Polar Body Diagnosis Research Articles

Do the risks of preimplantation genetic testing for aneuploidy outbalance the benefits?

Much has been achieved in the field of preimplantation genetic testing for aneuploidy (PGT-A; previously preimplantation genetic screening, PGS) in recent years, particularly with the novel and more precise methods for analyzing biopsied cells such as comparative genomic hybridization (CGH) and single-nucleotide polymorphism (SNP) arrays. Although these methods have been used in individual studies and shown an improvement in pregnancy and birth rates in some cases, the final results from prospective randomized studies are still awaited. Until these are available, the promising results from individual studies remain experimental. We also owe it to our patients to apply promising novel methods outside of studies, if a positive effect has been demonstrated. The same applies to the polar body diagnosis (PBD) practiced in Germany for aneuploidy screening. Thus, one can conclude that neither PGT-A by means of analyzing blastomeres nor polar body screening to indirectly investigate oocytes currently have a positive effect on pregnancy and birth rates. The large specialist societies, the American Society for Reproductive Medicine (ASRM) and the European Society of Human Reproduction and Embryology (ESHRE), also advise against routine use of these methods.

Pre-implantation and polar body diagnosis in cases of parental chromosomal translocations applying array-CGH.

Parents with a balanced chromosomal translocation show an increased risk of reduced fertility including spontaneous abortions and chromosomally unbalanced offspring. In the course of genetic counseling the parents frequently decide to seek artificial reproductive techniques. In vitro fertilization (IVF) offers the chance to perform Polar Body Diagnosis (PBD) or Preimplantation Diagnosis (PID). Female translocation carriers can opt for analysis of polar body, blastomeres or trophectoderm cells while male translocation carriers have only a choice between blastomere and trophectoderm diagnostic. In Germany, up to the year 2010 a restrictive “Embryo Protection Law” did allow only PBD excluding male translocation carriers from diagnostics. Thus, experience with PBD applying FISH or array-CGH was collected in cases of maternal translocations. For such cases, advantages and limits of array analysis as compared to the FISH approach will be presented. Furthermore, the resolution power of the BlueGnome 24sure and 24sure+ arrays will be shown. In particular we report here on the segregation pattern of maternal translocation chromosomes in 16 cases including 24 cycles and analyzing 97 first polar bodies. In addition to the distribution of the translocation chromosomes the number of segregation errors leading to aneuploidy in the 1st. polar body will be presented. Since a German high court ruled that PID of trophectoderm cells should be legal under very stringent conditions, the Embryo Protection Law had been slightly modified in 2011. In future this will allow the application of array analysis also in cases of paternal translocation.

Pregnancy and Birth After a Two-Step PGD: Polar Body Diagnosis for Hemophilia A and Array CGH on Trophectoderm Cells for Chromosomal Aberrations.

Objective: To demonstrate that a PGD program can be successfully established after the 2011 verdict of the German Bundestag concerning PGD. Material and Method: Eight years previously, the couple had had a daughter who suffered from clinically manifest hemophilia A due to an unbalanced X-inactivation, as well as microdeletion syndrome resulting in severe physical and mental disability. The couple wished to have a second child but refused the idea of a "trial" pregnancy. Given the indications for both, it was necessary to carry out polar body diagnosis (PBD) to rule out hemophilia A and, during the same cycle, a subsequent PGD on the blastocysts to rule out genetic aberrations. The PBD and PGD (trophectoderm biopsy, TEB) were performed after high-dosage ovarian stimulation and ICSI fertilization of the oocytes. A blastocyst was successfully transferred on day 6. Results: The patient conceived immediately. The pregnancy developed normally and the patient gave birth to a girl in the 40th week of pregnancy. Post-natal examinations showed that the baby is free from hemophilia A and is developing normally both physically and mentally. Conclusion: Establishment of a PGD program is now possible after legalization of PGD in Germany. It is possible to apply two investigative techniques in a single treatment cycle if multifactorial diagnosis is required.

