Embryonic soluble HLA-G as a marker of developmental potential in embryos

I. Noci1,5, B. Fuzzi1, R. Rizzo2, L. Melchiorri2, L. Criscuoli1, S. Dabizzi1, R. Biagiotti1, S. Pellegrini1, A. Menicucci3 and O.R. Baricordi2,4

1 Department of Gynaecology, Perinatal Medicine and Human Reproduction, University of Firenze, 3 Immunohematology and Transfusion Medicine, Careggi Hospital, 50134 Firenze, 2 Section of Medical Genetics, Department of Diagnostic and Experimental Medicine, and 4 Biotechnology Center, University of Ferrara, 44100 Ferrara, Italy

5 To whom correspondence should be addressed. Email: ivo.noci{at}unifi.it


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: In human reproduction, embryo implantation is complex and poorly understood. At present, no single markers are used in routine treatment to assay biochemical functions of the human embryo. Soluble human leukocyte antigen-G (sHLA-G) could be considered a possible marker of embryo developmental potential. It is localized primarily on the extravillous trophoblast, making this antigen a potential mediator of immune interaction at the maternal–fetal interface during gestation. METHODS: Soluble-HLA-G levels were evaluated by an enzyme-linked immunosorbent assay (ELISA) employing monoclonal antibody MEM-G9. It was evaluated in 318 media of single embryo cultures. We correlated the presence of sHLA-G with embryo morphology and the pregnancy obtained in that treatment cycle. RESULTS: No correlation was found between embryo morphology and sHLA-G levels. Pregnancy was observed only when the medium of at least one transferred embryo contained sHLA-G. In 26 out of 66 patients, none of the obtained embryos showed any detectable sHLA-G molecules and no pregnancy occurred. CONCLUSIONS: From our results, we propose sHLA-G as a potential marker of embryo development: the sHLA-G ELISA can be a useful biochemical assay in addition to embryo morphology in embryo selection for transfer in IVF treatment if there are other embryos with the same morphology.

Key words: embryo culture/embryo developmental potential/soluble HLA-G/pregnancy


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In human reproduction techniques, embryo implantation is complex and poorly understood. A significant number of embryos (≥70%) fail to implant, and only 14% of transferred embryos will give rise to a full-term infant. At present, there is another aspect to be taken into account: embryo selection for transfer is based only on morphological and cleavage criteria (Rijnders and Jansen, 1998Go; Desai et al., 2000Go; Gardner et al., 2000Go), but embryo viability is not strictly correlated with embryo quality (Ziebe et al., 1997Go; Balaban et al., 2001Go).

Both earlier and more recent papers have reported the importance of several molecules in the regulation of preimplantation embryo development and its metabolism (O'Neill et al., 1987Go; Gardner and Leese, 1993Go; Gardner et al., 2001Go; Hansis and Edwards, 2003Go). Nevertheless, not one of these metabolites has found routine clinical application in embryo selection for transfer, to our knowledge; only recently, Roudebush et al. (2001Go, 2002Go) proposed platelet-activating factor (PAF) as an indicator of embryo viability. This substance may be used as a marker for embryo selection because PAF production by human embryos seems to be related to pregnancy outcome. At present, this assay has not yet received routine application.

In this study, we investigate another possible marker of embryo developmental potential, soluble human leukocyte antigen-G (sHLA-G). Jurisicova et al., (1996)Go were the first to report findings on this marker: this study produced data on total mRNA and protein in human embryos. Recently, our study group evaluated the presence of sHLA-G molecules in supernatant cultures of early human embryos obtained by IVF (Menicucci et al., 1999Go), and our subsequent investigation showed that sHLA-G is a mandatory, but not sufficient, prerequisite for the development of pregnancy. In fact, in 101 patients studied, clinical pregnancy was obtained only if sHLA-G molecules were detected in the supernatant of growing embryos on the transfer day (Fuzzi et al., 2002Go).

