Superovulation with an anti-inhibin monoclonal antibody improves the reproductive performance of rat strains by increasing the pregnancy rate and the litter size.

Rats are multiparous rodents that have been used extensively in research; however, the low reproductive performance of some rat strains hampers the broader use of rats as a biomedical model. In this study, the possibility of increasing the litter size after natural mating in rats through superovulation using an anti-inhibin monoclonal antibody (AIMA) was examined. In outbred Wistar rats, AIMA increased the number of ovulated oocytes by 1.3-fold. AIMA did not affect fertilization and subsequent embryonic development, resulting in a 1.4-fold increase in litter size and a high pregnancy rate (86%). In contrast, conventional superovulation by eCG/hCG administration decreased the pregnancy rate to 6-40% and did not increase the litter size. In inbred Brown Norway rats, AIMA increased the litter size by 1.2-fold, and the pregnancy rate increased more than twice (86% versus 38% in controls). AIMA also increased the litter size by 1.5-fold in inbred Tokai High Avoiders and Fischer 344 rats. AIMA increased the efficiency of offspring production by 1.5-, 2.7-, 1.4-, and 1.4-fold, respectively, in the four rat strains. Thus, AIMA may consistently improve the reproductive performance through natural mating in rats, which could promote the use of AIMA in biomedical research.

Effect of microgravity on mammalian embryo development evaluated at the International Space Station.

Mammalian embryos differentiate into the inner cell mass (ICM) and trophectoderm at the 8-16 cell stage. The ICM forms a single cluster that develops into a single fetus. However, the factors that determine differentiation and single cluster formation are unknown. Here we investigated whether embryos could develop normally without gravity. As the embryos cannot be handled by an untrained astronaut, a new device was developed for this purpose. Using this device, two-cell frozen mouse embryos launched to the International Space Station were thawed and cultured by the astronauts under microgravity for 4 days. The embryos cultured under microgravity conditions developed into blastocysts with normal cell numbers, ICM, trophectoderm, and gene expression profiles similar to those cultured under artificial-1 g control on the International Space Station and ground-1 g control, which clearly demonstrated that gravity had no significant effect on the blastocyst formation and initial differentiation of mammalian embryos.

Intracytoplasmic sperm injection induces transgenerational abnormalities in mice.

In vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are 2 major assisted reproductive techniques (ARTs) used widely to treat infertility. Recently, spermatogonial transplantation emerged as a new ART to restore fertility to young patients with cancer after cancer therapy. To examine the influence of germ cell manipulation on behavior of offspring, we produced F1 offspring by a combination of two ARTs, spermatogonial transplantation and ICSI. When these animals were compared with F1 offspring produced by ICSI using fresh wild-type sperm, not only spermatogonial transplantation-ICSI mice but also ICSI-only control mice exhibited behavioral abnormalities, which persisted in the F2 generation. Furthermore, although these F1 offspring appeared normal, F2 offspring produced by IVF using F1 sperm and wild-type oocytes showed various types of congenital abnormalities, including anophthalmia, hydrocephalus, and missing limbs. Therefore, ARTs can induce morphological and functional defects in mice, some of which become evident only after germline transmission.

Lessons from the "Humanization of Childbirth" Projects: Qualitative analysis of seven projects funded by the Japan International Cooperation Agency.

The "Humanization of Childbirth" Project is one of the various maternity care models that respect women and their newborn children. For more than a quarter of a century, the Japan International Cooperation Agency (JICA) has been implementing technical cooperation projects worldwide that place the humanization of childbirth at the center of the concept. By reviewing the project reports, the following 11 key processes were found for the formulation and implementation of future projects for the humanized maternity care: i) project-finding/exploration of unmet needs, ii) identification of local key persons, iii) organization of a project team and a back-up committee, iv) development of an action plan, v) sharing of concepts, vi) development of local leadership, vii) organization of infrastructure, viii) final evaluation and wrap-up seminar, ix) ensuring sustainability, x) development of younger generation experts, and xi) sustainable and autonomous action.

Association between the quality of care and continuous maternal and child health service utilisation in Angola: Longitudinal data analysis.

