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Objectives: The objective of this study was to determine whether or not the angiotensin-converting enzyme insertion/deletion (ACE I/D), angiotensin II type 1 receptor (AT1R), and angiotensinogen (AGT) gene polymorphisms are associated with idiopathic recurrent spontaneous abortions (RSAs) in Korean women., Study Design: A total of 251 patients with unexplained consecutive pregnancy losses, and 126 healthy controls with at least one live birth and no history of pregnancy loss., Result: The odds ratios (ORs) of the ACE ID (OR=2.423; 95% confidence interval (CI)=1.417-4.142; p=0.001) and the ACE II (OR=2.050; 95% CI=1.143-3.675; p=0.018) for the ACE DD genotype were significantly different between patients with idiopathic RSA and controls; however, there were no significant differences between patients and controls with respect to the AT1R 1166A>C and AGT M235T polymorphisms. In a haplotype-based analysis of I-A (p=0.010), D-A (p=0.004), I-A-T (p=0.033), D-A-T (p=0.0005), and D-C-T (p=0.013) polymorphism pairs with synergistic effects derived by the MDR method in patients and in controls showed significant results., Conclusion: This study suggests that ACE, AT1R and AGT polymorphisms and haplotypes are a genetic determinant for the risk of idiopathic RSA in Korean women., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Prostaglandins participate in a variety of female reproductive processes, including ovulation, fertilization, embryo implantation and parturition. In particular, maternal prostacyclin (PGI(2)) is critical for embryo implantation and the action of PGI(2) is not mediated via its G-protein-coupled membrane receptor, IP, but its nuclear receptor, peroxisome-proliferator-activated receptor δ (PPARδ). Recently, several studies have shown that PGI(2) enhances blastocyst development and/or hatching rate in vitro, and subsequently implantation and live birth rates in mice. However, the mechanism by which PGI(2) improves preimplantation embryo development in vitro remains unclear. Using molecular, pharmacologic and genetic approaches, we show that PGI(2)-induced PPARδ activation accelerates blastocyst hatching in mice. mRNAs for PPARδ, retinoid X receptor (heterodimeric partners of PPARδ) and PGI(2) synthase (PGIS) are temporally induced after zygotic gene activation, and their expression reaches maximum levels at the blastocyst stage, suggesting that functional complex of PPARδ can be formed in the blastocyst. Carbaprostacyclin (a stable analogue of PGI(2)) and GW501516 (a PPARδ selective agonist) significantly accelerated blastocyst hatching but did not increase total cell number of cultured blastocysts. Whereas U51605 (a PGIS inhibitor) interfered with blastocyst hatching, GW501516 restored U51605-induced retarded hatching. In contrast to the improvement of blastocyst hatching by PPARδ agonists, PPAR antagonists significantly inhibited blastocyst hatching. Furthermore, deletion of PPARδ at early stages of preimplantation mouse embryos caused delay of blastocyst hatching, but did not impair blastocyst development. Taken together, PGI(2)-induced PPARδ activation accelerates blastocyst hatching in mice.

Although fenarimol is a widely used chlorinated fungicide applied to fruits and vegetables and is a suspected endocrine disrupter, transgenerational studies of low doses of fenarimol exposure are not currently available. The aims of this study are to address the effect of maternal exposure to low doses of fenarimol on the reproductive performance of offspring and to investigate the expression changes of genes associated with this effect. Pregnant mice were orally exposed to low doses (0, 2, 20, and 200 μg/kg body weight) of fenarimol during gestational and lactational periods, and their offspring were assessed. The body and organ weights and anogenital distance (AGD) of mice offspring (F1) maternally exposed to fenarimol were determined, and the reproductive performances of these mice were assessed by mating and ovarian follicular and sperm analyses. Fenarimol-exposed F1 mice had shortened AGDs, increased body weight with altered organ weights, increased number of pups, increased number of ovarian follicles, and enhanced sperm quality. Microarray data showed 82 upregulated and 743 downregulated genes in the ovaries of fenarimol-exposed mice, in which Cyp17a1, Cyp19a1, and ERβ were upregulated. In addition, Nobox, a pivotal gene required for proper folliculogenesis, was significantly increased in the ovaries of F1 mice. In conclusion, maternal exposure to fenarimol promotes normal reproductive function in female mouse offspring by increasing the expression levels of genes crucial for ovarian folliculogenesis, identifying fenarimol as a chemical that stimulates reproductive performance. Thus, consumption of fenarimol-contaminated diets by mothers may possibly alter normal reproductive function of offspring in humans and wildlife., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Objective: Laser-assisted intracytoplasmic sperm injection (LA-ICSI), also known as micro-opening or thinning of the zona pellucida (ZP) prior to ICSI, may help to reduce mechanical damage to the oocyte during the procedure. The aim of the present study was to evaluate and analyze the efficacy of our institutional LA-ICSI program, which features laser-assisted ZP thinning prior to ICSI, in comparison with conventional ICSI (C-ICSI), performed on patients with different clinical characteristics., Methods: Patients undergoing a total of 212 ICSI cycles were randomly divided into an LA-ICSI group (106 cycles) and a conventional ICSI group (106 cycles). To reduce tissue damage, we thinned the ZP by approximately 70%, using a laser, before ICSI. Patients thus treated formed the LA-ICSI group. Comparisons included the morphological quality of transferred embryos, blastocyst development of the remaining embryos, and clinical pregnancy, in terms of ICSI method and patient characteristics., Results: Fertilization, development of remaining embryos, and pregnancy rate were significantly higher in the LA-ICSI group compared with the C-ICSI group. Fertilization, embryonic development, and the pregnancy rate were all improved in younger patients (<38 years of age) and in those who underwent a low number of IVF-ET attempts (<3 trials). In addition, the pregnancy rate was increased in older patients., Conclusion: LA-ICSI may be useful in improving the chance of pregnancy in all ICSI patients.

Recently, a significant understanding of the molecular mechanisms regulating spermatogenesis has been achieved utilizing small RNA molecules (small RNAs), including small interfering RNAs (siRNAs), microRNAs (miRNAs), and Piwi-interacting RNAs (piRNAs) which emerged as important regulators of gene expression at the post-transcriptional or translation level. piRNAs are only present in pachytene spermatocytes and round spermatids, whereas miRNAs are expressed abundantly in male germ cells throughout spermatogenesis. This review is aimed at providing a glimpse of piRNAs and their interacting family proteins such as PIWIL1, PIWIL2, and PIWIL4 in spermatogenesis.

