1 Ciconia Fertility Clinic, Højbjerg, 2 Department of Obstetrics and Gynecology, Copenhagen University Hospital, Amtssygehuset in Herlev, DK-2730 Herlev, Denmark
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Abstract |
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Key words: embryo selection/IVF/ICSI/pregnancy/ZP thickness variation
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Introduction |
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Recently, some interest has been generated in studying a unique parameter for predicting clinical IVF outcomes based on thickness measurements of the zona pellucida (ZP) during fertilization and embryo transfers (Cohen et al., 1989; Bertrand et al., 1995
; Garside et al., 1997
; Loret De Mola et al., 1997
). Two lines of clinical evidence have significantly contributed to the relevance of these criteria for embryo selection. Firstly, initial evidence demonstrating that implantation rates of human embryos correlate with ZP thickness variation (ZPTV) and character, ranging from 10% for embryos with uniform thickness to 29% with thin or irregular ZP (Cohen et al., 1989
; Bertrand et al., 1995
). Secondly, an observation that some adverse influences of prolonged suboptimal embryo culture conditions, such as ZP thickening and hardening, leading to failure to hatch of approximately three-quarters of IVF embryos (Letterie, 1997
), could possibly be offset through microassisted embryo manipulation, like assisted hatching and zona thinning (DeFelici and Siracusa, 1982
; Cohen et al., 1992
).
Although most reports on this subject have highlighted the strong influence of ZP thickness of the transferred embryos on clinical IVF outcome, some observations have clearly suggested that variations in thickness of ZP of the embryo is a more reliable indicator for predicting IVF success (Cohen et al., 1989; Palmstierna et al., 1998
). Most of these studies were retrospective, non-randomized studies. To evaluate the benefit of sole ZPTV we conducted this pilot study as a randomized prospective study with the object of evaluating the ZPTV versus classic embryo score for selection of embryos prior to embryo transfer.
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Materials and methods |
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Randomization procedure and method
All embryos were recorded in the Fertility Quality Control system (FQC, Fercom, Denmark) prior to selection for embryo transfer on day 2 after fertilization. Every second day a specifically allocated embryologist (the same for the whole study) measured the ZPTV and selected the two embryos with the highest variation for transfer. Every other day the normal routines for embryo selection were used by any one of the trained embryologist staff, using criteria as previously described (Deschacht et al., 1988; Ziebe et al., 1997
). The stored images in this group were later assessed for ZPTV, but were not used in the embryo selection procedure.
Follicular stimulation
All women underwent controlled ovarian and follicular stimulation after pituitary down-regulation as previously described (Gabrielsen et al., 1996). Pituitary down-regulation was carried out by the gonadotrophin-releasing hormone agonist, buserelin acetate (Synarela®, Syntex, Denmark), administered by nasal spray three times a day from day 21 in the previous cycle. After confirmation of the down-regulation, ovarian stimulation was initiated 2 weeks later with 225 IU of FSH (Gonal-F®, Serono, Denmark; Puregon®, Organon, Denmark) administered s.c. daily. Follicular development was monitored by vaginal ultrasound measurements of the follicles. On the scheduled day of ovum pickup (leading follicles >18 mm in diameter), 10000 IU of human chorionic gonadotrophin, HCG (Profasi®, Serono A/S, Denmark) was administered subcutaneously, followed by ovum retrieval 36 h later by transvaginal ultrasound-guided follicle aspiration performed under i.v. sedation.
Oocyte preparation
The oocytes were incubated prior to insemination at 37°C in 5% CO2, individually in 4-well tissue culture dishes (Nunc, Denmark) containing the IVF medium containing 2% HSA, no serum (Medi-Cult, Denmark). When IVF procedure was used the oocytes were fertilized 34 h after the ovum retrieval with 1x105 motile spermatozoa per oocyte.
When ICSI was used, 3 h after ovum retrieval the oocytes were incubated in 80 IU hyaluronidase (Medi-Cult, Denmark) for 30 s, and the cumuluscorona complex was removed by repeated pipetting with a grass drawn pipette with an inner diameter of 0.1340.145 mm (SweMed Lab, Sweden). Each oocyte was transferred to a Falcon 1006 dish (Falcon, Denmark) in a separate droplet of 5 µl HEPES buffered Earle's balanced salt solution (Medi-Cult A/S, Denmark) and placed in the peripheral part of the dish. In the central part of the dish a droplet of 3 µl was placed containing polyvinylpyrrolidone (Scandinavian IVF, Sweden) and isolated spermatozoa. All were covered by light mineral oil (Scandinavian IVF). The morphology and maturation stage of the oocytes was assessed under an inverted microscope at x200 magnification (Nikon Diaphot 300 with Hoffmann modulation contrast) and each oocyte was recorded in the FQC system.
