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Viability of cumulus cells is associated with basal AMH levels in assisted reproduction
European Journal of Obstetrics & Gynecology and Reproductive Biology 183 (2014) 59–63
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Viability of cumulus cells is associated with basal AMH levels in assisted reproduction Thomas Ebner a,b,e,*, Omar Shebl a,b, Sandra Holzer c, Peter Oppelt a,b, Erwin Petek d, Gudrun Schappacher-Tilp e, Richard B. Mayer a,b a
Landes- Frauen- und Kinderklinik, Departement of Gynecological Endocrinology and Kinderwunsch Zentrum Linz, Krankenhausstr. 26–30, Linz, Upper Austria, Austria Johannes Kepler University, Faculty of Medicine, Altenberger Straße 69, Linz, Upper Austria, Austria c Technical University of Graz, Institute of Biochemistry, Petersgasse 12, Graz, Styria, Austria d Medical University of Graz, Institute of Human Genetics, Harrachgasse 21, Graz, Styria, Austria e Karl-Franzens-University Graz, Department for Mathematics and Scientific Computing, Heinrichstraße 36, Graz, Styria, Austria b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 March 2014 Received in revised form 30 July 2014 Accepted 10 October 2014
Objective(s): An interesting non-invasive approach to select embryos for transfer is analyzing the health state of somatic granulosa cells surrounding the oocyte addressing their mutual dependence. This prospective study was set up to analyse whether the DNA integrity of cumulus cells correlates with preimplantation development and basal AMH levels. Study design: Therefore, 56 patients who gave written consent were enrolled. Sequential denudation of the cumulus-oocyte-complexes was performed in order to separate corona radiata from outer cumulus cells. DNA integrity of both cell types was analysed using a modified chromatin dispersion test. Results: The percentage of viable corona radiata cells per patient showed a linear correlation to blastulation (P < 0.05). These innermost cells showed significantly lower rates of strand breaks (P < 0.01) as compared to outer cumulus cells. Age-corrected AMH was significantly associated with the DNA integrity of outer cumulus cells (P < 0.05). Conclusion(s): For the first time it could be shown that in fact clinical embryologists deal with two different entities of cumulus cells, inner and outer ones. It seems that any protective mechanism of the female gamete follows an outward gradient, so that negative effects, e.g. apoptosis, may impair outer cumulus cells first. Age-corrected AMH reflects quality of these outer cumulus cells.Keywords: AMH; Corona radiata cells; DNA fragmentation; Outer cumulus cells; SCD test ß 2014 Elsevier Ireland Ltd. All rights reserved.
Introduction Along the way to reaching the ultimate goal of assisted reproduction technologies, namely the reduction of multiple pregnancy rates by strictly limiting the number of embryos being transferred, clinicians and clinical embryologists have to face the problem of identifying the embryos/blastocysts with the best prognosis. Naturally, invasive techniques, such as polar body, embryo or trophectoderm biopsy most accurately reflect the actual state of
* Corresponding author at: Landes- Frauen- und Kinderklinik Linz, Kinderwunsch zentrum, Krankenhausstr. 26-30, A-4020 Linz, Austria. Tel.: +43 732 6923 0; fax: +43 732 6923 24604. E-mail address: [email protected] (T. Ebner). http://dx.doi.org/10.1016/j.ejogrb.2014.10.015 0301-2115/ß 2014 Elsevier Ireland Ltd. All rights reserved.
health of gametes and embryos, with the latter approach being the most reliable one, since it maximizes insight (e.g. including postzygotic errors) while minimizing mechanical harm due to the biopsy process. However, selection of the best embryo for transfer in routine IVF/ICSI programs is currently based on less invasive techniques such as morphological evaluation. Prediction of implantation can be enhanced if morphological information of gametes, zygotes, and embryos at different stages of preimplantation development is pooled [1]. Nevertheless, this non-invasive method is far from being a perfect predictor of implantation. Recently, adding time course of mitotic divisions to static morphological observations started the era of morphokinetics, a field that may provide additional help in specifying implantation behavior of embryos [2]. Another non-invasive approach is analyzing the health state of somatic granulosa cells surrounding the oocyte addressing their
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mutual dependence [3]. Since both cell types grow in an identical milieu and experience comparable conditions in terms of nutrition during folliculogenesis, somatic cells may serve as an indirect marker of the health of the female gamete. With respect to this, morphological evaluation of the cumulusoocyte-complex (COC) turned out to be rather imprecise and is of limited importance in predicting maturity and quality of oocytes [4]. Much rather, the rate of apoptosis in cumulus cells (CC) was successfully used for accurately predicting fertilization [5], blastulation [5], and pregnancy [6]. Recently, the transcriptomic profile of CC was detected and correlated with embryo potential and pregnancy outcome [7]. Furthermore, non-invasive markers of oocyte chromosome status and competence were derived from CC gene expression [8]. It appears that the number of candidate genes in human cumulus cells that might be predictive of pregnancy outcome increases regularly [9]. Alternatively, the relative telomere length in cumulus cells at the time of egg collection may be taken into account as a new biomarker for selecting highly competent oocytes and embryos [10]. This prospective study was set up to analyse the quality of CC as assessed by the integrity of DNA strands in patients of different prognosis (based on basal AMH). For calculating DNA fragmentation of both corona radiata and outer cumulus cells for the first time a modified chromatin dispersion (SCD) test, originally designed for spermatozoa [11], was applied to CC.
