Selective vitrification of euploid oocytes markedly improves survival, fertilization and pregnancy-generating potential.

Enthusiasm for oocyte cryopreservation has been limited by poor pregnancy rates per thawed metaphase II (MII) oocytes (<4%) and low implantation rates per embryos. The reasons relate to technical limitations in the freezing process, and the fact that <40% of oocytes are euploid and unable to produce 'competent' embryos. Comparative genomic hybridization was performed on the first polar body (PB-1) of 323 MII oocytes retrieved from 16 donors. Of these, 111 were euploid, and were vitrified. Seventy-five of 78 vitrified oocytes (96%) survived warming and were fertilized using intracytoplasmic sperm injection. Thirty-one (41%) subsequently developed into expanded blastocysts, of which no more than two were subsequently transferred per uterus to 16 out of 19 prospective embryo recipients. Twelve of 19 (63%) recipients produced 17 healthy babies (eight singletons, three twins, and one set of triplets) One twin pregnancy miscarried in the late first trimester The birth rate per transfer of a maximum of two blastocysts to 16 recipients was 75%. The implantation rate per vitrified euploid oocyte was 27%. This study showed a six-fold improvement in pregnancy rate per cryopreserved oocyte over previous reports and a marked improvement in implantation rate. If independently validated, this approach could open the door to commercial egg cryobanking, significantly expanding women's reproductive choices.

Artificial shrinkage of blastocoeles using either a micro-needle or a laser pulse prior to the cooling steps of vitrification improves survival rate and pregnancy outcome of vitrified human blastocysts.

BACKGROUND: Since we reported the first successful birth from a blastocyst vitrified using a cryoloop technique, our results showed that the survival rate of vitrified blastocysts was negatively correlated with the expansion of the blastocoele. We speculated that a large blastocoele may disturb the efficacy of vitrification. Therefore, we evaluated the effectiveness of artificial shrinkage (AS) of blastocoeles before vitrification, on increasing the survival rate of vitrified blastocysts. METHODS: Supernumerary expanded blastocysts on day 5 were vitrified after AS, which was performed by puncturing the blastocoele with a micro-needle, or by making a hole in the blastocoele with a laser pulse. After warming, viable blastocysts (confirmed by re-expansion of the blastocoele) were transferred to patients with hormone replacement cycle. We compared these data with those of our previous report where AS was not carried out. RESULTS: The survival rate was significantly higher (97.2%, 488/502) in this study than that of the previous report (86%). After 266 transferable cycles, 160 patients became pregnant (60.2%), which was significantly higher than our previous results (34.1%, 29/85). The implantation rate was 46.7% (209/448). CONCLUSIONS: Our results revealed that the survival rate and the pregnancy rate of vitrified expanded and hatching blastocysts can be improved by using AS to collapse the blastocele before vitrification.

Vitrification of human blastocysts using cryoloops: clinical outcome of 223 cycles.

BACKGROUND: The need to cryopreserve human blastocysts is increasing. The successful birth has been reported of a baby from a blastocyst vitrified using the cryoloop technique. The present study expands on this earlier report to confirm the effectiveness of this vitrification procedure. METHODS: In patients undergoing IVF at one of three clinics, supernumerary blastocysts on day 5 or 6 at various stages of development were vitrified using cryoloops. RESULTS: Of 725 vitrified blastocysts, 583 (80.4%) survived. After the transfer of 493 blastocysts in 207 cycles, 76 women (37%) became clinically pregnant. Among these women, 21 pregnancies ended in miscarriage, 23 healthy babies were born in 18 deliveries, and 37 pregnancies are ongoing. The survival rate of day 5 blastocysts (87%) was higher than that of day 6 blastocysts (55%), but implantation rates and pregnancy rates were not statistically significantly different. CONCLUSIONS: Clinical outcomes with 725 blastocysts and 207 transfers showed that vitrification using cryoloops is effective and practical for the cryopreservation of human blastocysts. Early blastocysts on day 5 seem to be the most suitable in terms of stage and age for cryopreservation, but developed and day 6 blastocysts can also be cryopreserved.

Births after vitrification at morula and blastocyst stages: effect of artificial reduction of the blastocoelic cavity before vitrification.

