Ovarian fecundity in patients with endometriosis can be estimated by the incidence of apoptotic bodies

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1 FERTILITY AND STERILITY VOL. 69, NO. 5, MAY 1998 Copyright 1998 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Ovarian fecundity in patients with endometriosis can be estimated by the incidence of apoptotic bodies Kenji Nakahara, M.D., Hidekazu Saito, M.D., Takakazu Saito, M.D., Mariko Ito, M.D., Nobuhiko Ohta, M.D., Toshifumi Takahashi, M.D., and Masahiko Hiroi, M.D. Department of Obstetrics and Gynecology, Yamagata University School of Medicine, Yamagata, Japan Objective: To investigate the incidence of apoptotic bodies in membrana granulosa in patients with endometriosis. Design: Retrospective analysis. Setting: Yamagata University Hospital. Patient(s): Fifty-eight normoovulatory women with endometriosis and 45 patients with male factor infertility underwent ovulation induction for IVF-ET with GnRH analogues and gonadotropins. Intervention(s): Patients underwent follicle aspiration after the administration of hcg. Main Outcome Measure(s): Recovered granulosa cells (GCs) were examined by fluorescence microscopy, and the incidence of apoptotic bodies was tabulated. These data and the other parameters associated with IVF results were analyzed on the basis of both the revised American Fertility Society (AFS) classification of endometriosis and the existence of. Result(s): The incidence of apoptotic bodies in membrana granulosa of patients with endometriosis was significantly higher than that of the control (male factor infertility) group and increased as the stage of the revised AFS classification advanced. The incidence of apoptotic bodies in membrana granulosa was significantly higher in patients with chocolate cysts than in those without. The patients with endometriosis had smaller numbers of developed follicles ( 15 mm), harvested oocytes, and mature oocytes than the male factor infertility patients. The existence of corresponded with a reduced number of both harvested oocytes and mature oocytes. Conclusion(s): Through the apoptosis of GCs, the existence of endometriosis may have negatively affected the follicle development and oocyte quality. (Fertil Steril 1998;69: by American Society for Reproductive Medicine.) Key Words: Apoptosis, apoptotic bodies, granulosa cells, in vitro fertilization, human, endometriosis, chocolate cyst Received June 23, 1997; revised and accepted November 25, Presented in part at the 53rd Annual Meeting of the American Society of Reproductive Medicine, Cincinnati, Ohio, October 18 22, Reprint requests: Kenji Nakahara, M.D., Department of Obstetrics and Gynecology, Yamagata University School of Medicine, Iida-Nishi, Yamagata City , Yamagata, Japan (FAX: ) /98/$19.00 PII S (98) In vitro fertilization and embryo transfer (IVF-ET) has been used to treat unexplained infertility and infertility due to causes such as tubal factor, male factor, and endometriosis. However, many investigators have reported that the outcome of IVF in patients with endometriosis is poor compared with the outcome in patients with other causes of infertility (1 4). The poor outcome associated with endometriosis may be due to mechanisms such as altered folliculogenesis (5), impaired fertilization (1, 4), embryo toxicity (6, 7), and defective implantation (2, 3, 8). Apoptosis, which is believed to be due to controlled cell death, was recently investigated intensively in various tissues with respect to both control and defense of living bodies (9). In various phases of apoptosis, the incidence of apoptotic bodies has been applied as one of the best morphological markers for prognosis of not only patients with neoplasms such as non- Hodgkin s lymphoma (10), prostatic intraepithelial neoplasms, and prostatic cancer (11) but also infertility patients undergoing IVF-ET treatment (12). Apoptotic bodies are revealed as cytoplasmic fragments containing condensed chromatin or fragments of condensed chromatin (10 12). During the process of follicular development, a limited number of follicles in the ovary continue to develop while most undergo atresia, which is associated with apoptosis (13). Apoptosis is found in the ovary not only during natural cycles but also during gonadotropin-stimulated cycles (14). Among various hormones associated with follicular development or atresia, gonado- 931

