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This article belongs to the Special Issue Novel Insights into Prenatal Genetic Testing. Background: Despite advances in routine prenatal cytogenetic testing, most anomalous fetuses remain without a genetic diagnosis. Exome sequencing (ES) is a molecular technique that identifies sequence variants across protein-coding regions and is now increasingly used in clinical practice. Fetal phenotypes differ from postnatal and, therefore, prenatal ES interpretation requires a large amount of data deriving from prenatal testing. The aim of our study was to present initial results of the implementation of ES to prenatal diagnosis in Polish patients and to discuss its possible clinical impact on genetic counseling. Methods: In this study we performed a retrospective review of all fetal samples referred to our laboratory for ES from cooperating centers between January 2017 and June 2021. Results: During the study period 122 fetuses were subjected to ES at our institution. There were 52 abnormal ES results: 31 in the group of fetuses with a single organ system anomaly and 21 in the group of fetuses with multisystem anomalies. The difference between groups was not statistically significant. There were 57 different pathogenic or likely pathogenic variants reported in 33 different genes. The most common were missense variants. In 17 cases the molecular diagnosis had an actual clinical impact on subsequent pregnancies or other family members. Conclusions: Exome sequencing increases the detection rate in fetuses with structural anomalies and improves genetic counseling for both the affected couple and their relatives. This research was funded by the Ministry of Health, granted to the Center of Postgradu- ate Medical Education, Poland, grant number Minigrant-501-1-106-44-20/MG4 to J.B., and by the National Science Centre, Poland, grant number Miniatura 2—Dec2018/02/X/NZ2/00709 to D.M. info:eu-repo/semantics/publishedVersion

Preeclampsia is a pregnancy disorder associated with shallow placentation, forcing placental cells to live in hypoxic conditions. This activates the transcription factor kappa B (NFκB) in maternal and placental cells. Although the role of NFκB in preeclampsia is well documented, its mechanism of activation in trophoblastic cells has been never studied. This study investigates the mechanism of NFκB activation in a first trimester trophoblastic cell line (HTR8/SVneo) stimulated by a medium containing serum from preeclamptic (PE) or normotensive (C) women in hypoxic (2% O2) or normoxic (8% O2) conditions. The results indicate that in HTR8/SVneo cells, the most widely studied NFκB pathways, i.e., canonical, non-canonical and atypical, are downregulated in environment PE 2% O2 in comparison to C 8% O2. Therefore, other pathways may be responsible for NFκB activation. One such pathway depends on the activation of NFκB by the p53/RSK1 complex through its phosphorylation at Serine 536 (pNFκB Ser536). The data generated by our study show that inhibition of the p53/RSK1 pathway by p53-targeted siRNA results in a depletion of pNFκB Ser536 in the nucleus, but only in cells incubated with PE serum at 2% O2. Thus, the p53/RSK1 complex might play a critical role in the activation of NFκB in trophoblastic cells and preeclamptic placentas.

Preeclampsia and hypertension complicate several pregnancies. Identifying women at risk of developing these conditions is essential to establish potential treatment modalities. Biomarkers such as C19MC microRNA in pregnant patients wopuld assist in defining pregnancy surveillance and implementing interventions. This study sought to analyze circulating C19MC microRNA as an early marker of hypertension and preeclampsia in pregnant patients. A systematic review was undertaken using the following registers: disease registries, pregnancy registries, and pregnancy exposure registries, and the following databases: PubMed, CINAHL, Web of Science, Scopus, and EMBASE. The risk of bias was assessed using the Cochrane technique. From the 45 publications retrieved from the registers and databases, only 21 were included in the review after the removal of duplicates, screening, and eligibility evaluation. All 210 publications had a low risk of bias and illuminated the potential use of circulating C19MC microRNA as an early marker of hypertension and preeclampsia in pregnant patients. Therefore, it was concluded that C19MC microRNA can be used as an early marker of gestational preeclampsia and hypertension.

