An efficient molecular genetic testing strategy for incontinentia pigmenti based on single-tube long fragment read sequencing.

Incontinentia pigmenti (IP) is a rare X-linked dominant neuroectodermal dysplasia that primarily affects females. The only known causative gene is IKBKG, and the most common genetic cause is the recurrent IKBKG△4-10 deletion resulting from recombination between two MER67B repeats. Detection of variants in IKBKG is challenging due to the presence of a highly homologous non-pathogenic pseudogene IKBKGP1. In this study, we successfully identified four pathogenic variants in four IP patients using a strategy based on single-tube long fragment read (stLFR) sequencing with a specialized analysis pipeline. Three frameshift variants (c.519-3_519dupCAGG, c.1167dupC, and c.700dupT) were identified and subsequently validated by Sanger sequencing. Notably, c.519-3_519dupCAGG was found in both IKBKG and IKBKGP1, whereas the other two variants were only detected in the functional gene. The IKBKG△4-10 deletion was identified and confirmed in one patient. These results demonstrate that the proposed strategy can identify potential pathogenic variants and distinguish whether they are derived from IKBKG or its pseudogene. Thus, this strategy can be an efficient genetic testing method for IKBKG. By providing a comprehensive understanding of the whole genome, it may also enable the exploration of other genes potentially associated with IP. Furthermore, the strategy may also provide insights into other diseases with detection challenges due to pseudogenes.

Clinical Application of Noninvasive Prenatal Testing for Pregnant Women with Assisted Reproductive Pregnancy.

OBJECTIVE: This paper analyzes the clinical significance of noninvasive prenatal testing (NIPT) for fetal chromosome aneuploidy in the screening of in vitro fertilization-embryo transfer (IVF) pregnancies. METHODS: The study subjects consisted of 3163 IVF-pregnant women who underwent NIPT at the Women's Hospital, School of Medicine, Zhejiang University and Taizhou Hospital, Zhejiang Province from February 2015 to June 2019. Fetal or neonatal karyotype analysis was carried out in high-risk patients, with subsequent follow-up on pregnancy outcomes. RESULTS: NIPT results of 3163 pregnant women suggested 20 cases of high-risk fetal chromosome aneuploidy, of which 2185 cases were a single pregnancy. Of the 13 cases of high-risk chromosome aneuploidy in single pregnancies, seven were true positive, and six were false positive according to fetal or newborn chromosomal karyotype diagnosis. Twin pregnancies accounted for 978 cases in which NIPT indicated seven cases of high-risk chromosome aneuploidy; six of these cases were true positive, and one case was false positive according to fetal or newborn chromosomal karyotype diagnosis. The specificity, positive predictive value, and false-positive rate of trisomy 21 syndrome in IVF single embryo NIPT were 99.86%, 62.5%, and 0.14%, respectively. The specificity, positive predictive value, and false-positive rate of trisomy 18 syndrome were 99.95%, 66.67%, and 0.05%, respectively. The specificity of trisomy 13 syndrome was 99.91%, and the false-positive rate was 0.09%. The specificity of trisomy 21 syndrome in IVF twin NIPT was 99.89%, the positive predictive value was 83.33%, and the false-positive rate was 0.11%. The specificity and positive predictive value of fetal trisomy 18 syndrome were 100.00%, and the false-positive rate of it were 0.00%. Sensitivity and false-negative rates were 100% in all cases. CONCLUSION: NIPT is an ideal prenatal test for IVF-pregnant women due to its high sensitivity and specificity in screening for fetal aneuploidy.

[Genetic Study and Prenatal Diagnosis of a Family with Thrombocytopenia-Absent Radius (TAR) Syndrome].

