First babies conceived with Automated Intracytoplasmic Sperm Injection.

RESEARCH QUESTION: Can an automated sperm injection robot perform Automated Intracytoplasmic Sperm Injection (ICSIA) for use in human IVF? DESIGN: The ICSIA robot automated the sperm injection procedure, including injection pipette advancement, zona pellucida and oolemma penetration with piezo pulses, and pipette removal after sperm release. The robot was first tested in mouse, hamster and rabbit oocytes, and subsequently using discarded human oocytes injected with microbeads. A small clinical pilot trial was conducted with donor oocytes to study the feasibility of the robot in a clinical setting. The ICSIA robot was controlled by engineers with no micromanipulation experience. Results were compared with those obtained with manual ICSI conducted by experienced embryologists. RESULTS: The ICSIA robot demonstrated similar results to the manual procedure in the different animal models tested as well as in the pre-clinical validations conducted in discarded human oocytes. In the clinical validation, 13 out of 14 oocytes injected with ICSIA fertilized correctly versus 16 out of 18 in the manual control; eight developed into good-quality blastocysts versus 12 in the manual control; and four were diagnosed as chromosomally normal versus 10 euploid in the manual control. Three euploid blastocysts from the ICSIA robot group have been transferred into two recipients, which resulted in two singleton pregnancies and two babies born. CONCLUSIONS: The ICSIA robot showed high proficiency in injecting animal and human oocytes when operated by inexperienced personnel. The preliminary results obtained in this first clinical pilot trial are within key performance indicators.

First pilot study of maternal spindle transfer for the treatment of repeated in vitro fertilization failures in couples with idiopathic infertility.

OBJECTIVES: To gain insights into the technical feasibility of maternal spindle transfer (MST) applied in the context of repeated in vitro fertilization (IVF) failures for the treatment of idiopathic infertility. DESIGN: A prospective pilot study. SETTING: IVF center. PATIENT(S): Twenty-five infertile couples with multiple previous unsuccessful IVF cycles (range, 3-11), no previous pregnancy, and no history of mitochondrial DNA (mtDNA) disease participated. The study focused on women <40 years, with previous IVF attempts characterized by a pattern of low fertilization rates and/or impaired embryo development. Couples with severe male-factor infertility were not eligible. Oocyte donors with previous successful IVF outcomes were matched with patients according to standard practice. INTERVENTION(S): We performed MST by transferring metaphase II spindles from the patients' oocytes into the previously enucleated donor oocytes, followed by intracytoplasmic sperm injection, in vitro embryo culture, blastocyst biopsy, and vitrification. Only euploid blastocysts were considered for embryo transfer. MAIN OUTCOME MEASURE(S): Outcome measures included oocyte fertilization, blastocyst development, clinical pregnancy and live birth, incidence of mitochondrial carryover and potential mtDNA reversal, as well as general health of the children born. RESULT(S): Twenty-eight MST cycles produced 6 children (19 embryo transfers, 7 clinical pregnancies). Pediatric follow-up of the children, performed at intervals from birth to 12-24 months of age, revealed their development to be unremarkable. DNA fingerprinting confirmed that the nuclear DNA of MST children was inherited from both parents, without any contribution from the oocyte donor. For 5 of the children, mtDNA was derived almost exclusively (>99%) from the donor. However, 1 child, who had similarly low mtDNA carryover (0.8%) at the blastocyst stage, showed an increase in the maternal mtDNA haplotype, accounting for 30% to 60% of the total at birth. CONCLUSION(S): This pilot study provides the first insights into the feasibility of applying MST for patients with idiopathic infertility and repeated IVF failures. Reconstructed oocytes produced embryos capable of implanting, developing to term and producing apparently healthy newborns/children. However, claims concerning the efficacy of MST with respect to infertility treatment would be premature considering the limitations of this study. Importantly, mtDNA reversal was detected in one child born after MST, a finding with possible implications for mitochondrial replacement therapies. CLINICAL TRIAL REGISTRATION NUMBER: Pilot trial registry number, ISRCTN11455145. The date of registration: 20/02/2018. The date of enrolment of the first patients: 18/03/2018.

