Polar Body Biopsy Research Articles

The effect of repeated biopsy on pre-implantation genetic testing for monogenic diseases (PGT-M) treatment outcome.

To study the outcome of repeated biopsy for pre-implantation genetic testing in case of failed genetic diagnosis in the first biopsy. The study group included 81 cycles where embryos underwent re-biopsy because there were no transferable embryos after the first biopsy: in 55 cycles, the first procedure was polar body biopsy (PBs) and the second cleavage-stage (BB); in 26 cycles, the first was BB and the second trophectoderm (BLAST) biopsy. The control group included 77 cycles where embryos underwent successful genetic diagnosis following the first biopsy, matched by maternal age, egg number, genetic inheritance type, and embryonic stage at the first biopsy. We measured genetic diagnosis rate, clinical pregnancy rates (PRs), live-birth rates (LBRs), gestational age, and birth weight. For repeated biopsy, genetic diagnosis was received in 67/81 cycles (82.7%); at a higher rate in PB + BB than in BB + BLAST (49/55, 89.1% and 18/26, 69.2% respectively, p= 0.055). Transferable embryos were found in 47 and 68 cycles in the study and the control groups. PRs/ET were 20/47 (42.6%) and 36/68 (52.9%) (p= 0.27), 16/36 (44.4%) following PB + BB, and 4/11 (36.4%) following BB + BLAST (p= 0.74). LBRs/ET were 13/47 (27.7%) in study group, and 28/68 (41.2%) in the controls (p= 0.14), 10/36 (27.8%) following PB + BB group, and 3/11 (27.3%) following BB + BLAST (p> 0.99). Gestational age and birth weight were similar in all groups. Re-biopsy of embryos when no genetic diagnosis could be reached following the first biopsy, achieved high rates of genetic diagnosis, pregnancies, and live births.

Chromosomal analysis in IVF: just how useful is it?

Designed to minimize chances of genetically abnormal embryos, preimplantation genetic diagnosis (PGD) involves in vitro fertilization (IVF), embryo biopsy, diagnosis and selective embryo transfer. Preimplantation genetic testing for aneuploidy (PGT-A) aims to avoid miscarriage and live born trisomic offspring and to improve IVF success. Diagnostic approaches include fluorescence in situ hybridization (FISH) and more contemporary comprehensive chromosome screening (CCS) including array comparative genomic hybridization (aCGH), quantitative polymerase chain reaction (PCR), next-generation sequencing (NGS) and karyomapping. NGS has an improved dynamic range, and karyomapping can detect chromosomal and monogenic disorders simultaneously. Mosaicism (commonplace in human embryos) can arise by several mechanisms; those arising initially meiotically (but with a subsequent post-zygotic 'trisomy rescue' event) usually lead to adverse outcomes, whereas the extent to which mosaics that are initially chromosomally normal (but then arise purely post-zygotically) can lead to unaffected live births is uncertain. Polar body (PB) biopsy is the least common sampling method, having drawbacks including cost and inability to detect any paternal contribution. Historically, cleavage-stage (blastomere) biopsy has been the most popular; however, higher abnormality levels, mosaicism and potential for embryo damage have led to it being superseded by blastocyst (trophectoderm - TE) biopsy, which provides more cells for analysis. Improved biopsy, diagnosis and freeze-all strategies collectively have the potential to revolutionize PGT-A, and there is increasing evidence of their combined efficacy. Nonetheless, PGT-A continues to attract criticism, prompting questions of when we consider the evidence base sufficient to justify routine PGT-A? Basic biological research is essential to address unanswered questions concerning the chromosome complement of human embryos, and we thus entreat companies, governments and charities to fund more. This will benefit both IVF patients and prospective parents at risk of aneuploid offspring following natural conception. The aim of this review is to appraise the 'state of the art' in terms of PGT-A, including the controversial areas, and to suggest a practical 'way forward' in terms of future diagnosis and applied research.

Impact of polar body biopsy on embryo morphokinetics\u2014back to the roots in preimplantation genetic testing?

