Follicular fluid progesterone downregulated HPGD and COX2 in granulosa cells via suppressing NF-ĸB in endometriosis†.

Increasing evidences showed that ovulatory dysfunction, possibly caused by luteinized unruptured follicular follicle syndrome (LUFS), is one of the reasons for endometriosis-related infertility. The present study was conducted to explore the potential effect of elevated progesterone in follicular fluid (FF) on ovulation in endometriosis. A prospective study including 50 ovarian endometriosis patients and 50 control patients with matched pairs design was conducted with alterations in FF and peritoneal fluid (PF) components identified by metabolomics analyses and differentially expressed genes in granulosa cells (GCs) identified by transcriptome analysis. Patients with endometriosis exhibited a significantly higher progesterone level in serum, FF, and PF. Granulosa cells from endometriosis patients revealed decreased expression of HPGD, COX-2, and suppressed NF-ĸB signaling. Similarly, progesterone treatment in vitro downregulated HPGD and COX2 expression and suppressed NF-ĸB signaling in granulosa tumor-like cell line KGN (Bena Culture Collection, China) and primarily cultured GCs, as manifested by decreased expressions of IL1R1, IRAK3, reduced pIĸBα/IĸBα ratio, and nucleus translocation of p65. On the contrary, TNF-α treatment increased expression of IL1R1, IRAK3, pIĸBα, p65, and HPGD in GCs. One potential p65 binding site was identified in the promoter region of HPGD by chromatin immunoprecipitation. In conclusion, we found that intrafollicular progesterone might downregulate HPGD and COX-2 in GCs via suppressing the NF-ĸB signaling pathway, shedding light on the mechanism underlying the endometriosis-related ovulatory dysfunction.

Embryo morphologic quality in relation to the metabolic and cognitive development of singletons conceived by in vitro fertilization and intracytoplasmic sperm injection: a matched cohort study.

BACKGROUND: Embryos with higher morphologic quality grading may have a greater potential to achieve clinical pregnancy that leads to a live birth regardless of the type of cleavage-stage embryos or blastocysts. Few studies have investigated the impacts of embryo grading on the long-term health of the offspring. OBJECTIVE: This pilot study aimed to examine the associations between embryo morphologic quality and the physical, metabolic, and cognitive development of singletons conceived by in vitro fertilization and intracytoplasmic sperm injection at preschool age. STUDY DESIGN: This matched cohort study included singletons born to infertile couples who underwent fresh cleavage-stage embryo transfer cycles with good- or poor-quality embryos from 2014 to 2016 at the reproductive center of the Women's Hospital, School of Medicine, Zhejiang University. A total of 144 children, aged 4 to 6 years, participated in the follow-up assessment from 2020 to 2021, and the response rate of poor-quality embryo offspring was 39%. Singletons in the good-quality embryo group were matched with singletons in the poor-quality embryo group at a 2:1 ratio according to the fertilization method and the children's age (±1 year). We measured the offspring's height, weight, body mass index, blood pressure, thyroid hormone levels, and metabolic indicators. Neurodevelopmental assessments were performed using the Chinese version of the Wechsler Preschool and Primary Scale of Intelligence, Fourth Edition, and the Adaptive Behavior Assessment System, Second Edition. We also collected data from the medical records. A linear regression model was used to analyze the association between embryo morphologic quality and offspring health outcomes. RESULTS: A total of 48 singletons conceived with poor-quality embryo transfer and 96 matched singletons conceived with good-quality embryo transfer were included in the final analysis. Age, sex, height, weight, body mass index, blood pressure, thyroid function, and metabolic indicators were comparable between the 2 groups. After adjustment for potential risk factors by linear regression model 1 and model 2, poor-quality embryo offspring exhibited a tendency toward higher free thyroxine levels than offspring of good-quality embryo transfers (beta, 0.22; 95% confidence interval, 0.09-0.90; beta, 0.22; 95% confidence interval, 0.09-0.91, respectively), but this difference was not clinically significant. Regarding neurodevelopmental assessments, there was no difference in the full-scale intelligence quotient based on the Wechsler Preschool and Primary Scale of Intelligence (109.96±12.42 vs 109.60±14.46; P=.88) or the general adaptive index based on the Adaptive Behavior Assessment System (108.26±11.70 vs 108.08±13.44; P=.94) between the 2 groups. The subindices of the 2 tests were also comparable. These findings remained after linear regression analysis. CONCLUSION: At 4 to 6 years of age, singletons born from poor-quality embryo transfers have comparable metabolic and cognitive development as those born from good-quality embryo transfers using fresh cleavage-stage embryos. The results of this pilot study indicate that poor-quality embryos that can survive implantation and end in live birth are likely to have a developmental potential comparable to that of good-quality embryos.

