Blastomere removal affects homeostatic control leading to obesity in male mice

in Reproduction
Authors:
Magdalena KotlarskaDepartment of Experimental Embryology, Institute of Genetics and Animal Biotechnology of the Polish Academy of Sciences, Jastrzębiec, Poland

Search for other papers by Magdalena Kotlarska in
Current site
Google Scholar
PubMed
Close
,
Dawid WiniarczykDepartment of Experimental Embryology, Institute of Genetics and Animal Biotechnology of the Polish Academy of Sciences, Jastrzębiec, Poland

Search for other papers by Dawid Winiarczyk in
Current site
Google Scholar
PubMed
Close
,
Wiesława FlorekDepartment of Experimental Embryology, Institute of Genetics and Animal Biotechnology of the Polish Academy of Sciences, Jastrzębiec, Poland

Search for other papers by Wiesława Florek in
Current site
Google Scholar
PubMed
Close
,
Marta ZiętekDepartment of Experimental Embryology, Institute of Genetics and Animal Biotechnology of the Polish Academy of Sciences, Jastrzębiec, Poland

Search for other papers by Marta Ziętek in
Current site
Google Scholar
PubMed
Close
,
Jolanta Pęczkowicz-SzyszkaDepartment of Experimental Embryology, Institute of Genetics and Animal Biotechnology of the Polish Academy of Sciences, Jastrzębiec, Poland

Search for other papers by Jolanta Pęczkowicz-Szyszka in
Current site
Google Scholar
PubMed
Close
,
Adrian Mateusz StankiewiczDepartment of Molecular Biology, Institute of Genetics and Animal Biotechnology of the Polish Academy of Sciences, Jastrzębiec, Poland

Search for other papers by Adrian Mateusz Stankiewicz in
Current site
Google Scholar
PubMed
Close
,
Rafał Radosław StarzyńskiDepartment of Molecular Biology, Institute of Genetics and Animal Biotechnology of the Polish Academy of Sciences, Jastrzębiec, Poland

Search for other papers by Rafał Radosław Starzyński in
Current site
Google Scholar
PubMed
Close
,
Roberta ArenaDepartment of Experimental Embryology, Institute of Genetics and Animal Biotechnology of the Polish Academy of Sciences, Jastrzębiec, Poland

Search for other papers by Roberta Arena in
Current site
Google Scholar
PubMed
Close
,
Gaspare DragoNational Research Council of Italy, Institute of Biomedical Research and Innovation, Laboratory of Environmental Epidemiology, Palermo, Italy

Search for other papers by Gaspare Drago in
Current site
Google Scholar
PubMed
Close
,
Silvestre SampinoDepartment of Experimental Embryology, Institute of Genetics and Animal Biotechnology of the Polish Academy of Sciences, Jastrzębiec, Poland

Search for other papers by Silvestre Sampino in
Current site
Google Scholar
PubMed
Close
, and
Jacek Andrzej ModlinskiDepartment of Experimental Embryology, Institute of Genetics and Animal Biotechnology of the Polish Academy of Sciences, Jastrzębiec, Poland

Search for other papers by Jacek Andrzej Modlinski in
Current site
Google Scholar
PubMed
Close

Correspondence should be addressed to S Sampino; Email: s.sampino@igbzpan.pl
Free access

Preimplantation embryos are particularly vulnerable to environmental perturbations, including those related to assisted reproductive technologies. Invasive embryo manipulations, such as blastomere biopsy, are applied worldwide in clinical settings for preimplantation genetic testing. Mouse models have previously shown that blastomere biopsy may be associated with altered phenotypes in adult offspring. The aim of the present study was to investigate the specific contribution of blastomere removal to the physiological, behavioral, and molecular regulators of energy homeostasis, as compared to sham manipulation (re-introducing the blastomere into the embryo after its removal) and in vitro culture. Mice derived from 8-cell embryos subjected to blastomere removal displayed: (i) higher body weight and adiposity, (ii) increased food intake and sucrose preference, (iii) decreased time of immobility in the tail suspension test, and (iv) resistance to weight loss after social isolation or following 3 days of physical exercise – compared to mice derived from sham biopsy or from in vitro-cultured embryos. Mice generated after blastomere removal also had increased circulating leptin and leptin gene expression in adipose tissue, as well as increased ghrelin receptor gene expression in the hypothalamus, compared to control mice. The effects of blastomere biopsy on offspring phenotype were sexually dimorphic, with females not being affected. These results indicate that blastomere deprivation, rather than other perturbations of the blastomere biopsy procedure, programs male embryos to develop physiological, behavioral, and molecular dysregulation of energy homeostasis, leading to postnatal obesity.

Abstract

Preimplantation embryos are particularly vulnerable to environmental perturbations, including those related to assisted reproductive technologies. Invasive embryo manipulations, such as blastomere biopsy, are applied worldwide in clinical settings for preimplantation genetic testing. Mouse models have previously shown that blastomere biopsy may be associated with altered phenotypes in adult offspring. The aim of the present study was to investigate the specific contribution of blastomere removal to the physiological, behavioral, and molecular regulators of energy homeostasis, as compared to sham manipulation (re-introducing the blastomere into the embryo after its removal) and in vitro culture. Mice derived from 8-cell embryos subjected to blastomere removal displayed: (i) higher body weight and adiposity, (ii) increased food intake and sucrose preference, (iii) decreased time of immobility in the tail suspension test, and (iv) resistance to weight loss after social isolation or following 3 days of physical exercise – compared to mice derived from sham biopsy or from in vitro-cultured embryos. Mice generated after blastomere removal also had increased circulating leptin and leptin gene expression in adipose tissue, as well as increased ghrelin receptor gene expression in the hypothalamus, compared to control mice. The effects of blastomere biopsy on offspring phenotype were sexually dimorphic, with females not being affected. These results indicate that blastomere deprivation, rather than other perturbations of the blastomere biopsy procedure, programs male embryos to develop physiological, behavioral, and molecular dysregulation of energy homeostasis, leading to postnatal obesity.

Introduction

Assisted reproductive technologies (ART) have contributed to the birth of millions of children worldwide in the last 40 years. Since the birth of Louise Brown in 1978, many ART have been introduced, including in vitro fertilization and embryo culture (Steptoe & Edwards 1978), intra-cytoplasmatic sperm injection (Palermo etal. 1992), and preimplantation genetic testing (PGT) (Handyside etal. 1990). Although ART are effective in treating subfertility, their initial establishment was not devoid of intense debate, mainly concerning ethical issues and eventual health consequences. Children conceived through ART are predominantly healthy (Hart & Norman 2013a,b); however, there is also evidence of increased risks of congenital abnormalities (Davies et al. 2012, Wen etal. 2012, Hansen etal. 2013), low birthweight (Helmerhorst etal. 2004, Dumoulin etal. 2010, Kleijkers etal. 2016), growth and developmental alterations (Koivurova etal. 2003, Kai etal. 2006), and negative health outcomes in children obtained from ART embryos (Ceelen etal. 2008, Sakka etal. 2010). Considering the rapid evolution of reproductive biotechnologies, including embryo selection and gene editing, systematic testing of ART’s effects on animal models would be beneficial to ensuring its safety in clinical applications.

PGT is mainly indicated to obtain genetically healthy children from disease-carrier couples and to dispose of aneuploid embryos prior to their transfer in utero. The diagnosis is achieved by harvesting embryonic cell/s, performing appropriate DNA analyses, then transferring only the embryos without genetic abnormalities. Embryonic cells can be biopsied in several ways, including blastomere biopsy (BB) from 8-cell embryos or trophectoderm biopsy (TB) from blastocysts. Recently, TB has been gradually supplanting BB in clinical practice because it is less invasive and more efficient in terms of embryonic survival and implantation rates (De Rycke etal. 2017). Nonetheless, the transition from BB to TB is still not complete, since TB requires substantial training and dedicated equipment, which may not be readily available to small IVF clinics. Epidemiological follow-ups of children born after BB have been generally reassuring with regard to its safety (Middelburg etal. 2011, Desmyttere etal. 2012, Thomaidis etal. 2012, Schendelaar etal. 2013, Winter etal. 2015, Sacks etal. 2016, Heijligers etal. 2018). However, the population size of these studies has been small, and they may be limited by selective biases related to intrinsic differences between PGT and control groups.

Although mouse and human embryos differ in many developmental features, controlled studies using mouse models have provided sound evidence that BB may disrupt developmental outcomes, leading to long-term effects on offspring phenotype, such as altered postnatal growth and atypical patterns of behavior (Yu etal. 2009, Zhao etal. 2013, Zeng etal. 2013, Sampino etal. 2014, Wu etal. 2014, Gu etal. 2018). However, the biological mechanisms underlying this phenomenon are not known. The BB procedure entails a series of manipulations, including embryo culture, perforation of the zona pellucida, and the removal of 1–2 cells, which can potentially disrupt cellular structures and embryonic homeostasis. To understand the specific contribution of the various procedural steps, we established three experimental groups of embryos: blastomere-deprived, sham-biopsied, or only in vitro-cultured. Gross morphology, cell count and distribution, and cell–cell adhesion integrity were examined in blastocysts. Physiological, behavioral, and molecular regulators of energy homeostasis, such as food intake, body weight, voluntary exercise, and the expression of several molecules in different tissues involved in the control of energy intake/expenditure, were monitored in the offspring. The results indicate that the absence of one single blastomere, rather than other perturbations occurring within the BB protocol, prompts embryonic reprogramming, leading to obesity in the derived male offspring.

Materials and methods

Ethical approval

All experiments were performed in accordance with the European Community regulation 86/609 and were approved by the Second Local Ethical Committee in Warsaw (approval no. WAW2/10/2017).

Experimental design

Mice offspring were generated from three experimental treatments of 8-cell embryos: (i) subjected to single blastomere biopsy, BB group; (ii) subjected to blastomere removal followed by its reintroduction, sham biopsy (SB) group; and (iii) unmanipulated, in vitro cultured controls, IVC group. Furthermore, four litters of naturally developed mice (in vivo group) were used as controls for body weight measurements. The resulting offspring were screened for phenotypic and molecular features related to energy homeostasis. Body and organ weights, food and sucrose intake, locomotion, voluntary exercise, and time of immobility in the tail suspension test were monitored as a measure of energy intake/expenditure. Furthermore, molecular regulators of energy homeostasis were examined in fat, hypothalamus, and blood tissues. Each experimental group included at least eight independent litters.

Animals

Mice were housed in 252 x 167 x 140 mm transparent polycarbonate cages, under a 12 h light:12 h darkness cycle, with 40–60% humidity, at 19–25°C, with free access to water and food. Hybrid C57BL/6J x CBA/H (B6CBAF1) 2- to 4-month-old females were hormonally stimulated, then mated with males of the same strain and age to produce embryos, which were collected 2 days after detection of the vaginal plug. The resulting 8 cell-stage embryos were subjected to BB or SB, then cultured in vitro until the blastocyst stage, or only in vitro cultured (IVC). Subsequently, the embryos were transferred to pseudo-pregnant Swiss albino foster females. The offspring derived from BB, SB, and IVC embryos were all subjected to body weight monitoring (n ≥ 19 mice/sex/group) and randomly assigned to different subgroups for analyses: (i) wheel and tail suspension tests (n ≥ 9 mice/sex/group) and (ii) food and sucrose intake (n ≥ 10 mice/sex/group); an independent cohort of mice was sacrificed for necropsyand molecular analyses (n = 9 males/group). All experiments were performed in accordance with European Community regulation 86/609 and were approved by the Second Local Ethical Committee in Warsaw (approval no. WAW2/10/2017).

