The prevalence of congenital anomalies: nationwide study in 2020 in Estonia.

OBJECTIVE: To assess the prevalence of congenital anomalies (CAs), chromosomal abnormalities and monogenic diseases among births and terminated pregnancies due to fetal anomalies (TOPFA) in 2020 in Estonia. Up to 2020 no data on prevalence of CAs in Estonia is reported. METHODS: For retrospective observational study data of all births and terminations of pregnancies after 12th gestational week from (i) the Estonian Medical Birth Registry, (ii) Abortion Registy, (iii) Health Insurance Fund and (iv) hospital records were linked. To calculate the total, live birth, stillbirth and TOPFA prevalence of CAs with 95% confidence intervals (CI), guidelines issued by EUROCAT, European network for the epidemiological surveillance of CAs, https://eu-rd-platform.jrc.ec.europa.eu/eurocat_en were followed. RESULTS: In 2020 the total prevalence of CAs, chromosomal abnormalities and monogenic diseases in Estonia was 378.6 per 10,000 births (95% CI 346.0, 413.5). The most prevalent CAs were heart defects, 163.7 cases per 10,000 births (95%CI 142.5, 187.2). The prevalence of chromosomal abnormalities and genetic diseases was 92.6 per 10,000 births (95%CI 76.8, 110.6), 80% of cases were among TOPFAs. No newborns with major aneuploidies (Trisomy 21, 18, 13, polyploidy) were reported in 2020. Live birth prevalence of CAs, including chromosomal abnormalities and genetic diseases was 258.4 per 10,000 live births (95%CI 231.5, 287.5) and stillbirth prevalence of CAs 0.8 per 10,000 births. CONCLUSIONS: The prevalence of CAs and genetic disorders in Estonia is one of the highest compared to prevalence reported by other European regions. It indicates to high population coverage with prenatal diagnostics in Estonia. Low number of major aneuploidies among live births may reflect good detection rate of major chromosomal abnormalities and cultural preferences.

Open versus laparoscopic appendectomy for acute appendicitis in pregnancy: a population-based study.

BACKGROUND: Laparoscopic appendectomy (LA) is the standard treatment for acute appendicitis (AA) in general population. However, the safety of LA during pregnancy has remained a matter of debate. The purpose of this study was to compare surgical and obstetrical outcomes in pregnant women who underwent LA vs. open appendectomy (OA) for AA. We hypothesized that LA results in improved surgical and obstetric outcomes during pregnancy. METHODS: Using a nationwide claim-based database in Estonia, a retrospective review of all cases of pregnant women undergoing OA or LA for AA from 2010 to 2020 was performed. Patient characteristics, surgical and obstetrical outcomes were analyzed. Primary outcomes were preterm delivery, fetal loss and perinatal mortality. Secondary outcomes included operative time, hospital length of stay (HLOS) and 30-day postoperative complications. RESULTS: Overall, 102 patients were included of whom 68 (67%) underwent OA and 34 patients (33%) LA, respectively. Patients in LA cohort had a significantly shorter length of pregnancy in terms of gestational weeks when compared to OA cohort (12 weeks versus 17 weeks, p = 0.002). Most of the patients in their 3rd trimester pregnancy were subjected to OA. Operative time in LA cohort was shorter than in OA cohort (34 min. versus 44 min., p = 0.038). HLOS in LA cohort was shorter than in OA cohort (2.1 days versus 2.9 days, p = 0.016). There were no differences between OA and LA cohorts in terms of surgical complications or obstetrical outcomes. CONCLUSIONS: Laparoscopic appendectomy for acute appendicitis was associated with a significantly shorter operative time and a shorter hospital length of stay while open and laparoscopic appendectomy cohorts experienced comparable obstetrical outcomes. Our findings support the laparoscopic approach for acute appendicitis in pregnancy.

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.

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.

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.

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.

Methylation allelic polymorphism (MAP) in chorionic gonadotropin beta5 (CGB5) and its association with pregnancy success.