Immunological microenvironment alterations in follicles of women with proven severe endometriosis undergoing in vitro fertilization.

The purpose of this study was to test the hypothesis that different cytokine profiles may exist in the follicular fluid of endometriosis (EM) patients undergoing in vitro fertilization (IVF), as these differences may provide insights into the pathogenesis of the disease. This was a cross-sectional study conducted at the reproductive center of a medical university hospital. The study included 49 patients receiving IVF. 20 infertile women with proven EM and 29 women without diagnosed EM (control group) were evaluated. Follicular fluid (FF) and serum were collected at the time of follicle aspiration and the concentrations of 38 cytokines were determined by multiplexed immunoassay. The results indicated that the levels of IL-4, IL-13, IL-3 and IL-1α were significantly increased in the FF of women with EM, while levels of IFN-γ, IL-17A, MDC and MIP-1α were decreased compared with in the control subjects. In conclusions, the immune microenvironment of the FF in patients with EM is altered. This may contribute to the pathologic mechanism responsible for the poor outcome of IVF in patients with EM.

The social adaptation of children born after ICSI compared with IVF-conceived children: a study from China.

We compared the social adjustment among Chinese children born after intracytoplasmic sperm injection (ICSI) vs those after in vitro fertilisation (IVF) and identify factors related to the adjustment. The social adaptation of 86 ICSI and 165 IVF conceived children of 4-6 years of age was assessed using the Infants-Junior Middle School Students' Social-Life Abilities Scale. There was no significant difference between the ICSI and IVF-conceived groups on the item of communication, self-dependence, locomotion, work skills, socialisation, self-management and total scores. Compared with routine IVF, ICSI does no harm to the social adaptation of children conceived through this technology.

[Conventional and molecular cytogenetic analyses of a derivative X chromosome in amniocentesis].

OBJECTIVE: To analyze the aberrant der(X) chromosome using conventional and molecular cytogenetic approaches in a fetus of second trimester and to discuss its clinical effect. METHODS: Conventional cytogenetic procedures (GTG and CBG banding) were performed on cultured amniotic fluid cells. Three-color fluorescence in situ hybridization (FISH) consisting of X chromosome enumeration probes(CEPX), CEPY and Tel Xp/Yp was further performed to study the aberrant der(X) chromosome. RESULTS: Der(X) was a rare X/Y translocation. The final karyotypes of the fetus was designated as: 46,X,der(X)t(X;Y)(p22.3;q11.2). ish der(X)t(X;Y)(p22.3;q11.2)(X/Ypter-, DXZ1+, DYZ1+)mat. CONCLUSION: The combination of FISH and conventional cytogenetic techniques is a powerful tool to determine derivative chromosome and to offer an accurate genetic counseling. Identification of Xp; Yq rearrangement can help estimate the risk of fetus abnormalities and give a more precise prognosis.

BNC1 Promotes Spermatogenesis by Regulating Transcription of Ybx2 and Papolb via Direct Binding to Their Promotor Elements.

