Polar Body Transfer Research Articles

P-167 The sperm parameters in doubling oocytes by the 1st polar body transfer technique: does it matter?

Abstract Study question Does sperm parameters affect the effectiveness of the 1st polar body (PB) transfer in oocytes and embryo doubling? Summary answer Certain sperm conditions affect the transfer technique efficiency. What is known already Previous studies have shown the effectiveness of the 1st polar body (PB) transfer. The technique allows for the modification of donor oocytes to increase the number of cells suitable for fertilization and at the same time preserve the genetic relatedness to the patient. We established that the effectiveness depends on the morphology of the 1stPB. We also showed that the yield of high-quality blastocysts does not depend on whether fresh or thawed donor oocytes are used. But it is also important to understand whether sperm parameters have a significant effect on the performance of the technique. Study design, size, duration The study was performed in the Medical Center IGR from December 2017 to December 2023 and involved 93 patients (mean age 39.7±5.4 years) who underwent the 1st PB transfer and had indicated sperm parameters used for fertilization, including: normozoospermia (NS, n = 43), asthenozoospermia (AS, n = 24), asthenoteratozoospermia (ATS, n = 20), oligozoospermia (OS, n = 5), cryptozoospermia (CS, n = 1). We evaluated the pronuclear scoring, number of embryos on the 3rd and the 5th post-fertilization day, euploidy according to sperm conditions. Participants/materials, setting, methods We used donor oocytes, patients’ oocytes and their 1stPBs obtained from 93 women and their partners sperm with defined parameters in accordance with WHO norms for 2021. The procedure of donor oocyte enucleation and the 1stPBs transfers was carried out using Nikon Ti Eclipse(Japan) inverted microscope, Saturn 3 laser console(UK). Preimplantation genetic testing for aneuploidy was performed using trophectoderm biopsy. Samples were diagnosed using Ion S5(USA). Statistical analysis was carried out using Chi-square test. Main results and the role of chance The total number of donor oocytes modified by the 1stPB of patient’ oocytes was 364, which were distributed as follows by sperm status: NS - 159 oocytes, AS - 103 oocytes, ATS - 77 oocytes, OS - 19 oocytes, and CS - 6 oocytes. The mean age of the female patients according to the groups was similar (39.6±4.9, 39.8±6.2, 40.0±3.8, 37.8±6.1,40, respectively). The number of 2PN pattern during pronuclear scoring was distributed among groups (NS:AS:ATS:OS:CS) with the following percentages 69.2:65.1:62.3:73.7:66.7, respectively. Among the developing embryos on the 3rdday the distribution was 46.6:46.5:36.4:57.9, respectively. No viable embryos were formed in the CS group, so the group was not included in further calculations. Statistically significant difference (SSD) was not found between the groups on the above-mentioned stages of development (p0.05). On the 5thday the number of developing embryos (percents) was distributed as follows: 23.3:28.2:11.7:31.6 for NS:AS:ATS:OS, respectively. It is worth noting that SSD was found between NS and ATS, OS and ATS groups (p < 0.05) and AS and ATS groups (p < 0.001). Percentage of euploid embryos relative to the number of examined blastocysts from groups NS:AS:ATS was 30.8:23.1:22.2, respectively, SSD wasn’t found between the groups(p > 0.05). Limitations, reasons for caution To increase performance of the 1st PB transfer technique it is also worth considering such parameters as sperm DNA fragmentation and level of sperm oxidative stress, which reflect the chromatin state of the male gamete. Wider implications of the findings The study showed that among patients with asthenoteratozoospermia, the yield of viable embryos was significantly lower compared to the other sperm conditions studied. This fact must be taken into account when predicting the result of using the 1st PB transfer technique. Trial registration number N/A

Earlier second polar body transfer and further mitochondrial carryover removal for potential mitochondrial replacement therapy.