OBJECTIVE: To analyze the potential genetic cause of thrombocytopenia-absent radius (TAR) syndrome in a family and provide prenatal diagnosis for them. METHODS: Genetic mutation analysis of the sporadic family with TAR syndrome was performed with chromosome microarray analysis (CMA), quantitative polymerase chain reaction (qPCR) and Sanger sequencing. DNA samples were collected from 4 members of the family, including the proband, her parents and her sister. CMA, qPCR and Sanger sequencing were performed to determine the pathogenic mutation and prenatal diagnosis of the fetus was made accordingly. RESULTS: The proband had a 378 kb genomic heterozygous deletion in 1q21.1, which contained RBM8 A and other genes. c.-21G>A mutation was also found in the RBM8 A of the proband. The above-mentioned microdeletion and mutation were inherited from the mother and father, respectively. Prenatal CMA suggested that the fetus carried a 378 kb microdeletion in 1q21.1, and DNA testing did not find c.-21G>A mutation. CONCLUSION: The heterozygous deletion in 1q21.1 and RBM8 A: c.-21G>A is considered to be the genetic etiology of TAR syndrome in the family. The study provides information for subsequent family genetic counseling and prenatal diagnosis.

Galectin-1 expression in the serum and placenta of pregnant women with fetal growth restriction and its significance.

BACKGROUND: This study aims to investigate galectin-1 (Gal-1) expression in the serum and placenta of pregnant women with fetal growth restriction (FGR) and its significance. METHODS: Thirty-one pregnant women with single-birth FGR but without comorbidities, eight pregnant women with FGR and preeclampsia (PE), and eight pregnant women with FGR and gestational diabetes mellitus (GDM) were enrolled as the study group, while 20 pregnant women with normal singleton pregnancy in the same period were enrolled as the control group. The serum Gal-1 level was detected using an enzyme-linked immunosorbent assay (ELISA), and Gal-1 expression in the placenta was detected by western blot. RESULTS: The results revealed that, compared with the control group, the serum Gal-1 level decreased in the women with FGR without comorbidities, and the difference was statistically significant (P < 0.001). Compared with the control group, the difference in serum Gal-1 expression in the FGR-PE group was not statistically significant (P = 0.29). The peripheral serum Gal-1 level decreased in the FGR-GDM group compared with the control group, and the difference was statistically significant (P < 0.001). The serum Gal-1 level was positively correlated with birth weight (r2 = 0.172, P < 0.01). Compared with the control group, the Gal-1 expression level decreased in the placenta of the pregnant women with FGR without comorbidities (P < 0.05). CONCLUSIONS: Gal-1 exhibits low expression in the serum and placenta of pregnant women with FGR. In addition, Gal-1 may be involved in the pathogenesis of FGR and could represent a new diagnostic marker of the disease.

The association between the two more common genetic causes of spermatogenic failure: a 7-year retrospective study.

Chromosomal abnormalities and Y chromosome microdeletions are considered to be the two more common genetic causes of spermatogenic failure. However, the relationship between chromosomal aberrations and Y chromosome microdeletions is still unclear. This study was to investigate the incidence and characteristics of chromosomal aberrations and Y chromosome microdeletions in infertile men, and to explore whether there was a correlation between the two genetic defects of spermatogenic failure. A 7-year retrospective study was conducted on 5465 infertile men with nonobstructive azoospermia or oligozoospermia. Karyotype analysis of peripheral blood lymphocytes was performed by standard G-banding techniques. Y chromosome microdeletions were screened by multiplex PCR amplification with six specific sequence-tagged site (STS) markers. Among the 5465 infertile men analyzed, 371 (6.8%) had Y chromosome microdeletions and the prevalence of microdeletions in azoospermia was 10.5% (259/2474) and in severe oligozoospermia was 6.3% (107/1705). A total of 4003 (73.2%) infertile men underwent karyotyping; 370 (9.2%) had chromosomal abnormalities and 222 (5.5%) had chromosomal polymorphisms. Karyotype analysis was performed on 272 (73.3%) patients with Y chromosome microdeletions and 77 (28.3%) had chromosomal aberrations, all of which involved sex chromosomes but not autosomes. There was a significant difference in the frequency of chromosomal abnormalities between men with and without Y chromosome microdeletions (P< 0.05).

