1 Departments of Reproductive Medicine, 2 Medical Statistics, Leiden University Medical Centre, PO Box 9600, 2300 RC Leiden, The Netherlands
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Abstract |
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Key words: cleavage/person performing ICSI/polar body/pregnancy rate/sperm injection
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Introduction |
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During the ICSI procedure the oocyte is fixed by a holding pipette in such a way that the polar body of the oocyte is at the 6 or the 12 o'clock position at the moment of injection. When the injection needle is entering the oocyte at the 3 o'clock position, the opening of the bevel of the needle is facing the 6 o'clock position. During injection it is checked if the tip of the needle is inside the cytoplasm by aspirating cytoplasm into the needle to make sure that the oocyte membrane is broken. During this aspiration of cytoplasm different structures might be damaged or lost. There might be a difference between the risk of losing or damaging structures in oocytes injected with the polar body at the 6 o'clock or the 12 o'clock position. In the human it can be assumed that the chromosomes of the oocyte are found in the periphery of the oocyte near the location of the first polar body (Sousa and Tesarik, 1994), and therefore the chance of damaging the meiotic spindle and/or disturbing the microtubule organization in the oocyte that has an important role in fertilization and embryonic development (Asch et al., 1995
) might differ with the position of the polar body relative to the opening of the injection needle. Those structures may be responsible for embryo development. Differences in damage of those structures might be expressed by differences in fertilization rates, cleavage rates, embryo quality, and pregnancy and abortion rates.
Differences between operators that perform the injections may also influence the ICSI outcome. Although the ICSI procedure itself is performed by a standard protocol, inter-individual differences in the injection technique itself may occur.
The effect of the ICSI procedure on oocyte characteristics and sperm characteristics after injection has been studied at both the cytological and developmental levels of oocytes and embryos (Payne et al., 1997). The effect of the position of the polar body relative to the opening of the injection needle at the moment of injection has been studied at the developmental level of oocytes and embryos (Nagy et al., 1995
; Blake et al., 1996
). However, those studies did not include pregnancy rates and abortion rates that resulted from the transfer of embryos originating from oocytes injected with the polar body at different positions relative to the opening of the injection needle. So far no studies have been published on variations between operators performing the injections.
In this study the influence of the position of the polar body during injection and of the person performing the injection on fertilization, cleavage, pregnancy, and delivery rates is examined.
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Materials and methods |
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The mean age of the female patients was 32.3 ± 4.7 years (range 2045) and of the male patients 36.3 ± 6.5 (range 2462).
The indication for undergoing ICSI treatment was poor semen parameters (i.e. <1x106 total number of morphologically normal, motile spermatozoa) or fertilization failure in previous conventional IVF despite normal spermatozoa parameters according to World Health Organization standards (WHO, 1987).
Ovarian stimulation
Ovarian stimulation was performed by a combination of a gonadotrophin-releasing hormone agonist, Decapeptyl (Ferring, Hoofddorp), The Netherlands; Synarel (Searle, Maarssen, The Netherlands), human menopausal gonadotrophin, Metrodin HP (Serono Benelux, Den Haag, The Netherlands); Pergonal (Serono Benelux), and human chorionic gonadotrophin (HCG), Profasi (Serono Benelux); Pregnyl (Organon, Oss, The Netherlands). Luteal phase supplementation was given by intravaginally administered progesterone, Progestan (Organon) and an HCG injection, Pregnyl (Organon) 6 days after oocyte retrieval.
Sperm preparation
Freshly ejaculated semen was allowed to liquefy. For seven patients, frozen semen was thawed (12 cycles). Volume was determined, concentration and percentage of motile spermatozoa were assessed in a Makler counting chamber and the total number of motile spermatozoa was calculated. HEPES-buffered Earle's medium with 0.5% human serum albumin was added to the semen sample and mixed by pipetting. Depending on the total number of motile spermatozoa, the mixed sample was pipetted on top of either a 1 ml 70 or 80% Percoll layer and centrifuged (800 g, 10 min). The supernatant was removed and the pellet was resuspended in HEPES-buffered Earle's medium. Depending on the total number of motile spermatozoa, this suspension was either pipetted on top of an 80% Percoll layer and then washed twice, first in the HEPES-buffered medium and the second time in Universal IVF medium (Medicult; Lucron, Milsbeek, The Netherlands), or washed twice in the medium after the first Percoll treatment, first in the HEPES-buffered medium and the second time in Universal IVF medium (Medicult). Volume, concentration, motility and the total number of motile spermatozoa were redetermined after processing. The spermatozoa were kept at 37°C in a CO2 incubator until ICSI took place.
