Hum. Reprod. Advance Access originally published online on October 19, 2006
Human Reproduction 2007 22(1):75-82; doi:10.1093/humrep/del363
Effects of urinary and recombinant gonadotrophins on gene expression profiles during the murine peri-implantation period
1 Division of Medical Pharmacology, Leiden Amsterdam Center for Drug Research/Leiden University Medical Center, Gorlaeus Laboratories and 2 Department of Gynaecology, Division of Reproductive Medicine and Department of Clinical Epidemiology, Leiden University Medical Center, Leiden, The Netherlands
3 To whom correspondence should be addressed at: Division of Medical Pharmacology, Leiden Amsterdam Center for Drug Research/Leiden University Medical Center, Gorlaeus Laboratories, PO Box 9502, 2300 RA Leiden, The Netherlands. E-mail: r.sibug{at}lacdr.leidenuniv.nl
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
BACKGROUND: Controlled ovarian stimulation (COS) with urinary gonadotrophins but not recombinant gonadotrophins, adversely affect the implantation process. In this study, we investigated the effects of urinary and recombinant gonadotrophins on gene expression profiles at implantation sites during the mouse peri-implantation period and the possible molecular mechanisms involved in the detrimental effects of urinary gonadotrophins using microarray technology. METHODS: Adult female CD1 mice were treated with (i) urinary human FSH (hFSH) and urinary HCG, (ii) recombinant hFSH and recombinant human LH or (iii) saline. Gene expression profiling with GeneChip mouse genome 430 2.0 arrays, containing 45 101 probe sets, was performed using implantation sites on embryonic day 5. Data were statistically analysed using Significance Analysis of Microarrays. Ten genes from the microarray analysis were selected for validation using quantitative RT–PCR (qRT–PCR). A parallel group of pregnant mice was allowed to give birth to study the effect of gonadotrophins on resorption. RESULTS: Urinary gonadotrophins differentially up-regulated the expression of 30 genes, increased resorption and reduced litter size, whereas recombinant gonadotrophins did not. Nine of the 10 genes were confirmed by qRT–PCR. CONCLUSIONS: Urinary gonadotrophins, but not recombinant gonadotrophins, exerted differential effects on gene expression during the murine peri-implantation period. These findings might contribute to improve protocols for COS, leading to higher successful pregnancy rates.
Key words: microarray/ovarian stimulation/peri-implantation period/recombinant gonadotrophins/urinary gonadotrophins
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Pregnancy outcome is dependent on the implantation of the blastocyst into the endometrial lining of the uterus which involves a series of co-ordinated and synchronized events in the development and maturation of the blastocyst and the development of the uterus into a receptive state (Psychoyos, 1973
). Successful implantation is of clinical importance because only a very low percentage of embryos transferred after IVF lead to successful pregnancies. This is partly because of genetic abnormalities (Simpson and Liebaers, 1996) and mainly because of the non-receptivity of the uterus for reasons that are still unknown (Edwards, 1994).
Gonadotrophins are routinely used in human assisted reproduction techniques and in the generation of transgenic animals. Two types of gonadotrophins are used nowadays in controlled ovarian stimulation (COS): recombinant gonadotrophins produced by recombinant DNA technology and urinary gonadotrophins extracted from urine of post-menopausal women. In rodents, recombinant gonadotrophins do not exert any effect on the implantation process (Sibug et al., 2005), whereas urinary gonadotrophins exert deleterious effects such as increased frequency of chromosomal abnormalities (Maudlin and Fraser, 1977; Elbling and Colot, 1985; Luckett and Mukherjee, 1986), increased pre- and post-implantation mortality (Beaumont and Smith, 1975; Ertzeid and Storeng, 1992) and impaired implantation and prolonged gestation period (Ertzeid et al., 1993; Ertzeid and Storeng, 2001; Sibug et al., 2002, 2005). In humans, COS with urinary gonadotrophins resulted in gross alteration in the expression of endometrial genes in comparison with natural cycles (Horcajadas et al., 2005). In addition, COS with both recombinant FSH and urinary hCG resulted in reduced integrin expression in the endometrium (Thomas et al., 2002). Integrins are expressed at the so-called ‘implantation window’ and are considered as useful markers for endometrial receptivity (Lessey et al., 1994). Hence, a reduction in their expression suggests a negative effect on pregnancy.