BNC1 is a transcription factor that is crucial for spermatogenesis and male fertility, although the underlying mechanism remains unclear. To study BNC1's specific role in spermatogenesis, we characterized a previously developed mouse model carrying a truncating mutation in Bnc1 (termed Bnc1+/tr for heterozygotes and Bnc1tr/tr for homozygotes) and found that the mutation decreased BNC1 protein levels and resulted in germ cell loss by apoptosis. Given that loss of functional Bnc1 is known to result in decreased expression of the spermatogenesis genes Ybx2 and Papolb, we aimed to explore whether and how BNC1 promotes transcription of Ybx2 and Papolb to mediate its role in spermatogenesis. We confirmed significant reduction in YBX2 and PAPOLB protein levels in testis tissue from Bnc1+/tr and Bnc1tr/tr males compared with wild-type mice (Bnc1+/+). Consistently, knockdown of Bnc1 led to downregulation of Ybx2 and Papolb in CRL-2196 cells in vitro. To investigate if BNC1 directly induces Ybx2 and Papolb gene expression, chromatin immunoprecipitation using mouse testicular tissue and luciferase reporter assays in HEK293 cells were used to identify functional binding of BNC1 to the Ybx2 and Papolb promoters at defined BNC1 binding sites. Taken together, this study reveals a mechanism for BNC1's role in spermatogenesis by directly binding to BNC1 binding elements in the promoter regions of both Ybx2 and Papolb and inducing transcription of these important spermatogenesis genes.

[Sperm chromosome analysis and preimplantation genetic diagnos is in an infertile male with mosaic trisomy 18].

OBJECTIVE: To analyze the numerical aberration rate of X, Y and chromosome 18 in sperms from an oligozoospermic male with mosaic trisomy 18 and to perform preimplantation genetic diagnosis (PGD) for the couple. METHODS: G-banding and fluorescence in situ hybridization (FISH) were performed on metaphase chromosome. Sperm was analyzed in three-color FISH with a probe mixture containing CEP18, CEPY and Tel Xq/Yq. A healthy man with normal semen parameters was used as control. RESULTS: Significant difference in the rates of disomy for chromosome 18 (0.63% vs. 0.16%) and the gonosomes (0.945% vs. 0.35%) and diploidy (0.87% vs. 0.31%) was found in the spermatozoa between the patient and the control. After four embryos were biopsied in one PGD cycle, two embryos with XY1818 and XX1818 were selected for implanting and clinical pregnancy was ongoing. CONCLUSION: Sperm-FISH allows further understanding of aneuploidy rate and accurate genetic counseling. FISHPGD was effective for patient with mosaic trisomy 18.

[Sperm-fluorescence in situ hybridization analysis in patients with pericentric inversions of Y chromosome].

OBJECTIVE: To analyze the sex chromosome meiotic segregation in inv(Y) patients by fluorescence in situ hybridization (FISH). METHODS: Conventional cytogenetic procedures (GTG and CBG banding) and FISH were performed on metaphase chromosome. Three-color FISH was performed on sperm samples using a probe mixture containing CEPX, Tel Xp/Yp and Tel Xq/Yq to investigate the sex chromosome segregation of five inv(Y) (p11.1q11.2) carriers. A healthy man with normal semen parameters was used as control. RESULTS: There was no statistical difference in the abnormal sex chromosome number and recombination frequencies in each spermatozoon from the patient in comparison with that in the control. CONCLUSION: There was no apparent sex chromosome abnormality in the sperm of the inv(Y) (p11.1q11.2) carriers. Sperm-FISH allows further understanding of the sex chromosome segregation pattern and an accurate genetic counseling.

[Genetic analysis of a complex chromosome rearrangement involving two chromosomes and four breakpoints in an azoospermic man].

OBJECTIVE: To perform genetic analysis of a complex chromosome rearrangement (CCR) 46,XY, t(3;11)(q27; q13), ins(11;3)(q13;p26p13) in an azoospermic man. METHODS: Peripheral blood lymphocytes we re obtained for karyotyping, and metaphases were studied by multicolor fluorescence in situ hybridization procedure, Y chromosomal microdeletions in the azoospermia factor (AZF) region were analyzed with multiplex polymerase chain reaction. RESULTS: The case was a complex chromosomal translocation between chromosomes 3 and 11 with four breakpoints, and accompanied with a band of chromosome 3 inserting into chromosome 11. No Y-chromosome microdeletions were identified at 6 STS sequences of the AZF loci. CONCLUSION: CCR can have a significant impact on male fertility. Molecular cytogenetic techniques may contribute to improving and personalizing reproductive counseling.

