Evolution of Minimally Invasive and Non-Invasive Preimplantation Genetic Testing: An Overview.

Preimplantation genetic testing (PGT) has become a common supplementary diagnοstic/testing tοol for in vitro fertilization (ΙVF) cycles due to a significant increase in cases of PGT fοr mοnogenic cοnditions (ΡGT-M) and de novο aneuplοidies (ΡGT-A) over the last ten years. This tendency is mostly attributable to the advancement and application of novel cytogenetic and molecular techniques in clinical practice that are capable of providing an efficient evaluation of the embryonic chromosomal complement and leading to better IVF/ICSI results. Although PGT is widely used, it requires invasive biopsy of the blastocyst, which may harm the embryo. Non-invasive approaches, like cell-free DNA (cfDNA) testing, have lower risks but have drawbacks in consistency and sensitivity. This review discusses new developments and opportunities in the field of preimplantation genetic testing, enhancing the overall effectiveness and accessibility of preimplantation testing in the framework of developments in genomic sequencing, bioinformatics, and the integration of artificial intelligence in the interpretation of genetic data.

Sperm Mitochondrial Content and Mitochondrial DNA to Nuclear DNA Ratio Are Associated with Body Mass Index and Progressive Motility.

BACKGROUND: Mitochondrial dysfunction is a risk factor in the pathogenesis of metabolic disorders. According to the energy requirements, oxidative phosphorylation and the electron transport chain work together to produce ATP in sufficient quantities in the mitochondria of eukaryotic cells. Abnormal mitochondrial activity causes fat accumulation and insulin resistance as cells require a balance between the production of ATP by oxidative phosphorylation (OXPHOS) in the mitochondria and the dissipation of the proton gradient to reduce damage from reactive oxygen species (ROS). This study aims to explore the relationship between the mitochondrial content of sperm and the ratio of mitochondrial DNA to nuclear DNA in relation to body mass index (BMI) and how it may affect the progressive motility of sperm cell. Understanding the relationships between these important variables will help us better understand the possible mechanisms that could connect sperm motility and quality to BMI, as well as further our understanding of male fertility and reproductive health. METHODS: Data were collected from 100 men who underwent IVF/ICSI at the University Hospital of Ioannina's IVF Unit in the Obstetrics and Gynecology Department. The body mass index (BMI) of the males tested was used to classify them as normal weight; overweight; and obese. Evaluations included sperm morphology; sperm count; sperm motility; and participant history. RESULTS: In the group of men with normal BMI, both BMI and progressive motility displayed a statistically significant association (p < 0.05) with mitochondrial DNA content, relative mitochondrial DNA copy number, and the mtDNA/nDNA ratio. Similar to this, there was a positive association between BMI and motility in the groups of men who were overweight and obese, as well as between the expression of mitochondrial DNA and the mtDNA/nDNA ratio, with statistically significant differences (p < 0.05). There was not a statistically significant difference observed in the association between the relative mtDNA copy number and BMI or motility for the overweight group. Finally, the relative mtDNA copy number in the obese group was only associated with motility (p = 0.034) and not with BMI (p = 0.24). CONCLUSIONS: We found that in all three groups, BMI and progressive motility exhibited comparable relationships with mitochondrial DNA expression and the mtDNA/nDNA ratio. However, only in the normal group and in the obese group, the relative mitochondrial DNA copy number showed a positive association with BMI and progressive motility.

Cancer Associated PRDM9: Implications for Linking Genomic Instability and Meiotic Recombination.

The PR domain-containing 9 or PRDM9 is a gene recognized for its fundamental role in meiosis, a process essential for forming reproductive cells. Recent findings have implicated alterations in the PRDM9, particularly its zinc finger motifs, in the onset and progression of cancer. This association is manifested through genomic instability and the misregulation of genes critical to cell growth, proliferation, and differentiation. In our comprehensive study, we harnessed advanced bioinformatic mining tools to delve deep into the intricate relationship between PRDM9F and cancer. We analyzed 136,752 breakpoints and found an undeniable association between specific PRDM9 motifs and the occurrence of double-strand breaks, a phenomenon evidenced in every cancer profile examined. Utilizing R statistical querying and the Regioner package, 55 unique sequence variations of PRDM9 were statistically correlated with cancer, from a pool of 1024 variations. A robust analysis using the Enrichr tool revealed prominent associations with various cancer types. Moreover, connections were noted with specific phenotypic conditions and molecular functions, underlining the pervasive influence of PRDM9 variations in the biological spectrum. The Reactome tool identified 25 significant pathways associated with cancer, offering insights into the mechanistic underpinnings linking PRDM9 to cancer progression. This detailed analysis not only confirms the pivotal role of PRDM9 in cancer development, but also unveils a complex network of biological processes influenced by its variations. The insights gained lay a solid foundation for future research aimed at deciphering the mechanistic pathways of PRDM9, offering prospects for targeted interventions and innovative therapeutic approaches in cancer management.

