Growth rate of human preimplantation embryos is sex dependent after ICSI but not after IVF

John C.M. Dumoulin1,3,5, Josien G. Derhaag1,3, Marijke Bras1, Aafke P.A. Van Montfoort1,3, Arnold D.M. Kester4, Johannes L.H. Evers1,3, Joep P.M. Geraedts2,3 and Edith Coonen1,3

1 Department of Obstetrics & Gynaecology and 2 Department of Clinical Genetics, Academic Hospital Maastricht, P.O.Box 5800, 6202 AZ Maastricht, 3 Research Institute Growth & Development (GROW) and 4 Department of Methodology and Statistics, University of Maastricht, P.O.Box 616, 6200 MD Maastricht, The Netherlands

5 To whom correspondence should be addressed. Email: jdum{at}sgyn.azm.nl


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: There is concern that IVF and/or ICSI might have an adverse effect on embryonic development via epigenetic alterations. Such alterations might also be involved in the sex-related growth differences in preimplantation embryos found in some animal species. In the present study we analysed cell numbers of human male and female surplus embryos that developed to the blastocyst stage after either IVF or ICSI in order to investigate possible sex-dependent differential growth rates. METHODS: Blastocysts resulting from surplus embryos obtained after either IVF or ICSI during a 5 year study period were analysed using fluorescence in situ hybridization (FISH). RESULTS: The number of cells and sex could be determined in 330 blastocysts collected from 92 IVF cycles and in 322 blastocysts collected from 121 ICSI cycles. Whereas female and male embryos originating from IVF showed comparable mean log cell numbers per embryo ± SEM (3.76±0.05 in 147 female and 3.72±0.04 in 183 male embryos), significant differences were observed in embryos originating from ICSI (3.57±0.05 in 162 female and 3.90±0.03 in 160 male embryos). The sex-related growth difference was significantly greater in ICSI than in IVF embryos. In a subset of 84 embryos, inner cell mass (ICM) and trophectoderm (TE) were analysed separately. A significantly higher mean log cell number of TE cells in ICSI male embryos was found as compared to their female counterparts (3.44±0.12 in 16 female and 3.90±0.11 in 29 male embryos), whereas this difference was not found in IVF embryos. CONCLUSION: A clear sex-related growth difference was found in human blastocysts originating from ICSI, but not in blastocysts originating from IVF. It is as yet unknown which mechanism is responsible for our findings. We hypothesize that the ICSI procedure might interfere with the process of imprinted X-inactivation.

Key words: chromosomal abnormalities/FISH/human blastocysts/ICSI/preimplantation development


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
IVF and ICSI are widely used assisted reproductive technologies worldwide. Reports from European registers show that in 2000 totals of 126 961 IVF cycles and 99 976 ICSI cycles were performed, and the percentage of infants born after assisted reproduction was shown to range between 1.0 and 3.8% of the total number of live-born in the reporting countries (Nyboe Andersen et al., 2004Go). Since the introduction of assisted reproduction there has been major concern about the safety of these technologies and possible adverse effects on the children conceived with assisted reproduction. Relatively high rates of adverse outcomes such as prematurity, low birthweight and perinatal mortality are found in assisted reproduction pregnancies compared to pregnancies in the general population (Olivennes et al., 2002Go). Although these adverse outcomes are strongly related to the high percentage of multiple pregnancies observed after assisted reproduction, they are also found in singleton pregnancies (Olivennes et al., 2002Go). Besides multiple births, several patient-related factors are involved in the increased risk of adverse outcome after assisted reproduction such as the infertile status of the IVF patient, maternal age, and parity (Olivennes et al., 2002Go). The role of the IVF technique and in vitro culture in particular is less clear. In animal studies, however, in vitro culture has been shown to be related to aberrant fetal and perinatal development (Niemann and Wrenzycki, 2000Go; Khosla et al., 2001Go).