BACKGROUND: In 1996, with the introduction of sequential media, we set up a programme of cryopreservation of supernumerary morulae (day 4) and blastocysts (day 5) using a vitrification procedure. Our results showed that the efficiency of the vitrification method was dependent on the stage of embryo development and was negatively correlated with the expansion of the blastocoele. We postulated that a large blastocoele might disturb cryopreservative potential due to ice crystal formation during the cooling step. We analysed therefore the effectiveness of reducing before vitrification the volume of the blastocoelic cavity. METHOD: Day 4 and day 5 embryos were vitrified in 40% ethylene glycol-18% Ficoll and 0.3 mol/l sucrose before plunging the straws directly into liquid nitrogen. Artificial shrinkage of the blastocyst was achieved after pushing a needle into the blastocoele cavity until it contracted. RESULTS: The survival rate post-thawing of day 4 and intact day 5 embryos was correlated with the volume of the blastocoele. In the control group only 20.3% blastocysts or expanded blastocysts survived as compared with 54.5 and 58.5% with morulae and early blastocyst respectively. After puncturing the blastocoelic cavity, an increase in the survival rate of up to 70.6% was noted. The pregnancy rates were improved after the artificial shrinkage procedure (20.5%) compared with the control intact blastocyst group (4.5%) (not significant). After reduction of the blastocoelic cavity, a significant increase in the implantation rate per vitrified blastocyst was observed (12.0 versus 1.4% P < 0.01). CONCLUSIONS: Our results showed that survival rates in cryopreserved expanded blastocysts could be improved by reducing the fluid content. This was presumably because mechanical damage caused by ice crystal formation was avoided. These observations should be considered when establishing a strategy and a protocol for cryopreservation of day 5 embryos.

Successful birth after transfer of vitrified human blastocysts with use of a cryoloop containerless technique.

OBJECTIVE: Clinical application of vitrification for the cryopreservation of human blastocysts. DESIGN: Clinical trial of vitrification of human blastocysts. SETTING: Private assisted reproductive technology clinic. PATIENT(S): Supernumerary blastocysts after fresh blastocyst transfer were vitrified for subsequent transfer. INTERVENTION(S): Culture of pronuclear embryos to the blastocyst stage in sequential media and subsequent vitrification of supernumerary blastocysts using a cryoloop technique. MAIN OUTCOME MEASURE(S): Clinical outcome after transfer of vitrified blastocysts. RESULT(S): A total of 60 vitrified blastocysts from 21 patients were warmed, and the survival rate at 2 hours after warming was 63%. Six clinical pregnancies were achieved after 19 transfers. One healthy baby was born, four pregnancies are ongoing, and one ended in miscarriage. CONCLUSION(S): Human blastocysts can be successfully vitrified by suspension on a small nylon loop and a direct plunge into liquid nitrogen. A delivery and ongoing pregnancies prove the safety of this method. This report documents the first successful pregnancy and delivery achieved by blastocyst vitrification using the cryoloop containerless technique.

Vitrification of human embryos based on the assessment of suitable conditions for 8-cell mouse embryos.

Experiments were conducted to find a suitable cryoprotectant and suitable procedure for vitrification of 8-cell mouse embryos. The method was then applied clinically to the cryopreservation of human embryos in our assisted reproduction programme. Mouse embryos were vitrified with 30 or 40% 1,2-propanediol (PROH), dimethylsulphoxide (DMSO), ethylene glycol, glycerol, or acetamide, each diluted with a solution containing 30% Ficoll plus 0.5 M sucrose. Embryos were exposed to the solutions for 0.5 or 2 min at 20 or 25 degrees C, cooled in liquid nitrogen and warmed rapidly. Embryo survival was assessed by in-vitro development. In PROH-, DMSO- and acetamide-based solutions, higher survival rates (29-82%) were obtained with less permeating conditions, suggesting that these cryoprotectants are considerably toxic. In glycerol- and ethylene glycol-based solutions, however, higher survival rates (74 and 92% respectively) were obtained with more permeating conditions, suggesting that these cryoprotectants are less toxic. Human embryos on days 2-3 were vitrified in an ethylene glycol-based solution (EFS40). Survival, assessed by the morphology, was higher in 4-cell embryos on day 2 and 8-cell embryos on day 3 than in 2-3-cell embryos on day 2 or 2-7-cell embryos on day 3. From 18 transfers, one ended with the delivery of healthy twin babies.

Experimental study of tracheal allotransplantation with cryopreserved grafts.