The major histocompatibility complex (MHC) genes, or recessive genes linked to MHC, appear to influence growth, development and reproduction, as well as susceptibility to gestational trophoblastic tumours and recurrent spontaneous miscarriages in humans (Ho et al., 1991Go). In addition to genes encoding classical class I HLAs (HLA-A, B and C), the MHC gene family also contains a set of non-classical class I genes. One member of this gene family is HLA-G. It differs from the other HLA class I genes in terms of three specific properties: (i) an alternative splicing of its primary transcript (mRNA) gives rise to four membrane-bound (HLA-G1, G2, G3 and G4) and three soluble (HLA-G5, G6 and G7) isoforms (Ishitani and Geraghty, 1992Go; Fujii et al., 1994Go; Kirszenbaum, et al., 1994Go); (ii) a limited polymorphism (Morales et al., 1993Go; Le Discorde et al., 2002Go); and (iii) a restricted pattern of expression in adult and embryonic human tissue. In the placenta, HLA-G is localized primarily on the extravillous (both invasive and chorionic membrane) trophoblast. Recent published data suggest that the expression of HLA-G protects cells against natural killer (NK) cell lysis (Rajagopalan and Long, 1999Go; Marchal-Bras-Goncalves et al., 2001Go; Riteau et al., 2001Go). Moreover, in vitro data show evidence that HLA-G downregulates the production of Th1 cytokines, interferon-{gamma} (IFN-{gamma}) and tumour necrosis factor-{alpha} (TNF-{alpha}), from decidual mononuclear cells and peripheral blood mononuclear cells; furthermore, HLA-G promotes the production of the Th2 cytokine interleukin (IL)-4 by peripheral blood mononuclear cells (Kanai et al., 2001Go). It is well known that Th1-type cytokines promote allograft rejection and that Th2-type cytokines inhibit Th1 responses thereby increasing the probabilities of fetal survival (Piccinni et al., 2001Go). Due to the biological effects of HLA-G on both NK cell activity and Th1/Th2 cytokine balance, we suggest that HLA-G, produced by trophoblast cells and present at the feto-maternal interface, plays an important role in the embryo implantation process and in the phenomenon of fetal allograft tolerance.

Moreover, the reported expression of HLA-G molecules in some solid tumours (Paul et al., 1998Go; Wiendl et al., 2002Go), virally infected cells (Onno et al., 2000Go; Lozano et al., 2002), transplanted organs (Lila et al., 2002Go; Creput et al., 2003Go), cutaneous inflammatory diseases (Khosrotehrani et al., 2001Go) and recently in the cerebral spinal fluid of multiple sclerosis patients (Fainardi et al., 2003Go) has enlarged the tissue distribution for both membrane and soluble HLA-G antigens (sHLA-G1/G5), suggesting a generalized tolerogenic function for HLA-G molecules.

In our previous investigation (Fuzzi et al., 2002Go), we employed the conventional embryo culture method (each well containing 1–4 oocytes/embryos), and were thus unable to evaluate the sHLA-G production of each single embryo. For this reason, we decided to investigate the presence of sHLA-G molecules in supernatants of singly cultured embryos, and we also evaluated the presence of sHLA-G in the culture medium of each transferred embryo. We correlated sHLA-G presence with embryo morphology and the pregnancy obtained in that treatment cycle. Furthermore, a small group of these patients undertook a second cycle, and we evaluated the results of sHLA-G assay in these cases separately. Our results induce us to propose sHLA-G as a potential marker of embryo viability in IVF programmes.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patients and IVF
The study was performed on a group of consecutively infertile patients who had undertaken IVF or ICSI at the Department of Gynecology, Perinatology and Reproductive Medicine at the University of Florence (September 2001 and July 2002). Only women who had at least one embryo available for embryo transfer were included; in addition, only one cycle per couple was considered. Following these criteria, 66 couples were recruited for the study. Clinical indications for IVF or ICSI were, respectively, tubal factor (40.7 and 10.2%), endometriosis (11.2 and 2.6%), unexplained infertility (33.3 and 5.1%), male factor (7.4 and 66.7%) and mixed causes (7.4 and 15.4%). This study was approved by the local ethics committee.

