To the Editor:
We have read with interest the paper by Kotze et al. (1), reporting the retrospective analysis of 2,040 patients for the expression of soluble HLA-G (sHLA-G) by day-2 embryos after intracytoplasmic injection. The data represent further confirmation of the role of sHLA-G molecule quantification in embryo culture supernatants as a marker for embryo selection (2). In pregnancy several tolerance mechanisms have been demonstrated to counteract the maternal immune response. Among these, the expression of HLA-G by invasive cytotrophoblasts has been shown to play a fundamental role in creating a tolerogenic condition at the feto-maternal interface (2).
By now, more than 15,000 embryo culture supernatants have been evaluated for sHLA-G expression, with a positive correlation with embryo implantation rate and pregnancy outcome. However, further research is needed in HLA-G investigation in assisted reproductive technology (ART). Three aspects should be taken into consideration: 1) recognition of a common sHLA-G detection protocol; 2) necessity to identify a standardized range for positivity, as reported by Kotze et al. (1); 3) comprehension of the factors involved in the differential expression of sHLA-G between equal stage embryos originating from the same woman (3). Theoretically, HLA-G expression in embryos may differ due to HLA-G polymorphisms that modify HLA-G expression (4). For this, a simultaneous quantification of sHLA-G levels in culture supernatants and the analysis of maternal HLA-G polymorphisms could help in the definition of correct range of positivity for embryo grading. Meanwhile, sHLA-G molecules have been identified in follicular fluids (FFs), where granulosa cells and monocyte/macrophage populations have indicated to be mainly responsible for sHLA-G presence (5). A significant relationship was observed between sHLA-G presence in FFs and sHLA-G levels in culture supernatants of the corresponding fertilized oocyte. Furthermore, the secretion of HLA-G antigens was associated with oocyte maturation stage. Overall, these data suggest that the identification of sHLA-G in FFs could mark oocyte maturation and embryos with an improved implantation rate.
We believe that all these data support a concrete investment in the use of sHLA-G molecule analysis in ART.
Roberta Rizzo, Ph.D., Olavio R. Baricordi, Ph.D.
Department of Medical Sciences, Section of Microbiology and Medical Genetics, University of Ferrara, Ferrara, Italy
1. Kotze D, Kruger TF, Lombrd C, Padayachee T, Keskintepe L, Sher G. The effect of the biochemical marker soluble human leukocyte antigen G on pregnancy outcome in assisted reproductive technology-a multicenter study. Fertil Steril 2013, In Press. DOI:pii: S0015-0282(13)02763-5.10.1016/j.fertnstert.2013.07.1977
2. Rizzo R, Vercammen M, van de Velde H, Horn PA, Rebmann V. The importance of HLA-G expression in embryos, trophoblast cells, and embryonic stem cells. Cell Mol Life Sci 2011;68:341-52.
3. Criscuoli L, Rizzo R, Fuzzi B, Melchiorri L, Menicucci A, Cozzi C, et al. Lack of histocompatibility leukocyte antigen-G expression in early embryos is not related to germinal defects or impairment of interleukin-10 production by embryos. Gynecol Endocrinol 2005;20:264-9.
4. Rizzo R, Hviid TV, Stignani M, Balboni A, Grappa MT, Melchiorri L, et al. The HLA-G genotype is associated with IL-10 levels in activated PBMCs. Immunogenetics 2005;57:172-81.
5. Rizzo R, Fuzzi B, Stignani M, Criscuoli L, Melchiorri L, Dabizzi S, et al. Soluble HLA-G molecules in follicular fluid: a tool for oocyte selection in IVF? J Reprod Immunol 2007;74:133-42.
Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2013.10.013
The authors respond:
We agree with the statements in the letter. We would like to use this opportunity to highlight a few problem areas regarding the sHLA-G assay. It has been postulated by Haviid et al. (1) that sHLA-G plays a pivotal role during the implantation process. The presence of sHLA-G in spent medium of individually cultured embryos and its influence on assisted reproductive technology outcomes have been reported by Sher et al. (2) and in a meta-analysis by Vercammen et al. in 2008 (3). The results of a prospective randomized trial (4) followed by a multicenter (5) approach confirmed our previous findings.
Lately the quest to find the best way to identify an embryo with the highest potential to develop into a live baby has been an issue of active debate. Notwithstanding the patient population that undoubtedly needs preimplantation genetic screening/preimplantation genetic diagnosis, there is a desperate need to establish criteria for a noninvasive method to be implemented in order to achieve the goal of identifying such embryo/s for transfer. We have shown that embryos transferred on day 3 (with good morphology) that have a positive expression of sHLA-G had superior outcome when compared with embryos that were transferred based on morphology only. That said, we further suggest that transferring blastocysts with a positive sHLA-G expression might be even more superior.
We have highlighted shortfalls in the current publication. To apply this assay effectively, all embryos have to be cultured and tracked individually in order to specifically identify the “correct” embryo/s for transfer. A positive aspect of applying this assay is the potential to transfer fewer (even a single blastocyst) embryos without compromising pregnancy outcome and simultaneously to reduce the risk of multiple pregnancies.
Due to the lack of standardization, it is our belief that a standardized sHLA-G assay with a specifically defined range (for positive sHLA-G expression) and standard “measuring units” should be established through international corroboration. This relatively inexpensive noninvasive approach could be a paradigm shift for identifying the most competent embryo/s for transfer and to achieve the above-mentioned goal.
Dirk Kotze, Ph.D.
Jones Institute, Norfolk, Virginia
Thinus F. Kruger, M.D., Ph.D., D.Sc.
Department of Obstetrics and Gynecology, Tygerberg Hospital and Stellenbosch University, Tygerberg, South Africa
1. Hviid TV, Hylenius S, Lindhard A, Christiansen OB. Association between human leukocyte antigen-G genotype and success of in vitro fertilization and pregnancy outcome. Tissue Antigens 2004;64:66-9.
2. Sher G, Keskintepe L, Nouriani M, Roussev R, Batzofin J. Expression of sHLA-G in supernatants of individually cultured 46-h embryos: a potentially valuable indicator of ’embryo competency’ and IVF outcome. Reprod Biomed Online 2004;9:74-8.
3. Vercammen MJ, Verloes A, Van de Velde H, Haentjens P. Accuracy of soluble human leukocyte antigen-G for predicting pregnancy among women undergoing infertility treatment: meta-analysis. Hum Reprod Update 2008;14:209-18.
4. Kotze DJ, Hansen P, Keskintepe L, Snowden E, Sher G, Kruger T. Embryo selection criteria based on morphology VERSUS the expression of a biochemical marker (sHLA-G) and a graduated embryo score: prediction of pregnancy outcome. J Assist Reprod Genet 2010;27:309-16.
5. Kotze D, Kruger TF, Lombrd C, Padayachee T, Keskintepe L, Sher G. The effect of the biochemical marker soluble human leukocyte antigen G on pregnancy outcome in assisted reproductive technology-a multicenter study. Fertil Steril 2013, In Press. DOI:pii: S0015-0282(13)02763-5. 10.1016/j.fertnstert.2013.07.1977
Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2013.10.014