The invasive nature of the ICSI technique has led to even more concern about its safety (Kurinczuk, 2003Go; Retzloff and Hornstein, 2003Go). Data from most large follow-up studies of children indicate that ICSI is, in general, a safe technique. Incidences of major and minor congenital malformations in children born after ICSI or after conventional IVF are reported to be similar, and in the range observed in the general population (Bonduelle et al., 2002Go; Van Steirteghem et al., 2002Go). Furthermore, no differences were found when comparing medical and developmental outcome of ICSI children up to 2 years of age with normally conceived children (Sutcliffe et al., 2001Go; Bonduelle et al., 2003Go). However, some less reassuring studies were published and concern has been raised about the potential dangers of the procedure. A slight but significant increase in de novo sex chromosomal aneuploidy and structural autosomal abnormalities in ICSI children was reported (Bonduelle et al., 2002Go; Van Steirteghem et al., 2002Go). This fact is probably related to the higher aneuploidy rate in the sperm of the subfertile men treated rather than to the ICSI procedure itself (Bonduelle et al., 2002Go). Furthermore, Hansen et al. (2002)Go reported an increased risk of major congenital malformations after ICSI. Also, preimplantation development after ICSI seems to be compromised. It has been reported by us (Dumoulin et al., 2000Go) and others (Shoukir et al., 1998Go; Miller and Smith, 2001Go) that embryos obtained after ICSI have a lower potential to develop into blastocysts as compared to embryos obtained after IVF. One possible reason for this impaired blastocyst development of ICSI embryos is that the sperm used for ICSI are usually obtained from subfertile men and have a relatively high risk of genetic abnormalities such as fragmented DNA and chromosomal abnormalities (see for references: Retzloff and Hornstein, 2003Go). However, the ICSI procedure in itself seems to contribute to a reduced capacity of blastocyst formation in comparison with conventional IVF, as suggested by studies in which sibling oocytes were subjected to ICSI or IVF using sperm from the same semen sample (Griffiths et al., 2000Go; Jeziorowski et al., 2002Go). Indeed, certain technical aspects of the injection procedure can affect subsequent embryonic development to the blastocyst stage (Dumoulin et al., 2001Go). In animal studies it has been demonstrated that ICSI results in abnormal sperm decondensation (Hewitson et al., 1999Go) and calcium oscillations not equivalent to those initiated after IVF (Kurokawa and Fissore, 2003Go). Other concerns about the safety of the ICSI procedure include the possible effect of ICSI on imprinted genes, of which many are involved in early development (Cox et al., 2002Go; DeBaun et al., 2003Go; Kurinczuk, 2003Go; Maher et al., 2003Go; Retzloff and Hornstein, 2003Go).

A phenomenon that has been speculated to be related with external stress factors inflicted upon embryos cultured in vitro is the finding that, in some species, male and female embryos develop at different rates during the preimplantation period (see for review: Kochhar et al., 2001Go). This sex-dependent differential growth rate is as yet not well understood. It is probably an in vitro artefact, as it has been shown that male and female embryos of several species develop at different rates in vitro but not in vivo (Peippo and Bredbacka, 1995Go; Kaminski et al., 1996Go; Kochhar et al., 2001Go). Several studies suggest that the sex of the embryo may influence the embryo response to environmental stress such as exposure to transient elevated temperatures (Edwards et al., 2001Go; Kochhar et al., 2001Go).

