Hum. Reprod. Advance Access originally published online on August 11, 2005
Human Reproduction 2005 20(12):3526-3531; doi:10.1093/humrep/dei240
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Leukocyte origin and profile in follicular aspirates at oocyte retrieval
1 University of Bristol, Department of Clinical Sciences South Bristol, Obstetrics and Gynaecology, St Michael’s Hospital, Southwell Street, Bristol BS2 8EG, UK and 2 Immunogenetics Unit, Department of Genetics, La Trobe University, Bundoora, Vic. 3086, Australia
3 To whom correspondence should be addressed. E-mail: mike.smith{at}bristol.ac.uk
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Abstract |
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BACKGROUND: Follicular aspirates represent a snapshot in time of conditions within the follicle at oocyte retrieval in women undergoing in vitro fertilization and embryo transfer. This clinical material has been much investigated and yet its cellular composition remains unclear. In this study we investigated the origin and profile of leukocytes found within follicular aspirates. METHODS: We performed morphological and immunohistochemical analyses of follicular aspirates and peripheral blood obtained concurrently at oocyte retrieval. RESULTS: There was no correlation between erythrocyte and leukocyte numbers in follicular aspirates. The profile of leukocyte subtypes within follicular aspirates was variable and differed significantly from the peripheral circulation in a significant proportion of the analysed samples. A subset of follicular aspirates displayed a marked increase in monocytes/macrophages and an apparent concomitant reduction in polymorphonuclear leukocytes compared with peripheral blood. CONCLUSIONS: Leukocytes within follicular aspirates cannot be accounted for solely as a result of blood vessel damage during oocyte retrieval. The variation in leukocyte subtypes observed in some follicular aspirates may reflect a coordinated infiltration of these cells, characteristic of progressive inflammatory responses in other systems. The possibility that leukocyte variation is indicative of follicular maturation deserves further investigation due to its potential relevance in optimizing oocyte selection.
Key words: follicular aspirates/IVF-ET/leukocyte profile/leukocyte origin/peripheral blood
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Introduction |
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The follicular aspirate has proved an invaluable source for the study of ovarian function, and yet the cellular composition of this clinically important material is not well defined. Whilst the primary cellular constituent of follicular aspirates is generally regarded as the granulosa cell, other cell types have also been identified. Most commonly reported amongst these non-granulosa cell populations are leukocytes, often accounting for more than half of all cells within follicular aspirates (Best et al., 1994
; Evagelatou et al., 1996; Lachapelle et al., 1996; Smith et al., 2000).
Previous studies (Hill et al., 1987; Droesch et al., 1988; Loukides et al., 1990; Beckmann et al., 1991; Best et al., 1994; Lachapelle et al., 1996) which have investigated leukocyte populations within follicular aspirates have tended to lack consensus. For example, in similar studies, Hill et al. (1987) reported a range of 0.1–2.5 x105 lymphocytes/ml of follicular aspirate sample, whereas Droesch et al. (1988) reported 2.14–2.79 x105 lymphocytes/ml of follicular aspirate. In terms of the proportions of leukocyte subtypes found within follicular aspirate samples, Loukides et al. (1990), reporting only upon monocytes/macrophages, found that these represented 5–15% of all cells. Similarly, Beckmann et al. (1991) reported that 6% of all cells in follicular aspirate samples were monocytes/macrophages and, additionally, that <2% of all cells were polymorphonuclear (PMN) leukocytes while some 10% were lymphocytes. Best et al. (1994) reported that macrophages represented 6–14% of all leukocytes in follicular aspirate samples, whereas lymphocytes represented 40–52%. Lachapelle et al. (1996) reported that the leukocyte population of follicular aspirate samples was made up of 22–44% lymphocytes.
These studies are inherently difficult to compare, however, not only due to differences in the reporting of different leukocyte subtypes but also because of variations in the preparation of follicular aspirate samples.
The origin of leukocytes within follicular aspirates is unclear. Beckmann et al. (1991) concluded that the appearance of leukocytes within follicular aspirates was solely a consequence of blood vessel damage occurring during oocyte harvesting. Whilst the presence of erythrocytes in follicular aspirates supports this notion, large proportions of leukocytes can nevertheless be present in these samples with little apparent erythrocyte contamination (M. P. Smith, unpublished data). Other studies suggest that leukocytes actively infiltrate the follicle prior to ovulation (Hill et al., 1987; Hill and Anderson, 1989; Loukides et al., 1990; Best et al., 1994; Runesson et al., 1996), although several analyses of follicles in situ have failed to identify these cells within the granulosa cell layer of apparently healthy follicles (Brannstrom et al., 1994; Best et al., 1996; Takaya et al., 1997; Chang et al., 1998; Suzuki et al., 1998).