Whole-Exome Sequencing Revealed Mutations of MED12 and EFNB1 in Fetal Agenesis of the Corpus Callosum.

Agenesis of the corpus callosum (ACC) is a birth defect in which the corpus callosum is either partially or completely missing. With recent advances in prenatal ultrasound, detection of ACC in obstetric practices is becoming more common. Etiologies of ACC include chromosome errors, genetic factors, prenatal infections, and other factors related to the prenatal environment. In an effort to elucidate more about the genetic influence in the pathogenesis of ACC, we identified, through whole-exome sequencing (WES), two gene mutations in two families with complete agenesis of the corpus callosum. These two mutations are located on chromosome X: one is a hemizygous missense mutation c.3746T>C (p. L1249P) in the gene mediator complex subunit 12 (MED12); the other one is a heterozygous missense mutation c.128+5G>C in gene ephrin B1 (EFNB1). Historically, early diagnosis of complete ACC during pregnancy has been difficult; however, WES has provided us with a creative avenue of diagnosis, combining identification of genetic mutations with prenatal imaging.

Comprehensive genetic diagnosis of patients with Duchenne/Becker muscular dystrophy (DMD/BMD) and pathogenicity analysis of splice site variants in the DMD gene.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by mutations in the DMD gene. The aim of this study is to identify pathogenic DMD variants in probands and reduce the risk of recurrence of the disease in affected families. Variations in 100 unrelated DMD/BMD patients were detected by multiplex ligation-dependent probe amplification (MLPA) and next-generation sequencing (NGS). Pathogenic variants in DMD were successfully identified in all cases, and 11 of them were novel. The most common mutations were intragenic deletions (69%), with two hotspots located in the 5' end (exons 2-19) and the central of the DMD gene (exons 45-55), while point mutations were observed in 22% patients. Further, c.1149+1G>A and c.1150-2A>G were confirmed by hybrid minigene splicing assay (HMSA). This two splice site mutations would lead to two aberrant DMD isoforms which give rise to severely truncated protein. Therefore, the clinical use of MLPA, NGS, and HMSA is an effective strategy to identify variants. Importantly, eight embryos were terminated pregnancies according to prenatal diagnosis and a healthy boy was successfully delivered by preimplantation genetic diagnosis (PGD). Early and accurate genetic diagnosis is essential for prenatal diagnosis/PGD to reduce the risk of recurrence of DMD in affected families.

Preimplantation genetic diagnosis and screening (PGD/S) using a semiconductor sequencing platform.

BACKGROUND: Recent advances in semiconductor sequencing platform (SSP) have provided new methods for preimplantation genetic diagnosis/screening (PGD/S). The present study aimed to evaluate the applicability and efficiency of SSP in PGD/S. METHODS: The artificial positive single-cell-like DNAs and normal single-cell samples were chosen to test our semiconductor sequencing platform for preimplantation genetic diagnosis/screening (SSP-PGD/S) method with two widely used whole-genome amplification (WGA) kits. A total of 557 single blastomeres were collected from in vitro fertilization (IVF) couples, and their WGA products were processed and analyzed by our SSP-PGD/S method in comparison with array comparative genomic hybridization (array-CGH). RESULTS: Our SSP-PGD/S method indicated high compatibilities with two commercial WGA kits. For 557 single blastomeres, our method with four million reads in average could detect 24-chromosome aneuploidies as well as microdeletion/microduplication of the size over 4 Mb, providing 100% consistent conclusion with array-CGH method in the classification of whether it was transplantable. CONCLUSIONS: Our studies suggested that SSP-PGD/S represents a valuable alternative to array-CGH and brought PGD/S into a new era of more rapid, accurate, and economic.

Detection of fetal duplication 16p11.2q12.1 by next-generation sequencing of maternal plasma and invasive diagnosis.

