Can expelled cells/debris from a developing embryo be used for PGT?

Background Preimplantation genetic testing (PGT) is offered to a wide range of structural and numerical chromosomal imbalances, with PGT- polymerase chain reaction (PCR), as the method of choice for amplifying the small DNA content achieved from the blastomere biopsy or trophectoderm (TE) biopsy, that might have a detrimental impact on embryonic implantation potential. Since human embryos cultured until Day-5–6 were noticed to expel cell debris/ fragments within the zona pellucida, we aimed to examine whether these cell debris/ fragments might be used for PGT, as an alternative to embryo biopsy. Methods Blastocysts, which their Day-3 blastomere biopsy revealed an affected embryo with single-gene defect, and following hatching leaved cell debris/fragments within the zona pellucida were analyzed. Each blastocyst and its corresponding cell debris/fragments were separated and underwent the same molecular analysis, based on multiplex PCR programs designed for haplotyping using informative microsatellites markers. The main outcome measure was the intra-embryo congruity of Day-3 blastomere biopsy and its corresponding blastocyst and cell debris/fragments. Results Fourteen affected embryos from 9 women were included. Only 8/14 (57.2%) of embryos demonstrated congruent molecular genetic results between Day-3 embryo and its corresponding blastocyst and cell debris/fragments. In additional 6/14 (42.8%) embryos, molecular results of the Day-3 embryos and their corresponding blastocysts were congruent, while the cell debris/fragments yielded no molecular diagnoses (incomplete diagnoses). Conclusions It might be therefore concluded, that in PGT cycles, examining the cell debris/fragments on Day-4, instead of Day-3 blastomere or Day-5 TE biopsies, is feasible and might avoid embryo biopsy with its consequent detrimental effect on embryos’ implantation potential. Whenever the latter results in incomplete diagnosis, TE biopsy should be carried out on Day-5 for final genetic results. Further large well-designed studies are required to validate the aforementioned PGT platform.

Pre-implantation Screening and Diagnosis

Through cellular biopsy of a developing embryo, genetic testing can be performed as part of the embryo selection phase of an in vitro fertilization (IVF) cycle. Preimplantation genetic screening (PGS) and preimplantation genetic diagnosis (PGD) allow embryos to be tested for genetic conditions on a chromosome and gene level, respectively, prior to implantation in the uterus and prior to pregnancy. Included in this review are indications for PGS and PGD, the biopsy and diagnostic methods that are most frequently utilized, advances in recent non-invasive technologies, and potential impacts that PGS/PGD and IVF may have on developing embryos. This review contains 5 figures, 1 table, and 53 references. Keywords: Preimplantation genetic diagnosis, preimplantation genetic screening, blastocyst biopsy, blastomere biopsy, advances in PGS/PGD, indications for PGS/PGD, non-invasive embryo biopsy, infertility

The Technological Advances in Embryo Selection and Genetic Testing: A Look Back at the Evolution of Aneuploidy Screening and the Prospects of Non-Invasive PGT

Since the birth of the first IVF baby, Louise Brown, in 1978, researchers and clinicians have sought ways to improve pregnancy outcomes through embryo selection. In the 1990s, blastomere biopsy and fluorescence in situ hybridization (FISH) were developed in human embryos for the assessment of aneuploidy and translocations. Limitations in the number of chromosomes that could be assayed with FISH lead to the development of comparative genomic hybridization (CGH); however, pregnancy rates overall were not improved. The later development of trophectoderm biopsy with comprehensive chromosome screening (CCS) technologies, as well as the subsequent development of next-generation sequencing (NGS), have shown much greater promise in improving pregnancy and live birth rates. Recently, many studies are focusing on the utilization of non-invasive preimplantation genetic testing (niPGT) in an effort to assess embryo ploidy without exposing embryos to additional interventions.

Blastomere removal affects homeostatic control leading to obesity in male mice

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.

