Ciconia Research and Development Aps, Copenhagen, Denmark

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1 FERTILITY AND STERILITY VOL. 77, NO. 3, MARCH 2002 Copyright 2002 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. in human cumulus cells in relation to zona pellucida thickness variation, maturation stage, and cleavage of the corresponding oocyte after intracytoplasmic sperm injection Erik Høst, M.Sc., a,c Anette Gabrielsen, M.Sc., b Svend Lindenberg, M.D., Ph.D., a,c and Steen Smidt-Jensen, M.D., Ph.D. a,d Ciconia Research and Development Aps, Copenhagen, Denmark Received May 29, 2001; revised and accepted September 5, Reprint requests: Erik Høst, M.Sc., Ciconia Research and Development Aps, Symbion Fruebjergvej 3, DK-2100, Denmark (FAX: ; eh@ciconia.dk). a Ciconia Research and Development Aps, University of Copenhagen, Copenhagen, b Ciconia Århus, University of Copenhagen, Copenhagen, c Department of Obstetrics and Gynaecology, Herlev Amtssygehus, University of Copenhagen, Copenhagen, d Department of Obstetrics and Gynaecology, Hvidovre Hospital, University of Copenhagen, Copenhagen, /02/$22.00 PII S (01) Objective: To assess the degree of apoptosis in the cumulus cells and the variation of the zona pellucida and the maturity and fertilization of the corresponding oocyte. Design: Retrospective study. Setting: Private fertility clinic. Patient(s): Fifty couples undergoing ICSI. Intervention(s): ICSI. Main Outcome Measure(s): Correlation between apoptosis in the cumulus cells and the zona pellucida thickness variation, maturation stage, fertilization rate, and embryo score. Result(s): This study demonstrated no correlation between apoptosis in cumulus cells and the thickness and variation of the zona pellucida in oocytes and embryos. The incidence of apoptosis was significantly higher in cumulus cells from empty zona pellucidas and germinal vesicle stage and metaphase I oocytes compared with metaphase II oocytes. Nonfertilized metaphase II oocytes showed significantly higher incidence of apoptosis compared with fertilized metaphase II oocytes. There was a correlation between embryo score and the zona pellucida thickness variation. Conclusion(s): in cumulus cells had no impact on the zona pellucida thickness and variation in oocytes and embryos. The zona pellucida thickness variation was positively correlated to good embryo score. A higher degree of apoptosis was seen in cumulus cells from immature oocytes compared with mature oocytes. Furthermore, apoptosis in cumulus cells impaired the fertilization rate of metaphase II oocytes after ICSI. (Fertil Steril 2002;77: by American Society for Reproductive Medicine.) Key Words:, cumulus, fertilization rate, maturation, zona pellucida eliminates comprised or superfluous cells. It is a complex process that involves a variety of different signaling pathways and results in a multitude of changes in the dying cell, and it plays an important role in the normal function of all tissues. The cell undergoes shrinkage, chromatin margination, membrane blebbing, nuclear condensation, segmentation, and division into apoptotic bodies, which may be phagocytosed (1, 2). In mammalian and human follicular development, apoptosis can be initiated in at least four different cell compartments, i.e., the techa cells, the granulosa cells, the cumulus cells, and the oocyte itself. Several studies demonstrated the presence of apoptosis in the granulosa and cumulus cells during the maturation of the oocyte-cumulus complex, specifically during the resumption of the meiosis (3 7). Other studies have demonstrated that healthy embryo development is based on a well-correlated and coordinated oocyte development before ovulation in spite of immature oocyte maturation process. Both in vitro and in vivo maturation of these oocytes are dependent on a nuclear and cytoplasmic maturation. The impact, still unknown, from the cumulus cells through gap junctions to the oocytes is of vital 511

