Article Blood clots in the cumulus oocyte complex predict poor oocyte quality and postfertilization

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1 RBMOnline - Vol 16 No Reproductive BioMedicine Online; on web 14 April 2008 Article Blood clots in the cumulus oocyte complex predict poor oocyte quality and postfertilization development Thomas Ebner PhD graduated with honours from the Paris Lodron University of Salzburg, Austria, in He then worked on his thesis in cancer research at the Institute for Pathology at the General Hospital, Salzburg until After a 2-year period as a researcher in the Division of Animal Physiology, Salzburg University he began his IVF career in Linz. His post-doctoral thesis on non-invasive selection at different stages of preimplantation development qualified him as a university lecturer in Salzburg. He has published more than 60 papers and his research interests are non-invasive selection processes in IVF, apoptosis, and culture media. Dr Thomas Ebner T Ebner 1,3, M Moser 1, O Shebl 1, M Sommergruber 1, C Yaman 2, G Tews 1,2 1 Landes-, Frauen- und Kinderklinik, IVF-Unit, Linz, Upper Austria, Austria; 2 General Hospital, Department of Gynecology and Obstetrics, Linz, Upper Austria, Austria 3 Correspondence: thomas.ebner@gespag.at Abstract Assessment of oocyte maturity and quality (morphological appearance) at the time of retrieval is difficult as the egg is obscured by a large cumulus mass that hinders adequate scoring. Since no data are available on the possible relationship between the cumulus oocyte complex (COC) and oocyte morphology, this prospective intracytoplasmic sperm injection study was set up in 87 consecutive patients. COC were grouped according to expansion of both corona radiata and cumulus matrix. Special emphasis was placed on recording morphological anomalies of COC (inclusion of blood clots and amorphous clumps). For all mature ovae, quality was assessed and preimplantation development followed up to blastocyst stage if fertilized. The risk of not harvesting an oocyte was higher in COC with blood clots compared with normal cumulus matrices (P = 0.004). COC expansion did not allow for prediction of either nuclear status or quality of the egg. The presence of blood clots within the cumulus matrix was associated with reduced oocyte quality (dense central granulation), fertilization rate and blastocyst formation, compared with unaffected COC (P < 0.05). It may be postulated that COC showing blood inclusions derive from poor quality follicles, which has a detrimental effect on oocyte quality and further cleavage to blastocyst stage. Consequently, mechanical removal of blood clots cannot rescue the corresponding embryo. Keywords: blastocyst formation, blood clots, central granulation, cumulus oocyte complex, oocyte quality Introduction Oocyte maturity and quality at the time of retrieval are difficult to assess as the egg is obscured by a large cumulus mass that hinders adequate scoring. While the importance of this is negligible in intracytoplasmic sperm injection (ICSI) due to a preceding denudation process, it is of importance in conventional IVF where harvested cumulus oocyte complexes (COC) are traditionally evaluated according to the appearance and expansion of the corona radiata and the cumulus complex (Veeck, 1986, 1990). Based on these criteria, oocytes within cumulus matrix are roughly categorized as either mature (metaphase II) or immature (prophase and metaphase I). In detail, an expanded and luteinized cumulus complex and a radiant corona radiata suggest completion of nuclear maturation, while the absence of expanded cumulus or corona cells is associated with total immaturity (prophase I). Thus, any intermediate COC, in terms of expansion, would correlate to metaphase I (Veeck, 1999). However, since the early years of assisted reproduction treatment it has been evident that assessment of maturation in stimulated cycles is rather imprecise (Laufer et al., 1984; Hammitt et al., 1992, 1993). The reported failure of adequate prognosis has more recently been confirmed by Rattanachaiyanont et al. (1999), who noted a discrepancy between the actual grade Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK

2 802 of the COC and the nuclear maturity of the corresponding egg. Interestingly, some 60% of presumed immature oocytes turned out to be at metaphase II, whereas approximately 8% of expected metaphase II eggs did not show a first polar body (Rattanachaiyanont et al., 1999). In contrast to oocyte maturity, data on oocyte quality on the basis of morphology and its possible dependence on COC morphology are scarce. Though some studies postulated a correlation between oocyte appearance and the morphology of the corresponding cumulus matrix, their evidential value is limited since fertilization rate (Ng et al., 1999), blastocyst formation and quality (Lin et al., 2003) as well as pregnancy rates (Ng et al., 1999) were used to retrospectively estimate oocyte appearance. In this respect, it has to be kept in mind that these publications exclusively dealt with IVF cases, which usually do not require oocyte denudation. In ICSI, however, a more explicit correlation between COC morphology and gamete maturity and quality can be obtained only a few hours after follicle aspiration. This is of special interest in those COC that show certain types of anomalies, such as blood clots (Motta et al., 1995) or amorphous clumps, both of which could indicate suboptimal follicular maturation. As far as is known, this is the first prospective ICSI study trying to correlate COC morphology and the quality and maturational status of the corresponding egg. Materials and methods In a 3-month study period, 87 consecutive ICSI patients were recruited for this prospective analysis of COC morphology and its possible prognostic value on oocyte maturity as well as quality. The mean age of the female patients was 34.2 ± 5.2 years. Approximately half of them (n = 44) suffered from primary infertility whereas the others had a history of at least one biochemical or clinical pregnancy (no information on COC morphology of previous cycles available). Though all couples were confronted with male factor infertility, 16 couples (18%) had a female indication as well (10 with tubal factors, four with endometriosis, and two with polycystic ovaries). No correlation between female indication and COC appearance could be observed. Since this study required a slightly altered time schedule compared with the routine procedure, Institutional Review Board approval was sought and granted. A total of 53 (60.9%) women were stimulated using an antagonist protocol. Thus, human menopausal gonadotrophin (Menogon; Ferring, Kiel, Germany) was started on day 2 of the cycle. In addition, a gonadotrophin-releasing hormone (GnRH) antagonist (Orgalutran; Organon, Vienna, Austria) was given after 5 6 days of stimulation, depending on the presence of a mm follicle in the ultrasound scan. The remaining 34 patients (39.1%) had their ovaries stimulated according to a long protocol. Therefore, down-regulation of the pituitary was achieved with the GnRH agonist buserelin (Suprecur; Aventis Pharma, Vienna, Austria) and stimulation was initiated with human menopausal gonadotrophin (Menogon). In all patients, ovulation was induced with 5000 IU human chorionic gonadotrophin (HCG, Pregnyl; Organon, Vienna, Austria), providing that the leading follicle had reached an appropriate diameter and serum oestradiol appeared adequate. Follicle aspiration (aspiration vacuum of 200 mmhg) was carried out transvaginally under ultrasound guidance 36 h after HCG administration using a one-way follicle puncture set (Reproline, Rheinbach, Germany) with an outer diameter of 1.4 mm. Immediately after oocyte retrieval, the COC collected were transferred to Universal IVF Medium (MediCult, Copenhagen, Denmark) and grouped according to their morphology. Instead of evaluating the maturity of the whole complex, corona radiata and cumulus were scored separately, essentially as reported by Rattanachaiyanont et al. (1999); however, for ease of analysis it was tightened when appropriate. It should be mentioned that the optical set-up in this study did not allow for adequate counting of cell layers (Ng et al., 1999), though no association between this parameter and oocyte appearance has been suggested (Rattanachaiyanont et al., 1999). According to the different expansion patterns (dense or radiant) of both cell types (corona and cumulus) four distinguishable grades were analysed: grade 1 (suspected mature), fluffy and radiant corona and cumulus with visible oocyte; grade 2, dense corona (oocyte hardly visible) but fluffy cumulus; grade 3, radiant corona (oocyte visible) but rather dense cumulus; grade 4 (suspected immature), dense corona and cumulus without visible oocyte (Figure 1). In addition, special emphasis