Comparison of pregnancy outcomes following fresh and electively frozen single blastocyst transfer in natural cycle and clomiphene-stimulated IVF cycles.

STUDY QUESTION: Are there any differences in live birth rates (LBR) following fresh blastocyst transfer in natural or clomiphene-stimulated cycles, or after elective blastocyst freezing in clomiphene-stimulated cycles followed by thawing and transfer at different time-points? SUMMARY ANSWER: Clomiphene citrate (CC) administration adversely affected the LBR after single fresh blastocyst transfer (SBT) in CC cycles compared with that in natural cycles, while this adverse effect of CC is not present when a single vitrified-warmed blastocyst transfer (SVBT) is performed in subsequent natural ovulatory cycles, regardless of the duration between CC administration and the day of SVBT. WHAT IS KNOWN ALREADY: CC affects uterine receptivity associated with a thinning of the uterine endometrium through an antioestrogenic effect. However, the duration that this adverse effect of CC on uterine endometrium persists after initial use is still unknown. STUDY DESIGN SIZE DURATION: A retrospective cohort study of 157 natural cycle IVFs followed by SBT and 1496 minimal ovarian stimulation with CC IVF cycles followed by SBT (n = 24) or SVBT (n = 1472) from January 2010 to December 2014 was conducted. SVBT cycles were classified into two groups according to the period between the last day of CC administration and the day of SVBT (A: ≤60 d and B: ≥61 d). All groups were then compared based on pregnancy outcomes (natural-SBT group: n = 157, CC-SBT group: n = 24, SVBT-A: n = 1143, SVBT-B: n = 329). PARTICIPANTS/MATERIALS SETTING METHODS: Women were aged 30-39 years at oocyte retrieval. In SVBT cycles, blastocysts were vitrified and warmed using a Cryotop safety kit. SVBT was performed in subsequent natural ovulatory cycles. The main outcomes were LBR and neonatal outcome, and both were compared among the groups. MAIN RESULTS AND THE ROLE OF CHANCE: The LBR in the CC-SBT group (29.2%, 7/24) was significantly lower compared with the natural-SBT (56.1%, 88/157) (P = 0.01) and SVBT-A (50.0%, 572/1143) (P = 0.04), but not SVBT-B (47.4%, 156/329), groups. Furthermore, multivariate logistic regression analysis revealed that the LBR was comparable among the natural-SBT and SVBT groups, but was significantly lower in the CC-SBT group (adjusted odds ratio: 0.324, 95% CI: 0.119-0.800, P = 0.01). No significant differences among all groups were observed for gestational age (P = 0.19), birthweight (P = 0.41) and incidence of malformation (P = 0.53). LIMITATIONS REASONS FOR CAUTION: In this study we analysed a biased sample, based on clinical judgement regarding endometrial thickness, and the study was limited by its retrospective nature. The low statistical power caused by the group size disparity was also a limitation, especially in the CC-SBT group. Although the outcome showing inferiority of CC-SBT compared to natural-SBT is consistent with general findings in the literature, further large-scale clinical studies, ideally RCTs, are necessary to validate our results and clarify the prolonged effect of CC in SVBT cycles on pregnancy and neonatal outcomes. WIDER IMPLICATIONS OF THE FINDINGS: Our observation suggests that CC administered in minimal ovarian stimulation cycles affects adversely the pregnancy outcomes when SBT is performed. Therefore, for a CC-based minimal stimulation IVF cycle, we suggest that frozen embryo transfer should be performed in a subsequent natural ovulatory cycle to avoid the possibility of implantation failure associated with CC administration. STUDY FUNDING/COMPETING INTERESTS: The authors have no conflicts of interest to declare. No external funding was either sought or obtained.

Heat incubation inactivates streptococcal exotoxins and recombinant cholesterol-dependent cytolysins: suilysin, pneumolysin and streptolysin O.

Streptococcus species release cholesterol-dependent cytolysins (CDCs), which are a main toxin, and their heat susceptibility is poorly understood. The aim of this study was to clarify the heat susceptibility of streptococcal exotoxins and CDCs. Streptococcal exotoxins were treated with heat incubation at 60 °C for 10 or 30 min. The Streptococcus suis exotoxin of serotypes 1 and 2 exhibited more than 50 % haemolytic activity, and all Streptococcus pneumoniae exotoxins exhibited more than 60 % haemolytic activity. During the thermolabile assay, the virulent streptococcal haemolytic activity remarkably decreased after being heated at 60 °C for 10 m. Then, streptococcal recombinant CDCs were produced and put through a thermolabile assay. The haemolytic activity of suilysin (SLY), pneumolysin (PLY) and streptolysin O (SLO) decreased more than 80 % after heat incubation. We also conducted a TER assay to evaluate the cell monolayer. The cell monolayer of all CDCs broke down, and the FITC-dextran translocated at 1 h post addition, while the CDCs treated with heating did not induce cell disruption. Moreover, the microscopy analysis demonstrated that CDCs treated with heating lost their activity. In conclusion, heat incubation induced the inactivation of streptococcal exotoxins and CDCs. Heat incubation plays a role in the degradation of the streptococcal exotoxin, and this result applies to the inhibition of streptococcal infection.