Functional association between telomeres, oxidation and mitochondria.

Prior research has substantiated the vital role of telomeres in human fertility. Telomeres are prerequisites for maintaining the integrity of chromosomes by preventing the loss of genetic material following replication events. Little is known about the association between sperm telomere length and mitochondrial capacity involving its structure and functions. Mitochondria are structurally and functionally distinct organelles that are located on the spermatozoon's midpiece. Mitochondria produce adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS), which is necessary for sperm motility and generate reactive oxygen species (ROS). While a moderate concentration of ROS is critical for egg-sperm fusion, and fertilization, excessive ROS generation is primarily related to telomere shortening, sperm DNA fragmentation, and alterations in the methylation pattern leading to male infertility. This review aims to highlight the functional connection between mitochondria biogenesis and telomere length in male infertility, as mitochondrial lesions have a damaging impact on telomere length, leading both to telomere lengthening and reprogramming of mitochondrial biosynthesis. Furthermore, it aims to shed light on how both inositol and antioxidants can positively affect male fertility.

Biological and Clinical Significance of Mosaicism in Human Preimplantation Embryos.

Applications and indications of assisted reproduction technology are expanding, but every new approach is under scrutiny and thorough consideration. Recently, groups of assisted reproduction experts have presented data that support the clinical use of mosaic preimplantation embryos at the blastocyst stage, previously excluded from transfer. In the light of published contemporary studies, with or without clinical outcomes, there is growing evidence that mosaic embryos have the capacity for further in utero development and live birth. Our in-depth discussion will enable readers to better comprehend current developments. This expansion into the spectrum of ART practices requires further evidence and further theoretical documentation, basic research, and ethical support. Therefore, if strict criteria for selecting competent mosaic preimplantation embryos for further transfer, implantation, fetal growth, and healthy birth are applied, fewer embryos will be excluded, and more live births will be achieved. Our review aims to discuss the recent literature on the transfer of mosaic preimplantation embryos. It also highlights controversies as far as the clinical utilization of preimplantation embryos concerns. Finally, it provides the appropriate background to elucidate and highlight cellular and genetic aspects of this novel direction.

Reverse Transcriptase Affects Gametogenesis and Preimplantation Development in Mouse.

BACKGROUND/AIM: The expression of reverse transcriptase (RT) in ovaries, testes, gametes and embryos highlights its critical role in cell growth and differentiation. We sought to investigate the effects of the potent RT inhibitor lamivudine in gametogenesis and mouse embryo preimplantation development. MATERIALS AND METHODS: Male and female FVB/N mice were treated with the reverse transcriptase inhibitor Lamivudine for seven consecutive weeks. Following treatment, mouse sperm parameters, testicular and ovarian morphology as well as post-IVF embryo development were evaluated. RESULTS: Lamivudine impaired the sperm parameters and the testicular structure in male mice, the number of primordial germ cells and primary oocytes in ovaries of female mice, and the embryos' morphology and development up to the blastocyst stage during in vitro culture. CONCLUSION: The administration of lamivudine affected the processes of spermatogenesis and oogenesis as well as the in vitro preimplantation development of mouse embryos.

Copy-number variation analysis in familial nonsyndromic congenital anomalies of the kidney and urinary tract: Evidence for the causative role of a transposable element-associated genomic rearrangement.

