Influence of environmental factors on facial pigmented spots: Epidemiological survey of women living in the northern and southern regions of Japan.

BACKGROUND: Several epidemiological studies have been conducted to understand the relationship between environmental factors (including chronic sun exposure) and clinical signs of pigmented spots. However, no quantitative analysis has focused on the adverse effects of the detailed features of pigmented spots, including their color intensity, size, and number on the cheek. This study was performed to elucidate the adverse effects of environmental factors on clinical signs of pigmented spots. METHODS: We conducted an epidemiological survey of 102 Japanese women in 2 regions of high and low sun exposure (southern and northern regions, respectively). Using image analysis of high-resolution digital facial photographs, individual pigmented spots were quantified according to color, size, and total number on the cheek. Each indicator was then compared between the groups. RESULTS: For the number of pigmented spots on the cheek, the age-related increase curve showed a large slope in the southern group. For the size of pigmented spots, no significant difference was found between the two groups, and large pigmented spots were observed on the cheek even in the northern group. For the color intensity of the spots, the southern group showed a marked age-related change; among older subjects, the pigmented spots were significantly darker in the southern than northern group. CONCLUSION: Our results may indicate that environmental factors, including chronic exposure to sunlight, mainly increase the number of pigmented spots and darkening of these spots. However, the occurrence of large pigmented spots may be related to intrinsic factors represented by heredity rather than environmental factors.

Development of a method for evaluating skin dullness: A mathematical model explaining dullness by the color, optical properties, and microtopography of the skin.

BACKGROUND: Skin dullness has long been a major concern of Japanese women. It is usually evaluated and judged visually by experts. Although several factors are recognized to play a role, it is unclear to what extent such physiological characteristics contribute to skin dullness. The purpose of this study is to establish an objective method for evaluation, which will assist in developing cosmetics products targeting skin dullness. METHODS: We conducted a skin measurement study on 50 Japanese women in their 30-50s, where skin dullness was visually assessed by a group of experts to obtain an average dullness score, and several skin parameters were obtained. We then developed a regression model that explains the visual assessment score using these physiological parameters. RESULTS: The results of partial least squares analysis of the dullness perception and physiological characteristics showed that skin dullness can be defined by colorimetric, optical, and skin surface microtopography parameters. Additionally, the contribution of each parameter to the model was determined. Our results suggest that dullness perception is highly affected by the melanin content and yellowness of the skin, followed by skin reddishness, roughness, and translucency score, whereas glossiness has less effect. Strikingly, the contribution ratio of each parameter varied among age groups. Furthermore, we confirmed that the predicted value of skin dullness increases with age. CONCLUSION: Our results will help the design of cosmetics targeting factors specific to age groups in developing effective solutions for skin dullness.

Prediction and association analyses of skin phenotypes in Japanese females using genetic, environmental, and physical features.

BACKGROUND: Skin characteristics show great variation from person to person and are affected by multiple factors, including genetic, environmental, and physical factors, but details of the involvement and contributions of these factors remain unclear. OBJECTIVES: We aimed to characterize genetic, environmental, and physical factors affecting 16 skin features by developing models to predict personal skin characteristics. METHODS: We analyzed the associations of skin phenotypes with genetic, environmental, and physical features in 1472 Japanese females aged 20-80 years. We focused on 16 skin characteristics, including melanin, brightness/lightness, yellowness, pigmented spots, wrinkles, resilience, moisture, barrier function, texture, and sebum amount. As genetic factors, we selected 74 single-nucleotide polymorphisms of genes related to skin color, vitamin level, hormones, circulation, extracellular matrix (ECM) components and ECM-degrading enzymes, inflammation, and antioxidants. Histories of ultraviolet (UV) exposure and smoking as environmental factors and age, height, and weight as physical factors were acquired by means of a questionnaire. RESULTS: A linear association with age was prominent for increase in the area of crow's feet, increase in number of pigmented spots, decrease in forehead sebum, and increase in VISIA wrinkle parameters. Associations were analyzed by constructing linear regression models for skin feature changes and logistic regression models to predict whether subjects show lower or higher skin measurement values in the same age groups. Multiple genetic factors, history of UV exposure and smoking, and body mass index were statistically selected for each skin characteristic. The most important association found for skin spots, such as lentigines and wrinkles, was adolescent sun exposure. CONCLUSION: Genetic, environmental, and physical factors associated with interindividual differences of the selected skin features were identified. The developed models should be useful to predict the skin characteristics of individuals and their age-related changes.