Material and methods After ethical approval had been received (#12HO5) participation in this prospective trial was offered to all patients searching for ICSI treatment in the Kinderwunsch Zentrum Linz. A total of 6 patients refused participation for private reasons. It should be noted that cases of severe endometriosis were not included since it was not clear if quality of COC/CC might have been affected a priori. Patients Ultimately, 56 patients (34.0 4.3 years) gave written consent and were enrolled in the analysis. These patients suffered from male infertility in 22 (39%) cases (including three cases of ICSI with cryopreserved testicular spermatozoa), five patients were diagnosed with bilateral tubal blockage (9%), and eight (14%) with polycystic ovary syndrome or mild forms of endometriosis. The remaining cases were either unexplained or a mixture of male and female indications (38%). Half of the cycles involved were cases of secondary sterility (n = 28). Mean values of basal FSH (8.2 4.2 IU/ml), LH (6.3 3.2 IU/ ml) and estradiol (48.3 21.2 pg/ml) were within normal ranges and so was the AMH (5.1 4.3 ng/ml). Basal AMH (cycle day 3) was analysed using an enzyme immunoassay (Instrumentation Laboratory & Beckman-Coulter, Vienna, Austria). The analytical sensitivity of this ELISA was estimated at 0.1 ng/ml. The intraassay coefficient of variation is 12.3% and the interassay one 14.2%. All female partners were stimulated according to either an antagonist (62.5%) or an agonist scheme (37.5%). In the first regimen, recombinant products (Elonva1 or Puregon1, MSD, Vienna, Austria) were administered in approximately two thirds of the cases (n = 23), while urinary products (Menopur1, Ferring, Vienna, Austria) were used for the rest. The antagonist used was Orgalutran1 (MSD). In the long protocol, however, down regulation was performed with Buserelin (Suprecur1, Sanofi-Aventis, Vienna, Austria) and the gonadotrophins used were Puregon1or Menopur1. Since cumulus cell quality was found to be comparable in GnRH agonist and antagonist protocol respective data were pooled [12].
Ovulation was induced with 10,000 IU hCG (Pregnyl1, MSD). Oocyte retrieval was carried out transvaginally under ultrasound guidance 36 h after ovulation induction. On average, stimulation took 10.1 2.0 days and the estradiol level at the day of ovulation induction was found to be 1886 1698 pg/ml. Processing of cumulus cells COC were collected in BM1 medium (Laboratoires Eurobio, Les Ulis, France) and cultured for 2 h before a modified denudation process was applied that facilitated separation of inner (corona radiata) and outer cumulus cells. It should be kept in mind that corona radiata cells might have been contaminated with some outer cumulus cells. However, since this was a standardized denudation procedure [13] it can be assumed that the degree of contamination is limited and the same for all probes. In detail, COC were incubated in 80 IU hyaluronidase (Origio, Ma˚løv, Denmark) for 15 s followed by a rather coarse denudation process in BM1 medium using glass pipettes of an approximate diameter of 200 mm. This combined treatment helped to isolate CC from the periphery but left corona radiata cells attached. These innermost cells of the COC were then harvested by additional mechanical fine tuning without further contact to hyaluronidase. In order to minimize contact with hyaluronidase two washing steps in BM1 medium were performed (5000 U/min, Biofuge 13, Heraeus Instruments, Hanau, Germany). Finally, two cohorts of cells (corona radiata cells and outer cumulus cells) per patient were available and incubated separately in 40 ml BM1 medium each. For later analysis, samples were stored in a freezer ( 20 8C). It is important to note that in patients with a limited number of COC (and, thus, with a smaller number of corona radiata cells available for analysis) only outer cumulus cells were harvested in order to allow for proper evaluation (n = 8). In preclinical studies it could be demonstrated that neither centrifugation nor freezing had a negative effect on DNA strand break rate of CC. Immediately prior to workup CC were thawed in batches with the examiner being blinded to patient number and, more importantly, to the source of the somatic cells. For determination of the percentage of DNA-damaged cells an improved SCD test, the so-called Halosperm1 assay (Halotech DNA SL, Tres Cantos, Spain), was used. Although it has originally been designed for spermatozoa [11], it can also be used for cumulus cells as well. Although the present SCD test is not validated for usage in somatic cells yet its application appears attractive considering the fact that both SCD test and other strand break tests such as TUNEL assay [14] do predict the same level of DNA fragmentation. The latter method which is a well acknowledged assay to analyse strand breaks in cumulus cells [5,15]. One drawback of this approach is that the SCD test, unlike single cell gel electrophoresis (Comet assay), does not allow for proper quantification of the extent of strand breaks, since it relies on simple diffusion of DNA fragments alone (no electrophoretic force applied). Furthermore, it is not automatically said that with standard SCD treatment somatic cell DNA causes the same type of haloes than sperm DNA since their state of DNA organization is different (e.g., sperm chromatin being highly condensed due to protamine incorporation). Indeed, a less extensive halo in DNAintact cumulus cells was observed in the present study as compared to DNA intact sperms [16]. A total of 30 ml CC suspension were mixed with 40 ml of liquid agarose. This mix was pipetted onto precoated slides and covered with small coverslips. In a refrigerator (4 8C, 5 min), a microgel was produced with the CC embedded within. After the coverslip was removed slides were incubated in an acidic solution (contains hydrochloric acid) for 7 min and immersed in a lysing solution (contains dithiothreitol) for another 25 min (indirect proof of strand
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breaks requires denaturation of DNA). After a washing step in distilled water the probes were dehydrated in three ascending concentrations of ethanol (70–90–100%) for 2 min each, air-dried, and stored at room temperature. Finally, slides were stained for light microscopic counting. Strong staining was preferred to better visualize the periphery of the dispersed DNA halos. Whenever possible, 500 cells per sample were scored under the 100 objective of the microscope. The absence of reference haloes in somatic cells required proper validation of cumulus cell haloes. A batch of CC was used as a negative probe (no acidic and lysing solution used) in order to determine a reference value for CC without a halo representing damaged cells (13.91 0.50 mm). Since in normally distributed data it is expected that within a range of three standard deviations (e.g., 1.5 mm) 99.7% of all measured values can be found a threshold of ca. 15.5 mm was set to distinguish between damaged and DNA intact CC. ICSI and in vitro culture Within 1 h after the denudation process ICSI was performed using spermatozoa that had been accumulated with a swim-up procedure. ICSI was performed using Microtech pipettes (Gynemed, Lensahn, Germany) with the polar body at the 6 o’clock position with a minimum of ooplasm being aspirated. Only MIIoocytes of adequate quality (eggs with clusters of smooth endoplasmic reticulum or large vacuoles were not injected) were considered for