Polycyclic aromatic hydrocarbons (PAHs) are a large family of environmentally prevalent toxic compounds generated from the combustion of organic materials and diesel exhaust. Humans and wild animals are exposed to PAHs mostly through dietary intake of contaminated food. Benzo[b]fluoranthene (B[b]F) is a common constituent of PAH complexes present in diverse types of food. B[b]F has been found in human milk, raising the demand for the need for risk assessment of offspring after maternal exposure to B[b]F. In the present study, pregnant mice were orally exposed to low doses (2-2000μg/kg body weight) of B[b]F during gestational and lactational periods, and their male offspring were assessed. Maternal B[b]F exposure disturbed normal sperm function in F1 offspring. To understand the molecular and cellular mechanisms by which the perinatal exposure to B[b]F decreased sperm quality, the testes of young adult F1 mice were examined for changes in expression of steroidogenesis-related and testicular apoptosis mediators and found that aryl hydrocarbon receptor, estrogen receptor α, and a set of proapoptotic proteins including Bax, Noxa, Bad, and Bim were significantly upregulated. Therefore, the current transgenerational animal study implies that consumption of PAH-contaminated diets by mothers may possibly influence their offspring to cause dysfunctional male reproductive function in humans., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Cryopreservation of mature eggs is a useful technique that can be applied in assisted reproductive technology. However, the method has some limitations, such as cryodamage induced by biophysical modifications during the cryopreservation process. To assess these biophysical damage, we analyzed the relationship between intracellular calcium ([Ca2+]i) oscillatory activity via type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) distribution after vitrification and efficiency of cryopreservation according to cryoprotectant (CPA) composition. In immunostaining, results of IP(3)R1 with eggs after the vitrification performed using ethylene glycol (EG) alone or EG + dimethylsulfoxide (DMSO) as CPAs, CPA-treated, and fresh eggs displayed a homogeneous IP(3)R1 distribution which is spread uniformly throughout cytoplasm with clusters on the cortex. However, after vitrification and warming process, more than 60% of eggs displayed a heterogeneous distribution which is non-uniform distribution with patches and disconnection of IP(3)R1. In 90-min incubation for recovery from cryodamage, eggs from the EG + DMSO group recovered from with a heterogeneous IP(3)R1 distribution to the homogeneous distribution, but not in EG alone group. In ICSI experiments, vitrified eggs in the EG-alone group presented significantly low blastocyst formation compared to those of the fresh and EG + DMSO groups. These results suggest that the vitrification process influences IP(3)R1 distribution, and subsequently, [Ca2+]i oscillatory activity and embryonic development. Accordingly, we propose that IP(3)R1 distribution and [Ca2+]i oscillatory activity are correlated with egg quality and developmental potential after vitrification, and may thus be applied as an effective indicator to evaluate the efficiency of oocyte cryopreservation methods.

Nanomaterials such as multi-walled carbon nanotubes (MWCNTs) are applied to various industrial products and thus may be released to soils, but their potential environmental impacts remain largely undetermined. We investigated the short-term effect of MWCNTs on the activity and biomass of microorganisms inhabiting two different soil types in an incubation study. Up to 5000 μg MWCNT g(-1) soil was applied and the activities of 1,4-β-glucosidase, cellobiohydrolase, xylosidase, 1,4-β-N-acetylglucosaminidase, and phosphatase and microbial biomass were measured. In both soil types, most enzyme activities showed a tendency to be repressed under 500 μg MWCNT g(-1) soil, and all enzymatic activities as well as microbial biomass C and N were significantly lowered under 5000 μg MWCNT g(-1) soil. Our results suggest that high concentrations of MWCNTs could lower the microbial activity and biomass in soils, and they may serve as an important guideline in regulating the release of MWCNTs to the soil environment., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Purpose: To compare the IVF outcomes of vitrification-thawed blastocyst transfer cycles utilizing different endometrial preparation methods., Methods: We retrospectively assessed IVF outcomes in 611 patients (648 cycles) who underwent blastocyst frozen embryo transfer (FET) between January 2007 and December 2009. All embryos had been cryopreserved by a vitrification method following a previous IVF cycle. Patients were prepared for transfer by using either the natural cycle (n = 310/Group 1), the natural cycle with ovulation induction employing human chorionic gonadotropin (n = 134/Group 2), or a hormonally manipulated artificial cycle with estrogen and progesterone supplementation (n = 204/Group 3)., Results: Multivariate logistic regression analysis showed a significant difference in clinical pregnancy rate between Groups 3 (30.4%) and 1 (41.9%) (odds ratio [OR], 0.567; 95% confidence interval [CI], 0.379-0.847, P = 0.006) whereas the difference between Groups 2 and 1 was not significant (41.8% vs. 41.9%; OR, 0.683; 95% CI, 0.435-1.073; P = 0.098). Other significant variables affecting clinical pregnancy rate were the number of embryos transferred, the grade of transferred embryos, and maximal endometrial thickness., Conclusion: The results showed that, using vitrification-thawed blastocyst transfer, employment of natural cycles with or without hCG treatment was associated with better outcomes than was the use of hormonally manipulated cycles.

Objective: The objectives of this study were to analyze efficacy of immature and mature mouse oocytes after vitrification and warming by applying various combinations of cryoprotectants (CPAs) and/or super-rapid cooling using slush nitrogen (SN(2))., Methods: Four-week old ICR female mice were superovulated for GV- and MII-stage oocytes. Experimental groups were divided into two groups. Ethylene glycol (EG) only group: pre-equilibrated with 1.5 M EG for 2.5 minutes and then equilibrated with 5.5 M EG and 1.0 M sucrose for 20 seconds. EG+dimethylsulfoxide (DMSO) group: pre-equilibrated with 1.3 M EG+1.1 M DMSO for 2.5 minutes and equilibrated with 2.7 M EG+2.1 M DMSO+0.5 M sucrose for 20 seconds. The oocytes were loaded onto grids and plunged into SN(2) or liquid nitrogen (LN(2)). Stored oocytes were warmed by a five-step method, and then their survival, maturation, cleavage, and developmental rates were observed., Results: The EG only and EG+DMSO groups showed no significant difference in survival of immature oocytes vitrified after warming. However, maturation and cleavage rates after conventional insemination were greater in the EG only group than in the EG+DMSO group. In mature oocytes, survival, cleavage, and blastocyst formation rates after warming showed no significant difference when EG only or EG+DMSO was applied. Furthermore, cleavage and blastocyst formation rates of MII oocytes vitrified using SN(2) were increased in both the EG only and EG+DMSO groups., Conclusion: A combination of CPAs in oocyte cryopreservation could be formulated according to the oocyte stage. In addition, SN(2) may improve the efficiency of vitrification by reducing cryoinjury.

In this report, we describe 10 male cases of complex chromosome rearrangements (CCRs) with fertility problems: seven of them showed impairment of spermatogenesis, oligoasthenoteratozoospermia or azoospermia; in the other three cases, recurrent abortions were observed. The CCRs were characterized by conventional fluorescence in situ hybridization (FISH) and multicolor FISH methods as well as by the routine G-banding technique. CCRs found in three cases with recurrent abortions were double two-way exchanges, which were the simplest forms of CCRs; three oligoastenoteratozoospermic cases were double two-way exchanges or three-way exchanges. However, the CCRs in four azoospermic cases were much more complicated forms of CCRs. From our results and a review of the literature, we conclude that the complexity of CCRs might affect the severity of spermatogenetic impairment rather than the number of chromosomes involved or the location of breakpoints., (Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To evaluate the effectiveness of liquid nitrogen (LN(2)) vapor (indirect contact method) for the storage of human semen., Design: Experimental study., Setting: University hospital-based fertility center., Patient(s): We evaluated 150 patients with normal sperm parameters., Intervention(s): Human semen (N = 120) was mixed with Semen Freezing Medium, divided into two groups, frozen, and stored in LN(2) or LN(2) vapor. Frozen semen from each group (n = 40) was thawed after storage for 1 week, 1 month, or 3 months and then analyzed. In the second experiment, semen (n = 30) was divided into four groups, frozen, and stored in LN(2) or stored 7 cm, 12 cm, or 17 cm above the surface of LN(2) for 1 week., Main Outcome Measure(s): The motility and viability of sperm were evaluated by basic analysis, the morphology was analyzed with use of staining, the DNA integrity was assessed with use of terminal deoxyuridine triphosphate nick end-labeling assays, and active mitochondria were detected with use of rhodamine 123 staining., Result(s): The LN(2) and LN(2) vapor groups did not differ with regard to sperm motility, viability, morphology, DNA integrity, or active mitochondria when the cryostorage periods were compared. Furthermore, the quality of sperm stored within 17 cm of the surface of LN(2) for 1 week did not change., Conclusion(s): The storage of human semen in LN(2) vapor, without direct contact with LN(2), may represent a useful alternative for the effective storage of human semen., (Copyright © 2010. Published by Elsevier Inc.)

