Abstract
Purpose
In our previous study, we confirmed that the supplementation of vitrified-warmed murine oocytes with autologous adipose stem cell (ASC)-derived mitochondria during intracytoplasmic sperm injection enhances post-fertilization developmental competence in mice. To ensure the safety of this technology, we conducted a thorough study in mice to investigate the potential presence of specific malformations in offspring developed from this approach.
Methods
A transgenerational comparative analysis was conducted on founder mice from embryos that developed after mitochondrial supplementation, and two subsequent generations. Reproductive performance, body growth rate, histopathological parameters, hematological parameters, daily activity patterns, and daily body temperature changes in male and female mice across these three generations were assessed in comparison to wild-type mice of the same age.
Results
Both male and female animals in all three generations showed comparable reproductive performance to the control group. Additionally, body growth rate by the age of 8 weeks were found to be comparable to controls across all three generations. Notably, no significant histopathological abnormalities were detected in vital organs, including the brain, heart, liver, kidneys, lungs, ovaries, and testes, in any individuals from the studied cohorts. The blood parameters were consistent with the control data. The continuous monitoring of activity and body temperature changes (both day and night) over a 1-week period revealed a pattern closely resembling that observed in the control animals.
Conclusion
Injection of ASC-mitochondria into oocytes may be a promising technique to support developmental potential without causing adverse epigenetic events in the offspring in mice. However, before considering clinical application, additional safety screening using larger animals or non-human primates is essential.
Supplementary Information
The online version contains supplementary material available at 10.1007/s10815-024-03137-2.
Keywords: Mitochondrial supplementation, Oocyte quality enhancement, Transgenerational safety, Adipose stem cell
Introduction
Due to the demands of modern lifestyles, couples often postpone their initial pregnancy, which may cause increased female infertility, with advanced female age and oocyte quality emerging as the predominant contributing factors. Other than maternal age, maternal obesity, maternal diabetes, environmental pollution, and interventions associated with assisted reproductive technology (ART) can also exert a negative impact on oocyte quality, leading to a reduction in female fertility [1–7]. Therefore, to preserve female fertility, the cryopreservation of germ cells, especially oocytes, is an essential technology in modern ART. However, the post-fertilization developmental potential of vitrified oocytes is known to be lower than that of fresh oocytes [7]. We previously showed that autologous adipose stem cell (ASC) mitochondrial energy transfer (ASCENT) into vitrified-warmed oocytes significantly improves embryonic adenosine triphosphate (ATP) levels and developmental potential [8]. However, the epigenetic changes caused by this kind of extra micromanipulation may leads to incompatible communication between the nuclear and mitochondrial genomes [9, 10].
Children born from cryopreserved fertilized ova are known to have higher birth weights than those born from ova without cryopreservation [11]. The increase in birth weight does not directly affect the health of the child, and cryopreservation continues to be used as an essential technique in the treatment of human infertility, with healthy children continuing to be born despite the apparent increase in birth weight. Furthermore, epigenetic changes, such as altered DNA methylation patterns in early embryos, can develop in response to various in vitro and in vivo environmental signals, resulting in adverse physiological or pathological outcomes in offspring [12, 13].
As with cryopreservation, maternal aging and obesity are risk factors that reduce the ability of oocytes to develop post-fertilization. This is mainly due to increased mitochondrial defects, and autologous mitochondrial supplementation of the oocytes has been proposed as an effective solution [14]. However, as with other ART techniques, autologous mitochondrial supplementation from adipose stem cells may also produce morbidity in the progeny. Therefore, to ascertain the potential for clinical application, the frequency and type of malformations that may result from autologous mitochondrial supplementation of oocytes should be confirmed through comprehensive preclinical studies using small, large animals and non-human primates.
In this study, we analyzed whether autologous mitochondrial supplementation of vitrified-warmed oocytes has adverse effects on subsequent generations, examining the safety of this strategy. We developed mitochondria-supplemented founder mice and crossed male founder mice and their female offspring with wild-type (WT) mice to produce two generations of progeny. We then performed a comparative analysis of reproductive performance, body growth rate, histopathological parameters, hematological parameters, daily activity patterns, and daily body temperature changes.
Materials and methods
ASCENT technology
During the mitochondria supplementation procedure, to attain the minimum required number of mitochondria to improve intra cytoplasmic sperm injection (ICSI) results, mitochondria isolated from 2.5 × 105 ASCs were used throughout the study [8]. At the final stage of mitochondria isolation, the mitochondrial pellet was kept in a standard respiration buffer with 0.5% human serum albumin until use. Approximately 2–3 pL of mitochondrial suspension and a spermatozoon were injected together (mito-ICSI) into an oocyte (Supplementary Fig. 1). As described in our previous study, founder offspring were developed following the transplantation of embryos after ASC mitochondria supplementation of the cryopreserved oocytes.