Infiltration of Bupivacaine and Triamcinolone in Surgical Wounds of Milligan-Morgan Hemorrhoidectomy for Postoperative Pain Control: A Double-Blind Randomized Controlled Trial.

BACKGROUND: Pain management after hemorrhoidectomy continues to be challenging. OBJECTIVE: The aim of this study was to evaluate the effectiveness of infiltration of bupivacaine hydrochloride and triamcinolone acetonide in surgical wounds of Milligan-Morgan hemorrhoidectomy for postoperative pain relief. DESIGN: A double-blind randomized controlled clinical trial was conducted between May 2015 and June 2019. SETTINGS: This study was carried out in a secondary referral hospital center in Spain. PATIENTS: Patients who underwent an elective Milligan-Morgan hemorrhoidectomy for grade III-IV hemorrhoids were included. INTERVENTIONS: Patients allocated in the intervention group received a single infiltration of bupivacaine and triamcinolone and patients allocated in the control group did not receive any intervention. MAIN OUTCOMES MEASURES: The primary end point was the median of maximum pain by visual analog scale during the 48 hours of hospital stay. Secondary end points were pain at follow-up, pain during defecation, analgesic requirements, wound healing rate, and complications. RESULTS: A total of 128 patients were randomly assigned (64 in each group). The median of maximum pain score at 48 hours was 3 points in the intervention group and 6 points in the control group, which was significantly different ( p < 0.05). No differences were observed regarding pain during defecation. Morphine requirements were significant lower in the intervention group (6.3% vs 21.9% patients; p = 0.01; 5 vs 23 cumulative doses; p = 0.01). Wound healing and complication rates were similar in both groups. LIMITATIONS: Use of placebo was not considered in the control group ("intervention group" vs "control group" clinical trial). Data regarding pain or other complications at 48 hours postoperatively were pooled and expressed as median of values. CONCLUSIONS: Infiltration of surgical wounds with bupivacaine and triamcinolone decreases postoperative pain and reduces morphine requirements in Milligan-Morgan hemorrhoidectomies. See Video Abstract at http://links.lww.com/DCR/B867 .Clinical trial registration: European Registry of Clinical Trials. EUDRACT 2014- 003043-35. INFILTRACIN DE BUPIVACANA Y TRIAMCINOLONA EN HERIDAS QUIRRGICAS DE HEMORROIDECTOMA DE MILLIGANMORGAN PARA EL CONTROL DEL DOLOR POSOPERATORIO ENSAYO CLNICO DOBLE CIEGO, ALEATORIZADO Y CONTROLADO: ANTECEDENTES:El manejo del dolor posterior a una hemorroidectomía sigue siendo un desafío.OBJETIVO:El objetivo de este estudio fue evaluar la efectividad de la infiltración de bupivacaína y triamcinolona en heridas quirúrgicas de hemorroidectomía de Milligan-Morgan para el alivio del dolor posoperatorio.DISEÑO:Se realizó un ensayo clínico controlado, aleatorizado, doble ciego entre mayo del 2015 y junio del 2019.ENTORNO CLINICO:Este estudio se llevó a cabo en un centro hospitalario de referencia secundaria en España.PACIENTES:Se incluyeron pacientes que se sometieron a una hemorroidectomía de Milligan-Morgan electiva por hemorroides de grado III-IV.INTERVENCIONES:Los pacientes asignados al grupo de intervención recibieron una única infiltración de bupivacaína y triamcinolona y los pacientes asignados al grupo de control no recibieron ninguna intervención.PRINCIPALES RESULTADOS RESULTADOS:El factor de valoración principal fue la mediana del dolor máximo según la escala análoga visual durante las 48 horas de estancia hospitalaria. Las variables secundarias fueron dolor durante el seguimiento, dolor durante la defecación, requerimientos de analgésicos, tasa de cicatrización de heridas y complicaciones.RESULTADOS:Se asignó aleatoriamente a un total de 128 pacientes (64 en cada grupo). La mediana de puntuación máxima de dolor a las 48 horas fue de 3 puntos en el grupo de intervención y de 6 puntos en el grupo de control con diferencias significativas (p < 0,05). No se observaron diferencias en cuanto al dolor durante la defecación. Las necesidades de morfina fueron significativamente menores en el grupo de intervención (6,3% versus 21,9% de los pacientes; p = 0,01; 5 versus 23 dosis acumuladas; p = 0,01). Las tasas de curación de heridas y complicaciones fueron similares en ambos grupos.LIMITACIONES:No se consideró el uso de placebo en el grupo de control (ensayo clínico: "grupo de intervención" versus "grupo control"). Los datos con respecto al dolor u otras complicaciones después de 48 horas postoperatorias se agruparon y expresaron como mediana de los valores.CONCLUSIONES:La infiltración de heridas quirúrgicas con bupivacaína y triamcinolona disminuye el dolor posoperatorio y reduce los requerimientos de morfina en la hemorroidectomía de Milligan-Morgan. Consulte Video Resumen en http://links.lww.com/DCR/B867 . (Traducción- Dr. Francisco M. Abarca-Rendon).Registro de ensayos clínicos: Registro europeo de ensayos clínicos. EUDRACT 2014- 003043-35.

