Blastocyst telomere length predicts successful implantation after frozen-thawed embryo transfer.

STUDY QUESTION: Do embryos with longer telomere length (TL) at the blastocyst stage have a higher capacity to survive after frozen-thawed embryo transfer (FET)? SUMMARY ANSWER: Digitally estimated TL using low-pass whole genome sequencing (WGS) data from the preimplantation genetic testing for aneuploidy (PGT-A) process demonstrates that blastocyst TL is the most essential factor associated with likelihood of implantation. WHAT IS KNOWN ALREADY: The lifetime TL is established in the early cleavage cycles following fertilization through a recombination-based lengthening mechanism and starts erosion beyond the blastocyst stage. In addition, a telomerase-mediated slow erosion of TL in human fetuses has been observed from a gestational age of 6-11 weeks. Finally, an abnormal shortening of telomeres is likely involved in embryo loss during early development. STUDY DESIGN SIZE DURATION: Blastocyst samples were obtained from patients who underwent PGT-A and FET in an IVF center from March 2015 to May 2018. Digitally estimated mitochondrial copy number (mtCN) and TL were used to study associations with the implantation potential of each embryo. PARTICIPANTS/MATERIALS SETTING AND METHODS: In total, 965 blastocysts from 232 cycles (164 patients) were available to investigate the biological and clinical relevance of TL. A WGS-based workflow was applied to determine the ploidy of each embryo. Data from low-pass WGS-PGT-A were used to estimate the mtCN and TL for each embryo. Single-variant and multi-variant logistic regression, decision tree, and random forest models were applied to study various factors in association with the implantation potential of each embryo. MAIN RESULTS AND THE ROLE OF CHANCE: Of the 965 blastocysts originally available, only 216 underwent FET. While mtCN from the transferred embryos is significantly associated with the ploidy call of each embryo, mtCN has no role in impacting IVF outcomes after an embryo transfer in these women. The results indicate that mtCN is a marker of embryo aneuploidy. On the other hand, digitally estimated TL is the most prominent univariant factor and showed a significant positive association with pregnancy outcomes (P < 0.01, odds ratio 79.1). We combined several maternal and embryo parameters to study the joint effects on successful implantation. The machine learning models, namely decision tree and random forest, were trained and yielded classification accuracy of 0.82 and 0.91, respectively. Taken together, these results support the vital role of TL in governing implantation potential, perhaps through the ability to control embryo survival after transfer. LIMITATIONS REASONS FOR CAUTION: The small sample size limits our study as only 216 blastocysts were transferred. The number was further reduced to 153 blastocysts, where pregnancy outcomes could be accurately traced. The other limitation of this study is that all data were collected from a single IVF center. The uniform and controlled operation of IVF cycles in a single center may cause selection bias. WIDER IMPLICATIONS OF THE FINDINGS: We present novel findings to show that digitally estimated TL at the blastocyst stage is a predictor of pregnancy capacity after a FET cycle. As elective single-embryo transfer has become the mainstream direction in reproductive medicine, prioritizing embryos based on their implantation potential is crucial for clinical infertility treatment in order to reduce twin pregnancy rate and the time to pregnancy in an IVF center. The AI-powered, random forest prediction model established in this study thus provides a way to improve clinical practice and optimize the chances for people with fertility problems to achieve parenthood. STUDY FUNDING/COMPETING INTERESTS: This study was supported by a grant from the National Science and Technology Council, Taiwan (MOST 108-2321-B-006-013 -). There were no competing interests. TRIAL REGISTRATION NUMBER: N/A.

MicroRNA-320a enhances LRWD1 expression through the AGO2/FXR1-dependent pathway to affect cell behaviors and the oxidative stress response in human testicular embryonic carcinoma cells.

BACKGROUND: Testicular cancer is fairly rare but can affect fertility in adult males. Leucine-rich repeats- and WD repeat domain-containing protein 1 (LRWD1) is a sperm-specific marker that mainly affects sperm motility in reproduction. Our previous study demonstrated the impact of LRWD1 on testicular cancer development; however, the underlying mechanisms remain unclear. METHODS: In this study, various plasmids associated with LRWD1 and miR-320a manipulation were used to explore the roles and regulatory effects of these molecules in NT2D1 cellular processes. A Dual-Glo luciferin-luciferase system was used to investigate LRWD1 transcriptional activity, and qRT-PCR and western blotting were used to determine gene and protein expression. RESULTS: The results suggested that miR-320a positively regulated LRWD1 and positively correlated with NT2D1 cell proliferation but negatively correlated with cell migration and invasion ability. In addition, the miRNA-ribonucleoprotein complex AGO2/FXR1 was shown to be essential in the mechanism by which miR-320a regulates LRWD1 mRNA expression. As miR-320a was required to regulate LRWD1 expression through the AGO2 and FXR1 complex, eEF2 and eLF4E were also found to be involved in miR-320a increasing LRWD1 expression. Furthermore, miR-320a and LRWD1 were responsive to oxidative stress, and NRF2 was affected by the presence of miR-320a in response to ROS stimulation. CONCLUSIONS: This is the first study showing the role of miR-320a in upregulating the testicular cancer-specific regulator LRWD1 and the importance of the AGO2/FXR1 complex in miR-320a-mediated upregulation of LRWD1 during testicular cancer progression.