Some of the patients studied underwent two repeated cycles, and we evaluated the results of HLA-G levels in these cases separately.

All patients received a similar stimulation regimen. Ovarian stimulation was induced using a recombinant FSH (Gonal F Serono Pharma, Bari, Italy; or Puregon, Organon Italia, Milan, Italy) protocol starting on the the third to the fifth day of the cycle after pituitary downregulation with 0.1 mg/day triptorelin (Decapeptyl 0.1, Ipsen, Milan, Italy) for 6–8 days prior to the expected period. Complete pituitary desensitization was confirmed by both low plasma estradiol <50 pg/ml and ultrasonic examination to exclude the presence of ovarian cysts and verify that endometrial thickness was <5 mm. HCG (10 000 IU) (Profasi HP, Serono Pharma) was administered when at least two follicles exceeded 17 mm in diameter. Oocyte recovery was performed by transvaginal ultrasound guidance and local analgesia 34–36 h after HCG administration. The luteal phase was supported with natural progesterone in oil, 50 mg/day (Prontogest 100 mg, Amsa, Florence, Italy) from the day of oocyte recovery. The embryo transfer was performed on the third day after oocyte collection. A single serum {beta}-HCG measurement was performed 12 days after embryo transfer. A clinical pregnancy was defined when an intra-uterine gestational sac with fetal heartbeat was detected via transvaginal ultrasound examination.

Collected oocytes were cultured in a 4-well multidish (Nunc, Roskilde, Denmark) with 600 µl of culture medium (IVF or M3, Medi Cult, Denmark) with the addition of serum substitute supplement (SSS, Irvine Scientific, Santa Ana, CA). Each well contained only one oocyte. IVF or ICSI techniques were used for insemination. Oocyte fertilization was observed 16–18 h after insemination under an inverted microscope (TM/300, Nikon, Japan), the fertilization rate was calculated and the zygotes were transferred into fresh medium. After 48 h in culture, embryo morphology was evaluated and the embryos were transferred into fresh medium; finally, after 72 h in culture, the embryos were again evaluated, they were scored and up to four of them were chosen for transfer according to morphological criteria. The embryos were evaluated for the number of blastomeres, regularity or irregularity of blastomers and fragmentation percentage. Each of these parameters was scored and the sum of them defined embryo quality as described in Table I.


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Table I. Morphological evaluation criteria employed for embryo quality grading (for explanation see the text)

 
On the day of transfer (72 h), 550 µl of supernatants were collected from each human embryo culture and, in some cases, they were also collected on the day before (48 h); samples were stored at –20°C until they were tested for the presence of sHLA-G.

HLA-G determination
Soluble-HLA-G levels in culture supernatants were assayed as previously reported (Fournel et al., 2000Go). Briefly, 96-microwell plates Nunc-Immuno Plate PolySorp were coated with monoclonal antibody MEM-G9 (Exbio, Praha, Czech Republic) that recognizes the HLA-G molecule in {beta}2-microglobulin-associated form (soluble HLA-G1 and HLA-G5 isoform) at a concentration of 20 µg/ml in 0.1 mol/l carbonate buffer pH 9.5 for 1 h at 37°C and then overnight at 4°C. After three washes with phosphate-buffered saline (PBS) containing 0.05% Tween-20 (PBS-Tween), plates were saturated with 100 µl of PBS containing 4% bovine serum albumin (BSA) overnight at 4°C. Undiluted supernatant samples were added to each well (50 µl) in triplicate. After incubation for 2 h at 37°C, plates were washed three times with PBS-Tween, and incubated for 1 h at 37°C with 50 µl of biotinylated monoclonal antibody W6/32 that recognizes a framework determinant expressed on {beta}2-microglobulin-associated HLA class I heavy chain (ATCC, Rockville, MD). Biotinylation of W6/32 monoclonal antibody was obtained using the EZ-Link Sulfo-NHS-LC Biotinylation Kit (Pierce, Rockford, IL). After five washes with PBS-Tween, 100 µl of extravidin–peroxidase (Sigma-Aldrich, Milano, Italy) were added and incubated for 15 min at 37°C. Then after five washes with PBS-Tween, 100 µl of o-phenyldiamine peroxidase substrate (Sigma-Aldrich) were added to each well and incubated for 15 min at room temperature. The concentration of sHLA-G was estimated by absorbance at 405 nm on an enzyme-linked immunosorbent assay (ELISA) Microplate Reader 400 (Packard, Meridien, CT). Supernatants from the HLA-G-transfected LCL 721.221, purified by affinity chromatography by using the anti-HLA class I monoclonal antibody W6/32, were utilized for the generation of standard calibration curves. The limit of sensitivity was 1 ng/ml.