Animals
All experiments were performed following the guidelines of the Institutional Animal Care Ethics Committee and Use Committee of Osaka City University in Osaka, Japan (experiment approval no. 22017). Female and male C57BL/6JJmsSlc and ICR mice (4–28 weeks) were purchased from SLC Japan. The mice were housed in a room maintained under regular controlled SPF environmental conditions, with a temperature of 22–24 °C and a 12-h light/dark cycle. For the development of founder mice after mitochondria supplementation and conventional ICSI, oocytes were collected from C57BL/6JJmsSlc mice (n = 25). For transgenerational comparative analysis, three mice from each generation and each gender, (week 15) or week 30 (male only) or week 40 (male, n = 1) were compared with three age- and gender-matched WT mice. The mice from each generation were separated and placed in different levels of the racks. To ensure sufficient space per animal, the number of mice did not exceed five per cage. All the mice were continuously behaviorally and physiologically examined by experienced animal care-takers who confirmed absence of any concerning external pathological conditions.
Development of founder mice and first and second generation of offspring from founder mice
To develop the founder mice, following embryo development using the C57BL/6JJmsSlc mouse oocytes subsequent to mito-ICSI, embryos at the two-cell stage were transferred to a day-0.5 pseudopregnant ICR mouse that had been successfully mated with a vasectomized male the night before transfer. The offspring born after the embryo transfer were defined as the founder mice. Upon reaching sexual maturity, the founder (F0) mice (between 10 and 15 weeks of age, male = 5 and female = 1) were paired for mating with either female (n = 8) or male (n = 1) WT mice to initiate the first generation (F1). Subsequently, to produce the second generation (F2), sexually mature female mice from the first generation (n = 6) were paired overnight with established stud males (n = 3), or males from the first generation (n = 1) were mated with sexually mature female WT mice (n = 3). For the control group of this study, commercially available WT mice of the same strain (n = 9) were employed. Successful mating was confirmed through the presence of a mating plug, indicating that copulation had occurred. Pregnant mice underwent natural parturition, and the offspring remained with their mothers until they reached the weaning age of 3 to 4 weeks. The process for generating subsequent generations is visually outlined in Fig. 1. In order to analyze litter sizes, the number of pups was counted in at least four distinct litters within each generation.
Fig. 1.
Development of transgenerational offspring and safety analytical criteria. Schematic diagram for the transgenerational offspring development criteria. The both sexes of founder mouse were mated with sexually mature respective counterpart WT animals to develop first generation and female animal of first generation were mated with sexually mature WT studs to develop second generation. All three generations were studied for their breeding potentials, body weight gain and macroscopic parameter, hematological parameters, metabolism and behavioral parameters, histopathological changes, and compared with age and sex-matched WT animals
Assessment of body weight, organ mass, and organ size
The body weight of the mice was assessed weekly, commencing from the first week after birth and persisting through the eighth week, separately for each gender. These measurements were obtained at approximately the same time each week to ensure consistency. Regarding organ size measurements, a cohort of three animals from each generation, as well as age-matched WT mice of both genders, were included in the study. Careful organ collection and size measurements were conducted under the supervision of experienced pathology professionals and the collected samples were transferred for histopathological analysis.
Histopathological assessment of mice
A comprehensive histopathological examination was conducted on randomly selected male founder mice at 30 weeks of age, as well as age-matched, randomly selected male WT mice (n = 6). Additionally, both male and female mice from the first and second generation and WT counterparts at 15 weeks of age were included in the analysis (n = 18). The histopathological evaluation included a wide range of organs, including the brain, heart, liver, gall bladder, spleen, thymus, pancreas, lung, trachea, adrenal glands, kidney, thyroids, salivary glands, associated lymph nodes, tongue, esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon), mesenteric lymph node, pituitary gland, bladder, ovaries, uterus, vagina, testis, seminal vesicle, and skin. The harvested organs were rinsed in phosphate-buffered saline (PBS) and preserved in 10% formalin before being sent to the Department of Oncologic Pathology at the Graduate School of Medicine, Mie University, Japan, for histopathological analysis. After overnight fixation in 10% formalin, the specimens underwent a thorough PBS rinse and were subjected to paraffin embedding. Sections measuring 5 µm thickness were prepared from the organs. Before analysis, all specimens underwent hematoxylin and eosin (H & E) staining. All the samples were anonymized with a number as described in the Supplementary File 1. The evaluation of tissue samples was carried out randomly by a professional pathologist who was blinded to the group designations, ensuring impartial assessment.
Ovarian follicle counting
The ovarian follicle numbers were quantified in both the first and second generations and compared with age-matched WT animals. It is important to note that follicle numbers in founder female animal was not measured, as founder female was utilized to produce the next generations. To determine the follicle numbers, four consecutive ovarian sections (prepared during the histopathological procedures), each measuring 5 µm in thickness and spaced at 40-µm intervals, were examined. The sections were stained with H & E and observed under a light microscope following a previously established procedure [15, 16]. To prevent the unintended counting of the same follicle multiple times, only those follicles containing a visible nucleated oocyte were included in the count. All follicles were systematically characterized based on their morphological features. Specifically, primordial follicles were identified as those comprising an oocyte enveloped by a single layer of flattened granulosa cells, while primary follicles were distinguished as an oocyte encircled by a single layer of cuboidal granulosa cells. Secondary follicles were defined as follicles in which the oocyte was surrounded by multiple layers of cuboidal granulosa cells but lacked an antrum, while the follicles surrounded by multiple layers of cuboidal granulosa cells and several small antra were considered early antral follicles and antral follicles were identified by the presence of an oocyte enveloped by several layers of cuboidal granulosa cells with a visible full antrum. To calculate the total volume of each section, the area of the section was multiplied by the thickness of the section. Follicle density (available per μm3) was subsequently analyzed by dividing the number of follicles in each stage within each section by the total volume of the respective ovarian section, as determined using the method described above.