Development and validation of an expanded targeted sequencing panel for non-invasive prenatal diagnosis of sporadic skeletal dysplasia.

BACKGROUND: Skeletal dysplasia (SD) is one of the most common inherited neonatal disorders worldwide, where the recurrent pathogenic mutations in the FGFR2, FGFR3, COL1A1, COL1A2 and COL2A1 genes are frequently reported in both non-lethal and lethal SD. The traditional prenatal diagnosis of SD using ultrasonography suffers from lower accuracy and performed at latter gestational stage. Therefore, it remains in desperate need of precise and accurate prenatal diagnosis of SD in early pregnancy. With the advancements of next-generation sequencing (NGS) technology and bioinformatics analysis, it is feasible to develop a NGS-based assay to detect genetic defects in association with SD in the early pregnancy. METHODS: An ampliseq-based targeted sequencing panel was designed to cover 87 recurrent hotspots reported in 11 common dominant SD and run on both Ion Proton and NextSeq550 instruments. Thirty-six cell-free and 23 genomic DNAs were used for assay developed. Spike-in DNA prepared from standard sample harboring known mutation and normal sample were also employed to validate the established SD workflow. Overall performances of coverage, uniformity, and on-target rate, and the detecting limitations on percentage of fetal fraction and read depth were evaluated. RESULTS: The established targeted-seq workflow enables a single-tube multiplex PCR for library construction and shows high amplification efficiency and robust reproducibility on both Ion Proton and NextSeq550 platforms. The workflow reaches 100% coverage and both uniformity and on-target rate are > 96%, indicating a high quality assay. Using spike-in DNA with different percentage of known FGFR3 mutation (c.1138 G > A), the targeted-seq workflow demonstrated the ability to detect low-frequency variant of 2.5% accurately. Finally, we obtained 100% sensitivity and 100% specificity in detecting target mutations using established SD panel. CONCLUSIONS: An expanded panel for rapid and cost-effective genetic detection of SD has been developed. The established targeted-seq workflow shows high accuracy to detect both germline and low-frequency variants. In addition, the workflow is flexible to be conducted in the majority of the NGS instruments and ready for routine clinical application. Taken together, we believe the established panel provides a promising diagnostic or therapeutic strategy for prenatal genetic testing of SD in routine clinical practice.

LRWD1 expression is regulated through DNA methylation in human testicular embryonal carcinoma cells.

BACKGROUND: Sperm growth and maturation are correlated with the expression levels of Leucine-rich repeat and WD repeat-containing protein 1 (LRWD1), a widely expressed protein in the human testicles. The decrease in LRWD1 cellular level was linked to the reduction in cell growth and mitosis and the rise in cell microtubule atrophy rates. Since DNA methylation has a major regulatory role in gene expression, this study aimed at exploring the effect of the modulation of DNA methylation on LRWD1 expression levels. RESULTS: The results revealed the presence of a CpG island up of 298 bps (- 253 ~ + 45) upon LRWD1 promoter in NT2/D1 cells. The hypermethylation of the LRWD1 promoter was linked to a reduction in the transcription activity in NT2/D1 cells, as indicated by luciferase reporter assay. The methylation activator, floxuridine, confirmed the decrease in the LRWD1 promoter transcriptional activity. On the other hand, 5-Aza-2'-deoxycytidine (5-Aza-dc, methylation inhibitor), significantly augmented LRWD1 promoter activity and the expression levels of mRNA and proteins. Furthermore, DNA methylation status of LRWD1 promoter in human sperm genomic DNA samples was analyzed. The results indicated that methylation of LRWD1 promoter was correlated to sperm activity. CONCLUSIONS: Thus, the regulation of LRWD1 expression is correlated with the methylation status of LRWD1 promoter, which played a significant role in the modulation of spermatogenesis, sperm motility, and vitality. Based on these results, the methylation status of LRWD1 promoter may serve as a novel molecular diagnostic marker or a therapeutic target in males' infertility.