Objectives: Recurrent reproductive loss (RPL) is a global health issue affecting a significant number of women. Approximately half of miscarriages have an unexplained etiology. Familial aggregation and twins studies prove that some cases of the RPL could have a genetic background. Recent evidences suggest that cytokines (e.g. IL-6, TNF alpha or TGF beta) and matrix metalloproteinases (MMP) are important for maintenance of pregnancy. Single gene polymorphisms (SNP), affecting these proteins production or their function may predispose to the loss of the pregnancy. The aim of this study was to evaluate the association between the following polymorphisms of IL6 (rs1800795), TNF (rs1800629), TGFB1 (rs1800471), MMP1 (rs1799750), MMP2 (rs2285053 and rs243865), MMP3 (rs35068180), MMP9 (rs3918242) and the recurrent pregnancy loss in polish population. Material and methods: Study subjects comprised of 67 patients with a history of recurrent pregnancy loss (≥ 2 miscarriages in history) and 75 controls. The distribution of genotypes for selected polymorphisms were determined by RFLP-PCR. Results: Maternal genotypes GG TNF, or 5A/5A MMP3 may be associated with the recurrent pregnancy loss. No association between the IL6, TGFB1, MMP1, MMP2, or MMP9 studied polymorphisms and the predisposition to miscarriage was found. Conclusions: This study demonstrated a possible association between rs1800629 TNF, rs35068180 MMP3 polymorphisms and recurrent pregnancy loss.

Although higher nuclear factor &kappa, B (NF&kappa, B) expression and activity is observed in preeclamptic placentas, its mechanism of activation is unknown. This is the first study to investigate whether the canonical, non-canonical, or atypical NF&kappa, B activation pathways may be responsible for the higher activation of NF&kappa, B observed in preeclamptic placentas. The study included 268 cases (130 preeclamptic women and 138 controls). We studied the expression of the genes coding for NF&kappa, B activators (NIK, IKK&alpha, IKK&beta, and CK2&alpha, ) and inhibitors (I&kappa, B&alpha, and I&kappa, B&beta, ) using RT-PCR in real time. The RT-PCR results were verified on the protein level using ELISA and Western blot. To determine the efficiency of the pathways, the ratios of activator(s) to one of the inhibitors (I&kappa, or I&kappa, ) were calculated for each studied pathway. The preeclamptic placentas demonstrated significantly lower IKK&alpha, but higher I&kappa, protein levels. In addition, the calculated activator(s) to inhibitor (I&kappa, ) ratios suggested that all studied pathways might be downregulated in preeclamptic placentas. Our results indicate that preeclamptic placentas may demonstrate mechanisms of NF&kappa, B activation other than the canonical, non-canonical, and atypical forms. In these mechanisms, inhibitors of NF&kappa, B may play a key role. These observations broaden the existing knowledge regarding the molecular background of preeclampsia development.

Congenital hypogonadotropic hypogonadism (CHH) is caused by dysfunction of hypothalamic gonadotropic-releasing hormone (GnRH) axis. The condition is both clinically and genetically heterogeneous with more than 40 genes implicated in pathogenesis. The goal of the present study was to identify causative mutations in CHH individuals employing 2 step procedure with a targeted NGS panel as first-line diagnostics and subsequently whole exome sequencing in unsolved cases. Known or novel potentially deleterious variants were found in 28 out of 47 tested CHH patients. Molecular diagnosis was reached in 19/47 CHH cases. In 13 cases monogenic variants were identified in ANOS1, FGFR1, GNRHR, CHD7, SOX10, and PROKR2, while 6 patients showed digenic or trigenic inheritance patterns. The achieved diagnostic rate was comparable to other studies on genetics of CHH. By evaluating and reporting more patients with CHH we make progress in unravelling its genetic complexity and move a step closer to personalised medicine.

The COVID-19 pandemic in 2020 affected the entire healthcare system in Poland, causing medical personnel to be relocated to other duties and limiting patients’ contacts with healthcare professionals. A large part of the planned diagnostics and treatment was delayed due to lack of equipment and personnel. Against this background, we analysed the implementation of the publicly funded prenatal screening programme (PSP) in Poland compared to the previous year. This is a cross-sectional study. We used nationwide datasets on the implementation of the prenatal testing programme over the period 2019–2020, datasets from the Statistics Poland on birth and the data on the development of the COVID-19 epidemic in Poland. In the year 2020, we observed a 12.41% decrease in woman enrolled to the programme compared to 2019. However, the decrease concerned only women under 35 years of age. With respect to the number of deliveries in the calendar year, the number of patients enrolled in the programme decreased by 3% (31% vs. 34%, p < 0.001). We also observed an increase in estriol measurements per the number of patients included in the programme, and a reduction in the number of PAPP-A tests in the first trimester, which proves an increased share of the triple test in the prenatal diagnosis of chromosomal aberrations. With respect to the number of deliveries, the number of amniocentesis procedures performed under PSP decreased by 0.19% (1.8% vs. 1.99%, p < 0.0001). In 2020, compared to the previous year, the number of patients included in the prenatal testing programme in Poland decreased. In terms of the number of births in Poland, the number of integrated screening tests also decreased, at the expense of increasing the percentage of triple tests. There were also significant reductions in the number of invasive diagnostic tests.