Spermatogenesis analysis
Spermatogenesis was analyzed in the founders (30 weeks) and, first and second generations (15 weeks) compared with WT animals of the same age. Testis tissue sections (prepared during the histopathological procedures) were evaluated employing standard H & E staining protocols. Spermatogenesis status was assessed through semi-quantitative methodology, Johnsen’s score [17, 18] for 100 seminiferous tubules within each cross-section, with all samples examined at an identical magnification. These individual scores were then aggregated to calculate the mean Johnsen’s score. In brief, under a 40 × magnification, seminiferous tubules from each animal were assessed and assigned a score ranging from 1 to 10 (Supplementary Table 1). This scoring system was based on the presence or absence of various germ cell types within the testicular seminiferous tubules, including spermatozoa, spermatids, spermatocytes, spermatogonia, germ cells, and Sertoli cells. The purpose was to evaluate the histological state of spermatogenesis. A higher Johnsen’s score signified a more favorable state of spermatogenesis, while a lower score indicated more severe dysfunction. Specifically, a score of 1 denoted complete inactivity with no epithelial maturation in the tubules, while a score of 10 was assigned to tubules exhibiting full epithelial maturation and maximum activity.
Locomotor activity and body temperature analysis of mice
Individual nanotag devices (Kissei Comtec Co. Ltd., Nagano, Japan) were implanted subcutaneously into randomly selected male and female mice belonging to the WT, first generation, and second-generation groups at the age of 15 weeks (n = 18), as well as 40-week-old male founder mice and age-matched WT mice (n = 2). The founder females’ daily locomotor and temperature analytical data were not measured as founder female was used to develop the next generation. The purpose of these implants was to continuously monitor locomotor activity and body temperature non-invasively. In brief, all implantation procedures were conducted under anesthesia, employing a combination of medetomidine (Me; Domitor® Nippon Zenyaku Kogyo Co., Ltd., Tokyo, Japan), midazoram (Mi: Dormicum®, Astellas Pharma Inc., Tokyo, Japan), and butorphanol (Bu; Vetorphale®, Meiji Seika Kaisha, Ltd., Tokyo, Japan), following established protocols. To ensure aseptic conditions, the dorsal area of each mouse was shaved and disinfected using a 70% alcohol solution. Subsequently, a 1.5-cm incision was carefully made in the dorsal skin. The nanotag device was then implanted subcutaneously, and the incision was closed using polyglycolic acid sutures (Matsuda Medical Industry Co. Ltd, Tokyo, Japan). Prior to sealing the surgical site completely, 3–5 drops of 100 mg/10 ml flumarin antibiotics were administered to prevent infection. Following the implantation procedure, each mouse was allowed to recover in an individual cage situated on a heated plate for a duration of 2–3 h. After this initial recovery period, the mice were returned to the conventional animal housing facility. One week later, once the mice had fully recovered, data collection commenced as per the instructions provided with the nanotag devices. Locomotor activity and subcutaneous body temperature data were automatically recorded and stored in the nanotag devices at 5-min intervals for eight consecutive days. On the eighth day, data collection was temporarily halted, and the accumulated data were transferred and stored separately for analysis. Following this, the mice were euthanized, and a postmortem examination was conducted to verify the animals’ well-being throughout the data collection period, ensuring that they were in good health.
Hematological parameter analysis
Whole blood samples were collected at the time of biopsy collection for histopathological analysis. The founder female animals were not assessed as female used to develop next generation. Samples were collected by puncturing the inferior vena cava of the mice. To prevent coagulation, ethylenediamine-N,N,N,N-tetraacetic acid (2Na(EDTA.2Na), Dojindo Laboratories, Kumamoto, Japan) was employed as an anticoagulant in a ratio of 20 µl of EDTA for every 0.5 mL of blood. These whole blood samples were subsequently utilized for a complete analysis of various hematological parameters, including white blood cell (WBC) count, red blood cell (RBC) count, hemoglobin (HGB) concentration, hematocrit (HCT) level, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), platelet (PLT) count, red cell distribution width–coefficient of variation (RDWCV), red cell distribution width standard deviation (RDWSD), procalcitonin (PCT) level, mean platelet volume (MPV), and platelet distribution width (PDW). The analyses were conducted using an automated blood cell analyzer (Celltac α, Nihon Kohden Co. Ltd, Tokyo, Japan), ensuring accuracy and precision in the measurement of hematological parameters.