RéSUMé: CONTEXTE: La croissance et la maturation des spermatozoïdes sont corrélées avec les niveaux d'expression de la protéine 1 riche en répétitions Leucine et contenant des répétitions WD (LRWD1), une protéine largement exprimée dans les testicules humains. La diminution du niveau cellulaire en LRWD1 a été liée à une réduction de la croissance et des mitoses cellulaires, et à une augmentation des taux d'atrophie des microtubules cellulaires. Puisque la méthylation de l'ADN joue un rôle régulateur majeur dans l'expression des gènes, cette étude visait à explorer l'effet de la modulation de la méthylation de l'ADN sur les niveaux d'expression de LRWD1. RéSULTATS: Les résultats ont révélé la présence d'un îlot CpP de 298 pbs (-253~+45) sur le promoteur de LRWD1dans les cellules NT2/D1. L'hyperméthylation du promoteur de LRWD1 était liée à une réduction de l'activité de transcription dans les cellules NT2/D1, comme indiqué par l'analyse de l'expression d'un gène rapporteur codant pour la luciférase. L'activateur de méthylation, la floxuridine, a confirmé la diminution de l'activité transcriptionnelle du promoteur de LRWD1. D'autre part, la 5-Aza-2'-déoxycytidine (5-Aza-dc, inhibiteur de méthylation), a significativement augmenté l'activité du promoteur de LRWD1 et les niveaux d'expression de l'ARNm et des protéines. En outre, le statut de méthylation de l'ADN du promoteur de LRWD1 dans les échantillons d'ADN génomique de sperme humain a été analysé. Les résultats ont indiqué que la méthylation du promoteur de LRWD1 était corrélée à l'activité des spermatozoïdes. CONCLUSIONS: Ainsi, la régulation de l'expression LRWD1 est corrélée avec le statut de méthylation du promoteur de LRWD1, qui a joué un rôle important dans la modulation de la spermatogenèse, de la mobilité et de la vitalité des spermatozoïdes. Sur la base de ces résultats, le statut de méthylation du promoteur de LRWD1 peut servir de nouveau marqueur diagnostic moléculaire ou de cible thérapeutique dans l'infertilité masculine.

Semen quality and insulin-like factor 3: Associations with urinary and seminal levels of phthalate metabolites in adult males.

Certain phthalates have adverse effects on male reproductive functions in animals, and potentially affect human testicular function and spermatogenesis, but little is known about the active mechanisms. We measured the urinary and seminal phthalate metabolites and explored their associations on insulin-like factor 3 (INSL3) and semen quality. Urine, blood, and semen samples were collected from the male partners of subfertile (n = 253) and fertile (n = 37) couples in a reproductive center in southern Taiwan. INSL3, reproductive hormones, semen-quality, and 11 phthalate metabolites in urine and semen were measured. There were significant correlations in the distribution pattern of metabolites, such as the relative contribution of low or high molecular weight phthalate metabolites. The significantly monotonic trends in semen volume, sperm concentration and motility were associated with increasing quartiles of INSL3 (all p-trend < 0.001). In adjusted regression models, increases in urinary phthalate metabolites levels were adversely associated with sperm concentration (monobenzyl phthalate [MBzP], mono-2-ethylhexyl phthalate [MEHP] and MEHP%), motility (MBzP and MEHP) and INSL3 (MBzP, MEHP and MEHP%) (all p < 0.01). Higher seminal phthalate metabolite levels were associated with decreases in sperm concentration (MEHP and mono-2-ethyl-5-hydroxyhexyl phthalate), motility (mono-ethyl phthalate [MEP] and di-(2-ethylhexyl) phthalate [DEHP] metabolites), normal morphology (MEP), and INSL3 (monomethyl phthalate and MEP) (all p < 0.05). Our data suggest that INSL3 secretion, reproductive hormone balance, and sperm production and quality might be simultaneously adversely affected for individuals excreting increasing levels of phthalates metabolites (especially di-ethyl phthalate, butylbenzyl phthalate, and DEHP) in urine and semen samples.

Phthalates might interfere with testicular function by reducing testosterone and insulin-like factor 3 levels.