Several gene expression profiling studies on molecular factors involved in the implantation process have been performed. However, genome-wide analysis to assess and compare the effects of urinary and recombinant gonadotrophins during the mouse peri-implantation period has not yet been done. Understanding the molecular impact of gonadotrophins is of great importance because this will not only contribute to a better understanding of the implantation process but may also improve COS protocols. Therefore, this study was undertaken to assess and compare the effects of urinary and recombinant gonadotrophins on the expression profiles of genes during the mouse peri-implantation period using microarray technology. Total RNA was extracted from the implantation site on embryonic day 5 (ED5), and gene expression profiles were studied. Expression of selected genes was validated using quantitative RT–PCR (qRT–PCR). The data show that stimulation with urinary gonadotrophins altered the gene expression profiles during the peri-implantation period, whereas the recombinant gonadotrophins did not exert any significant change. To our knowledge, this is the first study that compared the effects of recombinant and urinary gonadotrophins on gene expression profiles of implantation sites during the peri-implantation period.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Animals and experimental procedure
CD1 mice (8–10 weeks, Charles River, Germany) were housed five per cage upon arrival and allowed to acclimatize for 1 week. They had free access to food and water with a 12:12 dark : light cycle (lights on at 0700 h). All animal experiments were in accordance with the governmental guidelines for care and use of laboratory animals and approved by the Animal Care Committee of the University of Leiden.
The female mice (12 mice per group) were first injected (i.p.) with either of the following: (a) urinary human FSH (hFSH) (Metrodin; Serono, Coinsins, Switzerland), (b) recombinant hFSH (Gonal-F; Serono, Aubonne, Switzerland) or (c) saline at 12:00 pm, and 47 h later with urinary HCG l (Pregnyl; Organon, Oss, The Netherlands), recombinant human LH (Lhadi; Laboratoires Serono, Aubonne, Switzerland) or saline, respectively. All gonadotrophins were given in a concentration of 5I U in 0.1 ml saline. Based on this scheme, the following groups of mice were generated: (a) urinary gonadotrophin, (b) recombinant gonadotrophin and (c) saline (control). After the last injection, males were allowed to mate with the females (one male per two females). Successful mating was confirmed by the presence of a vaginal plug the following day (0800–0900 h). The day of vaginal plug detection was considered as ED0.
For microarray analysis, pregnant mice were injected with 0.1 ml 1% Chicago blue dye (Sigma-Aldrich, Steinheim, Germany) in the jugular vein under isoflurane anaesthesia to visualize the implantation sites (Psychoyos, 1973) on ED5. After allowing the dye to circulate for 10 min, the whole uterus with the embryos was dissected out and the animals were killed by decapitation. The uteri were cleared of fat tissue, immediately frozen in liquid nitrogen and stored at –80°C until further processing.
To assess pregnancy outcome, another set of saline-, urinary gonadotrophin- or recombinant gonadotrophin-treated pregnant mice (6–8 mice per group) were allowed to give birth and were sacrificed after delivery. The uteri were dissected out, and resorption (%) was determined from the difference between number of implantation sites and litter size.
Total RNA preparation and microarray hybridization
The site where the blastocyst was implanted (implantation site) was cut off on both sides from the adjoining intersites. Our previous studies showed that urinary gonadotrophins can delay implantation and decrease vascular permeability, and these are reflected by a smaller size and absence or less intensity of dye staining (Sibug et al., 2002, 2005). The expression profiles of genes exhibit a temporal pattern and some genes become active only at certain critical periods (Hamatani et al., 2004). To exclude these confounding effects of different developmental stages, we used only implantation sites with comparable size and dye staining. The position of implantation sites did not show a distribution pattern. Hence, they were randomly collected from the different parts of the uterus as long as they met the mentioned criteria.
Total RNA (from three to four implantation sites per mouse, pooled) was extracted using TRizol reagent (Invitrogen, Carlsbad, NM, USA) and used as a template for complementary DNA (cDNA) synthesis and biotinylated antisense cRNA preparation. The quality and amount of total RNA were characterized on nanochips using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). The synthesis of cDNA and cRNA, labelling, hybridization and scanning of the samples were performed as described by Affymetrix (www.affymetrix.com). GeneChip mouse genome 430 2.0 arrays, each containing 45 101 probe sets, (Affymetrix; Santa Clara, CA, USA) were used to hybridize the labelled cRNA. Four gene chips (n = 4 animals) per group were used giving rise to a total of 12 gene chips (n = 12 animals) for three groups. Each gene chip contained a sample from three to four pooled implantation sites from one mouse. Hybridization and scanning of the probe arrays were done at the Leiden Genome Technology Center (for details, see www.lgtc.nl) with Fluidics Station 450 and Gene Chip Scanner 3000.
Data analysis for microarray
Microarray Analysis Suite 5.0 (MAS5.0; Affymetrix) was used to estimate signal intensities plus signal reliabilities and to normalize the array signals by total intensity normalization (Quackenbush, 2002). Briefly, a given transcript is represented by a probe set consisting of 11 perfect/mismatch probe pairs. The software determines the degree of detection (detection call) based on the signal intensity, background noise and variation of the expression signals within the probe pairs. Present, marginal or absent calls have P 
0.04, 0.04–0.06 or 
0.06, respectively.