PurposePolar body biopsy (PBB) is a common technique in preimplantation genetic testing (PGT) to assess the chromosomal status of the oocyte. Numerous studies have been implemented to investigate the impact of biopsies on embryo development; however, information on embryo morphokinetics is still lacking. Hence, we investigated the impact of PBB on morphokinetic parameters in early embryo development.MethodsFour hundred four embryos (202 PBB, 202 control) were retrospectively analyzed. Patients were stimulated with a gonadotropin-releasing hormone antagonist ovarian hyperstimulation protocol. After fertilization check, embryos were incubated in a time-lapse incubator. The groups were matched for maternal age at time of oocyte retrieval.ResultsMean group times for reaching specific developmental time points showed no significant difference comparing embryos with PBB conducted and without. Likewise, further subdivision of the PBB group in euploid and aneuploid embryos revealed no differences in the early embryo morphokinetic development compared to the control group. Aneuploidy testing revealed a high prevalence of chromosomal aberrations for chromosomes 21, 4, 16, and 19.ConclusionsIn conclusion, PBB does not impact the morphokinetic parameters of the embryo development. PBB can be safely applied without the risk of impairing the reproductive potential of the embryo and can be highly recommended as safe and practicable PGT approach, especially in countries with prevailing restrictions regarding PGT analysis.

Advances in Preimplantation Genetic Testing for Monogenic Disease and Aneuploidy

Genetic testing of preimplantation embryos promises to prevent monogenic disease in children born to at-risk couples, the transfer of unbalanced embryos to patients carrying a balanced translocation, and the use of aneuploid embryos created during in vitro fertilization. Technologies have evolved from fluorescence in situ hybridization to next-generation-sequencing-based aneuploidy screening and allow for simultaneous testing of multiple genetic abnormalities in a single biopsy. The field has also shifted away from polar body or blastomere biopsy and toward trophectoderm biopsy as the new standard. This review describes the multitude of available platforms and methodologies used in contemporary preimplantation genetic testing.

The Impact of Biopsy on Human Embryo Developmental Potential during Preimplantation Genetic Diagnosis.

Preimplantation Genetic Diagnosis and Screening (PGD/PGS) for monogenic diseases and/or numerical/structural chromosomal abnormalities is a tool for embryo testing aimed at identifying nonaffected and/or euploid embryos in a cohort produced during an IVF cycle. A critical aspect of this technology is the potential detrimental effect that the biopsy itself can have upon the embryo. Different embryo biopsy strategies have been proposed. Cleavage stage blastomere biopsy still represents the most commonly used method in Europe nowadays, although this approach has been shown to have a negative impact on embryo viability and implantation potential. Polar body biopsy has been proposed as an alternative to embryo biopsy especially for aneuploidy testing. However, to date no sufficiently powered study has clarified the impact of this procedure on embryo reproductive competence. Blastocyst stage biopsy represents nowadays the safest approach not to impact embryo implantation potential. For this reason, as well as for the evidences of a higher consistency of the molecular analysis when performed on trophectoderm cells, blastocyst biopsy implementation is gradually increasing worldwide. The aim of this review is to present the evidences published to date on the impact of the biopsy at different stages of preimplantation development upon human embryos reproductive potential.

Deliveries from trophectoderm biopsied, fresh and vitrified blastocysts derived from polar body biopsied, vitrified oocytes

This longitudinal study reports preliminary findings of six patients who underwent first polar body biopsy followed by oocyte vitrification. All oocytes were warmed, inseminated by intracytoplasmic sperm injection and cultured to blastocyst. All suitable blastocysts underwent trophectoderm biopsy for aneuploidy screening, and supernumerary blastocysts were vitrified. Euploid blastocysts were transferred either fresh or in a subsequent programmed cycle. Of the 91 metaphase II oocytes, 30 had euploid first polar bodies. Development to blastocyst was more likely in oocytes with a euploid first polar body (66.7% versus 24.6%; P < 0.001). Nineteen euploid blastocysts were produced: 10 from oocytes with a euploid first polar body and nine from oocytes with an aneuploid first polar body. Five out of six patients (83%) had a live birth or ongoing pregnancy at the time of analysis. Eleven euploid blastocysts have been transferred and seven implanted (64%). Although the chromosomal status of the first polar body was poorly predictive of embryonic ploidy, an association was found between chromosomal status of the first polar body and development to blastocyst. Further study is required to characterize these relationships, but proof of concept is provided that twice biopsied, twice cryopreserved oocytes and embryos can lead to viable pregnancies.