Hormonal stimulation and embryo collection

B6CBAF1 females aged 2–4 months were hormonally stimulated using 5 IU/0.1 mL pregnant mare serum gonadotrophin (PMSG, MSD Animal Health, USA), followed by 5 IU/0.1 mL of human chorionic gonadotropin (hCG, MSD Animal Health, USA) 50 h after PMSG injection, then mated with 2- to 4-month-old males of the same strain. After overnight male–female co-housing, successful mating was confirmed by the presence of the vaginal plug (designated as 0.5 days post-coitum, dpc). At 2.5 dpc, 8-cell stage embryos were collected by flushing the oviducts with pre-warmed M2 medium, prepared as previously described (Nagy 2003). All embryos with no signs of fragmentation and a total number of 8 blastomeres were included in the study.

Micromanipulations and embryo in vitro culture

Embryos assigned to the IVC group were directly incubated during the subsequent 24 h in pre-equilibrated potassium simplex optimized medium (KSOM) (Sigma-Aldrich) under paraffin oil, at 37°C, in a humidified atmosphere of 5% CO2. The biopsy was performed as previously described (Sampino etal. 2014), with slight modifications. Briefly, embryos were transferred in a drop of M2 medium containing 5 mg/mL cytochalasin B (Sigma-Aldrich), under paraffin oil. For the BB group, a single, randomly selected blastomere was aspirated from each embryo and discarded using a tip-bevelled enucleation pipette. For the SB group, the blastomere was first aspirated, then reintroduced immediately into the embryo; only embryos with >8 intact blastomeres after 1 h of monitoring were included in the study. BB and SB embryos were then incubated until the blastocyst stage in the same conditions as those of for the IVC group.

Assessment of blastocyst gross morphology, cell count/distribution, and cell–cell adhesion integrity

Developmental rate and timing to the blastocyst stage, as well as blastocysts’ gross morphology, including the degree of fragmentation and the presence of appropriate blastocoel vacuolization, were examined to assess embryo quality. Only high-quality embryos were transferred to female recipients to obtain offspring. An independent cohort of embryos was generated to examine blastocyst cell count and distribution, as well as cell–cell adhesion integrity. The expressions of F-actin and E-cadherin were analyzed by phalloidin staining and immunostaining, respectively, at the blastocyst stage. Moreover, immunostaining for CDX2 was performed to label trophectodermal cells. Briefly, the zona pellucida was removed using acidified Tyrode’s solution. Embryos were fixed in 4% paraformaldehyde in PBS with 0.1% Tween 20 and 0.01% Triton X-100 overnight at 4°C, permeabilized in 0.55% Triton X-100 in PBS for 15 min, and blocked in 20% fetal bovine serum (Sigma-Aldrich) in PBS for 1 h. Primary antibodies were anti-E-cadherin (Zymed, USA; 18–0223) and anti-CDX2 (Biogenex, USA; MU392A-UC) at 1/100 dilution. Secondary Alexa Fluor-conjugated antibodies (Invitrogen) were incubated at a 1/500 dilution for 75 min. DNA was visualized using 5 μg/mL Hoechst 33342 (Molecular Probes). F-actin was visualized using 0.1 μg/mL phalloidin–tetramethylrhodamine B isothiocyanate (Sigma-Aldrich). Embryos were observed under a confocal microscope (Nikon A1R). Confocal images were analyzed using IMARIS 6.0.1 software (Bitplane AG, UK) and ImageJ. The total number of Hoechst-positive nuclei was counted and assigned to the inner cell mass (ICM) or trophectoderm (TE), depending on coexpression of CDX2. Intercellular junction molecules E-cadherin and F-actin were visually inspected by two independent observers, taking into account the: (1) continuity of E-cadherin and F-actin signals along intercellular edges, (2) presence of defined E-cadherin signal between TE cells on equatorial sections, and (3) distribution of F-actin signal in the TE and ICM.

Embryo transfer

Uterine embryo transfer was performed at 3.5 dpc to 2- to 4-month-old pseudo-pregnant Swiss albino females, which were mated with proven vasectomized males 3 days before the transfer, as previously described (Sampino etal. 2014). Recipient female mice were intraperitoneally anesthetized with xylazine and ketamine (10–16 and 80–120 mg/kg, respectively). The ovary, oviduct, and part of the uterine horn were exposed and immobilized with a clamp holding the ovarian fat pad. The uterine wall was punctured with a dissecting needle, and a thin, fire-polished glass pipette (diameter ~80 µm) was used to transfer 5–7 blastocysts in each uterine horn. After embryo transfer, females were first kept in their cages on a warm plate at 37°C overnight, then transferred to the colony and left undisturbed until delivery.

Assessment of offspring phenotype

Pups were weighed every other day from postnatal day 2 (P2) to P20. Physical landmarks, including eyes/ear opening, incisor eruption, and fur development, were scored during this period. After weaning, mice were weighed weekly. Adult mice aged 3–5 months were subjected to a screening battery of tests designed to examine behavioral domains related to energy homeostasis. Energy intake was examined by measuring food intake and sucrose preference in singly housed individuals. Conversely, distance moved in a 6-min open field test, time spent running a wheel in a 3-day voluntary exercise assay, and time spent moving in the tail suspension test were examined as measures of energy expenditure (Supplementary methods, see section on supplementary materials given at the end of this article). Behavioral tests were performed in a room separated from the colony. Mice were transferred to the testing room at least 2 h before the beginning of the tests to permit habituation. There was at least a 2-week interval between tests. Body weights were monitored daily before, during, and after food intake and wheel running assays. The experimenters performing the behavioral tests were blind to the experimental group.

Necropsy and tissue collection

A separate cohort of 3- to 4-month-old male mice of the BB, SB and IVC experimental groups was sacrificed and examined for the presence of tumors or gross morphological abnormalities, as well as to collect tissues. Analyses were performed only in males, which presented more substantial differences in body weight and other outcomes. Blood was collected immediately after decapitation in 1.5 mL Eppendorf tubes filled with 25 µL of heparin (50 UI) and centrifuged at 4°C to obtain plasma, which was stored at –80°C until further molecular analysis. Liver, kidneys with their fat pads, and perigonadal adipose tissue were dissected and weighed. Finally, the hypothalamus and gonadal fat pads were stored at –80°C after a brief wash in PBS until further molecular analyses.

ELISA

Leptin peptide concentration was measured in the plasma and gonadal fat pads of BB, SB, and IVC mice (n = 9 males/group) using an ELISA kit (Cloud-Clone Corp., USA). The total protein concentration in fat was determined by flow spectrophotometry in homogenized samples. The protein concentration in plasma was determined by the Bradford method using a Bio-Rad Protein Assay reagent (Bio-Rad). Measurements were made to determine the concentration in ng/mg of the protein in wet tissue (DU-68 Spectrophotometer, Beckman, USA). ELISAs were performed in 96-well plates in duplicate and were validated with controls of known concentration supplied by the manufacturer. Absorbance of the color enzyme reaction product was measured at a wavelength of 450 nm on an ELx800 (BioTek Instruments).

Quantitative real-time polymerase chain reaction

The expression level of the gene leptin (Lep) was measured in fat tissue, while the expression levels of leptin receptor (Lepr), growth hormone secretagogue receptor (Ghsr), neuropeptide Y (Npy), growth hormone (Gh), and hypocretin (Hcrt) were measured in the hypothalami of BB, SB, and IVC mice (n = 9 males/group) using quantitative real-time PCR (qPCR) synthesis, based on the SYBR Green dye. Total RNA was isolated using an RNAeasy Mini Kit (Qiagen), and cDNA was synthesized using oligo (dT) primers and a First Strand cDNA Synthesis Transcriptor kit (Roche Applied Science), according to the manufacturer’s instructions. RT reactions were performed with 1 μg of total RNA in a final volume of 20 μL. Succinate dehydrogenase complex flavoprotein subunit A (Sdha) and hydroxymethylbilane synthase (Hmbs) were selected as reference genes for normalization of gene expression based on a stability analysis of four reference genes (Sdha, Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein z, Hmbs, and beta-2 microglobulin) using the NormFinder program (Aarhus University Hospital, Denmark). Primer pairs were designed using Primer-BLAST (NCBI, USA) (www.ncbi.nlm.nih.gov/tools/primer-blast/), the RefSeq IV.80 database, and OligoAnalyzer 3.1 (https://www.idtdna.com/pages/tools/oligoanalyzer). Detailed information about the primers can be found in Supplementary Table 1. PCR was performed in a LightCycler 96 thermocycler (Roche Applied Science). All samples were analyzed in duplicate. The qPCR results were analyzed in accordance with Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines (Bustin etal. 2009).

Statistical analysis

Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software) and R version 3.5.1 (http://www.R-project.org/). The effects of treatment on preimplantation and early postnatal developmental outcomes were determined by Fisher’s exact test or one-way ANOVA. The effect of treatment and age on offspring body weight measurements was determined using two-way repeated measures ANOVA, followed by Bonferroni post hoc tests. Comparisons concerning behavioral measurements were made using one-way ANOVA, or nonparametric Kruskal-Wallis tests, followed by Bonferroni’s or Dunn’s multiple comparison tests, respectively. The effects of treatments and timepoints on food and sucrose intake were analyzed by two-way ANOVA, followed by Bonferroni’s test. A linear mixed model was used to study the effects of litter size and treatment on the pups’ body weight gain between P2 and P20. For adults, separately by gender, we assessed the effect of treatment on weekly weight gain using a mixed linear model for repeated measurements. The model was adjusted for litter size and mouse body weight at the previous week. For pups’ body weight and developmental landmarks, data were collapsed across the sexes. Protein expression data were analyzed by comparing fold changes among experimental groups using one-way ANOVA. For real-time PCR, one-way ANOVA was used if R ratios were normally distributed and showed equal variance between experimental groups, and a Kruskal–Wallis test was used if R ratios were not normally distributed. All values are reported as mean ± s.e.m. Differences were considered statistically significant at P < 0.05.

