Pronuclear morphology scoring and chromosomal status of embryos in severe male infertility

S. Kahraman1,5, Y. Kumtepe2, S. Sertyel1, E. Dönmez1, M. Benkhalifa1,3, N. Findikli1 and P. Vanderzwalmen4

1 Istanbul Memorial Hospital, Reproductive Endocrinology and ART Unit, Istanbul, Turkey, 2 Ataturk University, School of Medicine, Department of Obstetrics and Gynaecology, Erzurum, Turkey, 3 Advanced Technologies Laboratory, Paris, France and 4 Von Helmont Hospital, IVF Center, Brussels, Belgium


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: The study aim was to evaluate the relationship between pronuclei morphology scoring (PNMS) and the chromosomal complement of embryos in couples with severe male infertility undergoing ICSI. A total of 3116 pre-embryos was scored according to PNMS in 452 cycles. METHODS: Pre-embryos were classified into eight categories based on the alignment, size, linear or irregular distribution of pronuclear bodies (PNB), position and clarity of cytoplasmic halo and abutting of the pronucleus. These categories were subdivided into groups I and II according to the similarity and distribution of PNB. RESULTS: In total, 2574 pre-embryos formed by using ejaculated sperm, while 542 pre-embryos developed by injection of testicular sperm or round spermatids. More group II pre-embryos with markedly different morphology from group I were formed after ICSI with testicular sperm than with fresh ejaculated sperm (32.1 versus 22.7%, P < 0.01). Of 490 pre-embryos in which pronuclear morphology was evaluated, 263 were biopsied for preimplantation genetic diagnosis. The rate of chromosomal abnormality was higher in embryos developed from group II pre-embryos (52.2%) than in embryos developed from group I prezygotes (37.6%, P < 0.05). CONCLUSIONS: Group II pre-embryos had markedly different morphology from group I, and had a low rate of blastocyst formation and high risk of chromosomally abnormal embryos. When testicular sperm and round spermatids were used for ICSI, more group II pre-embryos and chromosomally abnormal embryos were produced than with ejaculated sperm. Pronuclear morphology was correlated with chromosomal complement, and impacted upon by the sperm source.

Key words: chromosomal complement/embryo development/preimplantation genetic diagnosis/pronuclei morphology/severe male infertility


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Finding effective and easily applicable criteria based on non-invasive examination for the selection of the most viable embryos is one of the most important issues in the practice of assisted reproductive technologies. As there is clearly a relationship between zygote morphology and the ability of an embryo to implant, screening embryos at the zygote and later stages could increase the implantation rates with both day 3 and day 5 transfers (Scott et al., 2000Go).

Numerous oocyte, pronuclear and embryo grading criteria have been proposed, including cumulus-coronal morphology (Ng et al., 1999Go), polar body aspect (Ebner et al., 1999Go, 2000Go), nuclear and cytoplasmic maturity or oocyte polarity (Garello et al., 1999Go), pronuclear morphology (Payne et al., 1997Go; Scott and Smith, 1998Go; Tesarik and Greco, 1999Go), and embryo morphology either at 2 or 3 days after oocyte retrieval (Puissant et al., 1987Go; Steer et al., 1992Go; Tan et al., 1992Go) or at the blastocyst stage (Gardner and Lane, 1998Go).

In one study (Ludwig et al., 2000Go), a scoring system was applied based on quality assessment of pronucleus (PN) stages and the morphological appearance of PN oocytes, in order to evaluate its impact on the pregnancy rate. These authors concluded that scoring of PN stage oocytes might allow the transfer of two selected PN oocytes in patients under German embryo protection law and could reduce multiple pregnancy rates without reducing implantation. Others (Salumets et al., 2001Go) observed that the cleavage rate was slower in zygotes with unpolarized PN than in those where one PN was polarized (P < 0.05). However, their controversial conclusion was that there were no significant differences in embryo quality or implantation/pregnancy rates between proposed zygote classes.

In three studies retrospective data was used to define the PN scoring systems (Scott and Smith, 1998Go; Tesarik and Greco, 1999Go; Tesarik et al., 2000Go). The pronuclei morphology scoring (PNMS) used by these authors was based on empirical observations correlated with pregnancy. The basis of this scoring system was a combination of pronuclear stage, nucleoli number and distribution and cytoplasmic appearance. A strong association was found between implantation and the equality of nucleoli size within each nucleus of the pronuclear embryos from which the transferred embryos developed. Subsequently, this scheme was redefined to a single observation system (Scott et al., 2000Go).

