The study of the efficiency of in vitro maturation of ovarian tissue oocytes in pediatric patients.

PURPOSE: Although recent in vitro maturation (IVM) studies in pediatric patients have demonstrated successful retrieval and maturation of oocytes, the studies included only a small number of premenarchal patients. In the present study, we examined the potential use of oocyte retrieval and maturation for pediatric patients who undergo ovarian tissue cryopreservation (OTC). METHODS: We retrospectively examined the clinical records of pediatric patients who underwent OTC at our institution between October 2015 and December 2022. Data on the age, primary disease, menstrual history, pre-procedure chemotherapy, anti-Müllerian hormone (AMH) level, number of oocytes collected ex vivo from ovarian tissue, and number of mature oocytes from IVM were examined. RESULTS: Data of 60 pediatric patients (aged 1 to 17 years) were included for analysis. Oocytes were retrieved from 36 patients; the oocytes of 18 of these patients could be cryopreserved. The IVM rate was significantly lower in the premenarchal patients than in the postmenarchal patients. The number of mature oocytes retrieved from IVM was higher in the no-chemotherapy group than in the chemotherapy group. A significant positive correlation was observed between the AMH level and the IVM outcomes. CONCLUSION: Oocyte retrieval and maturation in pediatric patients undergoing OTC is particularly useful in those not receiving chemotherapy. In patients receiving chemotherapy, the AMH level may be useful for predicting the IVM outcome. Activation of the oocyte maturation process in vivo in pediatric patients and better understanding of the major regulators of oocyte maturation are necessary to improve the utility of the IVM procedure.

Shortening the duration between ovarian removal and cryopreservation helps preserve fertility and maintain ovarian reserve after transplantation in mice.

OBJECTIVE: To evaluate the effect of storage of ovaries before cryopreservation on long-term fertility and ovarian reserve after transplantation in mice. DESIGN: Experimental study. SETTING: University hospital. ANIMAL(S): C57BL/6J and C57BL/6J-Tg (CAG-GFP) female mice. INTERVENTION(S): Storage and cryopreservation of mouse ovaries. Long-term fertility analysis of mice transplanted with thawed ovaries. MAIN OUTCOME MEASURE(S): Estrous cycles, number of live births, ovarian weight, and follicular counts of ovarian grafts. RESULT(S): At the first mating 3 months after ovarian transplantation, the mean number of live births was 2.6 ± 0.6 in the control group (no storage); in the storage groups, the mean number of live births was 2.9 ± 0.7 after 4 hours, 1.3 ± 0.5 after 8 hours, 0.2 ± 0.2 after 12 hours, and 0.8 ± 0.5 after 24 hours of storage; the difference from the control group was significant in the 12-hour storage group. At the second mating 6 months after ovarian transplantation, the mean number of live births was 1.8 ± 0.6 in the control group and 2.4 ± 0.6 and 0.3 ± 0.2 in the 4- and 8-hour storage groups, respectively; no live births occurred in the 12- and 24-hour storage groups. Seven months after ovarian transplantation, the numbers of primordial, primary, early secondary, and late secondary follicles were significantly lower in the 8-, 12-, and 24-hour storage groups than in the control group. CONCLUSION(S): In mice, shortening the storage time of ovaries before cryopreservation preserved fertility and ovarian reserve after transplantation, indicating that human ovaries might be cryopreserved immediately after harvesting or transported as quickly as possible to a cryopreservation facility to allow young patients with cancer to preserve long-term fertility and ovarian reserve.

Learning Curve of Surgeons Performing Laparoscopic Ovarian Tissue Transplantation in Women with Premature Ovarian Insufficiency: A Statistical Process Control Analysis.