Background: Many low- and middle-income countries (LMICs) prioritise minimising maternal, neonatal, and infant mortality. To improve maternal and child health, various evidence-based interventions have been introduced. Quality of care is pertinent while strengthening service utilisations. Achieving optimal-quality care is often marred with difficulties, such as inadequate skills and knowledge of health workers, poor fidelity to protocols, and poor user acceptance. Angola is a LMIC facing these problems. This study aimed to demonstrate the influence of health facilities' quality of care at antenatal care (ANC) on subsequent maternal, newborn and child health (MNCH) service utilisation in Angolan pregnant women. Methods: Population-based cohort data from the Maternal and Child Health Handbook (MCH-HB) effectiveness study were analysed. The original study was conducted among women who became pregnant between March and April 2019 in Benguela Province, Angola. Socioeconomic and MNCH service utilisation indicators were collected through interviewer-administered structured questionnaires. The indicator of quality of care was a composite measure that assessed the implementation of the MCH-HB based on the RE-AIM framework, mostly consisted of common factors related to delivery and management of MNCH services. A multivariate logistic regression analysis was performed between quality of care, socioeconomic factors, and service utilisation indicators among the intervention group participants who had at least one ANC visit. Results: Of the 3351 pregnant women who visited ANC at least once, 2911 without missing values among explanatory or dependent variables were included in the analysis. Among them, 2032 (69.8%) were exposed to optimal-quality ANC, and 2058 (70.7%), 1573 (54.0%), and 941 (32.3%) achieved ANC target, facility delivery, and vaccination target for six-month-old infants, respectively. Exposure to suboptimal-quality care at ANC was associated with lower odds for facility delivery (adjusted odds ratio (AOR) = 0.60, 95% CI = 0.49-0.73) and the achievement of the vaccination target (AOR = 0.43, 95% CI = 0.33-0.55). A low socioeconomic status was inversely associated with health service utilisation indicators. Conclusions: Health facilities' quality of care influences subsequent MNCH service utilisation. Therefore, simultaneous efforts to improve quality of care and the mobilisation of pregnant women and communities are essential for enhancing maternal and child health.

Derivation of embryonic stem cells from wild-derived mouse strains by nuclear transfer using peripheral blood cells.

Wild-derived mouse strains have been extensively used in biomedical research because of the high level of inter-strain polymorphisms and phenotypic variations. However, they often show poor reproductive performance and are difficult to maintain by conventional in vitro fertilization and embryo transfer. In this study, we examined the technical feasibility of derivation of nuclear transfer embryonic stem cells (ntESCs) from wild-derived mouse strains for their safe genetic preservation. We used leukocytes collected from peripheral blood as nuclear donors without sacrificing them. We successfully established 24 ntESC lines from two wild-derived strains of CAST/Ei and CASP/1Nga (11 and 13 lines, respectively), both belonging to Mus musculus castaneus, a subspecies of laboratory mouse. Most (23/24) of these lines had normal karyotype, and all lines examined showed teratoma formation ability (4 lines) and pluripotent marker gene expression (8 lines). Two male lines examined (one from each strain) were proven to be competent to produce chimeric mice following injection into host embryos. By natural mating of these chimeric mice, the CAST/Ei male line was confirmed to have germline transmission ability. Our results demonstrate that inter-subspecific ntESCs derived from peripheral leukocytes could provide an alternative strategy for preserving invaluable genetic resources of wild-derived mouse strains.

Association between the continuum of care and postpartum depression among Angolan mothers.

BACKGROUND: This study investigated the association between maternal and child health service utilization patterns and postpartum depression (PPD). METHODS: This study analyzed a dataset of women who participated in a randomized controlled trial to examine the effectiveness of the Maternal and Child Health Handbook in Angola. We defined probable PPD as an Edinburgh Postpartum Depression Scale (EPDS) score ≥ 10. The EPDS was administered at approximately 6 months postpartum. Service utilization patterns were defined using numbers of antenatal care (ANC), facility delivery, and vaccination visits by 6 months postpartum. The association between service utilization patterns and PPD was examined using logistic regression analyses adjusting for socioeconomic factors and parity. The continuum of care (CoC) complete pattern (four ANC/facility delivery/four vaccination) was used as a reference. RESULTS: The data of 7087 participants whose children were alive and aged 6 months or older at the endline survey were analyzed. Prevalence of PPD was 17.9 % in urban and 43.2 % in rural municipalities. In urban municipalities, dropouts from the CoC at delivery and after delivery had significantly higher odds of PPD (AOR = 1.45, 95 % CI = 1.00-2.10; AOR = 1.57, 95 % CI = 1.24-1.99). In rural municipalities, dropouts from the CoC after delivery (AOR = 1.60, 95 % CI = 1.12-2.28) had significantly higher odds of PPD. LIMITATIONS: The onset of depressive symptoms was not assessed. The EPDS was validated in some Portuguese speaking countries but not in Angola. CONCLUSION: PPD was associated with irregular service utilization patterns such as dropouts from the CoC. Therefore, CoC and mental health must be promoted simultaneously.

