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BACKGROUND: To find the obstetrical and delivery associated risk factors of antenatal and postnatal grade III intraventricular hemorrhage (IVH) or periventricular hemorrhagic infarction (PVHI) in preterm neonates. METHODS: A retrospective study of obstetric and delivery associated risk factors included neonates (<35 gestational weeks) with severe IVH/PVHI (n = 120) and a prospectively collected control group (n = 50). The children were divided into: (1) antenatal onset group (n = 27) with insult visible on cerebral ultrasonography within the first 12 hours of birth or periventricular cystic changes visible in PVHI within the first 3 days; (2) neonatal onset group (n = 70) with insult diagnosed after initial normal findings or I-II grade IVH, and (3) unknown time-onset group (n = 23) with insult visible at > 12 h of age. RESULTS: The mothers of the antenatal onset group had significantly more bacterial infections before delivery compared to the neonatal onset group: 20/27 (74.1%) versus 23/69 (33.3%), (odds ratio (OR) 5.7 [95% confidence interval 2.1–16]; p = 0.0008) or compared to the control group (11/50 (22%); OR 11 [2.8–42]; p = 0.0005). Placental histology revealed chorioamnionitis more often in the antenatal compared to the neonatal onset group (14/21 (66.7%) versus 16/42 (38.1%), respectively; OR 3.7 [1.18–11]; p = 0.025). Neonates with neonatal development of severe IVH/PVHI had significantly more complications during delivery or intensive care. CONCLUSIONS: Bacterial infection during pregnancy is an important risk factor for development of antenatal onset severe IVH or PVHI. In neonates born to mothers with severe bacterial infection during pregnancy, cerebral ultrasonography is indicated for early detection of severe IVH or PVHI. [ABSTRACT FROM AUTHOR]

Objective: to survey the views of midwives in Estonia about who they considered should have responsibility for carrying out certain aspects of antenatal care (ANC) in Estonia.Design, setting and study population: in collaboration with key stakeholder organisations, the authors developed eight statements on aspects of ANC and five combinations of possible professionals (including midwives obstetrician-gynaecologists and various combinations of the two) who could have responsibility for carrying out those aspects of ANC and included them in a self-administered questionnaire. The questionnaire was sent with a covering letter and stamped addressed return envelope to all 366 midwives in Estonia. Two postal reminders were sent to non-responders.Results: the response rate was 73.5%. There was no consensus among respondents about whose responsibility it was to diagnose pregnancy, carry out the risk assessment of a pregnancy, or carry out fetal monitoring during pregnancy. There was consensus among respondents that either midwives or obstetrician-gynaecologists could have responsibility for referring for further tests and examinations if a pregnancy was thought to be at risk. There was also consensus that counselling, biometry and blood pressure monitoring should be the sole responsibility of midwives.Key conclusions: despite national policy to shift ANC towards being midwifery-led and despite provisions in a European Directive permitting most roles in ANC to be performed autonomously by trained midwives, there is no consensus among Estonian midwives that all aspects of ANC should be their responsibility at present. Thorough research is required to establish which specific ANC roles Estonian midwives are not willing to take responsibility for, and to examine why they are not willing to take on such roles. [ABSTRACT FROM AUTHOR]

