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Screening oocytes by polar body biopsy
RBMOnline - Vol 13. No 1. 2006 104–109 Reproductive BioMedicine Online; www.rbmonline.com/Article/2181 com/Article/2181 on web 15 March 2005 com/Article/
Article Screening oocytes by polar body biopsy Dr Anja Dawson graduated from the University of Hamburg, Germany. She is currently about to finish her residency in the Department of Gynaecology and Obstetrics at the University Hospital Schleswig-Holstein, Campus Lübeck, Germany, headed by Professor Klaus Diedrich, before a further education in reproductive endocrinology and infertility is planned.
Dr Anja Dawson A Dawson, G Griesinger, K Diedrich1 IVF Unit, Department of Gynaecology and Obstetrics, University Hospital Schleswig-Holstein, Campus Luebeck, Ratzeburger Allee 160, Luebeck, Germany 1 Correspondence: Tel: +49 451 500 2134; Fax: +49 451 500 2160; e-mail: [email protected]
Abstract Preimplantation genetic aneuploidy screening performed by polar body biopsy has become a frequently used method, especially as in several countries only preconceptional genetic diagnosis is allowed. To penetrate the zona pellucida, mechanical, chemical and laser-assisted techniques have been introduced. In this paper, the advantages, disadvantages, efficacy and safety of these techniques are elucidated. Keywords: laser drilling, polar body biopsy, PZD, Tyrode’s acid, zona pellucida dissection
Introduction Preimplantation genetic diagnosis (PGD) is the earliest form of prenatal diagnosis, and can be performed on polar bodies or on embryonic cells. This diagnostic tool may be applied for individual genetic risks, or it is performed during IVF with the aim of increasing pregnancy rates and decreasing abortion rates (Kuliev and Verlinsky, 2002). Due to legal restrictions, PGD on embryonic cells is not allowed in countries such as Germany, Austria, Switzerland and Italy. Therefore, PGD on polar bodies (PB) has a special relevance for these countries, as being the only possibility for genetic testing prior to implantation. Both polar bodies represent extra-embryonic material, as they are extruded during meiosis I and after fertilization, and they are expected to have no biological role during embryonic development. Therefore, their removal should not interfere with fertilization or embryonic development.
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Polar body biopsy as a tool for the analysis of numerical chromosomal disorders was first introduced by Verlinsky et al. in 1990, and was later used for the detection of single gene disorders by analysing the first and the second polar body (Verlinsky et al., 1997). Further fields of application for polar body biopsy are translocation analyses (Munné et al., 1998) and the detection of X-linked disorders (Verlinsky et al., 2001b, 2006), as well as human leukocyte antigen (HLA) typing for a variety of disorders (Verlinsky et al., 2001a; Rechitsky et al.,
2004, 2006; Kuliev et al., 2005). Up to now, PGD has been used to test embryos for more than 100 genetic conditions including such fatal diseases as Tay−Sachs disease, Fanconi anaemia, cystic fibrosis or sickle cell anaemia (Baruch et al., 2005). Polar body biopsy was initially performed on first polar bodies only, from which the term ‘preconceptional genetic diagnosis’ derives, as it can be accomplished prior to the fusion of the pronuclei. Later studies have biopsied both polar bodies before syngamy, as this allows more relevant information to be retrieved. For PGD on polar bodies, several technical issues have to be considered. The successful retrieval of polar bodies is the prerequisite for further diagnosis, as damage to the oocyte during PB retrieval can lead to its loss. Although the mechanical opening of the zona pellucida (ZP) is preferred by Verlinsky’s group, most PGD centres use the laser technique, and the available procedures for ZP opening for PB retrieval are explained later in this paper. Another concern during the protocol of PB biopsy and PGD is that polar bodies are sometimes lost after retrieval and are therefore not available for the diagnosis of the corresponding oocyte. For PB aneuploidy screening, fluorescence in-situ hybridization (FISH) is the most frequently used method. There are several commercial kits available to detect 4–5 chromosomes (usually chromosomes 13, 16, 18, 21 and 22) in both polar bodies. A
Article - Screening oocytes by polar body biopsy - A Dawson et al. normal chromosomal distribution of the oocyte exists if two signals of each chromosome are detectable in PB1 and one signal in PB2 respectively. Using the conventional FISH method, only a limited number of chromosomes are detectable during one hybridization. Due to the limited time frame in countries such as Germany, a maximum of 2× 4–5 chromosomes out of 23 can be detected. Usually, chromosomes often found in aneuploid abortuses, in surviving trisomies or in women with balanced translocations are detected. This might leave chromosomal abnormalities undetected, which lead to developmental arrest before implantation. As an alternative to the FISH technique, polymerase chain reaction (PCR)-based aneuploidy testing is available. Both techniques are reliable, but the big advantage of the PCR method is that analysis of the copy number of the chromosomes and a PCR reaction for causative gene analysis can simultaneously be performed in one cell (Rechitsky et al., 2006). The PB biopsy and aneuploid testing has to be completed before syngamy. That leaves a time frame of 18–20 h after intracytoplasmic sperm injection (ICSI). A simultaneous biopsy of both polar bodies is performed 6–10 h after ICSI, followed by an immediate analysis via FISH, and the results are related to oocyte fertilization. The fertilized oocytes are transferred after further cultivation, cryopreserved or discarded. There are several disadvantages of polar body analysis compared with PGD on embryonic cells. One major concern is that only indirect information on the chromosomal stage of the oocyte can be achieved, leaving all paternal abnormalities undetected (the genotype of the oocyte is derived from the complement present in the polar body). Therefore, polar body analysis only allows the study of the female contribution to aneuploidy, single gene defects and segregation of maternal structural abnormalities in meiosis. Both first and second maternal meiotic errors can only be excluded if information of both polar bodies has been obtained (Angell, 1994). Post-zygotic (mitotic) errors are not detected and a gender determination or the detection of Y-linked disorders is not possible. The allelic dropout often leads to misdiagnosis in PGD and the risk of false