Effectiveness of the maternal and child health handbook for improving continuum of care and other maternal and child health indicators: A cluster randomized controlled trial in Angola.

Background: The maternal and child health (MCH) handbook is promoted as a tool for strengthening continuum of care. We assessed the effect of a MCH handbook intervention package on continuum of maternal and child health care and health outcomes for mother and child. Methods: We conducted an open-label, parallel two-arm cluster randomized controlled trial in Angola. We randomly assigned municipalities in Benguela province through block randomization to a group using a package of enhanced maternity care service (which included the MCH handbook distribution and its supplementary intervention) and another using usual care (two stand-alone home-based records). We included women who were pregnant at the beginning of the trial period and attended a public health care facility for maternity care services. Neither health care providers, study participants nor data assessors were masked, but the statistician was. The primary outcome was a measure of service utilization assessed via achievement of maternal behavior-based continuum of care at three months postpartum. We conducted an intention-to-treat analysis in women with available data. Results: We randomized 10 municipalities to either the intervention (five clusters) or control (five clusters) group. Of the 11 530 women approached between June 8, 2019, and September 30, 2020, 11 006 were recruited and 9039 included in the final analysis (82%; 3774 in the intervention group and 5265 in the control group). The odds for achievement of maternal behavior-based continuum of care in the intervention group was not significantly different from that in the control group (adjusted odds ratio (aOR) = 1.18, 95% confidence interval (CI) = 0.46-2.93) at three months postpartum. However, the odds of initiating antenatal care clinic use were significantly higher in the intervention group (odds ratio (OR) = 5.16, 95% CI = 2.50-10.67). No harms associated with the intervention were reported. Conclusions: Distribution of the MCH handbook and its supplementary interventions promoted initiation of antenatal care service use, but did not increase service utilization sufficiently enough for attainment of study defined maternal behavior-based continuum of care. Registration: ISRCTN20510127.

Development and implementation of the maternal and child health Handbook in Angola.

Although the maternal and child health (MCH) Handbook is widely used in many countries, its development and implementation process has not been sufficiently documented in scientific publications. This is a report of how the Angola MCH Handbook was developed, what challenges we encountered during its implementation and how they were solved. Leading the process was the MCH Handbook Committee set up to develop the MCH Handbook and implement the programme in liaison with various stakeholders. We developed the MCH Handbook through participatory workshops with the objective of producing user-responsive content and designs, foster healthy interaction and build common understanding among stakeholders. After pilot use in select health facilities, the MCH Handbook programme, which included training, monitoring and supervision, mothers' class and community awareness raising activities, was gradually implemented in three model provinces. Core members of the committee closely observed each step of the programme to identify challenges in each field, and revised the tool and programme throughout the process. As nationwide implementation of the MCH Handbook Programme progresses, it is important to continually identify challenges specific to different localities while taking measures to address them. In our experience, stakeholder involvement from the early planning and preparation stages was critical to ensure their continued commitment at later stages and for programme continuity. Our approach of tool development involving various stakeholders and flexible implementation strategies were key elements for user acceptance and programme sustainability that may be applicable for introduction of similar interventions in other settings.

Development of a new device for manipulating frozen mouse 2-cell embryos on the International Space Station.

Whether mammalian embryos develop normally under microgravity remains to be determined. However, embryos are too small to be handled by inexperienced astronauts who orbit Earth on the International Space Station (ISS). Here we describe the development of a new device that allows astronauts to thaw and culture frozen mouse 2-cell embryos on the ISS without directly contacting the embryos. First, we developed several new devices using a hollow fiber tube that allows thawing embryo without practice and observations of embryonic development. The recovery rate of embryos was over 90%, and its developmental rate to the blastocyst were over 80%. However, the general vitrification method requires liquid nitrogen, which is not available on the ISS. Therefore, we developed another new device, Embryo Thawing and Culturing unit (ETC) employing a high osmolarity vitrification method, which preserves frozen embryos at -80°C for several months. Embryos flushed out of the ETC during thawing and washing were protected using a mesh sheet. Although the recovery rate of embryos after thawing were not high (24%-78%) and embryonic development in ETC could not be observed, thawed embryos formed blastocysts after 4 days of culture (29%-100%) without direct contact. Thus, this ETC could be used for untrained astronauts to thaw and culture frozen embryos on the ISS. In addition, this ETC will be an important advance in fields such as clinical infertility and animal biotechnology when recovery rate of embryos were improved nearly 100%.