STUDY QUESTION: Do phthalates create a male reproductive hormone imbalance by down-regulating the secretion of testosterone and insulin-like factor 3 (INSL3)? SUMMARY ANSWER: Our study suggests that exposure to phthalates is related to a reduction in the secretion of testosterone and INSL3 in adult males. WHAT IS KNOWN ALREADY: There is evidence that exposure to phthalates, an abundant group of industrial plasticizers, negatively affects testosterone biosynthesis, but little is known about the mechanism in men. The hypothesis that exposure to phthalates reduces the levels of testosterone and INSL3, a marker of Leydig cell function, is underexplored. STUDY DESIGN, SIZE, DURATION: This case-control study of 176 men ran from 2010 to 2012. Infertile men were recruited through infertility clinics in Taiwan, fertile men were recruited from childbirth preparation classes and all were categorized based on the World Health Organization definition of infertility and by the diagnoses of obstetricians. PARTICIPANTS/MATERIALS, SETTING, METHODS: Urinary concentrations of 11 phthalate metabolites were measured, along with serum levels of FSH, LH, total testosterone (TT), estradiol, sex hormone-binding globulin and Inhibin B. Androgen status indices including free testosterone (fT) and the free androgen index (FAI) were calculated. The circulating INSL3 level was evaluated using a radioimmunoassay. Non-parametric analyses, trend tests and linear regression models were used. MAIN RESULTS AND THE ROLE OF CHANCE: Urinary mono-n-butyl phthalate (MnBP), mono-(2-ethylhexyl) phthalate (MEHP) and mono-2-ethyl-5-carboxypentyl phthalate were significantly higher in infertile than in fertile men. Serum Inhibin B, the Inhibin B : FSH ratio, the TT : LH ratio and INSL3 were significantly lower in infertile men. In multiple regression models controlled for potential confounders, there is an inverse association between urinary levels of mono-methyl phthalate (MMP), mono-iso-butyl phthalate (MiBP), MEHP, MEHP% and serum TT (P = 0.001, 0.007, 0.042 and 0.012, respectively). The inverse associations were also found between urinary levels of MiBP, monobenzyl phthalate (MBzP), MEHP, MEHP% and serum fT (P = 0.028, 0.017, 0.045 and 0.027, respectively); between urinary levels of MMP, MEHP, MEHP% and the TT : LH ratio (P = 0.004, 0.029 and 0.039, respectively); between urinary levels of MMP, MiBP, MnBP, MBzP, MEHP and the FAI (P = 0.002, 0.008, 0.037, 0.028, 0.042 and 0.016, respectively). Urinary MBzP and MEHP% were negatively associated with a decrease in serum INSL3 (P = 0.049 and <0.001). We also observed a strong inverse relationship between MEHP% quartiles and serum TT, fT, the TT : LH ratio and INSL3 (Ptrend = 0.003, 0.080, 0.002 and 0.012, respectively). Serum INSL3, TT, fT and the TT : LH ratio were lower for men in the highest MEHP% quartile than in the reference group (P = 0.007, 0.002, 0.090 and 0.001, respectively). LIMITATIONS, REASONS FOR CAUTION: A potential limitation is using a single urine and blood sample to predict urinary phthalate metabolites and reproductive hormone status over long periods. However, there is evidence that a single measure provides a reliable result in population studies. WIDER IMPLICATIONS OF THE FINDINGS: Non-occupational exposure to phthalates, including di-2-ethylhexyl phthalate, might lead to adverse effects on testicular/Leydig cell function and be of concern owing to the ubiquitous multisource exposure to phthalates among the general population. Although our findings are in agreement with recent experimental data, more studies are required to draw firm conclusions on the relation of INSL3 to phthalate exposure or testicular/Leydig cell function.

Enhanced myometrial autophagy in postpartum uterine involution.

OBJECTIVE: To understand the mechanisms of postpartum uterine involution, we investigated the uterine myometrial changes during pregnancy and the postpartum period. MATERIALS AND METHODS: Nine groups of uterine myometrial samples from mice (n = 4) were collected on gestational Day 0 (nonpregnant), Day 1, Day 2, Day 7, Day 14, and Day 21 and on postpartum Day 1, Day 2, and Day 7. Human samples of uterine myometrium on term (n = 1) and postpartum Day 1 (n = 2) were also collected. Ki-67 immunostaining was used to determine myometrial proliferation. For cell hypertrophy analysis, organelle proteins, ß-actin, prohibin, calnexin, and golgin-97 were analyzed by Western blotting. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and evaluation of activated caspase-3 expression by Western blot analysis assay were used to detect apoptosis. Autophagy was assayed via the evaluation of LC3 expression by Western blotting, immunohistochemistry, and autophagosomes by electron microscopy. RESULTS: Uterine myocytes proliferated during the early stage of gestation with a peak at Day 2, whereas myocyte hypertrophy with increased cellular organelle production occurred gradually in later stages of pregnancy. Postpartum autophagy developed abruptly in uterine myocytes without obvious apoptosis. CONCLUSION: Autophagy of myocytes may play an important role in uterine involution. These results have implications for our understanding of myometrial functional adaptations during pregnancy and the physiological role of autophagy in the uterine remodeling events in the postpartum period.

Characterization of 3-hydroxyisobutyrate dehydrogenase, HIBADH, as a sperm-motility marker.