OBJECTIVE: The objective of study is to report the feasibility of non-invasive prenatal screening (NIPS) combined with invasive detection by chromosomal analysis in identifying fetal duplication, providing clinical performance of NIPS on copy number variations (CNVs) detection. MATERIAL AND METHODS: NIPS was offered to a 35-year-old pregnant woman. Amniocentesis was performed to confirm the positive screening result. Fetal sample was detected by karyotyping, fluorescence in situ hybridization (FISH), and chromosomal microarray (CMA). Parental karyotyping was also conducted. RESULTS: NIPS result was positive for chromosome 16, indicating an extra copy of chromosome 16. FISH and chromosomal karyotyping revealed that the fetus had a marker chromosome derived from chromosome 16. CMA further demonstrated an approximately 19-Mb duplication in chromosome 16. The final fetal karyotype was 47,XY,+mar. ish der (16)(D16Z3+).arr 16p11.2q12.1 (30 624 186-49 696 337 × 3). Ultrasound scan and MRI showed some structure malformations. CONCLUSIONS: A protocol for CNVs detection by combining a series of genetic methods was presented in this study and a novel marker duplication 16p11.2q12.1 was reported. With the ability to identify subchromosomal deletions and duplications in fetus, NIPS could reduce the possibility of invasive diagnosis. The followed confirmation test for positive sample is necessary and ensures the accuracy of the diagnosis.

Detection of fetal subchromosomal aberration with cell-free DNA screening led to diagnosis of parental translocation: Review of 11344 consecutive cases in a university hospital.

BACKGROUND: Fetal chromosome aberrations and sub-chromosomal copy number variations (CNVs) are not rare. There are several ways to detect duplications and deletions; cell-free DNA screening (cfDNA screening) is nowadays an accurate and safe detection method. The objective of this study is to report the feasibility of cfDNA screening as an indicator of parental balanced chromosome translocation. RESULTS: From February 2015 to March 2016, cfDNA screening was offered to 11344 pregnant women. 137 out of 11344 individuals tested positive for aneuploidies using cfDNA screening were confirmed by karyotyping. 6 additional cases also tested positive for other deletion/duplication were confirmed by chromosomal microarray analysis (CMA). 11201 patients tested negative and 10342 of them were confirmed through interviews after delivery. Among the 137 cases that were screened positive in cfDNA screening, 91 were common trisomies (63 cases of trisomy 21, 25 cases of trisomy 18 and 3 cases of trisomy 13) and 46 cases were positive for sex-chromosomal abnormalities. In addition, 6 cases were positive for other deletion/duplication in which 2 were identified as terminal duplication and deletion on different chromosomes. The cfDNA screening findings were confirmed by CMA or karyotyping, and the origins of CNVs were validated afterward by karyotyping or fluorescence in situ hybridization (FISH) using parental blood samples. CONCLUSION: CfDNA screening may help identify deletions and duplications in fetus, which in some cases may indicate risk of a parent being a balanced rearrangement carrier, and that the diagnostic follow-up testing is necessary.

A feasible diagnostic approach for the translocation carrier from the indication of products of conception.

BACKGROUND: Chromosome translocations are rare but frequently associated with infertility. The objective of this study is to investigate the feasibility of using chromosomal microarray analysis (CMA) on products of conception (POC) samples as an indicator of parental balanced translocation. From January 2011 to December 2016, CMA using Affymetrix Cytoscan™750K array was performed on 1294 POC samples in our hospital. Karyotyping and fluorescence in situ hybridization (FISH) using parental blood samples were performed to validate the origin of subchromosomal copy number variations (CNVs). RESULTS: In the 1294 cases of POCs, we detected CNVs of terminal duplication and deletion that imply unbalanced translocation derivatives in 16 cases, and accurate diagnosis with the parental study was made in all the cases by karyotyping and/or FISH. In 10/16 (62.5%) of these cases, CNVs were inherited from one carrier parent of balanced translocation (Cases 1 to 10), while 6/16 (37.5%) cases occurred de novo (Cases 11 to 16). CONCLUSION: This study clearly illustrated the importance of the utilization of CMA on POC, followed by parental karyotyping and FISH to better characterize CNVs. This approach is especially useful for couples in whom one partner carries a cryptic/submicroscopic balanced translocation but has an apparently normal karyotype.