positive results has been estimated at 5% (Munné et al., 2002). The sequential biopsy of both polar bodies, although very helpful in the exclusion of meiotic errors, is very time consuming. However, a simultaneous biopsy of both polar bodies often results in the retrieval of an already degenerated first polar body, which can especially be a problem as the binding capacity of FISH DNA probes is decreased in polar bodies in opposition to embryonic cells, and also decreases with PB ageing. Furthermore, due to oocyte ageing, fertilization has to be performed before the final result of the polar body analysis is available. Another aspect is the decreased survival rate of cryopreserved 2 pronuclear (PN)-stage oocytes after zona pellucida opening for PB retrieval. A positive aspect of polar body biopsy is that there is no need for manipulation of the embryo with the risk of an embryonic impairment due to the withdrawal of blastomeres, and false positive results due to mosaicism after post-zygotic chromosomal misdistribution in embryonic cells are avoided. PGD on polar bodies seems to have little effect on improving implantation rates, but there seems to be a benefit for women with an increased maternal age regarding reduction of abortion rates (Grossmann et al., 2004). Furthermore, the likelihood of a child with trisomy 13, 18 or 21 is reduced using this diagnostic
tool for decision making. Several studies found no deleterious effects of the removal of the polar bodies on further embryonic development, and the clinical application in genetic evaluation of human oocytes resulted in clinical pregnancies and livebirths (Verlinsky and Kuliev, 1996). Additionally, a follow-up study on 100 children born after first and second polar body biopsy showed no adverse effects (Strom et al., 2000). Because most numerical chromosomal abnormalities originate from female meiosis, it has been suggested that polar body testing might be used to increase the efficacy of assisted reproduction techniques by a positive selection of euploid oocytes for further in-vitro culture and transfer to the womb. Since the chance of meiotic errors in oocytes increases with age, aneuploidy screening by polar body analysis has mostly been performed in women with advanced maternal age. Another patient group of poor IVF prognosis are those with recurrent IVF implantation failure. PGD for aneuploidy screening seems to improve IVF outcome in this group as well (Caglar et al., 2005), but no convincing data are available so far. In one study by Taranissi et al., the impact of maternal age in patients with recurrent implantation failure (RIF) on the outcome of PGD-aneuploidy screening (AS) cycles has been assessed (Taranissi et al., 2005). The authors found no difference between the two age groups (group A <40 years of age and group B >40 years) regarding the number of fertilized oocytes, but reported a significantly higher proportion of euploid oocytes/embryos, pregnancy and delivery rates per transfer in the younger age group, indicating a detrimental effect of rising maternal age in RIF patients. Munné et al. (2003) also showed the very poor prognosis of RIF patients with advanced maternal age not benefiting from PGD-AS. The first use of micromanipulation techniques was to assist oocyte fertilization (Cohen et al., 1988) and to increase the implantation rate by helping the hatching process (Cohen et al., 1990). Soon after, these techniques were used to biopsy and detect the genetic information of polar bodies or embryonic cells. Micromanipulation techniques include different techniques such as partial zona dissection, which is a mechanical method using a microneedle to open the zona pellucida (Malter and Cohen, 1989), the chemical digestion of the zona using acid (zona drilling) (Gordon et al., 1986) and laser zona drilling (Palanker et al., 1991; Neev et al., 1992; Germond et al., 1995). Several mechanical, chemical and laser techniques for drilling or penetrating the zona pellucida and performing polar body biopsy or blastocyst biopsy have been introduced and further improved in the last 15 years. Special attention has been given to the safety and efficacy of these procedures, as optimal performance obviously has a crucial impact on further embryo development. This paper summarizes the literature elucidating the safety and efficacy of these techniques.
Mechanical partial zona dissection Conventional partial zona dissection (PZD) involves a slit in the zona pellucida by means of a sharp closed microneedle, which results in a longitudinal incision, often irregularly shaped with openings that usually do not exceed 30–40 μm. One concern of
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Article - Screening oocytes by polar body biopsy - A Dawson et al. conventional PZD is that the slit may be too small or that it can be hardened during the fertilization process and might entrap the embryo during the hatching process (Cohen and Feldberg, 1991). Another form of direct penetration of the zona pellucida using a bevelled suctioning pipette has also been proven efficient for polar body biopsy, and allows use of the same pipette for the two procedures of zona dissection and polar body retrieval (Verlinsky and Cieslak, 1993). A refinement of conventional zona dissection is threedimensional PZD, in which two slits perpendicular to one another are made into the zona. The cross-shaped opening allows access to the perivitelline space and simultaneous removal of both polar bodies even when they are located in separate areas. Microtool requirements are one holding pipette and one microneedle. After the first slit opening the zona pellucida, the oocyte is vertically rotated and the second cut is performed (Cieslak et al., 1999). Regarding mechanical techniques for polar body biopsy, some observational studies have been performed to detect the safety of partial zona dissection (PZD). One study by Strom et al. (2000) on a cohort of 109 infants compared outcome in children after PZD with the literature data on ICSI children. Polar body biopsy was performed for both Mendelian disorders and aneuploidy screening. The authors found no differences between the two groups regarding gestational age, mode of delivery, perinatal mortality, birth weight and birth length, birth defects and post-natal growth. Another observational study by Horwitz et al. (unpublished abstract, 2005) on 576 infants showed no increase in birth defects in children after blastomere biopsy or polar body biopsy using the partial zona dissection. Genetic diagnosis was performed to elucidate aneuploidy and single gene disorders. For data analysis, the group used birth data and information on 413 babies supplied by the parents.