fertilization. Fertilization check was carried out 16–20 h post insemination. On day 1, only 2-pronuclear zygotes were kept in culture which was done in small groups using 30 ml of EmbryoAssist medium (Origio). On days 2 and 3 of preimplantation development, all embryos were scored according to number and size of blastomeres as well as presence of fragmentation and multinucleation. Embryos of those patients considered for day 5 transfer (n = 38) were transferred to fresh 30 ml droplets of BlastAssist medium (Origio) on the morning of day 3. In this subgroup of patients presence and degree of compaction was checked on day 4. The next day blastocysts were scored according to their expansion, and by quality of inner cell mass and trophectoderm, respectively. It is important to note that for early blastocysts (Gardner score 1 and 2) scoring of the two cell lineages was not possible by definition. Embryos were transferred at cleavage stage (day 3) in 12 cases; however, two patients had no embryos available for transfer. The rest of the patients went for blastocyst transfer but in 4 cases all blastocysts had to be vitrified because of threatening ovarian hyperstimulation syndrome. In these cases the first vitrified/ warmed cycle was included in the pregnancy data. All transfers were done in BlastAssist medium using Gynetics catheters (Emtrac Set 4.2, Gynemed, Lensahn, Germany). Statistical analysis Linear correlation between two metric variables was assessed by the Pearson product-moment correlation coefficient. Moreover, we used Spearman rank correlation to study rank correlation, the dependence between two variables when at least one variable is ordinal, or to study the dependence of two variables in the present of outliers. If one variable was dichotomous, we used the pointbiserial correlation coefficient. Finally, for elucidating possible multivariate dependences or non-linear effects we used multivariate correlation models and non-linear regression models. The level of significance was 5% for all tests. Parameters in non-linear models were tested individually for significance. Moreover, we further tested the alternative hypothesis that the non-linear
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regression model is a better fit than a constant model by appropriate F-tests. We used the method of linear regression to control linear confounding variables. A Kolmogorov–Smirnov onesample test was used to check whether variables were normally distributed. If the normality assumption was violated, we studied differences in mean by a Kruskal–Wallis test. Otherwise, differences in mean were assessed by t-tests. Throughout the manuscript R denotes the correlation coefficient and p the level of significance. Metric values are presented as mean standard deviation. Results Cycle outcome A total of 555 COC were collected from 56 patients; however, only 438 (78.9%) gametes were found to be mature. The rest was either at prophase I (10.8%) or metaphase I (7.2%). Thirty-five of the COC did not contain an oocyte. Out of the metaphase II eggs 278 showed no extra- or intracytoplasmic anomalies (63.5%). The fertilization rate was 314/438 (71.7%). Almost all zygotes (99.4%) had cleaved the following day with 65.7% of them being of good quality. At this particular day 2 of preimplantation development multinucleation was found in 41 embryos (13.1%). A similar rate of good quality embryos was observed on day 3 (64.7%). In 38 patients embryos were further cultivated to blastocyst stage which represented 230 day 3 embryos. Out of these, 189 (82.2%) showed signs of compaction on day 4 and 146 (63.5%) reached blastocyst stage on day 5. Two thirds (98/146) were good quality blastocysts. Fifty patients had fresh embryo transfer and four a vitrified/ warmed one. A total of 23/54 biochemical pregnancies were achieved (42.6%). No significant differences in pregnancy rates were seen with respect to transfer day and cryostorage, respectively. There were two miscarriages and one ectopic pregnancy, resulting in 20 live births (live birth rate 37%). One multiple gestation was diagnosed in 23 pregnant patients (4.4%). The corresponding implantation rate was 24/66 (36.4%). No correlation between AMH and the occurrence of implantation as well as pregnancy was observed. However, AMH significantly correlated with level of estradiol at the day of ovulation induction (P < 0.0001), number of COC (P = 0.0002), number of MII oocytes (P = 0.0001) and, consequently, with number of fertilized eggs (P = 0.0008) and cleaving embryos (P = 0.0006). DNA fragmentation of cumulus cells Overall, more than 39,000 CC were collected and analysed for strandbreaks. Significantly more outer cumulus cells were harvested per patient as compared to corona radiata cells (P < 0.01). Pooled CC data revealed that 60.2% of CC (corona radiata plus outer CC) showed intact DNA. Interestingly, outer CC showed significantly higher (P < 0.01) rates of strand breaks than corona radiata cells (Fig. 1). Moreover, the percentage of viable corona radiata cells showed linear correlation to the formation of blastocysts of the respective patient (R = 0.511, P = 0.0127). Neither a linear nor a non-linear relationship between AMH and the percentage of outer CC or corona radiata cells without strand breaks could be detected (P > 0.05). However, by using linear regression to eliminate the confounding variable ‘‘female age’’, a significant correlation (R = 0.3364, P = 0.0304) between age corrected AMH and the percentage of viable outer CC was observed (Fig. 2).
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Fig. 1. Box plot of the percentage of strand breaks of outer cumulus cells and corona radiata cells. The red line indicates the median; the box indicates the 25th and 75th percentile. The whiskers extend to the most extreme data points.
Comments To the best of our knowledge, this manuscript provides first evidence that patients with rather low AMH levels under controlled ovarian hyperstimulation produce COCs of reduced quality as assessed by the rate of cumulus cells showing strand breaks in their DNA. Although it cannot be proved that the type of strand breaks is related to apoptosis (e.g., single-stranded) this scenario is rather likely since present data nicely correspond to other investigations on the possible correlation between cumulus cells and apoptosis performed with TUNEL analysis [17]. Our previous findings indicated that AMH values diverging from the norm are not only associated with poorer oocyte quality [18] but also with the health of the corresponding somatic cells. Considering the mutual interdependence of AMH expressing somatic cells and gametes bilateral damage could be a common scenario in such cases. Since AMH is mainly a product of the granulosa cells from preantral and small antral follicles [19] the question arises whether somatic cells of preovulatory follicles can serve as a model at all? With respect to this it could be shown that AMH is also expressed in granulosa cells from mature follicles although to a lesser extent than in those from smaller follicles [20]. Interestingly, in preovulatory follicles containing a mature MII-oocyte cumulus cells from the COC had a significantly higher AMH expression than mural granulosa cells from the same follicle [20] but a lower production of AMH as compared to cumulus cells from follicles containing immature oocytes [21]. The latter observation is in accordance with a lower expression of AMH receptor 2 in COC from mature vs.