O-25 PGD for monogenic disorders on polar bodies and trophectoderm biopsies – potential impact of genetic factors and hormone treatment on the outcome of polar body diagnosis, “PBD”

O-25 PGD for monogenic disorders on polar bodies and trophectoderm biopsies – potential impact of genetic factors and hormone treatment on the outcome of polar body diagnosis, “PBD”

Geburt nach Eizellvitrifikation und Polkörperdiagnostik für Mukoviszidose

Fragestellung: Die Polkörperdiagnostik für monogene Erkrankungen wurde bisher an reifen Eizellen (Metaphase II) nach der Eizellgewinnung durchgeführt. Durch das neue Kryokonservierungsverfahren der Vitrifikation ist es möglich, Eizellen ohne Verlust der Befruchtungsfähigkeit einzufrieren. Kann an Eizellen nach vorangegangener Vitrifikation die Fertilisation und Polkörperdiagnostik erfolgreich mit nachfolgender Schwangerschaft und Geburt durchgeführt werden? Material und Methodik: Eine 33-jährige Anlageträgerin für zystische Fibrose (CF), die bereits ein Kind mit Mukoviszidose geboren hat, wurde zur CFTR-Polkörperdiagnostik stimuliert. Aufgrund unerwarteter technischer Probleme mit dem Testsystem für die Polkörperdiagnostik vor der Follikelpunktion wurden in Absprache mit der Patientin die gewonnenen 11 Metaphase-II-(MII)-Eizellen nach der Punktion vitrifiziert. Die Polkörperdiagnostik wurde 3 Monate später an den aufgetauten Eizellen im Rahmen eines künstlichen Zyklus durchgeführt. Ergebnisse: Acht MII-Eizellen waren nach dem Auftauen vital. Fünf dieser Eizellen ließen sich nach ICSI imprägnieren (sog. Vorkern- oder Pronukleusstadium). Zwei Vorkernstadien wurden wegen Mutationsnachweis und eine Eizelle wegen Verlusts des 2. Polkörperchens verworfen, sodass 2 Eizellen mit mütterlichem Wildtyp-Allel weiterkultiviert und transferiert wurden. Es konnte eine fortlaufende Einlingsschwangerschaft erzielt werden. Aufgrund einer schweren Gestose wurde die Patientin bereits in der 26. SSW von einem gesunden, aber unreifen männlichen Neugeborenen per Sectio entbunden. Schlussfolgerung: Nach unserer Kenntnis ist dies der erste Bericht einer erfolgreichen Polkörperdiagnostik für monogene Erkrankungen nach Vitrifikation von MII-Eizellen mit nachfolgender erfolgreicher Schwangerschaft.

Outcome of intracytoplasmic sperm injection with and without polar body diagnosis of oocytes

To compare the reproductive outcome of women undergoing intracytoplasmic sperm injection (ICSI) with or without polar body diagnosis of oocytes. Nonrandomized retrospective study. University-based human genetic institute in collaboration with a private fertility center. Six hundred seven women undergoing ICSI with polar body diagnosis and 591 women undergoing ICSI without polar body diagnosis at the same time in the same fertility center. Polar body testing of ICSI oocytes by five-color fluorescence in situ hybridization. Pregnancy rate (positive fetal heartbeats) and live-birth rate (of at least one child). The pregnancy and live-birth rates were significantly lower in women undergoing ICSI with polar body diagnosis than in women without polar body diagnosis. The negative effects of polar body diagnosis were evident in all analyzed subgroups, that is, women of different age groups, with one ICSI cycle, with transfer of a high-quality embryo, and with male factor infertility as indication for ICSI. Within the legal restrictions of the German embryo protection law aneuploidy testing of oocytes may not improve reproductive outcome.

Polar body biopsy: a viable alternative to preimplantation genetic diagnosis and screening

Polar body diagnosis (PBD) is a diagnostic method for the indirect genetic analysis of oocytes. Polar bodies are by-products of the meiotic cell cycle, which have no influence on further embryo development. The biopsy of polar bodies can be accomplished either by zona drilling or laser drilling within a very short time period. However, the paternal contribution to the genetic constitution of the developing embryo cannot be diagnosed by PBD. The major application of PBD is the detection of maternally derived chromosomal aneuploidies and translocations in oocytes. For these indications, PBD may offer a viable alternative to blastomere biopsy as the embryo's integrity remains unaffected, in contrast to preimplantation genetic diagnosis (PGD) by blastomere biopsy. The rapid pace of developments in the field of molecular diagnostics will also influence the advantages of PBD, and probably allow more general diagnostic applications in the future.