Vitrification of a small number of spermatozoa in normozoospermic and severely oligozoospermic samples

Despite broad utilization of sperm cryopreservation, little progress has been made to modify freezing protocols or to improve rates of sperm survival. Vitrification is an alternative method for freezing human spermatozoa without toxic permeable cryoprotectants (CPAs). The purpose of our study was to optimize the vitrification and post thaw recovery of a small number of spermatozoa using only nonpermeating CPAs in a closed straw system in normozoospermic and severely oligozoospermic samples. Individual motile spermatozoa (n = 295) were selected from semen samples of 15 normozoospermic and 10 severe oligozoospermia patients. Overall sperm recovery after vitrification was 80% (n = 236) with 80% (n = 189) viability and 41.5% (n = 98) retained post-warming motility. Two different loading techniques were compared to transfer selected spermatozoa into straws in preparation for vitrification: by spontaneous capillary action (CA) and with the aid of a polar body biopsy (PBB) pipette. There was evidence that the PBB loading technique increases the odds of spermatozoa recovery in both subsets (p = 0.01 and p = 0.04) in the normal and abnormal subsamples, respectively.

Laser microsurgery of cells by femtosecond laser scalpel and optical tweezers

This paper provides a brief overview of the current state of research in the field of laser microsurgery of preimplantation embryos. The combined system femtosecond laser tweezers-scalpel developed in JIHT RAS is described. A scheme of device and peculiarities of functioning its nodes as well as the ground for the choice of their parameters are presented. The results of the development of methodology for noncontact polar body biopsy of the preimplantation embryo are also presented to demonstrate the potentials of the combined multi-purpose system femtosecond laser tweezers-scalpel.

Live birth after PGD with confirmation by a comprehensive approach (karyomapping) for simultaneous detection of monogenic and chromosomal disorders

Preimplantation genetic diagnosis (PGD) for monogenic disorders has the drawback of time and cost associated with tailoring a specific test for each couple, disorder, or both. The inability of any single assay to detect the monogenic disorder in question and simultaneously the chromosomal complement of the embryo also limits its application as separate tests may need to be carried out on the amplified material. The first clinical use of a novel approach (‘karyomapping’) was designed to circumvent this problem. In this example, karyomapping was used to confirm the results of an existing PGD case detecting both chromosomal abnormalities and a monogenic disorder (Smith–Lemli–Opitz [SLO] syndrome) simultaneously. The family underwent IVF, ICSI and PGD, and both polar body and cleavage stage biopsy were carried out. Following whole genome amplification, array comparative genomic hybridisation of the polar bodies and minisequencing and STR analysis of single blastomeres were used to diagnose maternal aneuploidies and SLO status, respectively. This was confirmed, by karyomapping. Unlike standard PGD, karyomapping required no a-priori test development. A singleton pregnancy and live birth, unaffected with SLO syndrome and with no chromosome abnormality, ensued. Karyomapping is potentially capable of detecting a wide spectrum of monogenic and chromosome disorders and, in this context, can be considered a comprehensive approach to PGD.

Embryo Biopsy for Aneuploidy Detection in the General Infertility Population

Since report of the first live birth following preimplantation genetic screening (PGS) in 1995, the procedure and available technologies for aneuploidy detection have rapidly evolved. Through these efforts, the biology of meiotic and mitotic segregation errors has been partially elucidated. A process that began with polar body biopsy and four-color fluorescence in situ hybridization to detect copy number in a limited number of chromosomes is now hardly recognizable: current molecular methods permit high-density screening of the entire human genome for copy number variants, structural rearrangements, microdeletions, and polyploids to a resolution of 35 kilobases in less than 48 hours. Indeed, with the advent of real-time quantitative analyses of ploidy status that allow same-day trophectoderm biopsy with fresh transfer of a euploid blastocyst, the future is bright for PGS. Questions remain about how best to safely offer this technology to patients, and which patients, if any, will benefit from routine biopsy. Herein, we will review the limited available evidence for application of PGS in the general infertility population as an adjunct method to optimize live birth rates.