Image analysis of skin color heterogeneity focusing on skin chromophores and the age-related changes in facial skin.

BACKGROUND/PURPOSE: Heterogeneity with respect to skin color tone is one of the key factors in visual perception of facial attractiveness and age. However, there have been few studies on quantitative analyses of the color heterogeneity of facial skin. The purpose of this study was to develop image evaluation methods for skin color heterogeneity focusing on skin chromophores and then characterize ethnic differences and age-related changes. METHODS: A facial imaging system equipped with an illumination unit and a high-resolution digital camera was used to develop image evaluation methods for skin color heterogeneity. First, melanin and/or hemoglobin images were obtained using pigment-specific image-processing techniques, which involved conversion from Commission Internationale de l'Eclairage XYZ color values to melanin and/or hemoglobin indexes as measures of their contents. Second, a spatial frequency analysis with threshold settings was applied to the individual images. Cheek skin images of 194 healthy Asian and Caucasian female subjects were acquired using the imaging system. Applying this methodology, the skin color heterogeneity of Asian and Caucasian faces was characterized. RESULTS: The proposed pigment-specific image-processing techniques allowed visual discrimination of skin redness from skin pigmentation. In the heterogeneity analyses of cheek skin color, age-related changes in melanin were clearly detected in Asian and Caucasian skin. Furthermore, it was found that the heterogeneity indexes of hemoglobin were significantly higher in Caucasian skin than in Asian skin. CONCLUSION: We have developed evaluation methods for skin color heterogeneity by image analyses based on the major chromophores, melanin and hemoglobin, with special reference to their size. This methodology focusing on skin color heterogeneity should be useful for better understanding of aging and ethnic differences.

Microarray analysis of global gene expression in leukocytes following lithium treatment.

OBJECTIVES: To elucidate the molecular effects of lithium, we studied global gene expression changes induced by lithium in leukocytes from healthy subjects. METHODS: Eight healthy male subjects participated in this study. Lithium was prescribed for weeks to reach a therapeutic serum concentration. Leukocyte counts and serum lithium concentrations were determined at baseline (before medication), after 1 and 2 weeks of medication and at 2 weeks after stopping medication. Gene expression profiling was performed at each time point using Agilent G4112F Whole Human Genome arrays (The Agilent Technologies, Santa Clara, CA, USA). Expression of some candidate genes was also assessed by real-time polymerase chain reaction (PCR). RESULTS: Gene ontology analysis revealed that the cellular and immune responses to stimulus and stress indeed played a major role in the cellular response to lithium treatment. Pathway analysis revealed that the interleukin 6 pathway, the inhibitor of differentiation pathway, and the methane metabolism pathway were regulated by lithium. Using real-time PCR, we also confirmed that five candidate genes in these pathways were significantly changed, including suppressor of cytokine signaling 3 and myeloperoxidase. CONCLUSIONS: Our investigation suggests that the molecular action of lithium is mediated in part by its effects on the cellular and immune response to stimulus and stress followed by the interleukin 6, inhibitor of differentiation, and methane metabolism pathways.

Imaging of hemoglobin oxygen saturation ratio in the face by spectral camera and its application to evaluate dark circles.

BACKGROUND: Contact-type spectrophotometers have been widely used to measure skin color to determine the color values and melanin and hemoglobin contents. Recently, a spectral camera was introduced to evaluate two-dimensional color distribution. However, its application to skin color measurement has been limited. METHODS: The original spectral imaging system developed for facial skin consisted of a spectral camera and an original lighting unit for uniform irradiation of the face. The distribution of skin chromophores in the face, including melanin and oxy- and deoxyhemoglobin, was calculated from the reflectance data for each pixel of the spectral images. In addition, to create a mean spectral image of the group, a face morphing technology for spectral data was proposed. Using the system, we determined the characteristics of the dark circles around the eyes and also evaluated the effects of an anti-dark circle cosmetic. RESULTS: This system enabled the sensitive detection of skin chromophores in the face. Melanin content increased and hemoglobin oxygen saturation ratio decreased locally in the infraorbital areas of women with dark circles compared with those of women without dark circles. In addition, we were able to detect improvement in the dark circles after 6 weeks' use of anti-dark circle cosmetic products by visualizing the distribution of the relative concentrations of melanin and hemoglobin oxygen saturation ratio. CONCLUSION: Using a spectral camera, we developed a non-contact image-processing system that was capable of capturing a wide area of the face to visualize the distribution of the relative concentrations of skin chromophores in the face.