Accuracy of a combined score of zygote and embryo morphology for selecting the best embryos for IVF.

OBJECTIVE: To evaluate the accuracy of a scoring system combining zygote and embryo morphology in predicting the outcome of in vitro fertilization (IVF) treatment. METHODS: In a study group, 117 consecutive IVF or intracytoplasmic sperm injection (ICSI) cycles with embryo transfer were carried out and 312 embryos were scored using a combined scoring system (CSS) of zygote and embryo morphology before transplantation. In a control group, a total of 420 IVF or ICSI cycles were carried out and 1176 embryos were scored using a cumulative embryo score (CES). The effects of the combined scoring system on the embryo implantation rate and pregnancy rate per cycle were analyzed. RESULTS: Using the combined scoring system, the embryo implantation rate (27.6%) and the clinical pregnancy rate (48.7%) were significantly higher than those in the control group (20.8% and 38.6%, respectively). Also, the implantation rate of embryos scoring>or=70 (38.5%: 82 sacs/213 embryos) was significantly higher (P<0.001) than that of embryos scoring<70 (4%: 4 sacs/99 embryos). The pregnancy rate of patients with embryos scoring>or=70 using the combined scoring system (66.7%) was significantly higher (P<0.001) than that of patients with embryos scoring>or=20 using the cumulative embryo score (59.0%). CONCLUSION: The results suggest that selecting embryos with a high score (>or=70) using the combined scoring system could increase the implantation rate and pregnancy rate, and that using a scoring system combining assessments of human zygotes and pre-implantation embryos might predict IVF outcomes more accurately than using a cumulative embryo score.

[Preimplantation genetic diagnosis of translocation].

OBJECTIVES: To observe the genetic characteristics of chromosomes and the rates of implantation and pregnancy in couples of translocation carriers who undergo preimplantation genetic diagnosis (PGD) and to evaluate the significance of PGD in the treatment of translocation carriers. METHODS: Fluorescence in situ hybridization (FISH) was performed to analyze the embryos of 12 carriers of reciprocal translocation and 22 carriers of Robertsonian translocation. The results of diagnosis and the implantation and pregnancy rates were analyzed. RESULTS: A total of 253 embryos from 36 couples were retrieved and FISH was applied for the examination. The characteristics of chromosomes were diagnosed in 225 embryos and the rate of successful PGD was 88.9%. Fifty-eight embryos were found to have normal chromosome or balanced translocation and were transferred into the uterus. The rate of implantation was 36% (5/14) and 14% (6/44) and the rate of pregnancy was 4/9 and 26% (5/19) for carriers of Robertsonian translocation and reciprocal translocation, respectively. CONCLUSIONS: The FISH-based PGD is effective in the diagnosis of Robertsonian translocation and reciprocal translocation of embryos. It provides the possibility of a high rate of implantation and pregnancy, and avoids recurrent abortion and unwilling termination of pregnancy.

Preimplantation genetic diagnosis for Down syndrome pregnancy.

OBJECTIVE: To evaluate the effect of preimplantation genetic diagnosis (PGD) conducted for women who had Down syndrome pregnancy previously. METHODS: Trisomy 21 was diagnosed by using fluorescence in site hybridization (FISH) before embryo transfer in two women who had Down syndrome pregnancies. Each received one or two PGD cycles respectively. RESULTS: Case 1: one PGD cycle was conducted, two oocytes were fertilized and biopsied. One embryo is of trisomy 21 and the other of monosomy 21. No embryo was transferred. Case 2: two PGD cycles were conducted, in total, sixteen oocytes were fertilized and biopsied. Four embryos were tested to be normal, six of trisomy 21, and one of monosomy 21. Five had no signal. Four normal embryos were transferred but no pregnancy resulted. CONCLUSION: For couples who had pregnancies with Down syndrome previously, PGD can be considered, and has been shown to be an effective strategy.