Coenzyme Q10 Supplementation and Oocyte Aneuploidy in Women Undergoing IVF-ICSI Treatment.

BACKGROUNDThe age-related reduction in live-birth rate is attributed to a high rate of aneuploidy and follicle depletion. We showed in an animal model that treatment with Coenzyme Q10 (CoQ10) markedly improved reproductive outcome. The aim of this study was to compare the post-meiotic oocyte aneuploidy rate in in vitro fertilization (IVF) and intra cytoplasmic sperm injection (ICSI) patients treated with CoQ10 or placebo.METHODSWe conducted a double blind placebo controlled randomized trial that included IVF–ICSI patients 35–43 years of age. The patients were treated with either 600 mg of CoQ10 or an equivalent number of placebo caps. We compared the post-meiotic aneuploidy rate using polar body biopsy (PBBX) and comparative genomic hybridization (CGH). According to the power calculation, 27 patients were needed for each arm.RESULTSOwing to safety concerns regarding the effects of polar body biopsy on embryo quality and implantation, the study was terminated before reaching the target number of participants. A total of 39 patients were evaluated and randomized (17 CoQ10, 22 placebo), 27 were given the study medication (12 CoQ10, 15 placebo), and 24 completed an IVF–ICSI cycle including PBBX and embryo transfer (10 CoQ10, 14 placebo). Average age, base line follicle stimulating hormone (FSH), peak estradiol and progesterone serum level, as well as the total number of human menopausal gonadotropin (hMG) units—did not differ between the groups. The rate of aneuploidy was 46.5% in the CoQ10 group compared to 62.8% in the control. Clinical pregnancy rate was 33% for the CoQ10 group and 26.7% for the control group.CONCLUSIONNo significant differences in outcome were detected between the CoQ10 and placebo groups. However, the final study was underpowered to detect a difference in the rate of aneuploidy.

New advances of preimplantation and prenatal genetic screening and noninvasive testing as a potential predictor of health status of babies.

The current morphologically based selection of human embryos for transfer cannot detect chromosome aneuploidies. So far, only biopsy techniques have been able to screen for chromosomal aneuploidies in the in vitro fertilization (IVF) embryos. Preimplantation genetic diagnosis (PGD) or screening (PGS) involves the biopsy of oocyte polar bodies or embryonic cells and has become a routine clinical procedure in many IVF clinics worldwide, including recent development of comprehensive chromosome screening of all 23 pairs of chromosomes by microarrays for aneuploidy screening. The routine preimplantation and prenatal genetic diagnosis (PND) require testing in an aggressive manner. These procedures may be invasive to the growing embryo and fetus and potentially could compromise the clinical outcome. Therefore the aim of this review is to summarize not only the new knowledge on preimplantation and prenatal genetic diagnosis in humans, but also on the development of potential noninvasive embryo and fetal testing that might play an important role in the future.

Controlled implantation after preimplantation genetic screening by polar body biopsy and fish

Preimplantation genetic screening (PGS) wants to improve artificial reproductive technologies (ART), primarily by raising the rates of pregnancy, implantation and birth. At the moment there are two main approaches for PGS: biopsy of blastocysts, predominantly in the USA, and polar body biopsy, in Europe, both now combined with array comparative genome hybridization (aCGH). Improvement was contested for a long time. And aCGH is expensive when applied to the many oocytes per cycle in comparison to the few blastocysts per cycle.