We present recommendations on performance and safety measures of ultrasound examinations in obstetrics and gynecology during the SARS COV-2 pandemic. The statement was prepared based on the current knowledge on the coronavirus by the Ultrasound Section of the Polish Society of Obstetrics and Gynecology. It has to be noted that the presented guidance is based on limited evidence and is primarily based on experiences published by authors from areas most affected by the virus thus far, such as China, Singapore, Hong Kong, and Italy. We realize that the pandemic situation is very dynamic. New data is published every day. Despite the imposed limitations related to the necessity of social distancing, it is crucial to remember that providing optimal care in safe conditions should remain the primary goal of healthcare providers. We plan to update the current guidelines as the situation develops.

Anterior segment dysgenesis (ASD) encompasses a spectrum of ocular disorders affecting the structures of the anterior eye chamber. Mutations in several genes, involved in eye development, are implicated in this disorder. ASD is often accompanied by diverse multisystemic symptoms and another genetic cause, such as variants in genes encoding collagen type IV. Thus, a wide spectrum of phenotypes and underlying genetic diversity make fast and proper diagnosis challenging. Here, we used AMELIE, an automatic text mining tool that enriches data with the most up-to-date information from literature, and wANNOVAR, which is based on well-documented databases and incorporates variant filtering strategy to identify genetic variants responsible for severely-manifested ASD in a newborn child. This strategy, applied to trio sequencing data in compliance with ACMG 2015 guidelines, helped us find two compound heterozygous variants of the B3GLCT gene, of which c.660+1G>A (rs80338851) was previously associated with the phenotype of Peters plus syndrome (PPS), while the second, NM_194318.3:c.755delC (p.T252fs), in exon 9 of the same gene was noted for the first time. PPS, a very rare subtype of ASD, is a glycosylation disorder, where the dysfunctional B3GLCT gene product, O-fucose-specific β-1,3-glucosyltransferase, is ineffective in providing a noncanonical quality control system for proper protein folding in cells. Our study expands the mutation spectrum of the B3GLCT gene related to PPS. We suggest that the implementation of automatic text mining tools in combination with careful variant filtering could help translate sequencing results into diagnosis, thus, considerably accelerating the diagnostic process and, thereby, improving patient management.

Background. Preeclamptic pregnancies often present an intensified inflammatory state associated with the nuclear activity of NFκB. NEMO is an essential regulator of nuclear factor kappa B (NFκB) in cytoplasmic and nuclear cellular compartments. The aim of the present study is to examine the level and localization of the NEMO protein in preeclamptic and nonpreeclamptic placentas. Methods. The study includes 97 preeclamptic cases and 88 controls. NEMO distribution was analyzed immunohistochemically. Its localization in the nuclear and cytoplasmic fractions, as well as in total homogenates of placental samples, was studied by western blot and ELISA. Results. The western blot and ELISA results indicate a significant difference in NEMO concentration in the total and nuclear fractions between preeclamptic and control samples (p<0.01 and p<0.001, respectively). In the cytoplasmic complement, similar levels of NEMO were found in preeclamptic and control placentas. In addition, immunohistochemical staining revealed that the NEMO protein is mainly localized in the syncytiotrophoblast layer, with controls demonstrating a stronger reaction with NEMO antibodies. This study also shows that the placental level of NEMO depends on the sex of the fetus. Conclusions. The depletion of the NEMO protein in the cellular compartments of placental samples may activate one of the molecular pathways influencing the development of preeclampsia, especially in pregnancies with a female fetus. A reduction of the NEMO protein in the nuclear fraction of preeclamptic placentas may intensify the inflammatory state characteristic for preeclampsia and increase the level of apoptosis and necrosis within preeclamptic placentas.

