Małgorzata Szczepańska

Oxidative stress may be a piece in the endometriosis puzzle

OBJECTIVE To assess the total antioxidant potential of women with endometriosis-associated infertility, women with idiopathic infertility, and fertile controls. DESIGN Retrospective study. SETTING University medical hospital. PATIENT(S) Sixty-five women admitted for diagnostic laparoscopy. MAIN OUTCOME MEASURES Peritoneal fluid samples were analyzed for superoxide dismutase activity and total antioxidant status on spectrophotometry, glutathione peroxidase activity on enzyme-linked immunosorbent assay, and lipid peroxides levels on colorimetry. RESULT(S) Mean activity of superoxide dismutase, glutathione peroxidase, and total antioxidant status was lowest and lipid peroxide level was highest among infertile patients with endometriosis. Women with idiopathic infertility, in contrast, had the highest superoxide dismutase, glutathione peroxidase, and total antioxidant status activity and the lowest lipid peroxide level. CONCLUSION(S) High antioxidant potential is not a contributing factor in women with idiopathic infertility. Low total antioxidant status and low activity of antioxidant enzymes in the peritoneal fluid of infertile women with endometriosis probably do not influence fertility in these women, but these factors may play a role in the development of the disease.

Cranioectodermal Dysplasia, Sensenbrenner Syndrome, Is a Ciliopathy Caused by Mutations in the IFT122 Gene

Cranioectodermal dysplasia (CED) is a disorder characterized by craniofacial, skeletal, and ectodermal abnormalities. Most cases reported to date are sporadic, but a few familial cases support an autosomal-recessive inheritance pattern. Aiming at the elucidation of the genetic basis of CED, we collected 13 patients with CED symptoms from 12 independent families. In one family with consanguineous parents two siblings were affected, permitting linkage analysis and homozygosity mapping. This revealed a single region of homozygosity with a significant LOD score (3.57) on chromosome 3q21-3q24. By sequencing candidate genes from this interval we found a homozygous missense mutation in the IFT122 (WDR10) gene that cosegregated with the disease. Examination of IFT122 in our patient cohort revealed one additional homozygous missense change in the patient from a second consanguineous family. In addition, we found compound heterozygosity for a donor splice-site change and a missense change in one sporadic patient. All mutations were absent in 340 control chromosomes. Because IFT122 plays an important role in the assembly and maintenance of eukaryotic cilia, we investigated patient fibroblasts and found significantly reduced frequency and length of primary cilia as compared to controls. Furthermore, we transiently knocked down ift122 in zebrafish embryos and observed the typical phenotype found in other models of ciliopathies. Because not all of our patients harbored mutations in IFT122, CED seems to be genetically heterogeneous. Still, by identifying CED as a ciliary disorder, our study suggests that the causative mutations in the unresolved cases most likely affect primary cilia function too.

Reduced expression of HOXA10 in the midluteal endometrium from infertile women with minimal endometriosis

Recent human and animal studies have suggested that reduced HOXA10 expression in the implantation window of eutopic endometrium may contribute to infertility in women with endometriosis. Therefore, we examined the HOXA10 transcript, protein and HOXA10 promoter methylation levels in midluteal eutopic endometrium from 17 infertile women with minimal endometriosis and 15 healthy fertile women from a Polish cohort. Real-time quantitative PCR (RQ-PCR) and western blotting analysis revealed significantly lower levels of HOXA10 transcript (P=0.019) and protein (P=0.048) levels in eutopic endometrium from infertile women with endometriosis as compared to healthy fertile women. Moreover, sodium bisulfite sequencing of three HOXA10 CpG islands showed significantly higher methylation levels of genomic DNA from midluteal eutopic endometrium from infertile women with endometriosis as compared to healthy fertile women (P=0.006). We confirmed that DNA hypermethylation can be one of the potential molecular mechanisms silencing HOXA10 expression in the midluteal endometrium associated with infertility in women with endometriosis.

Polymorphic variants of CYP17 and CYP19A and risk of infertility in endometriosis

Endometriosis is recognized as an estrogen‐dependent disease. There are conflicting data demonstrating single nuclear polymorphisms (SNPs) of CYP17 and CYP19 steroidogenic genes as related to endometriosis risk. We assessed the CYP17 5′‐untranslated region ‐34 A/G (rs743572) and CYP19 Ex10 + C1558T (rs10046) SNPs in stage I–II endometriosis.