The RE-AIM framework-based evaluation of the implementation of the Maternal and Child Health Handbook program in Angola: a mixed methods study.

BACKGROUND: The World Health Organization recommends the Maternal and Child Health Handbook (MCH-HB) to promote health service utilization from pregnancy to early childhood. Although many countries have adopted it as a national health policy, there is a paucity of research in MCH-HB's implementation. Thus, this study aimed to evaluate the MCH-HB's implementation status based on the RE-AIM framework (Reach, Effectiveness, Adoption, Implementation, Maintenance), and identify facilitators of, and barriers to its implementation in Angola to understand effective implementation strategies. METHODS: A cross-sectional survey was conducted targeting all health facilities which implemented MCH-HB, subsamples of health workers, and officers responsible for the MCH-HB at the municipality health office. Using the 14 indicators based on the RE-AIM framework, health facilities' overall implementation statuses were assessed. This categorized health facilities into optimal-implementation and suboptimal-implementation groups. To identify barriers to and facilitators of MCH-HB implementation, semi-structured interviews were conducted among health workers and municipality health officers responsible for MCH-HB. The data were analyzed via content analysis. RESULTS: A total of 88 health facilities and 216 health workers were surveyed to evaluate the implementation status, and 155 interviews were conducted among health workers to assess the barriers to and facilitators of the implementation. The overall implementation target was achieved in 50 health facilities (56.8%). The target was achieved by more health facilities in urban than rural areas (urban 68.4%, rural 53.6%) and by more health facilities of higher facility types (hospital 83.3%, health center 59.3%, health post 52.7%). Through the interview data's analysis, facilitators of and barriers to MCH-HB were comprehensively demonstrated. MCH-HB's content advantage was the most widely recognized facilitator and inadequate training for health workers was the most widely recognized barrier. CONCLUSIONS: Strengthening education for health workers, supervision by municipality health officers, and community sensitization were potential implementation strategies. These strategies must be intensified in rural and lower-level health facilities.

Use of anti-inhibin monoclonal antibody for increasing the litter size of mouse strains and its application to in vivo-genome editing technology†.

The litter size of mouse strains is determined by the number of oocytes naturally ovulated. Many attempts have been made to increase litter sizes by conventional superovulation regimens (e.g., using equine or human gonadotropins, eCG/hCG but had limited success because of unexpected decreases in the numbers of embryos surviving to term. Here, we examined whether rat-derived anti-inhibin monoclonal antibodies (AIMAs) could be used for this purpose. When C57BL/6 female mice were treated with an AIMA and mated, the number of healthy offspring per mouse increased by 1.4-fold (11.9 vs. 8.6 in controls). By contrast, treatment with eCG/hCG or anti-inhibin serum resulted in fewer offspring than in nontreated controls. The overall efficiency of production based on all females treated (including nonpregnant ones) was improved 2.4 times with AIMA compared with nontreated controls. The AIMA treatment was also effective in ICR mice, increasing the litter size from 15.3 to 21.2 pups. We then applied this technique to an in vivo genome-editing method (improved genome-editing via oviductal nucleic acid delivery, i-GONAD) to produce C57BL/6 mice deficient for tyrosinase. The mean litter size following i-GONAD increased from 4.8 to 7.3 after the AIMA treatment and genetic modifications were confirmed in 80/88 (91%) of the offspring. Thus, AIMA treatment is a promising method for increasing the litter size of mice and may be applied for the easy proliferation of mouse colonies as well as in vivo genetic manipulation, especially when the mouse strains are sensitive to handling.

Mouse in vivo-derived late 2-cell embryos have higher developmental competence after high osmolality vitrification and -80°C preservation than IVF or ICSI embryos.