2 tropin-releasing hormone analogues (GnRH-a) have been reported to induce apoptosis in the rat ovary, whereas hmg and FSH block apoptosis (14). There have been few reports concerning apoptosis in IVF-ET programs. We previously reported that apoptosis could be detected in the ovaries of patients who underwent hyperstimulation for IVF (15). We also evaluated the incidence of apoptotic bodies of membrana granulosa on a per patient basis and showed that the incidence of apoptotic bodies resulted in a significant difference between the mural region and the cumulus region. The number of apoptotic bodies was significantly lower in patients with many oocytes retrieved than in those with few oocytes retrieved. In addition, the pregnant patient group had a significantly lower incidence of apoptotic bodies than the nonpregnant patient group (15). Moreover, individual follicles were analyzed with respect to both IVF results and the incidence of apoptotic bodies, and a lower incidence of apoptotic bodies resulted in better outcome (16). A significantly lower incidence of apoptotic bodies was observed in the follicles from which oocytes were retrieved than in those from which no oocytes were retrieved. A significantly lower incidence of apoptotic bodies was observed in follicles with fertilized oocytes than in those with unfertilized oocytes. In addition, a significantly lower incidence of apoptotic bodies was observed in the follicles with oocytes that became good embryos than in those with oocytes that became poor embryos (16). As shown above, the incidence of apoptotic bodies can be applied to evaluate ovarian function and oocyte quality. The aims of this study are: [1] to determine whether there is a difference among the incidence of apoptotic bodies in membrana granulosa of patients with stage I IV endometriosis on the basis of revised AFS classification; [2] to determine whether there is a difference in the incidence of apoptotic bodies in membrana granulosa of patients with endometriosis that have and those that do not have ( show the present state of endometriosis in the IVF treatment cycle); and [3] to determine whether the follicle development is affected according to the pathological state of endometriosis and the existence of. MATERIALS AND METHODS Patients and Follicle Aspiration Fifty-eight couples with endometriosis and a normal semen analysis were analyzed with respect to the incidence of apoptotic bodies in membrana granulosa between January 1995 and December 1995 at Yamagata University Hospital, Yamagata, Japan. The mean age of the study group was 35.5 years (range, 26 41). In addition, a control group of 45 couples with male factor infertility was used during the same period. The diagnosis of endometriosis was made laparoscopically for all patients, and they were treated surgically at the same time (e.g., aspiration of, adhesiolysis, and ablation or vaporization of endometriosis lesion). They were then treated with GnRH-a for 3 or 6 months. Those patients who had not conceived within 1 year from laparoscopy entered the IVF-ET program, and IVF-ET was performed at 3-month intervals. Patients who still had bilateral tubal adhesion due to endometriosis after surgical treatment and were not expected to conceive were immediately put on the IVF-ET schedule. The study was approved by the Yamagata University Hospital Committee for Research on Human Subjects. Written informed consent was obtained from all patients, and individual information about them was concealed. The ovarian hyperstimulation, follicle aspiration, and IVF protocol was described previously (12). When follicles of patients with endometriosis were aspirated, were not aspirated in the same syringe containing normal follicular fluid (FF). Cell Fixation and Quantification of Apoptotic Bodies Aspirated FF was transferred into tissue culture dishes (Falcon 3002; Becton Dickinson, Lincoln Park, NJ). Oocytecumulus cell complexes were isolated under a dissecting microscope (SZH-ILLB; Olympus, Tokyo, Japan) at magnification and put into an organ culture dish (Falcon 3037; Becton Dickinson) with human tubal fluid (HTF) medium (17). The oocyte-cumulus cell complexes were classified as mature or immature, as described previously (12, 18). Mural granulosa cell (GC) masses were put into another dish with HTF medium. The methods of fixing and staining GCs were described previously (12). Apoptotic changes of GCs start with condensation of chromatin followed by fragmentation of condensed chromatin. The apoptotic changes end when one apoptotic GC is fragmented into bodies containing some cytoplasm and condensed chromatin. These changes are examined by fluorescence microscopy as shown in our previous study (12). One thousand GCs were counted randomly at a magnification of 1,000 for each patient; the number of apoptotic cells out of these 1,000 GCs was calculated, and the percentage was calculated as the incidence of apoptotic bodies. Comparison Methods All patients with endometriosis had been diagnosed laparoscopically within 2 years before the IVF procedure, and the incidence of apoptotic bodies in membrana granulosa was tabulated. As a control group, 45 patients with male factor infertility were selected at random, and the incidence of apoptotic bodies in membrana granulosa was tabulated. On the basis of the revised American Fertility Society (AFS) classification of endometriosis, 58 patients with endometriosis were classified into stages I IV, and the incidence of apoptotic bodies in each group was analyzed. Those patients 932 Nakahara et al. Apoptosis and endometriosis Vol. 69, No. 5, May 1998