Fig. 2. Linear correlation (R = 0.3364) between age-corrected AMH values and the percentage of viable outer cumulus cells (P = 0.0304).
immature oocytes [12]. With this information taken together, using the DNA integrity status of cumulus cells of preovulatory COCs and correlating it with basal AMH appears reasonable. However, one technical flaw has to be pointed out which is the inability to collect a sufficient number of corona radiata cells for DNA-analysis in patients with a low number of COCs (exclusively patients with low AMH). In these rare cases only outer cumulus cells could be analyzed which luckily are the ones mostly being affected. Thus, the theoretical risk of an invalidation of the data set is minimized. A couple of patient related parameters were shown to be associated with basal AMH levels, amongst which female age [22], ovarian response [23], basal FSH [24] and medical indication [25] were shown to correlate with very low or high AMH values. Interestingly, patients of advanced age and presumably lower AMH showed cumulus cells that had a significant change in protein expression as compared to cumulus cells from younger patients showing normal AMH ranges [26,27]. In particular, genes involved in processing oxidative stress [24] and oxidative phosphorylation [27] were affected, which may ultimately result in an overall decline of mitochondrial capacity in cumulus cells of older patients. Furthermore, tropomyosin-related kinase, thought to mediate survival signals in order to maintain a viable COC, is underexpressed in patients with low AMH showing diminished ovarian reserve [28], thus promoting apoptosis in these patients. Present data confirm the poorer quality of (outer) cumulus cells in patient with low AMH once corrected for female age. It would be interesting to know if the same negative correlation would also hold true for corona radiata cells. On the other side of the AMH scale mainly PCO patients make up the IVF clientele. It has been published that these patients, usually showing the highest AMH values, present cumulus cells that significantly differ in gene expression compared to normal responders. Apart from genes involved in steroid metabolism, especially members of the epidermal growth factor-like and IGFlike families known to play a role in oocyte competence were affected [29,30]. Filali et al. [31] showed a difference in gene expression of anti-apoptotic BCL2 in unprimed polycystic ovaries. These authors documented a relationship of the said protein content and oocyte maturity as well as fertilization. Others report changes in gene expression related to energy production [32]. Although there is evidence from literature that cumulus cells of patients with high AMH levels might be harmed, this hypothesis is not supported by the present results. Most likely, the limited number of PCO patients (those who are thought to show impaired cumulus cells) included in the present study could have introduced a bias. Thus, quite unexpectedly, higher values of AMH were associated with better quality outer cumulus cells after adjustment for female age. A follow-up study exclusively dealing with PCO patients might bring this to light. A rather new finding is the present observation that COCs of normal and good responders consist of two separate entities;
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corona radiata cells and cumulus cells. More than a decade ago it had been hypothesized that not all cells within the COC are equivalent; much rather there are certain subpopulation of cumulus cells which results in a localized gradient of oocytesecreted factors in the COC. Indeed, in a bovine model Hussein et al. [17] demonstrated an inside-out gradient in gene expression of Bone Morphogenetic Proteins (BMP) 6, 7 and 15. The same phenomenon was observed in human COCs when German colleagues [33] found significant differences in RNA expression of Connexin 43 and BMP-15 between corona radiata and outer layer cumulus cells. The increased amount of oocyte-secreted BMP-15 localized in corona radiata cells [17,33] is thought to reflect the oocyte’s tendency to prevent cumulus cell apoptosis [17]. In other words, the anti-apoptotic action of the oocyte follows a gradient, e.g. the incidence of apoptosis was lowest in the inner most layer of cumulus cells and increased with increasing distance from the oocyte. One of the downstream BMP15/GDF9 targets in cumulus cells (PTX3) was claimed to predict oocyte health, particularly ongoing pregnancy rate [34]. The importance of the health state of the corona radiata cells is further supported by the yet unpublished positive correlation between the health state of corona radiata cells and the blastocyst formation rate. Since DNA fragmentation, e.g., during apoptosis, necessitates preceding DNA strand breaks it is consistent that our SCD data show a significantly reduced amount of DNA damaged cells in the corona radiata as compared to the outer layers. Obviously, any harm to the COC starts from the outside where the protective capacity of the oocyte is inadequate. To summarize, present data provide first evidence that a simple strand break test on somatic CC, particularly the innermost corona radiata cells, enables clinical embryologists to speculate on the fate of the associated oocyte/embryo, although, this assumption is based on an overall ‘‘state of health’’ of CC. However, since at the present this proof of principle study has been performed the consequent next step would be to apply SCD test on a per COC basis, thus, allowing strand break rate to be used as a prospective non-invasive selection criterion. This would definitely add a value in clinical management and counseling of patients provided that the efficiency of SCD for testing human cumulus cells is confirmed or replaced by a commercial product especially adapted for somatic cells. Condensation Based on DNA integrity of innermost cumulus cells (corona radiata) clinical embryologists may predict the fate of the associated oocyte/embryo. References [1] Ebner T, Moser M, Sommergruber M, Tews G. Selection based on morphological assessment of oocytes and embryos at different stages of preimplantation development: a review. Hum Reprod Update 2003;9:251–62. [2] Meseguer M, Herrero J, Tejera A, Hilligsøe KM, Ramsing NB, Remohı´ J. The use of morphokinetics as a predictor of embryo implantation. Hum Reprod 2011;26:2658–71. [3] Gilchrist RB, Lane M, Thompson JG. Oocyte-secreted factors: regulators of cumulus cell function and oocyte quality. Hum Reprod Update 2008;14:159–77. [4] Ebner T, Moser M, Shebl O, Sommergruber M, Yaman C, Tews G. Blood clots in the cumulus-oocyte complex predict poor oocyte quality and post-fertilization development. Reprod Biomed Online 2008;16:801–7. [5] Corn CM, Hauser-Kronberger C, Moser M, Tews G, Ebner T. Predictive value of cumulus cell apoptosis with regard to blastocyst development of corresponding gametes. Fertil Steril 2005;84:627–33. [6] Nakahara K, Saito H, Saito T, et al. The incidence of apoptotic bodies in membrane granulosa can predict prognosis of ova from patients participating in in vitro fertilization programs. Fertil Steril 1997;68:312–7. [7] Assou S, Haouzi D, Mahmoud K, et al. A non-invasive test for assessing embryo potential by gene expression profiles of human cumulus cells: a proof of concept study. Mol Hum Reprod 2008;14:711–9.