Background: We evaluated cytogenetic results occurring with first trimester pregnancy loss, and assessed the type and frequency of chromosomal abnormalities after assisted reproductive treatment (ART) and compared them with a control group. We also compared the rate of chromosomal abnormalities according to infertility causes in ICSI group., Methods: A retrospective cohort analysis was made of all patients who were referred to the Genetics Laboratory of Fertility Center of CHA Gangnam Medical Center from 2005 to 2009 because of clinical abortion with a subsequent dilation and evacuation (D&E) performed, and patients were grouped by type of conception as follows: conventional IVF (in vitro fertilization) (n = 114), ICSI (intracytoplasmic sperm injection) (n = 140), and control (natural conception or intrauterine insemination [IUI]) (n = 128). Statistical analysis was performed using SPSS software., Results: A total 406 specimens were referred to laboratory, ten abortuses were excluded, and in 14 cases, we did not get any spontaneous metaphase, chromosomal constitutions of 382 specimens were successfully obtained with conventional cytogenetic methods. Overall, 52.62% of the miscarriages were found to be cytogenetically abnormal among all patients, the frequency was 48.4% in the control group, 54.3% of miscarriages after ICSI and 55.3% after conventional IVF (p = 0.503). The most prevalent abnormalities were autosomal trisomy, however, nine (11.69%) sex chromosome aneuploidy were noted in the ICSI group vs. four (6.45%) and two (3.23%) cases in the conventional IVF group and control group. We compared chromosomal abnormalities of miscarriages after ICSI according to infertility factor. 55.71% underwent ICSI due to male factors, 44.29% due to non-male factors. ICSI group having male factors showed significantly higher risk of chromosomal abnormalities than ICSI group having non-male factors (65.8% vs. 34.2%, p = 0.009, odds ratio = 1.529, 95% CI = 1.092-2.141)., Conclusions: There is no increased risk of chromosomal abnormalities due to ART was found with the exception of a greater number of sex chromosomal abnormalities in the ICSI group with male factor infertility. Therefore, these alterations could be correlated with the underlying parental risk of abnormalities and not with the ICSI procedure itself.

Purpose: stem cell factor (SCF)/c-Kit regulates the proliferation and survival of germ cells or stem cells; however, little is known about the role of SCF/c-Kit in pre-implantation embryo development., Methods: using exogenous SCF supplementation and c-Kit siRNA injection, we investigated the role and mechanism of SCF/c-Kit in pre-implantation mouse embryos., Results: addition of soluble SCF to the culture medium improved blastocyst formation. c-Kit gene silencing reduced the rate of blastocyst formation and delayed embryonic development. The number of proliferating cells in c-Kit gene-silenced blastocysts decreased, whereas the number of apoptotic cells in blastocysts obtained from both experimental and the control groups was not affected. RT-PCR, immunostaining and western blotting revealed that proliferation-related Akt downstream targets were substantially affected by c-Kit gene silencing., Conclusion: SCF/c-Kit signaling through Akt downstream targets is likely involved in mediating the cleavage and proliferation of blastomeres during mouse pre-implantation embryogenesis.

Most couples with severe male factor infertility are treated with assisted reproduction technology and little has been known about the prognosis of severe male factor infertility itself. We investigated the prognosis of infertile male patients with severe oligozoospermia. Thirty-nine patients with severe nonobstructive oligozoospermia were followed more than 6 months without any medical or surgical intervention. Retrospective analyses of the natural sequence of the condition and influences on the future fertility potential of the study participants were conducted. Sperm concentration, motility, and morphology between first semen analysis and last semen analysis were not significantly different. However, during the follow-up period, 5 (12.8%) patients became azoospermic. In 7 (17.9%) patients, the sperm count declined to a severe level that could be detected only after centrifugation. Three patients underwent microdissection testicular sperm extraction (TESE) for sperm retrieval after confirmation of azoospermia. The sperm retrieval was successful only in 1 of the 3 patients. Therefore, male patients diagnosed with severe oligozoospermia should be informed about possible aggravation of their residual spermatogenesis function and the necessity of intermittent follow-up semen analyses. If follow-up semen tests show a declining tendency, sperm cryopreservation may be recommended for these patients. If azoospermia develops during the follow-up period, early TESE procedure should be considered to improve the chance of sperm retrieval.

Objectives: To investigate the natural courses of mild, moderate and severe idiopathic oligozoospermia, and which factors or semen variables were of utmost importance in predicting the courses., Methods: A total of 208 men (age 29-47years) who were diagnosed with mild, moderate and severe idiopathic oligozoospermia in a 9-year-period between January 2000 and December 2008 were followed up for more than 6months., Results: Overall, 16 (24.6%) of 65 patients with severe oligozoospermia developed azoospermia, whereas two (3.1%) patients with moderate oligozoospermia developed azoospermia and none of the patients with mild oligozoospermia developed azoospermia. Initial follicle stimulating hormone level and testicular volume between the subgroups were significantly different (P=0.0071 and 0.0039, respectively). The subgroup of patients who became azoospermic (n=18) showed statistically significant differences in terms of body mass index and the level of prolactin (PRL) from the subgroup that maintained the initial lingering sperm count (n=190; P=0.0086 and 0.0154, respectively). As the vitality of semen variables increased 1%, the risk of progression to azoospermia diminished by 0.892-fold, according to Cox's proportional hazards model analysis. A receiver operating characteristic curve analysis showed that the area under the curve was 0.755 and the sperm concentration value with the highest sensitivity and specificity was the reference value of 3-5 million/mL, with a sensitivity of 0.746 and specificity of 0.711 (P=0.01)., Conclusions: Patients with severe oligozoospermia should be warned of the possibility of becoming azoospermic and hence sperm freezing should be encouraged as early as possible., (© 2010 The Japanese Urological Association.)

Objective: To evaluate the safety, feasibility, and pregnancy outcomes of adnexal detorsion using simple aspiration following ovarian hyperstimulation in pregnancy., Study Design: Retrospective review of eight pregnant women who underwent ultrasound (US)-guided transvaginal aspiration to induce natural reduction of ovarian torsion following ovarian hyperstimulation., Results: Six of the eight patients were successfully treated with simple aspiration only, without any complications. Symptoms recurred in two patients, necessitating further laparoscopic detorsion. The outcome of the pregnancy was normal in seven women, with the eighth experiencing loss of one fetus from a twin pregnancy 2 days after laparoscopy. Normal ovarian blood flow was confirmed within one month after the treatment., Conclusion: US-guided transvaginal aspiration for adnexal detorsion in pregnant women following ovarian stimulation might serve as a simpler, less morbid and equally effective alternative to conventional treatment., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

Nobox is an oocyte-specific transcriptional regulator. Nobox deficiency disrupts early folliculogenesis and the expression of oocyte-specific genes in mice. In the present study, we found that peptidylarginine deiminase 6 (Pad6) was downregulated in Nobox-null ovaries. Pad6 is preferentially expressed in oocytes and its transcript is detectable at embryonic day 16.5. In addition, we identified one Nobox DNA-binding element (NBE) within the mouse Pad6 promoter. The NBE includes a core sequence TAATTA. Sequence-specific binding of Nobox to the TAATTA motif was confirmed. Nobox overexpression augmented transcriptional activity of a luciferase reporter driven by mouse Pad6. Our findings indicate that Nobox is a critical regulator that orchestrates oocyte-specific genes such as Pad6 during folliculogenesis., (Copyright 2010. Published by Elsevier B.V.)