Results

Preimplantation development

Neither BB nor SB influenced the ability of the embryos to develop to the blastocyst stage compared to IVC-derived embryos. Of the 291 embryos subjected to BB and placed in vitro after manipulation, 274 (94.34%) developed to the blastocyst stage. Similarly, the blastocyst rate was 96.89 and 95.38% in the SB and IVC groups, respectively. In all groups, the majority of blastocysts (86.94% for BB, 63.04% for SB, and 89.6% for IVC – no statistically significant difference) had proper morphology, with a clearly developed ICM and single blastocoel (Fig. 1A). The remaining blastocysts showed signs of cell degeneration and vacuolization, and the presence of a multi-cavitated blastocoel, thus were excluded from the long-term offspring study (Fig. 1B). The expression and distribution of the adhesion molecule E-cadherin were similar among the groups, as analyzed at the blastocyst stage. However, SB-derived embryos had weaker intercellular F-actin signal in both the ICM and the TE, compared to IVC- and BB-derived embryos (Fig. 1C). BB and SB blastocysts had fewer cells in both the ICM and TE compared to IVC controls, although the ICM/TE cell number ratio was not affected by either manipulation (Fig. 1D).

Figure 1
Figure 1

Preimplantation development to the blastocyst stage of BB, SB, and IVC embryos. Representative microphotographs of (A) high-quality blastocysts developed from BB, SB, and IVC embryos, compared to (B) low-grade blastocysts, which were excluded from the long-term study and not transferred, presenting multiple blastocoel cavities and signs of fragmentation and vacuolization. Scale bar = 0.40 mm. (C) Confocal images of IVC, BB, and SB blastocysts showing immunolocalization of E-cadherin (white signal) and F-actin (red signal). Scale bar = 0.20 mm. (D) Graphs showing the number of cells in the ICM and the TE, as well as the ICM/TE ratio. *P < 0.01 comparing IVC vs SB or BB with one-way ANOVA (n ≥ 9 embryos/experimental group).

Citation: Reproduction 161, 1; 10.1530/REP-20-0253

IVC, BB, and SB do not affect offspring survival or early postnatal development

For the BB group, 213 high-quality blastocysts were transferred to pseudo-pregnant females, while 136 were transferred for the SB group and 142 for the IVC group, resulting in 13, 8, and 9 pregnancies, respectively (Table 1). In total, 62 (29.1%), 39 (28.67%), and 41 (28.87%) embryos developed to term in the BB, SB, and IVC groups, respectively. None of these differences were deemed significant by Fisher’s exact test. No significant differences among the groups were observed regarding litter size, survival of the pups to P21, or physical developmental milestones (data not shown).

Table 1

Effects of blastomere biopsy (BB), sham biopsy (SB), and in vitro culture (IVC) on preimplantation and to-term development in mice.

Experimental groups No. of in vitro cultured embryos No. of obtained blastocysts (%) No. of transferred blastocysts (no. of recipients) No. of obtained offspring (%) Litter size No. of offspring surviving at P21 (%)
BB 291 274 (94.34%) 213 (16) 62 (29.1%) 4.54 ± 0.55 60 (96.77%)
SB 193 187 (96.89%) 136 (12) 39 (28.7%) 4.1 ± 0.35 39 (100%)
IVC 173 165 (95.38%) 142 (13) 41 (28.9%) 5.22 ± 0.88 40 (97.56%)

Blastomere deprivation, rather than sham biopsy, is associated with postnatal obesity in male offspring

The body weights at birth and during the first postnatal week were similar among the groups. Starting at P8, and during adolescence and adulthood, BB male mice showed increased body weight compared to SB and IVC controls (Fig. 2A, B and C). A significant effect of treatment was found in pups’ body weight (F(3,77) = 56.21; P < 0.0001), with post hoc analysis showing significant differences between BB pups vs SB and IVC controls. A similar effect of treatment was observed in adult males’ body weight (F(2,75) = 73.59, P < 0.0001), with BB-derived males showing increased body weight compared to both controls and increased weekly growth rate. In contrast, for adult females, the differences were significant only when comparing the BB group with in vivo groups. Furthermore, using a linear mixed model, we found that the effect of treatment on the pups’ growth rate between P2 and P20 remained significant, also considering litter size as a random effect (Supplementary Table 2). A similar scenario was observed for weekly body weight gain in adult male offspring, but not in females, using a linear mixed model for repeated measurements (Supplementary Table 3). The weight of the perigonadal fat depots was increased in BB males compared to IVC males, whereas the weight of kidneys with their fat pads was increased in the BB males compared to both SB and IVC sex-matched controls. The livers of BB males were also significantly increased in weight compared to IVC males (Fig. 2D). On the other hand, there were no significant differences in body or organ weights at most of the timepoints analyzed when comparing SB- and IVC-derived mice. Therefore, these data demonstrate that the absence of the blastomere, rather than micromanipulation-induced damage, is the triggering factor underlying postnatal obesity.

Figure 2
Figure 2

Blastomere removal causes increased body weight and adiposity in pups and male adult offspring. (A) Average body weight measured in 2- to 20-day-old pups derived from the different experimental treatments (BB, n = 62; SB, n = 39; IVC, n = 41; in vivo, n = 19). **P < 0.01 comparing BB vs all controls on days 8–20, and on days 10–20 comparing IVC and SB vs in vivo mice with two-way ANOVA. (B and C) Average body weight measured in 4- to 17-week-old male and female offspring, respectively (males: BB, n = 39; SB, n = 19; IVC, n = 20; in vivo, n = 10; females: BB, n = 21; SB, n = 19; IVC, n = 21; in vivo, n = 9). **P < 0.01 comparing BB vs SB in weeks 5–17; #P < 0.05 comparing SB vs IVC in weeks 15–17; §P < 0.01 comparing IVC vs in vivo-derived male mice; *P < 0.05 comparing BB vs in vivo in weeks 7–17. (D) Average weight of perigonadal fat, kidneys with their fat pad, and liver collected from male offspring of the different groups (n = 9/group). **P < 0.01 comparing BB vs IVC, and ***P < 0.01 comparing BB vs both controls with one-way ANOVA.

Citation: Reproduction 161, 1; 10.1530/REP-20-0253

Altered behavioral regulation of energy homeostasis in BB-derived male offspring

Food intake and daily body weight were measured in individually housed mice for 9 consecutive days. During the first 3 days after social isolation, IVC and SB male mice displayed body weight loss, whereas BB male mice gained weight (Fig. 3A). Cumulative food intake along 9 testing days was increased in BB male mice, compared to IVC and SB males (Fig. 3B). Locomotion in the open field test and wheel running activity were examined as behavioral markers of energy expenditure (Fig. 3C and D). The distances covered in the 6-min open field test were similar among the groups. In the wheel test, no significant effects of treatment were observed in time spent running, in time spent in and frequency of contact with the wheel, nor in latency to start running (data not shown). Interestingly, while SB and IVC mice displayed weight loss after 3 days of wheel testing, BB mice had a positive weight gain (Fig. 3E). Furthermore, mice were subjected to sucrose preference and tail suspension tests to examine their preference for a palatable, high-calorie drink and their body activity in an unescapable situation, respectively. Mice obtained from BB embryos showed an ~80% preference for the sucrose drinking source compared to water, whereas SB and IVC mice had a ~60% preference (Fig. 4A). In the tail suspension test, BB-derived mice spent more time moving compared to controls (Fig. 4B). No significant differences were observed among experimental groups when comparing female mice in behavioral measurements. Overall, these results demonstrate that the BB-induced obese phenotype in male offspring is associated with an altered behavioral regulation of energy homeostasis.

Figure 3
Figure 3

Blastomere removal influences daily body weight loss and food intake in male offspring after social isolation (n ≥ 10 mice/sex/group). (A) Daily weight changes along 9 days of monitoring after social isolation. **P < 0.01 comparing BB vs IVC, #P < 0.01 comparing BB vs SB, §P < 0.05 comparing SB vs IVC, with two-way ANOVA. (B) Average cumulative food intake after social isolation of adult male offspring. ***P < 0.001 comparing BB vs both controls with two-way ANOVA. (C) Distance traveled by the mice during a 6-min open field test. (D) Time spent running the wheel, measured during 25-min sessions/day during 3 consecutive days. (E) Changes in body weight between the first and the third testing day. *P < 0.05 comparing BB vs both controls with one-way ANOVA.

Citation: Reproduction 161, 1; 10.1530/REP-20-0253

Figure 4
Figure 4

Male mice subjected to blastomere removal display high sucrose preference in a 4-day two-bottle choice test and low time of immobility in the tail suspension test (n ≥ 10 mice/sex/group). (A) Sucrose preference was measured as the percentage of saccharine solution drunk by the mice compared to the total amount of liquid intake per day. *P < 0.05 comparing BB vs IVC on days 1–3, and BB vs SB on all 4 days, with two-way ANOVA. (B) Average time of immobility in a 6-min tail suspension test. *P < 0.05 comparing BB vs both controls with one-way ANOVA.

Citation: Reproduction 161, 1; 10.1530/REP-20-0253

Altered levels of molecular regulators of energy homeostasis in BB-derived male offspring

Circulating leptin was increased in BB compared to IVC mice (Fig. 5A) and compared to both control groups when measured in fat samples (Fig. 5B). There were no significant differences in leptin mRNA expression measured in fat tissue (Fig. 5C). Hypothalamic gene expression of the leptin receptor was comparable among the groups (Fig. 5D). Similarly, there were no statistically significant differences in hypothalamic mRNA expression of the genes neuropeptide Y (Npy), growth hormone (Gh), or hypocretin (Hcrt), which are also involved in the hypothalamic regulation of energy homeostasis (Fig. 5E, F and G). However, BB male mice showed decreased expression of the ghrelin receptor (Ghsr) in the hypothalamus compared to IVC controls (Fig. 5H).

Figure 5
Figure 5

Male mice subjected to blastomere removal display hyperleptinemia and high leptin expression in adipose tissues, as well as lower mRNA expression of ghrelin receptor in the hypothalamus (n = 9 males/group). (A and B) Leptin protein expression measured by ELISA in blood and fat tissues, respectively. *P < 0.05 and **P < 0.01 with one-way ANOVA or Kruskal–Wallis test. (C, D, E, F, G and H) mRNA levels of the leptin gene in the fat pads, and hypothalamic expression of Npy, Gh, Hcrt, and ghrelin receptor measured by quantitative real-time PCR. *P < 0.05 comparing BB vs IVC with Kruskal–Wallis test.

Citation: Reproduction 161, 1; 10.1530/REP-20-0253

Discussion

The present study shows that blastomere removal from 8-cell mouse embryos is associated with altered regulation of energy homeostasis and obesity in male offspring obtained after embryo transfer. The results indicate that the absence of the blastomere is the reprogramming factor that triggers the obese phenotype, rather than other manipulations. Male mice derived from blastomere-deprived embryos grew faster than controls (SB and IVC) beginning in the early postnatal days and displayed lifelong body weight gain, with increased visceral adiposity compared to controls. This phenotype was accompanied by higher levels of food intake and resistance to physiological weight loss following physical exercise, as observed in mouse models of human metabolic disorders (Ellacott etal. 2010).