Modified thin abdominal wall flap (glove flap) for the treatment of acute burns to the hands and fingers.

BACKGROUND: Burns to the dorsum of the fingers and hands require debridement and immediate coverage by skin flap at the earliest opportunity. In such situations, the conventional abdominal wall flap is still commonly used as it is a convenient and safe technique, but the foremost problem with this flap is that it is thick and therefore cosmetically unacceptable; it is also functionally not very suitable as the bulkiness of the digits prevents full range of motion. We have developed a modified thin abdominal flap (glove flap) which attains good results. METHODS: Incisions are made in the skin of the abdominal wall only where the hand is to be inserted and where each of the finger tips will be pulled through. The flap is undermined just under the skin to the depth that preserves the subcutaneous vascular networks to create a thin flap. The interdigital area of the flaps should not be undermined so as to create a glove-type pocket. The hand is then inserted in this subcutaneous pocket. After insertion of the injured hand for 10 to 14 days, the flap is resected and attached to the hand. RESULTS: Seven hands of 5 patients were treated by this technique and all the flaps survived safely. The function of the hands and fingers, including range of motion (ROM) in each joint, was successfully salvaged. The reconstructed hands and fingers were aesthetically pleasing. CONCLUSIONS: Although the abdominal wall flap is not a new technique, our modifications to this flap make it possible to acquire functionally and aesthetically better results. Although many excellent techniques such as perforator flaps have been reported recently, we conclude that the abdominal wall flap is still a very useful technique because it can be performed easily, safely and within a short time.

Treatment of ischial pressure ulcers with a posteromedial thigh fasciocutaneous flap.

This study describes the use of the posteromedial thigh fasciocutaneous flap for the treatment of ischial pressure sores. The authors prefer this flap because it is the fasciocutaneous flap nearest to the ischial region, it is easy to raise, and it causes no donor-site morbidity. In this study, 11 ischial pressure sores in 10 paraplegic patients were closed using the posteromedial thigh fasciocutaneous flaps. All flaps survived, although two caused distal necrosis; after these same two flaps were readvanced, they survived. After an average follow-up time of 77 months, seven of the 10 patients have had no recurrence of ulcers. This fasciocutaneous flap was previously described by Wang et al. However, this study revealed that the arrangement of the vascular pedicle was different from that described by Wang et al. To reveal the vascular supply of this flap, anatomic dissections were conducted. The source of circulation to this flap was the suprafascial vascular plexus, in addition to the musculocutaneous perforator. The dominant pedicle was the musculocutaneous perforator from either the adductor magnus muscle or the gracilis muscle. The key to safe elevation of this flap was the accurate outlining of the skin island directly over the vascular pedicle and the preservation of the proximal fascial continuity. Of the 11 flaps, two viability problems occurred. These partial flap losses resulted from the failure to properly include the perforator. It is the authors' conclusion that the width of the flap should be greater than 5 cm. In addition, it is safe to make a flap within a 1:3 base-to-length ratio in a fatty, diabetic patient. This posteromedial thigh fasciocutaneous flap was found to be a valuable alternative for reconstruction of primary or recurrent ischial pressure ulcers.

Color Doppler sonography in monitoring after free jejunum transplantation.

The authors assessed the usefulness of color Doppler imaging in the monitoring of vascular circulation after free jejunum transplantation. Seven male patients were examined daily with color Doppler sonography for the first postoperative week between 1999 and 2000. All sonographic examinations were performed with an SSD5500 ultrasound scanner. Arterial and venous signals were documented reliably in all patients. The flashing red spots around the serosal wall revealed the patency of the anastomosed artery and vein. No failures of the graft occurred. The presence of the color Doppler signals was considered sufficient to define vascular patency. The real-time monographic (B-mode) examination revealed the thickness and the plicae circulares of the jejunum wall. These monographic (B-mode) images supported the viability of the transplanted jejuna. The authors found color Doppler sonography to be a reliable and effective form of monitoring after free jejunum transplantation.