The second polar body(PB2) transfer in assisted reproductive technology is regarded as the most promising mitochondrial replacement scheme for preventing the mitochondrial disease inheritance owing to its less mitochondrial carryover and stronger operability. However, the mitochondrial carryover was still detectable in the reconstructed oocyte in conventional second polar body transfer scheme. Moreover, the delayed operating time would increase the second polar body DNA damage. In this study, we established a spindle-protrusion-retained second polar body separation technique, which allowed us to perform earlier second polar body transfer to avoid DNA damage accumulation. We could also locate the fusion site after the transfer through the spindle protrusion. Then, we further eliminated the mitochondrial carryover in the reconstructed oocytes through a physically based residue removal method. The results showed that our scheme could produce a nearly normal proportion of normal-karyotype blastocysts with further reduced mitochondrial carryover, both in mice and humans. Additionally, we also obtained mouse embryonic stem cells and healthy live-born mice with almost undetectable mitochondrial carryover. These findings indicate that our improvement in the second polar body transfer is conducive to the development and further mitochondria carryover elimination of reconstructed embryos, which provides a valuable choice for future clinical applications of mitochondrial replacement.

Early evidence of the artificial transfer/transplant of mitochondria to oocytes and zygotes by MitoCeption

Oocytes may carry mutations in their mitochondrial DNA (mtDNA) which affect fertility and embryo development leading to hereditary diseases or rejection. Mitochondrial replacement therapies (MRTs) such as polar body transfer, spindle transfer and pronuclear transfer, aim to change dysfunctional to normal mitochondria inside oocytes and zygotes resulting in healthier offspring. Even with promising results, MRTs techniques are invasive to oocytes and may negatively affect their viability and the success of the procedure. This article shows early evidence of the use of MitoCeption, a mitochondria transfer/transplant (AMT/T) technique to possibly induce the internalization of exogenous mitochondria in a dose-dependent manner to recipient oocytes in comparison to coincubation. By using human isolated mitochondria in a mix obtained from different donors we were able to identify their mtDNA in murine oocytes by qPCR. Fluorescence microscopy showed that exogenous and transferred mitochondria (MitoTracker ® Red) by MitoCeption were internalized in oocytes and zygotes (CellTracker® Green). After maintaining mitocepted zygotes to two-cell embryos, we transferred them to subrogate female mice and obtained healthy mice pups; however, without clear evidence of the maintenance of human mtDNA in the tissues of mice pups. These early results are puzzling, and they open the path to generate more research regarding the use of MitoCeption in comparison to coincubation in order to transfer mitochondria to oocytes using less invasive procedures.

P–234 Doubling oocytes using the 1st polar body: how it works according to patient’s age

Abstract Study question Does the 1st polar body (PB) transfer technique for oocytes and embryo doubling works at any patients’ age? Summary answer The transfer of the patient’s 1stPB to a donor oocyte shows an increase in the number of blastocysts in all studied age groups of patients. What is known already The problem of low ovarian reserve is one of the most difficult to solve by ART. Particular difficulties arise when patients are not ready to use donor oocytes. But transfer of the patient’s 1st polar body (PB) to donor oocyte cytoplasm has proven to be an effective method to help patients with low ovarian reserve and poor response. In confirmation of this, three healthy babies have been born to this date thanks to this technique. But the question remains open as until what age the application of this method is effective. It was evaluated in this study. Study design, size, duration The study was performed in the Medical Center IGR from March 2017 to January 2021 and involved 695 cells: 361 oocytes (group A) obtained from 85 patients (mean age 38.4±6.4 years) and 334 oocytes (group B) that were obtained received from 47 donors (mean age 27.0±3.6). We evaluated the number of high quality blastocysts (HQB) from maternal and modified oocytes by the 1st PB transfer and blastocysts euploidy in the different age groups. Participants/materials, setting, methods We used patients’ oocytes obtained from 85 women with low ovarian reserve and poor response. Donor oocytes have been previously enucleated and modified by the transfer of patients’ 1stPB with further fertilization. The procedure was carried out using Nikon Ti Eclipse(Japan) inverted microscope, Saturn 3 laser console(UK). Preimplantation genetic testing for aneuploidy was performed using trophectoderm biopsy. Samples were diagnosed using Ion S5(USA). Statistical analysis was carried out using Chi-square test and Fisher’s exact test. Main results and the role of chance In the group A there were 93 HQB (25.8%) that formed from 361 original patients’ oocytes and 70 HQB (20.9%) developed from 334 modified oocytes (group B) on the post-fertilization day 5 or 6. The statistically significant difference (SSD) was not found between the groups on this parameter (p > 0.05). Also, the data were grouped according to the age of the patients, namely group I (23–30 years) that included 13 patients, group II (31–39 years) – 28 patients, group III (40–48 years) – 44 patients. In group I there were 12 HQB (25.0%) that formed from 48 original patients’ oocytes and 14 HQB (29.9%) developed from 47 modified oocytes, in group II - 50 HQB (38.5%) that formed from 130 original patients’ oocytes and 31 HQB (27.2%) developed from 114 modified oocytes, in group III - 31 HQB (16.9%) that formed from 183 original patients’ oocytes and 25 HQB (14.5%) developed from 173 modified oocytes. The analysis showed SSD between the number of patients’ blastocysts in groups II and III (p < 0.001), as well as between the number of blastocysts from modified oocytes in groups II and III (p < 0.005), I and III (p < 0.05). There was not SSD among the number of euploid embryos. Limitations, reasons for caution With increasing age of patients, the morphological quality of their oocytes deteriorates, which is reflected in the structure of the polar bodies. In turn, the quality of the patient’s polar body plays a major role in the successful modification of the donor’s oocyte. Wider implications of the findings: : Results of analysis shows that even in the group of advanced maternal age it is possible to increase the yield of high qualitative blastocysts at least by 14,5% using the1st PB transfer technique. At the same time, the biological relatedness of patient with the future child is maintaining. Trial registration number Not applicable

