Affect Preimplantation Development Research Articles

O-071 Paternal body mass index affects early embryo development but does not impact clinical outcomes: a timelapse study of 7,656 embryos in oocyte donation cycles

Abstract Study question Does paternal body mass index (BMI) affect preimplantation development and clinical outcomes in oocyte donation cycles? Summary answer High paternal BMI was associated with delays in early development and poorer embryo quality on day 3, but this did not adversely affect pregnancy outcomes. What is known already Maternal obesity is known to negatively affect preimplantation development and clinical outcomes. While oocyte competence plays a major role in early embryogenesis, male obesity has been linked to poorer semen parameters and epigenetic alterations in sperm. Such characteristics may inherently compromise preimplantation development and ultimately impair reproductive success. Only a few reports have assessed the effects of male BMI on the progression of cell divisions and embryo morphokinetic patterns. However, current findings remain contradictory, while the effects of male obesity on fertility continue to be understudied. Study design, size, duration This retrospective study included 7,656 embryos from 1,342 oocyte donation ICSI cycles, performed between April 2018 and December 2021 across two fertility centres. Sperm originated from the partner (n = 1087) or donor (n = 255) and was either fresh (n = 460) or frozen (n = 835). Only the first fresh (n = 1,140) or frozen (n = 202) embryo transfer (ET) was considered. Our database included 1,089 single and 238 double ETs performed at either the cleavage (n = 424) or blastocyst stage (n = 903) of development. Participants/materials, setting, methods Males were stratified according to BMI, based on WHO guidelines, defined as: normal weight (BMI<24.9, n = 677); pre-obese, (BMI=25.0–29.9, n = 533); and obese (BMI>30, n = 132). Patients with severe male factor and preimplantation genetic testing cycles were excluded. Univariate analyses and logistic regression were used to evaluate associations between BMI, embryo morphokinetics and clinical outcomes. Analyses were adjusted for various confounders pertaining to patient demographics, sperm and cycle parameters. A p-value of < 0.05 was considered significant. Main results and the role of chance Overall, obese men displayed poorer sperm motility and underwent more day 2-4 embryo transfers and freeze-all cycles, compared to pre-obese and normal weight men (p < 0,05). Fertilization rates were not affected by male BMI (77.0% ± 18.9 normal weight; 77.4% ± 18.1 pre-obese; 75.3% ± 19.9 obese; p = 0.62), also in our adjusted analysis. Several morphokinetic parameters were delayed in embryos from pre-obese and obese patients, including pronuclei fading (tPNf, p = 0.0064) and division timings from the 2 to 5-cell stage (t2-t3-t4-t5, p < 0,05). Successively, the number of good quality cleavage embryos decreased significantly with higher BMI (normal weight, 62%; pre-obese, 59%; obese, 50.8%; p = 0.02). In contrast, developmental events past the 5-cell stage, including time to 8-cells (t8), blastulation (tSB) and expanded blastocyst (tB) were comparable amongst the groups (p > 0.05). Similarly, we observed no effect of male BMI on blastocyst quality (normal weight, 70.2%; pre-obese, 73.4%; obese, 75.5%; p = 0.06). Despite early developmental delays, we observed no significant effects on biochemical pregnancy (normal weight, 64.7%; pre-obese 64.1%; obese, 58.7%; p = 0.45), ongoing pregnancy (normal weight, 60.0%; pre-obese, 58.8%; obese, 52.9%; p = 0.35) and live birth rate (normal weight, 41.9%; pre-obese, 42.0%; obese, 34.9%; p = 0.27). Our adjusted analysis corroborated these findings. Limitations, reasons for caution The main limitation of this study is its retrospective design, which may not account for all confounders. Moreover, lifestyle factors, such as smoking or alcohol consumption were not considered. As such, results cannot be generalized to all patient populations. Wider implications of the findings Obese males show lower motile sperm counts and delayed cleavage development. While these changes impaired day 3 embryo quality, they did not affect blastocyst development and quality. That translates into comparable clinical outcomes, suggesting that specific counselling is not required in obese patients. Trial registration number not applicable

O-298 Poor semen parameters alter embryo morphokinetic patterns during preimplantation development but do not compromise clinical outcomes