2 importance for these processes (8, 9). It has been shown that fewer granulosa lutein cells are apoptotic in women who achieved pregnancy than in women who did not conceive after IVF (10). Standard morphological criteria used in evaluating embryo quality include the rate of division judged by the number of blastomeres, size, shape, symmetry, and cytoplasmic appearance of the blastomeres and the presence of anucleate cytoplasmic fragments (11). Recently, some interest has been generated in studying a unique parameter for predicting clinical outcome after IVF, based on the thickness and variation of the zona pellucida (ZP) (12). Reports on this subject highlighted the strong influence of the thickness of ZP and the ZP thickness variation (ZPTV) of the transferred embryos on outcome after IVF. Some have clearly suggested the ZPTV of the embryo as a good predictor for success after IVF (12 14). Previously, in a smaller study, we demonstrated a correlation between the degree of apoptosis-activated cumulus cells and the stage of maturation in the oocyte and the fertilization rate after ICSI (15). The purpose of this study was to evaluate the presence of cumulus cells with apoptosis of the corresponding oocyte and correlate these findings with the thickness and variation of ZP, maturation stage, fertilization rate, and embryo score. MATERIALS AND METHODS Patients The study population consisted of 50 consecutive couples who fulfilled the criteria for ICSI because of oligozoospermia (16) or previously failed fertilization by IVF, and who were referred to assisted reproduction at the Fertility Clinic, Ciconia, Århus, The number of recovered oocytecumulus complexes was 352 (median, ). The ages of the female patients varied between 26 and 43 years (median, ), and the ages of the males partners between 27 and 53 years (median, ). Ovarian hyperstimulation, long downregulation, GnRH, recfsh, hcg, and oocyte retrieval were performed as described elsewhere (17). The same protocol was used on the patients who failed conventional IVF and those who went directly to ICSI. The study was approved by the Institutional Review Board. Cumulus Cell Preparation, Analysis, and Assessment of A total of 352 oocyte-cumulus complexes were denudated separately 2 6 hours after retrieval in hyaluronidase 80 IU/mL (Medi-Cult, Jyllinge, Denmark). Nineteen oocytes were in the germinal vesicle phase, 25 were in metaphase I, and 292 were in metaphase II. Sixteen oocyte-cumulus complexes were scored as empty ZPs. The cumulus cells were collected and washed twice in Earle s balanced salt solution (Medi-Cult). The pellet with cumulus cells was then pipetted onto a Silan-coated microscope slide and a thin smear was prepared. Slides were air dried and fixed in 96% (w/v) ethanol for 5 minutes. For staining of the cumulus cells for the detection of apoptosis we used the Apoptag In Situ Detection Kit Peroxidase (INTERGEN) with some modifications (18). Briefly, the Apoptag kit is designed to label the free 3 -OH DNA termini in situ with chemically labeled and unlabeled nucleotides. The nucleotides contained in the reaction buffer are enzymatically added to the DNA by terminal deoxynucleotidyl transferase (TdT). TdT catalyzes a template-independent addition of nucleotide triphosphates to the 3 -OH ends of double or singlestranded DNA. The incorporated nucleotides from an oligomer composed of digoxigenin allowed the binding of an anti-digoxigenin antibody that is conjugated to peroxidase. The localized peroxidase enzyme then catalytically generates an intense signal from chromogenic substrates. and morphology were scored according to peroxidase/ DAB staining. To evaluate the degree of apoptosis, about 300 cumulus cells were analyzed on each slide, if possible, under the microscope by oil immersion with a magnification of times and bright-light field illumination. The slides were controlled by the same observer twice, as the slides were randomly reassessed. The intraobservation variation was found to be less than 2%. ZPTV Measurement The ZP thickness of all oocytes just before sperm injection and all embryos immediately before scheduled transfer on day 2 after pickup was recorded. The ZP thickness evaluations were performed directly under an inverted microscope (Nikon) equipped with a Hoffman modulation contrast optics using an ocular micrometer, calibrated to provide a direct value of the zona thickness (19). All ZPTV measurements were computed from the videocinematography recordings of the oocytes and embryos. The morphological images were recorded in a computerized database (FQC, Fercom, Copenhagen, Denmark) with a color videocamera (Panasonic) mounted on an inverted microscope with Hoffman modulation contrast (Nikon). The settings for microscopic observations were 200 magnification and bright-light field illumination. The ZP of each oocyte and embryo was subjected to three independent measurements, and calculation of the ZPTV was carried out in the FQC system. The ZPTV (in percent) within individual oocytes and embryos was calculated as described by Cohen et al. (12): 512 Høst et al. in cumulus cells and zona pellucida Vol. 77, No. 3, March 2002

