Tsung-Hsien Lee

Magnetic-activated cell sorting for sperm preparation reduces spermatozoa with apoptotic markers and improves the acrosome reaction in couples with unexplained infertility

BACKGROUND Couples with unexplained infertility (UI) tend to have low fertilization rates with current IVF procedures. Here, we attempted to identify spermatozoa with apoptotic markers in couples with UI and unsuccessful intrauterine insemination (IUI) and we investigated the efficiency and benefit of magnetic-activated cell sorting (MACS) for sperm preparation in such patients. METHODS Sixty couples with UI and two IUI failures were recruited. The sperm were prepared by conventional density gradient centrifugation (DGC) and divided into two aliquots. One aliquot was used as a control and the other was further processed by MACS (D + M). Apoptotic markers were identified using fluorescence-labeled dye and flow cytometry, including externalization of phosphatidylserine (EPS), disrupted mitochondrial membrane potential (MMP) and DNA fragmentation. The fertilization potential of prepared spermatozoa was analyzed by basic semen analysis, computer-aided sperm analysis and the induced acrosome reaction test (IART). RESULTS After DGC, spermatozoa showed 18.6% EPS, 28.3% disrupted MMP and 13.5% DNA fragmentation. Numbers of spermatozoa with apoptotic markers were significantly reduced by D + M, versus DGC alone (P < 0.001). Although the motility of spermatozoa was slightly decreased after MACS, most sperm motion characteristics were not impaired. Interestingly, the IART significantly improved after D + M, versus DGC alone, especially for the couples with a normal hemizona assay (P < 0.001). CONCLUSIONS The spermatozoa prepared by D + M showed a reduced level of apoptotic markers. Improvement in the IART suggests a high fertilization potential of the processed spermatozoa. The identification of apoptotic markers and use of MACS may be helpful in directing the management plan for patients with UI and multiple IUI failures.

Impact of female age and male infertility on ovarian reserve markers to predict outcome of assisted reproduction technology cycles

BackgroundThis study was designed to assess the capability of ovarian reserve markers, including baseline FSH levels, baseline anti-Müllerian hormone (AMH) levels, and antral follicle count (AFC), as predictors of live births during IVF cycles, especially for infertile couples with advanced maternal age and/or male factors.MethodsA prospective cohort of 336 first IVF/ICSI cycles undergoing a long protocol with GnRH agonist was investigated. Patients with endocrine disorders or unilateral ovaries were excluded.ResultsAmong the ovarian reserve tests, AMH and age had a greater area under the receiving operating characteristic curve than FSH in predicting live births. Furthermore, AMH and age were the sole predictive factors of live births for women greater than or equal to 35 years of age; while AMH was the major determinant of live births for infertile couples with absence of male factors by multivariate logistic regression analysis. However, all the studied ovarain reserve tests were not preditive of live births for women < 35 years of age or infertile couples with male factors.ConclusionThe serum AMH levels were prognostic for pregnancy outcome for infertile couples with advanced female age or absence of male factors. The predictive capability of ovarian reserve tests is clearly influenced by the etiology of infertility.

Evaluation of telomere length in cumulus cells as a potential biomarker of oocyte and embryo quality

STUDY QUESTION Is the relative telomere length in cumulus cells associated with embryo quality and the subjects age? SUMMARY ANSWER The relative telomere length in cumulus cells at the time of oocyte collection may be a new potential biomarker for selecting highly competent oocytes and good quality embryos. WHAT IS KNOWN ALREADY Telomeres play central roles in aging and in determining cell fate. In mammalian ovarian follicles, maturing oocytes are nurtured and supported by surrounding somatic cells, the mural granulosa and cumulus cells. STUDY DESIGN, SIZE, DURATION A total of 350 oocyte-cumulus complex samples were collected from 80 IVF cycles prospectively recruited for this study at the Lee Womens Hospital, Taichung, Taiwan. PARTICIPANTS/MATERIALS, SETTING, METHODS Cumulus cells were manually separated from the oocyte-cumulus complex under a microscope. DNA was extracted from cumulus cells and assessed for telomere length by real-time quantitative PCR. We analyzed telomere length relative to a single copy marker gene (36B4) to evaluate the effect of the real reproductive age of cumulus cells on oocyte and embryo development. MAIN RESULTS AND THE ROLE OF CHANCE The relative telomere length was longer in cumulus cells from mature oocytes compared with cumulus cells from immature oocytes, and in cumulus cells from good-quality embryos compared with cumulus cells from poor-quality embryos. The cut-off value of the T/S ratio between good and poor-quality embryos on embryonic Day 3 was 4.235. LIMITATIONS, REASONS FOR CAUTION Only a limited number of cumulus cells were measured for each oocyte and the corresponding embryo. WIDER IMPLICATIONS OF THE FINDINGS The relative telomere length in cumulus cells at the time of oocyte collection is predictive of highly competent oocytes and good-quality embryos but may not be sufficiently discriminating to be clinically useful. STUDY FUNDING/COMPETING INTEREST(S) National Science Council, Taiwan (NSC 97-2314-B-040-018). The authors have no conflicts of interest to declare.