Chemical zona drilling In contrast to the mechanical partial zona dissection, Tyrode’s acid (pH 2.3) creates a larger, rounder hole in the zona pellucida and its size is not always easy to control. The human zona pellucida is more resistant to acid treatment compared with mouse oocytes. Therefore higher amounts of Tyrode’s solution are needed to penetrate the human zona pellucida. For chemical zona drilling, oocytes or embryos are usually manipulated individually and an immediate washing is required to avoid higher pH differences (Cohen et al., 1992) as the local acidification of the culture medium or the deposition of acid into the perivitelline space may lead to cell lysis or possibly other, more subtle cell damage. For blastomeres, chemical drilling is normally done with separate pipettes using a double holder set-up: one pipette for drilling with an inner diameter of 5–7 μm and one pipette for aspiration with an inner diameter of 40 μm to avoid blastomere lysis. The Tyrode’s acid is loaded in the right pipette and released until an opening above the polar body is seen.
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Regarding the efficacy and safety of acid drilling some nonrandomized studies are available. One of the first studies focusing on acidic zona-drilling safety has been conducted by Talansky and colleagues (Talansky and Gordon, 1988). The group showed a higher fertilization and live birth rate after acidic drilling on mouse embryos. However, developmental abnormalities associated
with embryo loss, spontaneous chimerism and the possibility of conception of monozygotic twins were also encountered. Magli et al. (2004) combined polar body analysis with blastomere biopsy through the same opening in the zona pellucida, which was achieved by acid drilling. The effects of a combined procedure were evaluated retrospectively by comparing the results obtained in cycles where this strategy was applied with those cycles where either polar body biopsy or embryo biopsy was performed. The comparatively higher implantation rate in the blastomere biopsy group can be explained by the fact that only regularly dividing embryos were biopsied; therefore, chromosomally normal embryos with poor development have been sorted out already. However, no negative influence of chemical zona drilling on main outcome parameters could be observed. It is still not clear if acid exposure may interfere with further embryo development or implantation. The chemical digestion of the glycoprotein matrix of the human zona pellucida of oocytes for polar body biopsy may compromise the oocytes viability (Payne et al., 1991), and some authors have even described this technique as inappropriate (Gordon et al., 1988; Depypere and Leybaert, 1994).
Laser-assisted dissection Laser-assisted dissection as an alternative to chemical or mechanical zona penetration for embryo micromanipulation was first introduced in the early 1990s (Palanker et al., 1991; Neev et al., 1992). This technique was further improved and also used for polar body biopsy (Montag et al., 1997). Nowadays a 1.48 μm diode non-contact infrared laser is widely used for laser zona drilling, assisted hatching and for embryo biopsy, as it is considered to be unlikely to induce mutagenic changes in DNA contrary to UV lasers (Germond et al., 1995; Montag et al. 1998, 2004). Using electromagnetic waves, the laser beam induces only minimal superficial damage to the zona pellucida of oocytes or embryos. Lasers, which with longer wavelengths are less absorbed by water, seem to be more practicable, as they can be used in a non-contact mode. The opening of the zona pellucida by laser drilling is performed by exposing the surface of the ZP to laser light. The size of the hole can be chosen precisely by varying the exposure time, and the holes are much more precise than the holes produced by acidic drilling. Polar bodies can subsequently be withdrawn using blunt-ended glass capillaries. The efficacy and safety of laser drilling and laser-assisted polar body and blastomere biopsy has been the object of several studies. The use of a non-contact laser before embryo biopsy is an easy procedure, and several studies showed no adverse effects on embryo development or pregnancy rates (Boada et al., 1998; Germond et al., 1999; Joris et al., 2003). Selecting an area for laser drilling which has a sufficient distance to the next blastomere does not induce immediate laser damage (Chatzimeletiou et al., 2001). In a controlled, prospective study by Germond et al. (1995), the safety of laser drilling was evaluated. Laser drilling with openings of 4.5–20 μm at different sites has been performed on mouse oocytes and zygotes (B6D2F1). A group of 106 control
Article - Screening oocytes by polar body biopsy - A Dawson et al. embryos and 96 lasered embryos has been cultivated for 5−6 days and the embryos were transferred to foster mice (NMRI). From the lasered embryos, 93 offspring were followed up and cross-mated. The cytoplasmic changes of the oocytes and embryos have been assessed via electromicroscopy. The group found no impairment of the in-vitro or in-vivo development or of the fertility of the offspring. No lysis of oocytes occurred. The same group also determined the effect of laser-assisted zona pellucida dissection (Germond et al., 1996). Other evidence comes from follow-up studies on laser-assisted hatching of the zona pellucida (Kanyo and Konc, 2003). Another prospective, non-randomized study focused on the outcome of laser-assisted polar body biopsy and aneuploidy testing (Montag et al., 2004). Laser-assisted polar body biopsy has been performed in 140 IVF cycles of patients with an advanced maternal age (>35 years). The outcome of polar body biopsy was compared with a retrospective control group including all treatment cycles of the same time period. The group found a clinical pregnancy rate of 22.5% per embryo transfer in the biopsied group compared with 20.2% in the control group. The study provided no description of the follow up of the babies. The birth weights of the children in the polar body biopsy group were comparable with the control group. The numbers of children born after laser-assisted polar body biopsy reported in this study were too small to give a conclusive answer (Montag et al., 1998). Bergere et al. (2003, unpublished abstract) conducted a study on the safety of laser-assisted polar body biopsy. Eighty-three immature oocytes were incubated and matured in vitro and the first polar body was biopsied