Xenopus chip for single-egg trapping, in vitro fertilization, development, and tadpole escape.

Xenopus laevis is highly suitable as a toxicology animal model owing to its advantages in embryogenesis research. For toxicological studies, a large number of embryos must be handled simultaneously because they very rapidly develop into the target stages within a short period of time. To efficiently handle the embryos, a convenient embryo housing device is essential for fast and reliable assessment and statistical evaluation of malformation caused by toxicants. Here, we suggest 3D fabrication of single-egg trapping devices in which Xenopus eggs are fertilized in vitro, and the embryos are cultured. We used manual pipetting to insert the Xenopus eggs inside the trapping sites of the chip. By introducing a liquid circulating system, we connected a sperm-mixed solution with the chip to induce in vitro fertilization of the eggs. After the eggs were fertilized, we observed embryo development involving the formation of egg cleavage, blastula, gastrula, and tadpole. After the tadpoles grew inside the chip, we saved their lives by enabling their escape from the chip through reverse flow of the culture medium. The Xenopus chip can serve as an incubator to induce fertilization and monitor normal and abnormal development of the Xenopus from egg to tadpole.

Office hysteroscopy before first in vitro fertilization. A randomized controlled trial.

BACKGROUND: Implantation failure remains a mystery since decades. This procedure needs a "top quality embryo" and a "normal" uterine cavity. To assess uterine cavity before first in vitro fertilization (IVF), many diagnostic tools could be used. Hysteroscopy remains the gold standard to diagnose and treat intra-uterine anomalies. However, it is not clearly recommanded to offer an office hysteroscopy before first IVF when transvaginal ultrasound (TVUS) and hysterosalpingography (HSG) were normal. PURPOSE: This study aimed to assess the role of office hysteroscopy before first IVF when no intra-uterine anomalies are suspected. BASIC PROCEDURES: We conducted a randomized controlled trial including 171 women scheduled for their first IVF. Women were assigned to either Group I: office hysteroscopy before IVF or Group II: immediate IVF. We included women aged less than 40 years, having regular cycles, FSH levels less than10UI/l, antral follicular count ≥12, normal TVUS and HSG. Their body mass index (BMI) ranged from 19 to 30 kg/m2. We excluded patients known having severe endometriosis, polycystic ovarian syndrome (PCOS) and oocyte receivers. The primary outcome were livebirth rate and clinical pregnancy rate. MAIN FUNDINGS: Between january 2016 and september 2017, we randomly assigned 171 women to either Group I (n = 84) or Group II (n = 87). Hysteroscopy was done in the mid-follicular phase immediately before IVF. Baseline characteristics and IVF features were comparable between groups except for the IVF protocol. Live birth rate was 23,9% in Group I versus 19,3% in Group II. (p = 0,607). Clinical Pregnancy rate was 32,4% in Group I versus 21,7% in Group II. (p = 0,326). No statistical significance was observed for neither miscarriage rate nor multiple pregnancy rate. Hysteroscopy showed 30% unsuspected intra-uterine anomalies: 11 intra-uterine adhesions, 7 polyps, 7 clinical endometritis and one fibroid print. Therapeutic hysteroscopy was done only for 6 intra-uterine adhesions and 3 polyps. Other anomalies did not require operative hysteroscopy. Visual analog score during hysteroscopy was 4,69 +/-2,892. 5 women (6%) of Group I experienced discomfort during diagnostic hysteroscopy. Only one patient had vagal syncope. No further complications were observed. PRINCIPAL CONCLUSIONS: Office hysteroscopy before first IVF seems not improve IVF results. Minimal intra-uterine anomalies not diagnosed by transvaginal ultrasound and hysterosalpingography do not seem to reduce IVF results.

Advances of microfluidic technology in reproductive biology.

