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Polar body biopsy

      Polar body biopsy combined with array comparative genomic hybridization allows detection of maternal chromosomal aberrations. Although it has limitations, it can be seen as an alternative to blastomere and trophectoderm biopsy.

      Key Words

      Discuss: You can discuss this article with its authors and with other ASRM members at http://fertstertforum.com/montagm-polar-body-biopsy/
      Preimplantation genetic diagnosis (PGD) was implemented for diagnosing genetic diseases in known carriers before embryo transfer. Screening for chromosomal aneuploidies soon became a major indication for PGD, defined by the term “preimplantation genetic screening” (PGS). At that time PGD and PGS were both based on the biopsy and subsequent analysis of either blastomeres from day 3 embryos or polar bodies (PBs) from unfertilized or inseminated oocytes. The most obvious reason for PB biopsy was the legal condition in several countries. However, the controversial discussion about the benefit of PGS based on blastomere biopsy and fluorescence in situ hybridization (FISH) with 5–12 chromosomes led toward a reflection concerning the stage of biopsy (
      • Geraedts J.
      • Collins J.
      • Gianaroli L.
      • Goossens V.
      • Handyside A.
      • Harper J.
      • et al.
      What next for preimplantation genetic screening? A polar body approach!.
      ). The two alternatives discussed were PB and trophectoderm biopsy (
      • Harper J.
      • Coonen E.
      • De Rycke M.
      • Fiorentino F.
      • Geraedts J.
      • Goossens V.
      • et al.
      What next for preimplantation genetic screening (PGS)? A position statement from the ESHRE PGD Consortium Steering Committee.
      ). Meanwhile it is widely accepted that for PGS, both methods should be performed in combination with array comparative genomic hybridization (CGH) for all chromosomes. Obviously, PB biopsy has known restrictions because only the female part can be investigated, whereas trophectoderm biopsy uncovers both the maternal and the paternal impacts on the embryo itself. The present review summarizes the present state of PB biopsy.

      Diagnostic capabilities with polar bodies

      Polar bodies are by-products of the meiotic division of the oocyte. Removal of the first and/or second PB is an indirect approach allowing the genetic or chromosomal status of the oocyte to be inferred from that of the PB. Both PBs are not required for successful fertilization or normal embryonic development. Numerous reports have shown that PBs can be used for a large variety of investigations and diagnostic purposes. The range of procedures spans from genetic diseases to structural and numeric chromosome aberrations. However, PBs provide information only on the female part, which is critical in the case of certain monogenetic diseases. In a recessive disease, PB analysis will only distinguish whether the oocyte carries the normal or the affected allele. The state of the corresponding embryo will be determined by the paternal allele and can be normal, unaffected (carrier), or affected. If only PB diagnosis is done, oocytes identified with the affected allele will not be used although one-half of them could result in an unaffected carrier. This can be seen as an ethical dilemma.

      Polar body biopsy

       Opening of the Zona Pellucida

      Independently from the diagnostic goal, and like any other biopsy technique, PB removal requires access to the perivitelline space of the oocyte through an opening of the zona pellucida. In principle, there are three different methods available. One of the first methods used for blastomere biopsy, acidic tyrode solution, is not tolerated by the oocyte and may interfere with further embryo development. Thus opening the the zona can only be accomplished by mechanical (
      • Cieslak J.
      • Ivakhenko V.
      • Wolf G.
      • Sheleg S.
      • Verlinsky Y.
      Three dimensional partial zona dissection for preimplantation genetic diagnosis and assisted hatching.
      ) or by laser dissection (
      • Montag M.
      • van der Ven K.
      • Delacrétaz G.
      • Rink K.
      • van der Ven H.
      Laser-assisted microdissection of the zona pellucida facilitates polar body biopsy.
      ). In the hands of experienced embryologists both techniques work equally well, and laser-assisted biopsy requires less time for the overall procedure compared with the mechanical approach (
      • Magli M.C.
      • Montag M.
      • Köster M.
      • Muzi L.
      • Geraedts J.
      • Collins J.
      • et al.
      Implementing polar body biopsy and chromosome copy number analysis by array comparative genomic hybridisation: technical experiences from the ESHRE PGS Task Force pilot study.
      ) (Fig. 1). Mechanical and laser zona dissection create a permanent opening in the zona, and so the size or geometry of the opening has to be considered. The opening should not be too large, to avoid loss of blastomeres during embryo development, and it should not be too small, because this may trap the embryo during hatching at the blastocyst stage (
      • Montag M.
      • van der Ven H.
      Laser-assisted hatching in assisted reproduction.
      ).
      Figure thumbnail gr1
      Figure 1Course of laser-assisted polar body biopsy, shown in a sequence of images that were taken within a time interval of 2 minutes. After positioning of the oocytes with both polar bodies aligned in one focal plane (A), a precise opening is introduced by 2–3 laser shots (B). Through the opening a blunt biopsy capillary can be introduced to aspirate the polar bodies (C), which can then be released in a separate droplet in the same dish (D) for further processing.
      There is an on-going discussion on the safety of using a laser for biopsy. One publication reported a negative impact on embryo quality and development (
      • Levin I.
      • Almog B.
      • Shwartz T.
      • Gold V.
      • Ben-Yosef D.
      • Shaubi M.
      • et al.
      Effects of laser polar-body biopsy on embryo quality.
      ), whereas another study reported no differences in blastocyst development between mechanical and laser biopsy (
      • Macas E.
      • Xie M.
      • SChaufelberger S.
      • Merki-Feld G.S.
      • Stiller R.
      • Imthurn B.
      Vitrification of human single pronuclear oocytes following two approaches to polar body biopsy.
      ). According to data collection XI of the European Society for Human Reproduction and Embryology PGD consortium, laser is used in almost 75% of all PGS cycles, including those based on PB biopsy (
      • Goossens V.
      • Traeger-Synodis J.
      • Coonen E.
      • De Rycke M.
      • Moutou C.
      • Pehlivan T.
      • et al.
      ESHRE PGD Consortium data collection XI: cycles from January to December 2008 with pregnancy follow-up to October 2009.
      ).