Evaluation of the impact of laser-assisted hatching techniques on the hatching process of mouse blastocysts using time-lapse microscopy.

OBJECTIVE: To study the effect of zona opening (ZO) and 2 zona thinning (ZT) techniques on the hatching process of mouse embryos using a last-generation laser system and time-lapse microscopy (TLM). DESIGN: Prospective randomized study. SETTING: Private research center. ANIMALS: A total of 267 F1 hybrid (B6/CBA) mice embryos were included. INTERVENTION(S): Morulae were randomly selected and the zona pellucida (ZP) manipulated using a laser system according to 4 experimental groups: control (ZP intact, n = 59), ZO (25 µm hole, n = 70), ZT25 (25% perimeter thinned, n = 71), and ZT35 (35% perimeter thinned, n = 67). Embryo development was monitored by TLM until day 6. MAIN OUTCOME MEASURE(S): Time to first breach the ZP, hatching time, time to complete hatching, multiple breaching, multiple hatching, loss of cells, hole size, and embryo quality were analyzed. RESULT(S): No significant differences in the proportion of completely hatched embryos were found among groups. However, the time (average hours ± SD) to complete hatching was significantly delayed in the control group compared with all laser-treated groups: 118.3 ± 9.5 hours in the ZT25 group, 116.6 ± 8.7 hours in the ZT35 group, and 120.4 ± 9.9 hours in the ZO group. The applied laser techniques did not interfere with the quality of the blastocysts at day 5/6 of culture. CONCLUSION(S): ZO, ZT25, and ZT35 embryos hatched significantly earlier than the zona intact group without increasing the multiple hatching rates, suggesting an improvement of the hatching process. This study found that the pattern of the hatching process after ZT and ZO differs.

Maternal spindle transfer overcomes embryo developmental arrest caused by ooplasmic defects in mice.

The developmental potential of early embryos is mainly dictated by the quality of the oocyte. Here, we explore the utility of the maternal spindle transfer (MST) technique as a reproductive approach to enhance oocyte developmental competence. Our proof-of-concept experiments show that replacement of the entire cytoplasm of oocytes from a sensitive mouse strain overcomes massive embryo developmental arrest characteristic of non-manipulated oocytes. Genetic analysis confirmed minimal carryover of mtDNA following MST. Resulting mice showed low heteroplasmy levels in multiple organs at adult age, normal histology and fertility. Mice were followed for five generations (F5), revealing that heteroplasmy was reduced in F2 mice and was undetectable in the subsequent generations. This pre-clinical model demonstrates the high efficiency and potential of the MST technique, not only to prevent the transmission of mtDNA mutations, but also as a new potential treatment for patients with certain forms of infertility refractory to current clinical strategies.