was placed on recording morphological anomalies of COC since no ICSI data are currently available dealing with certain COC dysmorphisms and their potentially negative impact on the quality of the corresponding gamete. In detail, it was planned to screen for blood clots within the cumulus matrix (Daya et al., 1990) as well as amorphous clumps, possibly being a sign of postmaturity (Rattanachaiyanont et al., 1999). Though the nature of amorphous clumps/pycnotic nuclei is not fully understood, it can be assumed that these areas originate from cumulus cells. However, due to their relatively clear dissociation, it is easy to remove these anomalies using syringe needles or hand-drawn glass pipettes for manipulation. This is not the case for blood clots that sometimes adhere to COC because the thin strands of blood are somewhat interwoven with the cumulus matrix, making them more difficult to dislodge. Groups of COC of different grades were denuded (80 IU/ ml hyaluronidase, MediCult,) immediately after scoring (within 30 min after collection). Attempts were made to leave numerous cumulus cells attached during the denudation process (Ebner et al., 2006a) in order to compensate for the rather early manipulation, though no detrimental effect of early denudation on outcome has been reported (Van de Velde et al., 1998). ICSI was performed within half an hour after denudation according to routine technique (Ebner et al., 2001) using the 3 o clock position for injection. Though oocyte quality (Ebner et al., 2006b) was only checked in oocytes showing a first polar body, attempts were made to fertilize metaphase I gametes as well (but corresponding data were not incorporated into the present analysis). To facilitate analysis of morphological oocyte criteria on further outcome, these were classified as

3 a b c d Figure 1. Grading of cumulus oocyte complexes according to the expansion of corona radiata and cumulus matrix ( 40). (a) Grade 1 (suspected mature): fluffy and radiant corona and cumulus with visible oocyte; (b) Grade 2: dense corona (oocyte hardly visible) but fluffy cumulus; (c) Grade 3: radiant corona (oocyte visible) and rather dense cumulus; (d) Grade 4 (suspected immature): dense corona and cumulus without visible oocyte with blood clot (arrow). extracytoplasmic or cytoplasmic anomalies (Mikkelsen and Lindenberg, 2001). On the morning of day 1 (17 20 h after injection) regular fertilization was confirmed by the presence of two pronuclei and polar bodies. On the following days (days 2 and 3), embryos were scored in terms of number and size of blastomeres as well as for the presence of fragments and multinucleation, signs of compactation (day 4) and blastocyst development (day 5). The routine media for culture were Blastassist System Medium 1 (MediCult) until day 3 and Blastassist System Medium 2 in case of extended culture to day 5. During in-vitro culture, oocytes and corresponding embryos were cultured in groups according to their COC grade. The number of 8-cell embryos on day 3 was the key determinant for selecting day 3 (n = 63) or day 5 (n = 24) transfer. On the day of transfer, embryo/blastocyst selection was performed exclusively on the basis of the quality of the conceptus and not on COC morphology; thus, only a limited number of homogeneous transfers could be performed not allowing for adequate analysis of pregnancy data. In detail, six patients whose clinical parameters did not differ from those of other patients had exclusive transfers of embryos deriving from COC with blood clots; none of them became pregnant. It should be noted that distribution of COC grades was comparable between patients having day 3 and day 5 transfers. Comparisons were performed using chi-squared tests and t- tests. Statistical significance was defined as P < COC showing amorphous clumps did not show any deviation from those with normal cumulus masses, so they were pooled in order to increase sample number. Results In 87 patients, a total of 632 COC (7.3 ± 6.0 per patient) were collected. In 25 (4.0%) of these, no oocyte or only an empty zona pellucida was found within the cumulus mass. The latter phenomenon was found to be higher in the presence of blood clots (Table 1) due to difficulties during preparation. 803