3 TABLE 1 Comparison of the incidence of apoptotic bodies in membrana granulosa between patients in the endometriosis group and in the male factor group. Mean incidence ( SEM) of apoptotic bodies (%) Characteristic No. of patients Mean age ( SEM) (y) Mural GC masses Cumulus cell masses Male factor group (control) Endometriosis group * * Revised AFS stage I II III IV * P 0.01 (versus the male factor group). P 0.05 (versus stage I). also were classified with respect to the existence of chocolate cysts in the IVF treatment cycle and analyzed. In sum, we analyzed the other parameters associated with IVF results, including patient age, maximal follicle diameter, the number of follicles ( 15 mm), endometrial thickness on the day of hcg administration, the day of aspiration, the number of oocytes aspirated, and the number of mature oocytes. Statistical Analysis Data are presented as means SEM. Statistical methods included Wilcoxon s test and the Mann-Whitney U test. The Kruskal-Wallis test was applied for analysis of the incidence of apoptotic bodies among stages I IV of the revised AFS classification. Analysis of variance (ANOVA) and Scheffe s F test were applied as post hoc tests. A P of 0.05 was defined as statistically significant. RESULTS Comparison Among Stages I IV Groups of Revised AFS Classification The incidence of apoptotic bodies in membrana granulosa of patients with endometriosis and patients in the male factor group is shown in Table 1. The incidence of apoptotic bodies in membrana granulosa of the group of patients with endometriosis was significantly higher than that of the male factor group (P 0.01). When the patients with endometriosis were classified by the revised AFS classification, the incidence of apoptotic bodies in both mural GCs and cumulus cell masses increased as the stage of the revised AFS classification advanced, with the exception of the mural GC masses at stage IV. When compared with stage I, stages III and IV revealed a significantly higher incidence of apoptotic bodies in both cumulus and mural GC masses (cumulus: III, IV; P 0.05; mural: III; P 0.05; IV; P 0.01) (Table 1). Comparison in Terms of the Existence of Chocolate Cysts Comparison of the incidence of apoptotic bodies in membrana granulosa between patients both with and without is shown in Table 2. The incidence of apoptotic bodies in mural GC and cumulus cell masses of patients with was significantly higher than that of patients without (P 0.05 and P 0.01, respectively) (Table 2). Comparison of the Other Parameters Associated With IVF Results Patients with endometriosis had a smaller number of developed follicles than patients with male factor infertility; the difference in the number of developed follicles between the patients in stages II and III of endometriosis and the patients with male factor infertility was statistically significant. The number of retrieved oocytes and mature oocytes in patients with endometriosis was significantly smaller than that in patients with male factor infertility (P 0.05) (Table TABLE 2 Relationship of each characteristic of patients with endometriosis with and without chocolate cysts. Characteristic Positive for (n 31) Negative for (n 27) Mean age ( SEM) (y) Mean incidence ( SEM) of apoptotic bodies Mural region (%) * Cumulus region (%) * P 0.05 (versus the group without ). P 0.01 (versus the group without ). FERTILITY & STERILITY 933

4 TABLE 3 Comparison of parameters associated with IVF among each revised AFS stage of endometriosis. Variable Male factor group (control) Endometriosis stage I II III IV Endometriosis stages I IV No. of patients Mean age ( SEM) (y) Mean ( SEM) maximal follicle diameter (mm) Mean ( SEM) no. of follicles 15 mm * * Mean ( SEM) thickness of endometrium (mm) Mean ( SEM) day of follicle aspiration Mean ( SEM) no. of oocytes retrieved Mean ( SEM) no. of mature oocytes * P 0.05 (versus the male factor group). P 0.05 (versus the male factor group). P 0.05 (versus the male factor group). 