Most congenital anomalies of the kidney and urinary tract (CAKUT) are sporadic, but familial occurrence has been described, suggesting a genetic contribution. Copy­number variations (CNVs) were detected in patients with CAKUT to identify possible novel genomic regions associated with CAKUT. CNVs were investigated in 7 children with CAKUT from three unrelated families using array comparative genomic hybridization: female monozygotic twins with bilateral duplex collecting system/vesicoureteral reflux (VUR)/unilateral renal hypodyspasia (URHD); two male siblings with VUR/URHD; 3 male second cousins, one with bilateral VUR/URHD, one with bilateral VUR and one with ureterovesical junction obstruction (UVJO). Five patients had a normal constitution of CNVs, one had a duplication of 0.2 Mb on the 5q­arm (5q23.3), probably unrelated to CAKUT, and one with UVJO had a 1.4 Mb deletion on the 17q­arm (17q12) which includes a known CAKUT gene, HNF1B. The phenotype of HNF1B deletion was extended including renal magnesium wasting. A higher coverage in transposable elements (TEs) was found in the deleted region compared with the expected density in any random genomic region. Notably, the 5' breakpoint was mapped within a solo long terminal repeat (LTR) sequence. Moreover, highly similar members of solo LTR and mammalian interspersed repetitive (MIR) elements, as well as nucleotide sequence microhomology were detected at the breakpoint regions. In conclusion, the deletion detected in one patient suggests this genomic imbalance as causative for UVJO. A not very well known phenotype of HNF1B deletion resulting in both low urinary tract malformations and renal wasting of magnesium was described. The high load in TEs of the deleted region, the presence of highly similar elements, and the microhomology found at breakpoint regions may have contributed to the generation of the deletion. CNV analysis could reveal novel causative genomic regions in patients with CAKUT, and further studies in larger cohorts are needed.

The ovarian response to standard gonadotropin stimulation is influenced by AMHRII genotypes.

The aim of the current study was to explore whether anti-Müllerian hormone receptor II (AMHRII) genetic variants influence the hormonal profile and the ovarian response to standard gonadotropin stimulation of women undergoing medically assisted reproduction. Three hundred in vitro fertilization or intracytoplasmic sperm injection patients constituted the study population, while 300 women with at least one spontaneous pregnancy participated as controls. The follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2) and AMH levels were determined at the third day of the menstrual cycle. AMHRII 10A > G (rs11170555), 1749C > T (rs2071558) and -482A > G (rs2002555) polymorphisms were genotyped. The follicle and oocyte numbers, the follicle size and the clinical pregnancies were recorded. Regarding the AMHRII 1749C > T polymorphism, 1749CT women presented with higher total follicle and small follicle numbers compared to 1749CC women (p = 0.04 and p = 0.01, respectively). Whereas, as concerns the -482A > G polymorphism, -482AG women were characterized by higher total follicle and small follicle numbers comparing with -482AA women (p = 0.07 and p = 0.004, respectively). Finally, -482AG women presented with increased FSH levels compared to -482AA women (p < 0.05). However, no associations of AMHRII gene polymorphisms with serum AMH levels or clinical pregnancy rates were observed. AMHRII 1749C > T and -482A > G genetic variants were associated with the ovarian response to standard gonadotropin stimulation, affecting mainly the follicular growth.

ROBO2 gene variants in children with primary nonsyndromic vesicoureteral reflux with or without renal hypoplasia/dysplasia.

BACKGROUND: Primary nonsyndromic vesicoureteral reflux (VUR) and VUR with renal hypoplasia/dysplasia (VUR-RHD) are common congenital anomalies of the kidney and urinary tract (CAKUT). Sequence variations of the ROBO2 gene were investigated in children with nonsyndromic VUR or VUR-RHD. METHODS: Single-strand conformation polymorphism (SSCP) electrophoresis or multiple restriction fragment SSCP (MRF-SSCP), followed occasionally by direct sequencing, was used to screen 103 patients and 200 controls for nucleotide changes. Gene polymorphisms and transposable elements were investigated using bioinformatics. RESULTS: Two single-nucleotide polymorphisms were detected: IVS1-53 and IVS5-31. The frequency of A allele of IVS1-53G>A did not differ significantly between patients and controls. IVS1-53 does not affect mRNA splicing according to in silico analysis. IVS5-31A>G substitution was found in one patient, reported here for the first time in VUR. In silico results demonstrated alteration in two serine/arginine-rich (SR) protein-binding sites and two additional acceptor sites. The ROBO2 gene sequence was found to contain 25.9% transposable elements. CONCLUSION: ROBO2 variants were not found to be associated with nonsyndromic VUR or VUR-RHD, providing further evidence for genetic heterogeneity. The role of transposable elements in ROBO2 gene expression in CAKUT needs further investigation since they are generally considered to be mutagens.

Association of the (TAAAA)n repeat polymorphism of SHBG gene with the age at menopause in Greek postmenopausal women.