It is crucial to select the best quality embryos at as early a phase as possible to decide on their future development. It was concluded by some (Racowsky et al., 2000Go; Fisch et al., 2001Go) that predicting which cleaved embryos would form blastocysts could permit high success rates associated with blastocyst transfer to be achieved from day 3 embryo transfer. The graduated embryo score predicts blastocyst formation and pregnancy rate from cleavage-stage embryos.

Using a grading system to detect chromosomally abnormal embryos is a new idea. Many morphological abnormalities observed between the zygote and cleavage stages have been related to chromosome abnormalities (Munné and Cohen, 1998Go), while others (Sandalinas et al., 2001Go) reported that even though there is a strong selection against chromosomally abnormal embryos, the extension of culture to day 5 or day 6 cannot be used as a reliable tool to select against clinically relevant chromosome abnormalities such as trisomies.

In the present study, the relationship was investigated between PNMS and chromosomal complement in pre-embryos developed from the injection of ejaculated and testicular sperm or round spermatids obtained from severely oligoasthenoteratozoospermic or azoospermic males. The question was raised as to whether the sperm source would impact upon PNMS and the chromosomal status of embryos.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This retrospective study, which was conducted at the Istanbul Memorial Hospital ART and Genetics Center between February 2000 and March 2001, evaluated pre-embryos obtained from ICSI cycles performed for severe male infertility. The study was based on PNMS data and the chromosomal status of embryos developed from scored pre-zygotes. The PN scoring system used was based on previously published data, albeit with minimal modification (Tesarik and Greco, 1999Go; Tesarik et al., 2000Go).

Pituitary down-regulation was achieved by use of a GnRH agonist (Buserelin Suprefact®; Hoechst AG, Frankfurt, Germany), commencing on the 21st day of the menstrual cycle as the mid-luteal long protocol. Controlled ovarian hyperstimulation (COH) was achieved with FSH (MetrodinHP; Ares Serono Laboratories Co, Welwyn Garden City, UK) alone or in combination with hMG (Pergonal®; Ares Serono) in a step-down manner; 10000 units of HCG (Profasi®; Ares Serono) were administered to trigger ovulation, and oocytes were retrieved 36 h later.

An inverted microscope (Olympus IX70, Olympus Optical Co. Ltd., Japan) equipped with a Hoffman modulation contrast system (Modulation Optics Inc., Greenvale, NY, USA) was used to monitor embryo development during PNMS.

Only pre-embryos obtained from ICSI procedures were included in the study. The nuclear and cytoplasmic maturity and morphology of the oocytes were assessed before ICSI by removing the surrounding cumulus corona complex with either mechanical stripping or hyaluronidase treatment. Ejaculated and testicular sperm were obtained for ICSI from males with severe oligoasthenozoospermia and azoospermia.

Zygote scoring
Morphology scoring was based on the distribution and abutting PN and alignment and size of pronuclear bodies (PNB). At 18 h post ICSI the diameter and association of PN and number and distribution of PNB were evaluated by two observers, and images recorded using a digital recording system (Sony DV-CAM Digital Video Cassette Recorder, DSR30P, Japan) for additional confirmation. The zygotes were focused (using bright-field optics) on a monitor connected to an inverted microscope (Olympus IX70, Japan) equipped with a Hoffman modulation contrast system and a colour camera (Sony 3CCD Colour Video Camera, Japan). Zygotes were rotated (at x10 magnification) using a small round-tipped glass probe drawn by hand in order to observe the zona pellucida. The changing of focus is mandatory during observation of PN and PNB until the whole volume of both pronuclei is inspected. PN and polar bodies were checked at several focal points to eliminate superimposition of both abutted pronuclei.

Morphological parameters evaluated included the position and the size of pronuclei, the distribution and size of PNB in the pronucleus, and the halo. The pre-embryos were classified into eight categories based on the morphology of PN (Figure 1Go):



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Figure 1. Pre-embryos were classified into eight categories based on the alignment, size, linear or irregular distribution of pronuclear bodies, position and clarity of cytoplasmic halo and abutting of the PN. These categories were divided into subgroups. Group II had markedly different morphology from group I in terms of irregularity and size of PNB, clarity and equality of pronuclear halo and early syngamy.