[Expression of aquaporin 9 in granulosa cells of patients with polycystic ovary syndrome in IVF-cycles].

OBJECTIVE: To investigate aquaporin 9 (AQP9) mRNA and protein expression in antrum follicle and luteinizing granulosa cells of polycystic ovarian syndrome (PCOS) ovary, and its relation to follicular fluid steroids hormone levels during IVF cycles. METHODS: AQP9 mRNA expression on luteinizing granulosa cells in IVF cycles was detected by RT-PCR. AQP9 protein expression in antrum follicles of PCOS ovary and luteinizing granulosa cells was measured by immunohistochemistry. The concentrations of estradiol (E2), progesterone (P) and testerone (T) in follicular fluid were measured by radioimmunoassay (RIA). RESULT: The expression of AQP9 mRNA in luteinizing granulosa cells during IVF cycles was positive by RT-PCR. No significant differences in AQP9 mRNA levels in granulosa cells between PCOS and control group were found during IVF cycles. The expression level of AQP9 mRNA in large follicles was higher than that in small follicles, but not significantly. The immunoreactivity for AQP9 was localized in membrane and cytoplast of granulosa cells in antrum follicles from PCOS ovary and luteinizing granulosa cells during IVF cycles. Multiple regression analysis showed that AQP9 mRNA levels on granulosa cells were not correlated with E2, P and T levels in follicular fluid during IVF cycles. CONCLUSION: AQP9 may play an important role in the follicle development and antrum formation through water transport and AQP9 may be involved in the mechanism of follicle development in PCOS.

[In vitro maturation--n vitro fertilization--embryo transfer--frozen-thawed of human oocytes].

OBJECTIVE: To investigate the influence of different cycles, ovarian follicle size and IVM culture media on the number of retrieved immature oocytes, maturation rate, fertilization rate, embryo quality and implantation rate, pregnancy rate, delivery rate, survival and development of frozen-thawed embryos from IVM. METHODS: The oocytes were obtained by follicular aspiration from 19 women undergoing oocyte retrieval for in vitro maturation due to the possible risk of ovarian hyperstimulation in IVF-ET program. One patient was in natural cycle, four patients were in ovulation induction cycles with gonadotropine and fourteen patients is controlled ovarian stimulated cycles. All the oocytes retrieved from follicles with 10.0 - 13.5 mm in maximumdiameter were allowed to culture in medium M-199 (TCM 199) or HTF supplemented with other substance. RESULT: When there were nonuniform diameters of follicles and the diameter of largest oocyte exceeded 12 mm, the retrieval rate of oocytes, fertilization rate, and the number of high-quality embryos decreased. The high-quality embryos formation rate was higher for the oocytes cultured in TCM 199 medium than in HTF medium (P<0.01). After being frozen-thawed, the IVM embryos could achieve the same outcome when compared with the conventional IVF treatment. In addition, the offspring were healthy. CONCLUSION: When the nonuniform diameters of follicles and the diameter of largest oocyte exceeds 12 mm,the retrieval rate of oocytes, fertilization rate, and the number of high-quality embryos decreased. TCM199-based medium is better to improve the developmental potential and implantation rate of embryos derived from in vitro matured oocytes. After being frozen-thawed, the IVM embryos could achieve the same outcome when compared with the conventional IVF treatment. In addition, the offspring are healthy.

Aquaporin-2 expression in human endometrium correlates with serum ovarian steroid hormones.