Objectives: The aim of the present study was to improve efficiency of isolation and to optimize proliferative potential of human spermatogonial stem cells (SSCs) obtained from obstructive azoospermic (OA) and non-obstructive azoospermic (NOA) patients, and further, to characterize these cells for potential use in infertility treatment or study of reproductive biology., Materials and Methods: We have applied a cell-sorting method, using collagen and magnetic activated cell separation to overcome obstacles, developing a collection system, and simple long-term proliferation system, that yields large numbers of high-purity SSCs from obstructive OA and NOA patients., Results: SSCs derived from OA and NOA patients proliferated and maintained their characteristics for more than 12 passages (>6 months) in vitro. Moreover, the population of cells positive for the SSC-specific markers GFRalpha-1 and integrin alpha6, increased to more than 80% at passage 8., Conclusion: These finding may support the idea that in vitro propagation of SSCs could be a useful tool for infertility treatment and study of reproductive biology.

Objective: To describe a 46,XX male whose infertility is not accounted for by a translocation of the SRY gene to the X chromosome or to the autosomes., Design: Case report., Setting: Fertility Center of CHA Gangnam Medical Center, Seoul, South Korea., Patient(s): A 29-year-old male with normal male phenotype, in whom seminal analysis showed complete azoospermia., Intervention(s): Laboratory evaluations, radiologic studies, testicular biopsy, G-banding karyotype, in situ fluorescence hybridization, and polymerase chain reaction., Main Outcome Measure(s): Clinical and laboratory findings., Result(s): Peripheral blood culture for chromosome studies revealed 46,XX chromosome complement. Cytogenetic and molecular analyses excluded the presence of SRY gene. Radiologic studies displayed male structures without Müllerian ducts. Gonadal biopsy showed testicular Leydig cell hyperplasia., Conclusion(s): This is a very rare case of testicular differentiation in a 46,XX chromosomal constitution without SRY. This finding suggests that some unknown genes downstream participate in sex determination., (Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

We investigated the influence of stand density [938 tree ha(-1) for high stand density (HD), 600 tree ha(-1) for medium stand density (MD), and 375 tree ha(-1) for low stand density (LD)] on soil CO(2) efflux (R (S)) in a 70-year-old natural Pinus densiflora S. et Z. forest in central Korea. Concurrent with R (S) measurements, we measured litterfall, total belowground carbon allocation (TBCA), leaf area index (LAI), soil temperature (ST), soil water content (SWC), and soil nitrogen (N) concentration over a 2-year period. The R (S) (t C ha(-1) year(-1)) and leaf litterfall (t C ha(-1) year(-1)) values varied with stand density: 6.21 and 2.03 for HD, 7.45 and 2.37 for MD, and 6.96 and 2.23 for LD, respectively. In addition, R (S) was correlated with ST (R (2) = 0.77-0.80, P < 0.001) and SWC (R (2) = 0.31-0.35, P < 0.001). It appeared that stand density influenced R (S) via changes in leaf litterfall, LAI and SWC. Leaf litterfall (R (2) = 0.71), TBCA (R (2) = 0.64-0.87), and total soil N contents in 2007 (R (2) = 0.94) explained a significant amount of the variance in R (S) (P < 0.01). The current study showed that stand density is one of the key factors influencing R (S) due to the changing biophysical and environmental factors in P. densiflora.

In a previous study, we found SOHLH1 (spermatogenesis and oogenesis-specific basic helix-loop-helix 1) as the first testis-specific basic helix-loop-helix transcription factor essential for spermatogonial differentiation. SOHLH1 therefore represents an excellent candidate gene for testicular failure such as nonobstructive azoospermia (NOA). We analyzed whether there were mutations in the SOHLH1 gene in 96 Korean patients with NOA. The sequence analysis discovered three novel variations: one intronic variant (c.346-1G>A), and two nonsynonymous exonic variants (c.91T>C and c.529C>A) with known single nucleotide polymorphisms (SNPs), which included six intronic variants, two synonymous, and two nonsynonymous variants. We examined the consequences of mutations in SOHLH1 using in vivo and in vitro assays. Analysis of transcripts from minigenes carrying the c.346-1G>A revealed that splicing site variation leads to the partial deletion at a cryptic splicing site within exon 4. This deletion results in SOHLH1 with a truncated bHLH domain. Transient transfection assay showed that the SOHLH1 mutant with the truncated domain disrupted the transcriptional activity of KIT promoter, whereas two missense mutations harboring either p.Arg37Gln or p.Pro269Ser did not have a significant effect on its transactivation. Our findings indicate that a splice-acceptor site mutation that probably causes a nonfunctional SOHLH1 protein results in nonobstructive azoospermia by the lack of normal spermatogenesis., ((c) 2010 Wiley-Liss, Inc.)

Objective: To evaluate whether endometrial and subendometrial blood flow parameters measured using three-dimensional power Doppler ultrasound (3D PD-US) can predict pregnancy after IUI., Design: Prospective clinical study., Setting: Infertility center in a referral hospital., Patient(s): One hundred six women who underwent ovulation induction and IUI., Intervention(s): A color Doppler ultrasound and a 3D PD-US examination were performed on the day of IUI., Main Outcome Measure(s): Pulsatility index (PI), resistance index (RI), and systolic/diastolic (S/D) ratio of uterine artery, and vascularization index (VI), flow index (FI), and vascularization flow index (VFI) of the endometrium as well as those of subendometrial region. These measurements were analyzed in relation to IUI outcome (pregnant vs. nonpregnant)., Result(s): The pregnant group had higher endometrium VI, FI, and VFI scores than the nonpregnant group. In contrast, the subendometrial region VI, FI, and VFI scores did not differ between the groups, nor did the uterine artery PI, RI, and S/D. Pregnancies did not occur when endometrial blood flow had not been detected., Conclusion(s): Three-dimensional PD-US was useful for evaluating endometrial and subendometrial neovascularization in IUI cycles. Endometrial blood flow parameters may be useful predictors for pregnancy., (Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To develop an efficient freezing method suitable for large-scale cryopreservation of human embryonic stem cells (hESCs)., Design: Experimental study., Setting: Research institute., Patient(s): None., Intervention(s): Two genetically modified hESC lines, H9-EF1-GFP and CHA-hES3-EF1-GFP, were cryopreserved in cryovials using a combination of two equilibration methods (one-step and stepwise) and two cooling vehicles (cryo-container and program-controlled freezer). After thawing, the survival and differentiation rate were compared among groups., Main Outcome Measure(s): The hESC survival was assessed by alkaline phosphatase staining and differentiation status was determined by flow cytometry using an SSEA-4 antibody., Result(s): In both H9-EF1-GFP and CHA-hES3-EF1-GFP cells, the survival rate was highest in the group using stepwise equilibration and program-controlled freezer, and lowest in the group using one-step equilibration and cryo-container. In the groups using cryo-container, the survival and the frequency of undifferentiated cells in both cell lines was highly improved in a stepwise equilibration compared with one-step. Thawed hESCs were positively stained with pluripotent markers SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase. The karyotypes and expression of three germ layer markers in both cell lines were not changed after freezing/thawing., Conclusion(s): The stepwise equilibration of Knockout Serum Replacement and cryoprotectant during freezing and thawing resulted in higher survival rates by reducing osmotic damage irrespective of cooling vehicles., (Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