Mammalian embryos are most commonly cryopreserved in liquid nitrogen; however, liquid nitrogen is not available in special environments, such as the International Space Station (ISS), and vitrified embryos must be stored at -80°C. Recently, the high osmolarity vitrification (HOV) method was developed to cryopreserve mouse 2-cell stage embryos at -80°C; however, the appropriate embryo is currently unknown. In this study, we compared the vitrification resistance of in vivo-derived, in vitro fertilization (IVF)-derived, and intracytoplasmic sperm injection (ICSI)-derived mouse 2-cell embryos against cryopreservation at -80°C. The ICSI embryos had lower survival rates after warming and significantly lower developmental rates than the in vivo and IVF embryos. Further, IVF embryos had a lower survival rate after warming, but a similar rate to the in vivo embryos to full-term development. This result was confirmed by simultaneous vitrification of in vivo and IVF embryos in the same cryotube using identifiable green fluorescent protein-expressing embryos. We also evaluated the collection timing of the in vivo embryos from the oviduct and found that late 2-cell embryos had higher survival and developmental rates to full-term than early 2-cell embryos. Some early 2-cell embryos remained in the S-phase, whereas most late 2-cell embryos were in the G2-phase, which may have affected the tolerance to embryo vitrification. In conclusion, when embryos must be cryopreserved under restricted conditions, such as the ISS, in vivo fertilized embryos collected at the late 2-cell stage without long culture should be employed.

Mouse resources at the RIKEN BioResource Research Center and the National BioResource Project core facility in Japan.

The RIKEN BioResource Research Center (BRC) was established in 2001 as a comprehensive biological resource center in Japan. The Experimental Animal Division, one of the BRC infrastructure divisions, has been designated as the core facility for mouse resources within the National BioResource Project (NBRP) by the Japanese government since FY2002. Our activities regarding the collection, preservation, quality control, and distribution of mouse resources have been supported by the research community, including evaluations and guidance on advancing social and research needs, as well as the operations and future direction of the BRC. Expenditure for collection, preservation, and quality-control operations of the BRC, as a national core facility, has been funded by the government, while distribution has been separately funded by users' reimbursement fees. We have collected over 9000 strains created mainly by Japanese scientists including Nobel laureates and researchers in cutting-edge fields and distributed mice to 7000 scientists with 1500 organizations in Japan and globally. Our users have published 1000 outstanding papers and a few dozen patents. The collected mouse resources are accessible via the RIKEN BRC website, with a revised version of the searchable online catalog. In addition, to enhance the visibility of useful strains, we have launched web corners designated as the "Mouse of the Month" and "Today's Tool and Model." Only high-demand strains are maintained in live colonies, while other strains are cryopreserved as embryos or sperm to achieve cost-effective management. Since 2007, the RIKEN BRC has built up a back-up facility in the RIKEN Harima branch to protect the deposited strains from disasters. Our mice have been distributed with high quality through the application of strict microbial and genetic quality control programs that cover a globally accepted pathogens list and mutated alleles generated by various methods. Added value features, such as information about users' publications, standardized phenotyping data, and genome sequences of the collected strains, are important to facilitate the use of our resources. We have added and disseminated such information in collaboration with the NBRP Information Center and the NBRP Genome Information Upgrading Program. The RIKEN BRC has participated in international mouse resource networks such as the International Mouse Strain Resource, International Mouse Phenotyping Consortium, and Asian Mouse Mutagenesis and Resource Association to facilitate the worldwide use of high-quality mouse resources, and as a consequence it contributes to reproducible life science studies and innovation around the globe.

The Maternal and Child Health Handbook for Improving the Continuum of Care and Other Maternal and Child Health Indicators in Angola: An Implementation Study Protocol.