STUDY OBJECTIVE: To analyze patient safety in laparoscopic ovarian tissue transplantation surgery by tracking the rate of postoperative complications and the learning curves of the surgeons by statistical process control analysis. DESIGN: A retrospective study. SETTING: A university-affiliated hospital. PATIENTS: A total of 100 patients with premature ovarian insufficiency who underwent ovarian tissue cryopreservation by vitrification and then autologous transplantation of frozen-thawed ovarian tissues with in vitro activation. INTERVENTIONS: Ovarian tissue cryopreservation, in vitro activation, and transplantation. MEASUREMENTS AND MAIN RESULTS: We assessed the surgery complications, differences in total surgery time, transplantation time, and transplantation time per ovarian sheet in operations performed by 3 experienced laparoscopic surgeons. Surgeon A performed 80 operations; surgeon B, 29 operations; and surgeon C, 20 operations. Complications occurred in 1.55% of the procedures. Although all 3 surgeons' performance never fell below the unacceptable failure limit, only surgeon A became competent after 66 cases. CONCLUSION: The laparoscopic ovarian tissue transplantation surgery was generally safe given that the postoperative complications were infrequent (1.55%). Although the performance of all 3 surgeons was acceptable, only surgeon A attained the level of competency after 66 cases. The transplantation method may not be the key factor for reducing surgery time in this surgery. An efficient ovarian tissue transplantation team is more important in reducing the surgery time than the surgeon's surgical technique alone.

Longer time to extubation after general anesthesia with desflurane in patients with obstructive respiratory dysfunction: a retrospective study.

BACKGROUND: The prospect of patients with obstructive respiratory dysfunction undergoing surgery has increased with the growth in the elderly population; however, there have been few investigations about the recovery profile from volatile anesthesia. This study aimed to investigate the impact of obstructive respiratory dysfunction on recovery from desflurane anesthesia. METHODS: A retrospective cohort study included patients who underwent orthopedic lower limb surgery between September 2018 and March 2020. Patients were divided into two groups: those whose preoperative forced expiratory volume in 1 s/forced vital capacity ratio was <70% (obstructive respiratory dysfunction group, n = 180) or ≥70% (control group, n = 45). Time from discontinuation of desflurane to extubation (extubation time) was compared between the two groups. Univariate and multivariable Cox regression models were used to compare odds ratios for prolonged extubation (≥10 min). RESULTS: A total of 45 patients with obstructive respiratory dysfunction and 180 control patients were eligible for analysis. Extubation time was significantly longer in patients in the obstructive respiratory dysfunction group than those in the control group. In the multivariable Cox model, male sex (HR = 2.00, 95% CI 1.12-3.57; P = 0.020) and obstructive respiratory dysfunction (HR = 2.07, 95% CI 1.05-4.08; P = 0.036) were associated with prolonged extubation. CONCLUSIONS: This retrospective study indicated that extubation time was longer in patients with obstructive respiratory function than in patients without obstructive respiratory function. Male sex and obstructive respiratory function were factors that contributed to extubation time.

Oocyte retrieval after heterotopic transplantation of ovarian tissue cryopreserved by closed vitrification protocol.

PURPOSE: A device for closed vitrification was designed to reduce the risk of contamination and investigated on its efficacy for ovarian function recovery after cryopreservation and heterotopic transplantation. METHODS: Ovarian tissues from green fluorescence protein transgenic mice (10 GFP mice) were vitrified using the device, and warmed ovarian tissues were transplanted into the ovarian bursa region in wild-type female mice (6 mice). Fresh ovarian tissues were similarly transplanted as a control. After recovery of the estrous cycle, mice were mated with male mice. Ovarian tissues from six cynomolgus monkeys were vitrified and warmed with the device for autologous, heterotopic transplantation. Fresh tissue transplantation was not performed for the control. Ovarian function was examined by recovery of the hormonal cycle. Histological examination was conducted. RESULTS: The number of live pups per recipient mouse was not significantly different after transplantation of fresh or vitrified-warmed ovarian tissue, although the pregnancy rate was reduced with vitrified tissues. The hormonal cycle was restored in 5/6 monkeys after heterotopic transplantation of vitrified-warmed ovarian tissue. Follicles were harvested at eight sites in the omentum and 13 sites in the mesosalpinx. In vitro maturation (IVM)/IVF produced embryo but did not develop. CONCLUSIONS: Resumption of the hormonal cycles, follicle development, and oocyte retrieval from vitrified-warmed ovarian tissue transplants may indicate that the use of vitrification for ovarian tissue in a closed system has a potential of clinical application without the risk of contaminations. More detailed analyses of the effects of vitrification on ovarian tissue, such as gene expression patterns in oocytes and granulosa cells, may be needed for establishing a standard procedure for cryopreservation of ovarian tissues in human.

Effect of mouse ovarian tissue cryopreservation by vitrification with Rapid-i closed system.