Considering the above-mentioned data that in vitro culture conditions have been shown to result in sex-dependent growth rate differences during preimplantation embryonic development in animal species, it can be hypothesized that IVF and/or ICSI lead to sex-dependent differential growth rates also in human preimplantation embryos. Furthermore, in view of the fact that this phenomenon is probably stress-related, it can be hypothesized that ICSI more than IVF may lead to sex-dependent differences. For this reason, data obtained during the course of two earlier studies performed in our laboratory on chromosomal abnormalities at the blastocyst stage (Dumoulin et al., 2000Go, 2001Go; Derhaag et al., 2003Go; Coonen et al., 2004Go) were reanalysed in order to investigate possible growth rate differences in male and female human preimplantation embryos originating from either conventional IVF or ICSI.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Embryos and patients
During a period of ~5 years, starting in 1995, surplus embryos that were left over after transfer and that were not cryopreserved because of insufficient quality (see below) were obtained from couples undergoing IVF or ICSI for fertility treatment at the Academic Hospital Maastricht. Embryos that developed to the blastocyst stage were fixed when the patients had given written consent. ICSI treatment was performed in the majority of cases because of male subfertility, and in some cases because of failed fertilization in previous IVF cycles. Male subfertility was defined as a progressive motile sperm concentration of <3 x 106/ml in combination with <5% morphologically normal sperm, evaluated using strict criteria (Enginsu et al., 1992Go). Males presenting on several occasions with severe oligozoospermia (<5 x 106/ml total sperm density) were karyotyped, and, if an abnormal karyotype was found, excluded from treatment. Only ejaculated sperm were used for ICSI. A conventional in vitro insemination procedure was performed because of tubal factor subfertility, unexplained subfertility, mild male subfertility, and other causes of subfertility.

Culture procedures
IVF and ICSI procedures, as well as the culture procedure used, have been described in detail earlier (Dumoulin et al., 2000Go, 2001Go). Oocytes and embryos from each consecutive treatment cycle were alternately allocated to the use of either a commercially available medium (IVF-50TM; Vitrolife AB, Sweden), or ‘in-house’ prepared human tubal fluid (HTF) medium supplemented with 9% human serum protein solution and were cultured in 20 ml droplets covered by mineral oil. Oocytes were checked for the presence of pronuclei at 18–20 h after insemination or injection. The development of each individual embryo was followed by daily microscopic observation during which the number of cells was counted and the morphological aspect was noted. Embryo replacement was performed on the second or third day after oocyte retrieval. If available, two or three embryos, depending on the developmental stage and morphological appearance of the embryos, as well as on the age of the patient, were transferred to the uterus of the patient. After embryo transfer, any supernumerary embryos originating from normally fertilized [two pronucei (2PN)] zygotes were cultured until the third day after oocyte retrieval. Cryopreservation of supernumerary embryos was performed when, at 63–67 h after insemination, one or more embryos had reached the ≥8-cell stage, and when they were of good morphological quality (grades III and IV; Bolton et al., 1989Go). When cryopreservation was not performed, any surplus embryos were cultured for 2 or 3 more days.

During the long study period, several changes in the culture procedures were made that could have influenced embryonic development to the blastocyst stage. During the first 4 years of the study, surplus embryos were left from day 3 onwards in their original culture medium, while during the last year of the study, a medium designed for the culture of late preimplantation stage embryos to the blastocyst stage (G2.2 TM; Vitrolife AB) was used. During the first 2 years of the study, embryos were cultured separately, while during the rest of the study period, embryos were cultured communally with a maximum of five embryos per drop. During the first 2 years, oocytes and embryos were alternately allocated per set of two treatment cycles to culture either under an atmosphere of 5% O2, 5% CO2 and 90% N2, or 5% CO2 in air (Dumoulin et al., 1999Go), while during the last 3 years of the study, the reduced (5%) O2 concentration was used for all subsequent cycles.

FISH analysis of blastocysts resulting from the culture of surplus embryos
On the morning of day 5 after ovum retrieval, surplus embryos that developed to the full blastocyst stage were fixed and stained with 4',6-diamidino-2-phenylindole (DAPI) as described earlier (Coonen et al., 2004Go). The number of nuclei stained with DAPI was taken as the number of cells of the embryo. All other embryos, including those that had only just started to form a small blastocoelic cavity, were cultured for another day and were subsequently fixed on day 6 if they had developed to the full blastocyst stage. Embryos were subsequently analysed with fluorescence in situ hybridization (FISH) using directly labelled X-, Y- and 18-chromosome-specific DNA probes as described earlier (Coonen et al., 2004Go). In some embryos, the inner cell mass (ICM) and trophectoderm (TE) nuclei were differentially labelled before performing FISH (Derhaag et al., 2003Go). For evaluation of the chromosomal constitution, only blastocysts were taken into account that had a total cell count of ≥25 cells, and in which ≥75% of the cells gave clear FISH signals as an additional criterion. This study has been approved by the local ethics committee.