Intriguingly, the effects of blastomere removal on offspring phenotype were sex-specific, since female BB-derived offspring were statistically indistinguishable from SB and IVC controls. Sexual dimorphism contributes to subtle differences in developmental programming, health, and disease predisposition. The increased vulnerability of the male embryo/fetus to maternal stress, nutrition, and exposure to drugs and pollutants has been frequently reported (reviewed in Gabory etal. 2009), and also following manipulations of the embryonic microenvironment (Aiken & Ozanne 2013). Several lines of evidence suggest that the placenta may play a key role in the sex-specific response to environmental insults (Gallou-Kabani etal. 2010, Gabory etal. 2013, Saoi etal. 2020). BB has been shown to affect placental development and functions (Sugawara etal. 2012, Sato etal. 2014, Yao etal. 2016), although sex differences have not been explored. How BB affects male and female placental and fetal development differently may shed light on sex-specific vulnerabilities.

Male mice derived from embryos cultured in KSOM, including all in vitro cultured groups (IVC, SB, and BB), were heavier than mice that had developed completely in vivo. This result indicates that a 1-day exposure of mouse embryos to KSOM affects body weight and adiposity and that blastomere removal provides a further reprogramming hit, leading to an exacerbated obese phenotype. It is important to note that mice obtained by either sham biopsy or from embryos cultured only in vitro were statistically indistinguishable for most of the outcomes examined, suggesting that the micromanipulation itself does not significantly influence developmental programming if the blastomere is immediately returned to its embryonic environment. These results suggest that embryonic reprogramming occurring after BB is amenable to the absence of the single blastomere, rather than the physical damage inflicted to the embryo by the micromanipulation. Sugawara et al. had previously shown that the placental defects induced by BB are not observed in SB embryos (Sugawara etal. 2012), thus supporting the hypothesis that BB’s negative consequences are due to blastomere removal rather than other BB procedural perturbations. Conversely, previous studies did not investigate the effects of SB, whereas it was shown that mice derived from BB embryos display phenotypic changes compared to unmanipulated IVC control embryos (Yu etal. 2009, Yan etal. 2013, Sampino etal. 2014, Wu etal. 2014, Gu etal. 2018). Overall, the present study contributes to understanding the causal link between cell number integrity and how its reduction by BB may influence embryonic developmental programs.

The increased body weight and adiposity observed in BB male mice was accompanied by a subset of behavioral alterations linked to energy homeostasis regulation. For instance, food intake and sucrose preference were increased in BB mice, indicating a tendency toward higher energy intake. Moreover, although locomotion and wheel running, as measures of energy expenditure, were not affected by blastomere deprivation, the derived mice were resistant to losing weight and showed high food intake after social isolation, as well as steady body weight gain after 3 days of physical exercise, while control animals showed a decline in these parameters. Muscle metabolism and functions are known to influence energy allocation and consequently body weight gain/loss, strength, and motor coordination. BB may have concurrently affected muscle development, thus limiting the interpretation of some of the behavioral and physiological outcomes reported here. Nevertheless, BB mice also displayed increased time spent moving in the tail suspension test, thus indicating a tendency of BB mice to spend more time overcoming physiological immobility in an unescapable situation (Cryan etal. 2005). In contrast, immobility was observed at a higher rate after tail suspension in mice developed from IVC and SB control embryos. The short time of immobility and high preference for sucrose reflect low predispositions to depression- and anhedonia-like behaviors, respectively (Ferreira etal. 2018), indicating that blastomere removal may affect the development of the brain areas regulating those behaviors. A comprehensive understanding of the metabolic status induced by BB could be achieved by studying indirect calorimetry, including oxygen consumption and carbon dioxide production, respiratory exchange ratio and heat production, as well as monitoring glucose and lipid metabolisms.

At the molecular level, a higher expression of the hormone leptin was observed in the perigonadal adipose tissue of mice derived from biopsied embryos compared to both control groups. Although the mRNA expression of the leptin gene in fat tissues was not concurrently increased, the levels of circulating leptin were higher in BB mice compared to mice derived from IVC embryos. Common forms of obesity are typically associated with elevated leptin and resistance to leptin’s action on the hypothalamus (Frederich etal. 1995, Considine etal. 1996). Although the mice derived from blastomere-deprived embryos had hyperleptinemia, we found no differences in the mRNA expression of the hypothalamic leptin receptor gene nor of other homeostasis-related genes expressed in the hypothalamus. Nonetheless, BB mice had higher hypothalamic mRNA expression of the Ghsr gene, which is known to function as a ghrelin receptor (Yin etal. 2014). It has been shown that leptin and ghrelin levels may affect food intake, body weight, and energy expenditure, as well as behavior and emotions (Yamada etal. 2011, Huang etal. 2017). Considering this link, the behavioral phenotype of mice derived from blastomere-deprived embryos, associated with leptin and ghrelin alterations, suggest that blastomere removal may affect the development of the hypothalamus-adipose tissue axes and its functions.

How BB influences developmental programming and the postnatal phenotype is not well known. As early as the 4-cell stage, individual mouse blastomeres differ in their developmental fate (Piotrowska-Nitsche etal. 2005, Tarkowski etal. 2010, Mihajlović & Bruce 2017) and in their transcriptomic profile and epigenetic features (Torres-Padilla etal. 2007, Tang etal. 2011). Thus, the removal of one cell could potentially deprive the embryo of important developmental determinants for establishing a healthy phenotype. Biopsy is also likely to affect developing embryos by interfering with cell–cell adhesion (Damsky etal. 1983, Johnson etal. 1986) and gap and tight junctions (Ducibella & Anderson 1975, Lo & Gilula 1979, Lee etal. 1987). Our results demonstrate that BB does not affect cell adhesion integrity, although decreased F-actin expression was observed when the blastomere was reintroduced into SB embryos. In agreement with previous studies (Sugawara & Ward 2013, Sepulveda-Rincon etal. 2017), BB embryos had a decreased number of cells in both the ICM and the TE, compared to IVC embryos, although their gross morphology was not significantly influenced by the manipulation performed. Rather, embryonic reprogramming occurred, and the obese phenotype emerged in the derived offspring only when the blastomere was discarded. Since SB did not affect offspring phenotype, these results suggest that embryonic reprogramming takes place independent of blastocyst cell number and cell–cell adhesion integrity. BB deprives the embryo of 1/8 of its cellular assortment, leaving the remaining 7-cell embryo with one fewer unit collecting and producing energy supplies and one fewer unit to nourish. We speculate that the remaining blastomeres are forced to adjust their homeostatic pathways to adapt to the metabolic changes aroused by blastomere deprivation. The results presented here indicate that, eventually, such re-adaptation persists until postnatal life, affecting developmental outcomes related to important energy homeostasis regulators. Further studies will be necessary to ascertain whether BB causes metabolic imbalance already at preimplantation stages, as well as in placentas and fetal tissues.

Genetic diagnosis and screening of human preimplantation embryos have been widely implemented in the clinical setting for selection purposes. The results reported here indicate that the removal of one single blastomere from preimplantation embryos may predispose male mouse offspring to metabolic and behavioral disorders. However, mouse and human embryos differ in many of their metabolic and transcriptomic features, and blastomeres of the two species differ in their developmental fates and programming. Therefore, the effects of blastomere deprivation may be different in humans, whose specific characteristics may make the embryo resilient or vulnerable to environmental perturbations occurring during preimplantation stages. Moreover, the present study does not recapitulate the full protocol applied to humans in IVF clinics, which includes in vitro fertilization and embryo culture at earlier developmental stages. Nevertheless, the experiments presented here indicate that the removal of one cell may represents a reprogramming hit, such that depriving the embryo of one cell at such an early stage might affect the later metabolic development of a healthy phenotype. In light of this evidence, we suggest the full transition to less invasive approaches for PGT implementation in humans than BB (Magli etal. 2016, Shamonki etal. 2016, Farra etal. 2018), together with adequate risk assessment of the offspring’s health in randomized controlled trials (Kuiper etal. 2018).

Supplementary materials

This is linked to the online version of the paper at https://doi.org/10.1530/REP-20-0253.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported by the Polish National Science Center (2014/15/D/NZ4/04274 to S S) and by the Institute of Genetics and Animal Biotechnology of the Polish Academy of Sciences (S.III.1.3 to J A M). The funding agencies did not have any role in the design of the study; in the collection, analysis, nor interpretation of data; nor in the writing of the manuscript.

Author contribution statement

S S and J A M conceived and designed the study. M K, J P-S, and D W carried out the embryonic manipulations. M K, W F, M Z, R A, and S S carried out the behavioral studies. M K, W F, A M S, and R R S conducted the molecular analyses. D W and S S analyzed the blastocyst immunostaining. S S and G D carried out the statistical analyses. S S and J A M coordinated and supervised the study. S S and M K drafted the manuscript. J A M, A M S, and G D helped draft and critically revised the manuscript. All authors gave final approval for publication and agree to be held accountable for the work performed therein.

References

  • Aiken CE & Ozanne SE 2013 Sex differences in developmental programming models. Reproduction 145 R1R13. (https://doi.org/10.1530/REP-11-0489)

    • Search Google Scholar
    • Export Citation
  • Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW & Shipley GL et al. 2009 The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry 55 611622. (https://doi.org/10.1373/clinchem.2008.112797)

    • Search Google Scholar
    • Export Citation
  • Ceelen M, van Weissenbruch MM, Vermeiden JPW, van Leeuwen FE & Delemarre-van de Waal HA 2008 Cardiometabolic differences in children born after in vitro fertilization: follow-up study. Journal of Clinical Endocrinology and Metabolism 93 16821688. (https://doi.org/10.1210/jc.2007-2432)

    • Search Google Scholar
    • Export Citation
  • Considine RV, Sinha MK, Heiman ML, Kriauciunas A, Stephens TW, Nyce MR, Ohannesian JP, Marco CC, McKee LJ & Bauer TL 1996 Serum immunoreactive-leptin concentrations in normal-weight and obese humans. New England Journal of Medicine 334 292295. (https://doi.org/10.1056/NEJM199602013340503)

    • Search Google Scholar
    • Export Citation
  • Cryan JF, Mombereau C & Vassout A 2005 The tail suspension test as a model for assessing antidepressant activity: review of pharmacological and genetic studies in mice. Neuroscience and Biobehavioral Reviews 29 571625. (https://doi.org/10.1016/j.neubiorev.2005.03.009)

    • Search Google Scholar
    • Export Citation
  • Damsky CH, Richa J, Solter D, Knudsen K & Buck CA 1983 Identification and purification of a cell surface glycoprotein mediating intercellular adhesion in embryonic and adult tissue. Cell 34 455466. (https://doi.org/10.1016/0092-8674(83)90379-3)

    • Search Google Scholar
    • Export Citation
  • Davies MJ, Moore VM, Willson KJ, Van Essen P, Priest K, Scott H, Haan EA & Chan A 2012 Reproductive technologies and the risk of birth defects. New England Journal of Medicine 366 18031813. (https://doi.org/10.1056/NEJMoa1008095)

    • Search Google Scholar
    • Export Citation
  • De Rycke M, Goossens V, Kokkali G, Meijer-Hoogeveen M, Coonen E & Moutou C 2017 ESHRE PGD Consortium data collection XIV-XV: cycles from January 2011 to December 2012 with pregnancy follow-up to October 2013. Human Reproduction 32 19741994. (https://doi.org/10.1093/humrep/dex265)