Statistical analysis
The data screened for normality were analysed by independent Student's t-test for assessing the normality assumption and, when data indicated a markedly not normal distribution, a non-parametric test was used (Mann–Whitney U-test) (Tables II and VIII).


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Table II. Characteristics of the study population

 

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Table VIII. sHLA-G values in seven patients with two repeated cycles of IVF

 
A correlative study between sHLA-G levels and embryo morphology was performed using Yates corrected {chi}2 test (Table IV).


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Table IV. Correlative study results between embryo quality and the presence of sHLA-G in conditioned embryo culture medium on day 3

 
All statistical calculations were carried out using the software package SPSS for Windows (Version 6.0): P-values <0.05 were considered significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We conducted preliminary tests for sHLA-G antigens on a group of 20 supernatants from cultures with spermatozoa or oocytes alone. The gametes were kept in culture medium for 48–72 h, following the same embryonic culture procedure. No positive results for the presence of sHLA-G molecules were observed.

Table II shows the characteristics of the study population. No differences were observed between IVF and ICSI patients in any of the clinical parameters, with respect to basal constitution, stimulation regimen, ovarian response, oocyte–embryo patterns and sHLA-G secretion (Table III). Thus, we have considered IVF and ICSI patients as a homogenous group for statistical evaluation.


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Table III. sHLA-G secretion of the study population at 72 h in 318 samples evaluated

 
Figure 1 indicates when the samples of embryo culture medium were collected and the number of them assayed for sHLA-G levels (at 48 or 72 h): when sHLA-G was found, samples were grouped as sHLA-G positive; where sHLA-G was not found, they were grouped as sHLA-G negative. Sixty-six consecutive IVF/ICSI patients had one oocyte/embryo in each culture well, and were included in this study with their 318 embryos.



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Figure 1. Flow chart of the 318 samples of embryo culture medium assayed for sHLA-G.(sHLA-G (ng/ml) - NA: samples not available for assay).

 
A correlative study between sHLA-G levels present in conditioned embryo culture medium on day 3 and embryo morphology was performed. Embryos were scored and grouped into four classes, A, B, C or D. Table IV shows that embryo morphology does not correlate with the presence of sHLA-G in embryo culture medium, either in the total number of embryos obtained or in the number of transferred embryos. Similar results were obtained when the presence of sHLA-G was correlated with the number of blastomeres, their regular or irregular size, and the embryo fragmentation percentage, with reference to both the total number of obtained and transferred embryos (data not shown).

The 66 patients were grouped at 72 h. The sHLA-G of transferred embryos was not detectable in 26 out of 66 patients (39.4%) and no pregnancy occurred in this group. Instead, in 40 out of 66 patients (60.6%), sHLA-G was found (median 2.1 ng/ml; range 1.05–37 ng/ml; mean 4.17±6.78 ng/ml) in at least one transferred embryo, and nine clinical pregnancies occurred.