According to World Health Organization (WHO) reports about 70 million couples suffer from infertility all over the world. A lot of research groups are working on this issue and have made therapeutic approaches by integrating biology, medicine, genetics, chemistry, psychology, mechanic, and many other branches of science. However, these methods have their own pros and cons. Assisted Reproductive Technologies (ART) has appeared to solve infertility problems. In Vitro Fertilization (IVF), Intracytoplasmic Sperm Injection (ICSI), Intrauterine Insemination (IUI) are the most common and conventional technologies in this regard. There are at least two characteristics of microfluidics, mechanical and biochemical, which can be influential in the field of mammalian gamete and preimplantation embryo biology. These microfluidic characteristics can assist in basic biological studies on sperm, oocyte and preimplantation embryo structure, function and environment. Using microfluidics in sorting sperm, conducting different steps of oocyte selection and preparation, and transferring embryo by passing sub-microliter fluid through microchannels results in low cost and short time. The size and shape of microchannels and the volume of used fluid differs from non-human cells to human cells. The most progressions have been seen in animal models. Results suggest that microfluidic systems will lead to improved efficiencies in assisted reproduction.

Técnicas de Reproducción Humana asistida de alta complejidad / Human Reproduction Techniques high complexity assisted

INTRODUCCIÓN: La infertilidad es definida por la Organización Mundial de la Salud (OMS) como la patología del sistema reproductor caracterizada por la incapacidad de lograr un embarazo clínico después de 12 meses o más de relaciones sexuales no protegidas.[1] Las técnicas de alta complejidad son aquellos procedimientos que incluyen la manipulación de gametos, o embriones humanos para generar el embarazo. Entre las técnicas incluidas se encuentra la fertilización in-vitro (FIV) y la inyección intra-citoplasmática de espermatozoides (ICSI), transferencia de embriones y criopreservación de los mismos, la donación de ovocitos y embriones y el tratamiento de útero subrogado. [1,2]. En el año 2015 se reglamentó el acceso a las técnicas de reproducción humana asistida de alta complejidad en el Uruguay. La Ley Nº 19.167/013, los Decretos Nº 84/015 y N° 46/017, reglamentaron la financiación de las mencionadas técnicas.[3] Para el acceso a la cobertura las pacientes deberán tener una edad mayor de 18 y menor de 40, y deben cumplir con los criterios establecidos en la normativa de cobertura financiera.[2] El 17 de Agosto del 2020, mediante Decreto del Poder Ejecutivo se modifica la accesibilidad a las técnicas de Reproducción Humana Asistida, eliminado el incremento de los copagos según número de intentos, permitiendo un mayor alcance de la población objetivo.[4] Son tres las clínicas de fertilidad habilitadas en 2015 por el Ministerio de Salud Pública, a realizar procedimientos financiados por el Fondo Nacional de Recursos; Clínica Suizo Americana (CSA), Centro de Esterilidad Montevideo (CEM), y Centro de Reproducción Humana ( CERHIN). OBJETIVOS: Conocer el resultado de las técnicas de reproducción humana asistida de alta complejidad financiadas por el Fondo Nacional de Recursos desde el inicio de la cobertura. METODOLOGÍA: El presente es un estudio observacional retrospectivo, de una cohorte de pacientes usuarias de los procedimientos de RHA, basados en registros secundarios del Fondo Nacional de Recursos y Ministerio de Salud. De acuerdo a los objetivos específicos planteados se consideraron dos puntos de corte diferentes: para el 1er objetivo se tomaron aquellas mujeres incluidas en la cobertura financiera de las técnicas de reproducción humana asistida, desde el inicio de la cobertura en abril de 2015 hasta el 31 de diciembre de 2019. Para el análisis del segundo objetivo se evaluaron los nacidos vivos producto de las técnicas de reproducción humana asistida realizadas hasta el 31 de marzo de 2019 (Nacidos Vivos hasta diciembre de 2019). RESULTADOS: Caracterizar a las pacientes que hicieron uso del programa de RHA desde el inicio de cobertura hasta diciembre de 2019. La caracterización será en función de la edad, clínica de fertilidad, departamento de procedencia y tipo de prestador. CONCLUSIONES: Hasta diciembre de 2019 un total de 3.264 mujeres hicieron uso de las prestaciones financiadas por el Fondo Nacional de Recursos de técnicas de Reproducción Humana Asistida de alta complejidad, atribuyéndose a marzo de 2019 como resultado un total de 1.008 Nacidos Vivos producto de las técnicas financiadas (nacidos vivos hasta diciembre 2019). El corrimiento de la edad de inicio de la maternidad, ha hecho a las técnicas de Reproducción Asistida una opción en el tratamiento de la infertilidad de las parejas. Año a año, se incluyen aproximadamente 700 mujeres a la cobertura por el FNR, la actual disminución de los copagos y la extensión de edad de maternidad proyectan un número de mujeres a financiar creciente. La edad media de presentación ha variado en función del marco normativo, presentado al 2019 un promedio de 36,19 años (el más bajo desde el inicio de la cobertura), la modificación en la edad de presentación seguramente influya en resultados futuros tanto para gestaciones como para Nacidos Vivos. La evolución temporal muestra anualmente una tendencia creciente en la elección de una de las clínicas, observándose la siguiente distribución entre las clínicas con los siguientes porcentajes de distribución: CEM (año 2015 el 64.4%, en 2019 80,4%), CSA (año 2015 22,7%, en 2019 14.5%), Cerhin (año 2015 12.9%, en 2019 5.1%). La mayoría de las transferencias embrionarias evaluadas pertenecieron a transferencias en fresco (60.7%), superando el número de transferencias con embriones criopreservados. Los resultados mostraron una mayor tasa de parto en transferencias de criopreservados respecto a transferencias en fresco, en todos los grupos etarios e independiente de las clínicas de RHA esto debido a las mejores condiciones del endometrio al momento de la implantación. La tasa de parto en relación a la transferencia cae con el aumento de la edad materna, siendo el grupo menor a 35 años el que presentó mejores resultados. En los grupos etarios analizados Uruguay se presenta con una Tasa de parto por transferencia global mejor a la reportada por el resto de los países (según el registro de Red Lara). Respecto a las clínicas de Reproducción Asistida en las categorías analizadas, una de las clínicas presentó mejores tasas de parto por transferencias independiente de la edad materna con valores puntuales favorables en todos los rangos etarios analizados. Los resultados de Ovodonación obtenidos son similares a los obtenidos para ovocitos propios, siendo imputados el 22% de las transferencias realizadas y el 21% del total de los nacidos vivos adjudicables a las técnicas de reproducción Asistida. En un total de 1.008 nacimientos, se presentó una proporción de embarazos múltiples de 20,1% con variabilidad entre clínicas (19,3%-26,2%).

