagnosed HTN cases and population-based controls. The present report is focused on the clinical and epidemiological profile of HYPEST cases, and gender-specific effects on the pathophysiology of hypertension. METHODS: Current analysis was performed on 1,007 clinically diagnosed HTN patients (617 women and 390 men) aged 18-85 years. The hypertensives were recruited to the study by BP specialists at the North Estonia Medical Center, Centre of Cardiology, Tallinn or at the Cardiology Clinic, Tartu University Hospital, Estonia. Longitudinal BP data was extracted retrospectively from clinical records. Current and retrospective data of patient's medical history, medication intake and lifestyle habits were derived from self-administrated questionnaire and each variable was examined separately for men and women. Eleven biochemical parameters were measured from fasting serum samples of 756 patients. RESULTS: The distribution of recruited men and women was 39% and 61% respectively. Majority of Estonian HTN patients (85%) were overweight (BMI ≥ 25 kg/m2) and a total of 79% of patients had additional complications with cardiovascular system. In men, the hypertension started almost 5 years earlier than in women (40.5 ± 14.5 vs 46.1 ± 12.7 years), which led to earlier age of first myocardial infarction (MI) and overall higher incidence rate of MI among male patients (men 21.2%, women 8.9%, P < 0.0001). Heart arrhythmia, thyroid diseases, renal tubulo-intestinal diseases and hyperlipidemia were more prevalent in hypertensive women compared to men (P < 0.0001). An earlier age of HTN onset was significantly associated with smoking (P = 0.00007), obesity (BMI ≥ 30 kg/m2; P = 0.0003), increased stress (P = 0.0003) and alcohol consumption (P = 0.004). CONCLUSION: Understanding the clinical profile of HTN patients contributes to CVD management. Estonian hypertension patients exhibited different disease and risk profiles of male and female patients. This well-characterized sample set provides a good resource for studying hypertension and other cardiovascular phenotypes.

Genetically determined dosage of follicle-stimulating hormone (FSH) affects male reproductive parameters.

CONTEXT: The detailed role of FSH in contributing to male testicular function and fertility has been debated. We have previously identified the association between the T-allele of the FSHB promoter polymorphism (rs10835638; G/T, -211 bp from the mRNA start) and significantly reduced male serum FSH. OBJECTIVE: In the current study, the T-allele carriers of the FSHB -211 G/T single nucleotide polymorphism represented a natural model for documenting downstream phenotypic consequences of insufficient FSH action. DESIGN AND SUBJECTS: We genotyped rs10835638 in the population-based Baltic cohort of young men (n = 1054; GG carriers, n = 796; GT carriers, n = 244; TT carriers, n = 14) recruited by Andrology Centres in Tartu, Estonia; Riga, Latvia; and Kaunas, Lithuania. Marker-trait association testing was performed using linear regression (additive, recessive models) adjusted by age, body mass index, smoking, and recruitment center. RESULTS: Serum hormones directly correlated with the T-allele dosage of rs10835638 included FSH (additive model, P = 1.11 × 10(-6); T-allele effect, -0.41 IU/liter), inhibin-B (P = 2.16 × 10(-3); T-allele effect, -14.67 pg/ml), and total testosterone (P = 9.30 × 10(-3); T-allele effect, -1.46 nmol/liter). Parameters altered only among TT homozygotes were reduced testicular volume (recessive model, P = 1.19 × 10(-4); TT genotype effect, -9.47 ml) and increased serum LH (P = 2.25 × 10(-2); TT genotype effect, 1.07 IU/liter). The carrier status of rs10835638 alternative genotypes did not affect sperm motility and morphology, calculated free testosterone, serum SHBG, and estradiol concentrations. CONCLUSION: We showed for the first time that genetically determined low FSH may have wider downstream effects on the male reproductive system, including impaired testes development, altered testicular hormone levels (inhibin-B, total testosterone, LH), and affected male reproductive potential.