In Tables V and VI, the sHLA-G levels detected in culture medium of transferred and non-transferred embryos in patients with or without pregnancy are reported. In four cases (patients number 17, 27, 48 and 49), a large number of good quality embryos (type A or B) were available for embryo transfer and the choice of the embryos for transfer occurred randomly: in all four cases, no pregnancy occurred. The following sHLA-G assay showed that we had randomly transferred all embryos not producing sHLA-G, whereas the embryos producing sHLA-G (range 2–16 ng/ml) had not been used.


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Table V. sHLA-G values (ng/ml) detected in culture medium of transferred and non-transferred embryos in patients without pregnancies

 

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Table VI. sHLA-G values (ng/ml) detected in culture medium of transferred and non-transferred embryos in patients with pregnancies

 
Further evaluation was performed dividing the 66 patients into pregnant and not pregnant so we could investigate whether there were any differences in sHLA-G secretion levels. No differences were observed in any of the clinical parameters regarding basal constitution, stimulation regimen, ovarian response and oocyte–embryo patterns between these two groups of women (Table VII). Conversely, the statistical analysis (Mann–Whitney U-test) of the secretion of sHLA-G showed a slightly significant difference (P=0.045) between pregnant (median 2.07 ng/ml; range 0–22 ng/ml; mean 5.55±7.33 ng/ml) and non-pregnant patients (median 0 ng/ml; range 0–37 ng/ml; mean 2.45±5.54 ng/ml).


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Table VII. Clinical parameters evaluation in pregnant and non-pregnant women

 
Table VIII reports the results obtained in seven of the 66 patients who undertook a second treatment cycle. None of these patients showed the presence of sHLA-G in any of their embryo culture media in the first cycle, but in the second cycle some of them produced detectable sHLA-G levels and some of the positive embryos were transferred. It is worthy of note that four patients became pregnant.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Here we have presented our research on the individual production of sHLA-G by human embryos in IVF cultures, and we have evaluated the possibility that sHLA-G ELISA of IVF culture medium may be proposed as a marker of embryo developmental potential.

The first observation regards the time required to produce sHLA-G expression in embryo culture medium. On day 2, 8.9% of the supernatants showed sHLA-G secretion and 91.1% did not. Conversely, on day 3, 36.2% of the embryos produced a detectable amount of sHLA-G (range 1.05–37 ng/ml). We noticed that supernatants from cultures with spermatozoa or oocytes only did not show any detectable sHLA-G. Moreover, the findings of Tesarik et al. (1986)Go and Braude et al. (1988)Go suggest that the first two cell cycles of human embryogenesis are regulated at a post-transcriptional level, utilizing maternally inherited information, and that activation of the embryonic genome takes place between the 4- and 8-cell stages (~70 h after oocyte insemination) and is essential if both synthesis of proteins and further cleavage are to occur.

On the basis of these assumptions, we suggest that on day 2 of embryo culture, sHLA-G concentrations could be attributed to the activation of oocyte DNA after fertilization, but we also observed that this phenomenon did not occur frequently. In contrast, on day 3, about four out of 10 embryos showed detectable sHLA-G levels in their culture medium, probably due to activation of the embryonic genome. At present, our data for sHLA-G expression at the blastocyst stage in embryo culture medium are not available. In this regard, Jurisicova et al. (1996)Go reported that reverse transcription (RT)–PCR detected HLA-G heavy chain mRNA in 40% of 148 blastocysts tested. Conversely, no HLA-G mRNA was found by Hiby et al. (1999)Go, but they had studied only 11 embryos ranging from the 2-cell stage to blastocysts. Our data are consistent with the findings of Jurisicova et al. (1996)Go, and both sets of data suggest that about four out of 10 human embryos showed HLA-G from day 3 onwards. Furthermore, another study by Jurisicova et al. (1999)Go reported that in blastocysts HLA-G mRNA was higher (90%) compared with what had been reported previously, but a different RT–PCR approach was used and the embryos were pre-selected according to the expression of elongation factor EF-1{alpha}, which eliminates low transcriptional activity.