Embryo location in the uterus during embryo transfer: An in vitro simulation.

OBJECTIVE: To evaluate the location of transferred embryos under various parameters during embryo transfer in in vitro fertilization (IVF) by applying an in vitro experimental model for embryo transfer (ET). METHODS: Mock ET simulations were conducted with a laboratory model of the uterine cavity. The transfer catheter was loaded with a sequence of air and liquid volumes, including development-arrested embryos donated by patients. The transfer procedure was recorded using a digital video camera. An orthogonal design, including three independent variables (uterine orientation, distance of the catheter tip to the fundus, and injection speed) and one dependent variable (final embryo position), was applied. RESULTS: The uterine cavity was divided into six regions. The distribution of the transferred matter within the uterine cavity varied according to the uterine orientation. Medium speed-injected embryos were mostly found in the static region while fast- and slow-speed injected embryos were mostly found in the fundal region and the cervical-left region, respectively. The possibility of embryo separation from the air bubble increased from 11.1% in slow injection cases to 29.6% and 48.1% in medium and fast injection cases, respectively. CONCLUSION: The experimental model provides a new method for investigating ET procedures. Fast injection of embryos into a retroverted uterus may be more likely to result in embryo separation from the air bubble.

[The Embryo Toxicity Research of Manufacturing Water of Medical Device of in Vitro Fertilization Technology].

Water is an important component in liquid medical device products for human assisted reproductive technology. Water traits, conductivity, microbial limits, total organic carbon, easy oxides, heavy metal content, bacterial endotoxin and other indicators have an important impact on sperm, egg and embryo development in vitro, so for such products, the quality of water control is extremely important. The production water for producing such products is generally prepared by MilliQ purification system. In this research, we used four different types of water to fabricate the IVF liquids. It included deionized reverse osmosis water, ultra purified water and ultra purified water without endotoxin or nucleic acid, and compared with tap water. The in vitro rat embryo test system was used to study the embryotoxicity of this four different culture liquid production waters. From the result, the group of the super purified water without endotoxin and nucleic acid has the best result of the embryo formation rate, the number of total cell number and the inner cell number. This study proved the importance of removing endotoxin and nucleic acid from the water used for the preparation of the liquid products for assisted reproduction, and provided the basis for the selection of water quality for the liquid products for assisted reproduction.