Abstract Study question Does semen quality affect preimplantation development and clinical outcomes in ICSI cycles using donor oocytes? Summary answer Poor semen parameters led to altered morphokinetic parameters during preimplantation development but did not compromise blastocyst quality nor clinical outcomes in oocyte donation cycles. What is known already The quality of semen used for in vitro fertilization (IVF) may compromise the kinetics and morphology of developing embryos, ultimately impairing implantation and reducing live birth rates. Such effects are particularly pronounced when testicular or epididymal spermatozoa are used for IVF treatment. However, current studies assessing the impact of poor semen parameters on embryo development and clinical outcomes are often confounded by the use of oocytes from infertile patients. Performing such evaluations in oocyte donation cycles will provide more definitive insights into the role of semen during preimplantation development. Study design, size, duration This retrospective study included 616 embryos from 99 oocyte donation ICSI cycles, performed between February 2018 and July 2019. We compared embryo morphokinetics and clinical outcomes (cumulative pregnancy and live birth rates) across three groups with variable semen parameters. These included donor semen (control group, DD, n = 294), partner sperm obtained by testicular sperm extraction (TESE group, n = 72) and partner semen with altered parameters (including poor concentration, motility and morphology, ALT group, n = 250). Participants/materials, setting, methods We assessed several morphokinetic parameters, including second polar body extrusion (tPB2), pronuclei appearance (PN1a, PN2a), 2-3-4-5-8-cell stages (t2 to t8), start of blastulation (tSB) and blastocyst formation (tB). Kaplan-Meier survival curves were built for each parameter per study group. Survival curves were analysed by Cox regression. Qualitative parameters (even size PN and blastomeres, percent fragmentation, abnormal cleavages, blastocyst grade) were assessed using a Chi-Squared test, while Fisher’s exact test was used to evaluate clinical outcomes. Main results and the role of chance Interestingly, TESE embryos reached several developmental stages faster than the DD group: tPB2 (3.03 vs. 3.08, p < 0.001), PN1a (5.55 vs. 6.19, p < 0.001), PN2a (6.78 vs. 7.36, p < 0.001), tPNf (21.30 vs. 22.20, p = 0.001), t3 (34.02 vs. 35.79, p = 0.016), t4 (35.90 vs. 36.92, p = 0.015), t5 (46.70 vs. 48.07, p = 0.036), t8 (52.48 vs. 55.28, p = 0.049), tSB (85.53 vs. 94.62, p < 0.001) and tB (104.09 vs. 106.60, p = 0.002). ALT embryos were faster in early stages: PN1a (6.63 vs. 6.19, p = 0.005), PN2a (7.84 vs. 7.36, p < 0.001), t5 (48.12 vs. 48.07, p = 0.028), t8 (55.57 vs. 55.28, p = 0.032), but slower in the late stages: tSB (93.71 vs. 94.62, p = 0.001), tB (105.60 vs. 106.60, p = 0.002). Compared to DD, ALT and TESE embryos showed a lower rate of even pronuclei (ALT p = 4.6x10-06; TESE p = 8x10-05) and even blastomeres at the 2-cell (ALT p < 0.001; TESE p = 0.006) and 4-cell stage (ALT p = 0.004; TESE p = 0.005). Moreover, embryos derived from testicular sperm showed significantly higher fragmentation rates at the 8-cell stage (p = 0.005), while no significant differences in the frequency of irregular divisions nor in the number of top-quality blastocysts were detected in ALT and TESE. Importantly, cumulative live birth rates per cycle were similar across all study groups (DD 31/53, ALT 31/51, TESE 8/11; p > 0.05). Limitations, reasons for caution The main limitation resides in the retrospective nature of this study and the limited number of embryos in the TESE group. We also note variability in the testicular biopsy (obstructive and non-obstructive azoospermia) and non-normozoospermic diagnoses (oligozoospermia, teratozoospermia or asthenozoospermia), which may confound the analysis. Wider implications of the findings Although poor semen parameters significantly altered embryo morphokinetics, they did not compromise embryo quality, pregnancy or cumulative live birth rates, resulting in similar clinical outcomes as with double donation. Overall, our findings support the use of autologous sperm with donor oocytes, even in severe cases of male factor infertility. Trial registration number not applicable

Does in vitro fertilization affect the expression of miRNAs and their biogenesis pathway in preimplantation mouse embryos?