PURPOSE: Currently, open systems are mainly used for cryopreservation of ovarian tissue, oocytes, and embryos, but there is a potential risk of contamination. This study was performed to assess ovarian tissue cryopreservation by a closed vitrification system (Rapid-i vitrification system™), which is already used clinically for oocyte/embryo cryopreservation. METHODS: Ovaries of C57BL/6J mice were frozen and thawed by using the Rapid-i vitrification system™ (Rapid-i) followed by implantation into recipient mice. Hematoxylin-eosin staining was performed for histological examination of the frozen-thawed ovaries to assess follicle grade. Fertility after implantation of the ovaries was assessed from the live birth rate and the number of live pups. RESULTS: There was no significant difference in grade 1 primary follicles between fresh ovaries (control group, 94.2 ± 2.9%) and frozen-thawed ovaries (Rapid-i group, 87.1 ± 1.8%). However, there was a significant decrease in grade 1 early and late secondary follicles in the Rapid-i group compared with the control group. The live-birth rate was significantly lower in the Rapid-i group compared with the control group (29.2 vs. 83.3%, p < 0.05). On the other hand, there was no significant difference in the average number of live pups between the control group and the Rapid-i group (3 ± 0.4 vs. 2.7 ± 0.3). CONCLUSIONS: The Rapid-i seems to be effective for cryopreservation of mouse ovarian tissue. Under appropriate conditions, the Rapid-i could be employed for ovarian tissue cryopreservation and preservation of fertility in humans.

Aromatase inhibitor use during ovarian stimulation suppresses growth of uterine endometrial cancer in xenograft mouse model.

STUDY QUESTION: Could aromatase inhibitors (AI) be used to reduce risks of uterine endometrial cancer growth or recurrence during ovarian stimulation? SUMMARY ANSWER: In a xenograft mouse model of endometrial cancer, concomitant AI administration suppressed the growth of endometrial cancer during ovarian stimulation. WHAT IS KNOWN ALREADY: Recurrence and mortality rates of estrogen receptor-positive early breast cancer are reduced by long-term AI administration. Concomitant AI use for ovarian stimulation in patients with breast cancer is recommended for reducing estrogen-related potential risks. However, the efficacy of concomitant AI use for estrogen receptor-positive endometrial cancer have not been demonstrated conclusively by clinical or experimental animal studies. STUDY DESIGN, SIZE, DURATION: Forty nude mice xenografted with uterine endometrial cancer cells were allocated to four groups. Group 1: no ovarian stimulation (control). Group 2: ovarian stimulation. Group 3: AI administration + ovarian stimulation. Group 4: ovariectomy and ovarian stimulation. Tumor growth was evaluated during the 6-week treatment period. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ishikawa 3-H-12 uterine endometrial cancer cells (estrogen and progesterone receptors-positive) were transplanted into 6-week-old BALB/cSlc-nu/nu nude mice, followed by interventions 2 weeks later. MAIN RESULTS AND THE ROLE OF CHANCE: Compared to ovarian stimulation alone (Group 2), significant suppressions of tumor growth were observed in other three groups (Groups 1, 3 and 4, all at P < 0.05) and correlated with estrogen levels. AI administration had no apparent impact on embryo development. LIMITATIONS, REASONS FOR CAUTION: In this study, we examined the growth of endometrial cancer tumors using one endometrial cancer cell line. Clinical endometrial cancer or hyperplasia cells can have diverse origins and AI may not be effective against other cancer cell types. WIDER IMPLICATIONS OF THE FINDINGS: Concomitant AI use may provide a chance for safer childbirth by for patients with endometrial cancer or hyperplasia. STUDY FUNDING/CONPETING INTEREST(S): This study was supported by the Graduate Student Aid from the St. Marianna University School of Medicine. The authors declare no competing interests.

Rapid determination of total bromide in human serum using an energy-dispersive X-ray spectrometer.