OBJECTIVE: Tracheal reconstruction is necessary in patients with extensive tracheal stenosis caused by neoplasm, trauma, and congenital disease. We investigated the possibility of tracheal allotransplantation with cryopreserved grafts in a canine model. METHODS: A seven-ring section of thoracic trachea was removed in 19 adult mongrel dogs. In group A (n = 4), a five-ring tracheal autograft was implanted. In group B (n = 6), a five-ring allograft was implanted without immunosuppression. In group C (n = 9), a five-ring cryopreserved tracheal allograft was implanted without immunosuppression. Omentopexy wrapping around the grafts and both anastomotic sites was used in all animals. RESULTS: All grafts survived without any evidence of atrophy or stenosis in group A. All animals in group B died of severe airway obstruction within 1 month, and postmortem examination of these grafts showed epithelial defect and necrotic tracheal cartilage in the scar tissue. In group C, no animals died of asphyxia caused by severe stenosis of the grafts. The graft epithelium was no longer present 20 days after transplantation, and the graft was covered with regenerated epithelium within about 60 days after the operation. CONCLUSION: These findings show that cryopreserved tracheal allografts can be transplanted by means of omentopexy without immunosuppression and that cryopreservation may reduce tracheal allogenicity.

Origin of regenerated epithelium in cryopreserved tracheal allotransplantation.

BACKGROUND: Our previous study showed that a cryopreserved tracheal allograft could be transplanted using omentopexy without immunosuppression. The present study investigated, by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, whether the regenerated epithelia were of recipient origin or donor origin in a cryopreserved tracheal allotransplantation model. METHODS: Twenty-nine mongrel dogs were classified by preoperative peripheral blood PCR-RFLP analysis. The cryopreserved tracheal allografts were implanted into recipient animals that showed a different phenotype from donor animals. A small specimen of epithelia excised from the allograft of animals postmortem was analyzed with the modified PCR-RFLP method. RESULTS: The animals were separated into 16 phenotypes by preoperative PCR-RFLP results, and cryopreserved tracheal allografts transplanted into 8 animals. PCR-RFLP analysis of graft epithelia at 10 days after transplantation showed the donor blood phenotype and analysis of graft epithelia taken from the animals that survived more than 20 days after operation showed the recipient blood and epithelial phenotype. CONCLUSIONS: The donor epithelia in the grafts were no longer present within about 20 days after transplantation. The recipient epithelia migrated gradually from the anastomotic site, and the regenerated epithelia that are of recipient origin covered the allograft within about 50 days after transplantation.

Thoracoscopic operation for secondary pneumothorax under local and epidural anesthesia in high-risk patients.

BACKGROUND: Video-assisted thoracic operations usually require single-lung ventilation under general anesthesia. However, for high-risk patients with other underlying pulmonary diseases, one has to consider risks of general anesthesia itself. METHODS: Four high-risk patients (4 men; mean age, 73 years) with intractable secondary pneumothorax and other underlying pulmonary diseases were treated by video-assisted thoracic operations under local and epidural anesthesia. Absorbable polyglycolic acid sheets and fibrin glue were used to control the air leakage. RESULTS: The mean duration of the procedure was 108 minutes. Pain and cough reflex were well controlled, and spontaneous breathing and hemodynamics were well maintained during the operation. The mean duration of the postoperative chest drainage was 5 days. No significant postoperative complication was encountered. No pneumothorax had recurred at a mean follow-up of 16 months. CONCLUSIONS: Video-assisted thoracic operations can be performed safely under local and epidural anesthesia for the treatment of intractable secondary pneumothorax in high-risk patients. The air leakage can be controlled with the use of polyglycolic acid sheets and fibrin glue without bullectomy.

[PCR-RFLP analysis of epithelium in canine cryopreserved tracheal allograft].

In this study, we investigated whether the regenerated epithelia were recipient phenotype or donor phenotype using PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method. Preoperatively 24 mongrel dogs were classified to 14 types by PCR-RFLP result from peripheral blood. The PCR-RFLP result of peripheral blood agreed to that of recipient epithelia. The cryopreserved tracheal allotransplantation was performed among the five pairs in which we could distinguish donor from recipient by PCR-RFLP. The epithelia of graft at 10 days after transplantation showed donor phenotype, but the epithelia at postoperative 20 days or more showed recipient phenotype. These results showed that allogenic epithelium remained in early post-transplant time and was gradually omitted. The epithelia migrated gradually from the anastomotic site, and the graft was covered with regenerated epithelia showing recipient phenotype within about 50 post-transplant days.