In vitro fertilization (IVF) is a well-accepted procedure which has been utilized for the treatment of infertile patients. As embryos at early stages of development are very vulnerable, the IVF conditions may influence genetic and epigenetic regulation of preimplantation mouse embryo. We assessed the effect of IVF on the expression of developmental and implantation related miRNAs (miR-21, miR-93, miR-24, and let-7a), their common presumptive target (Stat3), and miRNA biogenesis pathway genes (Drosha, Dgcr8, Exportin-5, Dicer, and Ago2). in vivo 8-cell and blastocysts were compared to IVF embryos. Expression levels of miRNAs, Stat3, and miRNA biogenesis pathway genes were evaluated by qRT-PCR in in vivo (n = 8) and IVF (n = 4) embryos. The expression levels of let-7a and Stat3 were significantly reduced in IVF blastocyst when compared with in vivo (p = .004 and p = .009, respectively). Nevertheless, the IVF procedure did not influence the expression levels of miRNA biogenesis pathway components in 8-cell and blastocyst embryos. Downregulation of let-7a and developmental related transcription factor, Stat3, in IVF mouse blastocysts may affect preimplantation development and implantation of embryos. Moreover, the genes of the miRNA biogenesis pathway were not changed in preimplantation mouse embryos through the IVF procedure.

Oxygen concentration alters mitochondrial structure and function in in vitro fertilized preimplantation mouse embryos.

Does the oxygen concentration in the culture medium [either physiologic (5%) or atmospheric (20%)] affect mitochondrial ultrastructure and function in preimplantation mouse embryos generated by IVF? Embryos cultured in 20% oxygen show increased mitochondrial abnormalities compared to embryos cultured in 5% oxygen. ART are widely used and have resulted in the birth of more than 8 million children. A variety of media and oxygen concentrations are used to culture embryos. Embryos cultured under physiological O2 tension (5%) reach the blastocyst stage faster and have fewer alterations in gene expression when compared with embryos cultured under atmospheric oxygen conditions (20%). The mechanisms by which oxygen tension affects preimplantation development remain unclear, but mitochondria are believed to play an important role. The aim of this study was to evaluate how mitochondrial ultrastructure and function in IVF embryos were affected by culture under physiologic (5%) or atmospheric (20%) oxygen concentrations. Zygotes, 2-cell, 4-cell, morula and blastocyst were flushed out of the uterus after natural fertilization and used as controls. IVF was performed in CF1 x B6D2F1 mice and embryos were cultured in Potassium simplex optimized medium (KSOM) with amino acids (KAA) under 5% and 20% O2 until the blastocyst stage. Embryo development with the addition of antioxidants was also tested. Mitochondrial function was assessed by measuring mitochondrial membrane potential, reactive oxygen species (ROS) production, ATP levels, and the expression of selected genes involved in mitochondrial function. Mitochondria ultrastructure was evaluated by transmission electron microscopy (TEM). Embryos cultured under 20% O2 had fewer mitochondria and more vacuoles and hooded (abnormal) mitochondria compared to the other groups (P < 0.05). At the blastocyst stage the mitochondria of IVF embryos cultured in 20% O2 had lower mtDNA copy number, a denser matrix and more lamellar cristae than controls. Overall IVF-generated blastocysts had lower mitochondrial membrane potential, higher ROS levels, together with changes in the expression of selected mitochondrial genes (P < 0.05). ATP levels were significantly lower than controls only under 5% O2, with the 20% O2 IVF group having intermediate levels. Unexpectedly, adding antioxidant to the culture medium did not improve development. N/A. Findings in mice embryos might be different from human embryos. This study suggests that changes in the mitochondria may be part of the mechanism by which lower oxygen concentration leads to better embryo development and further emphasize the importance of mitochondria as a locus of reprogramming. This study was funded by R01 HD 082039 to PFR, the Department of Life, Health and Environmental Sciences, University of L'Aquila, Italy (RIA 2016-2018) and the Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, La Sapienza University of Rome, Italy (University grants 2016-2017). The authors declare no competing interests.

FRAP analysis of chromatin looseness in mouse zygotes that allows full-term development.