Efficiency comparison between two preimplantation genetic diagnostic methods for chromosomal translocation carriers

To compare the diagnostic efficiency between blastomere preimplantation genetic diagnosis (PGD) and polar body PGD for chromosomal translocation carriers. Group A had 8 cycles using whole painting probes for the first polar body diagnosis, while group B had 29 cycles using two subtelomeric probes and one centromeric probe for the blastomere diagnosis. The fertilization rate of group A was significantly lower than group B [66.1% (72/109) vs 85.2% (304/357), P < 0.05]. There was no significant difference in the successful biopsy rate between two groups. However, group A had a significantly higher loss rate during fixation and higher no signal rate after fluorescence in situ hybridization [FISH, 9.6% (12/104) vs 1.6% (4/252), 11.2% (10/89) vs 3.0% (7/233)]. Totally, the diagnostic efficiency in group A (72.5%, 79/109) was significantly lower than that in group B (89.8%, 230/256, P < 0.05). Although both the clinical pregnancy rate (3/7) and implantation rate (22.2%, 4/18) of group A were higher, the differences were not statistically significant (P > 0.05). Both methods can be used efficiently in the PGD for chromosomal translocation carriers. Blastomere PGD has a higher diagnostic rate.

Polar Body Diagnosis – A Step in The Right Direction?

Polar body diagnosis (PBD) is a new diagnostic method for the indirect genetic analysis of oocytes, which is carried out as part of in vitro fertilization. The biopsy of polar bodies is technically demanding and cannot be adopted uncritically in routine practice, in the absence of robust data to support this laboratory procedure. Selective literature review and analysis of own PBD data. The main application of PBD is the detection of chromosomal aneuploidies and maternally inherited translocations in oocytes. The major disadvantage of PBD is that the paternal contribution to the genetic constitution of the developing embryo cannot be evaluated. Moreover, the potential value of polar body biopsy for the diagnosis of monogenetic diseases is limited. The role of PBD in improving of success rates in assisted reproduction requires evaluation in further clinical trials. For maternal translocations, PBD can be used to reduce the risk of miscarriage. Rapid development in the field of molecular diagnostic and biopsy techniques will also influence PBD and will most likely allow wider application of this method in the near future.

The Optimization of Polar Body Diagnosis – A Consequence of the Embryo Protection Law

Couples wishing to have progeny want to rule out an increased health risk for their children. Most of them do everything possible to recognize a potentially elevated risk in good time. This is a natural, but also justified concern, and medicine has an obligation to help these couples. Sociological reasons are to a great extent responsible for the fact that women’s reproductive time window has become steadily smaller and age at childbirth has increased over the last few decades. While in the old Federal Republic of Germany 8.4% of children were born to women over 35 years of age in 1975, in 2002 this rate had increased to 15.4% in Germany and to as high as 17 % in 2006 (1). The probability of conception decreases rapidly in women from the age of 35 years onwards. Many couples wishing to have children then frequently pin their hopes on reproduction medicine and especially on in vitro fertilization (IVF) techniques and intracytoplasmic sperm injection (ICSI). The older age of these women has in turn led to an increased rate of numerical chromosome aberrations in the oocytes. In a 40-year-old woman, 50% to 70% of the mature oocytes are affected by aneuploidy (2, 3). Most numerical aberrations are associated with such severe developmental disorders that early embryonic demise is the result.

Neue Möglichkeiten der pränatalen genetischen Diagnostik inklusive Präimplantationsdiagnostik

During recent years the progress with the most important practical impact in prenatal diagnosis has been the implementation of first trimester risk screening for common aneuploidies leading to a much improved identification of pregnancies at risk. Molecular methods for a rapid, cost-effective, but selective aneuploidy diagnosis such as interphase FISH or QF-PCR have been around for years, do have their specific indications, but will unlikely replace conventional cytogenetic tools in routine diagnosis. They apparently do also play a role as marketing instruments in the competition among cytogenetic laboratories. The most thrilling issue for all cytogeneticists in the years to come will be the introduction of array-based methods in the prenatal routine diagnosis of chromosomal abnormalities. Polar body diagnosis has been the only option available for preimplantation genetic diagnosis in german speaking countries. The overwhelming majority of all professionals involved and many families concerned share the hope that the legal situation will improve in these countries to allow the examination of early embryos in high risk situations.