Statistics
Percentages of female and male embryos were compared to the expected percentages (50–50%) using {chi}2-test. The mean incidence of blastocyst formation for IVF and ICSI groups was analysed by unpaired Student's t-test. Because cell numbers have a positively skewed distribution and larger standard deviations in groups with a larger mean—as was to be expected because of the fact that cell numbers of developing embryos increase exponentially in time—a log transformation was applied before analysis (Altman, 1991Go; Burgoyne, 1993Go). The log-transformed cell numbers appeared to have a normal distribution, with a constant standard deviation over subgroups. The log-transformed data on blastocyst cell numbers were analysed by ANOVA using the Statistics Package for Social Sciences (SPSS) Version 11.5 (SPSS Inc., USA), followed by the Tukey multiple comparisons test as a post hoc analysis to determine where significant effects occurred. Furthermore, all variables that could possibly affect the number of cells per blastocyst: insemination method, embryo sex, day of fixation, day of first signs of cavitation, and several changes in culture procedures that unavoidably took place during the long study period, were entered into a multiple linear regression analysis model as covariates in order to assess their relative contribution to outcome. The interaction of sex and insemination method was used to test for the difference in sex-effect between ICSI and IVF.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A total of 798 IVF cycles and 1140 ICSI cycles were performed during the 5 year study period. In 343 IVF cycles (43%) and 513 ICSI cycles (45%), at least one surplus embryo was cultured to day 5 or 6, with mean numbers±SEM of surplus embryos per cycle of 4.61±0.19 in IVF cycles and 3.97±0.15 in ICSI cycles (unpaired Student's t-test: P<0.01). In the other 1082 cycles, either no fertilization took place, available embryos were all transferred, available surplus embryos were all cryopreserved, or no consent was obtained from the patient.

The incidence of blastocyst formation per cycle was significantly higher in the IVF group (33.7±1.8%) than in the ICSI group (25.2±1.4%) (P<0.001), resulting in a total number of 527 blastocysts in the IVF group and 509 in the ICSI group. In 652 blastocysts, fixation and FISH was succesful and sex and cell number could be determined. Of these, 330 blastocysts originated from 92 treatment cycles in which conventional IVF was performed, and 322 originated from 121 cycles in which ICSI was performed. The histogram from the total group of 652 blastocysts of which sex and cell number could be determined shows the clear skewed distribution of cell numbers, which was the reason for the log transformation of the data before statistical analysis (Figure 1).



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Figure 1. Histogram of cell numbers from 652 blastocysts.

 
Of the 330 IVF-derived blastocysts, the sex ratio was 147 (45%) female and 183 (55%) male ({chi}2-test: not significant), of the 322 blastocysts originating after ICSI, the sex ratio was 162 (50%) female and 160 (50%) male.

On days 2 and 3 of development after IVF or ICSI, upon microscopic examination no significant differences in cell numbers were found between male and female blastocyst stage embryos (Table I). However, as can be seen in Table II, the mean log cell number of male blastocysts after ICSI was significantly greater than that of female embryos, whereas this was not found after IVF. This was found both when the total group of blastocysts was analysed and when only blastocysts consisting of ≥25 cells were taken into consideration (Table II), which can be considered as the minimum number of cells of normally fertilized, expanded blastocysts on days 5 and 6 of development (Hardy et al., 1989Go). An interaction analysis confirmed that the sex difference was significantly greater in ICSI than in IVF.