       The Biopsy Procedure: When and How

      Recent investigations using polarization microscopy have shown that some oocytes presenting a first PB (PB1) may still be in telophase I owing to the presence of a connective spindle strand between the first PB and the oocyte (
      • Montag M.
      • Schimming T.
      • van der Ven H.
      Spindle imaging in human oocytes: the impact of the meiotic cell cycle.
      ). Such a spindle bridge is a remnant of the meiotic division and occurs during formation of PB1 as well as at the completion of the second meiotic division after extrusion of the second PB (PB2). The spindle bridge is present for only a limited time period, which is usually 1–2 hours after extrusion of the first or second PB. In view of this, PB biopsy should not be performed within too short a time period after their formation. As long as chromosomal material from the oocyte is still attached to these spindle fibers there is a risk of pulling this material out during biopsy, which may result in enucleating the oocyte.
      Removal of the first and second PB can be done at separate times (sequential approach) or at the same time (simultaneous approach). Simultaneous biopsy of the first and second PB requires only one manipulation and helps to reduce stress to the oocyte. It is best accomplished in a time window of 8–14 hours after fertilization. Too early biopsy bears the risk of spindle remnants in the second PB, and too late biopsy may result in a first PB that already started disintegration or degeneration. The latter problem is especially important if the analysis is based on FISH, because it may contribute to diagnostic failures (
      • Munné S.
      • Dailey T.
      • Sultan K.M.
      • et al.
      The use of first polar bodies for preimplantation diagnosis of aneuploidy.
      ).
      For simultaneous biopsy it can sometimes be difficult to distinguish PB1 and PB2 or to accurately separate PB1 and PB2 in case of fragmentation. Using single-nucleotide polymorphism (SNP)–based analysis of heterozygosity could help to identify and distinguish PB1 and PB2 (
      • Treff N.R.
      • Scott Jr., R.T.
      • Su J.
      • Campos J.
      • Stevens J.
      • Schoolcraft W.
      • et al.
      Polar body morphology is not predictive of its cell division origin.
      ). Otherwise, sequential biopsy may be preferred if a fragmented PB1 is already present at the time of injection.
      Regarding isolation of PBs for array-CGH, the timing of biopsy of PB2 seems to influence amplification results. It was reported that too early biopsy of PB2 (4–6 hours after ICSI) may slightly lower the amplification efficiency and that this effect disappeared after adjustment to later biopsy times (>8 hours after ICSI) (
      • Magli M.C.
      • Montag M.
      • Köster M.
      • Muzi L.
      • Geraedts J.
      • Collins J.
      • et al.
      Implementing polar body biopsy and chromosome copy number analysis by array comparative genomic hybridisation: technical experiences from the ESHRE PGS Task Force pilot study.
      ).
      There is ongoing discussion on the need to biopsy and analyze both PBs. A recent study showed that in younger women, PB1 is more prone than PB2 to meiotic errors causing aneuploidies, whereas the opposite holds true in older women (
      • Fragouli E.
      • Alfarawati S.
      • Goodall N.N.
      • Sánchez-Garcia J.F.
      • Colls P.
      • Wells D.
      The cytogenetics of polar bodies: insights into female meiosis and the diagnosis of aneuploidy.
      ). Therefore one could be tempted to conclude that analysis of PB1 is more important in younger women and PB2 in older women. However, a reliable diagnosis can only be based on the analysis of both PBs, and results from clinical studies on the concordance of PB1 and PB2 analysis and the corresponding oocyte give direct proof of this (
      • Magli M.C.
      • Montag M.
      • Köster M.
      • Muzi L.
      • Geraedts J.
      • Collins J.
      • et al.
      Implementing polar body biopsy and chromosome copy number analysis by array comparative genomic hybridisation: technical experiences from the ESHRE PGS Task Force pilot study.
      ,
      • Geraedts J.
      • Montag M.
      • Magli M.C.
      • Repping S.
      • Handyside A.
      • Staessen C.
      • et al.
      Polar body array CGH for prediction oft he status oft he corresponding oocyte. Part I: clinical results.
      ) (Fig. 2).
      Figure thumbnail gr2
      Figure 2Array-CGH–based PB diagnosis. (A) An example of a euploid polar body after array comparative genomic hybridization. Hybridization signals for all chromosomes are located within the standard deviation, indicative for no gains or losses. (B) A polar body with a loss of chromosome 9 and a gain for chromosomes 11 and 15, which indicates a potential gain of chromosome 9 and a loss of chromosomes 11 and 15 in the corresponding oocyte. (C) Analysis of a polar body from a translocation carrier. Besides the presence of an unbalanced translocation, visible by a gain of 2q and a loss of 4p, there is an additional loss of chromosome 7.