of recommendationsCorticosteroids should be administered to women at a gestational age between 24 +0 and 33 +6  weeks, when preterm birth is anticipated in the next seven days, as these have been consistently shown to reduce neonatal mortality and morbidity. (Strong-quality evidence; strong recommendation). In selected cases, extension of this period up to 34 +6  weeks may be considered (Expert opinion). Optimal benefits are found in infants delivered within 7 days of corticosteroid administration. Even a single-dose administration should be given to women with imminent preterm birth, as this is likely to improve neurodevelopmental outcome (Moderate-quality evidence; conditional recommendation).Either betamethasone (12 mg administered intramuscularly twice, 24-hours apart) or dexamethasone (6 mg administered intramuscularly in four doses, 12-hours apart, or 12 mg administered intramuscularly twice, 24-hours apart), may be used (Moderate-quality evidence; Strong recommendation). Administration of two "all" doses is named a "course of corticosteroids".Administration between 22 +0 and 23 +6  weeks should be considered when preterm birth is anticipated in the next seven days and active newborn life-support is indicated, taking into account parental wishes. Clear survival benefit has been observed in these cases, but the impact on short-term neurological and respiratory function, as well as long-term neurodevelopmental outcome is still unclear (Low/moderate-quality evidence; Weak recommendation).Administration between 34 + 0 and 34 + 6 weeks should only be offered to a few selected cases (Expert opinion). Administration between 35 +0 and 36 +6  weeks should be restricted to prospective randomized trials. Current evidence suggests that although corticosteroids reduce the incidence of transient tachypnea of the newborn, they do not affect the incidence of respiratory distress syndrome, and they increase neonatal hypoglycemia. Long-term safety data are lacking (Moderate quality evidence; Conditional recommendation).Administration in pregnancies beyond 37 +0  weeks is not indicated, even for scheduled cesarean delivery, as current evidence does not suggest benefit and the long-term effects remain unknown (Low-quality evidence; Conditional recommendation).Administration should be given in twin pregnancies, with the same indication and doses as for singletons. However, existing evidence suggests that it should be reserved for pregnancies at high-risk of delivering within a 7-day interval (Low-quality evidence; Conditional recommendation). Maternal diabetes mellitus is not a contraindication to the use of antenatal corticosteroids (Moderate quality evidence; Strong recommendation).A single repeat course of corticosteroids can be considered in pregnancies at less than 34 +0  weeks gestation, if the previous course was completed more than seven days earlier, and there is a renewed risk of imminent delivery (Low-quality evidence; Conditional recommendation).

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&#95;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.

Introduction and Hypothesis: Pelvic Floor Distress Inventory (PFDI-20) and the Pelvic Floor Impact Questionnaire (PFIQ-7) are reliable instruments for evaluating the quality of life in women with pelvic organ prolapse (POP). They have been translated and validated in many languages. The study was aimed at validating the Estonian translations of the PFDI-20 and PFIQ-7 tools., Methods: The questionnaires were translated into Estonian using a multistep translation method. A total of 132 women were enrolled: patients with diagnosed POP (n=57) were allocated to test-retest reliability analyses, and those with no POP signs (n=88) completed the questionnaire only once. The total scores of questionnaires and their subscales of both patient and reference groups were compared. Item response rate, floor and ceiling effects, corrected item-total correlations, internal consistency, and convergent and discriminant validity were analyzed. The study was approved by the Ethics Committee of Human Research of the University Clinic of Tartu, Estonia, and informed consent was obtained from each participant., Results: The translated questionnaires demonstrated good internal consistency (Cronbach's α values 0.77-0.93). The item response rate was 99%. Intra-class correlations (ICC) were strong for PFDI-20 and PFIQ-7 and their subscales ranged from 0.86 to 0.96. Construct validity of the tools demonstrated by manyfold higher scores among patients with POP compared with women without POP (p<0.0001)., Conclusions: The Estonian versions of the PFDI-20 and PFIQ-7 tools are reliable and valid instruments for assessing the quality of life in women with POP., (© 2023. The Author(s).)

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 3 rd 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., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ratnik, Rull, Aasmets, Kikas, Hanson, Kisand, Fischer and Laan.)

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., (© 2022 Walter de Gruyter GmbH, Berlin/Boston.)

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 ( I Ks ) 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.

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 CDY genes 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., Competing Interests: PH, LK, MP, EA, SR, MG, RF, MJ, OP, KP, PK, KR, YX, CT, ML No competing interests declared, (© 2021, Hallast et al.)

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., (© American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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.