Chromatin looseness, which can be analyzed by fluorescence recovery after photobleaching (FRAP) using eGFP-tagged core histone proteins, is an important index of the differentiation potential of blastomere cells and embryonic stem cells. Whether chromatin looseness is a reliable index of the developmental potential of embryos during ontogenesis is not known. As a necessary first step toward answering this question, we investigated whether FRAP-analyzed embryos are capable of normal preimplantation and full-term development. All tested concentrations (50, 100, and 250 ng/μL) of microinjected eGFP-H2B mRNA were sufficient for detecting differences in chromatin looseness between male and female pronuclei. After FRAP analysis, most of the zygotes developed into blastocysts. Importantly, a considerable number of offspring developed from the FRAP analyzed zygotes (32/78; 41.0%) and grew into healthy adults. The offspring of zygotes injected with 250 ng/μL of eGFP-H2B mRNA and bleached using 110 μW laser power for 5 s were not genetically modified. Interestingly, bleaching using a 3-fold stronger laser intensity for a 6-fold longer time did not cause toxicity during preimplantation development, indicating that bleaching did not critically affect preimplantation development. Finally, we confirmed that similar results were obtained using two different types of confocal laser-scanning microscopes. This FRAP protocol would be useful for investigating the association between chromatin looseness and development.

Conserved roles of fibroblast growth factor receptor 2 signaling in the regulation of inner cell mass development in bovine blastocysts.

A common process during preimplantation mammalian development is blastocyst formation, which utilizes signaling through fibroblast growth factor receptor 2 (FGFR2), yet the mechanisms through which FGFR2 signaling affect preimplantation development in bovine embryos remain incompletely understood. Here, we used RNA-interference to investigate the in vitro development, the frequency of blastomere apoptosis, and the mRNA expression of developmental marker genes in FGF receptor 2-knockdown (FGFR2-KD) bovine embryos. A reduction in FGFR2 mRNA did not affect preimplantation development or the frequency of apoptotic blastomeres, but did enhanced proliferation of the inner cell mass in blastocysts (P < 0.05)-which differs from the phenotype reported for bovine embryos using a pharmacological approach (treatment with the pan-FGFR blocker PD173074), but agrees with previous results obtained using mouse embryos. Moreover, the expression of an epiblast marker gene, NANOG, and a primitive endoderm marker gene, GATA6, remained unchanged, whereas the expression of another primitive endoderm marker gene, HNF4A, was significantly reduced in FGFR2-KD embryos. Therefore, FGFR2 signaling appears to be associated with the regulation of inner cell mass development and proliferation during blastocyst formation in cattle. Mol. Reprod. Dev. 83: 516-525, 2016. © 2016 Wiley Periodicals, Inc.

Morphokinetics of embryos developed from oocytes matured in vitro

In in vitro maturation (IVM) cycles primed with human chorionic gonadotropin (hCG), both immature and mature oocytes are retrieved from antral follicles sized 8-12mm. Using time-lapse microscopy, we compared the morphokinetic behavior of embryos developed from oocytes matured in vivo and in vitro, testing the hypothesis that IVM affects preimplantation development. Furthermore, we extended the morphokinetic analysis of these embryos by a comparison with embryos obtained in stimulated assisted reproduction technology (ART) cycles. In IVM cycles primed with follicle-stimulating hormone (FSH)/hCG, prior to sperm microinjection, oocytes surrounded by an expanded cumulus at retrieval and presumably mature (EC-MII) were incubated for 6h, while immature oocytes enclosed in a compact cumulus (CC) were matured in vitro for 30h. The morphokinetics of embryos selected for transfer or cryopreservation, derived from EC-MII and CC oocytes, were comparatively and retrospectively analyzed in terms of cleavage times (t2, t3, t4, t5, and t8) and intervals (cc2, cc3, s2, s3). For further comparison, the morphokinetics of embryos selected for transfer or cryopreservation (ICSI) or giving rise to ongoing pregnancies (model) in stimulated ART cycles was also assessed. The morphokinetic behavior of EC-MII and CC embryos was entirely comparable, as suggested by the absence of statistical differences in the averages of all cleavage times and intervals. Almost all cleavage and interval times were also similar between EC-MII, CC, ICSI, and model groups, with the exception of t4 and s2, which were delayed and longer, respectively, in embryos generated in IVM cycles (EC-MII and CC). These findings do not support the hypothesis that maturation in vitro affects embryo morphokinetics, while they suggest only marginal differences in the morphokinetics of embryos developed from oocytes matured in vivo and in vitro in IVM cycles and embryos developed from mature oocytes recovered in stimulated cycles.