      Transfer of polar bodies for FISH and array-CGH

      Transfer of PBs for FISH and array-CGH differs and requires adequate procedures. For FISH it is essential to transfer PBs under visual control onto a glass slide and within a very defined area. This is best accomplished by using the biopsy capillary with both PBs aspirated and transferring into a 0.1–0.2-μL drop of water that has been placed onto a clean slide with the use of the inverted injection microscope. It is essential to follow evaporation of the water droplet and drying of the PBs on the slide, which can be done on a stereomicroscope with good optical contrast and reasonably high magnification (×80–100). After drying, the area with the PBs must be encircled with the use of a diamond or tungsten pen on top of the slide, because this is the only way that guarantees finding that spot after hybridization and washing. Because PB1 and PB2 can be distinguished during evaluation of the FISH signals, both PBs can be placed in the same area. To work cost-effectively, several water droplets can be placed side by side as narrowly as possible within an area that can later be covered with a round coverslip of 5, 8, or 12 mm diameter to minimize the amount of hybridization probe needed. A detailed description of this procedure has been given (
      • Montag M.
      Polar body biopsy and its clinical application.
      ).
      For molecular-based chromosome enumeration protocols, such as array-CGH, both PBs must be transferred into separate reaction tubes and in a defined volume that fits the subsequent amplification protocol (
      • Magli M.C.
      • Montag M.
      • Köster M.
      • Muzi L.
      • Geraedts J.
      • Collins J.
      • et al.
      Implementing polar body biopsy and chromosome copy number analysis by array comparative genomic hybridisation: technical experiences from the ESHRE PGS Task Force pilot study.
      ). A standard setting for array-CGH is to prefill reaction tubes with 2.0–2.2 μL phosphate-buffered saline solution and to transfer PBs with 0.2–0.4 μL medium into this solution to give a final maximum volume of 2.4 μL. The transfer is best accomplished by using an unbreakable plastic capillary with a fine tip, similar to those used for oocyte denudation, that has a diameter ranging from 80 to 140 μm. With the use of a high-contrast stereomicroscope, PBs can be easily identified in the medium droplets and aspirated for immediate transfer. It is advisable to rinse the capillary after transfer to verify that the PB has been placed in the reaction tube, because this process can not be directly visualized owing to the plastic material of the tubes.

      Pitfalls of polar body biopsy

      A major problem of the PB approach is the interpretation of the results with FISH, especially for PB1. Whereas PB2 formation and aging can be precisely controlled because the time point of injection initiates the course of PB2 extrusion, the situation is different for PB1. At the time of ovum pick-up the majority of oocytes are already in metaphase II, but it is unknown at what time PB1 has been extruded and how long the chromatin of PB1 is already prone to an aging process. This aging process affects the quality of the DNA, the coherence of the chromatids, its dispersion after isolation on a slide, and, therefore, hybridization efficiency. In contrast, isolation and amplification of PB1 DNA for array-CGH can be accomplished with >90% success rates, even from PBs that show signs of degeneration.
      In view of the costs of array-CGH, the economic impact of PB biopsy has to be considered as well. Because both PBs need to be separately analyzed, costs are doubled. Bearing in mind the fact that not all oocytes develop into viable embryos is another drawback of PB biopsy.