The knowledge of genetic variants shaping human placental transcriptome is limited and they are not cataloged in the Genotype-Tissue Expression project. So far, only one whole genome analysis of placental expression quantitative trait loci (eQTLs) has been published by Peng et al. (2017) with no external independent validation. We report the second study on the landscape of placental eQTLs. The study aimed to generate a high-confidence list of placental cis -eQTLs and to investigate their potential functional implications. Analysis of cis -eQTLs (±100 kbp from the gene) utilized 40 placental RNA sequencing and respective whole genome genotyping datasets. The identified 199 placental cis -eSNPs represented 88 independent eQTL signals (FDR < 5%). The most significant placental eQTLs (FDR < 10 -5 ) modulated the expression of ribosomal protein RPL9, transcription factor ZSCAN9 and aminopeptidase ERAP2. The analysis confirmed 50 eSNP-eGenes pairs reported by Peng et al. (2017) and thus, can be claimed as robust placental eQTL signals. The study identified also 13 novel placental eGenes. Among these, ZSCAN9 is modulated by several eSNPs (experimentally validated: rs1150707) that have been also shown to affect the methylation level of genes variably escaping X-chromosomal inactivation. The identified 63 placental eGenes exhibited mostly mixed or ubiquitous expression. Functional enrichment analysis highlighted 35 Gene Ontology categories with the top ranking pathways "ruffle membrane" (FDR = 1.81 × 10 -2 ) contributing to the formation of motile cell surface and "ATPase activity, coupled" (FDR = 2.88 × 10 -2 ), critical for the membrane transport. Placental eGenes were also significantly enriched in pathways implicated in development, signaling and immune function. However, this study was not able to confirm a significant overrepresentation of genome-wide association studies top hits among the placental eSNP and eGenes, reported by Peng et al. (2017). The identified eSNPs were further analyzed in association with newborn and pregnancy traits. In the discovery step, a suggestive association was detected between the eQTL of ALPG (rs11678251) and reduced placental, newborn's and infant's weight. Meta-analysis across REPROMETA, HAPPY PREGNANCY, ALSPAC cohorts ( n = 6830) did not replicate these findings. In summary, the study emphasizes the role of genetic variation in driving the transcriptome profile of the human placenta and the importance to explore further its functional implications.

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.

Very few studies have analyzed how the composition of mother's microbiota affects the development of infant's gut and oral microbiota during the first months of life. Here, microbiota present in the mothers' gut, vagina, breast milk, oral cavity, and mammary areola were compared with the gut and oral microbiota of their infants over the first six months following birth. Samples were collected from the aforementioned body sites from seven mothers and nine infants at three different time points over a 6-month period. Each sample was analyzed with 16S rRNA gene sequencing. The gut microbiota of the infants harbored distinct microbial communities that had low similarity with the various maternal microbiota communities. In contrast, the oral microbiota of the infants exhibited high similarity with the microbiota of the mothers' breast milk, mammary areola and mouth. These results demonstrate that constant contact between microbial communities increases their similarity. A majority of the operational taxonomic units in infant gut and oral microbiota were also shared with the mothers' gut and oral communities, respectively. The disparity between the similarity and the proportion of the OTUs shared between infants' and mothers' gut microbiota might be related to lower diversity and therefore competition in infants' gut microbiota.

RNA degradation is a ubiquitous process that occurs in living and dead cells, as well as during handling and storage of extracted RNA. Reduced RNA quality caused by degradation is an established source of uncertainty for all RNA-based gene expression quantification techniques. RNA sequencing is an increasingly preferred method for transcriptome analyses, and dependence of its results on input RNA integrity is of significant practical importance. This study aimed to characterize the effects of varying input RNA integrity [estimated as RNA integrity number (RIN)] on transcript level estimates and delineate the characteristic differences between transcripts that differ in degradation rate. The study used ribodepleted total RNA sequencing data from a real-life clinically collected set ( n = 32) of human solid tissue (placenta) samples. RIN-dependent alterations in gene expression profiles were quantified by using DESeq2 software. Our results indicate that small differences in RNA integrity affect gene expression quantification by introducing a moderate and pervasive bias in expression level estimates that significantly affected 8.1% of studied genes. The rapidly degrading transcript pool was enriched in pseudogenes, short noncoding RNAs, and transcripts with extended 3' untranslated regions. Typical slowly degrading transcripts (median length, 2389 nt) represented protein coding genes with 4-10 exons and high guanine-cytosine content.-Reiman, M., Laan, M., Rull, K., Sõber, S. Effects of RNA integrity on transcript quantification by total RNA sequencing of clinically collected human placental samples., (© FASEB.)