For human embryonic stem cells (hESCs) to be used clinically, it is imperative that immune responses evoked by hESCs and their derivates after transplantation should be prevented. Human leukocyte antigens (HLA) and ABO blood group antigens are important histocompatibility factors in graft rejection. HLA matching between recipient and unrelated donors, in particular, is important in improving outcomes in hematopoietic cell transplantation (HCT). We have established and successfully maintained 29 hESC lines and analyzed the HLA and ABO genotypes of these lines. HLA-A, -B, -C and -DR (DRB1) genotyping was performed by polymerase chain reaction (PCR) sequence-based typing and ABO genotyping was carried out by PCR restriction fragment length polymorphism methods. To determine what proportion of the Korean population would be covered by these cell lines in organ transplantation, 27 cell lines with HLA-A, -B, and -DR data were evaluated for HCT (cord blood) donors and 28 cell lines with HLA-DR and ABO data were evaluated for solid organ (kidney) transplantation donors, and then compared the data with those from 6,740 donated cord bloods. When 2 HLA mismatches are allowed for HCT, as currently accepted for cord blood transplantation, it was estimated that about 16% and 25% of the possible recipients can find one or more donor cell lines with ≤2 mismatches at A, B, DRB1 allele level and at A, B antigen/DRB1 allele level, respectively. When HLA-DR antigen level matching and ABO compatibility was considered for solid organ (kidney) transplantation, it was estimated that about 29% and 96% of the possible recipients can find one or more ABO-compatible donor cell lines with 0 and 1 DR mismatches, respectively. We provided the first report on the HLA and ABO genotypes of hESC lines, and estimated the degree of HLA and ABO matching in organ transplantation for the Korean population.

Objective: To store embryos in LN(2) vapor, which eliminates direct contact with LN(2), is presented as an alternative method to protect embryos cryopreserved by vitrification or slow cooling from LN(2)-borne pathogens., Design: Basic animal research., Setting: University-affiliated hospital., Animal(s): Two-cell mouse embryos collected from superovulated ICR mice., Intervention(s): Embryos cryopreserved by vitrification or slow cooling were stored in LN(2) or LN(2) vapor and then thawed after 1 week, 1 month, or 6 months. Thawed embryos cultured to the blastocyst stage in vitro were evaluated or transferred into the uterus of foster mothers., Main Outcome Measure(s): Survival, apoptosis, and in vitro and in vivo development of freeze-thawed mouse embryos as a function of cryopreservation and cryostorage methods were determined., Result(s): After short- and long-term storage of vitrified and slow-cooled embryos, embryonic survival and development rate were the same for embryos stored in LN(2) vapor and in direct contact with LN(2). Cell numbers and apoptosis frequency were similarly indistinguishable between the two groups after storage of embryos for 6 months, and there were no differences in delivery rate or litter size after ET., Conclusion(s): The present study shows that embryos stored in LN(2) vapor retain full developmental potential. This storage system thus represents a useful alternative for safe and effective long-term storage of embryos that avoids direct contact with LN(2).

Background: We evaluated the usefulness of the hyomental distance (HMD) ratio (HMDR), defined as the ratio of the HMD at the extreme of head extension to that in the neutral position, in predicting difficult visualization of the larynx (DVL) in apparently normal patients, by examining the following preoperative airway predictors, alone and in combination: the modified Mallampati test, HMD in the neutral position, HMD and thyromental distance at the extreme of head extension and HMDR., Methods: Preoperatively, we assessed the five airway predictors in 213 adult patients undergoing general anesthesia with tracheal intubation. A single experienced anesthesiologist, blinded to the results of the airway evaluation, performed all of the direct laryngoscopies and graded the views using the modified Cormack and Lehane scale. DVL was defined as a Grade 3 or 4 view. The optimal cutoff points for each test were determined at the maximal point of the area under the curve in the receiver operating characteristic curve. For the modified Mallampati test, Class >or=3 was predefined as a predictor of DVL., Results: The larynx was difficult to visualize in 26 (12.2%) patients. In univariate analyses, the HMD and thyromental distance at the extreme of head extension and the HMDR were significantly related to DVL. The HMDR with the optimal cutoff point of 1.2 had greater diagnostic accuracy (area under the curve of 0.782), than other single predictors (P < 0.05), and it alone showed a greater diagnostic validity profile (sensitivity, 88%; specificity, 60%) than any test combinations., Conclusions: The HMDR with a test threshold of 1.2 is a clinically reliable predictor of DVL.

Objective: To report the survival rate of oocytes and the rate of successful pregnancies obtained from super-rapid cooling of oocytes using slush nitrogen (SN(2))., Design: Prospective clinical research., Setting: A university-affiliated hospital., Patient(s): Twenty-eight infertile women who underwent 30 cycles of IVF-ET using previously vitrified oocytes., Intervention(s): Oocytes were vitrified by super-rapid cooling using SN(2)., Main Outcome Measure(s): Morphological normality of thawed oocytes and clinical outcome., Result(s): In 30 cycles of ovarian stimulation for IVF, 364 surplus oocytes from 28 patients were vitrified using SN(2). Three hundred two (85.1% +/- 2.9%) of the oocytes survived after warming. Fertilization and cleavage rates were 77.4% +/- 3.5% (168/218) and 94.3% +/- 2.1% (158/168), respectively. Thirteen pregnancies (43.3%) resulted from 30 uterine transfers of 120 embryos with an implantation rate of 14.2% (17/120). There were no differences between the pregnancy rate after vitrification/warming and that obtained from routine noncryopreserved oocytes., Conclusion(s): The present report suggests that super-rapid cooling may improve the clinical efficacy of human oocyte vitrification and may be a valuable tool for human assisted reproductive technologies.

Background: This study evaluated the effect of vitrification using slush nitrogen (SN(2)) on cryopreservation of micromanipulated mouse embryos., Methods: The zona pellucida of 4-cell embryos was either left intact or dissected or dissected with biopsy of an intact blastomere. In a second study, a blastomere was destroyed and either removed (removed group) or not removed (remained group) prior to vitrification/freezing. The micromanipulated embryos were equilibrated and loaded into an open pulled straw (OPS), and plunged into liquid nitrogen (LN(2)) or SN(2)., Results: When using LN(2) vitrification, recovery and blastocyst formation rates of embryos were lower for zona pellucida-opened and/or blastomere-biopsied embryos compared with zona pellucida-intact embryos. Using SN(2) for vitrification resulted in increased survival and development of vitrified/warmed embryos in both the zona pellucida-opened and blastomere-biopsy groups. Similar results were observed when using embryos with a destroyed blastomere either removed or left remaining before vitrification. However, the number of total and apoptotic cells were similar for both LN(2) and SN(2). In addition, using SN(2) increased the rate of intact recovery and blastocyst formation in warmed hemi-8-cell embryos derived from the same embryo., Conclusions: These results suggest that vitrification using SN(2) is useful in cryopreservation of micromanipulated embryos obtained from a variety of programs, including assisted hatching, preimplantation genetic diagnosis and nuclear transfer.