Statistical analysis
All data are presented as mean ± standard error of the mean (SEM). Two-way analysis of variance (ANOVA) was used to compare the follicle numbers in each stage, and spermatogenesis scoring results were analyzed using one-way ANOVA. Additionally, unpaired Student’s t-test was used to compare groups. All the analyses were performed using GraphPad Prism software (Ver. 5, GraphPad Inc., San Diego, CA, USA). p < 0.05 was considered statistically significant.
Results
The effects of mitochondrial supplementation on body growth pattern of female and male offspring
All the physiological phenotypes were assessed by comparing them to age-matched and gender-matched WT animals. To establish the baseline for the initial body growth pattern, other than the WT animals, we utilized offspring resulting from the IVF procedure as an additional control group. To clarify the effect of mitochondrial transplantation to cryopreserved oocytes on other phenotypic traits of the offspring, we conducted physiological and anatomical assessments of females and males in each generation. First, we continually measured the individual weight of offspring from the first week of birth over eight consecutive weeks. Though the pattern of body growth was comparable, the average body weight of founder males and the IVF male offspring from week 1 to 8 was significantly higher than that of the WT control animals. The founder male pups and IVF male offspring were showing comparable average body weight over the period and at week 8 (Fig. 2a and Supplementary Fig. 2a). Male pups from the first and second generations showed a weight gain pattern comparable to that of WT male animals, with no body weight differences at f week 8 (Fig. 2b and c and Supplementary Fig. 2b & c). The body growth pattern of female founder pup was comparable to that of WT and IVF female pups (Fig. 2d and Supplementary Fig. 2d), and the IVF female pups showed significantly higher body weight at week 8 compared to the WT control group. Like male offspring in the first and second generations, female mice also showed a body growth pattern comparable to that of control animals over the period (Fig. 2e and f and Supplementary Fig. 2e & f). According to these data, though the offspring in the first and second generations born after mitochondrial transplantation showed normal growth potential, a tendency for getting more weight gain over the initial postnatal period was seen among male mice born after mitochondrial transplantation. However, according to a meta-analysis, higher body weight is common for pups born from IVF and ICSI, especially in mice [11]. Therefore, we suggest that this higher neonatal body weight gain may not have a direct relationship with the ASCENT.
Fig. 2.
The effects of mitochondrial supplementation on body growth pattern of female and male offspring. Body weight was measured weekly for each animal from the first week to the eighth week. Each week’s body weight was analyzed and compared using two-way ANOVA analysis and unpaired t-test analysis. a MMF0 (mito male founder, n = 5), b MM F1 (mito male generation 1, n = 7), c MM F2 (mito male generation 2, n = 8), d MF F0 (mito female founder, n = 1), e MF F1 (mito female generation 1, n = 8), f MM F2 (mito female generation 2, n = 8). The body weight gain in MM F0 male animals and IVF M animals were comparable to each other and significantly higher than in WT animals. MM F1, MM F2, MF F1, and MF F2 animals showed comparable body weight gain compared to age- and sex-matched WT animals. same WT and IVF animal’s data were utilized for all the comparisons (WT M n = 8, IVF M n = 6, WT F n = 5, IVF F n = 5). *(p < 0.05)
Mitochondrial supplementation does not have adverse effect of reproductive organ development and on the reproductive performances
The protocol for mito-ICSI and the control group was as described in our previous study [8]. Following the ICSI the oocytes were incubated as described previously and the zygotes that progressed to the two-cell stage of preimplantation development were transferred into pseudopregnant surrogate females. In this study, we obtained five male founders and one female founders from two transfers. Litter sizes are a significant indicator of fertility in multiparous species. Therefore, once both founder males and female had reached sexual maturity, they were mated with proven females or males to determine the fertility of both genders. The first litter from each of the founders produced the first generation with litter sizes of 5.71 ± 2.81 (mean ± SD). We mated the founders at least three times with WT animals to produce a further parity to use in a different analysis. Once the female animals from the first generation were mated with proven studs, they produced a mean litter size of 7.00 ± 1.41 (Fig. 3a). WT animals of the same age and strain were mated in-house and produced a mean litter size of 5.75 ± 0.47, which was comparable with the strain reference average litter sizes (5–11 pups per litter) [19], and with the founder and first generations.
Fig. 3.