Microinjection was performed according to Gabrielsen et al. (Gabrielsen et al., 1996). Following microinjection the oocytes were washed and transferred into 4-well tissue culture dishes containing well equilibrated IVF medium. On the following day the oocytes from the IVF procedure and the microinjection procedure were checked for the presence of pronuclei and recorded. The embryos were further cultured in the same dish until day 2, and all embryos were again recorded in the FQC system.
Sperm preparation
The sperm sample was collected 1 h before the ovum retrieval. The sample was allowed to liquefy. A basic semen analysis was performed (volume, sperm concentration, motility, morphology) and the spermatozoa were separated with a two-step (5580%) PureSperm® gradient (Cryos A/S, Denmark). The sperm sample used for IVF was adjusted to a final concentration of 2x106 spermatozoa/ml, and for ICSI to 0.2x106 spermatozoa/ml, where possible. Prepared spermatozoa were stored at 37°C in 5% CO2 until use.
Measurement of ZPTV
The ZP thickness of all embryos was recorded on day 2, immediately prior to the scheduled transfer. All ZP thickness evaluations were performed directly under an inverted microscope (Nikon) equipped with Hoffman modulation contrast optics using an ocular micrometer, calibrated to provide a direct value of the zona thickness (Garside et al., 1997). All ZP thickness variation measurements were computed from the videocinematography recordings of the embryos. The morphological images of all in-vitro cultured embryos, including the ones transferred, were recorded in a computerized database (FQC, Fercom, Denmark) (Figure 1
) with a colour video camera (Panasonic) mounted on an inverted microscope with Hoffman modulation contrast (Nikon). The setting for microscopic observations (x200 magnification) and bright field was kept constant during the study. The ZP of each embryo was subjected to three independent measurements and calculation of the ZPTV using the FQC system.
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Embryo transfer and pregnancy follow-up
The transvaginal ultrasound guided transfer procedure was carried out 48 h after ovum retrieval using an EdwardWallace embryo transfer catheter (Medical Systems, UK). Patients received daily luteal support with Progestan® (Organon, Denmark) vaginal pessaries three times a day. A positive serum HCG measurement confirmed pregnancy 14 days after the embryo transfer (biochemical pregnancy). A clinical pregnancy was confirmed by observation of viable gestation sac(s) at the first ultrasound examination, performed 5 weeks post transfer.
Data analysis and statistics
Comparisons between ZP thickness variations and pregnancy outcome were calculated by Student's t-test. For nominal data, a P value <0.05 was considered statistically significant. For comparison of sequential data, generating odds ratios (OR) and confidence interval (CI), we used MantelHaenzel 2 test.
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Results |
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The age range of the women was 2539 years (mean ± SD; 33.25 ± 3.94 and 33.03 ± 3.43 years), identical in both groups, as was the distribution between indications (female versus male factor). Clinical pregnancy rates according to the type of infertility category are listed in Table I.
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Discussion |
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The present prospective study could not demonstrate any differences in final outcome (i.e. pregnancy rate) using either classical embryo score or sole ZPTV measurement. Both methods provided identical pregnancy rates and implantation rates.
In several previous retrospective studies (Cohen et al., 1988; Palmstierna et al., 1998
) a beneficial effect using ZPTV score as a criterion for embryo selection has been claimed. The reason we could not demonstrate this in a prospective randomized study could be the relatively small number of observations and our cut-off limits for inclusion of patients. We did not include patients with a small number of embryos, or having embryo scores higher than 2.2. Thus, our randomization procedure was only used on patients having a relatively good prognosis for conceiving a baby regardless of the embryos used for transfer. However, examining the data collected from embryos with a relatively low quality, we found a significantly improved OR for pregnancy when ZPTV was used for embryo selection prior to embryo transfers.
Further, we confirmed that ZPTV measurement co-variates with pregnancy outcome as does classic embryo measurement for embryos with morphology of 2.1 or better.
In conclusion, the present study demonstrates a significantly better chance for pregnancy when ZPTV measurement is used, when only embryos with poor prognosis can be transferred (due to lack of embryos with a better morphology score).
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Notes |
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References |
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Submitted on January 25, 2001; accepted on June 27, 2001.