using the laser drilling procedure. The outcome was compared between four study groups: one control group with no drilling or biopsy, one laser-drilled group using the ZILOS C device (140 mW/ms), one group using the same device and performing the first polar body biopsy and the fourth group using a second laser (ZILOS tk, 180 mW/10.5 ms) followed by polar body biopsy. The authors detected no significant difference regarding oocyte lysis rate and chromosomal breakage rate between the four groups. The oocyte activation rate, detected via calcium ionophore treatment, was significantly lower using the ZILOS C, which reflects a safer procedure by using high power with a short duration. ZILOS C and ZILOS TK are both infrared lasers (1480 nm), but ZILOS TK has a higher full possible power (180 versus 140 mW for the ZILOS C). In both systems the duration of the exposure time can be adjusted (0.5 ms is the shortest possible time). A prospective and observational study by Grossmann et al. focused on the safety and efficacy of laser-assisted polar body biopsy. PB biopsy was performed on oocytes of 460 patients (Grossmann et al., 2004). The study group was compared with an unselected group and data were obtained using the German IVF Registry. Inclusion criteria for the study group was a maternal age >39 years, three or more previous performed IVF cycles or two or more previous abortuses. Chromosomes were detected on first and second polar body. In the study group the authors found a rate of abnormal chromosomes in 57.9% of all oocytes. The clinical pregnancy rate per oocyte retrieval in women aged 31–35 years was 22.7% in the study group and 27.6% in the control group. The corresponding clinical pregnancy rates in the age group of >40 years were 5.9 and 8.6% respectively. Abortion rates were lower in the study group
(28.6%) than in the control group (38.6%). There are a few studies comparing two techniques of zona drilling or penetration for polar body or blastomere biopsy available. One recent study by Chatzimeletiou et al. (2005) compared the safety of the use of a non-contact laser with the use of acidic zona drilling for human embryo biopsy. The group found no significant difference in the proportion of undamaged embryos or blastomeres between the two groups (75% after acid Tyrode’s versus 68% after laser drilling). The incidence of spindle abnormalities at blastocyst stage was not significantly different from controls and no significant difference in the incidence of chromosomal abnormalities and mosaicism between the two groups was observed. However, single biopsied blastomeres after laser drilling showed a greater tendency to form miniblastocysts and acid Tyrode’s drilling tended to retard blastocyst development. Another prospective study by Joris et al. (2003) compared the results of human embryo biopsy and the outcome of PGD after zona drilling using Tyrode’s acid medium or a laser. The study revealed fewer intact blastomeres after acid zona drilling (95.2%) than after laser zona drilling (98.3%, P = 0.02) whereas implantation and pregnancy rates did not differ. The laser method was described easier compared with the acid method. Malter and Cohen (1989) reinseminated initially unfertilized oocytes on day 1 following PZD (n = 60) or acid drilling using Tyrode’s acid (n = 51). The conclusion of the paper was that none of the Tyrode’s acid-exposed oocytes developed satisfactorily. Tyrode’s acid does not seem to jeopardize the fertilization process, but it does seem to interfere with further development. Two studies investigating different techniques for assisted hatching have been published. Hsieh et al. (2002) reported a higher effectiveness using laser-assisted hatching of human embryos compared with the chemical method regarding implantation and pregnancy rates in women with advanced maternal age. The authors concluded that laser zona drilling allows an easier, faster and safer micromanipulation of the zona pellucida. Another retrospective, not randomized study conducted by Balaban et al. (2002) compared the efficacy of four different techniques for assisted hatching. Partial zona dissection was used in 239, acid Tyrode in 191, diode laser hatching in 219 and pronase thinning in 145 IVF or ICSI cycles. The authors found no differences regarding implantation and pregnancy rates using each of the techniques compared with a control group of IVF or ICSI cycles without assisted hatching.
Considerations and conclusions The efficacy and safety of the different methods of zona drilling was evaluated in animal models first, and later feasibility and safety could be partly shown in humans as well. However, all methods of zona dissection and biopsy can harm the oocyte and impair further embryo development. In the hand of an experienced technician all methods for zona pellucida drilling or penetration seem to have a satisfying safety and efficacy. However, at present, no convincing evidence from an efficacy trial exists and a method of first choice has not been established yet, although several authors prefer laser-assisted drilling
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Article - Screening oocytes by polar body biopsy - A Dawson et al. as it seems to be an easier method. Concerns remain for all methods, as they might be associated with an impairment of the developmental potential of the oocyte. The safety of the procedures in terms of ongoing health of children born after polar body biopsy is not proven. Efficacy trials on polar body biopsy after acidic zona drilling are missing. Therefore, using Tyrode’s acid for polar body biopsy should be considered as an experimental procedure. The benefit of aneuploidy screening is probably at best small, since ‘chromosomally normal’ embryos do not implant in the majority of cases. Furthermore, cryopreservation efficacy after zona dissection is impaired and the risk of false-positive results (~5%) may lead to discarding of normal embryos. Whatever technique is used to open the zona pellucida polar body biopsy requires a high number of oocytes, which are not available in many advanced maternal age cases. That women of different age groups undergoing IVF have a benefit from PB biopsy regarding pregnancy and abortion rates still needs to be proven. The further improvement of techniques for the detection of single gene defects, translocations or for aneuploid testing of all chromosomes in both polar bodies will increase the application of polar body biopsy in countries with restricted laws.