Mitochondrial supplementation does not have adverse effect of reproductive organ development and on the reproductive performances. a The litter sizes of founder mouse and first generations (mean ± SEM), n = 5). b H&E-stained ovaries of wild-type (WT), mito female generation 1(MF F1), and mito female generation 2 (MF F2) were histologically similar at age 15 weeks, scale bar = 100 µm (low magnification), 20 µm (high magnification). Pr. F, primary follicle; SF, secondary follicle; EAF, early antral follicle; AF, antral follicle. c Ovarian follicle numbers WT, MF F1, and MF F2 at age 15 weeks. d Testicular mass according to body weight in WT, mito male generation 1 (MM F1) and, mito male generation 2 (MM F2) at week 15. e Representative histology of seminiferous tubules of each group (H&E-stained) × 20 magnification. f Spermatogenesis scoring according to Johnsen’s score in WT, MM F1, and MM F2 at week 15. g Testicular mass index. h Representative histology of seminiferous tubules. i Spermatogenesis of WT and MM F0 at week 30
To ascertain whether ASCENT had an impact on ovarian reserve or spermatogenesis, we conducted assessments encompassing the quantification of follicles at various developmental stages, including primordial, primary, secondary, early antral, and antral, as well as the determination of average testis weight and evaluation of spermatogenesis utilizing the Johnsen scoring system in 15-week-old female and male mice. Furthermore, we compared the phenotypic characteristics of the reproductive organs in both first and second generations of female and male mice subjected to ASCENT with those of age-matched WT animals. The low- and high-magnification sections are as shown in Fig. 3b, and did not show any observable abnormalities. In the case of follicle numbers, two-way ANOVA has been used to compare the follicle numbers in each stage and found no significant differences compared to WT animals (Fig. 3c).
The average weights of both testis of first and second generations at week 15 were comparable with those of their WT counterparts (Fig. 3d). The histological structure and spermatogenesis in mice were evaluated by the Johnsen’s scoring system. All the analyses were performed by two blinded professional pathologists who were unaware of the experimental conditions and group parameters. The seminiferous tubules were graded from 1 to 10 as described in Supplementary Table 1, according to the number and density of germ cells from their lumens. The sections were standardized based on Johnsen scores of the seminiferous tubule cross-sections of WT, first- and second-generation mice as shown in Fig. 3e. The scoring results were analyzed by using one-way analysis of variance (ANOVA) analysis and found that no significant differences between three groups (Fig. 3f). We also analyzed spermatogenesis in founder male mice at week 30 and compared them with age-matched WT animals. The founder mice’s testicular weight and spermatogenesis were comparable with those of WT mice (Fig. 3g–i). These results imply that ASCENT to cryopreserved oocytes does not adversely affect reproductive performances regardless of gender.
Macroscopic assessment, organ mass index, and histopathological analysis confirmed that mitochondrial supplementation does not have adverse effects on anatomical development
During sample collection for histopathological analysis, we systematically measured the weight and dimensions of major organs, such as the heart, liver, kidney, lung, spleen, reproductive organs, and gastrointestinal tract. We then evaluated the organ sizes in relation to individual body weight. Notably, the average sizes and average organ mass index of each generation and both sexes did not exhibit any statistically significant differences. (Supplementary Figs. 3–5 and Supplementary Tables 2, 3 and 4).
In a prior mouse study, mitochondrial supplementation to oocytes from oogonial stem cells resulted in the thickening of heart valves with myxomatous stroma in both the first and second generations of offspring. Consequently, we conducted an extensive histopathological examination of founder, first-generation, and second-generation male and female animals. Tissues from age-matched and gender-matched WT animals were analyzed as the control group. We specifically checked for any structural anomalies, tumor development, or atypical immune cell infiltration during the histopathological screening. The histopathological findings confirmed that, overall, the organs in all mice subjected to mitochondrial supplementation exhibited a generally normal histopathological profile when compared to both the control mice and the existing literature, for both genders (Supplementary File 1).
Offspring born after mitochondrial supplementation have normal hematological parameters
Hematological parameters are an important adjunct to both clinical medicine and biomedical research, which can be readily influenced by the physiological conditions, age, sex and strain of the animals, and heteroplasmic mitochondrial supplementation [20]. In our study, we employed autologous mitochondria as the intervention source, and the animals were maintained in a controlled environment. Therefore, in theory, the risk of encountering the aforementioned heteroplasmy-related abnormalities should be minimal. Nevertheless, we conducted a comprehensive analysis of hematological parameters in both first- and second-generation male and female animals and compared them with WT animals. Further, we analyzed the hematological parameters of founder male mice at the age of 30 weeks and compared them with age-matched WT male animals. In our investigation, all the parameters analyzed in founder mice showed no significant differences to those in WT animals (Fig. 4). Furthermore, we conducted an analysis of hematological parameters in both male and female animals from the first and second generations, comparing them with their respective WT counterparts at 15 weeks of age. In both male and female animals, no statistically significant differences were observed (Table 1). These findings collectively affirm that ASC mitochondrial transplantation does not appear to induce hematological disorders in offspring across generations.
Fig. 4.
Offspring born after mitochondrial supplementation have normal hematological parameters. Hematological parameters were measured and compared in WT and F0 male animals at week 30 and no significant difference found in any of parameters. White blood cell (WBC), red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet (PLT), red cell distribution width–coefficient of variation (RDWCV), red cell distribution width standard deviation (RDWSD), procalcitonin (PCT), mean platelet volume (MPV), platelet distribution width (PDW)
Table 1.