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Reprod Biomed Online 2009;19(Suppl. 4):71–84. [32] Kenigsberg S, Bentov Y, Chalifa-Caspi V, Potashnik G, Ofir R, Birk OS. Gene expression microarray profiles of cumulus cells in lean and overweight-obese polycystic ovary syndrome patients. Mol Hum Reprod 2009;15:89–103. [33] Van der Ven K, Montag M, Drengner C, Ko¨ster M, van der Ven H. Differential gene expression profiles in cells of the corona radiata and outer cumulus oophorus in relation to oocyte competence. Hum Reprod 2009;Suppl. 1(14):i32. [34] van Tol HT, Vernooij JC, Colenbrander B, Gutknecht D, Macklon NS, Roelen BA. Expression of leptin receptor mRNA in cumulus cells is correlated with expression of PTX3. Reprod Biomed Online 2010;20:741–50.
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European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Viability of cumulus cells is associated with basal AMH levels in assisted reproduction Thomas Ebner a,b,e,*, Omar Shebl a,b, Sandra Holzer c, Peter Oppelt a,b, Erwin Petek d, Gudrun Schappacher-Tilp e, Richard B. Mayer a,b a
Landes- Frauen- und Kinderklinik, Departement of Gynecological Endocrinology and Kinderwunsch Zentrum Linz, Krankenhausstr. 26–30, Linz, Upper Austria, Austria Johannes Kepler University, Faculty of Medicine, Altenberger Straße 69, Linz, Upper Austria, Austria c Technical University of Graz, Institute of Biochemistry, Petersgasse 12, Graz, Styria, Austria d Medical University of Graz, Institute of Human Genetics, Harrachgasse 21, Graz, Styria, Austria e Karl-Franzens-University Graz, Department for Mathematics and Scientific Computing, Heinrichstraße 36, Graz, Styria, Austria b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 March 2014 Received in revised form 30 July 2014 Accepted 10 October 2014
Objective(s): An interesting non-invasive approach to select embryos for transfer is analyzing the health state of somatic granulosa cells surrounding the oocyte addressing their mutual dependence. This prospective study was set up to analyse whether the DNA integrity of cumulus cells correlates with preimplantation development and basal AMH levels. Study design: Therefore, 56 patients who gave written consent were enrolled. Sequential denudation of the cumulus-oocyte-complexes was performed in order to separate corona radiata from outer cumulus cells. DNA integrity of both cell types was analysed using a modified chromatin dispersion test. Results: The percentage of viable corona radiata cells per patient showed a linear correlation to blastulation (P < 0.05). These innermost cells showed significantly lower rates of strand breaks (P < 0.01) as compared to outer cumulus cells. Age-corrected AMH was significantly associated with the DNA integrity of outer cumulus cells (P < 0.05). Conclusion(s): For the first time it could be shown that in fact clinical embryologists deal with two different entities of cumulus cells, inner and outer ones. It seems that any protective mechanism of the female gamete follows an outward gradient, so that negative effects, e.g. apoptosis, may impair outer cumulus cells first. Age-corrected AMH reflects quality of these outer cumulus cells.Keywords: AMH; Corona radiata cells; DNA fragmentation; Outer cumulus cells; SCD test ß 2014 Elsevier Ireland Ltd. All rights reserved.
Introduction Along the way to reaching the ultimate goal of assisted reproduction technologies, namely the reduction of multiple pregnancy rates by strictly limiting the number of embryos being transferred, clinicians and clinical embryologists have to face the problem of identifying the embryos/blastocysts with the best prognosis. Naturally, invasive techniques, such as polar body, embryo or trophectoderm biopsy most accurately reflect the actual state of
* Corresponding author at: Landes- Frauen- und Kinderklinik Linz, Kinderwunsch zentrum, Krankenhausstr. 26-30, A-4020 Linz, Austria. Tel.: +43 732 6923 0; fax: +43 732 6923 24604. E-mail address: [email protected] (T. Ebner). http://dx.doi.org/10.1016/j.ejogrb.2014.10.015 0301-2115/ß 2014 Elsevier Ireland Ltd. All rights reserved.
health of gametes and embryos, with the latter approach being the most reliable one, since it maximizes insight (e.g. including postzygotic errors) while minimizing mechanical harm due to the biopsy process. However, selection of the best embryo for transfer in routine IVF/ICSI programs is currently based on less invasive techniques such as morphological evaluation. Prediction of implantation can be enhanced if morphological information of gametes, zygotes, and embryos at different stages of preimplantation development is pooled [1]. Nevertheless, this non-invasive method is far from being a perfect predictor of implantation. Recently, adding time course of mitotic divisions to static morphological observations started the era of morphokinetics, a field that may provide additional help in specifying implantation behavior of embryos [2]. Another non-invasive approach is analyzing the health state of somatic granulosa cells surrounding the oocyte addressing their
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mutual dependence [3]. Since both cell types grow in an identical milieu and experience comparable conditions in terms of nutrition during folliculogenesis, somatic cells may serve as an indirect marker of the health of the female gamete. With respect to this, morphological evaluation of the cumulusoocyte-complex (COC) turned out to be rather imprecise and is of limited importance in predicting maturity and quality of oocytes [4]. Much rather, the rate of apoptosis in cumulus cells (CC) was successfully used for accurately predicting fertilization [5], blastulation [5], and pregnancy [6]. Recently, the transcriptomic profile of CC was detected and correlated with embryo potential and pregnancy outcome [7]. Furthermore, non-invasive markers of oocyte chromosome status and competence were derived from CC gene expression [8]. It appears that the number of candidate genes in human cumulus cells that might be predictive of pregnancy outcome increases regularly [9]. Alternatively, the relative telomere length in cumulus cells at the time of egg collection may be taken into account as a new biomarker for selecting highly competent oocytes and embryos [10]. This prospective study was set up to analyse the quality of CC as assessed by the integrity of DNA strands in patients of different prognosis (based on basal AMH). For calculating DNA fragmentation of both corona radiata and outer cumulus cells for the first time a modified chromatin dispersion (SCD) test, originally designed for spermatozoa [11], was applied to CC.