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Paper based on contribution presented at the International Serono Symposium ‘How to improve ART outcome by gamete selection’ in Gubbio, Italy, October 7–8, 2005. Received 2 December 2005; refereed 12 December 2005; accepted 5 April 2006.
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Article Screening oocytes by polar body biopsy Dr Anja Dawson graduated from the University of Hamburg, Germany. She is currently about to finish her residency in the Department of Gynaecology and Obstetrics at the University Hospital Schleswig-Holstein, Campus Lübeck, Germany, headed by Professor Klaus Diedrich, before a further education in reproductive endocrinology and infertility is planned.
Dr Anja Dawson A Dawson, G Griesinger, K Diedrich1 IVF Unit, Department of Gynaecology and Obstetrics, University Hospital Schleswig-Holstein, Campus Luebeck, Ratzeburger Allee 160, Luebeck, Germany 1 Correspondence: Tel: +49 451 500 2134; Fax: +49 451 500 2160; e-mail: [email protected]
Abstract Preimplantation genetic aneuploidy screening performed by polar body biopsy has become a frequently used method, especially as in several countries only preconceptional genetic diagnosis is allowed. To penetrate the zona pellucida, mechanical, chemical and laser-assisted techniques have been introduced. In this paper, the advantages, disadvantages, efficacy and safety of these techniques are elucidated. Keywords: laser drilling, polar body biopsy, PZD, Tyrode’s acid, zona pellucida dissection
Introduction Preimplantation genetic diagnosis (PGD) is the earliest form of prenatal diagnosis, and can be performed on polar bodies or on embryonic cells. This diagnostic tool may be applied for individual genetic risks, or it is performed during IVF with the aim of increasing pregnancy rates and decreasing abortion rates (Kuliev and Verlinsky, 2002). Due to legal restrictions, PGD on embryonic cells is not allowed in countries such as Germany, Austria, Switzerland and Italy. Therefore, PGD on polar bodies (PB) has a special relevance for these countries, as being the only possibility for genetic testing prior to implantation. Both polar bodies represent extra-embryonic material, as they are extruded during meiosis I and after fertilization, and they are expected to have no biological role during embryonic development. Therefore, their removal should not interfere with fertilization or embryonic development.
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Polar body biopsy as a tool for the analysis of numerical chromosomal disorders was first introduced by Verlinsky et al. in 1990, and was later used for the detection of single gene disorders by analysing the first and the second polar body (Verlinsky et al., 1997). Further fields of application for polar body biopsy are translocation analyses (Munné et al., 1998) and the detection of X-linked disorders (Verlinsky et al., 2001b, 2006), as well as human leukocyte antigen (HLA) typing for a variety of disorders (Verlinsky et al., 2001a; Rechitsky et al.,
2004, 2006; Kuliev et al., 2005). Up to now, PGD has been used to test embryos for more than 100 genetic conditions including such fatal diseases as Tay−Sachs disease, Fanconi anaemia, cystic fibrosis or sickle cell anaemia (Baruch et al., 2005). Polar body biopsy was initially performed on first polar bodies only, from which the term ‘preconceptional genetic diagnosis’ derives, as it can be accomplished prior to the fusion of the pronuclei. Later studies have biopsied both polar bodies before syngamy, as this allows more relevant information to be retrieved. For PGD on polar bodies, several technical issues have to be considered. The successful retrieval of polar bodies is the prerequisite for further diagnosis, as damage to the oocyte during PB retrieval can lead to its loss. Although the mechanical opening of the zona pellucida (ZP) is preferred by Verlinsky’s group, most PGD centres use the laser technique, and the available procedures for ZP opening for PB retrieval are explained later in this paper. Another concern during the protocol of PB biopsy and PGD is that polar bodies are sometimes lost after retrieval and are therefore not available for the diagnosis of the corresponding oocyte. For PB aneuploidy screening, fluorescence in-situ hybridization (FISH) is the most frequently used method. There are several commercial kits available to detect 4–5 chromosomes (usually chromosomes 13, 16, 18, 21 and 22) in both polar bodies. A
Article - Screening oocytes by polar body biopsy - A Dawson et al. normal chromosomal distribution of the oocyte exists if two signals of each chromosome are detectable in PB1 and one signal in PB2 respectively. Using the conventional FISH method, only a limited number of chromosomes are detectable during one hybridization. Due to the limited time frame in countries such as Germany, a maximum of 2× 4–5 chromosomes out of 23 can be detected. Usually, chromosomes often found in aneuploid abortuses, in surviving trisomies or in women with balanced translocations are detected. This might leave chromosomal abnormalities undetected, which lead to developmental arrest before implantation. As an alternative to the FISH technique, polymerase chain reaction (PCR)-based aneuploidy testing is available. Both techniques are reliable, but the big advantage of the PCR method is that analysis of the copy number of the chromosomes and a PCR reaction for causative gene analysis can simultaneously be performed in one cell (Rechitsky et al., 2006). The PB biopsy and aneuploid testing has to be completed before syngamy. That leaves a time frame of 18–20 h after intracytoplasmic sperm injection (ICSI). A simultaneous biopsy of both polar bodies is performed 6–10 h after ICSI, followed by an immediate analysis via FISH, and the results are related to oocyte fertilization. The fertilized oocytes are transferred after further cultivation, cryopreserved or discarded. There are several disadvantages of polar body analysis compared with PGD on embryonic cells. One major concern is that only indirect information on the chromosomal stage of the oocyte can be achieved, leaving all paternal abnormalities undetected (the genotype of the oocyte is derived from the complement present in the polar body). Therefore, polar body analysis only allows the study of the female contribution to aneuploidy, single gene defects and segregation of maternal structural abnormalities in meiosis. Both first and second maternal meiotic errors can only be excluded if information of both polar bodies has been obtained (Angell, 1994). Post-zygotic (mitotic) errors are not detected and a gender determination or the detection of Y-linked disorders is not possible. The allelic dropout often