Epidemiological survey of the quantity and anatomical position of facial pigmented spots in relation to age in 454 Japanese women.

Facial pigmented spots are one of the phenotypes of skin aging, but no quantitative analysis of spot features such as color intensity, size, anatomical position, and number on the cheek has yet been performed. In the current study, we conducted an epidemiological survey of 454 Japanese women in their 20s to 70s and analyzed age-related changes and site differences of facial pigmented spots. Using image analysis of high-resolution digital facial photographs, 4912 individual pigmented spots were quantified according to color, size, anatomical position, and total number on the cheek. As a result of color analysis, the color intensity of individual pigmented spots increased with aging, significantly so between ages 30s and 50s. The age-related increase in melanin index of facial spots was confirmed in all sites but did not significantly differ between facial subregions. Regarding the size of pigmented spots, the frequency of large spots increased with age, and large spots were detected in all facial sites. The total number of pigmented spots on the entire cheek increased with aging, significantly so between the 20s and 40s. The number of pigmented spots tended to increase from the region near the canthi to the lower cheeks. The number of spots was markedly increased in the buccal regions compared with the infraorbital and zygomatic regions. The data and methodology presented in the current study can link the state of facial pigmentation with the various factors involved in the histological development of pigmented spots, opening new possibilities in the fields of skin pharmacology and dermatology.

Lithium decreases VEGF mRNA expression in leukocytes of healthy subjects and patients with bipolar disorder.

OBJECTIVES: Vascular endothelial growth factor (VEGF) is thought to be involved in the pathophysiology of mood disorders and the target of antidepressants. The aim of this study was to elucidate molecular effects of lithium on VEGF expression by using leukocytes of healthy subjects and patients with bipolar disorder. METHODS: Eight healthy male subjects participated in the first study. Lithium was prescribed for 2 weeks, enough to reach therapeutic serum concentration. Leukocyte counts and serum lithium concentrations were determined at baseline, at 1- and 2-week medication, and at 2 weeks after stopping medication. VEGF mRNA levels were also examined in nine lithium-treated bipolar patients and healthy controls in the second study. RESULTS: In the first study, leukocyte counts were significantly increased at 2 weeks compared with those at baseline and were normalized after 2 weeks. VEGF mRNA levels were significantly decreased at 2 weeks and after 2 weeks compared with those at baseline. Consistent with the first study, VEGF mRNA levels were significantly decreased in the lithium-treated bipolar patients compared with healthy controls. CONCLUSIONS: Our investigation suggests that VEGF mRNA expression may be useful as a peripheral marker of the effects of lithium.

Th1 response during ribavirin and interferon-alpha combination therapy in chronic hepatitis C.

Ribavirin and interferon-alpha induce Th1 polarization of human CD4+ T cells. The study was conducted to investigate the whether cellular immune response during ribavirin/interferon-alpha therapy is associated with viral eradication by examining mRNA expression of molecules relevant to Th1 and Th2 polarization in CD4+ cells of 13 patients with chronic hepatitis C (seven patients with sustained viral response and six with transient response). Peripheral CD4+ T lymphocytes at 0, 4 and 24 weeks of treatment were tested. There were no significant differences in the mRNA levels at each point of time of the treatment between patients with sustained viral response and those with transient response. The percent increase in mRNA level of the IL-12R beta2 chain from the baseline to the end of the treatment was significantly higher in patients with sustained viral response (15.3+/-6.1%) than in those with transient response (-1.6+/-4.7%, p<0.05). There was no significant difference in percent changes in level of IL-12R beta1 chain mRNA between the two groups. In conclusion, the results of this study indicate that the increase of Th1 response is related to the inflammatory activity in the liver and possibly to ribavirin and interferon-alpha therapy. It is also suggested that the measurement of Th1 response has the potential to distinguish patients with relapse from those with sustained virus response.

Effects of in vivo exposure to eggs with sperm-immobilizing antibodies in follicular fluid on subsequent fertilization and embryo development in vitro.