Since the bromide preparations useful in the treatment of intractable infantile epilepsy show a tendency to accumulate in the body, they may cause chronic toxicosis. To prevent this, determination of the bromide ion concentration in the serum is essential. After establishing a simple and rapid technique using energy-dispersive X-ray spectroscopy for the analysis of the serum total bromide level, we applied this technique in a clinically diagnosed epilepsy patient. The standard curve for total bromide showed linearity (r=0.999) in the range of 10-2000 microg/ml, and the lowest detection limit was 5 microg/ml. The mean recovery rate of bromide added to reference serum to yield a concentration of 50 microg/ml was 93.5% (n=5, coefficient of variation=9.1%). Analysis took only 20 min. On analysis of the serum of a 10-year-old girl whose treatment was initiated with orally administered potassium bromide 1.0 g/kg, a good correlation was found between the total bromide level obtained with energy-dispersive X-ray spectroscopic analysis and the level of bromide ions determined by ion-exchange HPLC. The determination of serum total bromide by rapid energy-dispersive X-ray spectroscopic analysis is a useful method of monitoring to prevent bromide poisoning.

In vivo receptor binding of benidipine and amlodipine in mesenteric arteries and other tissues of spontaneously hypertensive rats.

The present study was undertaken to characterize the in vivo 1,4-dihydropyridine (DHP) receptor binding of long-acting 1,4-DHP calcium channel antagonists in the mesenteric artery and other tissues of SHR. In vivo specific binding of (+)-[3H]PN 200-110 in the SHR mesenteric artery was significantly (36.6-49.7 %) reduced 1-8 h after oral administration of benidipine (1.84 micromol/kg). A greater reduction in (+)-[3H]PN 200-110 binding in the mesenteric artery was observed at a higher dose (5.53 micromol/kg) of this drug. This dose of benidipine also reduced significantly the in vivo specific (+)-[3H]PN 200-110 binding in the aorta but not in the myocardium and cerebral cortex. Following oral administration of amlodipine (17.6 micromol/kg), a significant (51.7-94.2 %) reduction in (+)-[3H]PN 200-110 binding was seen at 1-18 h in the mesenteric artery and at 1-12 h in the aorta. Only a slight reduction in myocardial and cerebral cortical (+)-[3H]PN 200-110 binding was seen following amlodipine administration. In contrast, oral administration of nifedipine (28.9 micromol/kg) reduced markedly in vivo (+)-[3H]PN 200-110 binding in all the tissues of SHR at 1-6 h, and the degree and time-course of the reduction did not differ significantly among the tissues. The area under the curve (AUC) for the receptor occupancy vs time was calculated from the reduction rate (%) of in vivo specific (+)-[3H]PN 200-110 binding. The ratios of the AUCmesenteric artery to AUCaorta or AUCmesenteric artery to AUCmyocardium after oral administration of benidipine and amlodipine were greater than the corresponding value for nifedipine. The degree and time-course of arterial receptor occupancy by benidipine and amlodipine agreed well with those of their hypotensive effects in the conscious SHR. In conclusion, the present study demonstrates that benidipine and amlodipine may occupy, in a more selective and sustained manner, 1,4-DHP receptors in arterial tissues than in other tissues of SHR, and thus, such receptor binding specificity may be responsible for the long-lasting hypotensive effects of these drugs.

Gas chromatography-mass spectrometry analysis of 4-O-methylpyridoxine (MPN) in the serum of patients with ginkgo seed poisoning.

The 4-O-methylpyridoxine (MPN) present in the seeds of the Ginkgo biloba (maidenhair tree) has anti-vitamin B6 actions, and ginkgo seed poisoning can induce convulsions. We developed a specific quantitative method using gas chromatography-mass spectrometry for the analysis of MPN in human serum. The trifluoroacyl (TFA) derivative of MPN was obtained by treating MPN with trifluoroacetic anhydride at 50 degrees C for 5 min and remained stable for 6 h. The calibration curve of standard MPN obtained in the selective ion mode using the base ion (m/z 343) was linear between 100 pg and 10 ng, and the detection limit was 50 pg. The full mass spectrum of 100 pg of the TFA derivative of MPN was obtained easily. MPN was extracted from the serum with the use of a C18 solid-phase extraction cartridge. The recovery rate of MPN added to the serum at a concentration of 0.1 microg/mL was 90.0%.

[Simultaneous determination of propanil, carbaryl and 3,4-dichloroaniline in human serum by HPLC with UV detector following solid phase extraction].