Expression of HOXA-10 and HOXA-11 in the endometria of women with idiopathic infertility

In fertile women, HOXA-10 and HOXA-11 expression rises during the luteal phase, with the peak occurring during the implantation window, and stays at a high level until the end of the cycle. We evaluated the transcript and protein levels of HOXA-10 and HOXA-11 in the endometria of patients with idiopathic infertility (n = 15) and control patients (n = 10). The amounts of mRNA were determined by reverse transcription and real-time quantitative PCR. The protein levels were evaluated by Western blotting analysis. Using immunohistochemical techniques, we compared the localization of HOXA-10 and HOXA-11 proteins in the implantation window between the study and control groups. We observed statistically significantly decreased HOXA-10 and HOXA-11 transcript levels (p = 0.003, p = 0.012 respectively) in infertile patients compared to controls. There was no significant decrease in HOXA-10 protein levels between these groups (p = 0.074). However, we observed a significantly higher level of HOXA-11 protein in the endometria of infertile patients compared to controls (p = 0.015). HOXA-10 and HOXA-11 proteins were localized in the nuclei of the endometrial stromal cells. Immunohistochemical analyses did not reveal differences between amounts of HOXA-10 and HOXA-11 protein levels in infertility and control groups. Our results suggest that HOXA-10 and HOXA-11 gene expression in the endometrium during the implantation window may not be altered in patients with idiopathic infertility.

Polymorphic variants of folate and choline metabolism genes and the risk of endometriosis-associated infertility.

OBJECTIVE Endometriosis has been considered an epigenetic disease. Single nucleotide polymorphisms (SNPs) located in genes encoding enzymes of the folate and choline metabolism may affect DNA methyltransferase activity. STUDY DESIGN We studied 16 SNPs in 12 folate and choline metabolism genes, including BHMT (rs7356530 and rs3733890), BHMT2 (rs625879), CBS (844ins68), CHDH (rs893363 and rs2289205), CHKA (rs7928739), MTHFD1 (rs2236225), MTHFR (rs1801133), MTR (rs1805087), MTRR (rs1801394), PCYT1A (rs712012 and rs7639752), PEMT (rs4244593 and rs4646406) and TCN (rs1801198) in one hundred and sixty-three infertile women with minimal endometriosis and one hundred and fifty fertile women. RESULTS There were no significant differences between genotype and allele frequencies of these gene variants in infertile women with endometriosis (n=163) and controls (n=150). The lowest, but not statistically significant, p values of the trend test were observed for the CBS 844ins68 and MTR rs1805087 (ptrend=0.0527 and ptrend=0.0771, respectively) polymorphisms. However, the exhaustive multifactor dimensionality reduction analysis revealed an epistatic interaction between rs1801133 of MTHFR and rs4244593 of PEMT in endometriosis-associated infertility (p=0.0240). CONCLUSIONS Our results showed moderate evidence for the contribution of SNPs located in genes encoding folate and choline metabolism enzymes to infertility in women with endometriosis.

Polymorphic variants in vitamin D signaling pathway genes and the risk of endometriosis-associated infertility.

It has recently been reported that vitamin D blood plasma levels are associated with reduced risk of endometriosis. The present study aimed to investigate whether the vitamin D binding protein (GC), vitamin D receptor (VDR), and retinoid X receptor (RXR) gene variants may be genetic risk factors for endometriosis‑associated infertility. The subjects consisted of 154 women with endometriosis‑associated infertility and 347 controls. Using polymerase chain reaction restriction fragment length polymorphism and high resolution melt techniques, the GC rs1155563, rs2298849 and rs7041; RXRA rs10881578, rs10776909 and rs749759; VDR BsmI rs1544410; and FokI rs2228570 single nucleotide polymorphisms (SNPs) were investigated in the patients with endometriosis and the healthy controls. The results indicated that no significant differences were observed between the genotype and allele frequencies of all experimental SNPs in the vitamin D signaling pathway genes in women with endometriosis-associated infertility and controls. However, a significant association was present between the A‑T haplotype, consisting of VDR rs1544410 and rs222857 minor alleles, and endometriosis-associated infertility [OR=1.659 (1.122‑2.453), P=0.011]. The results of the present study suggested that VDR gene variants act as genetic risk factors for endometriosis‑associated infertility.