      Predictive value of polar body diagnosis

      The growing interest in PB biopsy and diagnosis (
      • Geraedts J.
      • Collins J.
      • Gianaroli L.
      • Goossens V.
      • Handyside A.
      • Harper J.
      • et al.
      What next for preimplantation genetic screening? A polar body approach!.
      ) has stimulated further studies on the value of this technology. Whereas some studies reported a high or acceptable correlation for predicting aneuploidy based on PB analysis (
      • Geraedts J.
      • Montag M.
      • Magli M.C.
      • Repping S.
      • Handyside A.
      • Staessen C.
      • et al.
      Polar body array CGH for prediction oft he status oft he corresponding oocyte. Part I: clinical results.
      ,
      • Christopikou D.
      • Tsorva E.
      • Economou K.
      • Shelley P.
      • Davies S.
      • Mastrominas M.
      • et al.
      Polar body analysis by array comparative genomic hybridization accurately predicts aneuploidies of maternal meiotic origin in cleavage stage embryos of women of advanced maternal age.
      ), others question the accuracy of PB diagnosis owing to the high incidence of postzygotic errors (
      • Capalbo A.
      • Bono S.
      • Spizzichino L.
      • Biricik A.
      • Baldi M.
      • Colamaria S.
      • et al.
      Sequential comprehensive chromosome analysis on polar bodies, blastomeres and trophoblast: insight into female meiotic errors and chromosomal segregation in the preimplantation window of embryo development.
      ). Both studies were done on array-CGH, but recent data showed that quantitative PCR has a much higher accuracy than array-CGH (
      • Treff N.R.
      qPCR based CCS.
      ). At present we can not exclude that the approach of analyzing DNA from a single PB may be more prone to methodologic impact than trophectoderm biopsies with multiple cells.
      Another controversial topic is reciprocal chromosome aneuploidies in PBs. A recent study showed that this situation mostly gives rise to normal euploid embryos (
      • Forman E.J.
      • Treff N.R.
      • Stevens J.M.
      • Garnsey H.M.
      • Katz-Jaffe M.G.
      • Scott Jr., R.T.
      • et al.
      Embryos whose polar bodies contain isolated reciprocal chromosome aneuploidy are almost always euploid.
      ). Furthermore, the birth of a healthy child has been reported from an oocyte with reciprocal aneuploid PBs (
      • Scott Jr., R.T.
      • Treff N.R.
      • Stevens J.
      • Forman E.J.
      • Hong K.H.
      • Katz-Jaffe M.G.
      • et al.
      Delivery of a chromosomally normal child froma n oocyte wuth reciprocal aneuploid polar bodies.
      ). These finding will affect future decisions on how to proceed with such diagnostic results, provided that the diagnosis is based on a quantitative evaluation that allows differentiating between chromatid- or chromosome-derived aneuploidies.

      Conclusion

      PB biopsy has been proven to be sufficiently effective for the diagnosis of structural and numeric chromosome aberrations in human oocytes with the use of FISH (
      • Montag M.
      • van der Ven K.
      • Dorn C.
      • van der Ven H.
      Outcome of laser-assisted polar body biopsy.
      ,
      • Verlinsky Y.
      • Tur-Kaspa I.
      • Cieslak J.
      • Bernal A.
      • Morris R.
      • Tranissi M.
      • et al.
      Preimplantation testing for chromosomal disorders improves reproductive outcome of poor-prognosis patients.
      ) and array-CGH (
      • Geraedts J.
      • Montag M.
      • Magli M.C.
      • Repping S.
      • Handyside A.
      • Staessen C.
      • et al.
      Polar body array CGH for prediction oft he status oft he corresponding oocyte. Part I: clinical results.
      ). Nevertheless, the use of PB biopsy and array-CGH for PGS is still a matter of debate because of cost-effectiveness, the high incidence of postmeiotic aneuploidies that are undetectable by the PB approach (
      • Capalbo A.
      • Bono S.
      • Spizzichino L.
      • Biricik A.
      • Baldi M.
      • Colamaria S.
      • et al.
      Sequential comprehensive chromosome analysis on polar bodies, blastomeres and trophoblast: insight into female meiotic errors and chromosomal segregation in the preimplantation window of embryo development.
      ), and arguable diagnostic accuracy (
      • Treff N.R.
      qPCR based CCS.
      ).

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