 



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Figure 2. Group I: (A) Aligned nucleoli, cytoplasmic halo, abutting pronuclei; (B) clear halo, abutting PN, aligned, small nucleolus; (C) clear halo, abutting PN, large nucleolus scattered irregularly; (D) clear halo, abutting PN, small nucleolus scattered irregularly. Group II: (E) Clear halo, abutting PN, aligned nucleolus irregular in size; (F) clear halo, abutting PN nucleoli irregular in size and distribution; (G) blurred halo, separate PN, irregularly scattered, unclear nucleoli; (J) early syngamy.

 
Oocyte incubation prior to ICSI was carried out in IVF-50 Medium (Gamete 100; Scandinavian IVF Science, Gothenburg, Sweden) at 37°C in an atmosphere of 5% in air. ICSI was executed as described previously (Kahraman et al., 1999Go). Assessment of fertilization was made 18 h after sperm injection. Further embryonic development was observed from day 2 to day 5.

Day 3 Scoring
Embryo quality was evaluated using a grading system (Puissant et al., 1987Go). On day 3, embryos were scored as grade I–V according to certain morphological criteria. Grade I exhibited embryos with more than seven cells, <10% fragmentation and good cell-to-cell contact; grade II presented embryos with more than seven cells, 10–15% fragmentation; grade III had slow-growing embryos with five to seven cells with 20% fragmentation or uneven blastomere size; grade IV had slow-growing embryos with four to six or more cells but with >20% fragmentation or uneven blastomere size; and grade V embryos had four or less cells or were grossly fragmented.

Embryo biopsy
The indications for preimplantation genetic diagnosis (PGD) (n = 65 cycles) included advanced maternal age (>=37 years, n = 38), repeated implantation failure (>=2 cycles, n = 18) and recurrent early abortions during previous ICSI attempts (>=2, n = 9). Embryos with seven or more cells (grade I or II) on day 3 were selected for biopsy. Zona drilling was carried out using a 1.48 µ diode laser (Fertilase®; MTM, Medical Technologies, Montreux SA, Switzerland). Three pulses of 6 µs duration were used for zona opening and a regular opening of 25–30 µm was created to allow placement of the blastomere biopsy pipette (Cook IVF, Queensland, Australia). Only one blastomere from each suitable embryo with clear nucleus was biopsied. Biopsied embryos were cultured overnight, both to observe their future development and to allow for a sufficient period for the fluorescence in-situ hybridization (FISH) procedure. For the multicolour FISH analysis, five DNA probes were used for the simultaneous detection of chromosomes 13, 18, 21, X and Y (Figure 3Go). Embryos were classified as complex abnormal when two or more chromosomes had an abnormal count but were not completely polyploid or haploid. All blastomeres of abnormal embryos not being transferred were fixed and evaluated by the FISH procedure to confirm the diagnosis. Abnormal FISH results in embryos developed from group II-scored pre-embryos are shown in Figure 3Go.



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Figure 3. Abnormal FISH results for chromosomes 13, 18, 21, X, Y in embryos developed from group II-scored pre-embryos. (A) Trisomy 13, Monosomy 21; (B) Triploidy; (C) Trisomy X; (D) Trisomy 18, Monosomy 21.

 
Blastocyst scoring
Embryos containing less than six blastomeres on the afternoon of day 3 were accepted as slow-growing embryos, and PN scored pre-embryos were followed up to 12:00 h on day 5 whenever possible, depending on their developmental capacity. Each embryo was categorized according its morphology either during the pre-embryo period or during further embryo development. If no increase in blastomere numbers was recorded during 48 h follow-up, embryos were defined as arrested.

Blastocyst classification was carried out at 12:00 h on day 5 depending on the presence of blastocoele or the sign of early cavitation, defined as trophectoderm with sufficient cells to form a continuous layer without a single cell stretching or flattening on the surface, and a well-defined and organized, tightly packed inner cell mass with more than 60 cells. If none of the embryos presented this morphology, they were cultured for one more day and then re-scored.

Statistical analysis
Results were expressed as mean (± SD) and percentages. Statistical analysis of the data was performed using the {chi}2-test and Pearson correlation analysis where appropriate.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The mean age of female and male partners was 33.7 ± 4.82 and 36.84 ± 6.46 years respectively. A total of 3116 pre-embryos was scored according to PNMS in 452 ICSI cycles. In total, 2574 pre-embryos were formed by using ejaculated sperm for ICSI, while 542 embryos were formed by injection of testicular sperm or round spermatids.

In 321 cycles, PN scored pre-embryos (n = 1679) were cultured until day 3, while in 131 cycles (n = 1437) the further development of embryos was observed until day 5 as these embryos had more blastomeres and a higher mitotic index on day 3. The fertilization rate was similar in the two groups (70.2 versus 74.0%).