4 The risk of harvesting no oocyte was significantly higher if blood clots were present within the COC (P = 0.004). The oocytes found were at metaphase II in 78.5% of the cases (n = 496), at metaphase I in 9.3% (n = 59) and completely immature in 8.2% (n = 52). With respect to anomalies in the cellular matrix, blood clots were present in 97 COC (15.3%) and amorphous clumps in 83 (13.1%). Interestingly, neither presence of blood clots (Table 1) nor amorphous clumps were related to morphology of the COC on the basis of cumulus and/or corona expansion, i.e. grades 1 4 had 16%, 10%, 9%, and 15% of blood clots, respectively. The presence of blood clots or amorphous clumps was not related to demographic and stimulation data except the expected correlation (P = ) between the possibility of having at least one COC with blood clots and oestradiol concentration on the day of ovulation induction (1660 ± 1254 pg/ml oestradiol in blood clot positive cycles versus 900 ± 772 pg/ml in negative ones). COC morphology and oocyte maturity Of all normal COC expected to contain metaphase II gametes (group 1) almost 90% were actually mature; but approximately 3% showed a germinal vesicle (Table 1). In the other extreme case (group 4), an immature egg was predicted; however, only half of the gametes showed a germinal vesicle but 25% had already completed nuclear maturation, as indicated in Table 1. Intermediate degrees of expansion did not differ significantly, but COC with a fluffy corona radiata (group 3) tended to allow for a better prediction of MII oocytes than COC with a fluffy cumulus complex (group 2). COC morphology and oocyte quality Table 2 indicates that oocyte morphology was not predicted by the radiation and expansion of the corona/cumulus matrix. This is supported by the fact that comparable rates of normal gametes were found in the four groups analysed, which allowed for pooling these data. Focusing on the presence of anomalies and their possible association with oocyte quality, it was found that in the group of COC with amorphous clumps (n = 83), a similar rate of normal oocytes was found as compared with COC without dysmorphisms. Blood clot positive COC, on the other hand, yielded a significantly lower number of normal oocytes as indicated in Table 2 (P = 0.043). In detail, in these COC a significantly higher percentage of oocytes showing dense central granulation was found (24.3%) as compared with COC without any anomaly (8.7%) (P < 0.001). COC morphology and fertilization outcome All groups analysed showed similar fertilization rates. In detail, 74.0%, 64.0%, 87.5% and 75.0% of the MII oocytes in groups 1 4, respectively, showed two pronuclei. The same was observed for MI oocytes (25.0%, 40.0%, 25.0% and 50.0%). Table 3 indicates that MII oocytes from COC presenting blood clots had a significant lower fertilizability compared with oocytes from normal COC (P < 0.05). No correlation could be observed between the four different grades of COC and further cleavage behaviour up to day 4. Blood clots in the COC, however, led to significantly lower blastulation rates as compared with all other COC (Table 3). Table 1. Relationship between cumulus oocyte complex morphology and maturity of the corresponding gamete evaluated within half an hour after collection. COC n Metaphase II Metaphase I Prophase I No oocyte morphology or empty group zona pellucida Group (89.1) 27 (6.4) 10 (2.4) 9 (2.1) Group (51.1) 10 (21.3) 10 (21.3) 3 (6.4) Group (71.4) 4 (19.0) 1 (4.8) 1 (4.8) Group (26.1) 9 (19.6) 22 (47.8) 3 (6.5) Groups a (82.7) 50 (9.7) 43 (8.4) 16 b (3.1) (without blood) Groups a (72.2) 9 (9.3) 9 (9.3) 9 b (9.3) (with blood) Values in parentheses are percentages; values with the same superscript letter are significantly different. a P < 0.05; b P <

5 Table 2. Correlation between cumulus oocyte complex morphology and quality of the corresponding metaphase II gamete evaluated immediately after denudation. Gamete parameter Group 1 Group2 Group 3 Group 4 Groups 1 4 with blood Total Normal 243 (64.8) 11 (45.8) 10 (66.6) 9 (75.0) 36 (51.4) c Cytoplasmic anomalies ser 9 (2.4) 0 (0.0) 1 (6.7) 0 (0.0) 1 (1.4) Vacuoles 22 (5.9) 2 (8.3) 3 (20) 0 (0.0) 4 (5.7) Central granulation 31 (8.3) 5 (20.8) 0 (0.0) 1 (8.3) 17 (24.3) d Incorporations a 43 (11.5) 4 (16.7) 1 (6.7) 0 (0.0) 3 (4.3) Extracytoplasmic anomalies Ovoid shape 8 (2.1) 1 (4.2) 0 (0.0) 1 (8.3) 2 (2.9) Discolouration 11 (2.9) 0 (0.0) 0 (0.0) 1 (8.3) 4 (5.7) PVS anomalies b 8 (2.1) 1 (4.2) 0 (0.0) 0 (0.0) 3 (4.3) Values in parentheses are percentages; PVS = perivitelline space; ser = aggregation of smooth endoplasmic reticulum. a