Mitochondria: emerging therapeutic strategies for oocyte rescue.

As the vital organelles for cell energy metabolism, mitochondria are essential for oocyte maturation, fertilization, and embryo development. Abnormalities in quantity, quality, and function of mitochondria are closely related to poor fertility and disorders, such as decreased ovarian reserve (DOR), premature ovarian aging (POA), and ovarian aging, as well as maternal mitochondrial genetic disease caused by mitochondrial DNA (mtDNA) mutations or deletions. Mitochondria have begun to become a therapeutic target for infertility caused by factors such as poor oocyte quality, oocyte aging, and maternal mitochondrial genetic diseases. Mitochondrial replacement therapy (MRT) has attempted to use heterologous or autologous mitochondria to rebuild healthy state of oocyte by increasing the amount of mitochondria (e.g., partial ooplasm transfer, autologous mitochondrial transfer), or to stop the transmission of mtDNA diseases by replacing abnormal maternal mitochondria (e.g., pronuclei transfer, spindle transfer, polar body transfer). Among them, autologous mitochondrial transfer is the most promising therapeutic technology as of today which does not involve using a third party, but its clinical efficacy is controversial due to many factors such as the aging phenomenon of germ line cells, the authenticity of the existence of ovarian stem cells (OSC), and secondary damage caused by invasive surgery to patients with poor ovarian function. Therefore, the research of optimal autologous cell type that can be applied in autologous mitochondrial transfer is an area worthy of further exploration. Besides, the quality of germ cells can also be probably improved by the use of compounds that enhance mitochondrial activity (e.g., coenzyme Q10, resveratrol, melatonin), or by innovative gene editing technologies which have shown capability in reducing the risk of mtDNA diseases (e.g., CRISPR/Cas9, TALENTs). Though the current evidences from animal and clinical trials are not sufficient, and some solutions of technical problems are still needed, we believe this review will guide a new direction in the possible clinical applied mitochondrial-related therapeutic strategies in reproductive medicine.