Normal hematological parameters are found in first and second offspring generations born after mitochondria supplementation
| Female | WBC (102/uL) | RBC (104/uL) | HGB (g/dL) | HCT (%) | MCV (fL) | MCH (pg) | MCHC (g/dL) | PLT (103/uL) | RDWCV (%) | RDWSD (fL) | PCT (%) | MPV (fL) | PDW (%) |
| WTF | 53.66 ± 6.62 | 798 ± 33.5 | 12.16 ± 0.84 | 35.9 ± 1.78 | 45 ± 0.92 | 15.26 ± 0.75 | 33.9 ± 2.05 | 60.95 ± 18.628 | 13.8 ± 0.29 | 24.83 ± 0.54 | 0.31 ± 0.08 | 5.08 ± 0.26 | 16.08 ± 1.2 |
| MF F1 | 55.17 ± 18.23 | 752.17 ± 133.7 | 11.78 ± 2.06 | 34.47 ± 5.78 | 45.93 ± 1.56 | 15.73 ± 1.12 | 34.2 ± 1.40 | 67.68 ± 13.94 | 14.28 ± 1.16 | 26.28 ± 2.66 | 0.34 ± 0.06 | 5.15 ± 0.40 | 16.15 ± 0.94 |
| MF F2 | 61 ± 5.43 | 904.2 ± 106.85 | 13.54 ± 1.39 | 39.2 ± 3.47 | 44.42 ± 0.62 | 15 ± 0.41 | 33.76 ± 0.73 | 66.42 ± 15.88 | 13.7 ± 0.46 | 24.34 ± 0.69 | 0.34 ± 0.08 | 5.16 ± 0.055 | 16.04 ± 0.57 |
| Male | WBC (102/uL) | RBC (104/uL) | HGB (g/dL) | HCT (%) | MCV (fL) | MCH (pg) | MCHC (g/dL) | PLT (103/uL) | RDWCV (%) | RDWSD (fL) | PCT (%) | MPV (fL) | PDW (%) |
| WTM | 63 ± 25.81 | 813.67 ± 60.39 | 12.17 ± 0.76 | 36.55 ± 2.84 | 44.9 ± 0.53 | 14.97 ± 0.42 | 33.33 ± 0.82 | 88.35 ± 6.35 | 14.02 ± 0.19 | 25.18 ± 0.58 | 0.45 ± 0.3 | 5.08 ± 0.2 | 15.81 ± 1.25 |
| MM F1 | 66.67 ± 15.7 | 795.67 ± 144.78 | 11.73 ± 2.2 | 34.53 ± 6.31 | 43.37 ± 0.06 | 14.77 ± 0.32 | 33.97 ± 0.76 | 53.27 ± 33.70 | 13.4 ± 0.35 | 23.27 ± 0.64 | 0.27 ± 0.16 | 5.07 ± 0.15 | 15.5 ± 0.95 |
| MM F2 | 64.2 ± 13.02 | 915.4 ± 48.04 | 13.82 ± 0.58 | 41.14 ± 1.70 | 45.38 ± 0.41 | 15.26 ± 0.15 | 33.58 ± 0.23 | 72.44 ± 7.076 | 13.2 ± 0.34 | 23.94 ± 0.65 | 0.34 ± 0.028 | 4.78 ± 0.164 | 15.04 ± 0.38 |
Hematological parameters were measured and compared in WT, F1, and F2 female and male animals at week 15 and no significant difference found in any of parameters
White blood cell (WBC), red blood Cell (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet (PLT), red cell distribution width–coefficient of variation (RDWCV), red cell distribution width standard deviation (RDWSD), procalcitonin (PCT), mean platelet volume (MPV), platelet distribution width (PDW)
First- and second-generation offspring born after mitochondrial supplementation show normal locomotor activity and body metabolism pattern
Complete locomotor activity has traditionally been analyzed as an indicator of the effect of these kind of intervention in animal models. A subcutaneous nano tag was implanted in male and female animals of each generation and age-matched control animals. After full recovery from nano-tag implantation, the locomotor activity counts and subcutaneous body temperature were continuously and simultaneously measured for 8 days. Since the animal house maintains controlled environment, all measurements of each group of mice were collected separately once the mice were 15 weeks old.
Mice showed the highest locomotor activity during the dark period, and regardless of gender, two peaks of activity during the dark period were observed for all three groups of animals. Similarly, all the mice showed relatively higher activity few hours after the light period started. Further, all the groups showed the lowest locomotor activity during the light period. However, all mice were showed fluctuations in activity during the light period, especially with the entry of researchers and the maintenance staff into the animal house (Fig. 5a and c). Interestingly, the average activity counts throughout dark and light periods were comparable in the first and second generations and WT animals despite the gender (Fig. 5b and d). The body temperature was also measured during the same period, and interestingly, all the animals showed body temperature changes in parallel with their activities. All the animals showed highest body temperature during the dark period, and lower temperatures and fluctuation with activities during the light period (Fig. 5e and g). And the average body temperature in the dark and light periods was comparable between three groups despite the gender (Fig. 5f and h). Regarding the founder male, locomotor activity and body temperature were analyzed in a male mouse from the study group and an age-matched WT mouse at 40 weeks. The results revealed that both the founder mouse and WT mouse exhibited activity patterns and body temperature changes consistent with those typically observed in mice, indicating no significant deviations from normal behavior or physiology in the founder mouse (Supplementary Fig. 6a & b). Nonetheless, it is important to note that locomotor activity and body temperature exhibited considerable variability, particularly in females, influenced by environmental factors and individual characteristics, such as the stage of the estrus cycle. Consequently, according to these findings we suggest that mitochondrial transplantation into oocytes does not have an adverse impact on the general behavior and body temperature of the offspring.