The correlation of PN scores with the source of sperm used for ICSI (ejaculated sperm, testicular sperm or spermatid cells) was evaluated. More group II scored pre-embryos (32.1 versus 22.7%) were formed by ICSI using testicular and round spermatids than by using fresh ejaculated sperm (P < 0.01). The proportion of G- and J-scored pre-embryos, with significantly different morphology in terms of distribution of PNB, clarity and equality of PN halo (G) and early syngamy (J) than the other categories, was greater in testicular sperm- and round spermatid-injected oocytes (P < 0.05).

The number of blastomeres was evaluated in day 3 embryos. Those with fewer than six blastomeres on day 3 were identified as slow-growing, and the proportion of these at 66–70 h after ICSI was significantly greater in group II than in group I (42.7 versus 31.3%, P < 0.01). The proportion of embryos with six to eight blastomeres after 66–70 h was greater in group I than in group II (54.9 versus 49.2%; P < 0.001), and a similar situation was seen among embryos with more than eight blastomeres (13.8 versus 8.1%). A higher cleavage rate, with more than eight cells on day 3, was seen in embryos developed from group I score zygotes (50.4%) compared with only 28.2% in those from Group II (P < 0.01).

A higher proportion of good blastocysts was seen in group I than in group II (43.5 versus 20.9%; P < 0.001), while more PN stage-arrested pre-embryos (13.4 versus 3.5%) and arrested or degenerated embryos (48.5 versus 36.6%) were observed in group II than in group I (P < 0.001).

No correlation was found between PN morphology and other parameters, including oocyte morphology, duration of ovulation induction, the total dose of gonadotrophin ampoules used, body mass index (BMI) and the peak estradiol levels. However, a correlation was identified between PN scored pre-embryos and the source of sperm used for ICSI, PN stage arrested pre-embryos, further embryo development and the rate of chromosomally abnormal embryos (P < 0.05) (Table IGo).


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Table I. Pronuclear (PN) morphology scoring and further embryo development
 
A total of 2142 embryos developed by the injection of either fresh ejaculated or testicular sperm, while a total of 964 embryos were obtained by using frozen ejaculated and testicular sperm. No correlation was found between the fresh and frozen ejaculated and testicular sperm in terms of PNMS.

In 26 out of 452 cycles, round spermatid injections were used as they were the only possible germ cells for ICSI obtained from simultaneous testicular sperm extraction (TESE). In 26 cycles, a total of 251 metaphase II (MII) oocytes was injected and the fertilization rate with 2PN was 24.6%. Among 77 pre-embryos, 61 had group II pattern (79.2%) and only 16 (11.8%) had group I pattern (P < 0.01). Although no statistically significant difference was observed between the proportion of arrested zygotes in the two groups (18.7 versus 14.3%), a significantly higher proportion of cleavage-arrested and slow-growing embryos was observed in embryos developed from group II pre-embryos (Table IIGo; P < 0.05). A high incidence of G and J scored pre-embryos was observed. At day 3, only five cleaving embryos (8.2%) developed from group II zygotes compared with 31.2% from group I.


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Table II. Correlation between PN scoring and further embryo development in round spermatid injection (ROSI) cases
 
In 65 cycles, PGD was performed for aneuploidy screening. Among 490 scored zygotes evaluated, a total of 263 embryos were biopsied, and the chromosomal abnormality rate was 41.3%. A total of 161 embryos was found to be chromosomally normal and they were transferred, while 102 embryos were chromosomally abnormal. The distribution of PNMS and chromosomal status are presented in Table IIIGo. The proportion of chromosomally abnormal embryos was higher in group II (56.2%) compared with embryos developed from group I scored zygotes (37.6%; P < 0.05). The major chromosomal abnormality was aneuploidy in both groups I and II. Likewise, the rate of complex aneuploidy and tetraploidy was similar in both groups, though a higher rate of haploidy was observed in group II (P < 0.05) (Figure 4Go). The low number of biopsied embryos which developed from group II was due to a high proportion of arrested pre-embryos and slow-growing embryos being unsuitable for embryo biopsy, but the difference was statistically significant (P < 0.05)


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Table III. Chromosomal status of embryos developed from group I and group II scored pre-embryos (n = 65)
 


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Figure 4. Distribution of chromosomal abnormalities in embryos developed from group I and group II scored pre-embryos.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pronuclear morphology and several factors reflecting embryo preimplantation development—namely the occurrence of zygote arrest, cleavage arrest and cleavage-stage embryo morphology—were evaluated in severe male infertility cases undergoing ICSI procedures. Furthermore, a group of embryos developed from scored pre-embryos was evaluated to assess a possible correlation between the chromosomal complement of cleaved embryos for fertilization and PN scoring and the source of the sperm (Tesarik and Greco, 1999Go; Scott et al., 2000Go). The present results confirm previously published data in terms of PNMS and further embryo development, despite the relationship between PNMS, the source of sperm used for ICSI and chromosomal status of corresponding embryos being evaluated for the first time.