Time-lapse technology for embryo culture and selection.

Culturing of human embryos in optimal conditions is crucial for a successful in vitro fertilisation (IVF) programme. In addition, the capacity to assess and rank embryos correctly for quality will allow for transfer of the potentially 'best' embryo first, thereby shortening the time to pregnancy, although not improving cumulative pregnancy and live birth rates. It will also encourage and facilitate the implementation of single embryo transfers, thereby increasing safety for mother and offspring. Time-lapse technology introduces the concept of stable culture conditions, in connection with the possibility of continuous viewing and documenting of the embryo throughout development. However, so far, even when embryo quality scoring is based on large datasets, or when using the time-lapse technology, the morphokinetic scores are still mainly based on subjective and intermittent annotations of morphology and timings. Also, the construction of powerful algorithms for widespread use is hampered by large variations in culture conditions between individual IVF laboratories. New methodology, involving machine learning, where every image from the time-lapse documentation is analysed by a computer programme, looking for patterns that link to outcome, may in the future provide a more accurate and non-biased embryo selection.

The Embryo Toxicity Research of Manufacturing Water of Medical Device of in Vitro Fertilization Technology / 中国医疗器械杂志

Water is an important component in liquid medical device products for human assisted reproductive technology. Water traits, conductivity, microbial limits, total organic carbon, easy oxides, heavy metal content, bacterial endotoxin and other indicators have an important impact on sperm, egg and embryo development in vitro, so for such products, the quality of water control is extremely important. The production water for producing such products is generally prepared by MilliQ purification system. In this research, we used four different types of water to fabricate the IVF liquids. It included deionized reverse osmosis water, ultra purified water and ultra purified water without endotoxin or nucleic acid, and compared with tap water. The in vitro rat embryo test system was used to study the embryotoxicity of this four different culture liquid production waters. From the result, the group of the super purified water without endotoxin and nucleic acid has the best result of the embryo formation rate, the number of total cell number and the inner cell number. This study proved the importance of removing endotoxin and nucleic acid from the water used for the preparation of the liquid products for assisted reproduction, and provided the basis for the selection of water quality for the liquid products for assisted reproduction.

The Influence Of Intrauterine Pressure On Embryo Retention In A Catheter After Embryo Transfer.

The retention of the embryo in the transfer catheter after embryo transfer (ET) during in vitro fertilization is a very common phenomenon, encountered by even the most experienced operators, and embryos retained in the transfer catheter or its sleeve require a repeat transfer. The exact mechanism of embryo retention has not been explained. Therefore, the present study aimed to investigate the mechanism of embryo retention in the catheter during embryo transfer by using a transparent uterus model equipped with pressure sensors and a video recorder. The results indicate that pressure changes in the uterine cavity during ET can influence the distribution of the transferred fluid containing the embryo. Under certain conditions, the transferred fluid can flow backward in the catheter, which may lead to retention of the embryo in the catheter.

From laparotomy to laparoscopy to in vitro fertilization.

Before the modern era of in vitro fertilization, reproductive surgery to deal with pelvic disease was the key intervention in the management of infertility. A series of clinical observations and animal experiments led to the development of microsurgical principles, which were applicable to all forms of gynecologic surgery. The evolution of endoscopy permitted minimally invasive approaches to most pelvic pathology. Assisted reproductive techniques now have primacy in the management of infertility, but women deserve to have fertility-enhancing or fertility-sparing surgery performed by a surgeon with relevant training. Thus, we have an obligation to maintain formal training programs in reproductive surgery.

Influence of human embryo cultivation in a classic CO2 incubator with 20% oxygen versus benchtop incubator with 5% oxygen on live births: the randomized prospective trial.