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Table I. Number of cells at day 2 and day 3 of male and female surplus embryos that subsequently developed into blastocysts

 

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Table II. Mean log cell number of male and female blastocysts in which sex could be determined subdivided in two groups on the basis of cell number

 
For both the IVF and ICSI treatment group, the sex of the embryo together with the following possible confounding factors were entered into a multiple linear regression analysis model: culture medium (HTF, Vitrolife IVF-50TM, Vitrolife G2.2TM), oxygen concentration (5 or 20%), culture in groups or single, day of blastocyst fixation (5 or 6), and first day of blastocoel formation (5 or 6) in order to assess their relative contribution to outcome. Regression analysis showed that sex of the embryo was an independent and highly significant factor associated with blastocyst cell number in the ICSI group only. Also, in an interaction analysis, the sex difference was shown to be significantly greater in ICSI than in IVF. To further illustrate this, data were subdivided into several different groups of interest and analysed using ANOVA. As can be seen in Tables III and IV, the difference between the mean log cell number of male and female embryos is significant in most subgroups only after ICSI.


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Table III. Mean cell number of male and female blastocysts confirmed by cell number (≥25 cells) subdivided in the different culture media used

 

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Table IV. Mean cell number of male and female blastocysts confirmed by cell number (≥25 cells) subdivided in different experimental groups according to the day of blastocoel formation and the day of fixation

 
In a subgroup of 84 blastocysts, the mean log cell number in the two compartments ICM and TE in each embryo were analysed (Table V). The number of ICM cells in IVF-derived male embryos was significantly lower than in ICSI-derived male embryos. In ICSI-derived embryos, the number of TE cells in male embryos was significantly higher than in female embryos, while in IVF-derived embryos, comparable numbers of cells were found in male and female embryos.


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Table V. Mean log cell number±SEMa in the inner cell mass (ICM) and trophectoderm (TE) compartments of male and female blastocysts originating either from IVF or from ICSI

 
In 443 blastocysts with ≥25 cells, ≥75% of cells had interpretable FISH signals, and chromosomal constitution could be determined. The difference between male and female embryos in the mean percentage of cells per blastocyst that were found to be disomic for both sex chromosomes as well as chromosome 18 was similar in the IVF group whereas it almost reached significance (P=0.050, unpaired Student's t-test) in the ICSI group (Table VI).


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Table VI. Chromosomal constitution of male and female blastocysts in the IVF and ICSI groups

 

    Discussion
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 Materials and methods
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Our study shows clearly that, after ICSI treatment, embryos of the male sex that develop to the blastocyst stage consist of significantly more cells than female blastocysts (Tables II V). This difference between male and female embryos was not found in embryos originating from conventional IVF treatment. Since significance in ICSI and non-significance in IVF per se does not imply that ICSI embryos behave differently from IVF embryos, we explicitly confirmed the greater sex difference in the ICSI group than in the IVF group using an interaction model. The sex-related growth difference between embryos originating from ICSI developed from the fourth day of development onwards, as no differences were found on the second and third day of development (Table I). It must be noted that for the present study, surplus embryos were used, i.e. embryos that were left over after transfer and that were not cryopreserved because of insufficient quality. It is unknown whether or not good quality embryos originating from IVF and ICSI show comparable sex-related growth differences.

The number of cells of a blastocyst is dependent on many variables such as the day after fertilization, the quality of the embryo and several culture-related parameters. The rather low number of cells per blastocyst in the present study is probably the result of the use of only minor quality surplus embryos (being all untransferred embryos that were considered unsuitable for cryopreservation because of their low cell number at the third day of development and/or their major fragmentation), as well as from the use of suboptimal media for blastocyst development. Indeed, when one of the recently developed sequential media to support the development of embryos during the late preimplantation stages was used, the number of cells per blastocyst was considerably higher (Table IV).

Unavoidably in such a long study period of 5 years, several changes in the culture procedures took place that could affect embryo growth and blastocyst formation. As mentioned above, the culture medium used from day 3 onwards was changed in the last year of the study period to a medium designed for the culture of late preimplantation stage embryos as stated by the manufacturer. Furthermore, the oxygen concentration in the gas phase was changed from ambient (~20%) to reduced (5%) concentrations (data not shown), and single culture of embryos was changed to group culture (data not shown). Therefore, to evaluate these potential confounding factors, a linear regression analysis was performed. The use of medium designed for culture of late preimplantation embryos as well as the combination of day 5 being the first day of blastocoel formation and day 6 being the day of fixation were found to be significantly associated with the number of cells per blastocyst. The sex of the embryo was shown to be an independent and highly significant factor associated with the number of cells per blastocyst in the ICSI group.