Material and methods After ethical approval had been received (#12HO5) participation in this prospective trial was offered to all patients searching for ICSI treatment in the Kinderwunsch Zentrum Linz. A total of 6 patients refused participation for private reasons. It should be noted that cases of severe endometriosis were not included since it was not clear if quality of COC/CC might have been affected a priori. Patients Ultimately, 56 patients (34.0 4.3 years) gave written consent and were enrolled in the analysis. These patients suffered from male infertility in 22 (39%) cases (including three cases of ICSI with cryopreserved testicular spermatozoa), five patients were diagnosed with bilateral tubal blockage (9%), and eight (14%) with polycystic ovary syndrome or mild forms of endometriosis. The remaining cases were either unexplained or a mixture of male and female indications (38%). Half of the cycles involved were cases of secondary sterility (n = 28). Mean values of basal FSH (8.2 4.2 IU/ml), LH (6.3 3.2 IU/ ml) and estradiol (48.3 21.2 pg/ml) were within normal ranges and so was the AMH (5.1 4.3 ng/ml). Basal AMH (cycle day 3) was analysed using an enzyme immunoassay (Instrumentation Laboratory & Beckman-Coulter, Vienna, Austria). The analytical sensitivity of this ELISA was estimated at 0.1 ng/ml. The intraassay coefficient of variation is 12.3% and the interassay one 14.2%. All female partners were stimulated according to either an antagonist (62.5%) or an agonist scheme (37.5%). In the first regimen, recombinant products (Elonva1 or Puregon1, MSD, Vienna, Austria) were administered in approximately two thirds of the cases (n = 23), while urinary products (Menopur1, Ferring, Vienna, Austria) were used for the rest. The antagonist used was Orgalutran1 (MSD). In the long protocol, however, down regulation was performed with Buserelin (Suprecur1, Sanofi-Aventis, Vienna, Austria) and the gonadotrophins used were Puregon1or Menopur1. Since cumulus cell quality was found to be comparable in GnRH agonist and antagonist protocol respective data were pooled [12].
Ovulation was induced with 10,000 IU hCG (Pregnyl1, MSD). Oocyte retrieval was carried out transvaginally under ultrasound guidance 36 h after ovulation induction. On average, stimulation took 10.1 2.0 days and the estradiol level at the day of ovulation induction was found to be 1886 1698 pg/ml. Processing of cumulus cells COC were collected in BM1 medium (Laboratoires Eurobio, Les Ulis, France) and cultured for 2 h before a modified denudation process was applied that facilitated separation of inner (corona radiata) and outer cumulus cells. It should be kept in mind that corona radiata cells might have been contaminated with some outer cumulus cells. However, since this was a standardized denudation procedure [13] it can be assumed that the degree of contamination is limited and the same for all probes. In detail, COC were incubated in 80 IU hyaluronidase (Origio, Ma˚løv, Denmark) for 15 s followed by a rather coarse denudation process in BM1 medium using glass pipettes of an approximate diameter of 200 mm. This combined treatment helped to isolate CC from the periphery but left corona radiata cells attached. These innermost cells of the COC were then harvested by additional mechanical fine tuning without further contact to hyaluronidase. In order to minimize contact with hyaluronidase two washing steps in BM1 medium were performed (5000 U/min, Biofuge 13, Heraeus Instruments, Hanau, Germany). Finally, two cohorts of cells (corona radiata cells and outer cumulus cells) per patient were available and incubated separately in 40 ml BM1 medium each. For later analysis, samples were stored in a freezer ( 20 8C). It is important to note that in patients with a limited number of COC (and, thus, with a smaller number of corona radiata cells available for analysis) only outer cumulus cells were harvested in order to allow for proper evaluation (n = 8). In preclinical studies it could be demonstrated that neither centrifugation nor freezing had a negative effect on DNA strand break rate of CC. Immediately prior to workup CC were thawed in batches with the examiner being blinded to patient number and, more importantly, to the source of the somatic cells. For determination of the percentage of DNA-damaged cells an improved SCD test, the so-called Halosperm1 assay (Halotech DNA SL, Tres Cantos, Spain), was used. Although it has originally been designed for spermatozoa [11], it can also be used for cumulus cells as well. Although the present SCD test is not validated for usage in somatic cells yet its application appears attractive considering the fact that both SCD test and other strand break tests such as TUNEL assay [14] do predict the same level of DNA fragmentation. The latter method which is a well acknowledged assay to analyse strand breaks in cumulus cells [5,15]. One drawback of this approach is that the SCD test, unlike single cell gel electrophoresis (Comet assay), does not allow for proper quantification of the extent of strand breaks, since it relies on simple diffusion of DNA fragments alone (no electrophoretic force applied). Furthermore, it is not automatically said that with standard SCD treatment somatic cell DNA causes the same type of haloes than sperm DNA since their state of DNA organization is different (e.g., sperm chromatin being highly condensed due to protamine incorporation). Indeed, a less extensive halo in DNAintact cumulus cells was observed in the present study as compared to DNA intact sperms [16]. A total of 30 ml CC suspension were mixed with 40 ml of liquid agarose. This mix was pipetted onto precoated slides and covered with small coverslips. In a refrigerator (4 8C, 5 min), a microgel was produced with the CC embedded within. After the coverslip was removed slides were incubated in an acidic solution (contains hydrochloric acid) for 7 min and immersed in a lysing solution (contains dithiothreitol) for another 25 min (indirect proof of strand
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breaks requires denaturation of DNA). After a washing step in distilled water the probes were dehydrated in three ascending concentrations of ethanol (70–90–100%) for 2 min each, air-dried, and stored at room temperature. Finally, slides were stained for light microscopic counting. Strong staining was preferred to better visualize the periphery of the dispersed DNA halos. Whenever possible, 500 cells per sample were scored under the 100 objective of the microscope. The absence of reference haloes in somatic cells required proper validation of cumulus cell haloes. A batch of CC was used as a negative probe (no acidic and lysing solution used) in order to determine a reference value for CC without a halo representing damaged cells (13.91 0.50 mm). Since in normally distributed data it is expected that within a range of three standard deviations (e.g., 1.5 mm) 99.7% of all measured values can be found a threshold of ca. 15.5 mm was set to distinguish between damaged and DNA intact CC. ICSI and in vitro culture Within 1 h after the denudation process ICSI was performed using spermatozoa that had been accumulated with a swim-up procedure. ICSI was performed using Microtech pipettes (Gynemed, Lensahn, Germany) with the polar body at the 6 o’clock position with a minimum of ooplasm being aspirated. Only MIIoocytes of adequate quality (eggs with clusters of smooth endoplasmic reticulum or large vacuoles were not injected) were considered for