Infertility is a growing problem that affects millions of people worldwide. Medical procedures known as in vitro fertilization (IVF) help many individuals experiencing infertility to have children. Typically in IVF, a woman's egg cells are collected, fertilized with sperm from a chosen male and grown for a few days in a laboratory, before returning them to the woman's body to continue to develop. However, there are some women whose egg cells cannot develop into a healthy baby after they have been fertilized. Many of these patients use egg cells from donors, instead. This greatly improves the chances of the IVF treatment being successful, but the resultant children are not genetically related to the intended mothers. Previous studies suggested that a cell compartment known as the cytoplasm plays a crucial role in allowing fertilized egg cells to develop normally. A new technique known as maternal spindle transfer, often shortened to MST, makes it possible to replace the entire cytoplasm of a compromised egg cell. This is achieved by transplanting the genetic material of the compromised egg cell into a donor egg cell with healthier cytoplasm that has previously had its own genetic material removed. Using this technique, it is possible to generate human egg cells for IVF that have the genetic material from the intended mother without the defects in the cytoplasm that may be responsible for infertility. However, it is not clear whether this approach would be a safe and effective way to treat infertility in humans. Costa-Borges et al. applied MST to infertile female mice and found that the technique could permanently correct deficiencies in the cytoplasms of poor quality egg cells, allowing the mice to give birth to healthy offspring. Further experiments studied the offspring and their descendants over several generations and found that they also had higher quality egg cells and normal levels of fertility. These findings open up the possibility of developing new treatments for infertility caused by problems with egg cells, so experiments involving human egg cells are now being performed to evaluate the safety and effectiveness of the technique.

Parameters of the Mouse Embryo Assay that affect detection of peroxides in mineral oil.

RESEARCH QUESTION: Can culture conditions influence the sensitivity of a Mouse Embryo Assay and its potential to detect peroxide-related toxicity in mineral oil samples? DESIGN: Protein type and concentration, embryo density and culture dish design were selected as the variables in the culture system with the potential to influence the assay's sensitivity. Fresh 1-cell mouse embryos were cultured under mineral oil samples with known peroxide concentrations. Protein type (human serum albumin [HSA] + α/ß-Globulins versus HSA versus bovine serum albumin [BSA]), concentration (5 mg/ml versus 0.5 mg/ml), embryo density (25 versus 3 µl/embryo) and culture dish (Petri versus micro-well dish) were adjusted to define the culture conditions with the highest sensitivity. RESULTS: High concentrations of peroxides can be easily detected by current quality control standards. However, for oil samples with a lower concentration of peroxides, supplementing the culture medium with 5 mg/ml of HSA + alpha/beta-globulins or with HSA resulted in an increased detection of embryo toxicity compared with when BSA was used as the protein supplement. The sensitivity of the assay was greatly reduced when embryos were cultured in groups and when certain micro-well dishes were used. CONCLUSIONS: Current quality control protocols may not be sensitive enough to identify low concentrations of peroxides, which, if undetected, can increase over time and become potentially harmful during gamete and embryo culture. The different parameters established in this study allow the sensitivity of the Mouse Embryo Assays to be optimized to specifically detect peroxides in mineral oil samples prior to their release into the market and their broad use in human IVF.

Pre-clinical validation of a closed surface system (Cryotop SC) for the vitrification of oocytes and embryos in the mouse model.

Vitrification is currently a well-established technique for the cryopreservation of oocytes and embryos. It can be achieved either by direct (open systems) or indirect (closed systems) contact with liquid nitrogen. While there is not a direct evidence of disease transmission by transferred cryopreserved embryos, it was experimentally demonstrated that cross-contamination between liquid nitrogen and embryos may occur, and thus, the use of closed devices has been recommended to avoid the risk of contamination. Unfortunately, closed systems may result in lower cooling rates compared to open systems, due to the thermal insulation of the samples, which may cause ice crystal formation resulting in impaired results. In our study, we aimed to validate a newly developed vitrification device (Cryotop SC) that has been specifically designed for being used as a closed system. The cooling and warming rates calculated for the closed system were 5.254 °C/min and 43.522 °C/min, respectively. Results obtained with the closed system were equivalent to those with the classic Cryotop (open system), with survival rates in oocytes close to 100%. Similarly, the potential of the survived oocytes to develop up to good quality blastocysts after parthenogenetic activation between both groups was statistically equivalent. Assessment of the meiotic spindle and chromosome distribution by fluorescence microscopy in vitrified oocytes showed alike morphologies between the open and closed system. No differences were found either between the both systems in terms of survival rates of one-cell stage embryos or blastocysts, as well as, in the potential of the vitrified/warmed blastocysts to develop to full-term after transferred to surrogate females.