SummaryOur objective was to assess the effect of benchtop incubators with low oxygen concentrations on the clinical and embryological parameters of our patients. We conducted a prospective, randomized, opened controlled trial on infertile patients in stimulated cycles. In total, 738 infertile patients were assessed for eligibility and, after final exclusions, 230 patients were allocated either to a 5% O2 group (benchtop incubator) or a 20% O2 group (classic incubator). Finally, 198 patients in the 5% O2 group and 195 in the 20% O2 group were analysed. The outcomes measured were fertilization rate, clinical pregnancy rate, and live birth rate. The primary outcome - live birth rate per all transfers - did not show any improvement in the 5% oxygen group over the 20% oxygen group (25.3% versus 22.6%, P=0.531), but the number of day 5 blastocysts was significantly higher (P=0.009). Fertilization rate did not show any beneficial effect of reduced oxygen (5%) (73.4%±22.4% versus 74.6%±24.0%, P=0.606) per all transfers but there was statistically significant difference in the day 5 SET subgroup (85.3±15.1 versus 75.1±17.5; P=0.004). Clinical pregnancy rate showed results in favour of the 5% oxygen group for all subgroups (day 3: 23.7% versus 21.1%, P=0.701; day 5 SET: 35.0% versus 30.6%. P=0.569) but showed statistical significance only in the day 5 SET subgroup (51.1% versus 29.8%; P=0.038). Culturing of embryos in benchtop incubators under low oxygen produced more blastocysts and therefore was a better alternative for embryo selection, which resulted in higher pregnancy rates. To achieve higher live birth rates, embryo quality is not the only factor.

Comparison of 36 assisted reproduction laboratories monitoring environmental conditions and instrument parameters using the same quality-control application.

RESEARCH QUESTION: Assisted reproduction laboratories record instrument performance periodically. No standardized guidelines have been produced for this activity despite mandatory auditing systems in several countries. This study of 36 laboratories in 12 different countries was conducted to assess differences and similarities between quality assurance programmes using an adaptable cloud-based quality-control app for instrument monitoring. DESIGN: A total of 36 deidentified IVF laboratories that subscribed to the same quality-assurance application were studied. Data were evaluated based on instrument types allocated to 10 domains: incubators, gas tanks, warming surfaces, refrigerators and freezers, cryo-storage, environment, water purification, peripheral equipment, checklists and miscellaneous. RESULTS: The incubator domain constituted the greatest proportion of parameters (35%), followed by surface warming instruments at 15%. Most incubator O2 readings were monitored between 4.5 and 5.5%, and between 5.5 and 6.5% for CO2. The altitude of the laboratory was poorly correlated with the CO2 setting. Incubator display and measured values of gases and temperature by built-in sensors vary considerably compared with third-party sensors. A quality-control diligence score or mean average data points was calculated for each laboratory. This score is independent of number of instruments or laboratory size. Higher scores were associated with laboratories in countries with government regulations and mandatory auditing systems. CONCLUSIONS: Major differences exist in instrument monitoring practices among laboratories. Although incubator monitoring is the largest domain, many other sensitive instruments are diligently monitored by most laboratories. International standardization and guidelines are needed.

IVF-on-a-Chip: Recent Advances in Microfluidics Technology for In Vitro Fertilization.

In vitro fertilization (IVF) has been one of the most exciting modern medical technologies. It has transformed the landscape of human infertility treatment. However, current IVF procedures still provide limited accessibility and affordability to most infertile couples because of the multiple cumbersome processes and heavy dependence on technically skilled personnel. Microfluidics technology offers unique opportunities to automate IVF procedures, reduce stress imposed upon gametes and embryos, and minimize the operator-to-operator variability. This article describes the rapidly evolving state of the application of microfluidics technology in the field of IVF, summarizes the diverse angles of how microfluidics has been complementing or transforming current IVF protocols, and discusses the challenges that motivate continued innovation in this field.

Effect of Surgical Adhesive on the Uterus of Rabbits Following Occlusion.