64 Impaired Post-Implantation Development Following Blastomere Biopsy is Associated with Placental Hypomethylation

The removal of one cell from cleaving embryos (blastomere biopsy, BB) is the main component of pre-implantation genetic diagnosis (PGD), a diagnostic test aimed to select only healthy embryos before transfer in utero. Studies on animals have suggested BB as risk factor for impaired development during both pre- and postnatal life. However, we still have incomplete knowledge on the real side effects of BB and the mechanisms underlying them. The present study was designed to evaluate whether BB or miscellaneous factors (in vitro culture, IVC; and embryo transfer, ET) may affect developmental potential of conceptuses obtained following BB (i.e. pregnancy rate/loss, fetal/placental weight, feto:placenta ratio) and placental epigenetic programming. To this aim, 3-month-old C57 females, naturally mated without hormonal stimulation, were used as donors. At 2.5 days post-coitum (dpc), 8-cell stage embryos were collected and subjected to BB in PBS (without Ca2+/Mg2+) supplemented with 0.4% BSA and antibiotics. Embryos were cultured in KSOM medium in an incubator at 37.5°C and 5% CO2. Two in vitro control groups were used: (1) IVC, embryos cultured in vitro, not subjected to biopsy; (2) ET, blastocysts collected at 3.5 dpc and directly transferred into recipient females. At 3.5 dpc, 10 to 12 blastocysts were transferred into pseudo pregnant females (n ≥ 7/group). An additional control group consisted of naturally conceived pregnancy (n = 8, in vivo control, CTR). At 18.5 dpc, conceptuses were collected and subjected to gross morphological evaluations (n > 20 conceptuses/group). Placentae (n ≥ 4/group) were analysed for 5-methylation and hydroxymethylation content by ELISA assay. Decimal variables were analysed using the Mann-Whitney test, and percentages were analysed with the Fisher exact test. Assessment of development to term revealed reduced survival rate in BB, IVC, and ET v. CTR (45.23, 47.82, 60, and 98.15% respectively; P < 0.05) and increased number of stillborn/congenital anomalies in BB v. CTR fetuses (9.5 v. 1.5%; P < 0.05). Gross morphological observations displayed increased placental weight in BB, IVC, ET v. CTR (0.15, 0.17, 0.16 and 0.11 g, respectively; P < 0.0001) and reduced fetal weight in BB v. IVC, ET, and CTR groups (0.88, 1.09, 1.16, and 1.15 g, respectively; P < 0.05). Also, a significant reduction of feto:placenta ratio was detected in BB, IVC, ET v. CTR (5.9, 6.3, 8.1 v. 10.4; P < 0.05). The ELISA assays on placentae revealed significant reduction of 5-methylcytosine content in BB v. CTR (22.24 v. 33.55%; P < 0.05) but no differences in hydroxymethylcytosine. Altogether, our results showed that BB is associated with impaired development to term (reduced fetal weight, increased risk of perinatal mortality, and congenital anomalies) and hypomethylation in full-term placentae. Thus, our preliminary study suggests that BB, rather than IVC and ET, to be a factor of risk for proper development in utero through perturbation of developmental epigenetic programming. Further analysis will be necessary to better characterise the epigenetic profile in both fetuses and placentae obtained following BB.

Embryo biopsy and development: the known and the unknown

Preimplantation genetic diagnosis (PGD) has been introduced in clinical practice as a tool for selecting ‘healthy’ embryos before their transfer in utero. PGD protocols include biopsy of cleaving embryos (blastomere biopsy (BB)) or blastocysts (trophectoderm biopsy (TB)), followed by genetic analysis to select ‘healthy’ embryos for transfer in utero. Currently, TB is replacing the use of BB in the clinical practice. However, based on the European Society of Human Reproduction and Embryology Preimplantation Genetic Diagnosis Consortium reports, BB has been used in >87% of PGD cycles for more than 10 years. An exhaustive evaluation of embryo biopsy (both BB and TB) risks and safety is still missing. The few epidemiological studies available are quite controversial and/or are limited to normalcy at birth or early childhood. On the other hand, studies on animals have shown that BB can be a risk factor for impaired development, during both pre- and postnatal life, while little is known on TB. Thus, there is an urgent need of focused researches on BB, as it has contributed to give birth to children for more than 10 years, and on TB, as its application is significantly growing in clinical practice. In this context, the aim of this review is to provide a complete overview of the current knowledge on the short-, medium- and long-term effects of embryo biopsy in the mouse model.