The reliability of the data was ensured by excluding probe sets with absent call on all 12 chips. Then the P-values of the detection calls generated by Affymetrix were used to calculate the mean P-value of each probe set per experimental group (n = 4). Probe sets with a mean P 0.06 were called group Absent (gA), P = 0.04–0.06 were called group Marginal (gM) and P 0.04 were called group Present (gP). Probe sets that were gA in all groups (3gA), gM in all groups (3 gM), gA in two groups and gM in one group (2gA + 1 gM) and gA in one group and gM in the other two groups (1gA + 2 gM) were also excluded. Consequently, only probe sets with at least 1gP were considered reliable for statistical analysis. Significance Analysis of Microarrays (SAM) (Tusher et al., 2001) was used to identify responsive genes. SAM is a distribution-free test, which allows the user to control the false discovery rate (FDR), i.e. the relative number of false positives generated. An FDR of 63% and a fold change of 1.5 were set to generate the number of significant genes. An FDR of 63% was chosen because lower FDRs resulted in a very limited number of genes. A fold change of 1.5 was chosen because pilot studies showed that fold changes <1.5 were difficult to validate with qRT–PCR. Gene Ontology Biological Process classifications were obtained using the NetAffx Analysis Center (www.affymetrix.com), allowing genes with a similar biological process to be grouped together.
qRT–PCR
The specificity of the genes obtained from the microarray analysis was tested by qRT–PCR, using the same total RNA samples used for array hybridization. For each gene, four mice/group were used and run in duplicates. Total RNA was DNase-treated followed by cDNA synthesis. Briefly, the following were added together in a total volume of 20 µl: 10 µl of total RNA (1 µg), 1.5 µl of oligo dT (0.5–1 µg/µl; Invitrogen), 1 µl (10 mM) dNTP (Invitrogen), 2 µl 10x M-Moloney Murine Leukemia Virus (MuLV) reaction buffer (Finnzymes; Espoo, Finland), 1 µl RNaseOUT (40 U/µl, Invitrogen) and 4.5 µl of M-MuLV Reverse Transcriptase (200 U/µl; Finnzymes). For the negative controls, RNase-free water was added instead of the Reverse Transcriptase enzyme. The mixture was incubated for 1 h at 42°C and then the enzyme inactivated at 70°C for 15 min.
Primers were generated using the Primer3 Input program (primer3_www.cgi v 0.2). Primers were designed in the same region as the probe set on the gene chip. The selected primers were controlled for specificity using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). The primers used are summarized in Table I. Serial dilutions of a known amount of cDNA sample were used for the standard curves of the genes. The housekeeping gene 
-actin was used for normalization because it was not affected by the different gonadotrophin treatments. The LightCycler kit (LightCycler® FastStart DNA MasterPlus SYBR Green I; Roche, Mannheim, Germany) was used to prepare the qRT–PCR mix. In each PCR, 5 µl of cDNA, 2 µl of forward primer (10 µM), 2 µl of reverse primer (10 µM) and 4 µl of the LightCycler kit mix were added in a 20-µl reaction mixture. The samples, in duplicates, were amplified with an annealing temperature of 60°C using the LightCycler 2.0 (Roche). The protocol used to amplify all the genes was 45 cycles. However, the threshold cycle (Ct) values used to compute for the concentration of the different genes varied and, in general, were within the range of 19–28.
|
The concentrations and melting temperatures of the different samples for each gene were calculated using the LightCycler Software 4 (Roche). For every sample, the ratio of expression was determined by dividing the concentration of the target gene by that of
-actin. The computed ratios were then statistically tested with one-way analysis of variance and the post hoc least significant difference test. Significance was accepted with P 0.05. |
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Resorption
The degree of decidualization in the primary and secondary decidual zone and the developmental stage of the embryos were comparable in all the three groups (Figure 1).
|
The frequency of embryo loss was significantly higher in the urinary gonadotrophin-treated group (n = 7) in comparison with the saline group (n = 11) (32.3 ± 9.9 versus 5.2 ± 2.4%, respectively; Figure 2). No significant difference was observed between the recombinant gonadotrophin-treated group (n = 6) and the saline group (n = 11) (8.3 ± 2.3 versus 5.2 ± 2.4%, respectively). Litter size was significantly lower in urinary gonadotrophin-treated group than in saline-treated group but not in the recombinant-treated group (5.4 ± 1.3, 12.2 ± 1.0 and 12.0 ± 0.7, respectively).
|
Microarray expression profiling
Filtering out all probe sets with absent calls on all 12 chips resulted in 29373 of 45101 probe sets. Subsequent exclusion of probe sets containing 3 gM, 3gA, 2 gM + 1gA and 1 gM + 2gA led to 20966 genes, which comprise only 46% of all probe sets present in the chip and were used for further analysis. SAM analysis of the microarray data showed that a set of genes was differentially expressed between the urinary gonadotrophin and the control groups during the peri-implantation period. At an FDR of 63%, stimulation with urinary gonadotrophins significantly up-regulated the expression of 30 genes (Table II) and did not significantly down-regulate any gene. The recombinant gonadotrophins did not alter the gene expression profiles at all.