Number of somatic mutations in the mitochondrial D-loop region indicates poor prognosis in breast cancer, independent of TP53 mutation

The objective of this study was to investigate whether somatic mutations in the mitochondrial DNA (mtDNA) D-loop region correlate with known prognostic factors, namely, age, tumor size, lymph node status, metastasis, tumor-node-metastasis stage, lymphovascular invasion, and status of the progesterone receptor, estrogen receptor, ERBB2 (alias HER2/neu), and TP53 proteins (as determined by immunohistochemistry) and to investigate their relationship, if any, to TP53 mutations in human breast cancer. Thirty breast tumors without BRCA mutation, along with adjacent nontumorous tissues, were genotyped for the mtDNA D-loop region and for the promoter as well as the coding region of the TP53 gene. Clinicopathological parameters were recorded and assessed. In all, 17 somatic mtDNA D-loop mutations were identified, in 13 of 30 tumor samples (43%); two mutations were novel: 544C>T and 16510A>C. Four TP53 mutations were found in six tumor samples (20%), and two (c.437G>A and c.706T>C) were novel. Only progesterone receptor status correlated with the number of somatic mtDNA D-loop mutations (likelihood chi-square test; P < 0.05). Somatic mutations in the mtDNA D-loop and in TP53 were independent of each other (Fishers exact test; P > 0.05). These results suggest that the number of somatic mtDNA D-loop mutations may be an indicator of poor prognosis through a mechanism independent of TP53.

The association between microenvironmental reactive oxygen species and embryo development in assisted reproduction technology cycles.

This study was designed to determine the relevance between the levels of reactive oxygen species (ROS) in microenvironment (follicular fluid or culture media) and the embryo development in IVF/ICSI cycles. A total of 466 follicles from 174 IVF/ICSI cycles were collected for this study. The ROS levels in monofollicular fluid and spent culture media were evaluated by chemiluminescence assay with luminol as a probe. The results demonstrated that it is in ICSI cycles that elevated ROS levels in follicular fluid were associated with day 3 poor embryo quality. The ROS levels in spent culture media were correlated with advanced degree of fragmentation. In addition, ROS levels in culture media, instead of in follicular fluid, were negatively correlated with implantation potential of embryos. The ROS levels in culture media may be viewed as an embryo metabolic marker and function as an adjuvant criterion for embryo selection.

Delineation of an Isodicentric Y Chromosome in a Mosaic 45,X/46,X,idic(Y)(qter-p11.3:: p11.3-qter) Fetus by SRY Sequencing, G-banding, FISH, SKY and Study of Distribution in Different Tissues

Many factors such as genetic, developmental and hormonal are involved in mammalian sex determination. The relative importance and the mutual interactions among those factors are obscure. Study of cytogenetic mosaicism involving sex chromosomes may help to further unravel the mysterious process. We report a fetus with a mosaic karyotype, 45,X/46,X,idic(Y)(qter-p11.3::p11.3-qter), with unambiguous male external genitalia and a defect in the interventricular septum of the heart. Genotype of this fetus was extensively studied by technologies including sequencing of SRY (sex-determining region on the Y chromosome) gene, G-banding, FISH (fluorescence in situ hybridization) and SKY (spectral karyotyping). A markedly higher percentage of Y-containing cells was observed in the gonads (55%) than in the amniotic fluid (17%) and placental villi (11%), which was considered to be the major reason why the fetus did not have ambiguous genitalia.