In vitro maturation (IVM) programme has developed a culture technique for supporting the growth and maturation of immature oocytes since late of 1980s and has become important in ART methods. The establishment of an IVM programme can yield many advantages. Patients suffering from congenital or postnatal reproductive disorders, such as premature ovarian failure (POF) or polycystic ovarian syndrome (PCOS), can achieve pregnancies by transfer of viable embryos derived from IVM and in vitro fertilisation (IVF) systems. However, the pregnancy rate of IVM-IVF programme for PCO patients was still low. This disadvantage was caused by poor maturation of human immature oocytes and no synchronisation with implantation windows. Recently, application of FSH/hCG priming or optimal maturation medium for proper cytoplasmic maturation improves the success rate of IVM procedures. Indeed, increasing oocyte viability by employing optimised IVM system would also reduce ethical concerns about the disposal of retrieved oocytes with low developmental competence, and concerns about the abuse of the manipulation of human embryos.

Purpose: This study investigated the effects of HB-EGF on expression of integrin alphanubeta3 and implantation of embryos., Methods: Two-cell embryos were recovered and cultured with or without 10 ng/mL HB-EGF for 96h. Expression of integrin alphanubeta3 in cultured embryos was examined by real time-RT-PCR and immunofluorescence analysis; embryos were cultured with or without HB-EGF, then transferred into the uteri of pseudo-pregnant female mice in order to analyze their implantation rate., Results: HB-EGF improved embryonic hatching and outgrowth during extended culture, and up-regulated expression of integrin alphanubeta3 in both the preimplantation embryo and outgrowing blastocyst. Also, integrin alphanubeta3 subunits were localized at the pericellular borders and cell-cell contact areas. The number of successful implantation sites of transferred HB-EGF-treated embryos in the uterus was increased when compared to number of implantation sites with non-treated controls., Conclusions: HB-EGF may improve implantation by accelerating expression of integrin alphanubeta3 in peri-implantation mouse embryos.

Background: The purpose of this study was to establish the culture conditions required to isolate, identify and expand male germ stem cell-like cells (GSC-LC) from the testicular tissue of patients with non-obstructive azoospermia (NOA)., Methods and Results: Testicular tissues obtained from patients (two with maturation arrest (MA, n = 2) and Sertoli cell-only syndrome (SCOS, n = 11) were dissociated and plated into gelatin-coated dishes. After 2-4 weeks, cultures from both MA patients (100%) and four SCOS patients (36.3%) exhibited multicellular colonies, which proliferated successfully until passage 10. GSC-LC in the colonies displayed alkaline phosphatase activity, as well as Oct-4 and integrin b1 expression after every passage. After the fifth passage, GSC-LC were differentiated by encapsulation in calcium alginate and further cultivation. At 2 and 6 weeks, cells expressed c-Kit, Scp3, testis-specific histone protein 2B (TH2B), and transition protein (TP)-1. Fluorescence in situ hybridization additionally disclosed a few tetraploid and haploid cells at 6 weeks. Human oocytes were activated in the absence of artificial activation and cleaved after the injection of presumptive spermatids., Conclusions: Our novel culture system may be useful for diagnosing the existence of germ cells and facilitating the treatment of NOA patients.

Objective: To assess the obstetric outcome of pregnancies resulting from in vitro maturation (IVM) and IVF-ET of immature oocytes retrieved from women with polycystic ovary syndrome (PCOS)., Design: Prospective observational study., Setting: University fertility clinic., Patient(s): One hundred thirty-nine women undergoing 203 IVM treatment cycles., Intervention(s): Immature oocyte recovery from unstimulated ovaries. In vitro oocyte maturation and fertilization. Fresh ET and assessment of obstetric outcomes in the pregnant women., Main Outcome Measure(s): Pregnancy and obstetric outcome., Result(s): Forty-one pregnancies were obtained in 187 ETs, resulting in a pregnancy rate of 21.9%. Except for three patients lost to follow-up in these pregnancies, the abortion and live birth rates were 36.8% (14 of 38) and 63.2% (24 of 38), respectively. The mean (+/-SD) gestational age and birth weight at delivery for singletons were 38.4 +/- 2.0 weeks (range, 33-41.6 weeks) and 3,252 +/- 516 g (1,750-4,100 g), respectively. For twins these were 36.7 +/- 1.9 weeks (34.6-39 weeks) and 2,361 +/- 304 g (1,900-2,990 g), respectively. Pregnancy complications occurred in five patients (13.2%); these included preterm labor (n = 3) and placenta previa (n = 2). Two patients (5.3%) had a major congenital anomaly diagnosed by ultrasonography., Conclusion(s): The abortion rate, gestational age and birth weight at delivery, and obstetric complications of pregnancies conceived by IVM-ET in women with PCOS were comparable with those of other women with PCOS being treated by conventional IVF-ET. In vitro maturation followed by IVF-ET seems to be a useful treatment option for women with PCOS, thus avoiding the risk of ovarian hyperstimulation syndrome.

Previously, bone morphogenetic protein-7 (BMP-7) was suggested as a factor that may act to facilitate the transition of follicles from primordial stage to the pool of developed primary, preantral, and antral follicles (Lee et al. 2001: Biol Reprod 65:994-999.). Thus, aim of the present study was to evaluate effect(s) of BMP-7 on the primordial-primary follicle transition. Neonatal mouse ovaries were cultured in the presence or absence of 100 mIU/ml FSH with various doses of BMP-7 (0, 10, and 100 ng/ml). After 4-day culture period, number of follicles was counted and the expression of transcripts for FSH receptor (FSHR), kit ligand (KL), and c-kit was measured by RT-PCR. BMP-7 alone at 100 ng/ml concentration stimulated follicle development with concurrent increase of mRNA for FSHR. BMP-7 alone down-regulated KL expression however, the ratio between KL1 and KL2 was increased. There was no change in the c-kit mRNA expression. Results of the present study suggest that the BMP-7 is one of the factors involved in primordial-primary follicle transition in the mouse ovary and it may play a role in expression of FSHR for further follicular development.

Purpose: To report two cases of successful spouse pregnancies which were conceived with long-term cryopreserved spermatozoa that had been collected prior to the male patients receiving a bone marrow transplant., Methods: The first case is the pregnant wife of a 25-year-old man with chronic myelogenous leukemia, whose semen was collected before bone marrow transplant and then cryopreserved, thawed, and then injected into the wife's eggs via ICSI. The second case is a 28-year-old man with severe aplastic anemia who became a father after his wife's eggs were fertilized via ICSI with thawed spermatozoa., Results: These two cases were achieved pregnancies., Conclusions: These cases support research that men with malignancy have the chance of fathering their own genetic children. Therefore, it is important to increase the awareness of clinicians, oncologists, and patients to the new developments in preserving fertility for cancer patients.