Cathepsin B Inhibitor, E-64, Affects Preimplantation Development, Apoptosis and Oxidative Stress in Pig Embryos

Cathepsin B is abundantly expressed peptidase of the papain family in the lysosomes, and closely related to the cell degradation system such as apoptosis, necrosis and autophagy. Abnormal degradation of organelles often occurs due to release of cathepsin B into the cytoplasm. Many studies have been reported that relationship between cathepsin B and intracellular mechanisms in various cell types, but porcine embryos has not yet been reported. Therefore, this study evaluated the effect of cathepsin B inhibitor (E-64) on preimplantation developmental competence and quality of porcine embryos focusing on apoptosis and oxidative stress. The expression of cathepsin B mRNA in porcine em-bryos was gradually decreased in inverse proportion to E-64 concentration by using real-time RT-PCR. When putative zygotes were cultured with E-64 for 24 h, the rates of early cleavage and blastocyst development were decreased by increasing E-64 concentration. However, the rate of blastocyst development in 5 μM treated group was similar to the control. On the other hand, both the index of apoptotic and reactive oxygen species (ROS) of blastocysts were sig-nificantly decreased in the 5 μM E-64 treated group compared with control. We also examined the mRNA expression levels of apoptosis related genes in the blastocysts derived from 5 μM E-64 treated and non-treated groups. Expre-ssion of the pro-apoptotic Bax gene was shown to be decreased in the E-64 treated blastocyst group, whereas expre-ssion of the anti-apoptotic Bcl-xL gene was increased. Taken together, these results suggest that proper inhibition of cathepsin B at early development stage embryos improves the quality of blastocysts, which may be related to not only the apoptosis reduction but also the oxidative stress reduction in porcine embryos.

Sex-specific embryonic origin of postnatal phenotypic variability

Preimplantation developmental plasticity has evolved in order to offer the best chances of survival under changing environments. Conversely, environmental conditions experienced in early life can dramatically influence neonatal and adult biology, which may result in detrimental long-term effects. Several studies have shown that small size at birth, which is associated with a greater risk of metabolic syndrome, is largely determined before the formation of the blastocysts because 70%-80% of variation in bodyweight at birth has neither a genetic nor environmental component. In addition, it has been reported that adult bodyweight is programmed by energy-dependent process during the pronuclear stage in the mouse. Although the early embryo has a high developmental plasticity and adapts and survives to adverse environmental conditions, this adaptation may have adverse consequences and there is strong evidence that in vitro culture can be a risk factor for abnormal fetal outcomes in animals systems, with growing data suggesting that a similar link may be apparent for humans. In this context, male and female preimplantation embryos display sex-specific transcriptional and epigenetic regulation, which, in the case of bovine blastocysts, expands to one-third of the transcripts detected through microarray analysis. This sex-specific bias may convert the otherwise buffered stochastic variability in developmental networks in a sex-determined response to the environmental hazard. It has been widely reported that environment can affect preimplantation development in a sex-specific manner, resulting in either a short-term sex ratio adjustment or in long-term sex-specific effects on adult health. The present article reviews current knowledge about the natural phenotypic variation caused by epigenetic mechanisms and the mechanisms modulating sex-specific changes in phenotype during early embryo development resulting in sex ratio adjustments or detrimental sex-specific consequences for adult health. Understanding the natural embryo sexual dimorphism for programming trajectories will help understand the early mechanisms of response to environmental insults.

Roles of One-carbon Metabolism in Preimplantation Period

One-carbon metabolism (OCM) can be seen as integrated metabolic pathways centered on the metabolism of two nutritional substances, folate and methionine. Mammalian oocytes and preimplantation embryos express almost all enzymes that participate in OCM, suggesting that they can independently metabolize OCM nutrients. A deficiency or excess of OCM nutrients and their metabolites during in vitro culture affects preimplantation development of mammalian embryos. Recent in vivo studies have demonstrated that specific OCM dietary interventions during the periconceptional (mainly oocyte growth and preimplantation) period can cause epigenetic alterations in DNA of offspring and program the long-term consequences in their health in adulthood. The epigenetic processes are likely to be implicated in the effects of OCM nutrients; however, understanding their effects at the level of specific genes and their implications in assisted reproductive technology will require further investigations.