Genome transfer for the prevention of female infertility caused by maternal gene mutation

Poor oocyte quality is associated with early embryo developmental arrest and infertility. Maternal gene plays crucial roles in the regulation of oocyte maturation, and its mutation is a common cause of female infertility. However, how to improve oocyte quality and develop effective therapy for maternal gene mutation remains elusive. Here, we use Zar1 as an example to assess the feasibility of genome transfer to cure maternal gene mutation–caused female infertility. We first discover that cytoplasmic deficiency primarily leads to Zar1-null embryo developmental arrest by disturbing maternal transcript degradation and minor zygotic genome activation (ZGA) during the maternal-zygotic transition. We next perform genome transfer at the oocyte (spindle transfer or polar body transfer) and zygote (early pronuclear transfer or late pronuclear transfer) stages to validate the feasibility of preventing Zar1 mutation–caused infertility. We finally demonstrate that genome transfer either at the oocyte or at the early pronuclear stage can support normal preimplantation embryo development and produce live offspring. Moreover, those pups grow to adulthood and show normal fertility. Therefore, our findings provide an effective basis of therapies for the treatment of female infertility caused by maternal gene mutation.

Three-parent babies: Mitochondrial replacement therapies.

The mitochondria are intracellular organelles, and just like the cell nucleus they have their own genome. They are extremely important for normal body functioning and are responsible for ATP production - the main energy source for the cell. Mitochondrial diseases are associated with mutations in mitochondrial DNA and are inherited exclusively from the mother. They can affect organs that depend on energy metabolism, such as skeletal muscles, the cardiac system, the central nervous system, the endocrine system, the retina and liver, causing various incurable diseases. Mitochondrial replacement techniques provide women with mitochondrial defects a chance to have normal biological children. The goal of such treatment is to reconstruct functional oocytes and zygotes, in order to avoid the inheritance of mutated genes; for this the nuclear genome is withdrawn from an oocyte or zygotes, which carries mitochondrial mutations, and is implanted in a normal anucleated cell donor. Currently, the options of a couple to prevent the transmission of mitochondrial diseases are limited, and mitochondrial donation techniques provide women with mitochondrial defects a chance to have normal children. The nuclear genome can be transferred from oocytes or zygotes using techniques such as pronuclear transfer, spindle transfer, polar body transfer and germinal vesicle transfer. This study presents a review of developed mitochondrial substitution techniques, and its ability to prevent hereditary diseases.

13. THE CHROMOSOMAL SET ANALYSIS OF BLASTOCYSTS OBTAINED FROM MODIFIED DONOR OOCYTES BY THE PATIENT'S 1ST POLAR BODY TRANSFER

Introduction Transfer of the patient's 1st polar body (PB) to donor oocyte cytoplasm has proven to be an effective method to help patients with low ovarian reserve and poor response. This technique allows doubling the number of oocytes suitable for fertilization, while maintaining the biological relationship of patient with the future child. The use of transfer in our practice has shown the ability of modified oocytes to form high morphological quality blastocysts and their euploidy have been evaluated in this study. Materials and Methods The study was performed in the Medical Center IGR from March 2017 to December 2018 and involved 330 cells: 168 oocytes (group A) obtained from 39 patients (mean age 40.7±5.3 years) and 162 oocytes (group B) that were received from 28 donors (mean age 28.2±2.4). Donor oocytes have been pre-enucleated and modified by the transfer of patients’ 1stPB with further fertilization. The procedure was made using Nikon Ti Eclipse (Japan) inverted microscope, Saturn 3 laser console (UK). Preimplantation genetic testing for aneuploidy (PGT-A) was performed using trophectoderm (TE) biopsy on the post-fertilization hours 120 or 144. Samples were diagnosed using Ion S5 by Thermo Fisher Scientific(USA). The samples’ ploidy was verified by the FISH. We evaluated the number of euploid and aneuploid blastocysts from maternal and modified oocytes by the 1st PB transfer. Statistical analysis was carried out using Shapiro-Wilk test for normality and Chi-square test. Results In the group A there were 50 blastocysts (29.8%) that formed from 168 original patients’ oocytes and 29 blastocysts (17.9%) developed from 162 modified oocytes (group B). 45 blastocysts from group A and 27 blastocysts from group B were biopsied for PGT-A. The number of euploid embryos was 11 (24.4%) and 6 (22.2%) in the group A and group B, respectively, without statistically significant difference (SSD). Among the aneuploidies, single (only one chromosome was involved in nondisjunction) and complex (several chromosomes involved in nondisjunction) chromosomal anomalies were encountered. In the group A 13 (38.2%) samples carried single chromosomal disorder and 21 (61.8%) samples had complex quantitative abnormalities, in the group B these numbers reached 9 (47.4%).and 10 (52.6%) samples, respectively. Among the aneuploid embryos 79 chromosome abnormalities were found in A group samples and 38 numerical aberration in B group samples.. The following types of chromosomal abnormalities were detected in groups: autosome trisomy (50/63.3% vs. 25/65.8% samples), autosome monosomy (23/29.1% vs.6/15.8% samples), numerical violation of sex chromosomes (6/7.6% vs. 4/10.5% samples), polyploidy (0 vs. 2/5.3% samples), mosaicism (1/1.3% vs. 0 samples) in the group A and the group B, respectively. No SSD was found between the groups (p>0.05). Conclusions The results of our study demonstrated that the 1st PB transfer does not affect the chromosomal set of embryos obtained from modified oocytes in comparison with the original oocytes. This technique is useful to increase the number of female germ cells and avoid the full oocytes donation especially for patients with low ovarian reserve and for poor responders.