|
Based on gene ontology classification, the majority of the differentially expressed genes have biological functions related to the implantation process such as immunoregulation, cell adhesion, transcription, intracellular signalling, receptor activity and metabolism (Table II). However, there are a number of genes whose biological functions and relevance to the implantation are still unknown.
qRT–PCR
The following 10 genes were selected based on fold change and/or putative function in implantation process and tested using qRT–PCR for confirmation: epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (Efemp1), small proline-rich protein 2F (Sprr2f), kelch domain containing 4 (Klhdc4), histocompatibility 2, K1, K region (H2-K1), embigin (Emb), interferon-induced protein with tetratricopeptide repeats 3 (Ifit3), 2'–5' oligoadenylate synthetase-like 2 (Oasl), fibrinogen-like protein 2 (Fgl2), -2 microglobulin (B2m) and growth differentiation factor 10 (Gdf10). Except for Klhcd4, all the rest showed enhanced gene expression upon treatment with urinary gonadotrophins, confirming the microarray dataset. Note that the fold changes in the expression levels were higher in qRT–PCR than in the microarray analysis (Figure 3).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
The implantation process is a complex process orchestrated and synchronized by the development of the embryo and preparation for receptivity of the uterus. Different sets of genes undergo activation/deactivation at different stages of development to pave the way for a successful implantation of the blastocyst in the uterine lining. In this study, stimulation with urinary gonadotrophins, but not recombinant gonadotrophins, exerted differential effects on gene expression during the peri-implantation period. These gene expression effects might be related to negative effects on implantation because urinary gonadotrophins compared with saline treatment also increased resorption resulting in a small litter size.
These results are in accordance with our previous findings (Sibug et al., 2002, 2005) in which we demonstrated that urinary gonadotrophins led to a delayed implantation, reduced vascular permeability, reduced expression of the vascular endothelial growth factor system, retarded growth and prolonged gestational period. In contrast, the recombinant gonadotrophins did not exert any effect on these parameters. The qRT–PCR results clearly confirmed the microarray data with nine of the 10 genes selected for independent testing showing a change in expression with both methods. The validation of nine of the 10 genes is higher than that one would expect from an FDR of 63%. In general, the fold changes observed in the qRT–PCR are higher than those observed in the microarray data, a phenomenon that is often observed when comparing array hybridization and qRT–PCR results (Rajeevan et al., 2001; Bangur et al., 2002). Two of the 10 genes showed more than 2–fold difference (5.1x for Efmp1 and 3.2x for H2K-1) but for the majority the qRT–PCR results and microarray data corresponded well. This difference in fold changes between the microarray and qRT–PCR data is not unique in our hands. In fact, even larger differences, ranging from 9x to 21x, were reported by Rajeevan et al. (2001) and Bangur et al. (2002) and reflect the difference when using different techniques to assess messenger RNA expression.
Gene expression profiling studies in the developing oocyte and uterus before and during the peri-implantation period show that expression profiles of genes are dynamic and exhibit a spatio-temporal pattern (Yoshioka et al., 2000; Hamatani et al., 2004; Wang et al., 2004). Several genes are turned on and off at several stages of the developing oocyte until the activated blastocyst by waves of activation and deactivation. Subsequently, genes become active only at certain critical periods (Hamatani et al., 2004). Furthermore, it is possible that the activated and deactivated genes may have downstream effects on other genes. It is, therefore, possible that the changes in the gene expression profiles with urinary gonadotrophins on ED5 might be the result of a series of changes that took place in the developing oocyte and uterus before and until this time point.
Independent effects on the developing oocyte and uterus can also occur. It has been shown, for example, that unfertilized oocytes from ovulated female hamsters had an aberrant microfilament formation in comparison with the non-treated (Lee et al., 2005). In vitro experiments in rodents showed that the quality of developing oocytes/blastocysts is adversely affected by urinary gonadotrophins (Elbling and Colot, 1985; Edgar et al., 1987; Ertzeid and Storeng, 1992; Van der Auwera and D’Hooghe, 2001). Embryo transfer experiments in mice showed that prenatal loss of embryos and abnormal embryonic development after stimulation with urinary gonadotrophins are predominantly induced by effects of the hormone treatment on the maternal uterine environment (Elmazar et al., 1989; Spielmann and Vogel, 1993). Clinical data on COS also indicate that gonadotrophins might exert their effects only in the uterus. Birthweight of singletons conceived by implanting a cryopreserved embryo is significantly higher than birthweight after a fresh embryo transfer (Wennerholm, 2000; Wang et al., 2005). Because cryopreserved embryo transfer occurs predominantly in a natural cycle, while fresh embryo transfer in a hormonally induced state, one may hypothesize that COS might only influence uterine receptivity. Regardless of frozen or fresh embryos, it appeared that uterine environments were affected by the urinary gonadotrophin treatment, resulting in the outcome of pregnancy success.