Experimental data reported that sHLA-G antigens mediate inhibitory effects on CD8+ lymphocytes and NK cells (Le Gal et al., 1999Go; Navarro et al., 1999Go); all these data suggest a pivotal role for sHLA-G in the mechanism of maternal tolerance against trophoblast cells (Hunt et al., 2000Go); therefore, considering embryo implantation as a tissue transplant, we can infer that only embryos expressing sHLA-G activated this expedient to escape rejection. Our data show that only four out of 10 human embryos from IVF cycles expressing sHLA-G permitted fetal allograft tolerance.

It is beyond the scope of the present report to study the modality of embryo implantation. Data in the available literature show the presence of a complex network of hormones, cytokines and cells in the implantation process, and they focus on the importance of the production of leukaemia-inhibitory factor (LIF), IL-4, IL-10 and macrophage colony-stimulating factor (M-CSF) by the decidual cells (Delage et al., 1995Go; Li et al., 1998Go; Piccinni, 2002Go). Many researchers have investigated these aspects, and their results, in fertile women and women with recurrent miscarriage, are contradictory at best (Laird et al., 2003Go). On the contrary, to our knowledge, no data are available in the current literature on the permissive role of some of the cytokines in relation to the phenomenon of fetal allograft tolerance at the time of implantation.

The second aspect on which we focused was to investigate if sHLA-G produced by embryos in culture correlates with embryo morphology. Our results (Table IV) do not show any statistical correlation between embryo morphology and sHLA-G levels detected in embryo culture supernatants. Therefore, sHLA-G production by the embryo and embryo morphology are to be considered two different parameters. In fact, as shown in Table IV, sHLA-G was produced (59.1 and 68.9%) or not produced (56.7 and 70%) in good quality obtained or transferred embryos (score A), respectively.

The third question can be stated as follows: is there any correlation between sHLA-G embryo production and clinical pregnancy obtained in that particular treatment cycle? In our cases, sHLA-G was present in at least one of the transferred embryos in the pregnant women (Table VI), whereas pregnancy never occurred when the embryos transferred did not reveal the presence of sHLA-G by ELISA (n=26) (Table V). Summing our present (n=26) and previous (n=26; Fuzzi et al., 2002Go) data, 52 women were sHLA-G negative and no pregnancy occurred. Therefore, the absence of sHLA-G in supernatants of IVF embryos has a completely negative predictive value (100%) in obtaining a clinical pregnancy.

On the other hand, only nine out of 40 patients obtained a clinical pregnancy with transferred embryos producing sHLA-G (Tables V and VI), therefore the sHLA-G assay has a low positive predictive value. In this regard, it is necessary to take into account at least three other variables if clinical pregnancy is to be reached. (i) Chromosomal embryo assessment: pre-implantation genetic diagnosis programs (PGD) evidenced that up to 70% of embryos were affected by chromosomal abnormalities (Pehlivan et al., 2003Go; Shenfield et al., 2003Go); the low rate of newborns affected by chromosomal abnormalities showed that these conceptuses had not been implanted. Thus many sHLA-G-positive embryos do not evolve into pregnancy because they may have chromosomal abnormalities. Further multicentric studies will clarify this aspect. (ii) Endometrial receptivity: the literature has evidenced that endometrium morphology is significantly modified in assisted reproduction treatment cycles, and this could imply implantation failure (Noci et al.,1997Go; Lass et al., 1998Go). It is possible that a percentage of sHLA-G-positive cases which do not result in pregnancy may be connected to altered endometrial receptivity. In our study population, only the morphological assessment of the endometrium (by ultrasound investigation) was evaluated and did not reveal any differences (data not shown). (iii) In an IVF programme, the embryo transfer technique may also be one of the most significant variables in achieving pregnancy. Therefore, it is not surprising in our opinion that in some cases a clinical pregnancy did not occur when sHLA-G-positive embryos were transferred.