OBJECTIVE: To assess the potential association of the pentanucleotide (TAAAA)n repeat polymorphism in the promoter of SHBG gene with the age at menopause in a Greek female population. STUDY DESIGN: Cross-sectional study. Two hundred and ten postmenopausal women aged 46-63 years were enrolled. The age at the last menstrual period and anthropometric parameters were recorded in all participants. Blood sampling for genotyping of the (TAAAA)n polymorphism of SHBG gene was performed. MAIN OUTCOME MEASURE(S): Frequency and association of the (TAAAA)n alleles with age at menopause. RESULTS: The alleles with seven and eight TAAAA repeats were associated with the age at menopause. The age at menopause was higher in carriers than in non-carriers of the (TAAAA)7 allele (50.2±3.1 years vs. 48.0±4.8 years, p=0.026). Furthermore, the age at menopause was lower in women carrying the (TAAAA)8 allele (47.5±4.8 years) than in women not carrying this allele (48.8±4.4 years, p=0.048). CONCLUSIONS: The (TAAAA)7 and (TAAAA)8 alleles of the SHBG (TAAAA)n polymorphism may contribute to variation in the timing of natural menopause in postmenopausal women of Northwestern Greece.

Association of the A1330V and V667M polymorphisms of LRP5 with bone mineral density in Greek peri- and postmenopausal women.

UNLABELLED: Wnt signaling through low-density lipoprotein receptor-related protein 5 (LRP5) is an important determinant of bone mass regulation. OBJECTIVE: To explore the influence of two LRP5 single nucleotide polymorphisms (SNPs) A1330V and V667M on bone mineral density (BMD) and serum levels of osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand (RANKL) and bone metabolic markers in a Greek female population. STUDY DESIGN: Two hundred and nine postmenopausal and twelve perimenopausal women aged 40-63 years were enrolled. All participants underwent spinal BMD evaluation. Genotyping of A1330V and V667M polymorphisms was performed by real-time polymerase chain reaction. Levels of OPG, soluble RANKL (sRANKL) and bone metabolic markers were measured. RESULTS: As regards A1330V SNP, women carrying CT/TT genotypes had lower spinal BMD than women with CC (p<0.0001). Regarding V667M SNP, spinal BMD was lower in women with GA/AA than in women with GG genotypes (p<0.0001). These differences remained significant after adjustment for age, years since menopause and body mass index. The A1330V and V667M polymorphisms were in strong linkage disequilibrium. A significant interaction between A1330V and V667M SNPs on spinal BMD was revealed. The haplotype with both risk alleles of the two SNPs (AT) conferred more risk for low BMD than the haplotypes with one risk allele (GT or AC) or the haplotype-reference (GC) (p=0.046, p=0.045, and p=0.010, respectively). No effect was observed on circulating OPG, sRANKL levels and bone metabolic markers. CONCLUSIONS: These findings demonstrate that the A1330V and V667M polymorphisms are associated with low BMD in peri- and postmenopausal Greek women.

Gender association of the angiotensin-converting enzyme gene with ischaemic stroke.

We examined the association of the NG011648 polymorphism (insertion/deletion) of the angiotensin-converting enzyme (ACE) gene with ischaemic stroke occurrence, subtype of ischaemic stroke and ischaemic stroke patients' gender. Patients with first ever ischaemic stroke were recruited prospectively in a period of 18 months. Controls were matched with the patients for age, gender, and known risk factors for stroke. Demographic data, medical history, and vascular risk factors were collected. Genotypes were determined by polymerase chain reaction (PCR) and restriction enzyme analysis. Stroke and control groups were compared in regard to the prevalence of the NG011648 polymorphism. One hundred and seventy-six patients with ischaemic stroke and 178 controls were recruited and genotyped for NG011648 polymorphism (I/D) of the ACE gene. No significant difference in allele and genotype distributions emerged between control and patient groups, nor in the two subtype groups of lacunars and large artery atherosclerosis. After the data were stratified by gender, a low incidence of II homozygosity in female patients versus female controls (p = 0.05) and male patients (p = 0.013, Z score: -2.49) was found. Our results indicate that I/D polymorphisms may have a role in stroke onset, in respect to gender, with a possible favourable effect of II genotype in females.

Association of serum and follicular fluid SHBG levels and SHBG (TAAAA)n polymorphism with follicle size in women undergoing ovarian stimulation.