Fig. 5.
First- and second-generation offspring born after mitochondrial supplementation show normal locomotor activity and body metabolism pattern a Continuous locomotor activity counts of WT F, MF F1, and MF F2 were recorded at 5-min intervals over 1 week. The average values for each specific time of day were analyzed. The period from 8:00 AM to 8:00 PM (Zeitgeber time [ZT] 0–12) was the light period, and the period from 8:00 PM to 8:00 AM (Zeitgeber time [ZT]12–24) was the dark period. b Total average activity counts of WT F, MF F1, and MF F2 animals during light and dark periods. c and d Same as a and b, the daily locomotor activity and average activities of light and dark period of WT M, MM F1, and MM F2 at week 15. e Average daily body temperature changes were measured in WT F, MF F1, and MF F2 at week 15 at 5-min intervals over 1 week. f Total average temperature counts during light and dark periods of WT F, MF F1, and MF F2 animals. g and h Same as female animals’ body temperature values of male animals (WT M, MM F1, and MM F2) at week 15
Discussion
Interventions in assisted reproductive technology (ART) could potentially lead to the establishment or maintenance of epigenetic alterations that can affect embryo development and offspring health [21–24]. Though we previously showed that autologous adipose stem cell (ASC) mitochondrial energy transfer (ASCENT) into vitrified-warmed oocytes significantly improves embryo development capacity, the potential for abnormalities to arise in offspring as a consequence of mitochondrial supplementation needed evaluation. In this study, to validate the safety of ASCENT, we undertook comparative transgenerational analyses of various parameters in the immediate and two consecutive generations of offspring born after ASCENT. The results confirmed that all assessed parameters are comparable with those of WT animals, affirming the safety profile of ASCENT in mice.
Previous reports suggested that the use of ART results in offspring with higher birthweights and an increased rate of weight gain in the early life stages [11, 25]. In this study, the body growth patterns from week 1 to 8 in the founder, first-generation, and second-generation cohorts were found to be consistent with those observed in both WT and IVF animals. However, it is noteworthy that the rate of body weight gain observed each week was similar to that of IVF animals but significantly exceeded that of WT animals. This observation could potentially represent a shared phenotype among offspring born following ART manipulations [11, 25]. Interestingly, the body growth pattern and weekly body weight gain in both male and female animals in first-generation and second-generation mice were comparable to those of WT animals. These findings suggest that the increased weight gain observed in the founder mice may not be attributable to ASCENT.
The ratio of internal organ mass to total body size can be correlated to basal metabolic rate and could be altered under pathological conditions [26, 27]. Therefore, we analyzed the major organ mass index (organ weight relative to body weight) in male founder animals at the age of 30 weeks, as well as in male and female animals from generations 1 and 2 at 15 weeks. Our findings indicated that the major organ masses in the ASCENT group were comparable to those of WT animals of the same age and sex (Supplementary Figs. 3–5). Additionally, the macroscopic observations of the body and other organs in the group of animals supplemented with ASC mitochondria showed comparability to WT animals (Supplementary Tables 2, 3 and 4). These findings suggest that the supplementation of ASC mitochondria does not appear to lead to significant pathological deformities in the offspring.
In rodent species, litter size is a strong determinant of neonatal growth and long-term metabolic health [28]. As a positive impact of the mitochondrial supplementation of oocytes from oogonial stem cells, mice show a transgenerational upsurge in fecundity through increased litter size along with increased primordial follicles numbers [25]. In contrast, in our study with ASCENT, the litter sizes did not increase in a transgenerational manner, and the litter sizes of both founder and the first-generation were comparable with those of age-matched WT animals. Additionally, the first- and second-generation offspring’s ovaries exhibited comparable number of ovarian follicles to age-matched WT animals. In addition, male fertility depends upon the testes’ capacity to produce a substantial quantity of healthy spermatozoa through spermatogenesis [29, 30]. Various abnormalities of spermatogenesis can occur, with predominant anomalies in spermatozoa and sperm concentration, impaired sperm motility, or atypical morphology [31, 32]. We utilized Johnsen’s criteria to assess spermatogenesis in both the founder mice and the first and second generations. The founder mice exhibited consistently normal spermatogenesis even at the age of 30 weeks. Furthermore, the first and second generations displayed normal spermatogenesis by 15 weeks, comparable to that in age-matched WT animals. These findings confirm that ASCENT may not induce transgenerational gametogenesis defects in offspring mice.