Polymorphic variants of DNMT3A and the risk of endometriosis

OBJECTIVE Overexpression of DNA methyltransferase 3A (DNMT3A) and aberrant methylation of various genes in eutopic endometrium have been demonstrated in women with endometriosis. We aimed to study whether DNMT3A polymorphisms could be a genetic risk factor for endometriosis and endometriosis-related infertility. STUDY DESIGN We studied 5 SNPs (rs2289195, rs7590760, rs13401241, rs749131 and rs1550117) located in the DNMT3A gene in 357 women with endometriosis and 640 controls. RESULTS We did not observe significant differences between genotype and allele frequencies of rs2289195, rs7590760, rs13401241, rs749131 and rs1550117 SNPs in women with endometriosis, endometriosis-related infertility, and controls. The lowest p values of the trend test were observed for DNMT3A rs1550117 in endometriosis and endometriosis-related infertility (p(trend)=0.049 and p(trend)=0.055, respectively). CONCLUSIONS Our results did not supply evidence for the contribution of SNPs located in DNMT3A to either endometriosis or endometriosis-related infertility.

Use of ex utero intrapartum treatment procedure in fetal neck and high airway anomalies – report of four clinical cases

Abstract Purpose: To present antenatal management and use of ex utero intrapartum treatment (EXIT) in different fetal neck and high airway anomalies. Material and Methods: We have presented four different cases of fetal neck or airway pathology which were indications for EXIT, at our department. Results: In three cases of fetal neck tumors, the primary precise antenatal diagnoses of tumors were confirmed after birth. The airways of all three fetuses were properly secured during EXIT by laryngologist. All these newborns survived. In the fourth case, a primary, antenatal diagnosis of congenital high airway obstruction syndrome due to severe trachea obstruction was not confirmed after birth. Finally, due to complete trachea dysgenesis, neither tracheoscopy nor tracheostomy was done during EXIT and the baby died. Conclusion: Despite a failure of intrapartum treatment in the fourth case, we strongly recommend this procedure for deliveries of fetuses with a suspicion of airway obstruction.

HSD3B2, HSD17B1, HSD17B2, ESR1, ESR2 and AR expression in infertile women with endometriosis

OBJECTIVES The development of endometriosis is associated with changes in the expression of genes encoding the 3β-hydroxysteroid dehydrogenase type II (HSD3B2) and 17β-hydroxysteroid dehydrogenase type II (HSD17B2), estrogen receptors 1 (ESR1) and 2 (ESR2) and the androgen receptor (AR). However, little is known about the expression of HSD3B2, HSD17B1, HSD17B2, ESR1 ESR2 and AR during the endometrial phases in eutopic endometrium from infertile women with endometriosis. MATERIAL AND METHODS Using RT-qPCR analysis, we assessed the expression of the studied genes in the follicular and luteal phases in eutopic endometrium from fertile women (n = 17) and infertile women (n = 35) with endometriosis. RESULTS In the mid-follicular eutopic endometrium, we observed a significant increase in HSD3B2 transcript levels in all infertile women with endometriosis (p = 0.003), in infertile women with stage I/II endometriosis (p = 0.008) and in infertile women with stage III/IV endometriosis (p = 0.009) compared to all fertile women. There was a significant increase in ESR1 tran-scripts in all infertile women with endometriosis (p = 0.008) and in infertile women with stage I/II endometriosis (p = 0.019) and in infertile women with stage III/IV endometriosis (p = 0.023) compared to all fertile women. In the mid-luteal eutopic endometrium, we did not observe significant differences in HSD3B2, HSD17B1, HSD17B2, ESR1, ESR2 and AR transcripts between infertile women with endometriosis and fertile women. CONCLUSIONS Observed significant increase in HSD3B2 and ESR1 transcripts in follicular eutopic endometrium from infer-tile women with endometriosis may be related to abnormal biological effect of E2 in endometrium, further affecting the development of human embryos.