The increased expression of Fgl2 (synonym prothrombinase) might be involved in the increased resorption observed in the mice treated with urinary gonadotrophins. Fgl2 is an enzyme which converts prothrombin to thrombin and has been shown to be required for normal reproduction in mice (Clark et al., 2004). Interestingly, it has been correlated with spontaneous abortion in rodents (Clark et al., 1999a,b) and recurrent spontaneous abortions in humans (Knackstedt et al., 2001). These abortive effects might be because of immunoregulatory processes taking place during pregnancy and might be explained by the Th1/Th2 paradigm. According to this paradigm, the balance of pro-inflammatory T helper (Th)1 cytokines and anti-inflammatory Th2 cytokines and the local dominance of the Th2 cytokines, which protects the semi-allogenic embryo against both adaptive cell-mediated immune reaction, non-specific innate inflammatory and phagocytic responses, are critical for a successful pregnancy (Wegmann et al., 1993). Th1 cytokines are known to up-regulate Fgl2 and via thrombin leads to activation of polymorphonuclear leukocytes. These, in turn, trigger inflammation and fibrin deposition leading to embryonic death in mice (Clark et al., 1998, 2001). The presence of Fgl2 in the decidua and trophoblasts of aborted but not in control tissue (Clark et al., 1999a) suggests that even normal looking implantation sites treated with urinary gonadotrophins can experience higher gene expression resulting in resorption. The validity of the Th1/Th2 paradigm to solely explain pregnancy has been challenged (Chaouat et al., 2004) but nevertheless, its involvement in embryo loss observed in this study cannot be excluded. On the contrary, an apoptotic effect of Fgl2 might be involved. Fgl2 has also a direct apoptosis-inducing effect on cells, and apoptosis of trophoblast cells is a feature of spontaneous abortion (Knackstedt et al., 2001).
Implantation of the blastocyst involves adhesion, attachment and invasion of the uterine wall. Adhesion of the blastocyst on the uterine lining requires acquisition of attachment competence and functional expression of complementary adhesion-promoting molecules at the cell surface of the blastocyst and uterine lining (Carson et al., 1994). Blastocyst attachment and invasion of the uterine lining are accompanied by extensive degradation and remodelling of the extracellular matrix (ECM). The latter is regulated by a balance of metalloproteinases (MMP) and their inhibitors (Alexander et al., 1996). The cell adhesion molecules, Efemp1 and Emb, are among the genes which were significantly up-regulated by urinary gonadotrophins on ED5. Efemp1 might have affected the implantation process via strong binding with tissue inhibitor of metalloproteinases-3 (Timp-3) (Klenotic et al., 2004). Timp-3 suppresses ECM degradation (Klenotic et al., 2004) and acts as a major MMP inhibitor in the murine uterus by restricting invasion to the implantation site (Leco et al., 1996). Hence, although indirect, it is not preposterous to assume that the urinary gonadotrophin-induced Efemp1 expression might have hampered the implantation process by inhibiting ECM degradation via Timp-3. Emb, on the contrary, is a glycoprotein that promotes integrin-mediated cell substratum adhesion and regulates cell/ECM interaction during development. Its presence in the uterus of non-treated pregnant mice in considerable amounts (Huang et al., 1990) implies a significant role in the implantation process. As one would likely assume that its increased expression would have beneficial consequences, we cannot offer a possible mechanism on how it can negatively affect the implantation process.
The implantation process elicits a pro-inflammatory reaction, and the mechanism whereby the semi-allograft embryo escapes maternal immune response during pregnancy is still unknown (Billington, 1992). Reese et al. (2001) suggested that, during blastocyst implantation, a large number of immune-related genes are absent, and those present have reduced expression and remain inactive with the onset of implantation. The enhanced expression of the immune response genes H2-K1, Ifti3, B2m and Gdf10 is in accordance with this suggestion and might have contributed to the increased resorptions observed in the urinary gonadotrophin-treated animals. It should be noted that the inflammatory reaction might not be elicited solely by the semi-allogenic embryo though. The urinary gonadotrophins are not as pure as the recombinant gonadotrophins, and therefore, the presence of degradation products cannot be ruled out. This suggestion is supported by the finding that degradation products are present in urinary HCG (Wehmann and Nisula, 1980).
We do not know the significance of the alteration in expression of the gene Sprr2f in the present study. Sprr2f is a structural component of the cornified cell envelope of stratified squamous epithelia. Its distinct expression in the uterus (Song et al., 1999; Reese et al., 2001) is difficult to explain because the uterus does not contain stratified squamous epithelia. On the contrary, Sprr2f is not the only differentially expressed gene whose function is in question in the present study. As shown in Table II, there are a number of altered genes whose role in the implantation process remains to be discovered.