Effects of pre-pregnancy body mass index and gestational weight gain on neonatal birth weight.

To evaluate the effects of maternal pre-pregnancy body mass index (pre-BMI) and gestational weight gain (GWG) on neonatal birth weight (NBW) in the population of Chinese healthy pregnant women, attempting to guide weight control in pregnancy. A retrospective cohort study of 3772 Chinese women was conducted. The population was stratified by maternal pre-BMI categories as underweight (<18.5 kg/m2), normal weight (18.5-23.9 kg/m2), overweight (24.0-27.9 kg/m2), and obesity (≥28.0 kg/m2). The NBW differences were tested among the four groups, and then deeper associations among maternal pre-BMI, GWG, and NBW were investigated by multivariate analysis. NBW increased significantly with the increase of maternal pre-BMI level (P<0.05), except overweight to obesity (P>0.05). The multivariate analysis showed that both pre-BMI and GWG were positively correlated with NBW (P<0.05). Compared with normal pre-BMI, underweight predicted an increased odds ratio of small-for-gestational-age (SGA) and decreased odds ratio for macrosomia and large-for-gestational-age (LGA), and the results were opposite for overweight. With the increase of GWG, the risk of SGA decreased and the risks of macrosomia and LGA increased. In addition, in different pre-BMI categories, the effects of weight gain in the first trimester on NBW were different (P<0.05). NBW is positively affected by both maternal pre-BMI and GWG, extreme pre-BMI and GWG are both associated with increased risks of abnormal birth weight, and maternal pre-BMI may modify the effect of weight gain in each trimester on NBW. A valid GWG guideline for Chinese women is an urgent requirement, whereas existing recommendations seem to be not very suitable for the Chinese.

Identification of novel PKD1 and PKD2 mutations in a Chinese population with autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most frequently inherited renal diseases caused by mutations in PKD1 and PKD2. We performed mutational analyses of PKD genes in 49 unrelated patients using direct PCR-sequencing and multiplex ligation-dependent probe amplification (MLPA) for PKD1 and PKD2. RT-PCR analysis was also performed in a family with a novel PKD2 splicing mutation. Disease-causing mutations were identified in 44 (89.8%) of the patients: 42 (95.5%) of the patients showed mutations in PKD1, and 2 (4.5%) showed mutations in PKD2. Ten nonsense, 17 frameshift, 4 splicing and one in-frame mutation were found in 32 of the patients. Large rearrangements were found in 3 patients, and missense mutations were found in 9 patients. Approximately 61.4% (27/44) of the mutations are first reported with a known mutation rate of 38.6%. RNA analysis of a novel PKD2 mutation (c.595_595 + 14delGGTAAGAGCGCGCGA) suggested monoallelic expression of the wild-type allele. Furthermore, patients with PKD1-truncating mutations reached end-stage renal disease (ESRD) earlier than patients with non-truncating mutations (47 ± 3.522 years vs. 59 ± 11.687 years, P = 0.016). The mutation screening of PKD genes in Chinese ADPKD patients will enrich our mutation database and significantly contribute to improve genetic counselling for ADPKD patients.

Bone morphogenetic protein-15 in follicle fluid combined with age may differentiate between successful and unsuccessful poor ovarian responders.