What is the role of maternally provided Cdx2 mRNA in early mouse embryogenesis?

Genetic knockout studies in the mouse model first revealed the essential role of zygotically derived Cdx2 transcription factor during the later stages of blastocyst formation, characterized by a lack of functioning trophectoderm. However, the extent to which the potential provision of maternally derived Cdx2 affects preimplantation development has proved much less simple to address. Within the last year, two reports have been published arguing for and against a distinct functional role for maternal Cdx2. This commentary aims to discuss the approaches, results and interpretations of both studies in an attempt to resolve the apparent conflict and to constructively advance collective understanding.

Embryonic genotype and inbreeding affect preimplantation development in cattle

Infertility in cattle herds is a growing problem with multifactorial causes. Embryonic genotype and level of inbreeding are among the many factors that can play a role on reproductive efficiency. To investigate this issue, we produced purebred and crossbred bovine embryos by in vitro techniques from Holstein oocytes and Holstein or Brown Swiss semen and analyzed several cellular and molecular features. In the first experiment, purebred and crossbred embryos, obtained from abattoir oocytes, were analyzed for cleavage, development to morula/blastocyst stages, amino acid metabolism and gene expression of developmentally important genes. The results indicated significant differences in the percentage of compacted morulae, in the expression of three genes at the blastocyst stage (MNSOD, GP130 and FGF4) and in the utilization of serine, asparagine, methionine and tryptophan in day 6 embryos. In the second experiment, bovine oocytes were collected by ovum pick up from ten Holstein donors and fertilized with the semen of the respective Holstein sires or with Brown Swiss semen. The derived embryos were grown in vitro up to day 7, and were then transferred to synchronized recipients and recovered on day 12. We found that purebred/inbred embryos had lower blastocyst rate on days 7-8, were smaller on day 12 and had lower expression of the trophoblast gene PLAC8. Overall, these results indicate reduced and delayed development of purebred embryos compared with crossbred embryos. In conclusion, this study provides evidence that embryo genotype and high inbreeding can affect amino acid metabolism, gene expression, preimplantation development and therefore fertility in cattle.

Impact of embryonic expression of enhanced green fluorescent protein on early mouse development

The impact of embryonic enhanced green fluorescent protein (EGFP)-expression on development is not clear. In this study, we comprehensively assessed EGFP-expression pattern and its effect on early mouse development, following pronuclear-microinjection of the EGFP-transgene, containing chicken-β-actin promoter and cytomegalovirus enhancer. Preimplantation embryos exhibited differential EGFP-expression patterns. While blastocyst development of non-expressing embryos was 77.3 ± 1.8%, that of expressing embryos was only 43.9 ± 1.6% ( P<0.0001). Developmental competence of embryos negatively correlated ( r=−0.99) with the levels of EGFP-expression. Faint-, moderate-, and intense-expressing embryos developed to 83.1 ± 5.3%, 50 ± 5%, and 9.5 ± 3.9% blastocysts, respectively ( P<0.002). Interestingly, blastocysts expressing faint–moderate levels of EGFP were developmentally competent through the post-implantation period and delivered viable transgenic ‘green’ mice, following embryo transfer. These results indicate that hyper-expression of EGFP affects preimplantation development and faint–moderate level of its expression is compatible with normal embryogenesis in the mouse.

No differences in sheep somatic cell nuclear transfer outcomes using serum-starved or actively growing donor granulosa cells.

The aim of this study was to compare serum-starved and non-starved donor cells in sheep nuclear transfer with a special emphasis on cloning outcomes. Sheep oocytes, derived either in vivo or in vitro, were fused with cultured serum-starved or actively growing adult granulosa cells. Resulting blastocysts were transferred to recipients fresh or after vitrification, and subsequent pregnancies followed to term. Donor cell treatment did not significantly affect preimplantation development, pregnancy rates, fetal loss or neonate survival rates. Of 22 lambs born, ten survived the immediate perinatal period but all succumbed at various timepoints within the first few weeks of life. The results of the study suggest that the use of serum-starved cells offers no advantages or disadvantages to cloning outcomes. Neither were significant differences in outcomes observed when using either in vivo- or in vitro-derived oocytes or embryos transferred fresh or after vitrification. Yet, these results continue to highlight problems associated with somatic cell cloning as indicated by offspring mortality. It remains unclear whether the high offspring mortality in the current study was related to species, associated with the cell lines used or the result of other causes.