The aim of the present study was to examine the expression of aquaporin-2 (AQP2), a member of the water channel family aquaporins (AQPs), in human uterine endometrium and its modulation of ovarian steroid hormone at the proliferative and secretory phases. Western blot, immunohistochemistry, and RT-PCR were employed in the present study. Western blot revealed a 29-kDa band that represented AQP2 in human endometrium. The expression of AQP2 in endometrium was confirmed by RT-PCR and immunohistochemical results. The immunohistochemical analysis demonstrated that AQP2 was prominent in luminal and glandular epithelial cells of endometrium. The levels of endometrial AQP2 expression changed during the menstrual cycle and were higher in the secretory endometrium than in the proliferative endometrium. A significantly high level of AQP2 was detected at the mid-secretory phase. There was a positive correlation between the levels of the endometrial AQP2 expression and the concentrations of the serum 17beta-estradiol (E2) or/and progesterone (P4). These data for the first time corroborate that AQP2 is expressed in human endometrium and that the expression of AQP2 in human endometrium might be regulated by E2 or/and P4. The changed expression of AQP2 at different phases of the menstrual cycle may be essential to reproductive physiology in human. The high level of endometrial AQP2 expression was observed at the mid-secretory phase, the time of embryo implantation, suggesting that AQP2 might play physiological roles in the uterine receptivity.

Effects of preimplantation embryos on expressions of DNA methyltransferase 1 in mouse oviduct epithelial cells.

OBJECTIVE: To investigate the effects of mouse preimplantation embryos on the expressions of DNA methyltransferase 1(Dnmt1) of mouse oviduct epithelial cells. METHODS: The histological location of Dnmt1 protein was detected by immunohistochemical staining and the expression levels of Dnmt1 mRNA and protein in mouse oviduct were assayed by real-time reverse transcription-PCR(RT-PCR) and Western blotting in both pregnant and pseudopregnant mice at the 2-cell, 4-cell and 8-cell stages. RESULTS: The expressions of Dnmt1 protein were mainly located in the epithelial cells of mouse oviduct. It was found that during all three stages, the expression levels of Dnmt1 mRNA in the epithelial cells of the pregnant mice were significantly lower than those in the pseudopregnant mice (P< 0.05), and the level of Dnmt1 protein expression in the pregnant mice was significantly decreased as compared with that in pseudopregnant mice at the 4-cell stage. CONCLUSION: Expressions of both Dnmt1 mRNA and protein in the epithelial cells of mouse oviduct could be regulated by mouse preimplantation embryos, which might play an important role in the expression changes of some genes in oviduct epithelial cells during the preimplantation period.

[Protein and mRNA expression of aquaporin-1 in epithelial ovarian tumors and its clinic significance].

OBJECTIVE: To investigate protein and mRNA expression of aquaporin-1 (AQP1) in epithelial ovarian tumors and its clinic significance. METHODS: The protein and mRNA expressions of AQP1 were measured by immunohistochemical technique, western blot and RT-PCR in 65 cases of epithelial ovarian tumors and 13 cases of normal ovary tissue. RESULTS: AQP1 located in microvascular and small vessel epithelial cells. The protein and mRNA expressions of AQP1 in ovarian cancer (0.39 +/- 0.12, 0.93 +/- 0.51, respectively) and ovarian borderline tumors (0.43 +/- 0.21, 0.95 +/- 0.34, respectively) were significantly higher than that of ovarian benign tumors (0.27 +/- 0.13, 0.51 +/- 0.41, respectively; P < 0.05) and normal ovary tissue (0.24 +/- 0.13, 0.34 +/- 0.29, respectively; P < 0.05). Of all ovarian cancers, expression of AQP1 in cases with ascites more than 1000 ml (0.46 +/- 0.13, 1.25 +/- 0.57, respectively) was higher than that of ascites less than 1 approximately 499 ml (0.35 +/- 0.11, 0.75 +/- 0.45, respectively; P < 0.05). CONCLUSION: Over-expression of AQP1 plays an important role in development of epithelial ovarian tumors, and may be related with formation of ascites of ovarian carcinoma.

[Polymorphism in insulin receptor gene exon 17 in women with polycystic ovary syndrome].