The notion that leukocytes are resident in the follicle prior to ovulation appears predominantly based on reported differences between the profiles of leukocyte subtypes in follicular aspirates and peripheral blood (Hill et al., 1987; Hill and Anderson, 1989; Droesch et al., 1988; Loukides et al., 1990; Best et al., 1994; Runesson et al., 1996). In a more recent study, however, Giuliani et al. (1998) reported that leukocyte populations change in the peripheral circulation on the day of ovulation and that this alteration is greater in women undergoing IVF and embryo transfer. Thus, to compare accurately leukocyte populations between peripheral blood and the follicular aspirate, samples must be obtained concurrently. In only one study, however, have peripheral blood and follicular aspirate samples been obtained at the same time, and then only in five (20%) of the samples examined (Loukides et al., 1990). Thus, it is not clear whether a significant difference exists between leukocyte profiles of the peripheral circulation and follicular aspirates.
The potential role of leukocytes during the ovulatory process has been the subject of several reviews, all of which suggest that these cells may be important modulators of ovarian function (Espey, 1980, 1994; Bukulmez and Arici, 2000; Brannstrom and Enskog, 2002). Thus, it is clear that the leukocyte compartment of follicular aspirates deserves further attention.
We are intrigued by the presence of leukocytes within follicular aspirates and hypothesize that, if these cells are present simply as a consequence of blood vessel damage during oocyte harvesting, this will be reflected by a clear relationship between the numbers of erythrocytes and leukocytes in individual aspirates. In that event, an increase in the number of erythrocytes would be expected to correlate with an increase in the number of leukocytes and vice versa. In addition, we also hypothesize that a significant functional role for leukocytes in the periovulatory follicular milieu will be reflected by an alteration in the distribution of leukocyte sub-types in follicular aspirates compared to that of the peripheral circulation. In this preliminary study, therefore, we obtained samples from women (age range 26–36 years) with apparently normal ovarian function in order to assess the likely source and profile of these cells in follicular aspirates by (i) comparing leukocyte:erythrocyte ratios within individual samples obtained from 13 women, and (ii) comparing the leukocyte profile of aspirates and peripheral blood drawn from 14 aspirate donors at the time of oocyte harvesting.
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Materials and methods |
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Ethics approval for this study was granted by the Local Research Ethics Committee of the United Bristol Healthcare Trust. Informed consent was gained from sample donors.
Collection of follicular aspirates
Aspirates from individual follicles were obtained using ultrasound guided transvaginal aspiration of periovulatory follicles for oocyte retrieval from women undergoing IVF treatment at the University of Bristol, Centre for Reproductive Medicine, using a standardized treatment protocol of pituitary desensitization with buserelin, a gonadotrophin-releasing hormone agonist, and ovarian stimulation as described by Hull et al. (1992). In each case, aspirates were collected from one follicle per individual. Samples taken for analysis of leukocyte:erythrocyte ratios and those taken for comparison of leukocyte profiles in aspirates and peripheral blood were obtained from separate groups of women. Samples were obtained from women undergoing IVF-embryo transfer due to male factor infertility and with apparently normal ovarian function; that is, normal circulating hormone levels, an absence of anti-sperm antibodies and regular menstrual history. Volumes were similar for each sample (between 2 and 3 ml) and were stored at room temperature until transfer to the laboratory at St Michael’s Hospital, Bristol, usually within 1 h of collection.
Collection of peripheral blood
Approximately 5 ml of peripheral blood was obtained, immediately prior to oocyte retrieval, by venepuncture and collection into tubes coated with ethylenediamine tetraacetic acid, disodium salt (EDTA) to prevent clotting. Samples were stored at room temperature until transfer to the laboratory at St Michael’s Hospital, Bristol, usually within 1 h of collection.