Purpose: To report two azoospermic patients with reciprocal X-autosome translocations., Methods: Cytogenetic analysis utilizing GTG-banding and Yq microdeletions shown by polymerase chain reaction (PCR) with 12 sequence-tagged site (STS) markers for Y chromosome microdeletions., Results: Cytogenetic analysis showed one man with 46,Y,t(X;19)(q22;q13.3) and the other with 46,Y,t(X;8)(p22;q11). Neither had any Yq microdeletions shown. The patient with 46,Y, t(X;8)(p22;q11) showed a slightly lower than normal testosterone level. By NCBI-Blast search, we found four testis-specific genes, t-complex-associated-testis-expressed 1-like (TCTE1L), Ferritin, heavy polypeptide-like 17 (FTHL17), Testis expressed sequence 13A (TEX13A), and Testis expressed sequence 13B (TEX13B) located near breakpoints on X chromosome. FTHL17, TEX13A, and TEX13B are spermatogonially-expressed, germ-cell-specific genes., Conclusion: This is the first clinical report of azoospermia with reciprocal X-autosome translocations on Xp22 and q22. These translocations on Xp22 and q22 may be direct genetic risk factors for azoospermia.

Objective: To assess the usefulness of the vitrification method in clinical practice., Design: Clinical study of vitrification of human oocytes., Setting: A university-affiliated hospital., Patient(s): Thirty-four patients who agreed to undergo additional IVF-ET after the failed fresh cycle using the previously vitrified/thawed oocytes., Intervention(s): Surplus oocytes from the IVF-ET patients were vitrified for the next cycle., Main Outcome Measures: Morphologic normality of post-thawed oocytes, fertilization, cleavage, pregnancy, and implantation rate., Result(s): Overall morphological survival and fertilization rates of vitrified/liquefied oocytes were 68.6% (325/474) and 71.7% (142/198), respectively. Pregnancy rate and implantation rate per embryo transfer were 21.4% (6/28) and 6.4% (8/125), respectively. All pregnancies resulted in the delivery of healthy babies (1 twin and 5 singletons/6 pregnancies)., Conclusion(s): The feasibility of the vitrification method for human oocytes was confirmed by our clinical results. Subsequent studies on vitrification and thawing procedures should be undertaken for further optimizing the vitrification method.

The most prevalent problem in cancer therapy is the regrowth and metastasis of malignant cells after standard treatment with surgery, radiation, and/or chemotherapy. Gene therapy approaches have suffered from the inadequate transduction efficiencies of replication-defective vectors that have been used thus far. Replication-competent vectors, particularly adenoviruses that cause cytolysis as part of their natural life cycle, represent an emerging technology that shows considerable promise as a novel treatment option, particularly for locally advanced or recurrent cancer. A number of oncolytic adenoviruses that are designed to replicate selectively in tumor cells by targeting molecular lesions inherent in cancer, or by incorporation of tissue-specific promoters driving the early genes that initiate viral replication, are currently being tested in clinical trials. The results of these clinical trials indicate that, in its current form, oncolytic adenovirus therapy shows the best results and achieves an enhanced tumoricidal effect when used in combination with chemotherapeutic agents such as cisplatin, leucovorin and 5-fluorouracil. Nevertheless, each of the oncolytic adenoviruses in current use exhibits characteristic shortcomings, and there is still considerable room for improvement. Current strategies for improving the selectivity and efficacy of oncolytic adenoviruses include molecular engineering of tumor cell-specific binding tropism, selective modifications of viral early genes and incorporation of cellular promoters to achieve tumor-specific replication, augmentation of anti-tumor activity by incorporation of suicide genes, and manipulation of the immune response.

Study Objective: To evaluate fertility outcome and benefit of laparoscopic tubal anastomosis compared with laparotomy., Design: Retrospective study (Canadian Task Force classification II-2)., Setting: University hospital., Patients: Eighty-one women requesting reversal of sterilization. Fertility outcome was analyzed in 76 patients for a minimum of 6 months., Intervention: Laparoscopic tubal anastomosis in 37 women and abdominal tubal anastomosis in 44., Measurements and Main Results: In both groups anastomosis was performed in two layers with four stitches using microsurgical technique. Overall pregnancy rates were 80.5% in the laparoscopy and 80.0% in the laparotomy group. The mean interval from operation to pregnancy was similar in the two groups (p = 0.9). Mean operating time was significantly longer for laparoscopy (201.9 +/- 33.8 min) than for laparotomy (148.7 +/- 32.5 min), including diagnostic laparoscopy. However, mean hospital stay was shorter for laparoscopy than for laparotomy (3.3 +/- 2.0 vs 6.1 +/- 0.6 days, p <0.05)., Conclusion: Laparoscopic tubal anastomosis is less invasive and could be an alternative to laparotomy for reversal of tubal sterilization. Advanced laparoscopic equipment and much experience could enhance the pregnancy rate and reduce operating time.

The establishment of a long-term preservation system for mammalian oocytes is important for the development of both biological and medical sciences. A number of efforts have been made to develop this system. In human reproductive medicine, the development of an oocyte cryopreservation system can improve the efficacy of the current assisted reproductive technology (ART) for infertile patients with severe reproductive disorders. In this article, the technical development of cryopreservation programs for human oocytes and its biological background were reviewed. Clinical outcome after the use of this technology was further introduced.

Objective: To improve the efficacy of an IVF-ET program for unstimulated patients with polycystic ovary syndrome (PCOS) with the use of culture for oocyte maturation., Design: Prospective studies with the comparison of different ET procedures from March 1995 through February 1998., Setting: University-affiliated hospital., Patient(s): Ninety-four cycles in 64 consenting patients with PCOS., Intervention(s): Immature oocytes were retrieved from unstimulated patients with PCOS and subsequently cultured and fertilized in vitro. Zygote intrafallopian transfer (ZIFT), uterine ET, or a combined approach of ZIFT + uterine ET was subsequently performed., Main Outcome Measure(s): Laboratory and clinical data., Result(s): Among 1, 280 immature oocytes (13.6 +/- 7.5 oocytes per patient) retrieved, 89% (1,139) were morphologically normal, and 62.2% (708/1,139) of the normal oocytes matured in vitro after culture for 48 hours. When intracytoplasmic sperm injection was performed, 68% (481/708) developed to the normal pronuclear stage, and 88.1% of the embryos cocultured with Vero cells (266/302) cleaved. Eighty-five ET cycles were conducted and pregnancy was established in 23 cycles (27.1%), which consisted of 8 after uterine ET and 15 after a combined approach. Seventeen patients delivered 20 normal infants., Conclusion(s): The IVF-ET method using no ovarian stimulation followed by in vitro maturation culture can be a feasible assisted reproductive technology for treatment of PCOS with various complications.

Objective: To evaluate the fertility outcome after laparoscopic tubal anastomosis for reversal of sterilization., Design: Retrospective clinical study., Setting: A private practice affiliated with a university medical school., Patient(s): Two hundred two women who desired reversal of tubal sterilization., Intervention(s): Laparoscopic tubal anastomosis., Main Outcome Measure(s): The cumulative pregnancy rate (PR) and factors that influenced the fertility outcome., Result(s): The cumulative PR in the 186 patients for whom follow-up data were available was 60.3%, 79.4%, and 83.3% at 6, 12, and 18 months after operation, respectively. Five patients (3.2%) had ectopic pregnancies; one of these patients subsequently conceived normally. There were no statistically significant differences in the PR according to the sterilization method used, the site of the tubal anastomosis, or the length of the fallopian tube after surgery. The intrauterine PR was 87.1% (149/171) with bilateral anastomosis and 60% (9/15) with unilateral anastomosis. The PR decreased with increasing patient age (mean [+/- SD], 35+/-3.6 years) but was still 70.6% (12/17) in patients aged 40-45 years., Conclusion(s): Our findings suggest that laparoscopic tubal anastomosis is a highly successful procedure. This less invasive approach could be considered the procedure of choice in patients who desire reversal of tubal sterilization.

