Presence, but not type or degree of extension, of a cytoplasmic halo has a significant influence on preimplantation development and implantation behaviour

T. Ebner1,3, M. Moser1, M. Sommergruber1, U. Gaiswinkler1, R. Wiesinger1, M. Puchner1 and G. Tews1

1 Women’s General Hospital, IVF Unit, Lederergasse 47, A-4010 Linz, Upper Austria, Austria

3 To whom correspondence should be addressed. e-mail: thomas.ebner{at}gespag.at


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Since there is considerable disagreement in grading cytoplasmic haloes, this prospective study was set up to evaluate if certain subtypes of haloes are related to further development. METHODS: Out of a total of 152 patients, 713 zygotes could be checked for the formation of a halo. Where present, haloes were subdivided into concentric (symmetric) and polar (asymmetric) types. In addition, each halo was measured accurately to see if the extension of the halo might influence further development. In parallel, pronuclear patterns were checked. RESULTS: Halo-positive zygotes did not differ from halo-negative ones in terms of embryo quality and blastocyst formation rate. However, quality of blastocysts (assessed by their inner cell mass consistency) was significantly increased (P < 0.001) if a halo appeared at zygote stage. This phenomenon was not related to type of halo or degree of halo. In terms of pronuclear pattern, pattern 0 (0A, 0B) led to significantly more blastocysts (P < 0.001) of better quality (P = 0.002) compared with patterns 1–5. A stepwise logistic regression showed no relationship between different halo types and pronuclear pattern 0. CONCLUSIONS: The present study indicates that any halo has a positive prognostic value on blastocyst quality, irrespective of the fact that it is light or extreme, polar or concentric. In addition, the developmental advantage of pattern 0 is confirmed.

Key words: blastocyst formation/blastocyst quality/halo/pronuclear pattern/zygote morphology


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Since recent data indicate that cleavage stage morphology seems not to be an appropriate indicator of further preimplantation development (Rijnders and Jansen, 1998Go; Graham et al., 2000Go), the need for alternative prognostic parameters arises. Such morphological screening should be applied early in development in order to avoid a possible negative impact of prolonged in-vitro culture on embryo or blastocyst morphology (De Vos and Van Steirteghem, 1999Go).

In this respect, much research has been done at zygote stage (for review see Ebner et al., 2003bGo). Based on empirical observations and previously published data (Wright et al., 1990Go; Payne et al., 1997Go) Scott and Smith (1998Go) were the first to establish a day 1 score encompassing pronuclear morphology, cytoplasmic appearance and progression to first cleavage. Since this scoring system is rather complex and requires multiple observations, it has not gained acceptance in routine laboratory work. However, a limited number of studies applied it with great success to predict blastocyst formation (Zollner et al., 2002Go) and implantation behaviour (Ludwig et al., 2000Go).

The idea of pronuclear pattern analysis was further refined by Tesarik and Greco (1999Go) who focused exclusively on the interpronuclear synchrony of nucleoli (number and distribution). The effectiveness of this single-observation scoring was supported by several other groups, indicating clearly that early cleavage behaviour (Balaban et al., 2001Go), embryo morphology (Wittemer et al., 2000Go) and pregnancy rate (Montag and Van der Ven, 2001Go) may be predictable to a certain degree. The latter has been questioned by Salumets et al. (2001Go) who found no correlation between pronuclear patterns and treatment outcome.

One disadvantage of the Scott system (Scott and Smith, 1998Go) is that embryologists evaluating zygotes according to the work of Tesarik and Greco (1999Go) do not take into account appearance of a cytoplasmic halo. This manifestation of a microtubule-mediated withdrawal of mitochondria and other cytoplasmic components to the perinuclear region was found to be positively correlated with embryo quality (Salumets et al., 2001Go; Scott, 2003Go). In addition, there is evidence that halo-negative zygotes tend to form blastocysts less often (Zollner et al., 2002Go). This would be in line with the finding that cycles with halo-positive zygotes show increased pregnancy rates compared with cycles without (Stalf et al., 2002Go).

However, there is a certain disagreement between most of the studies dealing with cytoplasmic appearance. Some studies did not distinguish between several types of haloes, thus pooling symmetrical and polar haloes (Scott and Smith, 1998Go; Salumets et al., 2001Go) whereas others presuppose that symmetrical (Stalf et al., 2002Go) or extreme haloes (Zollner et al., 2002Go) are abnormal.