OBJECTIVE: Sex hormone-binding globulin (SHBG) is the main transport protein of sex steroids. Recently, it has been found to be produced by granulosa lutein cells, suggesting a local role of SHBG in the ovary. The aim of this study was to investigate whether serum and follicular fluid SHBG levels and SHBG (TAAAA)(n) polymorphism are related to follicle size and pregnancy rate in women undergoing in vitro fertilisation. METHODS: The study population consisted of 154 women with tubal and/or male-factor infertility undergoing IVF/ICSI and follicular fluid with oocytes from small (diameter ≤12 mm) and large (diameter ≥18 mm) follicles were studied. Genotyping of SHBG (TAAAA)(n) polymorphism was performed in peripheral blood samples. Serum and follicular fluids were used for hormones determination. RESULTS: Women with short allele genotypes (with less than 8 TAAAA repeats) had higher number of small follicles compared to women with long allele genotypes (5.6 ± 3.9 vs. 3.5 ± 3.2 small follicles, p < 0.003). Follicular fluid SHBG levels correlated positively with serum SHBG levels (p < 0.001) and with the total number of follicles (p < 0.02). Furthermore, small follicles had higher follicular fluid SHBG concentration compared to large follicles (102.9 ± 35.0 nmol/l vs. 85.85 ± 34.88 nmol/l, p < 0.028). CONCLUSION: SHBG levels and the SHBG (TAAAA)(n) polymorphism are associated with follicle size.

Absence of mutations in the HOXA11 and HOXD11 genes in children with congenital renal malformations.

Experimental studies have shown that homeobox genes are essential for the development of the kidney and urinary tract. Hoxa11/Hoxd11 double mutant mice demonstrate renal agenesis or hypoplasia. Since, to our knowledge, these genes have never been examined for alterations in humans with congenital anomalies of the kidney and urinary tract (CAKUT), we investigated whether mutations of HOXA11/HOXD11 genes are associated with non-syndromal congenital renal parenchymal malformations. DNA samples from 26 unrelated children with unilateral renal agenesis (URA), 20 with renal hypodysplasia (RHD) and 13 with multicystic dysplastic kidney (MCDK) were included in the study. Exons 1 and 2 of the HOXA11/HOXD11 genes were amplified individually by polymerase chain reaction (PCR) using 12 unique oligonucleotide primers. Single-strand conformation polymorphism (SSCP) analysis of overlapping polymerase chain reaction products was performed. SSCP analysis revealed no variant band shifts in the samples of the amplified segments of the 59 patients, suggesting lack of either mutation or polymorphisms. Our findings do not support the hypothesis that mutations in the HOXA11/HOXD11 coding regions are involved in the pathogenesis of human non-syndromal congenital renal parenchymal malformations. Further studies are necessary, since other genes known to affect nephrogenesis, as well as genetic and environmental factors, may be involved.

Non-invasive first-trimester detection of paternal beta-globin gene mutations and polymorphisms as predictors of thalassemia risk at chorionic villous sampling.

OBJECTIVE: The objective was to evaluate the beta-globin gene mutations and polymorphisms in cell-free fetal DNA in the early first trimester (7-9th weeks' gestation) for the prediction of thalassemia risk at chorionic villous sampling (CVS). STUDY DESIGN: Beta-globin gene mutations and polymorphisms were analyzed in 97 carrier families and 100 control couples. Using conventional PCR-DGGE we carried out cell-free fetal DNA analysis in 37 couples in whom only the father was an IVSI-110 carrier. RESULTS: Beta-globin gene mutations have 80% information content in contrast to 39% of polymorphisms. By non-invasive early first-trimester identification of the paternally transmitted IVSI-110 mutation, we reached a sensitivity and specificity of 96 and 100%, respectively. Although the detection rate of the Y chromosome in male fetuses was as high as 100%, beta-thalassemia allele drop-out cannot be excluded. CONCLUSIONS: Even though there is high sensitivity in non-invasive paternally transmitted beta-thalassemia mutation detection, intense effort must be made to avoid misdiagnoses before the clinical application of this approach.

Angiotensin II type 2 receptor gene polymorphism in Caucasian children with a wide spectrum of congenital anomalies of the kidney and urinary tract.