Aims: It has been shown that supplementation of patients' sera that contains sperm-immobilizing antibodies results in failure of fertilization and embryo development in vitro. The present study was carried out to investigate if exposing retrieved eggs to a high number of sperm-immobilizing antibodies in the follicular fluid (FF) in vivo affected subsequent fertilization and embryo development in vitro, even if they were washed with an antibody-free culture medium. Methods: Patients' sera and their FF were collected in 15 in vitro fertilization-embryo transfer (IVF-ET) or intracytoplasmic sperm injection-embryo transfer (ICSI-ET) treatment cycles from 11 infertile women with sperm-immobilizing antibodies in their sera. Quantitative sperm-immobilizing antibody titers (SI50 titers) in the sera and FF were evaluated. The fertilization rate, good-quality embryo rate and implantation rate by IVF-ET were compared between infertile patients having higher (10≤) SI50 titers and lower (<10) SI50 titers in their FF. Results: There was a significant correlation in the SI50 titers between the patients' sera and their FF (P < 0.0001). After thoroughly washing the collected eggs in culture medium without the patient's serum before IVF, there was no difference in the fertilization rate in the patients with high (10≤) and low (<10) SI50 titers in their FF (P = 0.62). However, the good-quality embryo rate in the patients with a high SI50 titer was significantly lower than patients with a low antibody titer (P < 0.05). There was no significant difference in the implantation rate between the two groups (P = 0.33). Conclusions: Similar amounts of sperm-immobilizing antibodies existed in the patients' FF and in their sera. ICSI did not seem to be necessary in patients having the antibodies if their sera were not supplemented in the culture media. Even with careful manipulation of eggs, it might be suggested that the harmful effects of sperm-immobilizing antibodies on embryo development cannot be completely avoided, especially in patients with high SI50 titers in the FF. (Reprod Med Biol 2006; 5: 137-143).

Severe ovarian hyperstimulation syndrome in a 42-year-old woman with successful pregnancy after intracytoplasmic sperm injection embryo transfer.

Ovarian hyperstimulation syndrome (OHSS) is an iatrogenic complication of ovulation induction for infertile women. It has been shown that patients suffering from OHSS are generally young because OHSS depends on the patient's ovarian reserve. Therefore, women ≥40 years of age seldom suffer from the severe form of OHSS. In the present study, we report a case of severe OHSS that occurred in a 42-year-old woman with a successful pregnancy after intracytoplasmic sperm injection (ICSI)-embryo transfer. She had been diagnosed as having polycystic ovary syndrome (PCOS). After 5 cycles of unsuccessful treatment with gonadotropins plus intrauterine insemination for her husband's asthenozoospermia, the treatment with assisted reproductive technology (ART) was indicated. In the third ART attempt, the patient was treated by ICSI-embryo transfer (ET) and she developed severe OHSS at 4 weeks' gestation. On admission, marked hemoconcentration, oligouria and hypo-albuminemia were diagnosed. A continuous autotransfusion system of ascites, which was developed to expand circulating plasma volume without exogenous albumin, was carried out for 5 h at a rate of 100-200 mL/h once a day for a total of 5 days. The course of the pregnancy was uneventful. At 37 weeks' gestation, a healthy baby boy weighing 3336 g was born by cesarean section when the patient was 43 years of age. The postoperative course was also uneventful. To the best of our knowledge, the present report describes the oldest woman showing severe OHSS. (Reprod Med Biol 2005; 4: 265-269).

Successful pregnancy following conservative treatment of massive ascites associated with acute Chlamydia trachomatis peritonitis.

It is well known that Chlamydia trachomatis causes acute and chronic pelvic inflammatory disease including salpingitis. We describe a case of successful pregnancy following conservative treatment of massive ascites associated with acute Chlamydia trachomatis peritonitis. In this present case, we conservatively treated a woman with acute chlamydial salpingitis accompanied with marked ascites and an adnexal mass that simulated a malignant neoplasm. Elevated CA125 and CA19-9 also suggested a malignancy at the time of diagnosis, however following treatment they decreased to below the cut-off value, and were useful in identifying the efficacy of medical treatment. The patient subsequently became pregnant after infertility treatment and underwent a normal vaginal delivery. We conclude that the possibility of Chlamydia trachomatis peritonitis should be considered when a patient presents with ascites and an adnexal mass in sexually active women. (Reprod Med Biol 2004; 3: 217-221).

Infertile women without sensitization to an appropriate amount of sperm do not produce sperm-immobilizing antibodies in their sera.