It has been shown that several proteins of the ribosomal RNA (rRNA) processing machinery are detected in the PNB that are scattered throughout the nucleus in early G1 of the cell cycle (Benavente et al., 1987Go). PNB appear to contain preassembled nucleolar complexes that are mainly involved in the processing steps of the pre-rRNAs. It has been proposed that fusion of PNB into nucleoli is dependent upon the resumption of transcription (Sheer et al., 1993). Therefore, the appearance of nucleoli and pronuclear structure is dependent on an early wave of pronuclear transcriptional activity (Tesarik and Kopency, 1989a, 1990) and on the presence of ooplasmic factors appearing during oocyte maturation (Tesarik and Kopecny, 1989bGo).

These processes may be at the origin of molecular abnormalities which appear later in embryo development between the 4- and 8-cell stages (Tesarik, 1987Go). The observation of scored pre-embryos to the blastocyst stage makes it possible to recognize a subpopulation of good morphology embryos that were highly capable of reaching the blastocyst stage and implanting successfully after transfer. Blastocyst formation in group I was statistically significantly higher than that of group II (43.5 versus 20.9%; P < 0.01). Thus, the developmental capacity of zygotes is associated with PN morphology, thereby confirming previously published data (Ludwig et al., 2000Go; Tesarik and Greco, 1999Go) which showed a higher development capacity of zygotes with `normal' pattern at the 2- to 4-cell stage, and other data (Scott et al., 2000Go) relating to development to the blastocyst stage.

The correlation between PN pattern and the source of sperm was also evaluated. Infertile males who are candidates for ICSI have an increased frequency of chromosomally abnormal sperm karyotypes (Chandley, 1979Go). Furthermore, FISH analysis for chromosomes 1, 12, 13, 21, XX, YY and XY in 255 613 sperm demonstrated significant increase in chromosomes 1, 13, 21 and XY disomy in infertile men compared with control donors (Martin, 2001Go). It is likely that some subsets of male infertility have an elevated risk of sperm chromosomal abnormalities, and men with oligospermia or azoospermia are particularly at risk. It appears that any perturbation during spermatogenesis can be associated with an increased frequency of chromosomal abnormalities (Martin, 2001Go). More group II scored embryos were formed by ICSI using testicular sperm and round spermatids than using fresh ejaculated sperm. It is well known that sperm quality influences not only fertilization but also subsequent embryo development. In humans, these paternal effects have been shown to affect the speed of embryo cleavage and morphology, the rate of in-vitro blastocyst formation and implantation rates after embryo transfer, after both conventional IVF and ICSI (Vanderzwalmen et al., 1991Go; Parinaud et al., 1993Go; Janny and Menezo, 1994Go; Hammadeh et al., 1996Go; Sanchez et al., 1996Go; Shaukir et al., 1998), though the mechanism underlying these phenomena is not known. In particular, it remains to be determined whether the paternal effects become manifest only after the major activation of embryonic gene expression, which occurs between the 4- and 8-cell stages in humans, or earlier (Tesarik et al., 1986Go; Braude et al., 1988Go).

In 26 cycles, pre-embryos developed from round spermatid injections were evaluated for PNMS in cases where no sperm were found on the day on which TESE and ICSI were performed simultaneously. At present, few data are available which focus on the chromosomal abnormalities of embryos developed from round spermatid injection, though a greater number of G- and J-scored embryos developed in cases with round spermatid injection. Hence, caution is needed when using immature testicular cells due to the risk of meiotic errors arising in the germinal cells from men with severely impaired spermatogenesis. According to our unpublished data on DNA analysis of unfertilized oocytes after round spermatid injection, the majority of the intact oocytes were blocked at MII with premature chromosome condensation, while the remainder were blocked at early syngamy with an asynchrony of DNA decondensation. Unlike mature sperm, round spermatids have a decondensed nucleus, and high rates of fertilization failure, embryo blocking and implantation failure were seen in round spermatid injection cases. A high rate of G and J scores in pre-embryos with two clearly distinct PN and early syngamy were observed when round spermatids were used for ICSI. The resultant syngamy might be of very short duration and disappear rapidly as the zygote enters the mitotic phase of the first cleavage division (Tesarik, 1998Go).