Human zygote reconstruction by spindle, polar body or pronuclear transfer to treat repeated embryo fragmentation or embryo developmental arrest: The future is now

A common and very difficult issue to overcome as a clinical embryologist during an IVF cycle is the event of severe embryonic fragmentation or, more rarely, full developmental arrest. The current approach to bypass these phenomena is oocyte donation, but this raises several ethical concerns from the couple’s side, since the offspring will bear only 50% of the biological characteristics of the parents. Apart from spindle and polar body transfer, the emerging technique in order to overcome these obstacles is called Pronuclear Transfer (PNT). PNT is safest to perform with improved results than spindle or polar body transfer. In PNT, the two parental pronuclei are being transferred into a donor’s enucleated zygote and after a fusion process has been achieved, the newly constructed embryo may result into a newborn bearing both the paternal and maternal biological characteristics. This process opens a whole new horizon in assisted reproduction and will probably replace the traditional oocyte donation in the form we know it today.

Компетенція кріоконсервованих ооцитів після енуклеації ядра та трансферу першого полярного тіла в ооплазму

Компетенція кріоконсервованих ооцитів після енуклеації ядра та трансферу першого полярного тіла в ооплазму

Assisted reproductive technologies to prevent human mitochondrial disease transmission.

Mitochondria are essential cytoplasmic organelles that generate energy (ATP) by oxidative phosphorylation and mediate key cellular processes such as apoptosis. They are maternally inherited and in humans contain a 16,569-base-pair circular genome (mtDNA) encoding 37 genes required for oxidative phosphorylation. Mutations in mtDNA cause a range of pathologies, commonly affecting energy-demanding tissues such as muscle and brain. Because mitochondrial diseases are incurable, attention has focused on limiting the inheritance of pathogenic mtDNA by mitochondrial replacement therapy (MRT). MRT aims to avoid pathogenic mtDNA transmission between generations by maternal spindle transfer, pronuclear transfer or polar body transfer: all involve the transfer of nuclear DNA from an egg or zygote containing defective mitochondria to a corresponding egg or zygote with normal mitochondria. Here we review recent developments in animal and human models of MRT and the underlying biology. These have led to potential clinical applications; we identify challenges to their technical refinement.

Polar body transfer restores the developmental potential of oocytes to blastocyst stage in a case of repeated embryo fragmentation.