Background and Aim: The factors that affect sperm immobilizing antibody production in some women are not fully understood. This study was performed to investigate if production of sperm immobilizing antibodies in women is associated with their husbands' sperm count. Methods: The sperm immobilization test (SIT) was performed on 221 infertile women whose husbands had normal semen characteristics according to the criteria by WHO; 160 patients were treated by intracytoplasmic sperm injection (ICSI) because of poor semen characteristics, and 1013 virgin female children acted as the controls. Results: A significant difference of the incidence in SIT was observed between the virgin female children and the women whose husbands had a normal sperm count (P < 0.0001). There was also a significant difference of the incidence in SIT between the women treated by intracytoplasmic sperm injection (ICSI) because of a severe male factor and those whose husbands had a normal sperm count (P < 0.05). Conclusions: These results indicate that production of sperm immobilizing antibodies in women begins after they have been exposed to a large enough amount of sperm. However, the precise amount of sperm required to produce the antibodies is not clarified by the present study. (Reprod Med Biol 2003; 2: 105-108).

Meta-analyses of blood homocysteine levels for gender and genetic association studies of the MTHFR C677T polymorphism in schizophrenia.

Previous studies suggest that elevated blood homocysteine levels and the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism are risk factors for schizophrenia. However, the effects of gender and MTHFR C677T genotypes on blood homocysteine levels in schizophrenia have not been consistent. We first investigated whether plasma total homocysteine levels were higher in patients with schizophrenia than in controls with stratification by gender and by the MTHFR C677T genotypes in a large cohort (N = 1379). Second, we conducted a meta-analysis of association studies between blood homocysteine levels and schizophrenia separately by gender (N = 4714). Third, we performed a case-control association study between the MTHFR C677T polymorphism and schizophrenia (N = 4998) and conducted a meta-analysis of genetic association studies based on Japanese subjects (N = 10 378). Finally, we assessed the effect of plasma total homocysteine levels on schizophrenia by a mendelian randomization approach. The ANCOVA after adjustment for age demonstrated a significant effect of diagnosis on the plasma total homocysteine levels in all strata, and the subsequent meta-analysis for gender demonstrated elevated blood homocysteine levels in both male and female patients with schizophrenia although antipsychotic medication might influence the outcome. The meta-analysis of the Japanese genetic association studies demonstrated a significant association between the MTHFR C677T polymorphism and schizophrenia. The mendelian randomization analysis in the Japanese populations yielded an OR of 1.15 for schizophrenia per 1-SD increase in plasma total homocysteine. Our study suggests that increased plasma total homocysteine levels may be associated with an increased risk of schizophrenia.

DNA methylation signatures of peripheral leukocytes in schizophrenia.

Schizophrenia (SCZ) is a complex psychiatric disease with a lifetime morbidity rate of 0.5-1.0 %. To date, aberrant DNA methylation in SCZ has been reported in several studies. However, no comprehensive studies using medication-free subjects with SCZ have been conducted. In addition, most of these studies have been limited to the analysis of the CpG sites in CpG islands (CGIs) in the gene promoter regions, so little is known about the DNA methylation signatures across the whole genome in SCZ. Genome-wide DNA methylation profiling (485,764 CpG sites) of peripheral leukocytes was conducted in the first set of samples (24 medication-free patients with SCZ and 23 non-psychiatric controls) using Infinium HumanMethylation450 Beadchips. Second, a monozygotic twin study was performed using three pairs of monozygotic twins that were discordant for SCZ. Finally, the data from these two independent cohorts were compared. A total of 234 differentially methylated CpG sites that were common between these two cohorts were identified. Of the 234 CpG sites, 153 sites (65.4 %) were located in the CGIs and in the regions flanking CGIs (CGI: 40.6 %; CGI shore: 13.3 %; CGI shelf: 11.5 %). Of the 95 differently methylated CpG sites in the CGIs, most of them were located in the promoter regions (promoter: 75.8 %; gene body: 14.7 %; 3'-UTR: 2.1 %). Aberrant DNA methylation in SCZ was identified at numerous loci across the whole genome in peripheral leukocytes using two independent sets of samples. These findings support the notion that altered DNA methylation could be involved in the pathophysiology of SCZ.

Extended range near-infrared imaging of water and oil in facial skin.