leads to misdiagnosis in PGD and the risk of false positive results has been estimated at 5% (Munné et al., 2002). The sequential biopsy of both polar bodies, although very helpful in the exclusion of meiotic errors, is very time consuming. However, a simultaneous biopsy of both polar bodies often results in the retrieval of an already degenerated first polar body, which can especially be a problem as the binding capacity of FISH DNA probes is decreased in polar bodies in opposition to embryonic cells, and also decreases with PB ageing. Furthermore, due to oocyte ageing, fertilization has to be performed before the final result of the polar body analysis is available. Another aspect is the decreased survival rate of cryopreserved 2 pronuclear (PN)-stage oocytes after zona pellucida opening for PB retrieval. A positive aspect of polar body biopsy is that there is no need for manipulation of the embryo with the risk of an embryonic impairment due to the withdrawal of blastomeres, and false positive results due to mosaicism after post-zygotic chromosomal misdistribution in embryonic cells are avoided. PGD on polar bodies seems to have little effect on improving implantation rates, but there seems to be a benefit for women with an increased maternal age regarding reduction of abortion rates (Grossmann et al., 2004). Furthermore, the likelihood of a child with trisomy 13, 18 or 21 is reduced using this diagnostic
tool for decision making. Several studies found no deleterious effects of the removal of the polar bodies on further embryonic development, and the clinical application in genetic evaluation of human oocytes resulted in clinical pregnancies and livebirths (Verlinsky and Kuliev, 1996). Additionally, a follow-up study on 100 children born after first and second polar body biopsy showed no adverse effects (Strom et al., 2000). Because most numerical chromosomal abnormalities originate from female meiosis, it has been suggested that polar body testing might be used to increase the efficacy of assisted reproduction techniques by a positive selection of euploid oocytes for further in-vitro culture and transfer to the womb. Since the chance of meiotic errors in oocytes increases with age, aneuploidy screening by polar body analysis has mostly been performed in women with advanced maternal age. Another patient group of poor IVF prognosis are those with recurrent IVF implantation failure. PGD for aneuploidy screening seems to improve IVF outcome in this group as well (Caglar et al., 2005), but no convincing data are available so far. In one study by Taranissi et al., the impact of maternal age in patients with recurrent implantation failure (RIF) on the outcome of PGD-aneuploidy screening (AS) cycles has been assessed (Taranissi et al., 2005). The authors found no difference between the two age groups (group A <40 years of age and group B >40 years) regarding the number of fertilized oocytes, but reported a significantly higher proportion of euploid oocytes/embryos, pregnancy and delivery rates per transfer in the younger age group, indicating a detrimental effect of rising maternal age in RIF patients. Munné et al. (2003) also showed the very poor prognosis of RIF patients with advanced maternal age not benefiting from PGD-AS. The first use of micromanipulation techniques was to assist oocyte fertilization (Cohen et al., 1988) and to increase the implantation rate by helping the hatching process (Cohen et al., 1990). Soon after, these techniques were used to biopsy and detect the genetic information of polar bodies or embryonic cells. Micromanipulation techniques include different techniques such as partial zona dissection, which is a mechanical method using a microneedle to open the zona pellucida (Malter and Cohen, 1989), the chemical digestion of the zona using acid (zona drilling) (Gordon et al., 1986) and laser zona drilling (Palanker et al., 1991; Neev et al., 1992; Germond et al., 1995). Several mechanical, chemical and laser techniques for drilling or penetrating the zona pellucida and performing polar body biopsy or blastocyst biopsy have been introduced and further improved in the last 15 years. Special attention has been given to the safety and efficacy of these procedures, as optimal performance obviously has a crucial impact on further embryo development. This paper summarizes the literature elucidating the safety and efficacy of these techniques.
Mechanical partial zona dissection Conventional partial zona dissection (PZD) involves a slit in the zona pellucida by means of a sharp closed microneedle, which results in a longitudinal incision, often irregularly shaped with openings that usually do not exceed 30–40 μm. One concern of
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Article - Screening oocytes by polar body biopsy - A Dawson et al. conventional PZD is that the slit may be too small or that it can be hardened during the fertilization process and might entrap the embryo during the hatching process (Cohen and Feldberg, 1991). Another form of direct penetration of the zona pellucida using a bevelled suctioning pipette has also been proven efficient for polar body biopsy, and allows use of the same pipette for the two procedures of zona dissection and polar body retrieval (Verlinsky and Cieslak, 1993). A refinement of conventional zona dissection is threedimensional PZD, in which two slits perpendicular to one another are made into the zona. The cross-shaped opening allows access to the perivitelline space and simultaneous removal of both polar bodies even when they are located in separate areas. Microtool requirements are one holding pipette and one microneedle. After the first slit opening the zona pellucida, the oocyte is vertically rotated and the second cut is performed (Cieslak et al., 1999). Regarding mechanical techniques for polar body biopsy, some observational studies have been performed to detect the safety of partial zona dissection (PZD). One study by Strom et al. (2000) on a cohort of 109 infants compared outcome in children after PZD with the literature data on ICSI children. Polar body biopsy was performed for both Mendelian disorders and aneuploidy screening. The authors found no differences between the two groups regarding gestational age, mode of delivery, perinatal mortality, birth weight and birth length, birth defects and post-natal growth. Another observational study by Horwitz et al. (unpublished abstract, 2005) on 576 infants showed no increase in birth defects in children after blastomere biopsy or polar body biopsy using the partial zona dissection. Genetic diagnosis was performed to elucidate aneuploidy and single gene disorders. For data analysis, the group used birth data and information on 413 babies supplied by the parents.