Technique for selective microangiography of skin flaps with high contrast and fine detail.

The technical factors of microangiography were studied, and the authors found that the main limiting factors in resolution were the injection pressure of the contrast medium and the thickness of the materials. A gentle manual hand pressure injection, with the aid of visual monitoring under the operating microscope, were keys to obtaining beautiful microangiograms.

Intradermal stitch blepharoplasty for orientals: does It disappear?

Intradermal stitch blepharoplasty is one of the most frequently performed aesthetic surgeries in Japan. The major disadvantage of this method is the general feeling that it is nonpermanent. Mutou and Mutou [1] first published this technique in 1972. From 1957 to 1997, they have performed 20,098 cases of this procedure. The aim of this study was to furnish a retrospective analysis of a failure rate after intradermal double-eyelid operations that had been performed by a single surgeon (Y. Mutou). We evaluated consecutive patients (male 115, female 1,457) from 1986 to 1993 who underwent intradermal blepharoplasty at Sapporo Chuo Keisei Geka Clinic. Fifty patients (male 3, female 47) revisited our clinic complaining of the "disappearance" or loss of the fold. The estimated disappearing rate was 3.43%. Thirty percent of the disappearance occurred within 1 year. Eighty-eight percent of the disappearances occurred within 5 years postoperatively.

Successful preservation of human skin by vitrification.

The vitrification technique was applied to the preservation of human skin. This technique was simple, and no expensive equipment was needed. Split-thickness human skins from 8 patients were immersed in vitrification solution for 10 minutes at room temperature, immediately plunged into a liquid nitrogen tank, and cryopreserved for 3 weeks. The vitrification solution consisted of 40% ethylene glycol (vol/vol) and phosphate buffered saline solution that contained 30% Ficoll 70 (vol/vol; Wako Junyaku, Co, Tokyo, Japan) and 0.5 mol/L sucrose. The viability of vitrified and cryopreserved skin was evaluated with the trypan blue dye exclusion test, the methyl-thiazoldiphenyl-tetrazolium (MTT) colorimetric assay, and a culture test of the keratinocytes obtained from vitrified skin. The results of the trypan blue dye exclusion test showed 87.4% of viable cells, and MTT developed an average 0.817 absorbance. When vitrified skin was compared with 4 degrees C refrigerated skins after 3 weeks of storage, the difference of viability was significant both on the trypan blue dye exclusion test (P < .05) and on the MTT assay (P < .01). However, there was no significant difference in the viability of vitrified skins compared with fresh skin. Furthermore, keratinocytes from vitrified skin grew uneventfully in culture test. We used these vitrified skin allografts for patients with flame burns and electric burns. These allografts took well in both cases and promoted wound healing. We concluded that the vitrification method for skin preservation is simple and reliable, and this method could contribute to skin banking.

Immunosuppressive effect on T cell activation by interleukin- 16-cDNA-transfected human squamous cell line.

It is well known that it is difficult to induce an immunotolerance with allogeneic skin transplantation. We attempted to find the immunosuppressive protocol for prolonging skin allograft rejection by using interleukin-16 because IL-16 is considered one of the natural ligands to CD4 molecules. First we examined whether synergistic immunosuppressive effects of recombinant IL-16 plus anti-CD4 mAbs are induced in mixed lymphocyte reaction (MLR). Next we used IL-16-cDNA-transfected OSC-20 (human oral squamous cell carcinoma cell line) as an in vitro model of the epidermal keratinocyte equivalent and examined whether this transfectant could inhibit the activation of allogeneic T cells. Our data indicated that IL-16 clearly inhibited human MLR and that IL-16 increased synergistically the immunosuppressive effect of anti-CD4 mAb. We also used IL-16 transfectant and this produced more than 50 ng/ml of IL-16 in the supernatant by which human MLR was significantly inhibited. Furthermore, this transfectant also inhibited the activation of allogeneic lymphocytes stimulated directly with transfectant cells. These results indicated that the IL-16-producing allogeneic skin graft might have a local immunosuppressive action that would prolong graft survival.

Razor-assisted treatment of axillary osmidrosis.

Shaving off the sweat glands and hair follicles with a single-use safety razor is described.

Cloning and expression of the gene encoding flavodoxin from Desulfovibrio vulgaris (Miyazaki F).

