The impact of the zona pellucida thickness variation of human embryos on pregnancy outcome in relation to suboptimal embryo development. A prospective randomized controlled study

A. Gabrielsen1,3, S. Lindenberg1,2 and K. Petersen1

1 Ciconia Fertility Clinic, Højbjerg, 2 Department of Obstetrics and Gynecology, Copenhagen University Hospital, Amtssygehuset in Herlev, DK-2730 Herlev, Denmark


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: The study represents a prospective randomized controlled study evaluating zona pellucida thickness variation (ZPTV) measurements versus conventional selection of embryos using classic embryo score criteria, prior to embryo transfer in human IVF/intracytoplasmatic sperm injection (ICSI). METHODS: Eighty-six patients having >=3 embryos, with a classic embryo score of <=2.2, were allocated to either ZPTV measurement or classic embryo morphology score before embryo transfer. The technician selecting embryos using classic embryo scoring was not aware of the ZPTV measurement results. Of the embryos allocated to ZPTV measurements, only the embryos with the highest ZPTV were transferred. RESULTS: We found no differences in the pregnancy rate per embryo transfer between the two groups (34.4 versus 35.7%). Neither did the implantation rates differ. However, significantly better results were obtained when ZPTV was used as the selection criteria in cases where all embryos had an asynchrony in development or a high embryo score (i.e. were of poorer quality) by classical evaluation (odds ratio = 2.51, confidence interval = 0.33–198). CONCLUSIONS: Using a normally-developed embryo with an optimal embryo score, no beneficial effect of using ZPTV measurement was seen. However, when only less optimal embryos were available to select for transfer, ZPTV provided a x2.5 increase in the chance of achieving a clinical pregnancy.

Key words: embryo selection/IVF/ICSI/pregnancy/ZP thickness variation


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Success during IVF treatment, as determined by clinical implantation and ensuing pregnancy culminating in the live birth of healthy offspring, is dependent on a complicated interplay of several clinical and non-clinical variables, including patient's age, etiology of infertility, ovulation induction protocols, endometrial receptivity and gamete/embryo quality. Although several extensive reports have appeared in the last 15 years on the subject of embryo grading prior to embryo transfer (Puissant et al., 1987Go; Visser and Fourie, 1993Go; Ziebe et al., 1997Go), a lack of consensus amongst embryologists regarding current embryo grading systems (Rosenboom and Vermeiden, 1995Go; Ziebe et al., 1997Go) emphasizes the need for further identification of reliable parameters for embryo quality assessment.

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., 1989Go; Bertrand et al., 1995Go; Garside et al., 1997Go; Loret De Mola et al., 1997Go). 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., 1989Go; Bertrand et al., 1995Go). 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, 1997Go), could possibly be offset through microassisted embryo manipulation, like assisted hatching and zona thinning (DeFelici and Siracusa, 1982Go; Cohen et al., 1992Go).

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., 1989Go; Palmstierna et al., 1998Go). 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patient selection
A total of 86 consecutive patients were admitted to IVF or intracytoplasmic sperm injection (ICSI). Inclusion criteria were age <40 years and >=3 embryos on day 2 after fertilization with an embryo score of <=2.2 (Deschacht et al., 1988Go). Embryo selection for transfers was based on scoring criteria (Deschacht et al., 1988Go) comprising rate of embryo development, uniformity of blastomeres and degree of anucleate fragmentation (Ziebe et al., 1997Go). The indication for ICSI was, in all cases, a raw sperm count with <2 x 106/ml living spermatozoa.

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., 1988Go; Ziebe et al., 1997Go). 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., 1996Go). 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 3–4 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 cumulus–corona complex was removed by repeated pipetting with a grass drawn pipette with an inner diameter of 0.134–0.145 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., 1996Go). 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 (55–80%) 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., 1997Go). 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 1Go) 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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Figure 1. Photograph of zona pellucida thickness measurement of an embryo using FQC system.

 
The thickness of the ZP variations (Zvar) in percentage within individual embryos was calculated as described by Cohen et al., by taking the difference between the average and the greatest thickness of the ZP, and dividing the result by the average thickness, then multiplying by 100 (Cohen et al., 1989Go). To minimize experimental bias, the same technician just prior to embryo transfer performed all zona measurements and video recordings.


Embryo transfer and pregnancy follow-up
The transvaginal ultrasound guided transfer procedure was carried out 48 h after ovum retrieval using an Edward–Wallace 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 Mantel–Haenzel {chi}2 test.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A total of 86 embryo transfers were allocated to this study. Of these, 54 were allocated to ZPTV measurements and embryo selection based on these findings, and 32 were allocated to conventional embryo score and transfer, without knowing the results of the ZPTV. The randomization by day of embryo transfer, and not by patient, influenced this distribution.

The age range of the women was 25–39 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 IGo.


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Table I. Clinical pregnancy rates in different infertility categories
 
Concerning ZPTV measurement and the relation to clinical pregnancy rate, we found a significantly increased rate for both selection methods when embryos had ZPTV >20% (81.8 and 92.3%) versus embryos <20% ZPTV (Table IIaGo), although this was not influenced by the selection method used. Thus, ZPTV was neither more nor less efficient for the selection of good embryos. The same was true of the implantation rate, which was identical for both selection groups (34.4 and 35.7% with ZPTV >20% versus 13.6 and 4.5% with ZPTV <20%) (Table IIaGo).