Background: Reducing maternal, neonatal, and infant mortality tops the health targets of sustainable development goals. Many lifesaving interventions are being introduced in antenatal, delivery, and postnatal care. However, many low- and middle-income countries (LMICs) have not reached maternal and child health targets. The Maternal and Child Health Handbook (MCH-HB) is recommended as a home-based record to promote a continuum of care from pregnancy to early childhood, and is gaining increasing attention among LMICs. Several countries have adopted it as national health policy. To effectively utilize the MCH-HB in LMICs, implementation needs to be considered. Angola is an LIMC in Sub-Saharan Africa, where maternal and child health indicators are among the poorest. The Angolan Ministry of Health adopted the MCH-HB program in its national health policy and is currently conducting a cluster randomized controlled trial (MCH-HB RCT) to evaluate its impact on the continuum of care. This study aimed to evaluate implementation status, and barriers and facilitators of MCH-HB program implementation in Angola. Methods: To evaluate implementation status comprehensively, the RE-AIM (reach, effectiveness, adoption, implementation, and maintenance) framework will be used. Four components other than effectiveness will be investigated. A cross-sectional survey will be conducted targeting all health facilities and officers in charge of the MCH-HB at the municipality health office in the intervention group after the MCH-HB RCT. Data from the cross-sectional survey, secondary MCH-HB RCT data, and operational MCH-HB RCT records will be analyzed. Health facilities will be classified into good-implementation and poor-implementation groups using RE-AIM indicators. To identify barriers to and facilitators of MCH-HB implementation, semi-structured interviews/focus group discussions will be conducted among health workers at a sub-sample of health facilities and all municipality health officers in charge of MCH-HB in the intervention group. The Consolidated Framework for Implementation Research will be adopted to develop interview items. Thematic analysis will be performed. By comparing good-implementation and poor-implementation health facilities, factors that differ between groups that contribute to successful implementation can be identified. Discussion: This study's findings are expected to inform MCH-HB implementation policy and guidelines in Angola and in other countries that plan to adopt the MCH-HB program.

Easy and quick (EQ) sperm freezing method for urgent preservation of mouse strains.

Cryopreservation of mouse spermatozoa is widely used for the efficient preservation and safe transport of valuable mouse strains. However, the current cryopreservation method requires special containers (plastic straws), undefined chemicals (e.g., skim milk), liquid nitrogen, and expertise when handling sperm suspensions. Here, we report an easy and quick (EQ) sperm freezing method. The main procedure consists of only one step: dissecting a single cauda epididymis in a microtube containing 20% raffinose solution, which is then stored in a -80 °C freezer. The frozen-thawed spermatozoa retain practical fertilization rates after 1 (51%) or even 3 months (25%) with the C57BL/6 J strain, the most sensitive strain for sperm freezing. More than half of the embryos thus obtained developed into offspring after embryo transfer. Importantly, spermatozoa stored at -80 °C can be transferred into liquid nitrogen for indefinite storage. As far as we know, our EQ method is the easiest and quickest method for mouse sperm freezing and should be applicable in all laboratories without expertise in sperm cryopreservation. This technique can help avoid the loss of irreplaceable strains because of closure of animal rooms in emergency situations such as unexpected microbiological contamination or social emergencies such as the COVID-19 threat.

Supply-Side Barriers to the Use of Public Healthcare Facilities for Childhood Illness Care in Rural Zambia: A Cross-Sectional Study Linking Data from a Healthcare Facility Census to a Household Survey.

Child mortality due to malaria and diarrhea can be reduced if proper treatment is received timely at healthcare facilities, but various factors hinder this. The present study assessed the associations between the use of public healthcare facilities among febrile/diarrheal children in rural Zambia and supply-side factors (i.e., the distance from the village to the nearest facility and the availability of essential human resources and medical equipment at the facility). Data from the Demographic and Health Survey 2018 and the Health Facility Census 2017 were linked. Generalized linear mixed models were used to assess the associations, controlling for clustering and other variables. The median distances to the nearest facility were 4.5 km among 854 febrile children and 4.6 km among 813 diarrheal children. Children who were over 10 km away from the facility were significantly less likely to use it, compared to those within 5 km (fever group: odds ratio (OR) = 0.36, 95% confidence interval (CI) = 0.20-0.66; diarrhea group: OR = 0.30, 95% CI = 0.18-0.51). The availability of human resources and equipment was, however, not significantly associated with facility use. Poor geographic access could be a critical barrier to facility use among children in rural Zambia.

Equilibrium vitrification of mouse embryos using low concentrations of cryoprotectants.