Nitric oxide modulates mitochondrial activity and apoptosis through protein S-nitrosylation for preimplantation embryo development

PurposePrevious studies reported that patients with endometriosis had excess nitric oxide (NO) in the reproductive tract and poor embryo development in IVF cycles. This study aims to elucidate the effects of NO on early embryo development.MethodsZygotes from superovulated B6CBF1 mice were cultured to blastocysts in a variety of media. Sodium nitroprusside (SNP) and NG-nitro-L-arginine (LNA) were added to the culture medium as a NO donor and a NO synthase inhibitor, respectively. The localization and fluorescence intensity of S-nitrosylated (SNO) proteins within 2-cell stage embryos were analyzed with confocal microscopy. Apoptosis and ATP production in the blastocysts were measured.Result(s)Subsequent to NO exposure, the SNO proteins mainly colocalized with the mitochondria and endoplasmic reticulum and the intensity of SNO proteins increased. The addition of a quanylate cyclase inhibitor and a cyclic GMP mimic agent induced nonsignificant changes in SNO proteins, whereas addition of a superoxide scavenger or a reduced form of glutathione rescued the embryos from the effects of NO. However, superoxide scavenger supplementation resulted in decreased blastocyst ATP production.Conclusion(s)Elevated NO exerts deleterious effects on embryo development, possibly through protein S-nitrosylation in the mitochondria and endoplasmic reticulum. Including glutathione as a component in the culture medium might counteract this effect.

The association between polypronucleate zygote formation with certain motion characteristics of sperm and IVF outcome

PurposeTo determine the efficiency of sperm motion characteristics as predictors for normal (2PN) and polypronulceate (PPN) zygotes in IVF.MethodsA retrospective cohort analysis for a total of 230 couples undergoing IVF treatment in a single infertility center.Result(s)Subsequent to semen analysis and hemizona assay, unexpected fertilization failure would appear to have occurred only extremely rarely (1/236, 0.4%). The rate of PPN, however, did arise and appeared to be related to certain sperm motion characteristics, such as lateral head displacement and concentration of progressive motile sperm. Interestingly, the patients featuring a high PPN rate (>20%) was associated with a greater pregnancy rate than those featuring a low PPN rate (<20%).ConclusionThe sperm motion characteristics examined herein could be utilized to predict the rate of PPN in IVF. In order to enhance the rate of 2PN and maintain the relative high rate of clinical pregnancy, an efficient method needs further investigation and development.

Effects of Synthetic Serum Supplementation in Sperm Preparation Media on Sperm Capacitation and Function Test Results

Albumin supplementation of culture media induces sperm capacitation in assisted reproduction technique cycles. Synthetic serum supplementation is clinically used to replace albumin for preventing transmission of infectious agents. However, the effects of synthetic serum supplementation on sperm capacitation have rarely been investigated. Spermatozoa from 30 men with normal basic semen analysis results were collected, divided into five aliquots, and cultured in capacitating conditions in four combinations of two synthetic serum supplements, serum substitute supplement (SSS) and serum protein substitute (SPS), and two fertilization media, Quinns Advantage™ Fertilization (QF) and human tubular fluid (HTF) media. Reactive oxygen species (ROS) levels in spermatozoa were measured through chemiluminescence. Furthermore, acrosome reaction and western blotting for tyrosine phosphorylation were used to evaluate sperm capacitation. HTF+SSS had significantly higher ROS levels than QF+SPS did (11,725 ± 1,172 versus 6,278 ± 864 relative light units). In addition, the spermatozoa cultured in QF+SPS had lower motility, acrosome reaction rates, and tyrosine phosphorylation levels compared with those cultured in HTF+SSS. In conclusion, the effects of synthetic serum supplementation on sperm capacitation varied according to the combination of media. These differences may lead to variations in spermatozoon ROS levels, thus affecting sperm function test results.

Rapid prenatal confirmation of LIT1 hypomethylation using a novel quantitative method (E-Q-PCR) in fetuses with Beckwith-Wiedemann syndrome impressed with ultrasonography

We described a simplified and high-performance test (E-Q-PCR) for rapid assessment of the DNA methylation status at LIT1, a major genetic locus of Beckwith-Wiedemann syndrome (BWS). The E-Q-PCR test can detect and quantify the methylation changes between BWS fetuses and unaffected individuals in aminocytes as well as in lymphocytes and can be completed in 1 working day, and thus is a useful method for prenatal molecular diagnosis of BWS.