Does polar body biopsy (PBB) predict the outcome of vitrified oocytes (OV): preliminary results of PBB, OV, thaw, trophectoderm biopsy (TEBX) and embryo transfer (ET) with ongoing pregnancies

Does polar body biopsy (PBB) predict the outcome of vitrified oocytes (OV): preliminary results of PBB, OV, thaw, trophectoderm biopsy (TEBX) and embryo transfer (ET) with ongoing pregnancies

Polar body (PB) based aneuploidy screening is significantly less predictive of the reproductive potential of embryos than embryo biopsy based techniques

PB biopsy (bx) for aneuploidy screening has been proposed as a less invasive alternative to embryo bx. However, unlike embryo based screening, the predictive value of PB screening for embryonic reproductive potential has not been established. This study seeks to compare PB and embryo based aneuploidy screening.

Oocyte morphology on day 0 correlates with aneuploidy as detected by polar body biopsy and FISH

For better selection of oocytes and embryos, preimplantation genetic screening (PGS) was introduced. As from the beginning of IVF, morphology was used as selection criteria; we investigated the combination of both. If there was a correlation between phenotype and genotype, invasive PGS might be replaced. Therefore, 104 cycles with PGS were done by biopsy of the first polar body and FISH with five chromosomes. Morphology of the oocyte was recorded digitally and noted for 12 categories in 4-13 values; evaluation of the chromosomes was noted for five chromosomes in five values. Morphology and genetics were correlated to each other. Correlations between morphology and genetics for day 0 were found: oocytes with an irregular or dark zona are less probable to have a normal chromosome 13 (80 vs. 53 %, p = 0.001). A medium amount of detritus in the perivitelline space makes it more probable to have a normal chromosome 18 (94 vs. 78 %, p = 0.001). A halo in the cytoplasm makes it less probable to be euploid for chromosome 22 (56 vs. 75 %, p = 0.018). For day 1, pattern "1, 2, 3 and fine" in the pronuclei makes it more probable to be euploid for chromosome 22 (78 vs. 63 %, p = 0.002). There are correlations between the oocyte genome and its morphology also on day 0. These correlations are not sufficient to replace PGS.

Sham-controlled implantation after preimplantation genetic screening by polar body biopsy and FISH

Preimplantation genetic screening wants to improve artificial reproductive technologies, primarily by raising the rates of pregnancy, implantation and birth. We investigated if embryos derived from oocytes detected euploid for five chromosomes implant better than those which were biopsied but where the genetic detection failed. They were nevertheless transferred, thus serving as a sham control. From 2004 to 2008 we performed 104 cycles of PGS with laser biopsy of the first polar body and FISH with five chromosomes. It was offered to all patients with eight or more oocytes, free of charge. The average female age was 36 years. If no euploid oocytes were available, not detected oocytes were transferred. In 104 cycles 99 embryo transfers (95 %) were performed, resulting in 28 pregnancies (27 %), 20 births (71 %) and 8 miscarriages (29 %). The implantation rate in the euploid group was 19 vs. 13 % in the not detected group (n.s.). This trend was the same independent of age and embryo morphology. The pregnancy rate does not differ significantly from the national average. The trend in better implantation rates of euploid oocytes justifies a continuation of studies in this matter.

Preimplantation genetic diagnosis and the biopsy technique: Important considerations

Preimplantation genetic diagnosis allows to test the genetic status of embryos prior to implantation. In order to obtain genetic material, on which carry out a genetic diagnosis, a procedure named embryo biopsy is required. In the last two decades, embryo biopsy at the cleavage stage has been the mostly performed procedure. However, recently, alternative methods allowing the retrieval of a larger number of cells (blastocyst stage biopsy), or representing a valid alternative to overcome ethical issues (polar body biopsy) have obtained increasing consensus. This article reviews different methods of embryo biopsy and points out their positive and negative aspects.

Does First Polar Body Array CGH Analysis Correlate with a Cleavage Stage Embryo Biopsy in IVF-ICSI Cycles and Preimplantation Genetic Screening