BACKGROUND: The counselling of poor ovarian responders about the probability of pregnancy remains a puzzle for gynaecologists. The aim of this study was to optimise the management of poor responders by investigating the role of the oocyte-derived factor bone morphogenetic protein-15 (BMP-15) combined with chronological age in the prediction of the outcome of in-vitro fertilisation-embryo transfer (IVF-ET) in poor responders. METHODS: A retrospective study conducted in a university hospital. A total of 207 poor ovarian responders who reached the ovum pick-up stage undergoing IVF/intracytoplasmic sperm injection (ICSI) with three or fewer follicles no less than 14 mm on the day of oocyte retrieval were recruited from July 1, 2008 to December 31, 2009. Another 215 coinstantaneous cycles with normal responses were selected as controls. The BMP-15 levels in the follicular fluid (FF) of the 207 poor responders were analysed by western blot. Based on the FF BMP-15 level and age, poor responders were sub-divided into four groups. The main outcome measures were the FF BMP-15 level, implantation rate, pregnancy rate, and live birth rate. RESULTS: The implantation rate (24.2% vs. 15.3%), chemical pregnancy rate (40% vs. 23.7%), clinical pregnancy rate (36.5% vs. 20.4%) and live birth rate (29.4% vs. 15.1%) in the high BMP-15 group were significantly higher than those in the low BMP-15 group. Furthermore, poor responders aged less than or equal to 35 years with a higher FF BMP-15 level had the best implantation, pregnancy and live birth rates, which were comparable with those of normal responders. CONCLUSIONS: Our study suggests a potential role of BMP-15 in the prediction of the IVF outcome. A high FF BMP-15 combined with an age less than or equal to 35 years may be used as a potential indicator for repeating IVF cycles in poor ovarian responders.

Endoplasmic reticulum stress induced by oxidative stress in decidual cells: a possible mechanism of early pregnancy loss.

Early pregnancy loss (EPL) is one of the most common complications of human reproduction. Combined with our previous proteomic studies on villous and decidual tissues of EPL, we found that alterations of the proteins involved in oxidative stress (OS), unfolded protein response (UPR) and proteolysis presented a complex and dynamic interaction at the maternal-fetal interface. In the present study, we developed a cell model of OS using normal decidual cells to examine cell viability and expression levels of proteins related to endoplasmic reticulum stress (ER stress) and UPR. We found that glucose regulated protein 78 (GRP 78) and ubiquitinated proteins were significantly up-regulated in hydrogen peroxide (H(2)O(2)) treated decidual cells in a dose-dependent manner. Excessive OS could influence proper function of UPR by decreasing VCP in decidual cells, thereby leading to cell damage as well as inhibition of cell growth and activation of apoptosis. Furthermore, when pretreated with MG 132, a pharmacological inhibition of the proteasome, the H(2)O(2) treated decidual cells became less viable and could not up-regulate the expression level of GRP 78 to resolve the protein-folding defects, which indicating that malfunction of UPR in decidual cells might aggravate the inhibitory effect of OS in decidual cells. The present results reveal that abnormal protein profiles associated with OS induced ER stress and malfunction of UPR might be involved in the development of EPL, and OS and ER stress are potential targets for pregnant care and prognosis in normal pregnancy and its disorders.

Insufficient maintenance DNA methylation is associated with abnormal embryonic development.

BACKGROUND: Early pregnancy loss (EPL) is a frustrating clinical problem, whose mechanisms are not completely understood. DNA methylation, which includes maintenance methylation and de novo methylation directed by DNA methyltransferases (DNMTs), is important for embryo development. Abnormal function of these DNMTs may have serious consequences for embryonic development. METHODS: To evaluate the possible involvement of DNA methylation in human EPL, the expression of DNMT proteins and global methylation of DNA were assessed in villous or decidua from EPL patients. The association of maintenance methylation with embryo implantation and development was also examined. RESULTS: We found that DNMT1 and DNMT3A were both expressed in normal human villous and decidua. DNMT1 expression and DNA global methylation levels were significantly down-regulated in villous of EPL. DNMT3A expression was not significantly changed in the EPL group compared to controls in either villous or decidua. We also found that disturbance of maintenance methylation with a DNMT1 inhibitor may result in a decreased global DNA methylation level and impaired embryonic development in the mouse model, and inhibit in vitro embryo attachment to endometrial cells. CONCLUSIONS: Our results demonstrate that defects in DNA maintenance methylation in the embryo, not in the mother, are associated with abnormal embryonic implantation and development. The findings of the current study provide new insights into the etiology of EPL.