Purpose/Aim of the Study: The present study investigated the effect of surgical adhesives on the uterus of rabbits and the histomorphology alterations following occlusion, to improve the clinical treatment of abnormal fallopian tube with surgical adhesives for in vitro fertilization and embryo transfer (IVF-ET). Materials and Methods: The experimental rabbits received laparotomy and occlusion of the uterus by surgical adhesive adjacent to the two fallopian tubes, while the control rabbits only received laparotomy. The body weight, hysterosalpingography, and histomorphology were measured to evaluate the uterine occlusion at 1 and 6 months after surgery. Results: There was no significant difference in the mortality rate or body weight between the experimental and control groups. In the experimental group, 38 uterine cavities were identified in 19 rabbits, of which 97.37% were occluded, with expanded uterine cavity and tissue oppression at 1 month after surgery. In total, 33 uterine cavities out of the 36 in the control group were occluded, with proliferation of new stratified epithelial cells observed at 6 months after surgery. In the control group, 20 uterine cavities of 10 rabbits were observed to be absent of occlusion at 1 month after surgery, while 18 uterine cavities in the remaining 9 rabbits were also absent of occlusion at 6 months after the surgery. Conclusion: Surgical adhesives are effective in occluding the uterus of rabbits without adverse effects, supporting their potential clinical use to treat the occlusion in abnormal fallopian tubes prior to IVF-ET.

Growth and Differentiation Factor-9 Supplementation Affects Viability and Morphology of Preantral Follicles in Equine Ovarian Fragments During Short-term in vitro Culture

Abstract This study aimed to evaluate different concentrations of growth and differentiation factor-9 (GDF-9) on the development and maintenance of equine preantral follicle morphology during short-term in vitro culture. Ovaries (n=5) from five mares were collected from a local slaughterhouse and transported to the laboratory, where nine fragments (5x5x1mm) were procured from each ovary. One fragment from each was immediately fixed and submitted for histological analysis (control group; D0). The other eight fragments were cultured in situ for two (D2) or six (D6) days in MEM+ or MEM+ supplemented with GDF-9 at different concentrations (i.e., 50, 100 and 200 ng/mL the GDF-9). After culturing with different concentrations of GDF-9 for 2 or 6 days, the fragments were processed for histological analysis. After two days of cultivation, we observed an increase in the percentage of developing follicles for 0 (MEM+), 50, 100 and 200 ng/mL GDF-9 compared to control (D0; P<0.05). When we evaluated all treatments that preserved follicular integrity, the GDF-9 concentration of 100 ng/mL presented results superior to those of the other cultures (P<0.05). While, at six days of culture, the concentration of 200 ng/mL of GDF-9 appeared to be more efficient in providing development compared to MEM+ (P<0.05). The percentage of morphologically intact follicles in the 6 days culture samples treated with 50 ng/mL of GDF-9 indicated that this concentration was effective in maintaining the integrity of the follicle (P<0.05). We conclude, therefore, that graduated GDF-9 addition to the medium ensure follicular development and is sufficient maintain the architecture.

An oviduct-on-a-chip provides an enhanced in vitro environment for zygote genome reprogramming.

Worldwide over 5 million children have been conceived using assisted reproductive technology, and research has concentrated on increasing the likelihood of ongoing pregnancy. However, studies using animal models have indicated undesirable effects of in vitro embryo culture on offspring development and health. In vivo, the oviduct hosts a period in which the early embryo undergoes complete reprogramming of its (epi)genome in preparation for the reacquisition of (epi)genetic marks. We designed an oviduct-on-a-chip platform to better investigate the mechanisms related to (epi)genetic reprogramming and the degree to which they differ between in vitro and in vivo embryos. The device supports more physiological (in vivo-like) zygote genetic reprogramming than conventional IVF. This approach will be instrumental in identifying and investigating factors critical to fertilization and pre-implantation development, which could improve the quality and (epi)genetic integrity of IVF zygotes with likely relevance for early embryonic and later fetal development.

Validation of the blood gas analyzer for pH measurements in IVF culture medium: Prevent suboptimal culture conditions.

Measurement of pH in IVF-media using the blood gas analyzer (BGA) requires validation, because IVF-media is outside the intended scope of the BGA. To determine whether the Siemens Rapidpoint 500 BGA is suitable for pH measurements in IVF-media this study will validate the BGA and assess its accuracy. In this method comparison study, the pH of over three hundred IVF-media samples was measured with the BGA and a pH electrode (Hanna pH checker). The precision of both the BGA and the pH electrode were excellent (coefficient variation <1.4%). However, the closeness of agreement between measured values of both devices were not equivalent to each other in the tested IVF-media, showing 15% to 85% accordance between devices. The pH measured with the blood gas analyzer was also significantly higher in the tested media, compared to that measured by the pH electrode. One of the tested media did not reach its target pH when it was measured with the BGA, even at 9% CO2. The results show that the validated blood gas analyzer produces excellent results in terms of precision but not in terms of accuracy. Inaccurate measurement may lead to misinterpretation of results and consequently to suboptimal culture conditions. Therefore, each laboratory is encouraged to perform a validation of their BGA.