PURPOSE: Asthenozoospermia is a major cause of male infertility. However, the molecular mechanisms underlying sperm-motility defects remain largely unknown in the majority of cases. In our previous study, we applied a proteomic approach to identify unknown proteins that were downregulated in spermatozoa with low motility compared to spermatozoa with good motility. Several sperm motility- related proteins have been identified. In this study, 3-hydroxyisobutyrate dehydrogenase (HIBADH), one of the proteins identified using the proteomic tools, is further characterized. METHODS: Reverse-transcription polymerase chain reactions (RT-PCR), western blotting, and immunofluorescence assays (IFA) were preformed to investigate the expression pattern. The enzymatic activity of HIBADH was evaluated in sperm with good (>50 %), moderate (< 50 %) and lower motility (< 20 %). RESULTS: Using RT-PCR, we found that transcripts of HIBADH are enriched in the cerebellum, heart, skeletal muscle, uterus, placenta, and testes of male humans. In western blotting, it is expressed in the placenta, testes, and spermatozoa. During spermiogenesis, HIBADH is located at the mid-piece (a specialized development from the mitochondria) of elongating, elongated, and mature sperm. The enzymatic activity of HIBADH in sperm with moderate and lower motility were significantly reduced compared with good motility (P<0.0001 and P<0.05, respectively). CONCLUSIONS: Our study indicated that HIBADH is involved in the mitochondrial function of spermatozoa, and maintains sperm motility. It may serve as a sperm-motility marker.

Effects of rosiglitazone on the cardiovascular profile in postmenopausal women without diabetes mellitus: interplay of thiazolidinediones and hormone therapy.

OBJECTIVE: Thiazolidinediones have antiatherothrombotic effects on persons with diabetes. Hormone therapy among postmenopausal women has both positive and negative cardiovascular effects. However, the effects of rosiglitazone with or without concurrent long-term hormone therapy on the cardiovascular profile of nondiabetic postmenopausal women are unknown. METHODS: Thirty-eight nondiabetic postmenopausal women were enrolled in this double-blind and placebo-controlled study. Eighteen participants received 4 mg rosiglitazone, and 20 participants took placebo daily for 12 weeks. Global endothelial function and plasma biomarkers were measured. RESULTS: Baseline characteristics and parameters were similar between the groups. Rosiglitazone, but not placebo, significantly reduced leukocyte count and plasma levels of matrix metalloproteinase-9 and inhibited the elevation of plasma levels of plasminogen activator inhibitor-1 and tissue plasminogen activator (P < 0.05 for all). Most of the favorable effects provided by rosiglitazone were still present in participants with concurrent hormone therapy. Increased body weight and waist size as well as elevation of the plasma levels of total and low-density lipoprotein cholesterol were noted after rosiglitazone treatment among participants without concurrent hormone therapy. No significant change in the global endothelial function occurred in response to treatment in either group. CONCLUSIONS: Rosiglitazone treatment provided both protective and harmful cardiovascular effects in nondiabetic postmenopausal women. Concurrent hormone therapy resulted in the maintenance of the major beneficial effects while neutralizing the unfavorable effects of rosiglitazone.

SEPT12 mutations cause male infertility with defective sperm annulus.

Septins are members of the GTPase superfamily, which has been implicated in diverse cellular functions including cytokinesis and morphogenesis. Septin 12 (SEPT12) is a testis-specific gene critical for the terminal differentiation of male germ cells. We report the identification of two missense SEPT12 mutations, c.266C>T/p.Thr89Met and c.589G>A/p.Asp197Asn, in infertile men. Both mutations are located inside the GTPase domain and may alter the protein structure as suggested by in silico modeling. The p.Thr89Met mutation significantly reduced guanosine-5'-triphosphate (GTP) hydrolytic activity, and the p.Asp197Asn mutation (SEPT12(D197N)) interfered with GTP binding. Both mutant SEPT12 proteins restricted the filament formation of the wild-type SEPT12 in a dose-dependent manner. The patient carrying SEPT12(D197N) presented with oligoasthenozoospermia, whereas the SEPT12(T89M) patient had asthenoteratozoospermia. The characteristic sperm pathology of the SEPT12(D197N) patient included defective annulus with bent tail and loss of SEPT12 from the annulus of abnormal sperm. Our finding suggests loss-of-function mutations in SEPT12 disrupted sperm structural integrity by perturbing septin filament formation.

Use of an oxytocin antagonist in in vitro fertilization-embryo transfer for women with repeated implantation failure: a retrospective study.