With regard to embryos undergoing developmental arrest, their presence was markedly increased in zygote pattern group II compared with group I. G-scored pre-embryos exhibited an ill-defined, blurred halo with separate PN irregular in size, and this appearance most likely indicated delayed growth and fusion of the nuclei which may affect the developmental potential in these embryos. It has been suggested unequivocally that paternally derived problems of embryo cleavage during pre-implantation development can be traced back to a very early post-fertilization period, before the occurrence of the first cleavage division (Tesarik et al., 2002Go).

Different embryological parameters relating to every step of early embryo development are known to have some predictive value for implantation potential (Payne et al., 1997Go).

Selection could occur at the pronuclear stage where spatial distribution and polarity of the nuclei and organelles are critical parameters to be assessed (Scott and Smith, 1998Go), or at the blastocyst stage (Bavister and Boatman, 1997Go; Gardner and Lane, 1998Go; Tsirigotis, 1998Go). In this retrospective study, a pronuclear scoring system was used to classify zygotes into eight patterns with scores from A to J. Day 3 cleaved or blastocyst stage embryos which developed from pattern E to G zygotes were transferred when they were the only available embryos. For each zygote pattern, the subsequent embryological development was analysed. Zygotes of patterns A to D led to significantly more good quality embryos, with a higher rate of blastocyst formation, than embryos developing from patterns E to J (P < 0.01).

One group (Sandalinas et al., 2001Go) aimed to determine whether culture to blastocyst stage might be an effective tool to select against chromosomally abnormal embryos. Although many morphological abnormalities observed between the zygote and cleavage stages have been related to chromosome abnormalities (Munné and Cohen, 1998Go), not all morphologically abnormal embryos are chromosomally affected and aneuploidy is not associated with morphological abnormality (Sandalinas et al., 2001Go). In the present study, higher zygote stage arrest, embryo arrest, slow-growing embryos and chromosomally abnormal embryos were observed when immature sperm forms and round spermatids were used. Fertilization and further embryo development were not affected when testicular sperm were used for ICSI in cases with azoospermia. However, when round spermatids were used for ICSI, greater numbers of G and J score group II prezygotes and slow-growing embryos were obtained. Although E and F score pre-embryos were present in group II, the most dramatic morphological difference was noticeable in scores G and F, as not only PNB but also the size and clarity of PN were different. A higher proportion of chromosomal abnormality was observed in embryos developed from group II-scored pre-embryos than from group I-scored zygotes (56.2 versus 37.6%; P < 0.05). The rate of haploidy was also higher in group II (P < 0.05), but a low number of biopsied embryos developed from G to J scores were observed due to a high rate of arrested zygotes and slow-growing embryos.

In conclusion, the results of the present study further confirm that PNMS might represent an effective prognostic evaluation in the prediction of postzygotic and further embryo development to the blastocyst stage (Scott et al., 2000Go). Group II patterning, with significantly different morphology from group I, indicates a low rate of blastocyst formation and a high risk of arrested, slow-growing and chromosomally abnormal embryos.


    Notes
 
5 To whom correspondence should be addressed at: Istanbul Memorial Hospital, Reproductive Endocrinology and ART Unit, Piyalepasa Bulvari, 80270, Okmeydani, Istanbul, Turkey. E-mail: skahraman{at}superonline.com Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bavister, B.D. and Boatman D.E. (1997) The neglected human blastocyst revisited. Hum. Reprod., 12, 1607–1610.[ISI][Medline]

Benavente, R., Rose, K.M., Reimer, G., Hugle-Dorr, B. and Scheer, U. (1987) Inhibition of nucleolar reformation after microinjection of antibodies to RNA polymerase I into mitotic cells. J. Cell Biol., 105, 1483–1491.[Abstract]

Braude, P., Bolton, V. and Moore, S. (1988) Human gene expression occurs between the four and eight cell stages of preimplantation development. Nature, 332, 459–461.[ISI][Medline]

Chandley, A.C. (1979) The chromosomal basis of human infertility. Br. Med. Bull., 35, 181–186.[ISI][Medline]

Ebner, T., Moser, M., Yaman, C., Feichtinger, O., Hartl, J. and Tews, G. (1999) Elective transfer of embryos selected on the basis of first polar body morphology is associated with increased rates of implantation and pregnancy. Fertil. Steril., 72, 599–603.[ISI][Medline]