Furthermore, since all protein machinery responsible for mitochondrial DNA (mtDNA) replication and maintenance is encoded by nuclear DNA, it is plausible that commonly occurring nuclear variants can influence mtDNA heteroplasmy [33]. The presence mtDNA heteroplasmy may give rise to aberrant hematological parameters due to impaired communication between mtDNA genotypes and the nuclear DNA [20]. Though such errors are primarily associated with obvious mitochondrial heteroplasmy conditions, we comprehensively analyzed total blood hematological parameters in the founder and two successive generations and compared these results with the status of age-matched and gender-matched WT animals. Interestingly, all hematological parameters in the groups of animals supplemented with ASC mitochondria were found to be comparable to those of the WT animals (Fig. 4 and Table 1). Further, it is important to note that ART procedures take place during a crucially coordinated phase of epigenetic reprogramming. This phase is particularly susceptible to epigenetic abnormalities, which have the potential to lead to cardiometabolic disturbances, heightened cancer susceptibility, modified immune responses, and the emergence of various chronic health issues in children conceived through ART [11, 34]. Hence, we conducted a complete comparative histopathological examination of all tissues in the founder and two subsequent generations of animals to detect any anomalies, tumor development, or atypical immune cell infiltration (Supplementary File 1). Our analysis revealed no significant abnormal histopathological findings in the offspring mice born after ASCENT.
Furthermore, several studies have indicated a potentially higher prevalence of conditions such as depression, autism spectrum disorder, attention-deficit disorder, and behavioral variances among ART-conceived individuals compared to those conceived naturally [35–38]. This phenomenon could be attributed to the metabolic demand of stress and novel learning experiences, which may require the brain to use its full capacity, which could be limited in offspring conceived through ART [39]. The dynamic alterations in mitochondrial physiology in response to metabolic shifts hold the potential to establish a connection between behavior and physiological well-being, both in states of health and during the onset of disease [40]. In our study, we continuously compared locomotor activities over 1 week period for founder male and both genders of first- and second-generation animals, and matched WT animals, to identify any abnormal behavioral phenotypes during the dark and light periods of the day. Interestingly, all the animal groups showed similar activity patterns and average activities during the dark and light periods (Fig. 5a–d and Supplementary Fig. 6a). In addition, during the body’s metabolic processes, heat is generated, and as much as 60% of this generated heat must be allocated to maintaining the body temperature [41]. Hence, the measurement of body temperature could serve as a valuable screening tool for understanding metabolic phenotypes and potentially predicting the health status of animals [42]. In our study, along with the locomotor analysis, we monitored changes in body temperature over the same timeframe. The group comparison revealed no statistically significant alterations (Fig. 5e–h and Supplementary Fig. 6b), affirming that the mice in the ASCENT group exhibited average metabolic performances.
Limitations
To ensure a comprehensive understanding of the normal behavior of the offspring, more refined and detailed behavioral analyses may be necessary. Moreover, certain data points were unavailable in the founder mice dataset, due to the limited availability of founder mice.
Conclusion
In certain instances of female infertility, the utilization of autologous ASC mitochondrial supplementation appears to be a viable option for enhancing oocyte quality and developmental capacity. Our recent murine study shed light on the potential of adipose stem cell-derived mitochondrial transfer to improve the developmental potential of oocytes displaying suboptimal quality. In this comprehensive examination, we explored any potential transgenerational consequences from this approach, represented as ASCENT in mice. This study revealed that offspring developed after ASCENT exhibit average reproductive performance, normal growth patterns, standard hematological parameters, typical behavioral traits, regular metabolic profiles, and an absence of pathological phenotypes. Given these outcomes, we suggest that ASCENT could stand as a viable and secure means of enhancing oocyte quality and developmental potential in mice. However, to establish its clinical relevance, similar types of experiments in larger animals or non-human primates that closely mimic human physiological conditions are required. Furthermore, screening human ASC mitochondria profiles is crucial to understanding the feasibility of utilizing ASC mitochondria for supplementation in human oocytes. These steps are pivotal for elucidating the potential applications and safety considerations of such interventions in human reproductive medicine.
Supplementary Information
Below is the link to the electronic supplementary material.
Acknowledgements
The authors acknowledge the staff members of the animal center of the Faculty of Medicine, Osaka Metropolitan university who helped to conduct the research smoothly, as well as staff members of the Department of Oncologic Pathology, Graduate School of Medicine, Mie University, who helped to conduct histopathological analyses. And authors acknowledge the members in Cambridge Proofreading & editing LLC who helps to improve the English language in the manuscript.
Author contributions
S. U. K. G, S.H., and Y. M. (Yoshiharu Morimoto) designed the experiments. Y. M. (Yuki Miyamoto), A. K. and T. N. performed mitochondria supplementation and embryo transplantation. S. U. K. G., S. H., M. Y., H. K., Y. T., and H. M. performed animal breeding, nanotag insertion, and various sampling. M. I. and M. W. performed histopathological analysis. S. U. K. G., wrote the manuscript. All authors contributed to data interpretation and editing of the manuscript, and gave final approval for its publication.
Funding
Part of this work was supported by a grant from the Japan Society for the Promotion of Science (KAKENHI 20K09674 to S.H.).
Data availability
The data underlying this article will be shared on reasonable request to the corresponding authors.
Declarations
Competing interests
The authors declare no competing interests.
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Data Availability Statement
The data underlying this article will be shared on reasonable request to the corresponding authors.