Erythrocyte and leukocyte numbers within follicular aspirates
The volume of each aspirate was measured prior to assessment of erythrocyte numbers using a Neubauer haemocytometer. Each sample was then depleted of erythrocytes by incubation in erythrocyte lysis buffer (10 mM KHCO3, 150 mM NH4Cl, 0.1 mM EDTA, pH 8.0) for 5 min at 4°C, prior to resuspension in 1 ml phosphate-buffered saline/bovine serum albumin. In order to identify accurately leukocytes within follicular aspirates, a preparation of each sample was immunostained by an indirect immunoperoxidase method as described by Holmes et al. (1990), using tissue culture supernatant from the hybridoma cell line F10–89-4 (Dalchau et al., 1980), which secretes a monoclonal antibody recognising the CD45 antigen, exclusively expressed by leukocytes (Thomas, 1989). A minimum of 200 cells was counted in order to determine the number of leukocytes in each sample. The numbers of leukocytes and erythrocytes in each sample were compared and, as data were not normally distributed, statistical analysis was performed using Spearman’s rank correlation coefficient and statistical analysis software (Statsdirect, Camcode, Ashwell, UK).
Leukocyte subtypes in follicular aspirate and peripheral blood samples
Samples of individual follicular aspirates and corresponding peripheral blood were obtained from each of the women. A single droplet of peripheral blood or follicular aspirate was placed on a gelatine-coated glass microscope slide (Hendley, Loughton, UK) and smeared along the length of the slide. Samples were then air-dried for approximately 1 h at room temperature and fixed by immersion in ice-cold methanol for 10 min.
Follicular aspirate smears were subsequently immunostained with tissue culture supernatant from the hybridoma cell line F10-89–4 (Dalchau et al., 1980) as described above.
Differential leukocyte counts were performed independently in duplicate by co-authors M.P.S and G.R.F. Leukocytes were classified morphologically by nuclear shape, and nuclear:cytoplasmic ratio as PMN leukocytes (including neutrophils, basophils and eosinophils), monocytes/macrophages, and lymphocytes. Duplicate counts were generally consistent and did not differ by more than 12% in any sample. An average value for each leukocyte subtype in each sample was calculated from the combined cell counts. Comparison of leukocyte profiles (i.e. all three subtypes) between peripheral blood and follicular aspirates was performed with a 2 x 3 c2 contingency test of independence using Statsdirect. Numbers of erythrocytes and leukocytes within individual follicular aspirates were determined and compared using Spearman’s rank correlation coefficient.
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Results |
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Erythrocyte and leukocyte numbers within follicular aspirates
All samples were found to contain large numbers of both erythrocytes and leukocytes, with a range of 0.55–8.3 x 107 cells/sample for erythrocytes and a range for leukocytes of 0.3–5.85 x105 total cells/sample. There was no correlation between numbers of erythrocytes and leukocytes in follicular aspirates (P = 0.379, P > 0.05, n = 13) (Figure 1). Thus, high numbers of erythrocytes did not necessarily correlate with high numbers of leukocytes and, conversely, high numbers of leukocytes did not necessarily correlate with high numbers of erythrocytes.
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Leukocyte subtypes in follicular aspirates and peripheral blood
The profile of leukocyte subtypes within individual follicular aspirates was assessed morphologically and using the leukocyte-specific antibody anti-CD45. Examples of CD45-positive leukocyte subtypes found in follicular aspirates are illustrated in Figure 2. Distributions of these cells were compared with samples of peripheral blood as described in Materials and methods, using the 2 x 3 c2 contingency test of independence.
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The range and median values of leukocyte subtypes in peripheral blood and follicular aspirates are shown in Table I. In samples of peripheral blood, the observed range of leukocyte subtypes was found to be similar to that reported previously for normal healthy women (Hoffbrand and Pettit, 1993). By contrast, follicular aspirates exhibited a much wider variability in the distribution of leukocyte subtypes and this was particularly evident for PMN leukocytes and monocytes/macrophages.
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The distribution of leukocyte subtypes in paired samples of peripheral blood and follicular aspirates is shown in Figure 3. The profile, in terms of the relative proportions of PMN leukocytes, lymphocytes and monocytes/macrophages, was very similar in all peripheral blood samples examined (black bars in Figure 3). Some follicular aspirate samples (hatched bars in Figure 3) were found to display profiles characteristic of the corresponding paired peripheral blood sample (e.g. samples 1 and 10). Marked differences in the profiles of these cells, however, were evident in several follicular aspirate samples. Overall, statistical analysis revealed that there was a difference (P < 0.05) in the profile of leukocyte subtypes between peripheral blood and follicular aspirates in eight of the 14 paired samples examined (asterisks in Figure 3). The most interesting differences in profiles were observed in samples 2, 3, 4, 5 and 6, in which follicular aspirate samples showed a marked increase in the monocyte/macrophage population by comparison with the corresponding paired peripheral blood samples. Moreover, in each of these five follicular aspirate samples a corresponding decrease in the proportion of PMN leukocytes was consistently observed.