In view of this lack of uniformity, our prospective study was set up to investigate the actual influence of certain subtypes of haloes on preimplantation development of IVF and ICSI embryos. Haloes were measured accurately in order to see if a light or extreme halo effect would have any impact on subsequent developmental stages. In addition, a stepwise logistic regression model was used to combine cytoplasmic appearance and pronuclear pattern in predicting blastocyst formation and quality.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
During a 5 month period, 152 patients (mean ± SD age 33.8 ± 4.4 years) were chosen to be part of this prospective study. This cohort consisted of 40 consecutive IVF patients and 112 ICSI patients, with the latter being randomly selected by taking the last one or two ICSI cases per day. None of the patients suffered from polycystic ovaries, endometriosis or poor ovarian reserve.

All 152 cycles were treated with a conventional long protocol using a combination of GnRH agonist (Suprecur®; Hoechst, Germany) and an individually adjusted dose of hMG (Menogon®; Ferring, Germany) or recombinant FSH (Puregon®; Organon, Austria). Prior to ultrasound-guided follicle aspiration (36 h), 5000–10 000 IU of hCG were administered (Pregnyl®; Organon) to induce ovulation.

For conventional IVF, oocyte–cumulus complexes were incubated for 3 h in BM1 medium (NMS Bio-Medical, Switzerland) before being inseminated. In preparation for insemination, ejaculates from normozoospermic patients were incubated in a Zech glass capillary dish (Astro Med Tec, Austria) which localizes an adequate number of motile sperm without exposure to centrifugation stress. The Zech device consists of two concentric wells overlaid by a U-ring and a cover glass. In detail, progressive motile sperm migrate from the ejaculate in the outer well to concentrate in the medium-filled inner well by using a capillary bridge created by the overlying U-ring. After ~1–2 h a 500 µl sperm sample was taken from the central chamber, analysed and used for insemination.

In contrast to IVF, sperm for ICSI were separated from the ejaculate by using a swim-up technique. ICSI was done as previously suggested (Ebner et al., 2001Go).

At 16–18 h after injection, fertilization was assessed. This ICSI schedule differs slightly from the IVF one. In order to compensate for a possible accelerated development in ICSI cases (Nagy et al., 1998Go; Montag and Van der Ven, 2001Go), IVF zygotes were screened 18–20 h post insemination following mechanical removal of cumulus cell remnants.

After oocytes were controlled for the presence of two pronuclei, they were incubated individually in 10 µl drops of Blastassist System Medium 1 (MediCult, Denmark) to ensure exact follow-up of further development. Documentation was done using an imaging and archival software (Octax EyeWare®; MTG, Germany).

When one zygote had been separated from the other, pronuclear pattern (Tesarik and Greco, 1999Go; Montag and Van der Ven, 2001Go) and presence of a halo were documented. In parallel, all deviations from a presumed normal zygote morphology were recorded (vacuolization, unequal pronuclear size, dense cytoplasmic granulation).

Halo-positive zygotes were classified according to the type of halo as either showing a concentric (or symmetric) appearance (Figure 1A) or a polar (or asymmetric) one predominately located on one side of the zygote (Figure 1B). The latter type was further subdivided with respect to the location of the halo (next to or opposite the polar bodies). In order to differentiate halo-negative zygotes (Figure 2) and to evaluate the exact type of halo, zygotes were rotated by means of the holding pipette. Thus, three-dimensional structure of the halo formation could be estimated. In the case of a presumed polar halo appearing concentric in another plane (n = 16), the zygote was admitted to the polar group.



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Figure 1. (A) Zygote with pronuclear pattern 3 and concentric appearance of the halo (7 µm). (B) Zygote with pronuclear pattern 0B and a polar halo opposite the polar bodies (9 µm).

 


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Figure 2. Zygote with moderate granular cytoplasm but without halo.

 
Using the capability of the measuring support inside the Octax EyeWare, the extension of the halo could be easily measured. It should be noted that, in contrast to concentric haloes which show approximately the same extent along the cortex, asymmetric haloes taper towards the end. In this special case the halo was measured at its maximum extension.

