Limited relationships between reactive oxygen species levels in culture media and zygote and embryo development.

PURPOSE: Reactive oxygen species (ROS) are thought to play a critical role in the success of IVF. The relationships between oxidative stress parameters in culture media and IVF outcomes have not been extensively investigated. The objective of this study is to examine the relationships between early human embryonic parameters and levels of ROS in culture media. METHODS: This prospective study was conducted with 2633 spent culture media collected from patients undergoing conventional IVF (n = 101) and intracytoplasmic sperm injection (ICSI) (n = 60). Both fertilization and early culture were performed in universal IVF medium and G series medium. ROS levels were measured by chemiluminescence assays using luminol as the probe on days 1, 3, and 5 and determined the relationships of ROS levels with zygote condition, embryo quality, and clinical pregnancy rate. RESULTS: ROS levels per embryo in culture media on the corresponding days 1, 3, and 5 showed significant correlations between each pair in the total cohort. Similar results were observed in the IVF and ICSI groups, but day 1 and day 3 ROS levels were significantly higher in culture media of IVF than of ICSI embryos. ROS levels in culture medium were not significantly associated with embryo quality, blastocyst formation, or arrest. ROS levels on day 1 were similar in media of normally fertilized zygotes, unfertilized oocytes, and polyspermic zygotes and were not associated with delayed embryonic development, high fragmentation, blastocyst formation, or arrest after prolonged culture. ROS levels in media were not associated with the likelihood of conception. CONCLUSIONS: ROS levels in culture media may not be an effective indicator of embryo selection for IVF.

Analysis of androgen receptor and anti-Müllerian hormone pathways in human granulosa cells under luteinizing hormone treatment.

BACKGROUND: The objective of this study was to determine the gene expression profiles of the androgen/androgen receptor (AR) and anti-Müllerian hormone (AMH)/ Sry-related high-mobility group box 9 (SOX9) pathways in granulosa-luteal cells from patients undergoing standard in vitro fertilization (IVF) with or without recombinant luteinizing hormone (rLH) therapy. METHODS: Levels of reproductive hormones in the pre-ovulatory follicular fluid and the expression levels of LHR (luteinizing hormone receptor), AR, SOX9, AMH, AR-associated protein 54(ARA54)and ARA70 were determined in granulosa-luteal cells by real-time reverse-transcription PCR. The effects of androgen and rLH treatments on AR and AMH expression levels were also tested in vitro using HO23 cells. RESULTS: We collected 35 an 70 granulosa cell samples from patients cycled with and without rLH supplementation, respectively. The clinical outcomes were similar in patients who received rLH therapy and those who did not, though the pre-ovulatory follicular fluid levels of androstenedione, testosterone, and estradiol were significantly higher and progesterone was lower in the rLH supplementation group. Moreover, granulosa-luteal cell mRNA levels of LHR, AR, AMH, and SOX9 were significantly higher in the rLH supplementation group relative to the group that did not receive rLH supplementation. In addition, we observed significant correlations between LHR and AR mRNA expression and among AR, AMH, and SOX9 mRNA expression in granulosa-luteal cells from patients undergoing standard IVF treatment. CONCLUSIONS: Increased expression of LHR, AR, AMH, and SOX9 is characteristic of granulosa-luteal cells from IVF/ intracytoplasmic sperm injection (ICSI) patients receiving rLH supplementation.

Y-chromosome genes associated with sertoli cell-only syndrome identified by array comparative genome hybridization.

BACKGROUND: The precise contribution of each chromosome gene or gene family in achieving male fertility is still the subject of debate. Most studies have examined male populations with heterogeneous causes of infertility, and have therefore reached controversial or uncertain conclusions. This study uses Y-chromosome array-based comparative genomic hybridization (aCGH) to examine a population of males with a uniform sertoli cell-only syndrome (SCOS) infertility phenotype. METHODS: Initial analysis of gene copy number variations in 8 SCOS patients, with determination of the log-ratio of probe signal intensity against a DNA reference, was performed using the Y-chromosome NimbleGen aCGH. To confirm the role of candidate genes, real-time quantitative RT-PCR was used to compare 19 patients who had SCOS non-obstructive azoospermia with 15 patients who had obstructive azoospermia but normal spermatogenesis. RESULTS: Our initial aCGH experiments identified CDY1a and CDY1b double deletions in all 8 patients who had total germ cell depletion. However, 5 patients had DAZ1/2 and DAZ3/4 deletions, 1 patient had a DAZ2 and DAZ3/4 deletion, and 2 patients had no DAZ1/2 or DAZ3/4 deletions. Examination of testicular mRNA expression in another 19 patients with SCOS indicated all patients had no detectable levels of CDY1. CONCLUSIONS: Our findings indicate that CDY1 deletion in SCOS patients, and analysis of the expression of DAZ and CDY1 genes using aCGH and quantitative RT-PCR, may be useful to predict the presence of mature spermatozoa.

Up-regulation of SOX9 in sertoli cells from testiculopathic patients accounts for increasing anti-mullerian hormone expression via impaired androgen receptor signaling.

BACKGROUND: Testosterone provokes Sertoli cell maturation and represses AMH production. In adult patients with Sertoli-cells-only syndrome (SCOS) and androgen insensitivity syndrome (AIS), high level of AMH expression is detected in Sertoli cells due to defect of androgen/AR signaling. OBJECTIVE: We postulated that up-regulation of SOX9 due to impairment of androgen/AR signaling in Sertoli cells might explain why high level of anti-Mullerian hormone (AMH) expression occur in these testiculopathic patients. METHODS: Biological research of testicular specimens from men with azoospermia or mouse. The serum hormone levels were studied in 23 men with obstructive azoospermia, 33 men with SCOS azoospermia and 21 volunteers with normal seminograms during a period of 4 years. Immunohistochemical staining and reverse-transcription PCR were used to examine the relationships among AR, SOX9 and AMH expression in adult human and mouse testes. The ability of AR to repress the expression of SOX9 and AMH was evaluated in vitro in TM4 Sertoli cells and C3H10T1/2 cells. RESULTS: SCOS specimens showed up-regulation of SOX9 and AMH proteins but down-regulation of AR proteins in Sertoli cells. The mRNA levels of AR were significantly lower and the SOX9, AMH mRNA levels higher in all SCOS patients compared to controls (P< 0.05). The testosterone levels in the SCOS patients were within the normal range, but most were below the median of the controls. Furthermore, our in vitro cell line experiments demonstrated that androgen/AR signaling suppressed the gene and protein levels of AMH via repression of SOX9. CONCLUSIONS: Our data show that the functional androgen/AR signaling to repress SOX9 and AMH expression is essential for Sertoli cell maturation. Impairment of androgen/AR signaling promotes SOX9-mediated AMH production, accounts for impairments of Sertoli cells in SCOS azoospermic patients.