3 ZPTV [(ZP max ZP mean )/ZP mean ] 100. ICSI, Oocyte, and Embryo Culture The sperm sample was collected 1 hour before the ovum pickup. Then pure sperm preparation sperm cell counts were assessed in a Makler chamber according to the WHO manual (16). The study group was characterized as undergoing ICSI because of a sperm count less than 20 million spermatozoa/ml or previously failed fertilization by IVF. ICSI. Two to six hours after ovum pickup, cumulus cells were removed using hyaluronidase, 80 IU/mL (Medi-Cult), and a single gross morphologically normal spermatozoon was immobilized in polyvinylpyrrolidone (Scandinavian IVF, Gothenburg, Sweden) and injected into the denudated oocyte. Culture. After incubation for hours, the oocytes were checked for the presence of pronuclei. Preincubation and embryo culture were carried out in Earle s medium with 2% human serum albumin. On the morning of ET, embryo score was judged, which included the rate of division, the number of blastomeres, size, shape, symmetry, and cytoplasmic appearance of the blastomeres, and the presence of anucleate cytoplasmic fragments (11). A low embryo score (range, ) was correlated with good embryo quality (20). Embryos with the most optimal morphology score and the most advanced stage of development were selected for transfer. The Mann-Whitney rank sum test and Fisher s exact test were used. Values were considered statistically significant when P.05. RESULTS We found that the incidence of apoptosis was significantly higher in cumulus cells from empty ZPs and germinal vesicle stage and metaphase I oocytes compared with cumulus cells from metaphase II oocytes (P.0001). No correlation was found between the degree of apoptosis in the cumulus cells and the thickness and variation of the ZP of the oocytes before ICSI from the day of ovum pickup (Table 1). As seen in Table 2, the number of apoptosis-activated cumulus cells had an impact on the fertilization rate after ICSI in the metaphase II oocytes. A significantly lower number of cumulus cells with apoptosis was seen in those oocytes that fertilized compared with those that did not (P.0211). The ZP thickness and variation from the day of ovum pickup and the day of ET showed no significant difference between those metaphase II oocytes that fertilized and those that did not. However, embryos with a score 2.1 had a significantly higher ZP thickness variation compared with embryos with a score 2.1 (P.0018). in cumulus cells had no impact on the quality, i.e., score of the embryos. TABLE 1 Maturation stage of oocytes: the percentages of apoptosisactivated cumulus cells, ZP thickness, and variation. Numbers are given as mean SD. Oocyte maturation stage (n) Empty ZP (n 16) Germinal vesicle (n 19) Metaphase I (n 25) Metaphase II (n 292) a P cumulus cells The age of the female did not correlate with any of the main outcome measures after ICSI (P.05). The implantation rate in this study group was 21.6%. DISCUSSION ZP thickness ( m) ZPTV a a Høst. in cumulus cells and zona pellucida. Fertil Steril The oocyte-cumulus complex comprises an intimate relation between a cumulus cell syncytium in conjunction with the oocyte involving large gap junctions. The cumulus cells produce camp, which keeps the oocyte in a meiotic arrest. When stimulated with FSH, and before the LH peak, these gap junctions are open and allow even large molecules to move freely between the cumulus cells through the ZP into the oocyte. Therefore, the signaling and regulation of the apoptotic process can easily be distributed from each cell in the oocyte-cumulus complex. One can speculate whether oocytes with connected cumulus complexes that are apoptosic activated have influenced or compromised further developmental potential and condition of the ZP in the corresponding oocyte and embryo. In this study, we demonstrated two significant influences of apoptosis in cumulus cells on the development of the corresponding oocyte: 1. in the cumulus cells was correlated to the maturation stage of the corresponding oocyte. A significantly higher proportion of apoptosis-activated cumulus cells was found in immature oocytes, i.e., metaphase I and germinal vesicle oocytes, 38 hours after the LH peak (hcg injection). 2. In metaphase II oocytes, the degree of apoptosis in the adjacent cumulus cells had an impact on the fertilization rate of the corresponding oocyte after ICSI. Similar findings were reported by Høst et al. (15). Including these oocyte-cumulus complexes, the total amount was FERTILITY & STERILITY 513