We have previously shown an extensive load of somatic copy number variations (CNVs) in the human placental genome with the highest fraction detected in normal term pregnancies. Hereby, we hypothesized that insufficient promotion of CNVs may impair placental development and lead to recurrent pregnancy loss (RPL). RPL affects ~3% of couples aiming at childbirth and idiopathic RPL represents ~50% of cases. We analysed placental and parental CNV profiles of idiopathic RPL trios (mother-father-placenta) and duos (mother-placenta). Consistent with the hypothesis, the placental genomes of RPL cases exhibited 2-fold less CNVs compared to uncomplicated 1 st trimester pregnancies (P = 0.02). This difference mainly arose from lower number of duplications. Overall, 1 st trimester control placentas shared only 5.3% of identified CNV regions with RPL cases, whereas the respective fraction with term placentas was 35.1% (P = 1.1 × 10 -9 ). Disruption of the genes NUP98 (embryonic stem cell development) and MTRR (folate metabolism) was detected exclusively in RPL placentas, potentially indicative to novel loci implicated in RPL. Interestingly, genes with higher overall expression were prone to deletions (>3-fold higher median expression compared to genes unaffected by CNVs, P = 6.69 × 10 -20 ). Additionally, large pericentromeric and subtelomeric CNVs in parental genomes emerged as a risk factor for RPL.

Recurrent pregnancy loss (RPL) concerns ~3% of couples aiming at childbirth. In the current study, transcriptomes and miRNomes of 1 st trimester placental chorionic villi were analysed for 2 RPL cases (≥6 miscarriages) and normal, but electively terminated pregnancies (ETP; n = 8). Sequencing was performed on Illumina HiSeq 2000 platform. Differential expression analyses detected 51 (27%) transcripts with increased and 138 (73%) with decreased expression in RPL compared to ETP (DESeq: FDR P < 0.1 and DESeq2: <0.05). RPL samples had substantially decreased transcript levels of histones, regulatory RNAs and genes involved in telomere, spliceosome, ribosomal, mitochondrial and intra-cellular signalling functions. Downregulated expression of HIST1H1B and HIST1H4A (Wilcoxon test, fc≤0.372, P≤9.37 × 10 -4 ) was validated in an extended sample by quantitative PCR (RPL, n = 14; ETP, n = 24). Several upregulated genes are linked to placental function and pregnancy complications: ATF4, C3, PHLDA2, GPX4, ICAM1, SLC16A2. Analysis of the miRNA-Seq dataset identified no large disturbances in RPL samples. Notably, nearly 2/3 of differentially expressed genes have binding sites for E2F transcription factors, coordinating mammalian endocycle and placental development. For a conceptus destined to miscarriage, the E2F TF-family represents a potential key coordinator in reprogramming the placental genome towards gradually stopping the maintenance of basic nuclear and cellular functions.

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: β = 249.2 pg/mL; P = .014) and rs12678447 (G allele: minor allele frequency, 7%; β = 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.

One in five pregnant women suffer from gestational complications, prevalently driven by placental malfunction. Using RNASeq, we analyzed differential placental gene expression in cases of normal gestation, late-onset preeclampsia (LO-PE), gestational diabetes (GD) and pregnancies ending with the birth of small-for-gestational-age (SGA) or large-for-gestational-age (LGA) newborns (n = 8/group). In all groups, the highest expression was detected for small noncoding RNAs and genes specifically implicated in placental function and hormonal regulation. The transcriptome of LO-PE placentas was clearly distinct, showing statistically significant (after FDR) expressional disturbances for hundreds of genes. Taqman RT-qPCR validation of 45 genes in an extended sample (n = 24/group) provided concordant results. A limited number of transcription factors including LRF, SP1 and AP2 were identified as possible drivers of these changes. Notable differences were detected in differential expression signatures of LO-PE subtypes defined by the presence or absence of intrauterine growth restriction (IUGR). LO-PE with IUGR showed higher correlation with SGA and LO-PE without IUGR with LGA placentas. Whereas changes in placental transcriptome in SGA, LGA and GD cases were less prominent, the overall profiles of expressional disturbances overlapped among pregnancy complications providing support to shared placental responses. The dataset represent a rich catalogue for potential biomarkers and therapeutic targets.