    • Search Google Scholar
    • Export Citation
  • Desmyttere S, De Rycke M, Staessen C, Liebaers I, De Schrijver F, Verpoest W, Haentjens P & Bonduelle M 2012 Neonatal follow-up of 995 consecutively born children after embryo biopsy for PGD. Human Reproduction 27 288293. (https://doi.org/10.1093/humrep/der360)

    • Search Google Scholar
    • Export Citation
  • Ducibella T & Anderson E 1975 Cell shape and membrane changes in the eight-cell mouse embryo: prerequisites for morphogenesis of the blastocyst. Developmental Biology 47 4558. (https://doi.org/10.1016/0012-1606(75)90262-6)

    • Search Google Scholar
    • Export Citation
  • Dumoulin JC, Land JA, Van Montfoort AP, Nelissen EC, Coonen E, Derhaag JG, Schreurs IL, Dunselman GA, Kester AD & Geraedts JP et al. 2010 Effect of in vitro culture of human embryos on birthweight of newborns. Human Reproduction 25 605612. (https://doi.org/10.1093/humrep/dep456)

    • Search Google Scholar
    • Export Citation
  • Ellacott KLJ, Morton GJ, Woods SC, Tso P & Schwartz MW 2010 Assessment of feeding behavior in laboratory mice. Cell Metabolism 12 1017. (https://doi.org/10.1016/j.cmet.2010.06.001)

    • Search Google Scholar
    • Export Citation
  • Farra C, Choucair F & Awwad J 2018 Non-invasive pre-implantation genetic testing of human embryos: an emerging concept. Human Reproduction 33 21622167. (https://doi.org/10.1093/humrep/dey314)

    • Search Google Scholar
    • Export Citation
  • Ferreira MF, Castanheira L, Sebastião AM & Telles-Correia D 2018 Depression assessment in clinical trials and pre-clinical tests: a critical review. Current Topics in Medicinal Chemistry 18 16771703. (https://doi.org/10.2174/1568026618666181115095920)

    • Search Google Scholar
    • Export Citation
  • Frederich RC, Hamann A, Anderson S, Löllmann B, Lowell BB & Flier JS 1995 Leptin levels reflect body lipid content in mice: evidence for diet-induced resistance to leptin action. Nature Medicine 1 13111314. (https://doi.org/10.1038/nm1295-1311)

    • Search Google Scholar
    • Export Citation
  • Gabory A, Attig L & Junien C 2009 Sexual dimorphism in environmental epigenetic programming. Molecular and Cellular Endocrinology 304 818. (https://doi.org/10.1016/j.mce.2009.02.015)

    • Search Google Scholar
    • Export Citation
  • Gabory A, Roseboom TJ, Moore T, Moore LG & Junien C 2013 Placental contribution to the origins of sexual dimorphism in health and diseases: sex chromosomes and epigenetics. Biology of Sex Differences 4 5. (https://doi.org/10.1186/2042-6410-4-5)

    • Search Google Scholar
    • Export Citation
  • Gallou-Kabani C, Gabory A, Tost J, Karimi M, Mayeur S, Lesage J, Boudadi E, Gross MS, Taurelle J & Vigé A et al. 2010 Sex- and diet-specific changes of imprinted gene expression and DNA methylation in mouse placenta under a high-fat diet. PLoS ONE 5 e14398. (https://doi.org/10.1371/journal.pone.0014398)

    • Search Google Scholar
    • Export Citation
  • Gu L, Zhang J, Zheng M, Dong G, Xu J, Zhang W, Wu Y, Yang Y & Zhu H 2018 A potential high risk for fatty liver disease was found in mice generated after assisted reproductive techniques. Journal of Cellular Biochemistry 119 18991910. (https://doi.org/10.1002/jcb.26351)

    • Search Google Scholar
    • Export Citation
  • Handyside AH, Kontogianni EH, Hardy K & Winston RM 1990 Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature 344 768770. (https://doi.org/10.1038/344768a0)

    • Search Google Scholar
    • Export Citation
  • Hansen M, Kurinczuk JJ, Milne E, de Klerk N & Bower C 2013 Assisted reproductive technology and birth defects: a systematic review and meta-analysis. Human Reproduction Update 19 330353. (https://doi.org/10.1093/humupd/dmt006)

    • Search Google Scholar
    • Export Citation
  • Hart R & Norman RJ 2013a The longer-term health outcomes for children born as a result of IVF treatment. Part II – Mental health and development outcomes. Human Reproduction Update 19 244250. (https://doi.org/10.1093/humupd/dmt002)

    • Search Google Scholar
    • Export Citation
  • Hart R & Norman RJ 2013b The longer-term health outcomes for children born as a result of IVF treatment. Part I – General health outcomes. Human Reproduction Update 19 232243. (https://doi.org/10.1093/humupd/dms062)

    • Search Google Scholar
    • Export Citation
  • Heijligers M, van Montfoort A, Meijer-Hoogeveen M, Broekmans F, Bouman K, Homminga I, Dreesen J, Paulussen A, Engelen J & Coonen E et al. 2018 Perinatal follow-up of children born after preimplantation genetic diagnosis between 1995 and 2014. Journal of Assisted Reproduction and Genetics 35 19952002. (https://doi.org/10.1007/s10815-018-1286-2)

    • Search Google Scholar
    • Export Citation
  • Helmerhorst FM, Perquin DAM, Donker D & Keirse MJNC 2004 Perinatal outcome of singletons and twins after assisted conception: a systematic review of controlled studies. BMJ 328 261. (https://doi.org/10.1136/bmj.37957.560278.EE)

    • Search Google Scholar
    • Export Citation
  • Huang HJ, Zhu XC, Han QQ, Wang YL, Yue N, Wang J, Yu R, Li B, Wu GC & Liu Q et al. 2017 Ghrelin alleviates anxiety- and depression-like behaviors induced by chronic unpredictable mild stress in rodents. Behavioural Brain Research 326 3343. (https://doi.org/10.1016/j.bbr.2017.02.040)

    • Search Google Scholar
    • Export Citation
  • Johnson MH, Maro B & Takeichi M 1986 The role of cell adhesion in the synchronization and orientation of polarization in 8-cell mouse blastomeres. Journal of Embryology and Experimental Morphology 93 239255.

    • Search Google Scholar
    • Export Citation
  • Kai CM, Main KM, Andersen AN, Loft A, Chellakooty M, Skakkebaek NE & Juul A 2006 Serum insulin-like growth factor-I (IGF-I) and growth in children born after assisted reproduction. Journal of Clinical Endocrinology and Metabolism 91 43524360. (https://doi.org/10.1210/jc.2006-0701)

    • Search Google Scholar
    • Export Citation
  • Kleijkers SHM, Mantikou E, Slappendel E, Consten D, van Echten-Arends J, Wetzels AM, van Wely M, Smits LJM, van Montfoort APA & Repping S et al. 2016 Influence of embryo culture medium (G5 and HTF) on pregnancy and perinatal outcome after IVF: a multicenter RCT. Human Reproduction 31 22192230. (https://doi.org/10.1093/humrep/dew156)

    • Search Google Scholar
    • Export Citation
  • Koivurova S, Hartikainen AL, Sovio U, Gissler M, Hemminki E & Järvelin MR 2003 Growth, psychomotor development and morbidity up to 3 years of age in children born after IVF. Human Reproduction 18 23282336. (https://doi.org/10.1093/humrep/deg445)

    • Search Google Scholar
    • Export Citation
  • Kuiper D, Bennema A, la Bastide-van Gemert S, Seggers J, Schendelaar P, Mastenbroek S, Hoek A, Heineman MJ, Roseboom TJ & Kok JH et al. 2018 Developmental outcome of 9-year-old children born after PGS: follow-up of a randomized trial. Human Reproduction 33 147155. (https://doi.org/10.1093/humrep/dex337)

    • Search Google Scholar
    • Export Citation
  • Lee S, Gilula NB& Warner AE 1987 Gap junctional communication and compaction during preimplantation stages of mouse development. Cell 51 851860. (https://doi.org/10.1016/0092-8674(87)90108-5)

    • Search Google Scholar
    • Export Citation
  • Lo CW & Gilula NB 1979 Gap junctional communication in the preimplantation mouse embryo. Cell 18 399409. (https://doi.org/10.1016/0092-8674(79)90059-x)

    • Search Google Scholar
    • Export Citation
  • Magli MC, Pomante A, Cafueri G, Valerio M, Crippa A, Ferraretti AP & Gianaroli L 2016 Preimplantation genetic testing: polar bodies, blastomeres, trophectoderm cells, or blastocoelic fluid? Fertility and Sterility 105 676683.e5. (https://doi.org/10.1016/j.fertnstert.2015.11.018)

    • Search Google Scholar
    • Export Citation
  • Middelburg KJ, van der Heide M, Houtzager B, Jongbloed-Pereboom M, Fidler V, Bos AF, Kok J, Hadders-Algra MPGS Follow-up Study Group 2011 Mental, psychomotor, neurologic, and behavioral outcomes of 2-year-old children born after preimplantation genetic screening: follow-up of a randomized controlled trial. Fertility and Sterility 96 165169. (https://doi.org/10.1016/j.fertnstert.2011.04.081)

    • Search Google Scholar
    • Export Citation
  • Mihajlović AI& Bruce AW 2017 The first cell-fate decision of mouse preimplantation embryo development: integrating cell position and polarity. Open Biology 7 29167310. (https://doi.org/10.1098/rsob.170210)

    • Search Google Scholar
    • Export Citation
  • Nagy A 2003 Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory Press.