Efficiency of different incubation systems for the in vitro production of bovine embryos.

SummaryThis study aimed to compare the efficiency of different incubation systems for in vitro embryo production in bovine. Oocytes/embryos were cultured in three incubators: conventional - CONV, mini bench - MINI and portable - PORT. After in vitro maturation (IVM), oocytes were verified for maturation rate. The remaining structures were submitted to in vitro fertilization and culture to verify cleavage (day 2) and blastocyst (day 7) rates. Reactive oxygen species (ROS) were evaluated in post-IVM oocytes and embryos (days 2 and 7) using arbitrary fluorescence units (AFUs). No significant difference (P>0.05) was observed for maturation rate. The CONV system (74.0%) produced the highest cleavage rate (P0.05) to MINI (65.0%). The same pattern and differences were observed for blastocyst rate: CONV (33.3%), MINI (32.3%) and PORT (21.9%). ROS levels were not different (P>0.05) in post-IVM oocytes: CONV (35.6±4.5), MINI (29.4±4.0) and PORT (35.6±4.5). For day-2 embryos, ROS levels were higher (P0.05) was observed in blastocysts. In conclusion, although it produced high ROS levels at day 2 of culture, the MINI system was as efficient as the CONV system for blastocyst production. This option may be an interesting and economical for the in vitro embryo industry.

[Method validation of a critical parameter in human embryos culture: culture media pH]. / Validation de la mesure d'un paramètre critique en culture d'embryons humains : le pH.

In vitro human embryos culture depends largely on the atmospheric conditions within the incubators of the laboratory. The pH of culture media, an indirect reflection of the CO2 content inside these incubators, is a critical parameter. Collaboration between the biochemistry and reproductive biology departments enabled the automated measurement of the pH in the culture medium on a blood gas analyzer. This method has been validated and evaluated. It is applicable in all laboratories whatever the medium and the conditions of culture. It allows strict monitoring of this parameter for the optimization of the culture conditions necessary to improve the results of in vitro fertilization attempts.

A new, simple, automatic vitrification device: preliminary results with murine and bovine oocytes and embryos.

PURPOSE: This paper reports the use of a novel automatic vitrification device (Sarah, Fertilesafe, Israel) for cryopreservation of oocytes and embryos. METHODS: Mice oocytes (n = 40) and embryos (8 cells, n = 35 and blastocysts, n = 165), bovine embryos (2PN, n = 35), and MII oocytes (n = 84) were vitrified using this automated device. A total of 42 (2 cells) mice embryos, 20 (2PN) bovine embryos, and 150 MII bovine oocytes were used as fresh controls and grown to blastocysts. Upon rewarming, all were assessed for viability, cleavage, blastocyst, and hatching rates. RESULTS: Ninety-five % (38/40) of the mice MII oocytes regained isotonic volumes and all (100%) the surviving were viable. Rewarmed 8-cell mice embryos had 95% (33/35) blastulation rate and 80% (28/35) hatched. Rewarmed mice blastocysts had 97% survival rate (160/165) and 81% (135/165) hatched. Fresh control mice embryos had 100% (42/42) blastulation and 73% (21/42) hatching rates. Bovine embryos' survival was 100% with 54% (19/35) cleavage and 9% (3/35) blastulation rate. Fresh control bovine embryos had 65% (13/20) cleavage and 20% (4/20) blastulation rate. Vitrified bovine oocytes had 100% survival (84/84), 73% (61/84) cleavage, and 7% (6/84) blastocysts' rates; fresh control had 83% (125/150) cleavage and 11% (17/150) blastocysts' rates. CONCLUSION: This novel automatic vitrification device is capable to produce high survival rates of oocytes and embryos. We anticipate that as the demand for vitrification of gametes, embryos, and reproductive tissues increases worldwide, the availability of an automated vitrification device will become indispensable for standardization, simplification, and reproducibility of the entire process.