On the next 2 days (days 2 and 3) embryos were checked for the number and size of blastomeres as well as for the presence of fragments. Individual culture (10 µl drops) was prolonged in Blastassist System Medium 2 (MediCult) until day 3 or 5. In the blastocyst transfer group, signs of compaction were controlled on day 4. Blastocyst extension and quality was checked on day 5 according to the method of Gardner et al. (2000Go) with some slight modifications. In detail, all blastocysts showing a blastocoele that was not more than half of the volume of the embryo (grades 1 and 2 according to Gardner et al., 2000Go) were pooled. The highest degree of extension was found to be blastocysts which started to hatch from the zona pellucida (former grade 5). In terms of inner cell mass (ICM), all cell aggregates consisting of several or many tightly packed cells (grade A and B) were considered to be of good prognosis. For scoring the trophectoderm (TE), the best grade was assigned if many cells formed a cohesive or loose epithelium (former grades A and B). It should be noted that in all subgroups the number of blastocysts checked for quality differs slightly from the total number of blastocysts since some early blastocysts did not show a clearly distinguishable ICM and TE.

According to the number and quality of 8-cell embryos on day 3 (Racowsky et al., 2000Go) transfer was done either on day 3 (n = 80) or 5 (n = 72) in BM1 medium (NMS Bio-Medical, Switzerland) using an Edwards–Wallace Catheter (Smiths Industries, UK). Clinical pregnancy rate and implantation rate were calculated using pooled day 3 and day 5 results. Since selection of transferable embryos or blastocyst did not differ from routine methods, institutional review board approval was not sought.

Statistics
T-Test and {chi}2-test were used to analyse nominal variables in the form of frequency tables. Statistical significance was determined at P < 0.05.

Receiver operating characteristic (ROC) curves represent the probability of true-positive results (sensitivity) as a function of false-positive results (1 – specificity). Sensitivity as well as specificity were calculated for all metric values (extension of halo, µm) of the decision axis and combined with the area under the curve (AUC). The AUC (sensitivity/1 – specificity) shows whether the test is adequate (AUC close to 1) or inadequate (AUC close to 0.5).

A stepwise logistic regression model was used to see if any combination of pronuclear pattern (pattern 0, pattern 1–5) and halo (halo positive, halo negative) might serve as a prognostic tool in terms of blastocyst formation and quality.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In 152 patients a total of 1248 oocytes were harvested of which 1102 (88.3%) were found to be at metaphase II (MII) stage. Using either conventional IVF or ICSI 792 (71.9%) oocytes could be fertilized correctly (two-pronucleate, 2PN). There was no difference in patient characteristics, gonadotrophin response, fertilization rate and blastocyst formation rate between IVF and ICSI patients (Table I).


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Table I. Patient and treatment characteristics in IVF and ICSI
 
Zygote stage (day 1)
Out of 792 fertilized oocytes, 127 (16.0%) showed abnormal zygote morphology. In detail, 85 zygotes showed vacuoles, 17 zygotes had pronuclei of unequal size, and 25 zygotes revealed dense cytoplasmic granulation. Since frequency of abnormal morphology was comparable in both IVF zygotes (n = 35) and ICSI ones (n = 92) (P = 0.52), these data were pooled in order to increase sample size. Halo appearance was not influenced since 81.9% (104/127) of the affected zygotes showed symmetric or asymmetric haloes.

In 79 (10.0%) out of 792 zygotes halo-like structure and pronuclear pattern could not be checked since nucleoli had already disappeared at the time of evaluation. Consequently, only 713 zygotes could be scored and measured adequately. Table II gives detailed information on morphology of IVF and ICSI zygotes (pooling both normal and abnormal ones). There was no influence of the method of fertilization (IVF, ICSI) on number and type of halo (P = 0.642) or pronuclear pattern (P = 0.553).


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Table II. Zygote morphology in IVF and ICSI zygotes (morphologically abnormal zygotes included)
 
Extension of halo (polar and concentric) ranged from 2.0 to 27.0 µm in IVF zygotes and from 2.4 to 26 µm in ICSI zygotes. The degree of halo was not related to blastocyst formation (P = 0.104) or blastocyst quality (P = 0.128) (Figure 3).



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Figure 3. Receiver operating characteristic (ROC) curves for prognostic power of halo extension on (A) blastocyst formation (n = 347) and (B) blastocyst quality (n = 146; assessed as the consistence of the inner cell mass).