  • Palermo G, Joris H, Devroey P & Van Steirteghem AC 1992 Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 340 1718. (https://doi.org/10.1016/0140-6736(92)92425-f)

    • Search Google Scholar
    • Export Citation
  • Piotrowska-Nitsche K, Perea-Gomez A, Haraguchi S & Zernicka-Goetz M 2005 Four-cell stage mouse blastomeres have different developmental properties. Development 132 479490. (https://doi.org/10.1242/dev.01602)

    • Search Google Scholar
    • Export Citation
  • Sacks GC, Altarescu G, Guedalia J, Varshaver I, Gilboa T, Levy-Lahad E & Eldar-Geva T 2016 Developmental neuropsychological assessment of 4- to 5-year-old children born following preimplantation genetic diagnosis (PGD): a pilot study. Child Neuropsychology 22 458471. (https://doi.org/10.1080/09297049.2015.1014900)

    • Search Google Scholar
    • Export Citation
  • Sakka SD, Loutradis D, Kanaka-Gantenbein C, Margeli A, Papastamataki M, Papassotiriou I & Chrousos GP 2010 Absence of insulin resistance and low-grade inflammation despite early metabolic syndrome manifestations in children born after in vitro fertilization. Fertility and Sterility 94 16931699. (https://doi.org/10.1016/j.fertnstert.2009.09.049)

    • Search Google Scholar
    • Export Citation
  • Sampino S, Zacchini F, Swiergiel AH, Modlinski AJ, Loi P & Ptak GE 2014 Effects of blastomere biopsy on post-natal growth and behavior in mice. Human Reproduction 29 18751883. (https://doi.org/10.1093/humrep/deu145)

    • Search Google Scholar
    • Export Citation
  • Saoi M, Kennedy KM, Gohir W, Sloboda DM & Britz-McKibbin P 2020 Placental metabolomics for assessment of sex-specific differences in fetal development during normal gestation. Scientific Reports 10 9399. (https://doi.org/10.1038/s41598-020-66222-3)

    • Search Google Scholar
    • Export Citation
  • Sato BLM, Sugawara A, Ward MA & Collier AC 2014 Single blastomere removal from murine embryos is associated with activation of matrix metalloproteinases and Janus kinase/signal transducers and activators of transcription pathways of placental inflammations. Molecular Human Reproduction 20 12471257. (https://doi.org/10.1093/molehr/gau072)

    • Search Google Scholar
    • Export Citation
  • Schendelaar P, Middelburg KJ, Bos AF, Heineman MJ, Kok JH, La Bastide-Van Gemert S, Seggers J, Van den Heuvel ER & Hadders-Algra M 2013 The effect of preimplantation genetic screening on neurological, cognitive and behavioural development in 4-year-old children: follow-up of a RCT. Human Reproduction 28 15081518. (https://doi.org/10.1093/humrep/det073)

    • Search Google Scholar
    • Export Citation
  • Sepulveda-Rincon LP, Islam N, Marsters P, Campbell BK, Beaujean N & Maalouf WE 2017 Embryo cell allocation patterns are not altered by biopsy but can be linked with further development. Reproduction 154 807814. (https://doi.org/10.1530/REP-17-0514)

    • Search Google Scholar
    • Export Citation
  • Shamonki MI, Jin H, Haimowitz Z & Liu L 2016 Proof of concept: preimplantation genetic screening without embryo biopsy through analysis of cell-free DNA in spent embryo culture media. Fertility and Sterility 106 13121318. (https://doi.org/10.1016/j.fertnstert.2016.07.1112)

    • Search Google Scholar
    • Export Citation
  • Steptoe PC & Edwards RG 1978 Birth after the reimplantation of a human embryo. Lancet 2 366. (https://doi.org/10.1016/s0140-6736(78)92957-4)

    • Search Google Scholar
    • Export Citation
  • Sugawara A & Ward MA 2013 Biopsy of embryos produced by in vitro fertilization affects development in C57BL/6 mouse strain. Theriogenology 79 234241. (https://doi.org/10.1016/j.theriogenology.2012.08.007)

    • Search Google Scholar
    • Export Citation
  • Sugawara A, Sato B, Bal E, Collier AC & Ward MA 2012 Blastomere removal from cleavage-stage mouse embryos alters steroid metabolism during pregnancy. Biology of Reproduction 87 4, 19. (https://doi.org/10.1095/biolreprod.111.097444)

    • Search Google Scholar
    • Export Citation
  • Tang F, Barbacioru C, Nordman E, Bao S, Lee C, Wang X, Tuch BB, Heard E, Lao K & Surani MA 2011 Deterministic and stochastic allele specific gene expression in single mouse blastomeres. PLoS ONE 6 e21208. (https://doi.org/10.1371/journal.pone.0021208)

    • Search Google Scholar
    • Export Citation
  • Tarkowski AK, Suwińska A, Czołowska R & Ożdżeński W 2010 Individual blastomeres of 16- and 32-cell mouse embryos are able to develop into foetuses and mice. Developmental Biology 348 190198. (https://doi.org/10.1016/j.ydbio.2010.09.022)

    • Search Google Scholar
    • Export Citation
  • Thomaidis L, Kitsiou-Tzeli S, Critselis E, Drandakis H, Touliatou V, Mantoudis S, Leze E, Destouni A, Traeger-Synodinos J & Kafetzis D et al. 2012 Psychomotor development of children born after preimplantation genetic diagnosis and parental stress evaluation. World Journal of Pediatrics 8 309316. (https://doi.org/10.1007/s12519-012-0374-0)

    • Search Google Scholar
    • Export Citation
  • Torres-Padilla ME, Parfitt DE, Kouzarides T & Zernicka-Goetz M 2007 Histone arginine methylation regulates pluripotency in the early mouse embryo. Nature 445 214218. (https://doi.org/10.1038/nature05458)

    • Search Google Scholar
    • Export Citation
  • Wen J, Jiang J, Ding C, Dai J, Liu Y, Xia Y, Liu J & Hu Z 2012 Birth defects in children conceived by in vitro fertilization and intracytoplasmic sperm injection: a meta-analysis. Fertility and Sterility 97 1331 .e11337 .e1. (https://doi.org/10.1016/j.fertnstert.2012.02.053)

    • Search Google Scholar
    • Export Citation
  • Winter C, Van Acker F, Bonduelle M, Desmyttere S & Nekkebroeck J 2015 Psychosocial development of full term singletons, born after preimplantation genetic diagnosis (PGD) at preschool age and family functioning: a prospective case-controlled study and multi-informant approach. Human Reproduction 30 11221136. (https://doi.org/10.1093/humrep/dev036)

    • Search Google Scholar
    • Export Citation
  • Wu Y, Lv Z, Yang Y, Dong G, Yu Y, Cui Y, Tong M, Wang L, Zhou Z & Zhu H et al. 2014 Blastomere biopsy influences epigenetic reprogramming during early embryo development, which impacts neural development and function in resulting mice. Cellular and Molecular Life Sciences 71 17611774. (https://doi.org/10.1007/s00018-013-1466-2)

    • Search Google Scholar
    • Export Citation
  • Yamada N, Katsuura G, Ochi Y, Ebihara K, Kusakabe T, Hosoda K & Nakao K 2011 Impaired CNS leptin action is implicated in depression associated with obesity. Endocrinology 152 26342643. (https://doi.org/10.1210/en.2011-0004)

    • Search Google Scholar
    • Export Citation
  • Yao Q, Chen L, Liang Y, Sui L, Guo L, Zhou J, Fan K, Jing J, Zhang Y & Yao B 2016 Blastomere removal from cleavage-stage mouse embryos alters placental function, which is associated with placental oxidative stress and inflammation. Scientific Reports 6 25023. (https://doi.org/10.1038/srep25023)

    • Search Google Scholar
    • Export Citation
  • Yin Y, Li Y & Zhang W 2014 The growth hormone secretagogue receptor: its intracellular signaling and regulation. International Journal of Molecular Sciences 15 48374855. (https://doi.org/10.3390/ijms15034837)

    • Search Google Scholar
    • Export Citation
  • Yu Y, Wu J, Fan Y, Lv Z, Guo X, Zhao C, Zhou R, Zhang Z, Wang F & Xiao M et al. 2009 Evaluation of blastomere biopsy using a mouse model indicates the potential high risk of neurodegenerative disorders in the offspring. Molecular and Cellular Proteomics 8 14901500. (https://doi.org/10.1074/mcp.M800273-MCP200)

    • Search Google Scholar
    • Export Citation
  • Zeng Y, Lv Z, Gu L, Wang L, Zhou Z, Zhu H, Zhou Q & Sha J 2013 Preimplantation genetic diagnosis (PGD) influences adrenal development and response to cold stress in resulting mice. Cell and Tissue Research 354 729741. (https://doi.org/10.1007/s00441-013-1728-1)

    • Search Google Scholar
    • Export Citation
  • Zhao HC, Zhao Y, Li M, Yan J, Li L, Li R, Liu P, Yu Y & Qiao J 2013 Aberrant epigenetic modification in murine brain tissues of offspring from preimplantation genetic diagnosis blastomere biopsies. Biology of Reproduction 89 117. (https://doi.org/10.1095/biolreprod.113.109926)

    • Search Google Scholar
    • Export Citation

 

  • Collapse
  • Expand

     An official journal of

    Society for Reproduction and Fertility

 

  • View in gallery
    Figure 1

    Preimplantation development to the blastocyst stage of BB, SB, and IVC embryos. Representative microphotographs of (A) high-quality blastocysts developed from BB, SB, and IVC embryos, compared to (B) low-grade blastocysts, which were excluded from the long-term study and not transferred, presenting multiple blastocoel cavities and signs of fragmentation and vacuolization. Scale bar = 0.40 mm. (C) Confocal images of IVC, BB, and SB blastocysts showing immunolocalization of E-cadherin (white signal) and F-actin (red signal). Scale bar = 0.20 mm. (D) Graphs showing the number of cells in the ICM and the TE, as well as the ICM/TE ratio. *P < 0.01 comparing IVC vs SB or BB with one-way ANOVA (n ≥ 9 embryos/experimental group).

  • View in gallery
    Figure 2

    Blastomere removal causes increased body weight and adiposity in pups and male adult offspring. (A) Average body weight measured in 2- to 20-day-old pups derived from the different experimental treatments (BB, n = 62; SB, n = 39; IVC, n = 41; in vivo, n = 19). **P < 0.01 comparing BB vs all controls on days 8–20, and on days 10–20 comparing IVC and SB vs in vivo mice with two-way ANOVA. (B and C) Average body weight measured in 4- to 17-week-old male and female offspring, respectively (males: BB, n = 39; SB, n = 19; IVC, n = 20; in vivo, n = 10; females: BB, n = 21; SB, n = 19; IVC, n = 21; in vivo, n = 9). **P < 0.01 comparing BB vs SB in weeks 5–17; #P < 0.05 comparing SB vs IVC in weeks 15–17; §P < 0.01 comparing IVC vs in vivo-derived male mice; *P < 0.05 comparing BB vs in vivo in weeks 7–17. (D) Average weight of perigonadal fat, kidneys with their fat pad, and liver collected from male offspring of the different groups (n = 9/group). **P < 0.01 comparing BB vs IVC, and ***P < 0.01 comparing BB vs both controls with one-way ANOVA.

  • View in gallery
    Figure 3

    Blastomere removal influences daily body weight loss and food intake in male offspring after social isolation (n ≥ 10 mice/sex/group). (A) Daily weight changes along 9 days of monitoring after social isolation. **P < 0.01 comparing BB vs IVC, #P < 0.01 comparing BB vs SB, §P < 0.05 comparing SB vs IVC, with two-way ANOVA. (B) Average cumulative food intake after social isolation of adult male offspring. ***P < 0.001 comparing BB vs both controls with two-way ANOVA. (C) Distance traveled by the mice during a 6-min open field test. (D) Time spent running the wheel, measured during 25-min sessions/day during 3 consecutive days. (E) Changes in body weight between the first and the third testing day. *P < 0.05 comparing BB vs both controls with one-way ANOVA.

  • View in gallery
    Figure 4

    Male mice subjected to blastomere removal display high sucrose preference in a 4-day two-bottle choice test and low time of immobility in the tail suspension test (n ≥ 10 mice/sex/group). (A) Sucrose preference was measured as the percentage of saccharine solution drunk by the mice compared to the total amount of liquid intake per day. *P < 0.05 comparing BB vs IVC on days 1–3, and BB vs SB on all 4 days, with two-way ANOVA. (B) Average time of immobility in a 6-min tail suspension test. *P < 0.05 comparing BB vs both controls with one-way ANOVA.