Improved development of ICR mouse 2-cell embryos by the addition of amino acids to a serum-, phosphate-and glucose-free medium.

This study was conducted to evaluate how exogenous amino acids could affect preimplantation development of ICR mouse embryos. Two-cell embryos collected from naturally mated mice were cultured in amino acid-, glucose- and phosphate-free preimplantation (P)-1 medium. In Experiments 1, 19 amino acids (aa; 1% and 0.5% of MEM essential and nonessential amino acid solutions, respectively) were added to P-1 medium supplemented with either fatty acid-free bovine serum albumin (BSA; 3 mg/mL) or human follicular fluid (hFF; 10%). Regardless of BSA or hFF addition, embryo development to the morula (84 to 86% vs. 97 to 100%) and the blastocyst (54% vs. 93 to 94%) stages was significantly (P<0.05) enhanced by the addition of aa compared with no addition. In Experiment 2, the cell number of blastomeres and inner cell mass (ICM) cells in blastocysts and the ratio of ICM cell to trophectodermal cell (TE) were evaluated after aa addition. In both BSA- and hFF-containing P-1 medium, a significant increase in total blastomere number were found after aa addition (47 to 52 vs. 62 to 63 cells) compared with no addition. However, the ICM/TE ratio was not significantly affected by aa supplementation in both media, while ICM cell number was greatly increased after aa addition in hFF-containing medium (12 vs. 17 cells). When blastocysts were further cultured up to 162 hr post-hCG injection, development to the hatched blastocyst stage was significantly promoted by aa addition (0% vs. 11 to 20%) in both BSA- and hFF-containing media. In conclusion, aa significantly promote the preimplantation development to the hatched blastocyst stage and such effect mainly exerted on supporting blastomere proliferation.

Maternal caffeine consumption during pregnancy does not affect preimplantation development but delays early postimplantation growth in rat embryos.

Models for studying prenatal drug-induced intrauterine growth retardation (IUGR) have, without exception, measured growth-related factors in the postimplantation embryo, fetus or neonate. Therefore, it is not known whether effects of drug exposure on growth and metabolism begin early in the preimplantation embryo, or whether IUGR is exclusively a postimplantation phenomenon. The present study investigates whether caffeine, a drug known to induce a dose-dependent fetal IUGR, affects embryo development before and/or after implantation or is exclusively a fetal phenomenon. Preimplantation embryo assessment (with treatment from Days 2 to 4 of pregnancy) included glucose utilization, cell number evaluation and stage of development (morula to hatched blastocyst); whereas, postimplantation embryo assessment (treatment from Days 2 to 10, 10.5 or 11 of pregnancy) included somite number evaluation and extent of neural tube closure, as seen using scanning electron microscopy. Comparing control preimplantation embryos with those exposed to 30 and 60 mg kg(-1) caffeine did not reveal any effects of caffeine exposure, as assessed on Day 5 of gestation. However, postimplantation embryo development assessed on Day 12 of gestation revealed that caffeine exposure of 15 and 30 mg kg(-1) significantly reduced, at both dosage levels, somite number and the extent of neural tube closure. In addition, comparisons of control and experimental groups revealed that in the high-dose caffeine group the forebrain cavity was significantly enlarged and bounded by a reduced, irregularly aligned neuroepithelium. The findings suggest that IUGR is a phenomenon first identifiable during late postimplantation embryogenesis and continues in fetal life.