The use of CG instead of LH in the urinary group might have also contributed to the differential effects observed in this study. CG is normally used to mimic the effects of pituitary LH because they have a high degree of homology and bind to the same membrane receptors. However, their molecular composition differs (Wehmann and Nisula, 1980; Stokman et al., 1993), indicating that their biological actions may not be identical (Niswender et al., 1985). Data showing that treatment with LH produces better quality embryos with a higher implantation rate than CG in New Zealand rabbits (Peinado et al., 1995; Romeu et al., 1995) and a higher number of implantation sites with greater vascular permeability in mice (Sibug et al., 2005) support this assumption.
The urinary gonadotrophin-treated uteri/embryos evaluated in the present study appeared normal and will most likely have survived until birth and beyond despite the hormonal treatment. However, there was an up-regulation of 30 genes in the urinary gonadotrophin-treated implantation sites despite a similar degree of decidualization, developmental stage and vascular permeability in comparison with the controls. This indicates that even normal looking uteri/embryos were facing the negative effects of urinary gonadotrophins.
In summary, urinary gonadotrophins, but not recombinant gonadotrophins, increased resorption and altered gene expression profiles during the peri-implantation period. The altered expressions of these genes might be some of the underlying factors in increased resorption of embryos in females treated with urinary gonadotrophins. Although caution must be taken in interpreting these results and extrapolating them to the human situation, we believe that these findings warrant careful consideration because COS with urinary gonadotrophins in humans resulted in a dramatic disturbance in the expression of endometrial genes (Horcajadas et al., 2005). Moreover, these results might contribute to the improvement of COS leading to successful pregnancy. Further studies are necessary to determine whether the effects of urinary gonadotrophins on gene expression profiles during the peri-implantation process observed in this study exert long-term consequences in the offspring.
|
Acknowledgements |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
We thank Dr P.Hendriksen (TNO, Zeist) for statistical help and helpful discussion.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Alexander CM, Hansell EJ, Behrendtsen O, Flannery ML, Kishnani NS, Hawkes SP, Werb Z. (1996) Expression and function of matrix metalloproteinases and their inhibitors at the maternal-embryonic boundary during mouse embryo implantation. Development 122:1723–1736.[Abstract]
Bangur CS, Switzer A, Fan L, Marton MJ, Meyer MR, Wang T. (2002) Identification of genes over-expressed in small cell lung carcinoma using suppression subtractive hybridization and cDNA microarray expression analysis. Oncogene 21:3814–3825.[CrossRef][ISI][Medline]
Beaumont HM and Smith AF. (1975) Embryonic mortality during the pre- and post-implantation periods of pregnancy in mature mice after superovulation. J Reprod Fertil 45:437–448.[Abstract]
Billington WD. (1992) The normal fetomaternal immune relationship. Baillieres Clin Obstet Gynaecol 6:417–438.[CrossRef][ISI][Medline]
Carson DD, Rohde LH, Surveyor G. (1994) Cell surface glycoconjugates as modulators of embryo attachment to uterine epithelial cells. Int J Biochem 26:1269–1277.[CrossRef][ISI][Medline]
Chaouat G, Ledee-Bataille N, Dubanchet S, Zourbas S, Sandra O, Martal J. (2004) Reproductive immunology 2003: reassessing the Th1/Th2 paradigm? Immunol Lett 92:207–214.[CrossRef][ISI][Medline]
Clark DA, Chaouat G, Arck PC, Mittruecker HW, Levy GA. (1998) Cytokine-dependent abortion in CBA x DBA/2 mice is mediated by the procoagulant fgl2 prothrombinase [correction of prothombinase]. J Immunol 160:545–549.
Clark DA, Arck PC, Chaouat G. (1999a) Why did your mother reject you? Immunogenetic determinants of the response to environmental selective pressure expressed at the uterine level. Am J Reprod Immunol 41:5–22.
Clark DA, Ding JW, Chaouat G, Coulam CB, August C, Levy GA. (1999b) The emerging role of immunoregulation of fibrinogen-related procoagulant Fgl2 in the success or spontaneous abortion of early pregnancy in mice and humans. Am J Reprod Immunol 42:37–43.
Clark DA, Ding JWYuG, Levy GA, Gorczynski RM. (2001) Fgl2 prothrombinase expression in mouse trophoblast and decidua triggers abortion but may be countered by OX-2. Mol Hum Reprod 7:185–194.
Clark DA, Foerster K, Fung L, He W, Lee L, Mendicino M, Markert UR, Gorczynski RM, Marsden PA, Levy GA. (2004) The fgl2 prothrombinase/fibroleukin gene is required for lipopolysaccharide-triggered abortions and for normal mouse reproduction. Mol Hum Reprod 10:99–108.