CONTEXT: Increased epigenetic variability in the placenta may have evolved in response to its role in mediating the conflicting demands of the mother and fetus. One essential guardian of early pregnancy maintenance is the placental hormone human chorionic gonadotropin (HCG). OBJECTIVE: Among the four primate-specific duplicate HCGß-coding genes, chorionic gonadotropin-ß8 (CGB8) and chorionic gonadotropin-ß5 (CGB5) jointly contribute 62-82% of the total HCGß transcript pool. Because these genes share common features with known imprinted placenta-expressed loci, we addressed the role of epigenetic mechanisms affecting their action. DESIGN AND SUBJECTS: Parental origin of CGB5 and CGB8 transcripts and promoter methylation patterns were addressed in trophoblastic tissues from 23 mother-offspring duos and nine mother-father-offspring trios including the following: 1) third-trimester normal delivery at term (n = 14), 2) first-trimester elective termination of uncomplicated pregnancy (n = 10), and 3) first-trimester recurrent (≥3) miscarriage (n = 8). RESULTS: A normal uncomplicated pregnancy was characterized by balanced, biallelic expression of CGB5 and CGB8. However, in three (two recurrent miscarriage and one early elective termination of uncomplicated pregnancy) of nine genetically informative cases of CGB5, monoallelic expression of maternal alleles and hemimethylated gene promoters were identified. CONCLUSION: Our finding may represent a novel methylation allelic polymorphism or gain of imprinting in CGB5 promoter leading to expressional silencing of paternal alleles and increasing susceptibility to pregnancy loss. Aberrant methylation patterns in placenta may result from random reprogramming defects affecting normal implantation process. Alternatively, methylation allelic polymorphism in the placenta favoring the failure of pregnancy may arise as a response to cellular stress caused by, in general, aneuploidy or conditions in placental-maternal interface.

Genomics and genetics of gonadotropin beta-subunit genes: Unique FSHB and duplicated LHB/CGB loci.

The follicle stimulating hormone (FSH), luteinizing hormone (LH) and chorionic gonadotropin (HCG) play a critical role in human reproduction. Despite the common evolutionary ancestry and functional relatedness of the gonadotropin hormone beta (GtHB) genes, the single-copy FSHB (at 11p13) and the multi-copy LHB/CGB genes (at 19q13.32) exhibit locus-specific differences regarding their genomic context, evolution, genetic variation and expressional profile. FSHB represents a conservative vertebrate gene with a unique function and it is located in a structurally stable gene-poor region. In contrast, the primate-specific LHB/CGB gene cluster is located in a gene-rich genomic context and demonstrates an example of evolutionary young and unstable genomic region. The gene cluster is shaped by a constant balance between selection that acts on specific functions of the loci and frequent gene conversion events among duplicons. As the transcription of the GtHB genes is rate-limiting in the assembly of respective hormones, the genomic and genetic context of the FSHB and the LHB/CGB genes largely affects the profile of the hormone production.

Chorionic gonadotropin beta-gene variants are associated with recurrent miscarriage in two European populations.

CONTEXT: The incidence of recurrent miscarriage (RM) (>or=3 consecutive pregnancy losses) is estimated as 1-2% in fertile couples. Familial clustering of RM has suggested the contribution of a genetic component. OBJECTIVE: A low level of human chorionic gonadotropin (HCG) in maternal serum during the first trimester of the pregnancy is a clinically accepted risk factor for miscarriage. We sought to study whether variation in chorionic gonadotropin beta-subunit genes (CGBs) expressed in placenta may contribute to the risk of RM. DESIGN: Resequencing of CGB5 and CGB8, the two most actively transcribed loci of the four HCG beta-duplicate genes, was performed. SETTING: A case-control study involving two sample sets, from Estonia (n = 194) and Finland (n = 185), was performed. PATIENTS: RM patients (n = 184) and fertile controls (n = 195) participated in the study. RESULTS: From 71 identified variants in CGB5 and CGB8, 48 polymorphisms were novel. Significant protective effect was associated with two single nucleotide polymorphisms located at identical positions in intron 2 in both CGB5 [P = 0.007; odds ratio (OR) = 0.53] and CGB8 (P = 0.042; OR = 0.15), and with four CGB5 promoter variants (P < 0.03; OR = 0.54-0.58). The carriers of minor alleles had a reduced risk of RM. The haplotype structure of the CGB8 promoter was consistent with balancing selection; a rare mutation in CGB8 initiator element was detected only among patients (n = 3). In addition, three rare nonsynonymous substitutions were identified among RM cases as possible variants increasing the risk of recurrent pregnancy loss. CONCLUSION: The findings encourage studying the functional effect of the identified variants on CGB expression and HCG hormone activity to elucidate further the role of CGB variation in RM.