Incorporations include refractile bodies; b PVS anomalies include giant first polar bodies; c P < 0.05 compared with pooled groups 1 4 without blood clots; d P < compared with pooled groups 1 4 without blood clots. Table 3. Preimplantation development of cumulus oocyte complexes (COC) with or without blood clots. Parameter COC with COC without P-value blood clots blood clots Total MII oocytes 70 (72.2) 426 (79.6) NS 2 pronuclei 45 (64.3) 323 (75.8) Cleavage 45 (64.3) 313 (73.5) NS Good-quality day 2 embryos 19/45 (42.2) 153/313 (48.9) NS Good-quality day 3 embryos 21/45 (46.7) 154/314 (49.0) NS No. of blastocyst cultures Compacting day 4 10/30 (33.3) 66/178 (37.1) NS Blastocyst formation day 5 7/30 (23.3) 78/178 (43.8) Values in parentheses are percentages; MII = metaphase II; NS = not statistically significant. Discussion Within the cumulus oophorus, which protrudes into the follicular cavity, the oocyte is located surrounded by the corona radiata, the innermost layers of cumulus cells. Maturation and growth of the egg is dependent on the nurturing capacity of the follicle, especially of the cumulus granulosa cells. In this respect, bidirectional communication between gamete and somatic cells occurs via paracrine and gap-junctional signalling (for reviews, see Canipari, 2000; Sutton et al., 2003). The latter mode is facilitated since highly specialized cells of the corona radiata have cytoplasmic processes penetrating the zona pellucida and reaching the oolemma. At the points of contact, a network of gap junctions unites the corona cells with the ovum as a structural and functional syncytium, which is essential for gamete growth (Eppig, 1977). The rate of growth is likely to be directly related to the number of coupled somatic cumulus cells (Brower and Schultz, 1982) and follicle size (Han et al., 2006). Since the vast majority of trans-zonal processes withdraw successively around the time of resumption of meiosis (induced by the preovulatory surge of gonadotrophins), the lightmicroscopical appearance (Goud et al., 1998; Sato et al., 2007) changes from a rather compact corona radiata to a less dense one. In parallel, considerable production of hyaluronic acid causes further dispersion of cumulus cells resulting in COC 805

6 806 growth as nuclear maturation proceeds (Eppig, 1981; Chen et al., 1990; Han et al., 2006). While it is suspected that in natural cycles the pattern of changes in COC closely parallels the corresponding meiotic maturation of the egg (Ng et al., 1999), stimulated cycles show varying degrees of asynchrony between COC appearance and actual oocyte maturity (Laufer et al., 1984; Hammitt et al., 1992, 1993; Rattanachaiyanont et al., 1999). Since morphological changes and proliferation of cells of the cumulus oophorus were seen to differ between patients and between sibling oocytes of the same cohort (Gregory et al., 1994) a variety of scoring systems for COC have been introduced (Veeck, 1986; Wolf, 1988; Daya et al., 1990; Ng et al., 1999; Rattanachaiyanont et al., 1999; Lin et al., 2003; Balaban and Urman, 2006) in order to predict the maturational stage of the corresponding oocyte for proper timing of insemination. All of them dealt with the individual expansion of the cumulus mass and/or corona radiata, but only few took into consideration the possible negative effect of morphological COC deviations, e.g. presence of blood clots or amorphous clumps (Hyttel et al., 1986; Daya et al., 1990; Rattanachaiyanont et al., 1999). The present modified approach to grade COC according to the expansion of the corona and cumulus turned out to be insufficient in terms of predicting nuclear status of the corresponding gamete. This is in line with literature (Laufer et al., 1984; Hammitt et al., 1992, 1993); however, the present data provide the first evidence that adequate expansion of the corona radiata, i.e. oocyte visible within the COC (groups 1 and 3), is superior in predicting nuclear maturation of the egg compared with the appearance of the cumulus (groups 2 and 4). This supports a suspected correlation between corona appearance and cleavage behaviour (Daya et al., 1990). Early denudation allowed for a precise assessment of oocyte appearance immediately after retrieval (36 h after HCG). It should be noted that COC expansion did not allow for prediction of oocyte quality based on morphology, indicating that maturational processes of both gamete and somatic cells