fertilization. Fertilization check was carried out 16–20 h post insemination. On day 1, only 2-pronuclear zygotes were kept in culture which was done in small groups using 30 ml of EmbryoAssist medium (Origio). On days 2 and 3 of preimplantation development, all embryos were scored according to number and size of blastomeres as well as presence of fragmentation and multinucleation. Embryos of those patients considered for day 5 transfer (n = 38) were transferred to fresh 30 ml droplets of BlastAssist medium (Origio) on the morning of day 3. In this subgroup of patients presence and degree of compaction was checked on day 4. The next day blastocysts were scored according to their expansion, and by quality of inner cell mass and trophectoderm, respectively. It is important to note that for early blastocysts (Gardner score 1 and 2) scoring of the two cell lineages was not possible by definition. Embryos were transferred at cleavage stage (day 3) in 12 cases; however, two patients had no embryos available for transfer. The rest of the patients went for blastocyst transfer but in 4 cases all blastocysts had to be vitrified because of threatening ovarian hyperstimulation syndrome. In these cases the first vitrified/ warmed cycle was included in the pregnancy data. All transfers were done in BlastAssist medium using Gynetics catheters (Emtrac Set 4.2, Gynemed, Lensahn, Germany). Statistical analysis Linear correlation between two metric variables was assessed by the Pearson product-moment correlation coefficient. Moreover, we used Spearman rank correlation to study rank correlation, the dependence between two variables when at least one variable is ordinal, or to study the dependence of two variables in the present of outliers. If one variable was dichotomous, we used the pointbiserial correlation coefficient. Finally, for elucidating possible multivariate dependences or non-linear effects we used multivariate correlation models and non-linear regression models. The level of significance was 5% for all tests. Parameters in non-linear models were tested individually for significance. Moreover, we further tested the alternative hypothesis that the non-linear
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regression model is a better fit than a constant model by appropriate F-tests. We used the method of linear regression to control linear confounding variables. A Kolmogorov–Smirnov onesample test was used to check whether variables were normally distributed. If the normality assumption was violated, we studied differences in mean by a Kruskal–Wallis test. Otherwise, differences in mean were assessed by t-tests. Throughout the manuscript R denotes the correlation coefficient and p the level of significance. Metric values are presented as mean standard deviation. Results Cycle outcome A total of 555 COC were collected from 56 patients; however, only 438 (78.9%) gametes were found to be mature. The rest was either at prophase I (10.8%) or metaphase I (7.2%). Thirty-five of the COC did not contain an oocyte. Out of the metaphase II eggs 278 showed no extra- or intracytoplasmic anomalies (63.5%). The fertilization rate was 314/438 (71.7%). Almost all zygotes (99.4%) had cleaved the following day with 65.7% of them being of good quality. At this particular day 2 of preimplantation development multinucleation was found in 41 embryos (13.1%). A similar rate of good quality embryos was observed on day 3 (64.7%). In 38 patients embryos were further cultivated to blastocyst stage which represented 230 day 3 embryos. Out of these, 189 (82.2%) showed signs of compaction on day 4 and 146 (63.5%) reached blastocyst stage on day 5. Two thirds (98/146) were good quality blastocysts. Fifty patients had fresh embryo transfer and four a vitrified/ warmed one. A total of 23/54 biochemical pregnancies were achieved (42.6%). No significant differences in pregnancy rates were seen with respect to transfer day and cryostorage, respectively. There were two miscarriages and one ectopic pregnancy, resulting in 20 live births (live birth rate 37%). One multiple gestation was diagnosed in 23 pregnant patients (4.4%). The corresponding implantation rate was 24/66 (36.4%). No correlation between AMH and the occurrence of implantation as well as pregnancy was observed. However, AMH significantly correlated with level of estradiol at the day of ovulation induction (P < 0.0001), number of COC (P = 0.0002), number of MII oocytes (P = 0.0001) and, consequently, with number of fertilized eggs (P = 0.0008) and cleaving embryos (P = 0.0006). DNA fragmentation of cumulus cells Overall, more than 39,000 CC were collected and analysed for strandbreaks. Significantly more outer cumulus cells were harvested per patient as compared to corona radiata cells (P < 0.01). Pooled CC data revealed that 60.2% of CC (corona radiata plus outer CC) showed intact DNA. Interestingly, outer CC showed significantly higher (P < 0.01) rates of strand breaks than corona radiata cells (Fig. 1). Moreover, the percentage of viable corona radiata cells showed linear correlation to the formation of blastocysts of the respective patient (R = 0.511, P = 0.0127). Neither a linear nor a non-linear relationship between AMH and the percentage of outer CC or corona radiata cells without strand breaks could be detected (P > 0.05). However, by using linear regression to eliminate the confounding variable ‘‘female age’’, a significant correlation (R = 0.3364, P = 0.0304) between age corrected AMH and the percentage of viable outer CC was observed (Fig. 2).
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Fig. 1. Box plot of the percentage of strand breaks of outer cumulus cells and corona radiata cells. The red line indicates the median; the box indicates the 25th and 75th percentile. The whiskers extend to the most extreme data points.
Comments To the best of our knowledge, this manuscript provides first evidence that patients with rather low AMH levels under controlled ovarian hyperstimulation produce COCs of reduced quality as assessed by the rate of cumulus cells showing strand breaks in their DNA. Although it cannot be proved that the type of strand breaks is related to apoptosis (e.g., single-stranded) this scenario is rather likely since present data nicely correspond to other investigations on the possible correlation between cumulus cells and apoptosis performed with TUNEL analysis [17]. Our previous findings indicated that AMH values diverging from the norm are not only associated with poorer oocyte quality [18] but also with the health of the corresponding somatic cells. Considering the mutual interdependence of AMH expressing somatic cells and gametes bilateral damage could be a common scenario in such cases. Since AMH is mainly a product of the granulosa cells from preantral and small antral follicles [19] the question arises whether somatic cells of preovulatory follicles can serve as a model at all? With respect to this it could be shown that AMH is also expressed in granulosa cells from mature follicles although to a lesser extent than in those from smaller follicles [20]. Interestingly, in preovulatory follicles containing a mature MII-oocyte cumulus cells from the COC had a significantly higher AMH expression than mural granulosa cells from the same follicle [20] but a lower production of AMH as compared to cumulus cells from follicles containing immature oocytes [21]. The latter observation is in accordance with a lower expression of AMH receptor 2 in COC from mature vs.