Oocyte preparation
Between 0 and 4 h after oocytecumulus complex (OCC) collection the OCC were denuded of their surrounding cumulus cells by incubation in 80 IU/ml hyaluronidase (Hyase; IVF Science, Göteborg, Sweden) in HEPES-buffered Earle's medium for 20 s and by repeated pipetting of the OCC in and out of a hand-drawn Pasteur pipette. After denudation the oocytes were washed in HEPES-buffered Earle's medium with 0.5% human serum albumin and the maturation stage of the oocytes was checked; the oocytes which had extruded a polar body were selected for ICSI and transferred to Universal IVF medium (Medicult) droplets under mineral oil (Sigma, Brunschwig Chemie, Amsterdam, The Netherlands) until ICSI took place. Just before starting the ICSI procedure all oocytes were checked again for the presence of a polar body.
ICSI procedure
Microinjection was carried out on the heated stage of an inverted microscope (Olympus, IX70, Paes, Zoetermeer, The Netherlands), using Hoffman modulation optics at x300 magnification. The injection and holding pipette were obtained from Humagen (Gynotec, Malden, The Netherlands). They were connected to two microinjectors (IM-6; Narishige, Paes) which were fitted to two micromanipulators (Narishige) by Teflon tubing (CT-1; Narishige). Petri dishes (Falcon type 1006; Micronic, Lelystad, The Netherlands) were prepared with two central droplets of 3 µl polyvinylpyrrolidone (PVP) solution (Medicult) with one containing prepared spermatozoa and one to flush the injection pipette when necessary. Five droplets of 5 µl HEPES-buffered Earle's medium were arranged around these droplets, each containing one oocyte. All droplets were covered with mineral oil (3.5 ml/Petri dish).
A single motile spermatozoon from the central spermatozoa droplet was immobilized by pressing the tail of the spermatozoon against the bottom of the Petri dish until it stopped moving. The spermatozoon was then aspirated into the injection pipette, tail first. The Petri dish was then moved in order to visualize an oocyte in one of the surrounding droplets. The oocyte was firmly attached to the holding pipette. The position of the polar body was chosen without any preference for the 6 or 12 o'clock position and without any knowledge about the patient. The position was then recorded for each oocyte. The injection pipette always entered the oocyte at the 3 o'clock position with the opening of the bevel directed to the 6 o'clock position. The breakage of the oolemma was checked by gentle aspiration of cytoplasm into the pipette and the spermatozoon was injected into the cytoplasm. The person who performed the injection was recorded for each oocyte. This depended on a weekly work schedule. All injections were performed by two operators with equal ICSI experience.
After injection the oocyte was washed and incubated in well-equilibrated Universal IVF medium at 37°C in 5% CO2 in air.
Embryo transfer
Embryo transfer took place 3 days after oocyte retrieval. In our standard protocol two embryos were transferred. In some circumstances, depending on age and/or number of available embryos, one or three embryos were transferred. These transfers were excluded from this study.
For transfer, a 1 ml syringe was filled with IVF medium (Medicult) and connected to a Wallace catheter (SIMS Portex Ltd, Hythe, UK). After flushing the catheter the selected embryos were aspirated into the catheter. The catheter was passed through the cervical canal and into the uterine cavity. The embryos were slowly injected, after which the catheter was withdrawn gradually.
Assessment of fertilization parameters, embryo quality, and pregnancy
Fertilization was scored 1618 h after injection. Fertilization was considered normal when two pronuclei (PN) were present. The presence of no, one, and more than two PN was recorded as well as the number of degenerative oocytes.
For all oocytes, cleavage and the quality were evaluated at days 2 and 3 after injection. According to the number and size of blastomeres and the amount of fragmentation the embryos were assigned to four different quality types: type 1, equal-sized blastomeres and no fragmentation; type 2, <20% fragmentation; type 3, 2050% fragmentation; type 4, >50% fragmentation.
Pregnancy was defined by an increasing serum ß-HCG 50 IU/l at 15 days after oocyte retrieval. Spontaneous abortion was defined as pregnancy ending in a miscarriage up until 16 weeks after the last menstrual period. No ectopic pregnancy occurred in this study.
Statistical analysis
Pearson's 2 test and logistic regression were used to compare the proportions of fertilization, type of cleavage, and pregnancy and abortion rates.
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Results |
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Fertilization results
Logistic regression on the number of normally fertilized oocytes showed that both the person who performed the injections (P = 0.02) and the position of the polar body (P = 0.01) significantly affected the number of 2PN oocytes (Table I): the percentage of 2PN oocytes that developed in the 6 o'clock group was higher than in the 12 o'clock group and this effect was very similar for both operators. Operator 1 obtained higher fertilization rates than operator 2 for the 12 o'clock group as well as for the 6 o'clock group. To exclude the possibility that these effects were the result of a general gain in experience with time, the study period was divided into two. The second period showed higher fertilization rates for both operators and for both positions of the polar body. However, in both periods the same effects of the operator performing the injection and of the position of the polar body were present.