 
Cleavage stages (day 2–4)
On day 2 and 3 neither cleavage rate nor percentage of fragmentation was influenced by presence, type or extension of halo. However, the opposite finding could be observed by analysing pronuclear patterns with regard to cleavage morphology, since pattern 0 zygotes showed a significantly higher cleavage rate compared with patterns 1–5.

From a total of 416 embryos that could have reached the blastocyst stage (Table III), a cohort of 138 embryos (33.2%) showed developmental arrest on day 4, whereas 231 had compacted (55.5%) and 13 (3.1%) had already reached the morula stage. Day 4 appearance was unrelated to presence, type or extension of halo as well as pronuclear pattern.


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Table III. Preimplantation development (days 2–5) according to type of halo and pronuclear pattern in pooled IVF and ICSI zygotes
 
Blastocyst stage (day 5)
On the fifth day of in-vitro culture, a total of 201 embryos formed blastocysts (48.3%). Number of blastocysts was not related to halo presence but to pronuclear pattern, since pattern 0 gave significantly more blastocysts than patterns 1–5 (P < 0.001).

In addition, a significantly reduced percentage of zygotes with abnormal morphology (unequally sized pronuclei, dense cytoplasmic granulation, vacuolization) (P = 0.007) reached blastocyst stage compared with normal zygotes (33.9 versus 50.9%).

It was observed that quality of ICM, but not TE, was significantly worse (P < 0.001) in blastocysts derived from zygotes without a halo. A similar negative impact of pronuclear patterns 1–5 on quality of the ICM was found (P = 0.002).

A stepwise logistic regression revealed that none of the possible combinations of pronuclear pattern and halo was adequate to predict blastocyst formation and quality in vitro at a statistically significant level (P = 0.144).

Pregnancy and implantation rate
A total of 58 pregnancies was achieved out of 152 transfers (38.2%). Day 3 pregnancy rate (25/80; 31.3%) did not differ significantly from day 5 rate (33/72; 45.8%). The implantation rates were 16.9% (27/160) for day 3 and 23.5% (34/145) for day 5 (P = 0.135).

Table IV gives detailed information on pregnancy as well as implantation rates after homogeneous transfers with respect to halo formation and pronuclear pattern. Exclusive transfer of embryos which derived from halo-positive zygotes (P = 0.046) or zygotes with pronuclear pattern 0 (P = 0.035) led to significantly higher clinical pregnancy rates compared with transfers of embryos from ‘bad quality’ zygotes. In terms of implantation rate, the level of significance was not reached with respect to halo formation (P = 0.095) or with pronuclear pattern (P = 0.066).


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Table IV. Outcome of embryo and blastocyst transfers (pooled) according to halo presence and pronuclear pattern
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Payne et al. (1997Go) were the first to report a subplasmalemmal zone of translucent cytoplasm immediately prior to formation of the male and female pronucleus. Subsequently, this focal clearing within the cortical cytoplasm (cytoplasmic flare) often progresses to involve the entire cytocortex (halo). This phenomenon is thought to be the manifestation of a microtubule-organized translocation of mitochondria and other cytoplasmic components to the centre of the oocyte, since virtually no detectable mitochondria were found in the cortical region of fertilized oocytes. More likely, mitochondria concentrate in the perinuclear cytoplasm in an ellipsoidal mass (Van Blerkom et al., 2000Go).

The physiological role of mitochondrial redistribution in human zygotes is unknown but it may be speculated that clustering of mitochondria to perinuclear regions may be involved in cell cycle regulation (Barnett et al., 1996Go; Wu et al., 1996Go; Bavister and Squirrell, 2000Go), e.g. by means of calcium mobilization and ATP liberation (Sousa et al., 1997Go; Diaz et al., 1999Go; Van Blerkom et al., 2000Go). In addition, location of mitochondria next to the pronuclei would allow immature mitochondria, as seen in zygotes (Motta et al., 2000Go), to complete maturation presuming that some input from the nucleus is needed (Bavister and Squirrell, 2000Go).

In several species, mitochondrial relocation at zygote stage is associated with the competence to form blastocysts (Barnett and Bavister, 1996Go; Krisher and Bavister, 1997Go). However, in mice no such beneficial effect of mitochondrial clustering could be observed (Muggleton-Harris and Brown, 1988Go).