Is the severe pain after Milligan-Morgan hemorrhoidectomy still currently remaining a major postoperative problem despite being one of the oldest surgical techniques described? A case series of 117 consecutive patients.

INTRODUCTION: Surgery is the only curative method of hemorrhoidal disease. Currently the Milligan-Morgan hemorrhoidectomy is still considered the "gold standard", since it is the best performing technique. However, postoperative pain remains a major problem. We analize the postoperative analgesic requirements for this procedure in 117 patients. PRESENTATION OF CASES: Between 2012 and 2013, 117 consecutive patients undergoing Milligan-Morgan hemorroidectomy, with an analysis of sex, age, total vascular anal cushions removed, hospital stay, complications, and relation with postoperative analgesic requirements. Patients with documented allergy to NSAIDs or pyrazolones were excluded. Additionally 23 patients undergoing Milligan-Morgan hemorrhoidectomy associated to internal lateral sphincterotomy were also analyzed. RESULTS: The mean age of patients was 51.7 years. The 50.8% were women and 49.2% men. In 33.3% of cases, one vascular anal cushion was removed, 2 in 39.3%, and 3 in 27.4%. The average stay for the 3 groups was 2.0days. An analgesic dose average of 4.1 by day was given, with opioid requirements in 22.2% of cases. It was statistically significant that as more anal cushions were eliminated was higher the opioids need. No significant difference of opioids use was found regarding patients undergoing sphincterotomy as additional procedure. DISCUSSION: Postoperative pain after a Milligan-Morgan hemorrhoidectomy currently remains a problem for colorectal surgery teams. This involves the use of opioids comparable to other major surgeries, finally causing not negligible days of admission charge. A protocolized analgesic treatment, as we actually do in our center, should be implemented after a Milligan-Morgan hemorrhoidectomy for improving the postoperative period pain management.

Perforated Meckel's diverticulum containing a carcinoid tumor successfully treated by the laparoscopic approach: Case report.

Mekel's diverticulum is a gastrointestinal malformation. Occurs in one of every 40 patients. It is usually asymptomatic whereas complications can be developed in 2% to 4%. The report is based on a 41-year old male, who attended to emergency, complaining of right lower quadrant abdominal pain. Blood tests showed high level of inflammatory markers. With acute appendicitis as presumptive diagnosis, laparoscopy was performed. The intraoperative findings were: a perforated Mekel's diverticulum with normal cecal appendix. Mechanical diverticular resection was made. The patient was successfully recovered from surgery. Histopathology examination showed: Meckel's diverticulum perforated with acute inflammation and neuroendocrine tumor (G1) pT1. Mekel's diverticulum is rarely affected by inflammatory complications and just few cases are associated with tumors. However, has ever been described before, coexisting both situations, being our patient the first reported with this exceptional clinical presentation, and treated successfully by laparoscopic approach.

Endometriosis causing acute appendicitis complicated with hemoperitoneum.

Endometriosis is a painful disorder characterized by endometrial tissue outside the uterine cavity. It usually affects the pelvis, but in rare cases it might extend to other parts of the body. The report is based on a case of a 39-year-old woman, who presented symptoms of acute appendicitis and diagnosis confirmed with ultrasonography and a computed tomography scan procedures. Laparoscopic appendicectomy was performed. After entering the abdominal cavity, hemoperitoneum was discovered with no associated pelvic or abdominal lesions. Biopsy confirmed acute appendicitis with endometrial glands and stroma infiltrating its muscular tissue. Patient recovered without complications and was discharged 48 h after surgery. The appendix is rarely affected by endometrial infiltration. The literature describes an incidence of 0.8%, just a few cases have been associated with acute appendicitis; however, none of them are described to present hemoperitoneum, being our patient the first one reported with such complication.

Isquemia intestinal crónica por estenosis severa múltiple resuelta mediante cirugía endovascular en paciente de alto riesgo / Chronic intestinal ischemia due to multiple severe stenoses treated by endovascular surgery in a high risk patient

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