We aimed to determine the developmental potential of human reconstructed oocytes after polar body genome transfer (PBT) and to report the case of a woman with multiple cycles of severe embryo fragmentation. Fresh and cryopreserved first polar bodies (PB1s) were transferred to enucleated metaphase II oocytes (PB1T), while fresh PB2s were removed from fertilized oocytes and used instead of the female pronucleus in donor zygotes. Reconstructed oocytes underwent intracytoplasmic sperm injection (ICSI) and were cultured to blastocyst. Biopsied trophectoderm cells of PBT-derived blastocysts were screened for chromosomes by next-generation sequencing (NGS). Then, cryopreserved PB1T was carried out in one woman with a history of several cycles of extensive embryo fragmentation, and the blastocysts derived from PB1T were screened for aneuploidy but not transferred to the patient. There were no significant differences in the rates of normal fertilization and blastocyst formation between fresh and cryopreserved PB1T and control oocytes. Of the three fresh and three cryopreserved PB1T-derived blastocysts, two and one blastocysts exhibited normal diploidy respectively. In contrast, 17 PB2 transfers yielded 16 two pronuclei (2PN) zygotes with one normal and one small-sized pronucleus each and no blastocyst formation. In the female patient, 18 oocytes were inseminated by ICSI in the fourth cycle and the PB1s were biopsied. Although the embryos developed from the patient's own oocytes showed severe fragmentation, the oocytes reconstructed after PB1T produced three chromosomally normal blastocysts. Normal blastocysts can develop from human reconstructed oocytes after PB1T. The application of the first PB transfers may be beneficial to patients with a history of poor embryo development and excessive fragmentation.

Challenges Towards Establishing Germline Gene Therapy for Inherited Mitochondrial Diseases

Abstract: Mitochondrial DNA (mtDNA) mutation is associated with serious human disorders and affects multiple organs and tissues with high-energy requirements. Since the transmission of mtDNA is complex and is not fully understood, an accurate estimation of mtDNA disease transmission by preimplantation genetic diagnosis (PGD) or by prenatal diagnosis (PND) remains challenging. Recently, nuclear transfer techniques, including maternal spindle transfer (MST), pronuclear transfer (PNT) and polar body transfer (PBT), have shown the promising results. These methods avoid the transmission of mutated mtDNA from mother to offspring, and are collectively known as the mitochondrial replacement therapy (MRT). Further, the United Kingdom Parliament approved the Human Fertilisation and Embryology Authority (HFEA) to grant licenses for experimental use of MST and PNT in humans in 2015. Thus, a new era of assisted reproductive technology (ART), in which cures can be provided at the gamete or early zygote stages, is reali...

Polar bodies in assisted reproductive technology: current progress and future perspectives.

During meiotic cell-cycle progression, unequal divisions take place, resulting in a large oocyte and two diminutive polar bodies. The first polar body contains a subset of bivalent chromosomes, whereas the second polar body contains a haploid set of chromatids. One unique feature of the female gamete is that the polar bodies can provide beneficial information about the genetic background of the oocyte without potentially destroying it. Therefore, polar body biopsies have been applied in preimplantation genetic diagnosis to detect chromosomal or genetic abnormalities that might be inherited by the offspring. Besides the traditional use in preimplantation diagnosis, recent findings suggest additional important roles for polar bodies in assisted reproductive technology. In this paper, we review the new roles of polar bodies in assisted reproductive technology, mainly focusing on single-cell sequencing of the polar body genome to deduce the genomic information of its sibling oocyte and on polar body transfer to prevent the transmission of mtDNA-associated diseases. We also discuss additional potential roles for polar bodies and related key questions in human reproductive health. We believe that further exploration of new roles for polar bodies will contribute to a better understanding of reproductive health and that polar body manipulation and diagnosis will allow production of a greater number of healthy babies.

Polar body transfer in oocytes for nuclear DNA replacement without mitochondrial DNA

Polar body transfer in oocytes for nuclear DNA replacement without mitochondrial DNA

Polar Body Genome Transfer for Preventing the Transmission of Inherited Mitochondrial Diseases

Inherited mtDNA diseases transmit maternally and cause severe phenotypes. Currently, there is no effective therapy or genetic screens for these diseases; however, nuclear genome transfer between patients' and healthy eggs to replace mutant mtDNAs holds promises. Considering that a polar body contains few mitochondria and shares the same genomic material as an oocyte, we perform polar body transfer to prevent the transmission of mtDNA variants. We compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Reconstructed embryos support normal fertilization and produce live offspring. Importantly, genetic analysis confirms that the F1 generation from polar body transfer possesses minimal donor mtDNA carryover compared to the F1 generation from other procedures. Moreover, the mtDNA genotype remains stable in F2 progeny after polar body transfer. Our preclinical model demonstrates polar body transfer has great potential to prevent inherited mtDNA diseases.