4 TABLE 2 Metaphase II oocytes and fertilization and embryo score: the percentages of apoptotic cumulus cells and ZP thickness and variation on the day of ovum pickup (day 0) and the transfer day (day 2). Numbers are given as mean SD. cumulus cells ZP thickness, day 0 ( m) ZPTV day 0 ZP thickness, day 2 ( m) ZPTV day 2 Metaphase II oocytes, nonfertilized (n 107) a Metaphase II oocytes, fertilized (n 185) a Embryo score 2.1 (n 59) b Embryo score 2.1 (n 126) b a P P Høst. in cumulus cells and zona pellucida. Fertil Steril , which indicates [1] a general decay or controlled cell destruction of these cumulus complexes before ovum pickup and [2] that apoptosis in cumulus cells does have an influence on the embryo development. This might be caused by transfer of the apoptotic signals from the cumulus cells into the oocyte. Therefore, the timing of the gap junction closure between the cumulus cells and the oocytes and the initiation of apoptosis may be crucial temporal events in the oocyte and embryo development. Consequently, LH induction of the final maturation process of the immature oocyte could be compromised by apoptosis. Other authors also demonstrated that granulosa cells in the follicles from which oocytes were subsequently fertilized showed a significantly lower incidence of apoptotic bodies than those in follicles from which the oocytes did not fertilize. Furthermore, a correlation between apoptosis in the membrane granulosa cells and the quality of embryos was seen (6). The ZP is a thick extracellular coat surrounding all mammalian oocytes and preimplantation embryos. The ZP supports communication between oocytes and follicle cells during oogenesis, protects oocytes and embryos during development, and regulates interactions between ovulated oocytes and spermatozoa during and after fertilization (21). A significant linear decreasing relationship exists between the mean ZP thickness of each patient and the maximum E 2 level, which increases with the hmg dose. A relationship between the ZP thickness and cumulus maturity appeared to be nonsignificant (22). It has been shown in the developing pre-embryo that the ZP thickness and specifically that the thickness variation is of significant importance for the hatching process and production of living offspring (14). Thus, a dynamic change in the zona composition during embryogenesis is of importance. Specifically, during in vitro culture, zona hardening has been mentioned as a problem for hatching (23). One of the possible important modulators of the ZP composition during oogenesis and early embryo development could be the closely related corona radiata (i.e., cumulus granulosa cells). These cells penetrate the zona and regulate the oocyte development and resumption of meiosis (24). In this study, we analyzed the relation and impact of cumulus cell apoptosis at the time of ovum pickup, oocyte nuclear maturation, embryo development, and ZP thickness and variation. We did not find any evidence of a relationship between cumulus cell apoptosis, i.e., early controlled cell death, and the different status of the zona morphology. Thus, the cumulus cell retraction and apoptosis had no influence on the ZP; however, an indirect impact is possible. We observed that apoptosis in the cumulus cells is highly associated with retarded nuclear development of the oocyte or atresia. However, a low number of apoptosic-activated cumulus cells indicates an undisturbed cumulus-oocyte relation and a healthy ovum, which later induces ZP changes. In conclusion, apoptosis in cumulus cells had no impact on the ZP in the corresponding oocyte and embryo. However, the maturity and fertilization of the oocyte were correlated with apoptosis in the cumulus cells. Acknowledgments: The authors thank the technicians, Ciconia Århus, for helpful collection and preparation of cumulus cells. References 1. Darzynkiewicz Z, Juan G, Li X, Gorczyca W, Murakami T, Traganos F. Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis). Cytometri 1997;27: Kerr JFR, Harmon BV. Definition and incidence of apoptosis: an historical perspective. In: molecular basis of cell death. Cold Spring Harbor Laboratory Press, Vinatier D, Dufour Ph, Subtil D. : a programmed cell death involved in ovarian and uterine physiology. Eur J Obstet Gynecol Reprod Biol 1996;67: Billig H, Assarsson B, Björnheden D, Svensson E, Thelander H, Utter M. Atresia as an apoptotic process. Frontiers in Endocrinology 1995; 18: Billig H, Chun SY, Eisenhauer K, Hsueh AJ. Gonadal cell apoptosis: hormone-regulated cell demise. Hum Reprod Update 1996;2: Høst et al. in cumulus cells and zona pellucida Vol. 77, No. 3, March 2002

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