To correlate first polar body(PB1) biopsy array comparative genomic hybridization(aCGH) technology with cleavage stage(D3) biopsy results analyzed with either FISH or SNP array. Prospective clinical trial. 15 IVF patients underwent PB1 biopsy followed by a D3 biopsy. Between 09/11-04/12, 27 patients consented and 15 met the study's criteria and underwent PB1 biopsy and D3 biopsy. 11 patients completed their cycle with a total of 101 PB1 and corresponding D3 biopsies. All mature metaphase II (MII) oocytes were fertilized by ICSI 4-6 hours following oocyte retrieval. 2-4 hours later, PB1 biopsies were performed and frozen. D3 biopsy was then performed and analyzed using either 9-probe FISH(13,15,16,17,18,21,22,X,Y) or SNP 24 CGH(physician/patient preference). Clinical decisions were based solely on the results of D3 analysis.ET was performed on day-5. Concordance between PB1 and D3 results, as well as ongoing pregnancy rates and IVF cycle outcome. 83% of MII oocytes fertilized normally using ICSI. Concordance analysis of the first 96(5 embryos had incomplete results) PB1 and corresponding D3 revealed 67.33% partial or complete concordance and 32.7% discordance. When the 9-probes FISH was used partial concordance was expected. PB1 aCGH had a sensitivity of 82.8% and specificity of 55.7% compared to D3. The kappa statistics was 0.35. Implantation rate was 31% (average 2.3 embryos per transfer). 36% pregnancy rate per cycle started and 27% ongoing pregnancy rate (57% and 42%, respectively, in patients who had ET).Tabled 1PB1 biopsyBlastomere biopsy%NormalNormal28.71AbnormalAbnormal - identical6.93AbnormalAbnormal - partial concordance8.91AbnormalAbnormal - different17.82NormalAbnormal5.94AbnormalNormal26.73N/A4.95100%PB1-Clinically correct diagnosis67.33%PB1-Clinically incorrect diagnosis32.67% Open table in a new tab Tabled 1D3 biopsy + (Normal)D3 biopsy - (Abnormal)Polar body + (Normal)292756Polar body - (Abnormal)63440356196KAPPA = 0.35 (Fair agreement) with C.I. (0.14, 0.54) p value = 0.0001 Open table in a new tab KAPPA = 0.35 (Fair agreement) with C.I. (0.14, 0.54) p value = 0.0001 PB1 biopsy can not completely replace embryo biopsy for PGS as it doesn't appraise 2nd meiosis error and mitotic errors. Our study suggests, that PB1 aCGH can accurately predict oocyte chromosomal status in 2/3 of cases and completed within 12 hours. Aneuploidy(partly due to mosaicism) was the predominant cause of non-viability in our study group. PB1 biopsy should be used in certain clinical scenarios and selected patients.

Does first polar body array CGH analysis correlate with a cleavage stage embryo biopsy in IVF-ICSI cycles and preimplantation genetic screening

OBJECTIVE: To correlate first polar body(PB1) biopsy(Bx) aCGH technology with cleavage stage(D3) Bx results analyzed with FISH or SNP-array. DESIGN: Prospective clinical trial. Main Outcome: Concordance between PB1 and D3 results, ongoing pregnancy rates and IVF outcome. MATERIALS AND METHODS: Between 09/11-04/12, 27 patients(Pts) consented and 15 enrolled and underwent PB1 Bx followed by a D3 Bx.11 Pts completed their cycle with a total of 101 PB1 and corresponding D3 Bxs.All mature oocytes(MII) were fertilized by ICSI 4-6h post retrieval with PB1 Bx performed and frozen 2-4h thereafter.D3 Bx was then performed and analyzed using 9 probe FISH(13,15,16,17,18,21,22,X,Y) or 24 chromosomes SNP array(physician/Pts preference).Clinical decisions were based solely on D3 analysis.ET was performed on D5. RESULTS: 83% of MIIs fertilized normally.Concordance analysis of PB1 and corresponding D3 revealed 67.3% partial/complete concordance and 32.7% discordance(6% normal embryos possibly discarded or 26.7% abnormal embryos possibly transferred).When 9-probe FISH was used partial concordance was expected.PB1 had a 82% sensitivity and 55% specificity when compared to D3.Kappa statistic was 0.35[fair agreement], P1⁄40.0001(95% CI1⁄40.14,0.54) Implantation rate was 31%(average of 2.28 fresh embryos per ET). Pregnancy rate/cycle started was 36% with 27% ongoing(57% and 42% per cycle transferred).