Background. Metalloproteinases (MMPs) play a pivotal role during the process of trophoblast invasion and placentation. The appearance of five functional single-nucleotide polymorphisms (SNP) in the genes of the metalloproteinases most commonly implicated in the implantation process may influence the development of preeclampsia. Methods. Blood samples were collected from 86 mothers and 86 children after preeclampsia and 85 mothers and 85 children with uncomplicated pregnancies. The distribution of genotypes for −1607 1G/2G MMP1, −735 C/T MMP2, −1306 C/T MMP2, −1171 5A/6A MMP3, and −1562C/T MMP9 polymorphisms was determined by RFLP-PCR. Results. The occurrence of 1G/1G MMP1 or 5A/5A MMP3 genotype in the mother or 1G/1G MMP1 or 5A/6A MMP3 genotype in the child is associated with preeclampsia development. Moreover, simultaneous maternal and fetal 1G/1G homozygosity increases the risk of preeclampsia development 2.39-fold and the set of maternal 5A/5A and fetal 5A/6A MMP3 genotypes by over 4.5 times. No association between the carriage of studied MMP2 or MMP9 polymorphisms and the predisposition to preeclampsia was found. Conclusion. The maternal 1G/1G MMP1 and 5A/5A MMP3 and fetal 1G/1G MMP1 and 5A/6A MMP3 gene polymorphisms may be strong genetic markers of preeclampsia, occurring either individually or together.

Background Metalloproteinases (MMPs) play a pivotal role during the process of trophoblast invasion and placentation. The appearance of five functional single-nucleotide polymorphisms (SNP) in the genes of the metalloproteinases most commonly implicated in the implantation process may influence the development of preeclampsia. Methods Blood samples were collected from 86 mothers and 86 children after preeclampsia and 85 mothers and 85 children with uncomplicated pregnancies. The distribution of genotypes for −1607 1G/2G MMP1, −735 C/T MMP2, −1306 C/T MMP2, −1171 5A/6A MMP3, and −1562C/T MMP9 polymorphisms was determined by RFLP-PCR. Results The occurrence of 1G/1G MMP1 or 5A/5A MMP3 genotype in the mother or 1G/1G MMP1 or 5A/6A MMP3 genotype in the child is associated with preeclampsia development. Moreover, simultaneous maternal and fetal 1G/1G homozygosity increases the risk of preeclampsia development 2.39-fold and the set of maternal 5A/5A and fetal 5A/6A MMP3 genotypes by over 4.5 times. No association between the carriage of studied MMP2 or MMP9 polymorphisms and the predisposition to preeclampsia was found. Conclusion The maternal 1G/1G MMP1 and 5A/5A MMP3 and fetal 1G/1G MMP1 and 5A/6A MMP3 gene polymorphisms may be strong genetic markers of preeclampsia, occurring either individually or together.

The precise etiology of preeclampsia is unknown. Family studies indicate that both genetic and environmental factors influence its development. One of these factors is NFkB, whose activation depends on NEMO (NFkB essential modulator. This is the first study to investigate the association between the existence of single nucleotide variant of the NEMO gene and the appearance of preeclampsia. A total of 151 women (72 preeclamptic women and 79 controls) and their children were examined. Sanger sequencing was performed to identify variants in the NEMO gene in the preeclamptic mothers. The maternal identified variants were then sought in the studied groups of children, and in the maternal and child controls, using RFLP-PCR. Real-time RT-PCR was performed to assess NEMO gene expression in maternal blood, umbilical cord blood and placentas. The sequencing process indicated the existence of two different variants in the 3'UTR region of the NEMO gene of preeclamptic women (IKBKG:c.*368C>A and IKBKG:c.*402C>T). The simultaneous occurrence of the TT genotype in the mother and the TT genotype in the daughter or a T allele in the son increased the risk of preeclampsia development 2.59 fold. Additionally, we found that the configuration of maternal/fetal genotypes (maternal TT/ daughter TT or maternal TT/son T) of IKBKG:c.*402C/T variant is associated with the level of NEMO gene expression. Our results showed that, the simultaneous occurrence of the maternal TT genotype (IKBKG:c.*402C>T variants) and TT genotype in the daughter or T allele in the son correlates with the level of NEMO gene expression and increases the risk of preeclampsia development. Our observations may offer a new insight into the genetic etiology and pathogenesis of preeclampsia.