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Table IIa. Correlation of pregnancy rates with zona pellucida thickness variation (ZPTV) values of transferred embryos
 
However, using only the ZPTV regardless of embryo morphology, the predictive value appeared to be much improved (Table IIbGo). A ZPTV value >25% of transferred embryos resulted in a pregnancy rate of 50%, whereas 0% transfers of embryos with ZPTV value <15% resulted in clinical pregnancies.


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Table IIb. Correlation of clinical pregnancy rates with degree of zona pellucida thickness variation (ZPTV) of transferred embryos
 
The ZPTV was similar for clinical pregnancy rate in both groups, but was significantly higher than the ZPTV score of embryos in the non-pregnant group (Table IIIGo).


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Table III. Mean zona pellucida thickness variation (ZPTV) in conceptual and non-conceptual cycles
 
We were unable to demonstrate any correlation between embryo score and ZPTV; perhaps resulting from the low number of observations. However, a significant trend was seen when only poor embryos were chosen for embryo transfer. When the embryologist only had embryos of 2–3 cells or >4 cells, 50–52 h after fertilization, i.e. asynchrony in development (Cohen et al., 1989Go), we found an OR for pregnancy rate of 2.5 when ZPTV was used (OR = 2.51, CI = 0.33–198) (Table IVGo).


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Table IV. Correlation of embryo score with mean zona pellucida thickness variation (ZPTV) and mean zona pellucida (ZP) thickness on day 2 after fertilization
 
No impact of age was found on either ZPTV or ZP thickness (Table VGo).


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Table V. Mean zona pellucida thickness variation (ZPTV) and mean zona pellucida (ZP) thickness between different age groups on day 2 after fertilization
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Recently, ZP thickness of the transferred embryo has emerged as a potentially reliable parameter for embryo selection. During the last few years much interest has been generated in this subject, especially following investigations into the relationship between implantation rate and ZP thickness or variation in the ZP thickness of the transferred embryo (Cohen et al., 1989Go; Palmstierna et al., 1998Go). These reports suggested that patients transferred with embryos with thinner ZP had a better chance of successful implantation and pregnancy compared with those transferred with embryos with thicker ZP. Other reports have proposed ZPTV as a reliable marker for selecting thawed as well as fresh human embryos for transfers (Cohen et al., 1988Go). Cohen et al. conducted a retrospective analysis of ZP thickness of transferred embryos through video recordings and concluded that the variation in ZP thickness and not the ZPTV per se was a strong predictor of IVF outcome (Cohen et al., 1989Go). The study revealed that embryos with a minor variation in ZP thickness had an implantation rate of about 10%, which increased to 29% in embryos with higher ZP variation. In a subsequent study, Palmstierna reported enhanced implantation rates for each embryo with ZP thickness variation >25% (Palmstierna et al., 1998Go). In the same report ZP thickness variation exhibited high predictive values for pregnancy outcome and was thus considered an important indicator for good embryo quality.

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., 1988Go; Palmstierna et al., 1998Go) 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).


    Notes
 
3 To whom correspondence should be addressed at: Ciconia Fertility Clinic, Århus, DK-8270 Højbjerg, Denmark.E-mail: ag{at}ciconia.dk Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bertrand, E., Van den Bergh, M. and Englert Y. (1995) Does zona pellucida thickness influence the fertilization rate? Hum. Reprod., 10, 1189–1193.[Abstract]

Cohen, J., Wiemer, K.E. and Wright, G. (1988) Prognostic value of morphologic characteristics of cryopreserved embryos: A study using videocinematography. Fertil. Steril., 49, 827–834.[ISI][Medline]

Cohen, J., Inge, K.L., Suzman, K., et al. (1989) Videocinematography of fresh and cryopreserved embryos: A retrospective analysis of embryonic morphology and implantation. Fertil. Steril., 51, 820–827.[ISI][Medline]

Cohen, J., Alikani, M., Trowbridge, J. et al. (1992) Implantation enhancement by selective assisted hatching using zona drilling of human embryos with poor prognosis. Hum. Reprod., 7, 685–691.[Abstract]

DeFelici, M. and Siracusa, G. (1982) Spontaneous hardening of the zona pellucida of mouse oocytes during in vitro culture. Gamete Res., 6, 107–113.[ISI]

Deschacht, J., Devroey, P., Camus, M. et al. (1988) In-vitro fertilization with husband and donor sperm in patients with previous fertilization failures using husband sperm. Hum. Reprod., 3, 105–108.[ISI][Medline]

Gabrielsen, A., Petersen, K., Mikkelsen A., and Lindenberg, S. (1996) Intracytoplasmic sperm injection does not overcome an oocyte defect in previous fertilization failure with conventional in-vitro fertilization and normal spermatozoa. Hum. Reprod., 11, 1963–1965.[Abstract]

Garside, W.T., Loret De Mola, J.R., Bucci, J.A. et al. (1997) Sequential analysis of zona pellucida thickness during in vitro culture of human zygotes: correlation with embryo quality, age and implantation. Mol. Reprod. Dev., 47, 99–104.[ISI][Medline]

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Loret De Mola, J.R., Garside, W.T., Bucci, J. et al. (1997) Analysis of the human zona pellucida during culture: correlation with diagnosis and preovulatory hormonal environment. J. Assisted Reprod. Genet., 14, 332–336.[ISI][Medline]

Palmstierna, M., Murkes, D., Csemiczky, G. et al (1998) Zona pellucida thickness variation and occurrence of visible mononucleated blastomeres in preembryos are associated with a high pregnancy rate in IVF treatment. J. Assisted Reprod. Genet., 15, 70–75.[ISI][Medline]

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Submitted on January 25, 2001; accepted on June 27, 2001.