In case of poisoning by herbicide compounded with Propanil (DCPA) and Carbaryl (NAC), we attempted simultaneous solid-phase extractions of DCPA, NAC, and 3,4-dichloroaniline (DCA), a metabolite of DCPA, from the patient's serum, and quantitative analytical method using HPLC-UV detection. With this HPLC method, the quantitative detection limits in the serum are 0.005 microgram/ml for DCPA and DCA and 0.001 microgram/ml for NAC, and the UV spectra of all three compounds could easily be obtained using a diode-array detection limit of 0.05 microgram/ml. When the three compounds were added to serum at concentrations ranging from 0.1-10.0 micrograms/ml, the recovery rates were satisfactory at between 91.1% and 101.9%. On analysis of the serum of patient who had ingested Kusanon A Emulsion, the ingested substance apparently caused an increase in the DCA concentration, which led to the appearance of methemoglobinemia. The possibility that the DCA concentration might be used for prognostic purposes was suggested.

In vivo measurement of 1,4-dihydropyridine receptors in mesenteric arteries of spontaneously hypertensive rats and effect of nifedipine and cilnidipine.

The present study was undertaken to measure 1,4-dihydropyridine (DHP) receptor binding sites in vivo in the mesenteric artery and other tissues of spontaneously hypertensive rats (SHR) and to examine the effect of nifedipine and cilnidipine. Specific in vivo binding of (+)-[3H]PN 200-110 in the SHR mesenteric artery was dose dependently reduced by oral administration of nifedipine at relatively low doses. Oral administration of cilnidipine (6.09 micromol/kg) significantly reduced the specific in vivo binding of (+)-[3H]PN 200-110 in the mesenteric artery, aorta, and myocardium. A significant reduction in (+)-[3H]PN 200-110 binding was seen at 1-12 h in the mesenteric artery and at 1-7 h in the aorta and myocardium. In contrast, oral administration of nifedipine (28.9 micromol/kg) markedly reduced in vivo (+)-[3H]PN 200-110 binding in all tissues of SHR at 1-6 h, and the degree and time course of the reduction did not differ much among the tissues. The area under the curve (AUC) for receptor occupancy vs. time was calculated from the reduction rate (%) of specific in vivo (+)-[3H]PN 200-110 binding. The ratio (1.4 or 1.7) of the AUC(mesenteric artery) to AUCaorta or AUCmyocardium after oral administration of cilnidipine was greater than the corresponding value (1.1) for nifedipine. In conclusion, the present study demonstrates that cilnidipine, but not nifedipine, may occupy 1,4-DHP receptors in the small artery in a more selective and sustained manner than in other tissues of SHR, and thus such receptor binding specificity may be responsible for the long-lasting hypotensive effect of this drug.

Relationship between prostatic alpha(1)-adrenoceptor binding and reduction in intraurethral pressure following continuous infusion of KMD-3213 in rats.

The relationship between alpha(1)-adrenoceptor binding in rat tissues and pharmacodynamic effects of continuous infusion of KMD-3213 was examined. In vivo specific binding of [(3)H]KMD-3213 after continuous intravenous infusion of the ligand (100 pmol/kg/min for 10 min, followed by 30 pmol/kg/min for 60 or 90 min) differed largely among the tissues examined. Specific binding of [(3)H]KMD-3213 in aorta, heart, lung, and kidney was not different in terms of infusion time in the case of continuous infusion for 10, 70 and 100 min, whereas the binding in prostate, vas deferens, and submaxillary gland by 70- and/or 100-min infusion was significantly greater than that by the 10-min infusion. A similar extent of specific binding in the prostate was observed by the infusion (100 min) of a three-fold higher dose of [(3)H]KMD- 3213. Continuous intravenous infusion of KMD-3213 (100 pmol/kg/min for 10 min, followed by 30 pmol/kg/min) for 70 or 100 min significantly reduced the phenylephrine-induced increase in the mean blood pressure and that in the intraurethral pressure of anesthetized rats. Extent and time course of the KMD-3213 effect reduction in the phenylephrine-induced increase in intraurethral pressure were closely associated with those in prostatic [(3)H]KMD-3213 binding after continuous infusion of the corresponding dosage of the radioligand. The reduction in the phenylephrine-induced increase by the infusion of a three-fold higher dose of KMD-3213 was significantly greater in the case of the intraurethral pressure than in that of the mean blood pressure, thereby suggesting a greater selectivity for the alpha(1)-adrenoceptor in the lower urinary tract than for that in the vascular tissue. In conclusion, the present study has shown that specific binding of [(3)H]KMD-3213 in the rat prostate after the continuous intravenous infusion of the radioligand may be closely associated with the pharmacological effect of this drug on the lower urinary tract.