  • View in gallery
    Figure 5

    Male mice subjected to blastomere removal display hyperleptinemia and high leptin expression in adipose tissues, as well as lower mRNA expression of ghrelin receptor in the hypothalamus (n = 9 males/group). (A and B) Leptin protein expression measured by ELISA in blood and fat tissues, respectively. *P < 0.05 and **P < 0.01 with one-way ANOVA or Kruskal–Wallis test. (C, D, E, F, G and H) mRNA levels of the leptin gene in the fat pads, and hypothalamic expression of Npy, Gh, Hcrt, and ghrelin receptor measured by quantitative real-time PCR. *P < 0.05 comparing BB vs IVC with Kruskal–Wallis test.

  • Aiken CE & Ozanne SE 2013 Sex differences in developmental programming models. Reproduction 145 R1R13. (https://doi.org/10.1530/REP-11-0489)

    • Search Google Scholar
    • Export Citation
  • Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW & Shipley GL et al. 2009 The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry 55 611622. (https://doi.org/10.1373/clinchem.2008.112797)

    • Search Google Scholar
    • Export Citation
  • Ceelen M, van Weissenbruch MM, Vermeiden JPW, van Leeuwen FE & Delemarre-van de Waal HA 2008 Cardiometabolic differences in children born after in vitro fertilization: follow-up study. Journal of Clinical Endocrinology and Metabolism 93 16821688. (https://doi.org/10.1210/jc.2007-2432)

    • Search Google Scholar
    • Export Citation
  • Considine RV, Sinha MK, Heiman ML, Kriauciunas A, Stephens TW, Nyce MR, Ohannesian JP, Marco CC, McKee LJ & Bauer TL 1996 Serum immunoreactive-leptin concentrations in normal-weight and obese humans. New England Journal of Medicine 334 292295. (https://doi.org/10.1056/NEJM199602013340503)

    • Search Google Scholar
    • Export Citation
  • Cryan JF, Mombereau C & Vassout A 2005 The tail suspension test as a model for assessing antidepressant activity: review of pharmacological and genetic studies in mice. Neuroscience and Biobehavioral Reviews 29 571625. (https://doi.org/10.1016/j.neubiorev.2005.03.009)

    • Search Google Scholar
    • Export Citation
  • Damsky CH, Richa J, Solter D, Knudsen K & Buck CA 1983 Identification and purification of a cell surface glycoprotein mediating intercellular adhesion in embryonic and adult tissue. Cell 34 455466. (https://doi.org/10.1016/0092-8674(83)90379-3)

    • Search Google Scholar
    • Export Citation
  • Davies MJ, Moore VM, Willson KJ, Van Essen P, Priest K, Scott H, Haan EA & Chan A 2012 Reproductive technologies and the risk of birth defects. New England Journal of Medicine 366 18031813. (https://doi.org/10.1056/NEJMoa1008095)

    • Search Google Scholar
    • Export Citation
  • De Rycke M, Goossens V, Kokkali G, Meijer-Hoogeveen M, Coonen E & Moutou C 2017 ESHRE PGD Consortium data collection XIV-XV: cycles from January 2011 to December 2012 with pregnancy follow-up to October 2013. Human Reproduction 32 19741994. (https://doi.org/10.1093/humrep/dex265)

    • Search Google Scholar
    • Export Citation
  • Desmyttere S, De Rycke M, Staessen C, Liebaers I, De Schrijver F, Verpoest W, Haentjens P & Bonduelle M 2012 Neonatal follow-up of 995 consecutively born children after embryo biopsy for PGD. Human Reproduction 27 288293. (https://doi.org/10.1093/humrep/der360)

    • Search Google Scholar
    • Export Citation
  • Ducibella T & Anderson E 1975 Cell shape and membrane changes in the eight-cell mouse embryo: prerequisites for morphogenesis of the blastocyst. Developmental Biology 47 4558. (https://doi.org/10.1016/0012-1606(75)90262-6)

    • Search Google Scholar
    • Export Citation
  • Dumoulin JC, Land JA, Van Montfoort AP, Nelissen EC, Coonen E, Derhaag JG, Schreurs IL, Dunselman GA, Kester AD & Geraedts JP et al. 2010 Effect of in vitro culture of human embryos on birthweight of newborns. Human Reproduction 25 605612. (https://doi.org/10.1093/humrep/dep456)

    • Search Google Scholar
    • Export Citation
  • Ellacott KLJ, Morton GJ, Woods SC, Tso P & Schwartz MW 2010 Assessment of feeding behavior in laboratory mice. Cell Metabolism 12 1017. (https://doi.org/10.1016/j.cmet.2010.06.001)

    • Search Google Scholar
    • Export Citation
  • Farra C, Choucair F & Awwad J 2018 Non-invasive pre-implantation genetic testing of human embryos: an emerging concept. Human Reproduction 33 21622167. (https://doi.org/10.1093/humrep/dey314)

    • Search Google Scholar
    • Export Citation
  • Ferreira MF, Castanheira L, Sebastião AM & Telles-Correia D 2018 Depression assessment in clinical trials and pre-clinical tests: a critical review. Current Topics in Medicinal Chemistry 18 16771703. (https://doi.org/10.2174/1568026618666181115095920)

    • Search Google Scholar
    • Export Citation
  • Frederich RC, Hamann A, Anderson S, Löllmann B, Lowell BB & Flier JS 1995 Leptin levels reflect body lipid content in mice: evidence for diet-induced resistance to leptin action. Nature Medicine 1 13111314. (https://doi.org/10.1038/nm1295-1311)

    • Search Google Scholar
    • Export Citation
  • Gabory A, Attig L & Junien C 2009 Sexual dimorphism in environmental epigenetic programming. Molecular and Cellular Endocrinology 304 818. (https://doi.org/10.1016/j.mce.2009.02.015)

    • Search Google Scholar
    • Export Citation
  • Gabory A, Roseboom TJ, Moore T, Moore LG & Junien C 2013 Placental contribution to the origins of sexual dimorphism in health and diseases: sex chromosomes and epigenetics. Biology of Sex Differences 4 5. (https://doi.org/10.1186/2042-6410-4-5)

    • Search Google Scholar
    • Export Citation
  • Gallou-Kabani C, Gabory A, Tost J, Karimi M, Mayeur S, Lesage J, Boudadi E, Gross MS, Taurelle J & Vigé A et al. 2010 Sex- and diet-specific changes of imprinted gene expression and DNA methylation in mouse placenta under a high-fat diet. PLoS ONE 5 e14398. (https://doi.org/10.1371/journal.pone.0014398)

    • Search Google Scholar
    • Export Citation
  • Gu L, Zhang J, Zheng M, Dong G, Xu J, Zhang W, Wu Y, Yang Y & Zhu H 2018 A potential high risk for fatty liver disease was found in mice generated after assisted reproductive techniques. Journal of Cellular Biochemistry 119 18991910. (https://doi.org/10.1002/jcb.26351)

    • Search Google Scholar
    • Export Citation
  • Handyside AH, Kontogianni EH, Hardy K & Winston RM 1990 Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature 344 768770. (https://doi.org/10.1038/344768a0)

    • Search Google Scholar
    • Export Citation
  • Hansen M, Kurinczuk JJ, Milne E, de Klerk N & Bower C 2013 Assisted reproductive technology and birth defects: a systematic review and meta-analysis. Human Reproduction Update 19 330353. (https://doi.org/10.1093/humupd/dmt006)

    • Search Google Scholar
    • Export Citation
  • Hart R & Norman RJ 2013a The longer-term health outcomes for children born as a result of IVF treatment. Part II – Mental health and development outcomes. Human Reproduction Update 19 244250. (https://doi.org/10.1093/humupd/dmt002)

    • Search Google Scholar
    • Export Citation
  • Hart R & Norman RJ 2013b The longer-term health outcomes for children born as a result of IVF treatment. Part I – General health outcomes. Human Reproduction Update 19 232243. (https://doi.org/10.1093/humupd/dms062)

    • Search Google Scholar
    • Export Citation
  • Heijligers M, van Montfoort A, Meijer-Hoogeveen M, Broekmans F, Bouman K, Homminga I, Dreesen J, Paulussen A, Engelen J & Coonen E et al. 2018 Perinatal follow-up of children born after preimplantation genetic diagnosis between 1995 and 2014. Journal of Assisted Reproduction and Genetics 35 19952002. (https://doi.org/10.1007/s10815-018-1286-2)

    • Search Google Scholar
    • Export Citation
  • Helmerhorst FM, Perquin DAM, Donker D & Keirse MJNC 2004 Perinatal outcome of singletons and twins after assisted conception: a systematic review of controlled studies. BMJ 328 261. (https://doi.org/10.1136/bmj.37957.560278.EE)

    • Search Google Scholar
    • Export Citation
  • Huang HJ, Zhu XC, Han QQ, Wang YL, Yue N, Wang J, Yu R, Li B, Wu GC & Liu Q et al. 2017 Ghrelin alleviates anxiety- and depression-like behaviors induced by chronic unpredictable mild stress in rodents. Behavioural Brain Research 326 3343. (https://doi.org/10.1016/j.bbr.2017.02.040)

    • Search Google Scholar
    • Export Citation
  • Johnson MH, Maro B & Takeichi M 1986 The role of cell adhesion in the synchronization and orientation of polarization in 8-cell mouse blastomeres. Journal of Embryology and Experimental Morphology 93 239255.