Introduction: Annexin A5 is an essential component of placental integrity that may potentially mediate susceptibility to phenotypes of compromised pregnancy. A promoter haplotype termed M2 of the coding gene ANXA5 has been implicated in various pregnancy complications such as preeclampsia and recurrent pregnancy loss (RPL), however with inconclusive results., Study Subjects and Methods: A retrospective case-control study combining resequencing and restriction fragment length polymorphism (RFLP) analysis was undertaken in 313 women with unexplained RPL and 214 fertile women from Estonia and Denmark to estimate the RPL disease risk of the M2 haplotype in Northern Europe. Comparative prevalence of the studied ANXA5 genetic variants in human populations was estimated based on the 1000 Genomes Project (n = 675, whole-genome sequencing data) and the KORA S3 500K dataset of South German samples (n = 1644, genome-wide genotyping data)., Results: Minor allele frequency of common polymorphisms in ANXA5 promoter was up to two-fold lower among Estonian RPL subjects than fertile controls. The M2 haplotype was not associated with RPL and a trend for decreased prevalence was observed among RPL patients compared to controls both in Estonia (8.1% vs 15.2%, respectively) and Denmark (9.7% vs 12.6%). The high M2 prevalence in fertile controls was consistent with estimations for European and East Asian populations (9.6%-16.0%)., Conclusions: This study cautions to consider the M2 haplotype as a deterministic factor in early pregnancy success because: i) no RPL disease risk was associated with the haplotype in two clinically well-characterized RPL case-control study samples, ii) high prevalence of the haplotype among fertile controls and world-wide populations is inconsistent with the previously proposed severe impact on early pregnancy success, iii) weak impact of M2 haplotype on the production of ANXA5 protein has been established by others.

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.

Given the possible critical importance of placental gene imprinting and random monoallelic expression on fetal and infant health, most of those genes must be identified, in order to understand the risks that the baby might meet during pregnancy and after birth. Therefore, the aim of the current study was to introduce a workflow and tools for analyzing imprinted and random monoallelic gene expression in human placenta, by applying whole-transcriptome (WT) RNA sequencing of placental tissue and genotyping of coding DNA variants in family trios. Ten family trios, each with a healthy spontaneous single-term pregnancy, were recruited. Total RNA was extracted for WT analysis, providing the full sequence information for the placental transcriptome. Parental and child blood DNA genotypes were analyzed by exome SNP genotyping microarrays, mapping the inheritance and estimating the abundance of parental expressed alleles. Imprinted genes showed consistent expression from either parental allele, as demonstrated by the SNP content of sequenced transcripts, while monoallelically expressed genes had random activity of parental alleles. We revealed 4 novel possible imprinted genes (LGALS8, LGALS14, PAPPA2 and SPTLC3) and confirmed the imprinting of 4 genes (AIM1, PEG10, RHOBTB3 and ZFAT-AS1) in human placenta. The major finding was the identification of 4 genes (ABP1, BCLAF1, IFI30 and ZFAT) with random allelic bias, expressing one of the parental alleles preferentially. The main functions of the imprinted and monoallelically expressed genes included: i) mediating cellular apoptosis and tissue development; ii) regulating inflammation and immune system; iii) facilitating metabolic processes; and iv) regulating cell cycle.

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., (© 2014 The Authors. *Human Mutation published by Wiley Periodicals, Inc.)

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., (Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Heterodimeric hCG is one of the key hormones determining early pregnancy success. We have previously identified rare missense mutations in hCGβ 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/β dimer. Although the amount of the mutant hCGβ 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 β-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 hCGβ genes CGB5 and CGB8.