Previously, we developed a method for vitrification of mouse embryos in a near-equilibrium state using EFS35c, PB1 medium containing 35% (v/v) ethylene glycol, and 0.98 M sucrose. This method has advantages in both slow freezing and vitrification. However, since the vitrification solution in this method contains high concentrations of cryoprotectants and thus has high osmolality, the solution would injure oocytes and embryos with high sensitivity to chemical toxicity and high osmolality. In this study, we examined whether embryos could be vitrified in a near-equilibrium state using a solution containing low concentrations of cryoprotectants and thus with low osmolality. To investigate whether embryos were vitrified in a near-equilibrium state, 2-cell mouse embryos were vitrified with EDFS10/10a, PB1 medium containing 10% (v/v) ethylene glycol, 10% (v/v) DMSO, and 0.4 M sucrose, in liquid nitrogen, stored at -80 °C for 4-28 days, and warmed in water at 25 °C. The viability of the embryos was evaluated by the appearance of embryos after warming and developmental ability. When embryos were vitrified in liquid nitrogen using EDFS10/10a, the survival and developmental ability into blastocysts after storage at -80 °C for 7 days were high, indicating that embryos were vitrified in a near-equilibrium state. A high proportion of embryos vitrified with EDFS10/10a developed to term after transportation with dry ice, re-cooling in liquid nitrogen, and transfer to recipients. Therefore, new equilibrium vitrification developed in this study may be useful for oocytes and embryos that are highly sensitive to the toxicity of cryoprotectants and high osmolality.

Development of assisted reproductive technologies for Mus spretus†.

The genus Mus consists of many species with high genetic diversity. However, only one species, Mus musculus (the laboratory mouse), is common in biomedical research. The unavailability of assisted reproductive technologies (ARTs) for other Mus species might be a major reason for their limited use in laboratories. Here, we devised ARTs for Mus spretus (the Algerian mouse), a commonly used wild-derived Mus species. We found that in vitro production of M. spretus embryos was difficult because of low efficacies of superovulation with equine chorionic gonadotropin or anti-inhibin serum (AIS) (5-8 oocytes per female) and a low fertilization rate following in vitro fertilization (IVF; 15.2%). The primary cause of this was the hardening of the zona pellucida but not the sperm's fertilizing ability, as revealed by reciprocal IVF with laboratory mice. The largest number of embryos (16 per female) were obtained when females were injected with AIS followed by human chorionic gonadotropin and estradiol injections 24 h later, and then by natural mating. These in vivo-derived 2-cell embryos could be vitrified/warmed with a high survival rate (94%) using an ethylene glycol-based solution. Importantly, more than 60% of such embryos developed into healthy offspring following interspecific embryo transfer into (C57BL/6 × C3H) F1 female mice. Thus, we have devised practical ARTs for Mus spretus mice, enabling efficient production of embryos and animals, with safe laboratory preservation of their strains. In addition, we have demonstrated that interspecific embryo transfer is possible in murine rodents.

Impact of the Maternal and Child Health handbook in Angola for improving continuum of care and other maternal and child health indicators: study protocol for a cluster randomised controlled trial.

BACKGROUND: The Maternal and Child Health (MCH) handbook is an integrated home-based record (HBR) designed to record in a single document all the information regarding health services provided to a pregnant woman and her child. The MCH handbook has the potential to support continuity of care which is key to strengthening maternal, newborn and child health. However, there is a lack of an integrated system to manage the health of pregnant women and young children on an ongoing basis in Angola. Thus, the Angolan Ministry of Health is partnering with the Japan International Cooperation Agency to build the capacity of healthcare providers through trainings and implementation of the MCH handbook to improve service utilisation. In this study, we will estimate the impact of an intervention package including distribution of MCH handbook and its supplementary interventions to women, on the utilisation of services provided at healthcare facilities from pregnancy through the postnatal and early childhood period. METHODS: This study is a cluster randomised controlled trial involving public healthcare facilities across all the municipalities located in Benguela Province, Angola. All women who go to participating healthcare facilities and with confirmed pregnancy around the beginning of the trial period will be included in the study. Women will be randomised according to the municipality where their primary maternity and/or childcare services are located. The intervention package will consist of MCH handbook distribution at all public healthcare facilities, MCH handbook utilisation training for healthcare providers and community mobilisation for women on the use of the MCH handbook. The intervention will be administered to all women in the intervention arm while those in the control arm will continue the traditional use of two stand-alone HBRs. The primary outcome measure for this study is to compare the proportion of women who achieve a complete continuum of care in both study arms. DISCUSSION: The findings from the study are expected to form a basis for revising the current trial version of the Angola MCH handbook and provide a framework for policy guiding nationwide scale-up and distribution of the MCH handbook. TRIAL REGISTRATION: ISRCTN Registry ISRCTN20510127 . Registered on 4 June 2019.