[Four cases of thymoma associated with pure red cell aplasia].

Thymomas associated with pure red cell aplasia (PRCA) constituted only 3.3% of 122 thymomas treated in our department from 1963 to March, 1995. Four patients with this disease (2 men and 2 women, mean age: 58 years, range: 41 to 66 years old) are reported. Auto-antibodies were found in all 4 cases, increase of T cell in two, suppression of erythropoietin production in one, and antibody to EB virus in one. Operation was performed in all cases (two thymothymectomies, one extended thymothymectomy, and one thymomectomy), and 3 of 4 patients were in the progressive stage of Masaoka's classification (I, III, IVa, and IVb each). As for the pathological findings, mixed type was found in three cases and lymphocyte predominant type in one. Two patients died from radiation pneumonia. As for PRCA, postoperative therapy was effective (100%) in all patients.

[A clinicopathological study of adenosquamous carcinoma of the lung].

We studied, clinicopathologically, 34 cases of adenosquamous carcinoma (ASC) treated in our institution over the last 30 years. Its incidence was 2.3% of all primary lung cancers. Following findings were characteristics to this type of lung cancer, male dominant, old age, heavy smoker, comparatively large diameter, peripheral location, and normal level of tumor makers. The 5-year survival rates were 35.0% for ASC, 38.1% for squamous cell carcinoma (SCC), and 50.2% for adenocarcinoma (AC). The component of SCC was predominant or equal to that of AC in 27 (79.4%) ASC cases. The degree of cell differentiation was moderate and both SCC and AC components showed the same degree of cell differentiation in most cases. Lymph node metastasis was seen in 15 (48.4%) cases.

[A case of thymoma in an elderly patient associated with myasthenia gravis].

A case of thymoma in an elderly patient associated with myasthenia gravis (MG) was reported. A 81-year-old female with growing dim of eye was diagnosed as MG, and treated with Mestinon. Two months later, she was detected to have an anterior mediastinal tumor on chest rentogenography and CT. She was diagnosed as MG (Osserman type IIA) with thymoma by Tensilon test and high anti-acetylcholine-receptor antibody level. Extended thymothymectomy was performed and the tumor was encapsulated and not invasive (stage I, lymphocyte predominant type). Postoperative course was good and subjective symptoms were improved without medication.

Intracytoplasmic sperm injection, fertilization, and embryo transfer after retrieval of spermatozoa by testicular biopsy from an azoospermic male with testicular tubular atrophy.

OBJECTIVE: To achieve fertilization and cleavage by spermatozoa retrieved by testicular biopsy from a male with testicular tubular atrophy. DESIGN: Clinical trial. SETTING: Private reproductive institute. PATIENT: Azoospermic male with demonstrated testicular tubular atrophy and almost complete spermatogenic arrest. INTERVENTION: Open biopsy retrieval of testicular tissue and sperm followed by intracytoplasmic sperm injection. MAIN OUTCOME MEASURES: Fertilization and cleavage. RESULTS: One four- to six-cell embryo was formed after intracytoplasmic sperm injection of five eggs with extruded polar bodies by retrieved sperm. CONCLUSION: Intracytoplasmic sperm injection after testicular sperm aspiration may be attempted in cases with severely decreased spermatogenesis and result in fertilization, cleavage, and embryo transfer.

Hypoplastic left heart syndrome with a single coronary artery originating from the pulmonary artery.

A newborn male infant with hypoplastic left heart syndrome due to mitral and aortic atresia died on the third day of life. Autopsy revealed a single coronary artery originating from the pulmonary artery, reversed coarctation of the aorta, a coronary sinus type atrial septal defect and stenosis of the left subclavian artery. To our knowledge, hypoplastic left heart syndrome associated with a single coronary artery originating from the pulmonary artery has never been reported in the English or Japanese literature. This may be the first such case.

Incidence of autoimmune antibodies in failed embryo transfer cycles.