Scott and Smith (1998Go) were the first to attribute a certain prognostic value to the presence of a halo, but it was part of a complex scoring system and its direct impact on implantation ability can only be estimated. In detail, this group assigned a score of 5 to all zygotes with heterogeneous cytoplasm showing either an asymmetrical halo located predominantly on one side of the pronuclei or a symmetrical halo all along the cortex. Considering the fact that zygotes without any halo were given a score of 3 and other morphological parameters, e.g. early cleavage 24–26 h after insemination, a score of 10, it seems likely that this imbalance may have resulted in biased scores that rather reflect early cleavage behaviour (2/5 of the maximum score) than other parameters.

Due to their legal situation, some German groups had to modify the Scott and Smith (1998Go) score by simply focusing on pronuclear patterns and cytoplasmic constitution (Ludwig et al., 2000Go; Zollner et al., 2002Go). Interestingly, even without considering early cleavage, a significant correlation of zygote morphology to blastocyst formation (Zollner et al., 2002Go) and implantation (Ludwig et al., 2000Go) could be observed, indicating that besides pronuclear pattern, presence of a halo is of some prognostic value in terms of further development.

In literature, percentage of halo appearance in 2PN zygotes ranges from 67.7 to 88.7% (Demirel et al., 2001Go; Salumets et al., 2001Go; Stalf et al., 2002Go; Ebner et al., 2003aGo). The finding that most of the zygotes show a halo in the presence of different pronuclear patterns makes it difficult to draw conclusions about the actual influence on further in-vitro development. Subdivision of haloes might help to elucidate their prognostic role in IVF laboratories since there is a lack of agreement as to whether to pool all variations of haloes (Scott and Smith, 1998Go; Salumets et al., 2001Go) or to exclude some subtypes from analysis (Stalf et al., 2002Go; Zollner et al., 2002Go).

The present prospective study is the first attempt to standardize assessment of haloes by strictly grading (no halo, polar halo next to polar bodies, polar halo opposite the polar bodies, concentric halo) and measuring them.

Our data indicate that ICSI has no influence on halo formation or extension, which led us to pool ICSI and conventional IVF data. In addition, it can be concluded that a detailed grading of haloes is not required in IVF laboratories since zygotes with different subtypes of haloes did not differ in subsequent development. However, in the case of absence of a halo, similar percentages of blastocysts did form in vitro compared with halo-positive zygotes, which is in contrast to literature (Zollner et al., 2002Go). On the other hand, a significant negative influence of halo-negativity on blastocyst quality (ICM) could be shown. Analysed separately from halo appearance, prognostic potential of pronuclear pattern was much higher since numbers of cleavages on days 2 and 3 as well as number and quality of blastocyst were positively correlated with pattern 0.

However, stepwise logistic regression to filter out a possible combination of both morphological parameters (halo presence and pronuclear pattern), which might help to predict blastocyst formation and quality, failed to yield adequate results. These findings strongly suggest that halo formation and pronuclear pattern are two completely different features. This is supported by data from Payne et al. (1997Go) who reported the asynchronous appearance of cytoplasmic flare and pronuclei.

Though decision-making in terms of transfer was totally based on morphology at the day of transfer, resulting in some so-called mixed transfers, the number of homogeneous transfers in terms of halo formation (114/152) and pronuclear pattern (94/152) was adequate for statistical analysis. In the present study, exclusive transfer of embryos from halo-positive zygotes gave higher clinical pregnancy rates than embryos deriving from halo-negative zygotes. This is in line with the literature (Ludwig et al., 2000Go; Stalf et al., 2002Go) but is has to be kept in mind that the work of Salumets et al. (2001Go) is contradictory. In addition, the reported increase in clinical pregnancy rate after transfer of former zygotes with pronuclear pattern 0 (Tesarik and Greco, 1999Go; Wittemer et al., 2000Go; Balaban et al., 2001Go; Montag and Van der Ven, 2001Go), which could be supported by the present study, is questioned by the same group (Salumets et al., 2001Go).

However, based on our experience it may be recommended that, out of a pool of embryos or blastocysts, the ones that should be selected for transfer are those that show an ideal pronuclear pattern and any type of halo on day 2 of development.


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 Materials and methods
 Results
 Discussion
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Submitted on March 7, 2003; resubmitted on June 6, 2003; accepted on July 16, 2003.