OBJECTIVE: This retrospective study aimed to investigate the use of an oxytocin antagonist in improving the pregnancy outcome of in vitro fertilization-embryo transfer (IVF-ET) in patients with repeated implantation failure (RIF). MATERIALS AND METHODS: A total of 150 infertile couples with RIF undergoing IVF-ET were divided into three groups. Patients who did not receive atosiban were used as controls (Group 1; n=80). Forty patients received a single bolus dose (6.75mg, 0.9mL/vial) of atosiban before ET (Group 2), and 30 patients received a bolus dose of 6.75mg atosiban followed by infusion at 18mg/hr for 3 hours immediately after ET (Group 3). RESULTS: A significantly higher implantation rate (30.21%) was noted in Group 2 compared with Groups 1 and 3 (11.8% and 15.9%, respectively; p=0.0006). The clinical pregnancy rate of Group 2 (37.5%) was significantly higher than that of Groups 1 (12.5%) and 3 (20%) (p=0.0057). The live birth rate was significantly higher in Group 2 (35%) than in Groups 1 and 3 (10% and 16.67%, respectively; p=0.0031). CONCLUSION: These results suggest that IVF-ET using lower dosage of atosiban may improve pregnancy outcomes of patients with RIF.

Protein tyrosine phosphatase non-receptor type 14 is a novel sperm-motility biomarker.

PURPOSE: To understand the molecular basis of sperm-motility and to identify related novel motility biomarkers. METHODS: Two-dimensional electrophoresis (2DE) followed by Reverse-phase-nano-high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (RP-nano-HPLC-ESI-MS/MS) were applied to establish the human sperm proteome. Then the sperm proteome of moderate-motile human sperm fraction and that of good-motile human sperm fraction from pooled spermatozoa of forty normozoospermic donors (Group 1 subjects) were compared to identify the dysregulated proteins. Among these down-regulated proteins, Protein tyrosine phosphatase non-receptor type 14 (PTPN14) was chosen to reconfirm by Western blotting and semi-quantitative reverse transcription polymerase chain reaction. For clinical application, Western blotting and real-time reverse transcription polymerase chain reaction was performed to compare the expression level of PTPN14 in (Group 2 subjects) nine normozoospermic controls and thirty-three asthenozoospermic patients (including 21 mild asthenozoospermic cases and 12 severe cases). Finally, bioinformatic tools prediction and immunofluorescence assay were performed to elucidate the potential localization of PTPN14. RESULTS: The expression levels of three proteins were observed to be lower in the moderate-motile sperm fraction than in good-motile sperm of group 1 subjects. Among three proteins with persistent down-regulation in the moderate-motile sperm, we reconfirmed that the expression level of PTPN14 was significantly lower in both mRNA and protein levels from the moderate-motile sperm fraction. Further, down-regulation of PTPN14 was found at the translational and transcriptional level in the asthenozoospermic men. Finally, Bioinformatic tools prediction and immunofluorescence assay showed that PTPN14 maybe predominantly localized at the mitochondria in the midpiece of human ejaculated sperm. CONCLUSIONS: Proteomics tools were applied to identify three possible sperm motility-related proteins. Among these proteins, PTPN14 was highly likely a novel sperm-motility biomarker and a potential mitochondrial protein.

SEPT12 deficiency causes sperm nucleus damage and developmental arrest of preimplantation embryos.

Oocytes fertilized with spermatozoa obtained from Septin 12+/- chimeric mice failed to develop beyond the morula stage after IVF and intracytoplasmic sperm injection because of significant DNA defects in the spermatozoa. Given that SEPT12 is expressed at the edge of the sperm nucleus in both humans and mice, we hypothesized the vital roles of Septin 12 in sperm head shaping, nuclear DNA condensation, and early embryonic development.

Polymorphisms of endocrine gland-derived vascular endothelial growth factor gene and its receptor genes are associated with recurrent pregnancy loss.