Edgar DH, Whalley KM, Mills JA. (1987) Effects of high-dose and multiple-dose gonadotropin stimulation on mouse oocyte quality as assessed by preimplantation development following in vitro fertilization. J In Vitro Fertil Embryo Transf 4:273–276.[CrossRef][ISI][Medline]
Edwards RG. (1994) Implantation, interception and contraception. Hum Reprod 9:985–995.
Elbling L and Colot M. (1985) Abnormal development and transport and increased sister-chromatid exchange in preimplantation embryos following superovulation in mice. Mutat Res 147:189–195.[ISI][Medline]
Elmazar MM, Vogel R, Spielmann H. (1989) Maternal factors influencing development of embryos from mice superovulated with gonadotropins. Reprod Toxicol 3:135–138.[CrossRef][ISI][Medline]
Ertzeid G and Storeng R. (1992) Adverse effects of gonadotrophin treatment on pre- and postimplantation development in mice. J Reprod Fertil 96:649–655.[Abstract]
Ertzeid G and Storeng R. (2001) The impact of ovarian stimulation on implantation and fetal development in mice. Hum Reprod 16:221–225.
Ertzeid G, Storeng R, Lyberg T. (1993) Treatment with gonadotropins impaired implantation and fetal development in mice. J Assist Reprod Genet 10:286–291.[CrossRef][ISI][Medline]
Hamatani T, Daikoku T, Wang H, Matsumoto H, Carter MG, Ko MS, Dey SK. (2004) Global gene expression analysis identifies molecular pathways distinguishing blastocyst dormancy and activation. Proc Natl Acad Sci USA 101:10326–10331.
Horcajadas JA, Riesewijk A, Polman J, van Os R, Pellicer A, Mosselman S, Simon C. (2005) Effect of controlled ovarian hyperstimulation in IVF on endometrial gene expression profiles. Mol Hum Reprod 11:195–205.
Huang RP, Ozawa M, Kadomatsu K, Muramatsu T. (1990) Developmentally regulated expression of embigin, a member of the immunoglobulin superfamily found in embryonal carcinoma cells. Differentiation 45:76–83.[CrossRef][ISI][Medline]
Klenotic PA, Munier FL, Marmorstein LY, Anand-Apte B. (2004) Tissue inhibitor of metalloproteinases-3 (TIMP-3) is a binding partner of epithelial growth factor-containing fibulin-like extracellular matrix protein 1 (EFEMP1). Implications for macular degenerations. J Biol Chem 279:30469–30473.
Knackstedt M, Ding JW, Arck PC, Hertwig K, Coulam CB, August C, Lea R, Dudenhausen JW, Gorczynski RM, Levy GA, et al. (2001) Activation of the novel prothrombinase, fg12, as a basis for the pregnancy complications spontaneous abortion and pre-eclampsia. Am J Reprod Immunol 46:196–210.
Leco KJ, Edwards DR, Schultz GA. (1996) Tissue inhibitor of metalloproteinases-3 is the major metalloproteinase inhibitor in the decidualizing murine uterus. Mol Reprod Dev 45:458–465.[CrossRef][ISI][Medline]
Lee ST, Kim TM, Cho MY, Moon SY, Han JY, Lim JM. (2005) Development of a hamster superovulation program and adverse effects of gonadotropins on microfilament formation during oocyte development. Fertil Steril 83:Suppl 1, 1264–1274.
Lessey BA, Castelbaum AJ, Buck CA, Lei Y, Yowell CW, Sun J. (1994) Further characterization of endometrial integrins during the menstrual cycle and in pregnancy. Fertil Steril 62:497–506.[ISI][Medline]
Luckett DC and Mukherjee AB. (1986) Embryonic characteristics in superovulated mouse strains. Comparative analyses of the incidence of chromosomal aberrations, morphological malformations, and mortality of embryos from two strains of superovulated mice. J Hered 77:39–42.
Maudlin I and Fraser LR. (1977) The effect of PMSG dose on the incidence of chromosomal anomalies in mouse embryos fertilized in vitro. J Reprod Fertil 50:275–280.[Abstract]
Niswender GD, Roess DA, Sawyer HR, Silvia WJ, Barisas BG. (1985) Differences in the lateral mobility of receptors for luteinizing hormone (LH) in the luteal cell plasma membrane when occupied by ovine LH versus human chorionic gonadotropin. Endocrinology 116:164–169.[Abstract]
Peinado JA, Molina I, Pla M, Tresguerres JA, Romeu A. (1995) Recombinant-human luteinizing hormone (r-hLH) as ovulatory stimulus in superovulated does. J Assist Reprod Genet 12:61–64.[CrossRef][ISI][Medline]
Psychoyos A. (1973) Hormonal control of ovoimplantation. Vitam Horm 31:201–256.[Medline]
Quackenbush J. (2002) Microarray data normalization and transformation. Nat Genet 32:Suppl, 496–501.