The evolution and genomic landscape of CGB1 and CGB2 genes.

The origin of completely novel proteins is a significant question in evolution. The luteinizing hormone (LHB)/chorionic gonadotropin (CGB) gene cluster in humans contains a candidate example of this process. Two genes in this cluster (CGB1 and CGB2) exhibit nucleotide sequence similarity with the other LHB/CGB genes, but as a result of frameshifting are predicted to encode a completely novel protein. Our analysis of these genes from humans and related primates indicates a recent origin in the lineage specific to humans and African great apes. While the function of these genes is not yet known, they are strongly conserved between human and chimpanzee and exhibit three-fold lower diversity than LHB across human populations with no mutations that would disrupt the coding sequence. The 5'-upstream region of CGB1/2 contains most of the promoter sequence of hCGbeta plus a novel region proximal to the putative transcription start site. In silico prediction of putative transcription factor binding sites supports the hypothesis that CGB1 and CGB2 gene products are expressed in, and may contribute to, implantation and placental development.

IgG, IgA and IgM antibodies against FSH: serological markers of pathogenic autoimmunity or of normal immunoregulation?

PROBLEM: Autoimmune mechanisms are often involved in causing infertility. Among the possible targets of autoantibodies, the follicle-stimulating hormone (FSH) which regulates the follicular maturation in human ovary is a promising candidate. We aimed to study whether anti-FSH-antibodies might be involved in different clinical types of infertility. METHOD OF STUDY: The study group consisted of 178 patients (75 with polycystic ovary syndrome (PCOS), 103 with endometriosis) and 75 pregnant women. Female blood donors formed the control group (n = 85). Indirect enzyme-linked immunosorbent assay tests were performed using purified FSH as antigens and a synthetic peptide corresponding to the 78-93 region (V14D) of the human FSH beta-chain. CONCLUSION: We showed that anti-FSH-antibodies were present in controls and their production decreased during pregnancy. Endometriosis and PCOS were associated with higher values of anti-FSH-immunoglobulin (Ig)A, anti-V14D-IgA, and endometriosis with anti-V14D-IgG. Our data suggest that anti-FSH-IgA could be a marker of ovarian disorders that cause infertility.

Expression of beta-subunit of HCG genes during normal and failed pregnancy.

BACKGROUND: One of the first proteins synthesized by a conceptus is HCG. The receptor-binding beta-subunit of HCG (HCGbeta) is encoded by highly homologous CGB, CGB5, CGB7 and CGB8 genes. The function of two additional gene copies, CGB1 and CGB2, is still unknown. We aimed to compare the expression of individual CGB genes during normal pregnancy and in cases of recurrent miscarriage (RM) and ectopic pregnancy (EP). METHODS: A semiquantitative RT-PCR with fluorescent-labelled primers coupled with the gene-specific restriction and quantification was used. RESULTS: The summarized expression of HCGbeta genes was high throughout the pregnancy, and moderately correlated with HCG in serum. In cases of RM, reduced hormone values were consistent with low mRNA levels, whereas for EP no reduction in transcriptional activity was detected. CGB1and CGB2 showed a considerable expression peak during the first trimester, both in normal and ectopic pregnancy, but not for RM. CONCLUSIONS: In cases of RM, low HCG could result from expression failure of HCGbeta genes, whereas in EP the problems other than the transcriptional failure contribute to reduced hormone levels. The expression patterns of CGB1 and CGB2 suggest their putative role in the implantation stage.