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Retrospective analysis revealed no distinguishing features in terms of age, the number of follicles, follicular size (where recorded) or estradiol levels between women with significantly different peripheral blood and follicular aspirate leukocyte profiles and those whose profiles were more similar.
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Discussion |
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Leukocytes can represent a considerable fraction of the cells found within follicular aspirates, and yet their significance to the periovulatory microenvironment is unclear. In an attempt to clarify the origins and likely importance of these cells, we firstly investigated the relationship between erythrocytes and leukocytes within follicular aspirate samples. If leukocytes were present within follicular aspirates simply due to blood vessel damage, one would reasonably expect to find a clear correlation between the numbers of these cells in this clinical sample, such that comparatively high numbers of erythrocytes would be associated with high numbers of leukocytes and vice versa. An absence of such a correlation, however, might indicate that leukocytes actively accumulate around and/or within the follicle at this time. This study found that the numbers of erythrocytes and leukocytes varied independently in follicular aspirates. On this basis it therefore seems unlikely that leukocytes are present in follicular aspirates solely as a consequence of blood vessel damage. Despite this, however, the presence of erythrocytes within follicular aspirates indicates that a degree of blood vessel damage does occur during oocyte harvesting.
The second aim of this study was to investigate the proportions of leukocyte subtypes found within follicular aspirates at oocyte retrieval. Thus, we compared the profile of leukocyte subtypes between individual follicular aspirates and paired samples of peripheral blood obtained at oocyte retrieval. Significant differences in the proportions of leukocyte subtypes between these samples might indicate active leukocyte infiltration of the periovulatory follicle and highlight their relative importance to the ovulatory process.
Comparing leukocyte subtype profiles between paired samples of follicular aspirates and peripheral blood revealed significant differences in eight of the 14 samples examined, as indicated by asterisks in Figure 3. This further supports the earlier conclusion that the presence of leukocytes within follicular aspirates is not solely due to blood vessel damage.
Interestingly, in five of the follicular aspirates examined, there was a marked increase in the proportion of monocytes/macrophages and an apparently concomitant reduction in the proportion of PMN leukocytes compared with those in the corresponding peripheral blood samples. The significance of this variation in the profiles of leukocyte subtypes within this intriguing subgroup of follicular aspirates is unclear. It is tempting to speculate, however, that this subgroup may represent follicles at a discrete developmental stage compared with those follicles where such a difference was not observed. Previously published reports suggest that variations in the relative proportions of PMN leukocytes and monocytes/macrophages may be indicative of the relative maturity of a given follicle. For example, prior to ovulation there appears to be an increase in the numbers of neutrophils within the thecal layers of follicles (Brannstrom et al., 1994), whilst macrophages actively infiltrate the newly formed corpora lutea (e.g. Hameed et al., 1995; Bukulmez and Arici, 2000). Thus, it might be speculated that the increase in the relative proportions of monocytes/macrophages compared with PMN leukocytes observed in several follicular aspirates during this study is indicative of an increase in follicular maturation.
The idea that variations in the profiles of leukocyte subtypes, particularly with regard to proportions of monocytes/macrophages, may be indicative of follicular maturity is also supported by the findings of Kawano et al. (2001), in which higher levels of monocyte chemoattractant protein were associated with follicles bearing mature oocytes compared with those bearing immature oocytes. Furthermore, in the broader context of an inflammatory response, to which the process of ovulation has long been compared (Espey, 1980, 1994), it is well established that subtypes of leukocytes infiltrate areas of inflammation in coordinated stages, such that PMN leukocytes are often the first to arrive at, and infiltrate, areas of inflammation, to be followed by monocytes/macrophages (Roitt et al., 1998).
To our knowledge, this study is the first to explore the relationship between numbers of leukocytes and erythrocytes in follicular aspirates, and also to examine the profiles of leukocyte subtypes in these samples and peripheral blood obtained concomitantly at oocyte harvesting. We report that leukocytes can appear independently of erythrocytes in follicular aspirates, indicating active infiltration of this population. In addition, in the majority of cases the profile of leukocytes is significantly different in follicular aspirates when compared with peripheral blood. The leukocyte population found within follicular aspirates clearly warrants further investigation and we anticipate that our observations will provide the basis for further detailed studies. The speculation that variations in the profile of leukocyte subtypes might be related to follicular maturation suggests that such studies may deliver clinically valuable information during IVF-embryo transfer.
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Submitted on January 15, 2005; resubmitted on January 17, 2005; accepted on July 11, 2005.
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