PROBLEM: The presence of antiphospholipid antibodies lupus anticoagulant (LAC), anticardiolipin antibody (ACA) as well as antinuclear antibody (ANA) has been associated with early spontaneous pregnancy loss and adverse pregnancy outcome. The purpose of this study was to investigate the possible role of autoimmune antibodies (LAC, ACA, and ANA) as a cause of implantation failure following embryo transfer (ET) after in vitro fertilization (IVF). METHOD: Three groups were studied: Group I, 56 patients who failed to conceive following ET; group II, 14 patients who have conceived following IVF-ET and delivered or are carrying an uncomplicated ongoing pregnancy; and group III, 69 patients who were new candidates for IVF-ET. RESULTS: Eighteen out of 56 (32.1%) of patients who failed to conceive following previous IVF-ET cycle (group I) tested positive for one or more of the autoimmune antibodies. None of the 14 patients of group II tested positive for autoimmune antibodies (P < .02). Seven out of the 69 patients (10%) of group III were found positive to one or more of the autoimmune factor. This rate is significantly lower than the rate of positive autoimmune antibodies detected in group I (P < .003). Fifteen patients of the 18 who tested positive for autoimmune antibodies and who had previously failed to conceive following ET underwent a subsequent IVF-ET cycle while being treated with prednisone and aspirin. Seven out of the 15 (46.6%) conceived and were able to sustain a clinical ongoing pregnancy. CONCLUSIONS: Patients receiving ET are carrying viable embryos within the intrauterine environment. Therefore, in this unique group of patients, failure to demonstrate a positive pregnancy test represents an implantation failure or a very early postimplantation loss. The results of this study suggest that periimplantation events may be affected by autoimmune antibodies. Very early miscarriage or implantation failure may be related to the same pathophysiological mechanism that causes recurrent miscarriages and is diagnosed incorrectly as infertility.

[An experience with surgical treatment for mucoepidermoid carcinoma of the lungs].

Mucoepidermoid carcinoma (MEC) of the lungs is thought to arise in the bronchial glands. It is a tumor that rarely develops and it has a low grade of malignancy. In this paper, we describe one case of infiltrative MEC, which we were able to diagnose preoperatively. Surgery revealed a high grade malignancy which is reported here with a discussion based on the related literature. The patient was a 63-year-old male who was referred to our hospital by another physician due to a cough and left chest pain. A simple chest X-ray revealed a tumor shadow and a fascicular shadow on its periphery in the upper left lobe. Bronchoscopy disclosed complete circumferential stenosis at B1+2,3 and reddening from this region to the main bronchus, but it was impossible to directly confirm the tumor. Pulmonary arterography did not depict the left upper pulmonary vein, but obstruction due to a tumor of that vein was observed. Given the above findings, under a diagnosis of infiltrative MEC, a left total lobectomy accompanied by a combined left atriectomy was performed. Although most cases of MEC have a low grade malignancy, there have been some reported cases with a very high grade of malignancy. Therefore, evaluation of the progress of this type of carcinoma by preoperative diagnosis as well as radical excision appropriate to lung cancer are considered to be important.

The angiotensin II antagonist saralasin inhibits ovulation in the perfused rat ovary.

OBJECTIVE: Our null hypothesis was that the angiotensin II antagonist saralasin does not reduce the number of ovulations in the rat ovarian perfusion model. STUDY DESIGN: Ovaries from pregnant mare's serum gonadotropin-stimulated immature rats were perfused with nutrient media to which luteinizing hormone and 3-isobutyl-1-methylxanthine had been added to induce ovulation. Test perfusions were treated with saralasin 1 mumol/L (n = 0.5) and compared with controls (n = 5) with the Student t test. Perfusions with both saralasin and angiotensin II and dose-response evaluations were performed. RESULTS: Saralasin-treated ovulations were 6.6 +/- 1.3 (mean + SEM) compared with 18.6 +/- 3.9, p < 0.02. The effects of saralasin could be reversed with the addition of an equimolar amount of angiotensin II. Dose-response evaluations showed a progressive inhibition of ovulation at 10(-8) to 10(-6) mol/L. CONCLUSION: The angiotensin II antagonist saralasin inhibits ovulation in a dose-dependent fashion; this effect is canceled by the addition of equimolar concentrations of angiotensin II.

Culdoscopic gamete tubal transfer: a new approach to gamete intrafallopian tubal transfer.

Twelve infertile couples who failed to conceive by previous infertility treatments and who qualified for culdoscopy had oocyte retrieval and gamete transfer through an operative culdoscopy method. Six patients achieved clinical pregnancy. Five are ongoing pregnancies, and one patient miscarried. There were no complications of the procedures.