Ebner, T., Yaman, C., Moser, M., Sommergruber, M., Feichtinger, O. and Tews, G. (2000) Prognostic value of first polar body morphology on fertilization rate and embryo quality in intracytoplasmic sperm injection. Hum. Reprod., 15, 427–430.[Abstract/Free Full Text]

Fisch, J.D., Rodriguez, H., Ross, R., Overby, G. and Sher, G. (2001) The Graduated Embryo Score (GES) predicts blastocyst formation and pregnancy rate from cleavage-stage embryos. Hum. Reprod., 16, 1970–1975.[Abstract/Free Full Text]

Garello, C., Baker, H., Rai, J., Montgomery, S., Wilson, P., Kennedy, C.R. and Hartshorne, G.M. (1999) Pronuclear orientation, polar body placement, and embryo quality after intracytoplasmic sperm injection and in-vitro fertilization: further evidence for polarity in human oocytes? Hum. Reprod., 14, 2588–2595.[Abstract/Free Full Text]

Gardner, D.K. and Lane, M. (1998) Culture of viable human blastocysts in defined sequential serum-free media. Hum. Reprod., 13 (Suppl. 3), 148–159.[Medline]

Hammadeh, M.E., al-Hasani, S., Stieber, M., Rosenbaum, P., Kupker, D., Diedrich, K. and Schmidt, W. (1996) The effect of chromatin condensation taniline blue staining and morphology (strict criteria) of human sperm on fertilization, cleavage and pregnancy rates in an intracytoplasmic sperm injection programme. Hum. Reprod., 11, 2468–2471.[Abstract]

Janny, L. and Menezo, Y.J. (1994) Evidence for a strong paternal effect on human preimplantation embryo development and blastocyst formation. Mol. Reprod. Dev., 38, 36–42.[ISI][Medline]

Kahraman, S., Akarsu, C., Cengiz, G., Dirican, K., Sozen, E., Can, B., Guven, C. and Vanderzwalmen, P. (1999) Fertility of ejaculated and testicular megalohead spermatozoa with intracytoplasmic sperm injection. Hum. Reprod., 14, 726–730.[Abstract/Free Full Text]

Ludwig, M., Schopper, B., Al-Hasani, S. and Diedrich, K. (2000) Clinical use of a pronuclear stage score following intracytoplasmic sperm injection: impact on pregnancy rates under the conditions of the German embryo protection law. Hum. Reprod., 15, 325–329.[Abstract/Free Full Text]

Martin, R.H. (2001) Association of reduced recombination with non-disjunction in human sperm and aneuploidy in male infertility. Abstract Book of Eleventh Annual Meeting of International Working Group on Preimplantation Genetics. May 15, Vienna, Austria.

Munné, S. and Cohen, J. (1998) Chromosome abnormalities in human embryos. Hum. Reprod. Update, 4, 842–855.[Abstract/Free Full Text]

Ng, S.T., Chang, T.H. and Wu, J.T.C. (1999) Prediction of the rates of fertilization, cleavage and pregnancy success by cumulus-coronal morphology in an in vitro fertilization program. Fertil. Steril., 72, 412–417.[ISI][Medline]

Payne, D., Flaherty, S.P., Barry, M.F. and Matthews, C.D. (1997) Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography. Hum. Reprod., 12, 532–541.[ISI][Medline]

Parinaud, J., Mieusset, R., Vieitez, G., Labal, B. and Richoilley, G. (1993) Influence of sperm parameters on embryo quality. Fertil. Steril., 60, 888–892.[ISI][Medline]

Puissant, F., Van Rysselberge, M., Barlow, P., Deweze, P. and Leroy, F. (1987) Embryo scoring as a prognostic tool in IVF treatment. Hum. Reprod., 2, 705–708.[Abstract]

Racowsky, C., Jackson, K.V., Cekleniak, N.A., Fox, J.H., Hornstein, M.D. and Ginsburg, E.S. (2000) The number of eight-cell embryos is a key determinant for selecting day 3 or day 5 transfer. Fertil. Steril., 73, 558–564.[ISI][Medline]

Salumets, A., Hyden-Granskog, C., Suikkari, A.M., Tiitinen, A. and Tuuri, T. (2001) The predictive value of pronuclear morphology of zygotes in the assessment of human embryo quality. Hum. Reprod., 16, 2177–2181.[Abstract/Free Full Text]

Sandalinas, M., Sadowy, S., Alikani, M., Calderon, G., Cohen, J. and Munné, S. (2001) Developmental ability of chromosomally abnormal human embryos to develop to the blastocyst stage. Hum. Reprod., 16, 1954–1958.[Abstract/Free Full Text]

Sanchez, R., Stalf, T., Khanaga, O., Turley, H., Gips, H. and Schill, W.B. (1996) Sperm selection methods for intracytoplasmic sperm injection (ICSI) and andrological patients. Andrology, 13, 228–233.