are not automatically synchronized. It has been suggested that this asynchrony could be due to a different sensitivity of cumulus cells and oocytes to gonadotrophin-induced maturation or to other intrafollicular factors (Laufer et al., 1984; Bar-Ami et al., 1989). Interestingly, those oocytes derived from COC showing distinct blood clots (but not with amorphous clumps) had a significantly higher rate of changes in cytoplasmic texture (Kahraman et al., 2000). This might be the reason for the present reduction in rates of fertilization and blastulation as compared with ovae from COC without blood clots. These observed declines are perfectly consistent with the hypothesis of Van Blerkom and Henry (1992) who suggested that anomalies developing early in maturation (e.g. extensive central granulation) may be associated with failed fertilization and aneuploidy (not analysed in this study). Any causal connection between blood clots and oocyte quality, however, would indicate that blood found within the cumulus mass is not necessarily an artefact caused by the ovarian puncture (Daya et al., 1990). This is further supported by the unpublished observation that a minor proportion of COC showing blood clots had actually been collected from follicular fluid contaminated by blood and the fact that red blood cells have also been reported in COC harvested from oviducts of unstimulated women (Motta et al., 1995). As the mentioned paper (Daya et al., 1990) was published more than 25 years ago, it could indeed be that follicle puncture sets used in those days were more invasive than nowadays. At least, there is evidence that technical details of aspiration procedure can change COC morphology (Bols et al., 1996, 1997). As a matter of fact, the observed (Daya et al., 1990) proportion of COC with blood inclusions (21%) was much higher than in the present study (15%). Apart from a suspected blocking function of blood clots preventing direct contact between spermatozoa and the zona pellucida (not relevant in ICSI cycles), Daya et al. (1990) blamed shifts in ph and temperature for the observed reduction in fertilization and cleavage. In addition, some negative impact could also be attributed to the production of reactive oxygen species induced by exogenous factors, e.g. blood components (Attaran et al., 2000; Guérin et al., 2001). Based on the present data, the presence of blood clots were not associated with atretic or luteinized follicles (no mucified COC observed) since demographic (low LH on the day of ovulation induction) and stimulation data were similar in all patients. To conclude, it may be postulated that COC showing blood inclusions derive from poor quality follicles that have a detrimental effect on oocyte quality and further cleavage to blastocyst stage. It is likely that these gametes are already harmed during folliculogenesis and, thus, their developmental capacity may not be retained by mechanical removal of blood clots. Based on preliminary implantation data, with no pregnancies after exclusive transfer of embryos/blastocysts deriving from COC showing blood clots, it may further be postulated that the implantation potential of such concepti could be impaired. Consequently, it can be suggested that affected COC should be cultured separately and corresponding embryos should only be transferred if no others of comparable quality are available. References Attaran M, Pasqualotto E, Falcone T et al The effect of follicular fluid reactive oxygen species on the outcome of invitro fertilization. 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Human Reproduction 13, Veeck LL 1999 The human oocyte. In: Veck LL (ed.) An Atlas of Human Gametes and Conceptuses. Parthenon Publishing, New York, London, pp Veeck LL 1990 The morphological assessment of human oocytes and early concepti. In: Keel BA, Webster BW (eds) Handbook of the Laboratory Diagnosis and Treatment of Infertility. CRC Press, Boca Raton, Boston, pp Veeck LL 1986 The morphologic estimation of mature oocytes and their preparation for insemination. In: Jones HW Jr, Jones GS et al. (eds) In-Vitro Fertilization Norfolk. Williams and Wilkins, Baltimore, p. 81. Wolf DP 1988 Oocyte quality and fertilization. In: Wolf DP In-Vitro Fertilization and Embryo Transfer. Plenum Press, New York, pp Declaration: The authors report no financial or commercial conflicts of interest. Received 11 October 2007; referred 16 November 2007; accepted 18 January