Growth differences between the sexes have long been known to be present in the fetal period and later. These differences are thought to be caused by the effects of androgens and other male-specific hormones synthesized by the testes. However, growth differences are also reported to exist between male and female embryos during the preimplantation period, well before the development of the fetal gonads. In several animal species, male embryos have been shown to grow faster than female embryos, as reflected either by a greater proportion of male embryos developing to the blastocyst stage at a certain point in time after fertilization (in the mouse: Tsunoda et al., 1985Go; Zwingman et al., 1993Go; sheep: Bernardi and Delouis, 1996Go; bovine: Pegoraro et al., 1998Go; Gutiérrez-Adán et al., 2001Go) or by a higher mean number of cells per blastocyst (in the mouse: Peippo and Bredbacka, 1995Go; bovine: Xu et al., 1992Go; Yadav et al., 1993Go). Few data exist on growth of male and female human preimplantation embryos. Unlike our findings, Ray et al. (1995)Go reported, in a relatively small study group of 39 male and 67 female human embryos presumably originating after conventional IVF, that on the second day of development, the number of cells of male embryos was slightly but significantly greater than that of female embryos (mean number of cells: 3.97±0.19 in male and 3.28±0.16 in female embryos). This difference seemed to be maintained up to the blastocyst stage, although the difference was not significant at that developmental stage (Ray et al., 1995Go). Indirect evidence of male embryos having a higher mean number of cells can be obtained from the results of several studies reporting significantly higher male:female ratios in infants born after IVF treatment (in which, as a standard, embryos with the fastest cleaving rates are transferred) compared to those born after spontaneous conception. This phenomenon was found both after transfers of cleavage stage embryos (Pergament et al., 1994Go; Tarín et al., 1995Go) as well as after blastocyst transfer (Ménézo et al., 1999Go; Milki et al., 2003Go).

In addition to differences in growth rate between male and female preimplantation embryos, differences in energy metabolism and metabolic activity were found. The expression of the housekeeping X-linked genes glucose-6-phosphate dehydrogenase (G6PD) and hypoxanthine phosphoribosyl transferase (HPRT) were found to be significantly higher in in vitro-cultured female embryos than in male embryos, both in the mouse (Epstein et al., 1978Go) and in the bovine (Gutiérrez-Adán et al., 2000Go; Lonergan et al., 2000Go; Peippo et al., 2002Go). The metabolic activity in in vitro-cultured male embryos was higher than in female embryos, as demonstrated by a significantly higher pyruvate and glucose uptake and lactate production in the human (Ray et al., 1995Go) and a 4-fold higher activity of the pentose-phosphate pathway (PPP) (Tiffin et al., 1991Go). Besides being involved in cell metabolism, G6PD and HPRT are also involved in controlling the amount of oxygen radicals in the cell (Rieger, 1992Go; Nicol et al., 2000Go). It has been hypothesized that the higher level of the X-linked oxygen radicals detoxifying enzymes in female embryos will lead to a lower level of oxygen radicals. As oxygen radicals also have a growth-stimulant effect, a lower level of oxygen radicals could be responsible for the observed retarded development (Rieger, 1992Go; Bredbacka and Bredbacka, 1996Go; Gutiérrez-Adán et al., 2000Go). In agreement with this hypothesis is the observation that the faster growth of male embryos is only found in the presence of glucose in the culture medium (Bredbacka and Bredbacka, 1996Go).