Scott, L.A. and Smith, S. (1998) The successful use of pronuclear embryo transfers the day after oocyte retrieval. Hum. Reprod., 13, 1003–1013.[Abstract]

Scott, L., Alvero, R., Leondires, M. and Miller, B. (2000) The morphology of human pronuclear embryos is positively related to blastocyst development and implantation. Hum. Reprod., 15, 2394–2403.[Abstract/Free Full Text]

Sheer, U., Thiry, M. and Goessens, G. (1983) Structure, function and assembly of the nucleolus. Trends Cell Biol., 3, 236–241.

Shoukir, Y., Chardonnens, D., Campana, A. and Sakkas, D. (1998) Blastocyst development from supernumerary embryos after intracytoplasmic sperm injection: a paternal influence ? Hum. Reprod., 13, 1632–1637.[Abstract]

Steer, C.V., Mills, C.L., Tan, S.L., Campbell, S. and Edwards, R.G. (1992) The cumulative embryo score: a predictive embryo scoring technique to select the optimal number of embryos to transfer in an in-vitro fertilization and embryo transfer programme. Hum. Reprod., 7, 117–119.[Abstract]

Tan, S.L., Royston, P. and Cambell, S. (1992) Cumulative conception and live birth rates after in-vitro fertilization. Lancet, 339, 1390–1394.[ISI][Medline]

Tesarik, J. (1987) Gene activation in the human embryo developing in vitro. In Feichtinger, W. and Kemeter, P. (eds), Future Aspects in Human In Vitro Fertilization. Springer-Verlag, Berlin, pp. 251–261.

Tesarik, J. (1998) Oocyte activation after intracytoplasmic injection of mature and immature sperm cells. Hum. Reprod., 13 (Suppl. 1), 117–127.

Tesarik, J. and Greco, E. (1999) The probability of abnormal preimplantation development can be predicted by a single static observation on pronuclear stage morphology. Hum. Reprod., 14, 1318–1323.[Abstract/Free Full Text]

Tesarik, J. and Kopecny, V. (1989a) Development of human male pronucleus: ultrastructure and timing. Gamete Res., 24, 135–149.[ISI][Medline]

Tesarik, J. and Kopecny, V. (1989b) Developmental control of the human male pronucleus by ooplasmic factors. Hum. Reprod., 4, 962–968.[Abstract]

Tesarik, J. and Kopecny, V. (1990) Assembly of the nucleolar precursor bodies in human male pronuclei is correlated with an early RNA synthetic activity. Exp. Cell Res., 191, 153–156.[ISI][Medline]

Tesarik, J., Kopecny, V., Plachot, M. and Mandelbaum, J. (1986) Activation of nucleolar and extranucleolar RNA synthesis and changes in the ribosomal content of human embryos developing in vitro J. Reprod. Fertil., 78, 463–470.

Tesarik, J., Junca, A.M., Hazout, A., Aubriot, F.X., Nathan, C., Cohen-Bacrie, P. and Dumont-Hassan, M. (2000) Embryos with high implantation potential after intracytoplasmic sperm injection can be recognized by a simple, non-invasive examination of pronuclear morphology. Hum. Reprod., 15, 1396–1399.[Abstract/Free Full Text]

Tesarik, J., Mendoza, C. and Greco, E. (2002) Paternal effects acting during the first cell cycle of human preimplantation development after ICSI. Hum. Reprod., 17, 184–189.[Abstract/Free Full Text]

Tsirigotis, M. (1998) Blastocyst stage transfer: pitfalls and benefits. Hum. Reprod., 13, 3285–3289.[Free Full Text]

Vanderzwalmen, P., Bertin-Segal, G., Geerts, L., Debauche, C. and Schoysman, R. (1991) Sperm morphology and IVF pregnancy rate: comparison between Percoll gradient centrifugation and swim-up procedures. Hum. Reprod., 6, 581–588.[Abstract]

Submitted on October 15, 2001; resubmitted on June 21, 2002; accepted on August 10, 2002.