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Cleavage results
A significant difference was found between the 12 o'clock and the 6 o'clock position of the 2PN embryos with regard to the quality of cleavage (2 = 10.52, df = 4, P = 0.032) (Table II
). Detailed analysis showed that this was caused by a lower number of type 4 embryos in the 12 o'clock group (
2 = 5.14, df = 1, P = 0.02). No differences were found between persons with regard to quality of cleavage in the group of 2PN embryos (
2 = 2.68, df = 4, P = 0.61).
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Two of the transfers with two embryos involved a mix of embryos originating from the injection by operator 1 and operator 2 and were therefore excluded from this analysis.
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Discussion |
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Overall fertilization percentages, cleavage results and pregnancy rates found in this study were similar to those found in our conventional IVF programme and to those reported by others (Tsirigotis et al., 1994; Gerris et al., 1995
; Nagy et al., 1995
; Palermo et al., 1995
; Hlinka et al., 1998
).
It was expected that aspiration towards the site of the polar body (6 o'clock position) would result in more damage of structures important for fertilization, assuming that the nuclear material is located near the polar body (Sousa and Tesarik, 1994). This was not confirmed by the results of this study. On the contrary, the fertilization rate was higher when the aspiration was closer to the polar body. Perhaps deposition of the sperm cell closer to the meiotic spindle is responsible for this result. One study (Blake et al., 1996
) supports this in which the highest fertilization rate was found when the sperm cell was injected adjacent to the meiotic spindle. The success of the injections could depend on the rotation of ooplasm by a correctly positioned sperm centrosome (Edwards and Beard, 1997
). However, another study (Nagy et al., 1995
) did not find a difference between the 6 and the 12 o'clock positions in the number of 2PN oocytes but in the quality of the embryos.
The percentage of multipronuclear ICSI oocytes in this study was 2.5%. This percentage is lower than that in our conventional IVF programme (7.9%). The difference in origin of those multipronuclear eggs might explain this observation. The multipronuclear oocytes in the ICSI procedure probably result from the incorporation of the second polar body (Palermo et al., 1996), while the majority of multipronuclear oocytes in the conventional IVF programme arise from dispermic fertilization. However, the position of the polar body did not affect the number of multipronuclear oocytes.
The number of oocytes developing only one pronucleus after ICSI (8.5%) was significantly (P = 0.02) higher than after conventional IVF (3.4%). It is probable that such oocytes are activated by ICSI resulting in the formation of one female pronucleus but fail to form a male pronucleus, although the spermatozoon may have contributed to activation. The formation of a male pronucleus might be impaired due to defects in the sperm cell itself, such as impaired microtubule nucleation and elongation and/or compromized sperm aster function (Asch et al., 1995). The number of 1PN oocytes might be influenced by PVP, which has a stabilizing effect on the disruption of the sperm plasma membrane. This disruption is needed to give the spermatozoa-associated oocyte-activating factor access to the sperm head as it is swelling (Hlinka et al., 1998
). The observation that significantly more 1PN oocytes develop from oocytes injected with the polar body at the 12 o'clock position in our study suggests that deposition of the sperm cell further from the meiotic spindle decreases the chance of normal fertilization, although activation may be achieved. Rotation of the ooplasm and the way the sperm centrosome is positioned may influence this result.
In agreement with the results of one study (Nagy et al., 1995), degeneration of injected oocytes seems to be independent of the position of the polar body during injection.
The difference in pregnancy rate between the 6 and the 12 o'clock polar body positions was almost solely the result of the significant interaction between the operator and the polar body position. This suggests that there are technical differences between people performing the injection, which are related to the position of the polar body. This might result in a difference in pregnancy rate depending on the position of the polar body. It was previously reported that the rate of development to the blastocyst stage is related to the person performing the injection (Bergers-Janssen et al., 1998). This supports our observations on interindividual differences. The latter may be related to subtle differences in injection technique that improve fertilization and pregnancy rates: the amount of cytoplasm that is sucked into the injection pipette, the force with which the injection pipette is pushed through the oocyte membrane, or the relative positioning of the sperm head inside the oocyte (e.g. in the more central or the peripheral ooplasm, far away from or in the vicinity of the second meiotic spindle). This study shows the importance of recording and evaluation of individual performances with regard to the ICSI technique.
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Acknowledgments |
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Notes |
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References |
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Submitted on March 26, 1999; accepted on June 17, 1999.