However, the sex related differential growth rate during the preimplantation period probably must be considered to be an in vitro artefact as it has been shown that male and female embryos develop at different rates in vitro but not in vivo in the mouse (Peippo and Bredbacka, 1995Go), bovine (Gutiérrez-Adán et al., 1996Go, 2001Go), and pig (Kaminski et al., 1996Go). Not all reported data indicate however that the sex-related growth differences are an in vitro artefact: in the mouse (Burgoyne, 1993Go) and in the pig (Cassar et al., 1995Go) also in vivo derived male blastocysts had significantly more cells as compared to female embryos. Recently, it was shown that in in vitro-cultured bovine embryos, female blastocysts produced double the amount of G6PD transcripts compared with male blastocysts, while in in vivo-produced embryos, female and male blastocysts produced equal amounts of G6PD (Wrenzycki et al., 2002Go), again an indication that sex-related embryonic differences are an in vitro artefact. The findings of a differential expression of X-linked genes between male and female preimplantation embryos cultured in vitro (Epstein et al., 1978Go; Gutiérrez-Adán et al., 2000Go; Lonergan et al., 2000Go; Peippo et al., 2002Go; Wrenzycki et al., 2002Go) but not in in vivo-derived embryos (Wrenzycki et al., 2002Go) implies that in the in vitro-produced female blastocysts, both X chromosomes were active whereas dosage compensation for this X-linked gene did occur in in vivo-generated embryos (Wrenzycki et al., 2002Go).

In mammals, one of the two X chromosomes in females (XX) will undergo a process of inactivation early in embryo development in order to ensure an equal gene dosage as in males (XY) (Lyon, 1961Go). In the human, X chromosome inactivation (XCI) has been estimated to start when the ICM consists of <6 cells (Monteiro et al., 1998Go). In the mouse, a dynamic multi-step process of XCI has been proposed (Huynh and Lee, 2001Go; Okamoto et al., 2004Go). After fertilization, during the early cleavage stages, the paternal X chromosome, after being initially active, undergoes imprinted inactivation from the 4-cell stage onwards (Okamoto et al., 2004Go). At the late blastocyst stage, this inactive state of the paternal X chromosome is reversed in all cells and the process of inactivating one of the X-chromosomes in females is repeated. In epiblast cells derived from the ICM and giving rise to the embryo proper, the imprint is erased and selection of the X chromosome to be inactivated is random. In the extra-embryonic tissues such as trophectoderm and yolk sac endoderm, the imprint is retained and the paternal X chromosome is preferentially silenced. DNA methylation plays an important role in imprinted XCI (see references in Goto and Monk, 1998Go). It was postulated that adverse in vitro culture conditions could give rise to aberrant epigenetic modifications in the genome, possibly by perturbation caused in the methylation process (Khosla et al., 2001Go; Young et al., 2001Go). Recent observations have suggested a link between IVF and/or ICSI and imprinting disorders, such Beckwith–Wiedemann syndrome and Angelman syndrome (Cox et al., 2002Go; Kurinczuk, 2003Go; Halliday et al., 2004Go). It was hypothesized by Cox et al. (2002)Go that imprinting defects found in ICSI children could be causally related to the technique of ICSI, resulting possibly from the introduction of the sperm acrosome and its digestive enzymes into the ooplasma, or from the mechanical stress that may damage cellular structures.

To conclude, our finding of a sex-related growth difference in blastocysts originating from ICSI, but not in blastocysts originating from IVF, raises again the issue of safety of the ICSI procedure. If the rapid, successive, imprinted inactivation and reinactivation of the paternal X chromosomes during preimplantation embryonic development, as was shown in the mouse, also existed in the human, it can be hypothesized that in female ICSI embryos this relatively unstable process is disturbed. This would result in more cells in an embryo in which temporarily both X chromosomes are active, which in turn could lead to the observed sex-related differences via an as yet unknown mechanism. The disturbance of the imprinted X-inactivation process in female ICSI embryos could be the result of some common feature in the sperm of men suffering from male factor subfertility, or the sperm injection technique itself might interfere with the process of the imprinted X inactivation.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on July 15, 2004; resubmitted on September 16, 2004; accepted on October 21, 2004.