BACKGROUND: Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) and its receptor genes [prokineticin receptor 1 (PKR1) and prokineticin receptor 2 (PKR2)] have been identified in the last decade and their expression is restricted to the steroidogenic glands (ovary, testis, adrenal gland and placenta). Their expression patterns also suggest a close relationship to early pregnancy. However, little information is available regarding the role of EG-VEGF and its receptors (PKR1 and PKR2) in recurrent pregnancy loss (RPL). This study was conducted to investigate the association between polymorphisms of EG-VEGF and its receptor genes (PKR1 and PKR2) and idiopathic RPL. METHODS: In this case-control study, 115 women with a history of idiopathic RPL and 170 controls were included. A total of 11 tag single nucleotide polymorphisms (SNPs) selected from EG-VEGF, PKR1 and PKR2 were genotyped. We further used multifactor dimensionality reduction (MDR) analysis to choose a best model and evaluate gene-gene interactions. RESULTS: Two tag SNPs of PKR1 (rs4627609, rs6731838) and one tag SNP of PKR2 (rs6053283) were significantly associated with idiopathic RPL (P < 0.05). The frequencies of haplotypes C-G and T-A of PKR1 and haplotype A-G-C-G-G of PKR2 were significantly increased in women with idiopathic RPL (P < 0.05); MDR tests revealed gene-gene interactions between three loci [EG-VEGF (rs7513898), PKR1(rs6731838), PKR2(rs6053283)] based on the association model (P = 0.008). The adjusted odds ratio of high- and low-risk genotype combinations in the three-locus model was 3.94 (95% confidence interval: 2.38-6.52). CONCLUSIONS: EG-VEGF receptor (PKR1, PKR2) gene polymorphisms and haplotypes were associated with idiopathic RPL. These three genes (EG-VEGF, PKR1 and PRK2) jointly contribute to RPL in the Taiwanese Han population.

Frontal cerebral blood flow changes after hormone replacement therapy in depressed postmenopausal women.

We investigated the effects of hormone replacement therapy (HRT) on frontal cerebral blood flow (CBF), depressive symptoms, and cognitive function in depressed postmenopausal women. Fourteen postmenopausal women with depressive symptoms underwent HRT, and seven controls not undergoing HRT were studied. We evaluated frontal CBF, expressed as frontal/cerebellum (F/C) ratio, using Tc-99m hexamethyl propylene amine oxime single photon emission computed tomography (Tc-99m HMPAO SPECT), cognitive function using the Mini-Mental Status Examination (MMSE), and depression using the HAD (Hospital Anxiety and Depression) scale. All studies were carried out at initial status and after 9 months. Single photon emission computed tomography was performed at rest and at activation during the Wisconsin Card Sorting Test (WCST). Initial frontal CBF was not different between groups. After 9 months, resting frontal CBF was similar between groups. However, activated frontal CBF was significantly higher in the HRT group than in controls (F/C ratio: 0.924+/-0.04 versus 0.853+/-0.05, P=0.007). Furthermore, the increase in the activated F/C ratio was inversely associated with years since menopause. Mini-Mental Status Examination scores improved after HRT, but depression scores did not. Hormone replacement therapy improved frontal CBF and cognitive function but not depression in postmenopausal women. The changes in frontal CBF were detected only during WCST activation and were most apparent during early postmenopausal years.

The expression level of septin12 is critical for spermiogenesis.

Septins belong to a family of polymerizing GTP-binding proteins that are required for many cellular functions, such as membrane compartmentalization, vesicular trafficking, mitosis, and cytoskeletal remodeling. One family member, septin12, is expressed specifically in the testis. In this study, we found septin12 expressed in multiple subcellular compartments during terminal differentiation of mouse germ cells. In humans, the testicular tissues of men with either hypospermatogenesis or maturation arrest had lower levels of SEPTIN12 transcripts than normal men. In addition, increased numbers of spermatozoa with abnormal head, neck, and tail morphologies lacked SEPT12 immunostaining signals, as compared with normal spermatozoa. To elucidate the role of septin12, we generated 129 embryonic stem cells containing a septin12 mutant allele with a deletion in the exons that encode the N-terminal GTP-binding domain. Most chimeras derived from the targeted embryonic stem cells were infertile, and the few fertile chimeras only produced offspring with a C57BL/6 background. Semen analysis of the infertile chimeras showed a decreased sperm count, decreased sperm motility, and spermatozoa with defects involving all subcellular compartments. The testicular phenotypes included maturation arrest of germ cells at the spermatid stage, sloughing of round spermatids, and increased apoptosis of germ cells. Electron microscopic examination of spermatozoa showed misshapen nuclei, disorganized mitochondria, and broken acrosomes. Our data indicate that Septin12 expression levels are critical for mammalian spermiogenesis.

CDC25 protein expression and interaction with DAZL in human corpus luteum.

OBJECTIVE: To determine whether the human corpus luteum exhibits CDC25 protein expression and its expression pattern, and identify the interaction with DAZL (deleted in azoospermia-like) in human luteal cells. DESIGN: Experimental study. SETTING: Medical research laboratory in a university hospital. PATIENT(S): Twelve human corpus luteum (CL), four in the early stage, four in the midstage, and four in the late stage of the luteal phase, respectively, and 10 granulosa-lutein cells from IVF patients. INTERVENTION(S): Immunohistochemical stain and Western blot were used to characterize the expression of CDC25 protein, and protein immunoprecipitation was used to identify the interaction of CDC25 with DAZL in human luteal cells. MAIN OUTCOME MEASURE(S): CDC25 protein expression pattern in different stages of luteal phase and interaction with DAZL. RESULT(S): In this study, we show evidence that CDC25 protein is express in granulosa-lutein cells from different stages of CL, and identified only the CDC25A interaction with DAZL in luteal cells. CONCLUSION(S): The CL is the final form of a developing follicle and is the major endocrine component of the ovary in maintaining early successful pregnancy. Expression of CDC25 protein throughout different stages of the ovarian cycles implies important functional roles in the regulation of female reproduction.