The gene encoding a flavodoxin of Desulfovibrio vulgaris (Miyazaki F) was cloned, and overexpressed in Escherichia coli. A 1.6-kbp DNA fragment, isolated from D. vulgaris (Miyazaki F) by double digestion with SalI and EcoRI, contained the flavodoxin gene and its regulatory region. An expression system for the flavodoxin gene under control of the T7 promoter was constructed in E. coli. The purified protein was soluble and exhibited a characteristic visible absorption spectrum. HPLC analysis of the recombinant flavodoxin revealed the presence of an identical FMN to that found in the native D. vulgaris flavodoxin, and its dissociation constant with FMN was determined to be 0.38 nM. In vitro H2 reduction analysis indicated that the recombinant flavodoxin is active, and its redox potential was determined to be E1 = -434 and E2 = -151 mV using methyl viologen and 2-hydroxy-1,4-naphthoquinone, respectively. Its redox behavior was also examined with the recombinant flavodoxin adsorbed onto a graphite electrode. The mutant, A16E, was also produced, which revealed the feature of a conserved Glu residue at the surface of the molecule.

A case of eosinophilic pustular folliculitis (Ofuji's disease) induced by patch and challenge tests with indeloxazine hydrochloride.

A 73-year-old male developed disseminated erythema over his entire body after exposure to indeloxazine hydrochloride, a cerebral activator. Patch testing with indeloxazine hydrochloride showed a positive reaction, and plaques, vesicles and pustules developed on the face after the patch test. These had the pathologic feature of eosinophilic pustular folliculitis (EPF, Ofuji's disease). A challenge test also provoked eruptions on the face, trunk, arms and legs, which were compatible with EPF. Moreover, both the patch and challenge tests with indeloxazine hydrochloride induced eosinophilia. This is the first report of drug allergy-induced EPF, where drug sensitivity induced an abnormal eosinophilic response mimicking EPF.

In vitro comparative study of the antitumor effects of human interferon-alpha, beta and gamma on the growth and invasive potential of human melanoma cells.

We have studied the effects of interferon (IFN)-alpha, beta, and gamma in vitro on the growth and invasive potential of human melanoma SK-MEL-118 cells. The antiproliferative effects of IFNs were assessed by a quantitative regrowth assay in which cells were treated with IFNs at concentrations of 10(2), 10(3) or 10(4) IU/ml for 3 days (until day 4) and then further incubated without IFNs for 7 days (until day 11). The growth inhibitory effect of each IFN on melanoma cells was dose- and time-dependent. Among these three types of IFNs, however, IFN-beta exerted the strongest inhibitory effect on cell growth. To assess the anti-invasive effect of each IFN on melanoma cells, we employed an in vitro assay system using matrigel-coated Transwell chambers. When cells were treated with 10(2), 10(3), or 10(4) IU/ml of the three types of IFNs for 24 hours, the amount of tritiated thymidine incorporated into melanoma cells were treated for 24 hours with 10(4) IU/ml of IFN-beta or gamma prior to the assay, the number of cells that invaded the filter decreased by 40%; this decrease was only 10% with the same amount of IFN-alpha. Simultaneous addition of IFNs during the invasion assay was not effective in any combination. Only when the cells were pretreated with IFNs, antiinvasive effects against melanoma cells were exerted. IFN-alpha was less inhibitory than IFN-beta or gamma on proliferation and not at all inhibitory on invasion. Considering both the antiproliferative and antiinvasive effects of IFNs, our results suggest that IFN-beta has the strongest antitumoral effect on human melanoma cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Up-regulation of ICAM-1 expression on human dermal fibroblasts by IFN-beta in the presence of TNF-alpha.

Unstimulated human fibroblasts show low or undetectable ICAM-1 expression. Interferon-beta (IFN-beta) at concentrations of 10, 100, and 1000 IU/ml in the presence of tumor necrosis factor-alpha (TNF-alpha) significantly increased the ICAM-1 expression of fibroblasts in a dose-dependent manner. Treatment with IFN-beta alone, however, did not up-regulate the ICAM-1 expression. Furthermore the attachment of peripheral blood mononuclear cells (PBMCs) to cytokine-treated fibroblasts was increased. This augmented attachment was partly inhibited by anti-ICAM-1 antibody. These results suggest that IFN-beta and TNF-alpha may cooperatively modulate the attachment of PBMCs in the dermis.

Elevated serum CA125 in progressive systemic sclerosis with pleural effusion.