Chemical zona drilling In contrast to the mechanical partial zona dissection, Tyrode’s acid (pH 2.3) creates a larger, rounder hole in the zona pellucida and its size is not always easy to control. The human zona pellucida is more resistant to acid treatment compared with mouse oocytes. Therefore higher amounts of Tyrode’s solution are needed to penetrate the human zona pellucida. For chemical zona drilling, oocytes or embryos are usually manipulated individually and an immediate washing is required to avoid higher pH differences (Cohen et al., 1992) as the local acidification of the culture medium or the deposition of acid into the perivitelline space may lead to cell lysis or possibly other, more subtle cell damage. For blastomeres, chemical drilling is normally done with separate pipettes using a double holder set-up: one pipette for drilling with an inner diameter of 5–7 μm and one pipette for aspiration with an inner diameter of 40 μm to avoid blastomere lysis. The Tyrode’s acid is loaded in the right pipette and released until an opening above the polar body is seen.
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Regarding the efficacy and safety of acid drilling some nonrandomized studies are available. One of the first studies focusing on acidic zona-drilling safety has been conducted by Talansky and colleagues (Talansky and Gordon, 1988). The group showed a higher fertilization and live birth rate after acidic drilling on mouse embryos. However, developmental abnormalities associated
with embryo loss, spontaneous chimerism and the possibility of conception of monozygotic twins were also encountered. Magli et al. (2004) combined polar body analysis with blastomere biopsy through the same opening in the zona pellucida, which was achieved by acid drilling. The effects of a combined procedure were evaluated retrospectively by comparing the results obtained in cycles where this strategy was applied with those cycles where either polar body biopsy or embryo biopsy was performed. The comparatively higher implantation rate in the blastomere biopsy group can be explained by the fact that only regularly dividing embryos were biopsied; therefore, chromosomally normal embryos with poor development have been sorted out already. However, no negative influence of chemical zona drilling on main outcome parameters could be observed. It is still not clear if acid exposure may interfere with further embryo development or implantation. The chemical digestion of the glycoprotein matrix of the human zona pellucida of oocytes for polar body biopsy may compromise the oocytes viability (Payne et al., 1991), and some authors have even described this technique as inappropriate (Gordon et al., 1988; Depypere and Leybaert, 1994).
Laser-assisted dissection Laser-assisted dissection as an alternative to chemical or mechanical zona penetration for embryo micromanipulation was first introduced in the early 1990s (Palanker et al., 1991; Neev et al., 1992). This technique was further improved and also used for polar body biopsy (Montag et al., 1997). Nowadays a 1.48 μm diode non-contact infrared laser is widely used for laser zona drilling, assisted hatching and for embryo biopsy, as it is considered to be unlikely to induce mutagenic changes in DNA contrary to UV lasers (Germond et al., 1995; Montag et al. 1998, 2004). Using electromagnetic waves, the laser beam induces only minimal superficial damage to the zona pellucida of oocytes or embryos. Lasers, which with longer wavelengths are less absorbed by water, seem to be more practicable, as they can be used in a non-contact mode. The opening of the zona pellucida by laser drilling is performed by exposing the surface of the ZP to laser light. The size of the hole can be chosen precisely by varying the exposure time, and the holes are much more precise than the holes produced by acidic drilling. Polar bodies can subsequently be withdrawn using blunt-ended glass capillaries. The efficacy and safety of laser drilling and laser-assisted polar body and blastomere biopsy has been the object of several studies. The use of a non-contact laser before embryo biopsy is an easy procedure, and several studies showed no adverse effects on embryo development or pregnancy rates (Boada et al., 1998; Germond et al., 1999; Joris et al., 2003). Selecting an area for laser drilling which has a sufficient distance to the next blastomere does not induce immediate laser damage (Chatzimeletiou et al., 2001). In a controlled, prospective study by Germond et al. (1995), the safety of laser drilling was evaluated. Laser drilling with openings of 4.5–20 μm at different sites has been performed on mouse oocytes and zygotes (B6D2F1). A group of 106 control
Article - Screening oocytes by polar body biopsy - A Dawson et al. embryos and 96 lasered embryos has been cultivated for 5−6 days and the embryos were transferred to foster mice (NMRI). From the lasered embryos, 93 offspring were followed up and cross-mated. The cytoplasmic changes of the oocytes and embryos have been assessed via electromicroscopy. The group found no impairment of the in-vitro or in-vivo development or of the fertility of the offspring. No lysis of oocytes occurred. The same group also determined the effect of laser-assisted zona pellucida dissection (Germond et al., 1996). Other evidence comes from follow-up studies on laser-assisted hatching of the zona pellucida (Kanyo and Konc, 2003). Another prospective, non-randomized study focused on the outcome of laser-assisted polar body biopsy and aneuploidy testing (Montag et al., 2004). Laser-assisted polar body biopsy has been performed in 140 IVF cycles of patients with an advanced maternal age (>35 years). The outcome of polar body biopsy was compared with a retrospective control group including all treatment cycles of the same time period. The group found a clinical pregnancy rate of 22.5% per embryo transfer in the biopsied group compared with 20.2% in the control group. The study provided no description of the follow up of the babies. The birth weights of the children in the polar body biopsy group were comparable with the control group. The numbers of children born after laser-assisted polar body biopsy reported in this study were too small to give a conclusive answer (Montag et al., 1998). Bergere et al. (2003, unpublished abstract) conducted a study on the safety of laser-assisted polar body biopsy. Eighty-three immature oocytes were incubated and matured in vitro and the first polar body was biopsied using the laser drilling