The A-1332G transition of the angiotensin II type 2 receptor (AT2R) gene was found to occur more often in males with ureteropelvic (UPJO) or ureterovesical junction obstruction (UVJO). However, other studies have shown controversial results. Tauhe frequency of this polymorphism was investigated in 275 Caucasian children (153 boys, 122 girls) with a wide spectrum of congenital anomalies both of upper (165) and lower (110) urinary tract system and in 200 controls (100 boys, 100 girls). Among the included malformations, renal agenesis and duplex collecting system (DCS) were studied for the first time. The frequency of the G allele did not differ among patients (193 of 397 total alleles, 48.6%) and controls (146 of 300, 48.7%). No significant difference was also found in the frequency of the G allele in subgroups of congenital uropathies compared with controls. When analysis was performed in males and females separately, no significant difference was found in the frequency of the G allele in male (45.1%) or female (50.8%) patients compared with male (57.0%) or female (44.5%) controls. Our data indicate that the AT2R gene A-1332G transition is not associated with the development of human congenital uropathies and further investigations should be carried out to unravel their etiology.

Impact of renin-angiotensin-aldosterone system genes on the treatment response of patients with hypertension and metabolic syndrome.

OBJECTIVE: To evaluate the influence of clinical, biochemical and genetic markers on the response to antihypertensive treatment in patients with essential hypertension and the metabolic syndrome (MetS). METHODS: Measurements of anthropometric indices, blood pressure (BP), and metabolic parameters were obtained from the medical records of 132 (77 women) newly diagnosed, untreated hypertensive patients. Renin-angiotensin-aldosterone system (RAAS) genes polymorphisms (including ACE I/D, angiotensinogen M235T, angiotensin II type 1 receptor [AT1-receptor] A1166C) were determined. Response to treatment was defined as BP less than 140/90 mmHg. RESULTS: Patients with MetS (n=60) had higher systolic BP and pulse pressure and a more atherogenic lipid profile than patients without MetS. The frequencies of the ACE and the AT1-receptor gene polymorphisms were similar between patients with and without MetS. Response to treatment was positively associated with pulse pressure, and the presence of the C allele as well as the AC genotype of the AT1-receptor gene and inversely with age after adjustment for confounding factors. CONCLUSIONS: RAAS genes distribution does not differ between hypertensive patients with and without the MetS. Higher baseline pulse pressure levels, the presence of the C allele and/or the AC genotype may be in favour of a better response to structured antihypertensive treatment in patients with MetS. However, these findings need to be evaluated in future studies.

Non-invasive prenatal detection of paternal origin hb lepore in a male fetus at the 7th week of gestation.

OBJECTIVE: To perform a reliable non-invasive prenatal detection of the Hb Lepore paternal mutation and determine the fetal gender in the first trimester of pregnancy. METHODS: DNA was extracted from a serum sample obtained from a pregnant woman at the mid first trimester of gestation. Hb Lepore-specific, mutant and normal, primers as well as Y-chromosome-specific STSs were used to carry out the analysis. RESULTS: Paternal Hb Lepore and the DYS14 and DYZ1 gene-specific sequences were detected in the serum sample obtained at the 7th week of pregnancy. None of the above sequences was detectable in the maternal peripheral blood cell DNA. CONCLUSION: Conventional polymerase chain reaction analysis of cell-free fetal DNA can be used to determine fetal gender and paternal Hb Lepore as early as the 7th week of pregnancy.

Interaction between the polymorphisms of the renin-angiotensin system in preeclampsia.

Preeclampsia is considered to be a multifactorial and multisystemic disorder with a genetic predisposition. Alterations in the renin-angiotensin system are considered to play a significant role in the pathogenesis of the disease. In order to investigate the possible association of the three most common polymorphisms of the renin-angiotensin system genes with preeclampsia we have examined 41 women with preeclampsia and 102 normotensive pregnant women. DNA samples were genotyped for the M235T polymorphism of the angiotensinogen gene (AGT), the insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme gene (ACE) and the A1166C polymorphism of the angiotensin II type 1 receptor gene (AT1R) by PCR. Allele and genotype frequencies of the AGT gene polymorphism differed between the two study groups. The TT genotype of the M235T polymorphism was significantly increased in women who developed preeclampsia (P<0.02). In addition, women with preeclampsia and TT genotype had more frequently the DD genotype or the 1166C allele than the control group showing a significant interaction between the genes. In conclusion, we found an association between the angiotensinogen variant 235T and preeclampsia as well as an interaction between the variant 235T and the two other genes studied.