The elevation of tumor markers in benign diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis, or diabetes mellitus has been reported recently. We had the opportunity to observe a female patient with progressive systemic sclerosis (PSS) and pleuritis who demonstrated a high level of CA125 in her pleural effusion and blood serum. The purpose of this report is to describe this case. We also investigated whether tumor markers are elevated in collagen disease. We measured the serum levels of CA125 and CA19-9 in our case of PSS with pleuritis, 27 female patients with collagen diseases including SLE, PSS, dermatomyositis and Sjögren syndrome, and 11 normal females as controls. Compared with the normal controls, there was no evident elevation of CA19-9 or CA125 levels in collagen diseases except in our case. Elevated serum CA125 may be one of the indicators of pleural effusion in collagen disease.

Novel mutations and deletions of the KIT (steel factor receptor) gene in human piebaldism.

Piebaldism is an autosomal dominant genetic disorder of pigmentation characterized by white patches of skin and hair. Melanocytes are lacking in these hypopigmented regions, the result of mutations of the KIT gene, which encodes the cell surface receptor for steel factor (SLF). We describe the analysis of 26 unrelated patients with piebaldism-like hypopigmentation--17 typical patients, 5 with atypical clinical features or family histories, and 4 with other disorders that involve white spotting. We identified novel pathologic mutations or deletions of the KIT gene in 10 (59%) of the typical patients, and in 2 (40%) of the atypical patients. Overall, we have identified pathologic KIT gene mutations in 21 (75%) of 28 unrelated patients with typical piebaldism we have studied. Of the patients without apparent KIT mutations, none have apparent abnormalities of the gene encoding SLF itself (MGF), and genetic linkage analyses in two of these families are suggestive of linkage of the piebald phenotype to KIT. Thus, most patients with typical piebaldism appear to have abnormalities of the KIT gene.

PTK1, a novel protein kinase required for proliferation of human melanocytes.

We have identified and characterized PTK1, a novel human non-receptor protein kinase. Partial PTK1 cDNA was initially isolated by RT-PCR from mRNA of normal human melanocytes. Northern blot hybridization detected a 3.8-kb PTK1 mRNA in all tissues and cell types studied. Nucleotide sequence analysis of a full-length PTK1 cDNA showed it to encode an 847-amino acid polypeptide with an amino-terminal SH3 domain, a kinase catalytic domain, a leucine zipper-like domain, and a carboxyl proline-rich domain. The PTK1 kinase domain contains motifs generally considered diagnostic of serine/threonine kinases but also contains amino acids highly conserved among the tyrosine kinases. This suggests that PTK1 is a serine/threonine kinase, but one very closely related to the tyrosine kinase superfamily. When normal human melanocytes were exposed to antisense PTK1 oligonucleotide, cell growth was inhibited, whereas a corresponding sense oligonucleotide had no inhibitory effect. These data indicate that PTK1 plays an important role in the proliferation of normal human melanocytes.

Tumor necrosis factor-alpha increased the integrin alpha 2 beta 1 expression and cell attachment to type I collagen in human dermal fibroblasts.

Cell adhesion molecules of human dermal fibroblasts play an important role in the processes of wound healing. The effects of tumor necrosis factor- alpha (TNF) on the expression of integrin beta 1 subfamily in human dermal fibroblasts were examined. TNF preferentially induced the expression of alpha 2 beta 1 integrins, receptors for collagen and laminin, in a time and dose dependent manner. Cell attachment to type I collagen increased by the treatment with TNF. However, cell attachment to fibronectin and laminin was not increased. This TNF-induced cell attachment could be reduced significantly by anti-integrin alpha 2 beta 1 antibody. Antibodies against receptors other than alpha 2 beta 1 integrin did not significantly reduce cell attachment. These data suggest that the increased attachment of human dermal fibroblasts to type I collagen appears to be mediated predominantly through the augmentation of integrin alpha 2 beta 1 expression by TNF.

The ramification of the superficial branch of the superior gluteal artery. Anatomical basis of a new gluteus maximus myocutaneous flap.

In order to design a new gluteus maximus myocutaneous flap, the ramification of the superficial branch of the superior gluteal artery was investigated in 56 sides of 33 Japanese cadavers. The superficial branch constantly divides into two main branches, which are called the ascending and transverse branches in this study. Of the ascending and transverse branches, one or both usually give off at least one well developed division running on the undersurface of the gluteus maximus muscle (98.2%). This division, which is called the intermediate branch in this study, generally reaches the superior edge of the muscle giving off only a couple of muscular branches and pierces the muscle and its deep fascia to supply skin (83.6%). Perforators of the intermediate branch constantly emerge from the fascia near the middle or lower one-third point on a line extending from the middle of the iliac crest to the tip of the greater trochanter.