Letters to the editor |
Reply: Origin of leukocytes and their profile in follicular aspirates
1 University of Bristol, Department of Clinical Sciences South Bristol, Obstetrics and Gynaecology, St Michael’s Hospital, Bristol, UK and 2 Immunogenetics Unit, Department of Genetics, La Trobe University, Bundoora, Victoria, Australia
3 To whom correspondence should be addressed at: University of Bristol, Obstetrics and Gynaecology, CSSB, Henry Wellcome LINE Laboratories, Whitson Street, Bristol BS1 3NY, UK. E-mail: mike.smith{at}bristol.ac.uk
Sir,
We appreciate Dr Akkoyunlu’s interest in our recent article on the origin of leukocytes and their profile in follicular aspirates at oocyte retrieval (Smith et al., 2005
).
Dr Akkoyunlu draws a comparison between our own study on harvested human follicular aspirates at oocyte retrieval and that of Akkoyunlu et al. (2004) on the presence of leukocytes and erythrocytes in the cumulus mass before and after thecal rupture in the mouse. On this basis, it is stated that we have ‘concluded that ... the presence of leukocytes and erythrocytes within follicular aspirates was not directly related to the vessel damage during oocyte harvesting’. This is an overstatement: in fact, our conclusion was somewhat more cautious. Erythrocytes do not normally extravasate: their presence in all the samples we examined indicates that a degree of blood vessel damage must have contributed to the composition of these follicular aspirates. Our finding that the numbers of leukocytes and erythrocytes varied independently in the aspirates led us to conclude simply that leukocytes were unlikely to be present solely as a result of vessel damage – we made no generic claim about whether blood vessel damage is the primary source of these cells or not. On the contrary, our study suggests that follicular aspirates are inherently variable in this respect: in some cases, blood vessel damage does not appear to account for all leukocytes present, whereas in others, the numbers of leukocytes originating from damaged vessels may mask any underlying contribution made by other mechanisms such as inflammation.
Our study used two separate groups of follicular aspirates: one to measure erythrocytes and leukocytes and a second independent group to assess leukocyte subtypes. Dr Akkoyunlu is critical of this experimental design, arguing that our study results would have been more convincing had these two groups been combined. In the first place, the collection and analysis of clinical material can impose constraints over and above those involving animal studies, and, in this case, only a limited number of samples were available. In addition, the two different phases of our study required the use of two different protocols for sample preparation. In the first group of aspirates, erythrocytes were counted before the samples were treated with erythrocyte lysis buffer to allow all CD45-positive leukocytes to be visualized. We omitted erythrocyte lysis buffer from the protocol used to prepare the second group of samples to optimize morphological preservation for the detailed analysis of leukocyte subtypes. In several cases, aspirates did not contain sufficient material for both sets of data to be readily obtained from the same sample. Despite this, sample numbers were clearly sufficient to satisfy rigorous statistical analysis; we therefore disagree with Dr Akkoyunlu’s assessment of our study results as less than convincing.
Dr Akkoyunlu suggests that specific antibodies could be used to identify leukocyte subtypes. In our study, we used standard morphological assessment of leukocyte subpopulations to accurately determine lymphocytes, monocytes/macrophages and polymorphonuclear leukocytes. The value of this approach is clearly illustrated in Figure 2 of our article, where the different leukocyte subsets are readily identifiable. Of course, we agree that the use of markers in addition to CD45 would allow further subtyping to be carried out. In this way, it would be possible, for instance, to determine the ratio of helper T to suppressor/cytotoxic T cells. Similarly, NK cells could have been enumerated using, for example, CD16 and CD56. However, the use of CD163, as suggested by Dr Akkoyunlu, would only have reinforced our morphological determination of tissue macrophages/activated monocytes.
If we had been asking about the inflammatory nature of the process leading to the presence of leukocytes in the aspirates, then fine-tuned analysis would indeed have been appropriate. Such an approach might, for example, reveal whether the numbers/proportions of macrophages correlate with any other parameters of the aspirate. This type of analysis would ideally require the simultaneous determination of cytokines, an exercise of some magnitude, and beyond the scope of the study. Our article describes in some detail the confusing and sometimes contradictory nature of the literature concerning the cellular composition of human follicular aspirates. In the light of this, our purpose, as we explicitly stated, was simply to establish whether leukocytes found in follicular aspirates might reflect blood contamination, any further studies being warranted only once this basic question had been satisfactorily resolved. For this purpose, the assessments we used were more than adequate.
From a clinical perspective, any information we can glean on follicular heterogeneity has potential relevance to the vexatious question of how we can optimize follicle selection during in vitro fertilization and embryo transfer. On the basis of our own observations as well as those described in other reports from the literature (e.g. Espey, 1980, 1994; Kawano et al., 2001), it seems perfectly reasonable to speculate that variations in the profile of leukocyte subtypes might relate to follicular maturity. Dr Akkoyunlu states that we should have included the measurement of different parameters that might reflect follicular maturation, such as hormone levels and follicular size. It would indeed be interesting if there are correlations between our own observations and other parameters. In that event, a link to follicular maturation would become a firm hypothesis rather than a mere speculation. Therefore, if Dr Akkoyunlu is suggesting that our observations themselves are weakened because they are not supported by including such data, then we cannot agree and reiterate that this was not the purpose of our study. If, on the contrary, it is being suggested that these studies are potentially valuable in future work, then we are happy to concur.
References
Akkoyunlu G, Korgun ET, Celik-Ozenci C, Seval Y, Demir R, Ustunel S. (2004) Distribution patterns of leucocyte subpopulations expressing different cell markers in the cumulus-oocyte complexes of pregnant and pseudopregant mice. Reprod Fertil Dev 15:389–395.[CrossRef][Medline]
Espey LL. (1980) Ovulation as an inflammatory reaction – a hypothesis. Biol Reprod 22:73–106.[CrossRef][Web of Science][Medline]
Espey LL. (1994) Current status of the hypothesis that mammalian ovulation is comparable to an inflammatory reaction. Biol Reprod 50:233–283.[Abstract]
Kawano Y, Kawasaki F, Nakamura S, Matsui N, Narahara H, Miyakawa I. (2001) The production and clinical evaluation of macrophage colony-stimulating factor and macrophage chemoattractant protein-1 in human follicular fluids. Am J Reprod Immunol 45:1–5.
Smith MP, Flannery GR, Randle BJ, Jenkins JM, Holmes CH. (2005) Leukocyte origin and profile in follicular aspirates at oocyte retrieval. Hum Reprod 20:3526–3531.
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