This group of patients produced a low clinical pregnancy rate probably due to several different negative factors such as previously failed IVF; others were low responders and some were 38 years of age or older (Table II).

Moreover, as previously reported, the mean value of sHLA-G levels in culture medium is statistically different, although as a borderline factor, between pregnant (5.55 ng/ml) and not pregnant women (2.45 ng/ml), with the non-parametric test (Mann–Whitney); on the other hand, pregnant and non-pregnant women did not differ regarding basal constitution characteristics, stimulation regimen, ovarian response and oocyte–embryo patterns (Table VII). It is interesting that six out of nine pregnancies occurred in patients who had had no previous failed IVF cycles.

These findings suggest the importance of the presence of sHLA-G for fetal allograft tolerance. However, receiver-operator characteristic (ROC) curve analysis was not able to evidence a cut-off value for sHLA-G (data not shown).

Another aspect which must be discussed regards patients presenting all sHLA-G-negative embryos, and this was the situation for about one-third of the patients in our study population (26 out of 66 patients). In none of these cases did pregnancy occur. At this point, our question is whether negative sHLA-G ‘couples’ exist or not: does the same couple always produce sHLA-G-negative embryos, without any chance of pregnancy, or is it possible that a subsequent cycle produces a different outcome, i.e. at least some sHLA-G-positive embryos? In this regard, we have investigated the presence of sHLA-G in supernatants of single embryo cultures from couples admitted to a second fertilization procedure, after a first cycle that showed the complete absence of detectable sHLA-G (none out of 31) in all obtained embryos. In the second IVF cycle, we found some positive sHLA-G embryo supernatants (15 out of 40) and four pregnancies occurred, one twin and the other three single (Table VIII). The high pregnancy rate which occurred in these patients with repeated cycles is encouraging, but this group was too small for statistical evaluation. These results suggest the previous lack of antigen modulation, which is independent of germinal defects, but which could also be ascribed to the occurrence of erroneous fertilization processes.

Finally, we have reported that in four cases (patients 17, 27, 48 and 49), 6–12 good quality embryos were available for embryo transfer, and the choice of embryos for transfer occurred randomly (see Table V). The sHLA-G assay performed later showed that we had randomly transferred only sHLA-G-negative embryos, whereas the positive sHLA-G embryos had not been used. In these four cases, no pregnancy occurred, but might have been obtained if a different embryo selection had taken place and if it had been based on both morphological and biochemical (sHLA-G) criteria. The findings of the study presented by Sher et al. (2004)Go are consistent with our group's results. We should note that this group used a different antibody, which can explain some of the differences in the results. MEM-G1 is accepted in immunohistology and western blotting applications, but it is not indicated for ELISA. For this reason, we used MEM-G9.

In conclusion, the present data confirm our previous results (Fuzzi et al., 2002Go) regarding the presence of sHLA-G in embryo culture media even when the embryos are cultured one per well, as reported herein. That pregnancy occurs only if sHLA-G reaches a detectable level in embryo culture media is also confirmed. The above-mentioned assay may be used together with embryo morphology in selecting embryos for transfer in IVF treatment where a number of embryos with the same morphology are present. As laboratory embryologists and infertility physicians, we are interested in receiving information about the viability of our embryos and about molecules which are connected with pregnancy. Moreover, from our preliminary data on repeated cycles, we can infer that a couple can produce sHLA-G-secreting embryo(s) in different cycles. This observation suggests that the parental genome arrangement plays an important role in the different probabilities of obtaining embryos which secrete sHLA-G; this hypothesis is supported by Rebmann et al. (2001)Go and Hviid et al. (2004aGo,bGo) in their studies.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We would like to thank our nurses, Candida De Carlo and Antonella Grupelli, for their invaluable collaboration, and Judith Siegel for linguistic help.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on October 6, 2003; resubmitted on July 26, 2004; accepted on September 29, 2004.