procedure. The outcome was compared between four study groups: one control group with no drilling or biopsy, one laser-drilled group using the ZILOS C device (140 mW/ms), one group using the same device and performing the first polar body biopsy and the fourth group using a second laser (ZILOS tk, 180 mW/10.5 ms) followed by polar body biopsy. The authors detected no significant difference regarding oocyte lysis rate and chromosomal breakage rate between the four groups. The oocyte activation rate, detected via calcium ionophore treatment, was significantly lower using the ZILOS C, which reflects a safer procedure by using high power with a short duration. ZILOS C and ZILOS TK are both infrared lasers (1480 nm), but ZILOS TK has a higher full possible power (180 versus 140 mW for the ZILOS C). In both systems the duration of the exposure time can be adjusted (0.5 ms is the shortest possible time). A prospective and observational study by Grossmann et al. focused on the safety and efficacy of laser-assisted polar body biopsy. PB biopsy was performed on oocytes of 460 patients (Grossmann et al., 2004). The study group was compared with an unselected group and data were obtained using the German IVF Registry. Inclusion criteria for the study group was a maternal age >39 years, three or more previous performed IVF cycles or two or more previous abortuses. Chromosomes were detected on first and second polar body. In the study group the authors found a rate of abnormal chromosomes in 57.9% of all oocytes. The clinical pregnancy rate per oocyte retrieval in women aged 31–35 years was 22.7% in the study group and 27.6% in the control group. The corresponding clinical pregnancy rates in the age group of >40 years were 5.9 and 8.6% respectively. Abortion rates were lower in the study group
(28.6%) than in the control group (38.6%). There are a few studies comparing two techniques of zona drilling or penetration for polar body or blastomere biopsy available. One recent study by Chatzimeletiou et al. (2005) compared the safety of the use of a non-contact laser with the use of acidic zona drilling for human embryo biopsy. The group found no significant difference in the proportion of undamaged embryos or blastomeres between the two groups (75% after acid Tyrode’s versus 68% after laser drilling). The incidence of spindle abnormalities at blastocyst stage was not significantly different from controls and no significant difference in the incidence of chromosomal abnormalities and mosaicism between the two groups was observed. However, single biopsied blastomeres after laser drilling showed a greater tendency to form miniblastocysts and acid Tyrode’s drilling tended to retard blastocyst development. Another prospective study by Joris et al. (2003) compared the results of human embryo biopsy and the outcome of PGD after zona drilling using Tyrode’s acid medium or a laser. The study revealed fewer intact blastomeres after acid zona drilling (95.2%) than after laser zona drilling (98.3%, P = 0.02) whereas implantation and pregnancy rates did not differ. The laser method was described easier compared with the acid method. Malter and Cohen (1989) reinseminated initially unfertilized oocytes on day 1 following PZD (n = 60) or acid drilling using Tyrode’s acid (n = 51). The conclusion of the paper was that none of the Tyrode’s acid-exposed oocytes developed satisfactorily. Tyrode’s acid does not seem to jeopardize the fertilization process, but it does seem to interfere with further development. Two studies investigating different techniques for assisted hatching have been published. Hsieh et al. (2002) reported a higher effectiveness using laser-assisted hatching of human embryos compared with the chemical method regarding implantation and pregnancy rates in women with advanced maternal age. The authors concluded that laser zona drilling allows an easier, faster and safer micromanipulation of the zona pellucida. Another retrospective, not randomized study conducted by Balaban et al. (2002) compared the efficacy of four different techniques for assisted hatching. Partial zona dissection was used in 239, acid Tyrode in 191, diode laser hatching in 219 and pronase thinning in 145 IVF or ICSI cycles. The authors found no differences regarding implantation and pregnancy rates using each of the techniques compared with a control group of IVF or ICSI cycles without assisted hatching.
Considerations and conclusions The efficacy and safety of the different methods of zona drilling was evaluated in animal models first, and later feasibility and safety could be partly shown in humans as well. However, all methods of zona dissection and biopsy can harm the oocyte and impair further embryo development. In the hand of an experienced technician all methods for zona pellucida drilling or penetration seem to have a satisfying safety and efficacy. However, at present, no convincing evidence from an efficacy trial exists and a method of first choice has not been established yet, although several authors prefer laser-assisted drilling
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Article - Screening oocytes by polar body biopsy - A Dawson et al. as it seems to be an easier method. Concerns remain for all methods, as they might be associated with an impairment of the developmental potential of the oocyte. The safety of the procedures in terms of ongoing health of children born after polar body biopsy is not proven. Efficacy trials on polar body biopsy after acidic zona drilling are missing. Therefore, using Tyrode’s acid for polar body biopsy should be considered as an experimental procedure. The benefit of aneuploidy screening is probably at best small, since ‘chromosomally normal’ embryos do not implant in the majority of cases. Furthermore, cryopreservation efficacy after zona dissection is impaired and the risk of false-positive results (~5%) may lead to discarding of normal embryos. Whatever technique is used to open the zona pellucida polar body biopsy requires a high number of oocytes, which are not available in many advanced maternal age cases. That women of different age groups undergoing IVF have a benefit from PB biopsy regarding pregnancy and abortion rates still needs to be proven. The further improvement of techniques for the detection of single gene defects, translocations or for aneuploid testing of all chromosomes in both polar bodies will increase the application of polar body biopsy in countries with restricted laws.
References
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