Parthenogenetic development ofMos-deficient mouse oocytes

Ovarian teratomas develop in Mos-/- mutant mice produced by homologous recombination. These teratomas are probably derived from oocytes that undergo spontaneous parthenogenetic activation within the ovaries. However, it is not clear how the activated eggs develop into teratomas since embryonic development beyond the four-cell stage was not observed either in vitro or in vivo. In this study, Mos-/- parthenotes derived from in vitro-matured oocytes were cultured using a recently developed medium, KSOM/AA, which promotes a high frequency of preimplantation development by normal embryos. In total, 5% of the Mos-/- oocytes developed to the blastocyst stage. Preimplantation-like and early postimplantation-like embryos were observed in the ovaries of 60-63-day-old Mos-/- mice. These observations support the hypothesis that Mos-/- teratomas are derived from parthenogenetically activated oocytes that undergo early embryonic development up to early postimplantation-like stages within the ovaries. Aberrant meiotic divisions commonly observed in Mos-/- oocytes in vitro may adversely affect preimplantation development and reduce the frequency of blastocyst formation even under the best culture conditions.

ENDO A cytokeratin expression in the inner cell mass of parthenogenetic mouse embryos.

During the preimplantation period of development, the first cellular polarization and diversification of the mouse embryo occurs. This process starts at the eight-cell stage and is directly driven by the cytoskeleton. Cell polarization finally leads to the first embryonic epithelium, the trophectoderm, characterized by the presence of cytokeratins. It has not been described whether genomic imprinting, an epigenetic modification of certain genes depending on the parent-of-origin, affects preimplantation development. However, implantation is one of the steps in which an exceptionally high mortality rate is observed in mouse parthenogenetic embryos, a phenomenon that may be influenced by a deficiency in trophectoderm differentiation. To assess this possibility we analyzed the expression of various cytoskeletal proteins in late preimplanted embryos. No differences were observed in the expression of microtubules and microfilaments, but surprisingly, the undifferentiated cells of the parthenogenetic inner cell mass showed distinct cytokeratin staining. This anomalous cytoskeleton expression may be considered as one of the earliest manifestation described to date of the effect of genomic imprinting in development.

Development of rat × mouse hybrid embryos produced by microsurgery

Experimental production of hybrid embryos between Mus musculus L. and Rattus norvegicus L. was achieved by nuclear transplantation using both ovulated oocytes in metaphase II and pronuclear zygotes. Recipient egg-cells were of mouse origin in all cases. The developmental potential of hybrids was examined in vivo. Nucleo-cytoplasmic hybrids resulting from the introduction of rat metaphase II chromosomes into enucleated mouse oocytes, which were subsequently activated, were regularly blocked at the 1- or 2-cell stage. Nuclear (genetic) hybrids produced by transfer of a rat nucleus (in the form of metaphase II chromosomes or a pronucleus) into a nucleated mouse recipient (oocyte or zygote) were capable of development to the 5- to 8-cell stage. Transplantation of rat cytoplasm alone to intact metaphase II oocytes, followed by oocyte activation, generated cytoplasmic hybrids which developed to the morula stage. In control experiments (nuclear transfer between mouse oocytes or zygotes), a high proportion of embryos formed morulae and blastocysts. These results demonstrate that the rat nucleus is incapable of functioning in mouse cytoplasm, that introduction of the rat genome into intact mouse egg-cells impairs normal development, and that transfer of foreign (rat) cytoplasm into mouse egg-cells affects preimplantation development of manipulated embryos.

Preimplantation diagnosis

For couples at risk of having children with an inherited disease, in vitro fertilization (IVF) and diagnosis at preimplantation stages of embryonic development would allow only unaffected embryos to be returned to the uterus. Any pregnancy should, therefore, be unaffected by the disease and the possibility of a termination following prenatal diagnosis at later stages of pregnancy eliminated. Biopsy of 1 or 2 cells at the 8-cell stage early on the third day post-insemination does not adversely affect preimplantation development and leaves 8–12 h for genetic analysis before transferring selected embryos later on the same day. Using the polymerase chain reaction (PCR) to amplify a DNA sequence specific for the male Y chromosome from single cells, the sex of biopsied embryos can be identified. Several pregnancies were successfully established after transfer of female embryos in couples at risk of X-linked recessive diseases, which only affect boys, resulting in the birth of several healthy girls. Methods for fluorescent detection of in situ hybridization of chromosome-specific DNA probes to embryo nuclei and DNA amplification of the specific regions of genes causing various diseases are now being developed which should allow preimplantation diagnosis of a range of chromosomal and single gene defects including cystic fibrosis one of the most common autosomal recessive diseases.