OBJECTIVE: To investigate insulin receptor (INSR) genotype exon 17 frequencies in women with polycystic ovary syndrome (PCOS) and to elucidate its role in the pathogenesis of PCOS. METHODS: The study involved 33 women with PCOS and 28 healthy control women who were genotyped for polymorphism of INSR gene exon 17 by single strand conformation polymorphism (SSCP) analysis. Body mass index (BMI), insulin sensitive index (ISI), the expression of INSR beta subunit, and serum concentration of luteinizing hormone (LH), total testosterone between the genotypes were compared. RESULTS: (1) The T-to-C mutation was observed in the INSR gene exon 17 (1008 bp). The frequency of the C/C genotype was significantly higher in patients (39%) than in the controls (11%) (P < 0.05). There was no significant difference in the distribution of genotypes between obese PCOS and non-obese PCOS, and between PCOS with insulin resistance (IR) and PCOS without IR. (2) In comparison of mutation genotype groups with wild genotype (T/T), ISI was significantly decreased (C/C genotype, P < 0.01; C/T genotype, P < 0.05), and no significant difference was observed in the other indices. CONCLUSIONS: There is polymorphism in INSR gene exon 17 in patients with PCOS. This variant leads to increased risk of IR in women with PCOS. It does not influence the expression of INSR beta subunit.

[Determination of interleukin-17 concentrations in peritoneal fluid of women with endometriosis].

OBJECTIVE: To investigate the role of interleukin-17 (IL-17) in the pathogenesis of endometriosis. METHODS: Enzyme-linked immunosorbent assay (ELISA) was used to detect the concentrations of IL-17 in peritoneal fluid of 36 patients with different stage endometriosis and 26 patients without endometriosis. RESULTS: The concentrations of IL-17 in peritoneal fluid of the patients with and without endometriosis were (5.7 +/- 1.9) ng/L and (5.3 +/- 1.4) ng/L, respectively, without significant difference between the two groups (P > 0.05). According to staging criteria of r-AFS, the concentrations of peritoneal IL-17 in the patients with stage I-II endometriosis (6.4 +/- 1.7) ng/L were significantly higher than those in the patients with stage III-IV endometriosis (5.1 +/- 1.8) ng/L and in the patients without endometriosis (P < 0.05). There was no difference with regard to peritoneal IL-17 concentrations between proliferative and secretory phases in the patients with or without endometriosis (P > 0.05). The levels of peritoneal IL-17 were significantly higher in the endometriosis patients with infertility (6.4 +/- 1.8) ng/L than in the endometriosis patients without infertility (5.1 +/- 1.8) ng/L (P < 0.05). CONCLUSION: IL-17 may play an important role in the pathogenesis of early endometriosis and pathophysiology of endometriosis-associated infertility.

Establishment of a transgenic cell line stably expressing human cytochrome P450 2C18 and identification of a CYP2C18 clone with exon 5 missing.

AIM: The human cytochrome P-450 2C18(CYP2C18) has been characterized. However, the protein has not been purified from liver and very little is known regarding the specific substrate of CYP2C18. In order to study its enzymatic activity for drug metabolism, the CYP2C18 cDNA was cloned and a stable CHL cell line expressing recombinant CYP2C18 was established. METHODS: The human CYP2C18 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR) from total RNAs extracted from human liver and cloned into pGEM-T vector. The cDNA segment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant plasmid of pREP9-CYP2C18 to Chinese hamster lung (CHL) cell. The enzyme activity of CYP2C18 catalyzing oxidation of tolbutamide to hydroxytolbutamide in postmitochondrial supernant(S9) fraction of the cell was determined by high performance liquid chromatography(HPLC). RESULTS: The amino acid sequence predicted from the cloned cDNA segment was identical to that of reported by Romkes et al (GenBank accession number: M61856, J05326). The S9 fraction of the established cell line metabolizes tolbutamide to hydroxytolbutamide. Tolbutamide hydroxylase activity was found to be 0.509+/-0.052 micromol x min(-1) x g(-1) S9 protein or 8.82+/-0.90 mol x min(-1) x mol(-1) CYP, but was undetectable in parental CHL cell. In addition, we have identified a CYP2C18 cDNA clone with exon 5 missing. CONCLUSION: The cDNA of human CYP2C18 was successfully cloned and a cell line, CHL-CYP2C18, efficiently expressing the protein of CYP2C18, was established. A spliced variant of CYP2C18 with exon 5 missing was identified in the cloning process.