Objective: To evaluate the developmental competence of vitrified human oocytes thawed using two different methods to establish an effective cryopreservation protocol., Design: In vitro model study., Setting: University-affiliated hospital., Patient(s): Patients who underwent a long protocol of ovarian stimulation with GnRH and gonadotropins., Intervention(s): Vitrified oocytes from the patients were thawed using either a four-step method with 2.5-minute intervals or a four-step method with 5-minute intervals., Main Outcome Measure(s): Morphologic normality, maturation, fertilization, and development of the oocytes to the blastocyst stage., Result(s): The two thawing methods did not significantly affect the morphologic normality (84%-100%), maturation (75%-100%), fertilization (38%-71%), polyspermy (more than three pronuclei; 0%-20%), or parthenogenetic activation (only female pronucleus; 0%-8%) of the vitrified oocytes. However, more of the vitrified oocytes developed to the two-cell (71%-100% versus 50%-67%), four-cell (71%-93% versus 0%-50%), eight-cell (46%-71% versus 0%), and blastocyst (23%-36% versus 0%) stages after thawing using the four-step method with 2.5-minute intervals than using the four-step method with 5-minute intervals., Conclusion(s): Vitrified human oocytes developed to the blastocyst stage with IVF. A four-step thawing method with 2.5-minute intervals was more effective in supporting preimplantation embryo development than a four-step thawing method with 5-minute intervals.

Objective: To evaluate fertility outcome after laparoscopic microsurgical tubal anastomosis., Design: A retrospective study., Setting: Infertility Medical Center affiliated with University Medical School., Patient(s): Fifty-four patients, who had previously undergone tubal sterilization, seeking reversal., Intervention(s): Laparoscopic microsurgical tubal anastomosis was performed., Main Outcome Measure(s): Pregnancy success was assessed., Result(s): The overall pregnancy rate (PR) was 77.5% (38/49) and 29 patients already have delivered healthy offspring. Pregnancy in seven patients is ongoing and one case ended in abortion. There was one case of ectopic pregnancy. The pregnancy success according to the method of previous tubal sterilization was 16 of 24 with the Fallope-ring method, 14 of 15 in cases of cauterization, and 8 of 10 in patients in whom the Pomeroy technique was used. The pregnancy success according to the site of anastomosis was 3 of 4 in cornual-isthmic, 4 of 5 in isthmic-isthmic, 26 of 35 in isthmic-ampulla, 3 of 3 in cornual-ampulla, and 2 of 2 in ampulla-ampulla. The pregnancy success according to the tubal length was 5 of 7 at a length < or = 4 cm, 3 of 5 at 5 cm, 15 of 17 at 6 cm, and 15 of 20 at lengths > or = 7 cm., Conclusion(s): Considering the high PR in our minimal follow-up period of 12 months, laparoscopic microsurgical tubal anastomosis could be an alternative procedure to microsurgical laparotomy in patients requesting reversal of tubal sterilization.

Objective: To investigate effects of 1,2-propanediol and freezing-thawing treatment on the maturation and developmental capacity of the human immature oocytes obtained from unstimulated ovaries., Design: Intact cumulus-enclosed immature oocytes collected from unstimulated ovaries were divided into three groups, such as no treatment as control (group 1), only 1,2-propanediol-treated (group 2), and cryopreserved group (group 3). Oocytes in group 1, group 2, and survived oocytes from cryopreservation in group 3 were cultured for 48 hours. A random selection of matured oocytes was inseminated with normal donor sperm to evaluate the fertilization and developmental capacity., Setting: Infertility Medical Center at the CHA General Hospital, Seoul, Korea., Patient(s): Oocytes were obtained from patients undergoing gynecological surgery., Main Outcome Measure(s): Rates of survival, maturation to metaphase II, fertilization, and cleavage., Result(s): Survival rate after freezing-thawing in group 3 was 55.1% (54/98). Oocytes were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal bovine serum, 10 IU/mL pregnant mare serum gonadotropin, and 10 IU/mL hCG. Maturation rates were 76.8% (63/82), 67.1% (47/70), and 59.3% (32/54) in the groups 1, 2, and 3, respectively. Maturation rate in group 3 was significantly lower than that of group 1. Fertilization rates were 90.5% (19/21), 81.0% (17/21), and 42.9% (6/14), and cleavage rates were 94.7% (18/19), 88.2% (15/17), and 16.7% (1/6) in groups 1, 2, and 3, respectively. Fertilization and cleavage rates of survived oocytes in group 3 also were significantly lower than those of groups 1 and 2., Conclusion(s): Results suggest that the pretreatment with 1.5 M 1,2-propanediol itself before the freezing has no inhibitory effect on the maturation, fertilization, and cleavage of human immature oocytes in vitro. However, the freezing-thawing procedure used had detrimental effects on the maturation and developmental capacity.

Immunosuppression by exogenous corticosteroids has been used to improve the rates of embryo implantation and pregnancy in in-vitro fertilization (IVF) patients who have micromanipulated embryos replaced. The present study was conducted, in a prospective design, to evaluate effects of corticosteroid on the pregnancy rate in IVF/embryo transfer patients who have non-micromanipulated embryos replaced. Infertile women < 40 years old with tubal factor were included in this study. Patients were grouped according to the different follicle stimulation protocols, and received various doses of 16 beta-methylprednisolone (0, 16 or 60 mg/day) for 4 days from the day of oocyte retrieval. The mean age, duration of infertility, length of folliculogenesis and serum oestradiol concentrations at the time of human chorionic gonadotrophin (HCG) injection were not significantly different between control and corticosteroid-treated groups of patients. Short-term immunosuppression by 16 beta-methylprednisolone administration did not show any effects on the pregnancy and miscarriage rates in IVF/embryo transfer patients. Also, immunosuppression showed no dose effects in any groups. There was no relationship between the types of follicle stimulation protocols and the effect of 16 beta-methylprednisolone. Therefore, we concluded that short-term immunosuppression by exogenous corticosteroids in IVF/embryo transfer patients who have embryos with intact zona pellucida replaced has neither positive nor negative effects on pregnancy rates.

This study describes the results with immature human follicular oocytes harvested from unstimulated ovaries, matured in vitro, fertilized, and transferred to an agonadal recipient. Two hundred seventy immature oocytes were aspirated from 23 ovaries removed for various gynecological indications from August 1988 to October 1989. The numbers of follicular oocytes collected from ovaries were compared by patients' ages and the stages of menstrual cycle. Immature oocytes in vitro were incubated with either mature follicular fluid (FF) or fetal cord serum (FCS). The maturation rate in the mature FF group was 55.8%, significantly higher than the 35.9% in the FCS group. In addition, mature FF group was shown to provide a significantly higher fertilization rate than the FCS group (81.0% versus 31.6%). More fertilized eggs developed into normal embryos in the nonstimulated cycle group than in stimulated cycles with routine treatment. Finally, five embryos were transferred to a woman with premature ovarian failure on day 18 of a steroid replacement cycle. She subsequently delivered healthy triplet girls. These results suggest that in vitro maturation of immature oocytes from unstimulated ovaries with mature follicular fluid could be used successfully in a donor oocyte program after in vitro fertilization.