Polar body diagnosis: A highly reliable tool to diagnose age related aneuploidies

Introduction: In humans, aneuploidy rates determined in newborns are clearly related to maternal age. Assisted reproductive treatment (ART) now allows to study age related aneuploidy rates of fertilised oocytes by polar body diagnosis (PBD) through fluorescence-in-situ-hybridisation (FISH).

Polar body diagnosis of 263 unfertilised oocytes: Frequent finding of aneuploidies

Introduction: Aneuploidies of human oocytes are presumed to be a major cause of fertilisation failure in assisted reproductive treatment (ART) cycles. As underlying mechanisms of origin, meiotic nondisjunction of bivalent chromosomes as well as unbalanced predivision of chromatids are discussed [1,2]. The purpose of this study was to evaluate the frequency of aneuploidies in oocytes remaining unfertilised during ART and to identify the underlying mechanism of formation by means of polar body diagnosis (PBD) using the fluorescence-in-situ-hybridisation (FISH) technique.

P-698: Polar body analysis of 314 unfertilized oocytes: What can we learn?

Aneuploidies of human oocytes are presumed to be a major cause of fertilization failure in assisted reproductive treatment (ART) cycles. Aneuploid oocytes result from meiotic segregation errors during oocyte maturation. As underlying mechanisms of origin, meiotic nondisjunction of bivalent chromosomes as well as unbalanced predivision of chromatids are discussed. The purpose of this study was to evaluate the frequency of aneuploidies in oocytes remaining unfertilized during ART and to identify the underlying mechanism of formation by means of polar body diagnosis (PBD) using the fluorescence-in-situ-hybridization (FISH) technique.

Polar body biopsy and aneuploidy testing by simultaneous detection of six chromosomes

To simultaneously detect six chromosomes in a single round of fluorescence in situ hybridization (FISH) during polar body diagnosis and aneuploidy testing in human in vitro fertilization (IVF) treatment. A commercially available five-color FISH probe was modified by an additional chromosome probe. This kit was first tested on lymphocyte spreads and then used for polar body diagnosis (PBD) in patients with advanced maternal age and repeated implantation failure. The outcome of IVF treatment was compared with a control group. All six chromosomes could be simultaneously detected and easily distinguished by FISH analysis. PBD and aneuploidy testing were performed in 75 treatment cycles and compared with 126 controls. The biochemical pregnancy rate was significantly higher in the PBD group (37.1% vs 22.9%, p < 0.05) and a trend was observed for higher clinical pregnancy and implantation rates (24.22% and 14.4% vs 18.62% and 10.8%, respectively) and lower abortion rates (20% vs 31.8%) following PBD. The simultaneous detection of six chromosomes in a single FISH round is possible and can be applied to PBD. This approach may present another step towards increasing the number of chromosomes for aneuploidy testing.

Polar Body Diagnosis for Hemophilia A Using Multiplex PCR for Linked Polymorphic Markers

Preimplantation genetic diagnosis (PGD) is usually performed on blastomeres. In Germany, the only possibility to perform PGD is by analysis of polar bodies. We performed PGD using polar bodies in a woman who is a carrier of hemophilia A. Multiplex PCR followed by nested fluorescent PCR for five linked polymorphic markers was established. From 11 analyzed polar bodies, only 1 showed alleles linked to the mutation. The corresponding oocyte was transferred and no pregnancy was established. As seen in other investigations, the rate of heterozygous first polar bodies is surprisingly high.

P▪51 Polar body diagnosis in translocation carriers

P▪51 Polar body diagnosis in translocation carriers

Chromosomal abnormalities in the first and second polar body

Aneuploidy free oocytes may be pre-selected by testing the first and second polar bodies removed from oocytes following their maturation and fertilization. We present here our experience on the application of the method in IVF cycles from patients of advanced maternal age. Overall, 5590 oocytes were obtained from 917 cycles and tested by polar body sampling and fluorescent in situ hybridization (FISH) analysis using specific probes for chromosomes 13, 16, 18, 21 and 22. FISH results were available in 4599 (82.2%) of 5590 oocytes studied, from which 2077(45.2%) were with aneuploidies. Thirty six point one percent of aneuploidies were of the first meiotic origin, and 29.3% of the second meiotic origin. Most errors in the first meiotic division were represented by chromatid errors. The transfer of embryos deriving from 2014 of 2520 aneuploidy free oocytes in 821 treatment cycles resulted in 182 (22.2%) clinical pregnancies and 140 healthy children born after confirmation of the polar body diagnosis. Polar body testing of oocytes provides an approach for pre-selection of aneuploidy free embryos, improving pregnancy rate in IVF patents of advanced maternal age.