Rajeevan MS, Ranamukhaarachchi DG, Vernon SD, Unger ER. (2001) Use of real-time quantitative PCR to validate the results of cDNA array and differential display PCR technologies. Methods 25:443–451.[CrossRef][ISI][Medline]
Reese J, Das SK, Paria BC, Lim H, Song H, Matsumoto H, Knudtson KL, DuBois RN, Dey SK. (2001) Global gene expression analysis to identify molecular markers of uterine receptivity and embryo implantation. J Biol Chem 276:44137–44145.
Romeu A, Molina I, Tresguerres JA, Pla M, Peinado JA. (1995) Effect of recombinant human luteinizing hormone versus human chorionic gonadotrophin: effects on ovulation, embryo quality and transport, steroid balance and implantation in rabbits. Hum Reprod 10:1290–1296.
Sibug RM, de Koning J, Tijssen AM, de Ruiter MC, De Kloet ER, Helmerhorst FM. (2005) Urinary gonadotrophins but not recombinant gonadotrophins reduce expression of VEGF120 and its receptors flt-1 and flk-1 in the mouse uterus during the peri-implantation period. Hum Reprod 20:649–656.
Sibug RM, Helmerhorst FM, Tijssen AM, De Kloet ER, de Koning J. (2002) Gonadotrophin stimulation reduces VEGF (120) expression in the mouse uterus during the peri-implantation period. Hum Reprod 17:1643–1648.
Simpson JL and Liebaers I. (1996) Assessing congenital anomalies after preimplantation genetic diagnosis. J Assist Reprod Genet 13:170–176.[CrossRef][ISI][Medline]
Song HJ, Poy G, Darwiche N, Lichti U, Kuroki T, Steinert PM, Kartasova T. (1999) Mouse Sprr2 genes: a clustered family of genes showing differential expression in epithelial tissues. Genomics 55:28–42.[CrossRef][ISI][Medline]
Spielmann H and Vogel R. (1993) Genotoxic and embryotoxic effects of gonadotropin hyperstimulated ovulation on murine oocytes, preimplantation embryos and term fetuses. Ann Ist Super Sanita 29:35–39.[Medline]
Stokman PG, de Leeuw R, van den Wijngaard HA, Kloosterboer HJ, Vemer HM, Sanders AL. (1993) Human chorionic gonadotropin in commercial human menopausal gonadotropin preparations. Fertil Steril 60:175–178.[ISI][Medline]
Thomas K, Thomson AJ, Sephton V, Cowan C, Wood S, Vince G, Kingsland CR, Lewis-Jones DI. (2002) The effect of gonadotrophic stimulation on integrin expression in the endometrium. Hum Reprod 17:63–68.
Tusher VG, Tibshirani R, Chu G. (2001) Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA 98:5116–5121.
Van der Auwera I and D’Hooghe T. (2001) Superovulation of female mice delays embryonic and fetal development. Hum Reprod 16:1237–1243.
Wang QT, Piotrowska K, Ciemerych MA, Milenkovic L, Scott MP, Davis RW, Zernicka-Goetz M. (2004) A genome-wide study of gene activity reveals developmental signaling pathways in the preimplantation mouse embryo. Dev Cell 6:133–144.[CrossRef][ISI][Medline]
Wang YA, Sullivan EA, Black D, Dean J, Bryant J, Chapman M. (2005) Preterm birth and low birth weight after assisted reproductive technology-related pregnancy in Australia between 1996 and 2000. Fertil Steril 83:1650–1658.[CrossRef][ISI][Medline]
Wegmann TG, Lin H, Guilbert L, Mosmann TR. (1993) Bidirectional cytokine interactions in the maternal-fetal relationship: is successful pregnancy a TH2 phenomenon? Immunol Today 14:353–356.[CrossRef][ISI][Medline]
Wehmann RE and Nisula BC. (1980) Characterization of a discrete degradation product of the human chorionic gonadotropin beta-subunit in humans. J Clin Endocrinol Metab 51:101–105.[ISI][Medline]
Wennerholm WB. (2000) Cryopreservation of embryos and oocytes: obstetric outcome and health in children. Hum Reprod 15:Suppl 5, 18–25.
Yoshioka K, Matsuda F, Takakura K, Noda Y, Imakawa K, Sakai S. (2000) Determination of genes involved in the process of implantation: application of GeneChip to scan 6500 genes. Biochem Biophys Res Commun 272:531–538.[CrossRef][ISI][Medline]
Submitted on February 14, 2006; resubmitted on May 1, 2006; resubmitted on July 10, 2006; resubmitted on July 24, 2006; accepted on July 28, 2006.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||