Anterior segment dysgenesis (ASD) encompasses a spectrum of ocular disorders affecting the structures of the anterior eye chamber. Mutations in several genes, involved in eye development, are implicated in this disorder. ASD is often accompanied by diverse multisystemic symptoms and another genetic cause, such as variants in genes encoding collagen type IV. Thus, a wide spectrum of phenotypes and underlying genetic diversity make fast and proper diagnosis challenging. Here, we used AMELIE, an automatic text mining tool that enriches data with the most up-to-date information from literature, and wANNOVAR, which is based on well-documented databases and incorporates variant filtering strategy to identify genetic variants responsible for severely-manifested ASD in a newborn child. This strategy, applied to trio sequencing data in compliance with ACMG 2015 guidelines, helped us find two compound heterozygous variants of the B3GLCT gene, of which c.660+1G>A (rs80338851) was previously associated with the phenotype of Peters plus syndrome (PPS), while the second, NM_194318.3:c.755delC (p.T252fs), in exon 9 of the same gene was noted for the first time. PPS, a very rare subtype of ASD, is a glycosylation disorder, where the dysfunctional B3GLCT gene product, O-fucose-specific β-1,3-glucosyltransferase, is ineffective in providing a noncanonical quality control system for proper protein folding in cells. Our study expands the mutation spectrum of the B3GLCT gene related to PPS. We suggest that the implementation of automatic text mining tools in combination with careful variant filtering could help translate sequencing results into diagnosis, thus, considerably accelerating the diagnostic process and, thereby, improving patient management.

The mechanism of preeclampsia and its way of inheritance are still a mystery. Biochemical and immunochemical studies reveal a substantial increase in tumor necrosis factor alpha, interleukin-1 beta, and interleukin-6 concentrations in the blood of women with preeclampsia. The level of these factors is regulated by nuclear facxtor-kappa B, whose activation in a classical pathway requires inhibitory kappa B kinase gamma (known as NEMO or IKBKG). Moreover, NEMO can schedule between cytoplasma and the nucleus. In the nucleus, IKBKG interacts with other proteins, and thus, it is implicated in the regulation of different gene expressions, which are related to cell cycle progression, proliferation, differentiation, and apoptosis.This is the first study investigating the association between the level of NEMO gene expression and the presence of preeclampsia. We tested the hypothesis that the simultaneous increase in NEMO gene expression both in the mother and her fetus may be responsible for the preeclampsia development. Moreover, the relationships between clinical risk factors of preeclampsia and the levels of NEMO gene expression in blood, umbilical cord blood, and placentas were investigated.A total of 91 women (43 preeclamptic women and 48 controls) and their children were examined. Real-time reverse transcription-polymerase chain reaction was used to assess the amount total NEMO messenger ribonucleic acid (mRNA) content and the mRNA level of each NEMO transcript from exons 1A, 1B, and 1C in maternal blood, umbilical cord blood, and placentas. Univariate analyses and correlation tests were performed to examine the association between NEMO gene expression and preeclampsia.Newborn weight and height, maternal platelet number, and gestational age (week of delivery) were lower in the group of women with preeclampsia than controls. NEMO gene expression level was found to be almost 7 times higher in the group of women with preeclampsia than healthy controls. The correlation analysis found that a simultaneous increase in the expression level of total NEMO mRNA in maternal blood and the mRNA for total NEMO (Rs = 0.311, P.05), transcripts 1A (Rs = 0.463, P.01), 1B (Rs = 0.454, P.01), and 1C (Rs = 0.563, P .001) in fetal blood was observed in preeclamptic pregnancies. In addition, the mRNA levels for total NEMO and transcripts 1A, 1B, and 1C were lower in placentas derived from pregnancies complicated by preeclampsia.Simultaneous increase of NEMO gene expression in maternal and fetal blood seems to be relevant for preeclampsia development. The results of our study also suggest that a decreased NEMO gene expression level in preeclamptic placentas may be the main reason for their intensified apoptosis.

A report on dichorionic/diamniotic pregnancy in which only one, female, fetus was infected with cytomegalovirus and presented with severe congenital diseases at birth. Infection of the fetus occurred after recurrent maternal infection. The second, male, fetus did not have CMV infection. The cesarean section was performed at the 38th week of gestation. The birth weight of the infected girl was 1680g, the main symptoms, beside dystrophy, concerned the central nervous system: microcephaly, brain atrophy, hydrocephalus, corpus callosum agenesis. She also had Turner syndrome symptoms. The viral load was highest in the urine 81.2 x10^6/ml, in the cerebro-spinal fluid 15.4x10^6/ml and lower in blood 0.38 x10^5/ml. The concentration of specific IgG was 308 U/ml. Specific IgM was not detected. Throughout hospitalization, the infection maintained only one viral genotype gB2. Despite treatment with ganciclovir (10 weeks) and foscarnet (2 weeks), the girl died at the age of 8 months. Novel molecular diagnostic techniques (nested and real time PCR) confirmed the congenital infection and were helpful in the monitoring of the infection and treatment efficacy.

490 Matrix Metalloproteinase 2, Timp1 and Timp-2 Concentrations in Tracheal Aspirate Fluid and Plasma of Preterm Infants Developing Bronchopulmonary Dysplasia