Preimplantation diagnosis of common aneuploidies in infertile couples of advanced maternal age.

Because age-related aneuploidies are mainly due to nondisjunction occurring in maternal meiosis, preimplantation genetic diagnosis (PGD) of aneuploidies may have considerable impact on the effectiveness of assisted reproduction technology (ART) for patients of advanced maternal age. Two different approaches have recently become available for PGD of chromosomal disorders, involving fluorescent in-situ hybridization (FISH) technique, which makes it possible to detect chromosome specific signals in interphase nuclei. One of these approaches is based on the cleavage stage biopsy and FISH analysis of one or two blastomeres, the other involving FISH analysis of polar bodies removed from the oocytes (Verlinsky et al. 1996). While both of these approaches have a great potential for preimplantation detection of common aneuploidies, each of them has its own limitations: the cleavage stage PGD may not reflect the chromosomal constitution of the embryo due to a high frequency of mosaicism in cleaving embryos (Delhanty et al, 1993; Munn6 et al, 1993; 1994; Harper et al., 1995), while PGD by polar body FISH analysis does not pick up paternally derived aneuploidies (Verlinsky et al, 1995; 1996a). However, because paternal contribution to the aneuploidy rates is minor compared to that originating in the maternal first and second meiotic divisions (Antonarakis et al, 1992; Hassold et al., 1993), and there is no evidence for the paternal age-related risk, the polar body diagnosis may allow detection and elimination from transfer of the majority of aneuploid embryos. Therefore, the polar body approach should make it possible to detect aneuploid oocytes in in-vitro fertilization (IVF) patients of advanced maternal age to prevent the transfer of embryos resulting from aneuploid oocytes (Verlinsky et al., 1995; 1996a; Munn6 et al., 1995). Accordingly, this should not only reduce the chances of an IVF couple of having a child with Down's syndrome and other common aneuploidies, but may also improve the efficiency of FVF as most aneuploid embryos are lost before or during implantation. The available experience on PGD of chromosomal aneuploidies reviewed by 5th Annual Meeting of International Working Group on Preimplantation Genetics has already demonstrated feasibility of both approaches (Verlinsky, 19%). The majority of the cases have been performed by polar body approach and involved 193 IVF patients of advanced maternal age (Verlinsky et al, 1996b). In these patients 1293 oocytes were biopsied and subjected to FISH analysis, with results of the first and/or second polar bodies available in 993 oocytes. Abnormal FISH patterns were observed in 328 (33%) oocytes based on the analysis of the first and/or second polar bodies. Of

Birth of healthy children after preimplantation diagnosis of common aneuploidies by polar body fluorescent in situ hybridization analysis

To perform preimplantation diagnosis of common aneuploidies by polar body analysis and fluorescent in situ hybridization technique using probes specific for chromosomes X, 18, and 13/21. The first and/or second polar bodies were removed and studied by fluorescent in situ hybridization to detect and avoid fertilization and transfer of oocytes with common aneuploidies. The Reproductive Genetics Institute's IVF program at Illinois Masonic Medical Center. One hundred ninety-three couples of advanced maternal age (34 to 46 years) under-going IVF treatment volunteered to be part of a clinical trial on preimplantation polar body diagnosis of common aneuploidies. Using micromanipulation procedures, the first and second polar bodies were removed after their extrusion from the oocytes. Fluorescent in situ hybridization signals specific for chromosomes X, 18, and 13/21. In 235 IVF cycles performed in 193 couples, 1,293 oocytes were biopsied and subjected to fluorescent in situ hybridization analysis, with fluorescent in situ hybridization results available in 993 oocytes (76.8%). Of 993 oocytes with fluorescent in situ hybridization results, 665 (67%) were predicted to be normal based on the chromosomes studied; 460 embryos resulting from these oocytes were transferred in 187 treatment cycles, resulting in 12 births of healthy children and 18 ongoing pregnancies after confirmation of the polar body diagnosis by chorionic villus sampling or amniocentesis. Polar body fluorescent in situ hybridization analysis may be used for preimplantation diagnosis of common aneuploidies in IVF patients of advanced maternal age.