    • Search Google Scholar
    • Export Citation
  • Kai CM, Main KM, Andersen AN, Loft A, Chellakooty M, Skakkebaek NE & Juul A 2006 Serum insulin-like growth factor-I (IGF-I) and growth in children born after assisted reproduction. Journal of Clinical Endocrinology and Metabolism 91 43524360. (https://doi.org/10.1210/jc.2006-0701)

    • Search Google Scholar
    • Export Citation
  • Kleijkers SHM, Mantikou E, Slappendel E, Consten D, van Echten-Arends J, Wetzels AM, van Wely M, Smits LJM, van Montfoort APA & Repping S et al. 2016 Influence of embryo culture medium (G5 and HTF) on pregnancy and perinatal outcome after IVF: a multicenter RCT. Human Reproduction 31 22192230. (https://doi.org/10.1093/humrep/dew156)

    • Search Google Scholar
    • Export Citation
  • Koivurova S, Hartikainen AL, Sovio U, Gissler M, Hemminki E & Järvelin MR 2003 Growth, psychomotor development and morbidity up to 3 years of age in children born after IVF. Human Reproduction 18 23282336. (https://doi.org/10.1093/humrep/deg445)

    • Search Google Scholar
    • Export Citation
  • Kuiper D, Bennema A, la Bastide-van Gemert S, Seggers J, Schendelaar P, Mastenbroek S, Hoek A, Heineman MJ, Roseboom TJ & Kok JH et al. 2018 Developmental outcome of 9-year-old children born after PGS: follow-up of a randomized trial. Human Reproduction 33 147155. (https://doi.org/10.1093/humrep/dex337)

    • Search Google Scholar
    • Export Citation
  • Lee S, Gilula NB& Warner AE 1987 Gap junctional communication and compaction during preimplantation stages of mouse development. Cell 51 851860. (https://doi.org/10.1016/0092-8674(87)90108-5)

    • Search Google Scholar
    • Export Citation
  • Lo CW & Gilula NB 1979 Gap junctional communication in the preimplantation mouse embryo. Cell 18 399409. (https://doi.org/10.1016/0092-8674(79)90059-x)

    • Search Google Scholar
    • Export Citation
  • Magli MC, Pomante A, Cafueri G, Valerio M, Crippa A, Ferraretti AP & Gianaroli L 2016 Preimplantation genetic testing: polar bodies, blastomeres, trophectoderm cells, or blastocoelic fluid? Fertility and Sterility 105 676683.e5. (https://doi.org/10.1016/j.fertnstert.2015.11.018)

    • Search Google Scholar
    • Export Citation
  • Middelburg KJ, van der Heide M, Houtzager B, Jongbloed-Pereboom M, Fidler V, Bos AF, Kok J, Hadders-Algra MPGS Follow-up Study Group 2011 Mental, psychomotor, neurologic, and behavioral outcomes of 2-year-old children born after preimplantation genetic screening: follow-up of a randomized controlled trial. Fertility and Sterility 96 165169. (https://doi.org/10.1016/j.fertnstert.2011.04.081)

    • Search Google Scholar
    • Export Citation
  • Mihajlović AI& Bruce AW 2017 The first cell-fate decision of mouse preimplantation embryo development: integrating cell position and polarity. Open Biology 7 29167310. (https://doi.org/10.1098/rsob.170210)

    • Search Google Scholar
    • Export Citation
  • Nagy A 2003 Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory Press.

  • Palermo G, Joris H, Devroey P & Van Steirteghem AC 1992 Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 340 1718. (https://doi.org/10.1016/0140-6736(92)92425-f)

    • Search Google Scholar
    • Export Citation
  • Piotrowska-Nitsche K, Perea-Gomez A, Haraguchi S & Zernicka-Goetz M 2005 Four-cell stage mouse blastomeres have different developmental properties. Development 132 479490. (https://doi.org/10.1242/dev.01602)

    • Search Google Scholar
    • Export Citation
  • Sacks GC, Altarescu G, Guedalia J, Varshaver I, Gilboa T, Levy-Lahad E & Eldar-Geva T 2016 Developmental neuropsychological assessment of 4- to 5-year-old children born following preimplantation genetic diagnosis (PGD): a pilot study. Child Neuropsychology 22 458471. (https://doi.org/10.1080/09297049.2015.1014900)

    • Search Google Scholar
    • Export Citation
  • Sakka SD, Loutradis D, Kanaka-Gantenbein C, Margeli A, Papastamataki M, Papassotiriou I & Chrousos GP 2010 Absence of insulin resistance and low-grade inflammation despite early metabolic syndrome manifestations in children born after in vitro fertilization. Fertility and Sterility 94 16931699. (https://doi.org/10.1016/j.fertnstert.2009.09.049)

    • Search Google Scholar
    • Export Citation
  • Sampino S, Zacchini F, Swiergiel AH, Modlinski AJ, Loi P & Ptak GE 2014 Effects of blastomere biopsy on post-natal growth and behavior in mice. Human Reproduction 29 18751883. (https://doi.org/10.1093/humrep/deu145)

    • Search Google Scholar
    • Export Citation
  • Saoi M, Kennedy KM, Gohir W, Sloboda DM & Britz-McKibbin P 2020 Placental metabolomics for assessment of sex-specific differences in fetal development during normal gestation. Scientific Reports 10 9399. (https://doi.org/10.1038/s41598-020-66222-3)

    • Search Google Scholar
    • Export Citation
  • Sato BLM, Sugawara A, Ward MA & Collier AC 2014 Single blastomere removal from murine embryos is associated with activation of matrix metalloproteinases and Janus kinase/signal transducers and activators of transcription pathways of placental inflammations. Molecular Human Reproduction 20 12471257. (https://doi.org/10.1093/molehr/gau072)

    • Search Google Scholar
    • Export Citation
  • Schendelaar P, Middelburg KJ, Bos AF, Heineman MJ, Kok JH, La Bastide-Van Gemert S, Seggers J, Van den Heuvel ER & Hadders-Algra M 2013 The effect of preimplantation genetic screening on neurological, cognitive and behavioural development in 4-year-old children: follow-up of a RCT. Human Reproduction 28 15081518. (https://doi.org/10.1093/humrep/det073)

    • Search Google Scholar
    • Export Citation
  • Sepulveda-Rincon LP, Islam N, Marsters P, Campbell BK, Beaujean N & Maalouf WE 2017 Embryo cell allocation patterns are not altered by biopsy but can be linked with further development. Reproduction 154 807814. (https://doi.org/10.1530/REP-17-0514)

    • Search Google Scholar
    • Export Citation
  • Shamonki MI, Jin H, Haimowitz Z & Liu L 2016 Proof of concept: preimplantation genetic screening without embryo biopsy through analysis of cell-free DNA in spent embryo culture media. Fertility and Sterility 106 13121318. (https://doi.org/10.1016/j.fertnstert.2016.07.1112)

    • Search Google Scholar
    • Export Citation
  • Steptoe PC & Edwards RG 1978 Birth after the reimplantation of a human embryo. Lancet 2 366. (https://doi.org/10.1016/s0140-6736(78)92957-4)

    • Search Google Scholar
    • Export Citation
  • Sugawara A & Ward MA 2013 Biopsy of embryos produced by in vitro fertilization affects development in C57BL/6 mouse strain. Theriogenology 79 234241. (https://doi.org/10.1016/j.theriogenology.2012.08.007)

    • Search Google Scholar
    • Export Citation
  • Sugawara A, Sato B, Bal E, Collier AC & Ward MA 2012 Blastomere removal from cleavage-stage mouse embryos alters steroid metabolism during pregnancy. Biology of Reproduction 87 4, 19. (https://doi.org/10.1095/biolreprod.111.097444)

    • Search Google Scholar
    • Export Citation
  • Tang F, Barbacioru C, Nordman E, Bao S, Lee C, Wang X, Tuch BB, Heard E, Lao K & Surani MA 2011 Deterministic and stochastic allele specific gene expression in single mouse blastomeres. PLoS ONE 6 e21208. (https://doi.org/10.1371/journal.pone.0021208)

    • Search Google Scholar
    • Export Citation
  • Tarkowski AK, Suwińska A, Czołowska R & Ożdżeński W 2010 Individual blastomeres of 16- and 32-cell mouse embryos are able to develop into foetuses and mice. Developmental Biology 348 190198. (https://doi.org/10.1016/j.ydbio.2010.09.022)

    • Search Google Scholar
    • Export Citation
  • Thomaidis L, Kitsiou-Tzeli S, Critselis E, Drandakis H, Touliatou V, Mantoudis S, Leze E, Destouni A, Traeger-Synodinos J & Kafetzis D et al. 2012 Psychomotor development of children born after preimplantation genetic diagnosis and parental stress evaluation. World Journal of Pediatrics 8 309316. (https://doi.org/10.1007/s12519-012-0374-0)

    • Search Google Scholar
    • Export Citation
  • Torres-Padilla ME, Parfitt DE, Kouzarides T & Zernicka-Goetz M 2007 Histone arginine methylation regulates pluripotency in the early mouse embryo. Nature 445 214218. (https://doi.org/10.1038/nature05458)

    • Search Google Scholar
    • Export Citation
  • Wen J, Jiang J, Ding C, Dai J, Liu Y, Xia Y, Liu J & Hu Z 2012 Birth defects in children conceived by in vitro fertilization and intracytoplasmic sperm injection: a meta-analysis. Fertility and Sterility 97 1331 .e11337 .e1. (https://doi.org/10.1016/j.fertnstert.2012.02.053)

    • Search Google Scholar
    • Export Citation
  • Winter C, Van Acker F, Bonduelle M, Desmyttere S & Nekkebroeck J 2015 Psychosocial development of full term singletons, born after preimplantation genetic diagnosis (PGD) at preschool age and family functioning: a prospective case-controlled study and multi-informant approach. Human Reproduction 30 11221136. (https://doi.org/10.1093/humrep/dev036)

    • Search Google Scholar
    • Export Citation
  • Wu Y, Lv Z, Yang Y, Dong G, Yu Y, Cui Y, Tong M, Wang L, Zhou Z & Zhu H et al. 2014 Blastomere biopsy influences epigenetic reprogramming during early embryo development, which impacts neural development and function in resulting mice. Cellular and Molecular Life Sciences 71 17611774. (https://doi.org/10.1007/s00018-013-1466-2)

    • Search Google Scholar
    • Export Citation
  • Yamada N, Katsuura G, Ochi Y, Ebihara K, Kusakabe T, Hosoda K & Nakao K 2011 Impaired CNS leptin action is implicated in depression associated with obesity. Endocrinology 152 26342643. (https://doi.org/10.1210/en.2011-0004)

    • Search Google Scholar
    • Export Citation
  • Yao Q, Chen L, Liang Y, Sui L, Guo L, Zhou J, Fan K, Jing J, Zhang Y & Yao B 2016 Blastomere removal from cleavage-stage mouse embryos alters placental function, which is associated with placental oxidative stress and inflammation. Scientific Reports 6 25023. (https://doi.org/10.1038/srep25023)

    • Search Google Scholar
    • Export Citation
  • Yin Y, Li Y & Zhang W 2014 The growth hormone secretagogue receptor: its intracellular signaling and regulation. International Journal of Molecular Sciences 15 48374855. (https://doi.org/10.3390/ijms15034837)

    • Search Google Scholar
    • Export Citation
  • Yu Y, Wu J, Fan Y, Lv Z, Guo X, Zhao C, Zhou R, Zhang Z, Wang F & Xiao M et al. 2009 Evaluation of blastomere biopsy using a mouse model indicates the potential high risk of neurodegenerative disorders in the offspring. Molecular and Cellular Proteomics 8 14901500. (https://doi.org/10.1074/mcp.M800273-MCP200)

    • Search Google Scholar
    • Export Citation
  • Zeng Y, Lv Z, Gu L, Wang L, Zhou Z, Zhu H, Zhou Q & Sha J 2013 Preimplantation genetic diagnosis (PGD) influences adrenal development and response to cold stress in resulting mice. Cell and Tissue Research 354 729741. (https://doi.org/10.1007/s00441-013-1728-1)

    • Search Google Scholar
    • Export Citation
  • Zhao HC, Zhao Y, Li M, Yan J, Li L, Li R, Liu P, Yu Y & Qiao J 2013 Aberrant epigenetic modification in murine brain tissues of offspring from preimplantation genetic diagnosis blastomere biopsies. Biology of Reproduction 89 117. (https://doi.org/10.1095/biolreprod.113.109926)

    • Search Google Scholar
    • Export Citation