Recently, near-infrared (NIR) imaging has been applied to detecting changes in skin hydration using the water OH band centered near 1460 nm. However, assigning changes in the intensity of the OH band near 1460 nm to changes in the skin's water content is complicated. Consequently, detection of small changes in facial skin water content is difficult. For highly sensitive imaging of facial skin water and oil, a near-infrared unit with a large detection range that includes the CH(3) and CH(2) stretching vibration modes at 1700-1800 nm and the strongest water bands centered near 1920 nm is required. In this study, an extended range indium gallium arsenide near-infrared camera was combined with a diffuse-illumination unit specifically developed for facial skin analysis. Images of water and oil in facial skin were obtained in real time using a combination of interference filters, such as 1950 ± 56 nm for water OH, 1775 ± 50 nm for oil CH, and 1300 ± 40 nm for background reflections. Clear near-infrared images were obtained with little mirror reflection. The water and oil content of facial skin could be evaluated even around the eyes, nose, and sides of the cheeks, which are areas that are difficult to analyze using current commercial devices. Differences were detected in the time-dependent changes of water and oil content in facial skin images obtained after the application of different types of moisturizer. The distribution of both water and oil in the facial skin could be visualized at the same time, and the images could be used to evaluate skin type and skin conditions.

Major complications and outcome of diagnostic and operative transvaginal hydrolaparoscopy.

AIM: Transvaginal hydrolaparoscopy (THL) has recently been developed as a less invasive alternative to conventional laparoscopy. There are some reports that described the usefulness and prognostic value of diagnostic THL in infertile women. Moreover, operative THL such as ovarian drilling for unovulatory women with polycystic ovarian syndrome (PCOS) to induce ovulation has also been found to be as effective as that by conventional laparoscopy. The risk of bowel injury and sepsis by transvaginal access with culdoscopy was higher than that with laparoscopy in the previous reports. The purpose of the present study was to examine the risk of diagnostic and operative THL according to two case studies with a literature review. METHODS: The authors carried out diagnostic or operative THL in 177 infertile women, aged 22-43 years. Major complications during THL and a review of the literature were analyzed. RESULTS: Two cases of bowel injury were diagnosed during diagnostic THL. No complication occurred during operative THL. In total, the incidence of bowel injury was 1.1%. The injuries were diagnosed during THL and treated expectantly under strict conditions in both cases. Ten studies in the literature reported a total of 4232 procedures, including 26 bowel injuries (0.61%) and one perforation of a retroflexed uterus (0.02%). CONCLUSIONS: The usefulness of THL for diagnostic and operative purposes is in no doubt. However, informed consent should be obtained and vigilance before and during THL should be maintained, although it can be done on an outpatient clinic basis.

Establishment of criteria for elective single embryo transfer at day 2 or day 3 by analyzing cases with successful implantation of all embryos transferred.

AIM: Elective transfer of two good-quality embryos has been used to avoid triplet or high-order multiple pregnancies. However, the rate of twin pregnancies has remained fairly unchanged. In the present study, criteria for elective single embryo transfer (eSET) at day 2 or day 3 were established by analyzing cases with successful implantation of all embryos transferred. METHODS: A total of 685 fresh or frozen-thawed embryo transfers following in vitro fertilization/intracytoplasmic sperm injection between April 2002 and March 2006 were performed. Only embryo transfers at day 2 or day 3, but not at blastocyst stage, were included. Successful implantation of all embryos transferred was obtained in 17 pregnancy cycles. RESULTS: Thirty-one gestational sacs with fetal heartbeats were obtained by a total of 31 embryo transfers in 17 infertile women. The average age was 32.6 years (23-38), and 14 (82.3%) of the 17 women were <36 years old. Fifteen (88.2%) of the 17 pregnancies were established at the first attempt of assisted reproductive technology (ART). Of the 17 women, eight (47.1%) women were multigravida and four (23.5%) women were multipara. The indications for ART or insemination methods did not seem to be related to the pregnancy results. Twenty-nine (93.5%) of 31 embryos implanted were considered good-quality embryos. Of the 17 fresh embryos transferred at day 2, 15 were at the 4-cell stage and two were at the 5-cell stage. Of the 11 fresh embryos transferred at day 3, one was at the 6-cell stage, two were at the 7-cell stage and eight were at the 8-cell stage. CONCLUSION: The criteria for eSET at day 2 or day 3 were established as follows: <36 years of age, a first treatment cycle and more than two good-quality embryos developed at least to the 4-cell stage at day 2, or 6-cell stage at day 3. Additionally, the past history of pregnancy or delivery should be considered, as patients positive for such history might have better implantation ability. eSET can be highly recommended to avoid twin pregnancies in subjects with the established criteria.