Association of USP26 haplotypes in men in Taiwan, China with severe spermatogenic defect.

AIM: To complete comprehensive haplotype analysis of USP26 for both fertile and infertile men. METHODS: Two hundred infertile men with severe oligospermia or non-obstructive azoospermia were subjected to sequence analysis for the entire coding sequences of the USP26 gene. Two hundred men with proven fertility were genotyped by primer extension methods. Allele/genotype frequencies, linkage disequilibrium (LD) characteristics and haplotypes of fertile men were compared with infertile men. RESULTS: The allele frequencies of five single nucleotide polymorphisms (370-371insACA, 494T>C, 576G>A, ss6202791C>T, 1737G>A) were significantly higher in infertile patients than control subjects. The major haplotypes in infertile men were TACCGA (28% of the population), TGCCGA (15%), TACCAA (8%), TGCCAA (6%), TATCAA (5%) and CATCAA (5%). The major haplotypes for the control subjects were TACCGA (58% of the population), CACCGA (7%), CATCGA (6%) and TGCCGA (5%). Haplotypes TGCCGA, TATCAA, CATCAA, CATCGC, TACCAA and TGCCAA were over-transmitted in patients with spermatogenic defect, whereas haplotypes TACCGA, CACCGA, and CATCGA were under-transmitted in these patients. CONCLUSION: Some USP26 alleles and haplotypes are associated with spermatogenic defect in the Han nationality in Taiwan, China.

Reduced frontal perfusion in depressed postmenopausal women: a SPECT study with WCST.

OBJECTIVES: To investigate frontal cerebral blood flow (CBF) in depressed postmenopausal women and its relation to cognitive function and the severity of depressive symptoms. METHODS: Regional CBF of 20 unmedicated depressed postmenopausal women was measured using Tc-99m HMPAO SPECT, both at rest and during frontal activation using the Wisconsin card sorting test (WCST). Frontal CBF was semi-quantified by comparing the radioactivity in the prefrontal region to the cerebellum (F/C ratio). We measured the severity of the symptoms of depression using the hospital anxiety and depression scale (HADS) and cognitive function using the mini-mental status examination (MMSE). RESULTS: At rest, there was no difference in frontal CBF between patients with moderate or severe (HADS> or =11) and patients with mild depressive symptoms (HADS<11). During the WCST, however, the HADS> or =11 group did not score as well as the HADS<11 group (P=0.03). The changes in F/C ratios were inversely correlated with HADS scores (r=-0.43, P=0.05) and positively correlated with MMSE scores (r=0.58, P=0.004). After adjusting for age, F/C ratios were significantly correlated with MMSE (P=0.002), but not with HADS scores. CONCLUSIONS: Frontal CBF did not increase in postmenopausal women with moderate/severe symptoms of depression during the WCST activation task, and reduced frontal CBF was related to the impairment of cognitive function. The combination of the functional activation test and SPECT imaging powerfully revealed this functional disease, which remains undetectable using more common baseline measurements.

DAZL protein expression in mouse preimplantation embryo.

OBJECTIVE: To evaluate the expression pattern of Dazl (deleted in azoospermia-like) protein in the mouse preimplantation embryo. DESIGN: Experimental study. SETTING: Medical research laboratory in a university hospital. ANIMAL(S): Twenty female 28- to 35-day-old FVB mice. INTERVENTION(S): Embryo collection at 1.5, 2.5, and 3.5 days postcoitus (plug date, 0.5 d postcoitus) to examine the Dazl protein expression from the two-cell embryo to the blastocyst. MAIN OUTCOME MEASURE(S): Dazl protein expression was analyzed by immunofluorescent staining. RESULT(S): There is abundant expression of Dazl protein in the cytoplasm of the blastomere. Strong fluorescent signals of Dazl protein expression were found in preimplantation embryo cytoplasm, including two-cell, eight-cell, morula, and blastocyst. CONCLUSION(S): By using an antibody raised against mouse Daz-like protein (Dazl), we showed that Dazl protein is present in all cleaving stages of the preimplantation embryo. This is the first report on the protein expression of a Dazl gene during embryogenesis in mice. However, further study is needed to evaluate the molecular functional role of Dazl.