Fig. 2. Linear correlation (R = 0.3364) between age-corrected AMH values and the percentage of viable outer cumulus cells (P = 0.0304).
immature oocytes [12]. With this information taken together, using the DNA integrity status of cumulus cells of preovulatory COCs and correlating it with basal AMH appears reasonable. However, one technical flaw has to be pointed out which is the inability to collect a sufficient number of corona radiata cells for DNA-analysis in patients with a low number of COCs (exclusively patients with low AMH). In these rare cases only outer cumulus cells could be analyzed which luckily are the ones mostly being affected. Thus, the theoretical risk of an invalidation of the data set is minimized. A couple of patient related parameters were shown to be associated with basal AMH levels, amongst which female age [22], ovarian response [23], basal FSH [24] and medical indication [25] were shown to correlate with very low or high AMH values. Interestingly, patients of advanced age and presumably lower AMH showed cumulus cells that had a significant change in protein expression as compared to cumulus cells from younger patients showing normal AMH ranges [26,27]. In particular, genes involved in processing oxidative stress [24] and oxidative phosphorylation [27] were affected, which may ultimately result in an overall decline of mitochondrial capacity in cumulus cells of older patients. Furthermore, tropomyosin-related kinase, thought to mediate survival signals in order to maintain a viable COC, is underexpressed in patients with low AMH showing diminished ovarian reserve [28], thus promoting apoptosis in these patients. Present data confirm the poorer quality of (outer) cumulus cells in patient with low AMH once corrected for female age. It would be interesting to know if the same negative correlation would also hold true for corona radiata cells. On the other side of the AMH scale mainly PCO patients make up the IVF clientele. It has been published that these patients, usually showing the highest AMH values, present cumulus cells that significantly differ in gene expression compared to normal responders. Apart from genes involved in steroid metabolism, especially members of the epidermal growth factor-like and IGFlike families known to play a role in oocyte competence were affected [29,30]. Filali et al. [31] showed a difference in gene expression of anti-apoptotic BCL2 in unprimed polycystic ovaries. These authors documented a relationship of the said protein content and oocyte maturity as well as fertilization. Others report changes in gene expression related to energy production [32]. Although there is evidence from literature that cumulus cells of patients with high AMH levels might be harmed, this hypothesis is not supported by the present results. Most likely, the limited number of PCO patients (those who are thought to show impaired cumulus cells) included in the present study could have introduced a bias. Thus, quite unexpectedly, higher values of AMH were associated with better quality outer cumulus cells after adjustment for female age. A follow-up study exclusively dealing with PCO patients might bring this to light. A rather new finding is the present observation that COCs of normal and good responders consist of two separate entities;
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corona radiata cells and cumulus cells. More than a decade ago it had been hypothesized that not all cells within the COC are equivalent; much rather there are certain subpopulation of cumulus cells which results in a localized gradient of oocytesecreted factors in the COC. Indeed, in a bovine model Hussein et al. [17] demonstrated an inside-out gradient in gene expression of Bone Morphogenetic Proteins (BMP) 6, 7 and 15. The same phenomenon was observed in human COCs when German colleagues [33] found significant differences in RNA expression of Connexin 43 and BMP-15 between corona radiata and outer layer cumulus cells. The increased amount of oocyte-secreted BMP-15 localized in corona radiata cells [17,33] is thought to reflect the oocyte’s tendency to prevent cumulus cell apoptosis [17]. In other words, the anti-apoptotic action of the oocyte follows a gradient, e.g. the incidence of apoptosis was lowest in the inner most layer of cumulus cells and increased with increasing distance from the oocyte. One of the downstream BMP15/GDF9 targets in cumulus cells (PTX3) was claimed to predict oocyte health, particularly ongoing pregnancy rate [34]. The importance of the health state of the corona radiata cells is further supported by the yet unpublished positive correlation between the health state of corona radiata cells and the blastocyst formation rate. Since DNA fragmentation, e.g., during apoptosis, necessitates preceding DNA strand breaks it is consistent that our SCD data show a significantly reduced amount of DNA damaged cells in the corona radiata as compared to the outer layers. Obviously, any harm to the COC starts from the outside where the protective capacity of the oocyte is inadequate. To summarize, present data provide first evidence that a simple strand break test on somatic CC, particularly the innermost corona radiata cells, enables clinical embryologists to speculate on the fate of the associated oocyte/embryo, although, this assumption is based on an overall ‘‘state of health’’ of CC. However, since at the present this proof of principle study has been performed the consequent next step would be to apply SCD test on a per COC basis, thus, allowing strand break rate to be used as a prospective non-invasive selection criterion. This would definitely add a value in clinical management and counseling of patients provided that the efficiency of SCD for testing human cumulus cells is confirmed or replaced by a commercial product especially adapted for somatic cells. Condensation Based on DNA integrity of innermost cumulus cells (corona radiata) clinical embryologists may predict the fate of the associated oocyte/embryo. References [1] Ebner T, Moser M, Sommergruber M, Tews G. Selection based on morphological assessment of oocytes and embryos at different stages of preimplantation development: a review. Hum Reprod Update 2003;9:251–62. [2] Meseguer M, Herrero J, Tejera A, Hilligsøe KM, Ramsing NB, Remohı´ J. The use of morphokinetics as a predictor of embryo implantation. Hum Reprod 2011;26:2658–71. [3] Gilchrist RB, Lane M, Thompson JG. Oocyte-secreted factors: regulators of cumulus cell function and oocyte quality. Hum Reprod Update 2008;14:159–77. [4] Ebner T, Moser M, Shebl O, Sommergruber M, Yaman C, Tews G. Blood clots in the cumulus-oocyte complex predict poor oocyte quality and post-fertilization development. Reprod Biomed Online 2008;16:801–7. [5] Corn CM, Hauser-Kronberger C, Moser M, Tews G, Ebner T. Predictive value of cumulus cell apoptosis with regard to blastocyst development of corresponding gametes. Fertil Steril 2005;84:627–33. [6] Nakahara K, Saito H, Saito T, et al. The incidence of apoptotic bodies in membrane granulosa can predict prognosis of ova from patients participating in in vitro fertilization programs. Fertil Steril 1997;68:312–7. [7] Assou S, Haouzi D, Mahmoud K, et al. A non-invasive test for assessing embryo potential by gene expression profiles of human cumulus cells: a proof of concept study. Mol Hum Reprod 2008;14:711–9.
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