Effects of In Vitro Maturation on Histone Acetylation in Metaphase II Oocytes and Early Cleavage Embryos.

In vitro maturation (IVM) of oocyte is an effective procedure for avoiding ovarian hyperstimulation syndrome in patients with polycystic ovaries (PCOS) during in vitro fertilization (IVF). To investigate the influences of IVM on epigenetic reprogramming and to search for the possible reasons for the lower rates of fertilization and cleavage in IVM oocytes, we examined the expression of two enzymes controlling histone acetylation, histone acetyltransferase GCN5 (GCN5) and histone deacetylase 1 (HDAC1), as well as their common target, acetyl-histone H3 (Ac-H3), in mouse metaphase II (MII) oocytes and preimplantation embryos. Results showed that IVM downregulated the protein expression of GCN5 in MII oocytes and two-cell embryos and changed the distribution of GCN5 in two-cell embryos. Expression of HDAC1 mRNA in MII oocytes and two-cell embryos decreased in the IVM group. However, none of these changes persisted after two-cell embryos. Levels of Ac-H3 in both oocytes and embryos remained unchanged after IVM. Our studies indicated that IVM could affect the protein and gene expression related to histone acetylation in oocytes and early cleavage embryos. By function of selection, parts of the changes could be recovered in late embryo development.

G546A polymorphism of growth differentiation factor-9 contributes to the poor outcome of ovarian stimulation in women with diminished ovarian reserve.

The growth differential factor-9 (GDF-9) gene, an oocyte-specific factor, was screened in 106 Chinese women with diminished ovarian reserve (DOR), and three single-nucleotide polymorphisms, c.G169A, c.C447T and c.G546A, were detected. We found GDF-9 c.G546A, but not c.G169A or c.C447T, to be correlated with the poor ovarian stimulation and in vitro fertilization outcomes in women with DOR.

Altered aquaporin expression in women with polycystic ovary syndrome: hyperandrogenism in follicular fluid inhibits aquaporin-9 in granulosa cells through the phosphatidylinositol 3-kinase pathway.

BACKGROUND: The present study was designed to evaluate whether the alteration of aquaporin-9 (AQP-9) expression in granulosa cells (GCs) of patients with polycystic ovary syndrome (PCOS) was associated with the hyperandrogenism in follicular fluid (FF). METHODS: We recruited infertile women with PCOS (n = 14) and infertile women with tubal blockage (controls, n = 31) for this study. We examined total testosterone (TT), free androgen index (FAI), sex hormone-binding globulin (SHBG), FSH, LH and estradiol in FF. Real-time PCR and western blotting were performed to assess AQP-9 expression in GCs, including effects of dihydrotestosterone (DHT) in vitro. RESULTS: AQP-9 protein was localized in the nucleus, cytoplasm and cell membrane of the human GCs. The TT, FAI and LH levels were all higher, and SHBG levels lower, in the FF of women with PCOS versus controls (P = 0.0145, 0.0001, 0.0191, 0.0001, respectively). AQP-9 mRNA level in GCs of patients with PCOS was tightly correlated with the TT, SHBG levels and FAI in FF (P = 0.0020, 0.0001, 0.0020, respectively). In vitro, DHT (10(-9) mol/l) decreased AQP-9 mRNA (lowest at 12 h) and protein levels in control GCs (P = 0.0005, 0.0247, respectively). The inhibitory effect of DHT on AQP-9 mRNA was attenuated by LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor (P = 0.0013). Fifty micromolar 4-(hydroxymercuri) benzoic acid sodium salt (PMB) and 10(-9) mol/l DHT blunted the swelling of GCs in hypotonic medium, respectively (P = 0.0350, 0.0027). CONCLUSION: Hyperandrogenism in FF of women with PCOS inhibited AQP-9 in GCs through the PI3K pathway.