3). When patients with endometriosis were classified into two groups, i.e., those with and without, the numbers of follicles developed, retrieved oocytes, and mature oocytes in patients with were smaller than those in patients without (developed follicle: P not significant; retrieved oocytes and mature oocytes: P 0.05) (Table 4). Pregnancies in Patients With Endometriosis Five of 58 patients became pregnant. The patients who were in the advanced stages of endometriosis (stages III and IV) in the previous laparoscopic examinations and the group without in the IVF treatment cycle had a TABLE 4 Comparison of parameters associated with IVF between endometriosis group with and endometriosis group without. Variable Endometriosis group Positive for Negative for No. of patients Mean age ( SEM) (y) Mean ( SEM) maximal follicle diameter (mm) Mean ( SEM) no. of follicles 15 mm Mean ( SEM) thickness of endometrium (mm) Mean ( SEM) day of follicle aspiration Mean ( SEM) no. of oocytes retrieved * Mean ( SEM) no. of mature oocytes * * P 0.05 (versus the group without ). higher pregnancy rate than the group in stages I and II in the previous diagnostic examinations and the group with chocolate cysts in the IVF treatment cycle (stage I: zero pregnancies in 8 cycles; stage II: one pregnancy in 15 cycles; stage III: one pregnancy in 19 cycles; stage IV: three pregnancies in 16 cycles; when were present, there was one pregnancy in 31 cycles; when were not present, there were four pregnancies in 27 cycles). However, this difference is not significant because of the small number of patients. The incidence of apoptotic bodies in the GCs of pregnant patients (n 5) was lower than in the GCs of nonpregnant patients (n 53) (mural GC masses: nonpregnant, 1.6% 0.2% (mean SEM); pregnant, 0.9% 0.1%, P 0.05; cumulus GC masses: nonpregnant, 0.40% 0.05%; pregnant, 0.30% 0.08%, not significant). DISCUSSION The purpose of this study was to determine the degree of disturbance for folliculogenesis in the ovaries of the patients with endometriosis undergoing the IVF-ET procedure with respect to the incidence of apoptotic bodies in membrana granulosa. This study shows that the incidence of apoptotic bodies in membrana granulosa of patients with endometriosis was increased as the stage of the revised AFS classification advanced. Our previous data showed that a higher incidence of apoptotic bodies correlates with a lower quality of oocytes in individual follicles (16). Thus, this study indicates that the quality of oocytes from patients with endometriosis undergoing IVF programs decreases in proportion to advancing stages of the revised AFS classification. In fact, when the result of oocyte donation was classified according to the origin of the oocytes donated, patients who received embryos derived from ovaries with edometriosis showed a significantly reduced implantation rate compared 934 Nakahara et al. Apoptosis and endometriosis Vol. 69, No. 5, May 1998

5 with the remaining groups (19). Taken together, these findings suggest that infertility in patients with endometriosis is probably related to alterations in the oocyte quality, which, in turn, decrease the ability of oocytes to fertilize, develop, and implant. The existence of is considered one of the indicators of endometriosis in the ovary. Chocolate cysts prove the existence of a more advanced stage of endometriosis than the nonexistence of. The existence of increased the apoptosis in the follicles and gave, in turn, the follicles an atretic status. Consequently, patients with endometriosis with had smaller numbers of follicles developed, oocytes harvested, and mature oocytes. The revised AFS classification is a previous laparoscopic diagnosis of a pathological condition in the pelvis or lower abdominal cavity of patients with endometriosis. This diagnosis was performed at a maximum of 2 years before IVF treatment. In the term between the last laparoscopic diagnosis and this IVF treatment, various treatments for endometriosis were used, including enucleation of endometrioma, GnRH-a treatment, and alcoholic fixation of endometrioma. Thus, in the IVF cycle, the pelvic status sometimes has been much altered from the status of the last laparoscopic diagnosis by those treatments. Therefore, although most pregnancies were established in the higher stage (stages III and IV) of the revised AFS classification, in terms of the existence of, most pregnancies were established in patients without chocolate cysts. This result is quite reasonable because the existence or nonexistence of in the IVF cycle reveals the present status of endometriosis, and the pregnancy outcome just reflected the present ovarian status of the ability of folliculogenesis. There is another reason why most pregnancies were established in the higher stages of the revised AFS classification. The revised AFS classification mainly consists of two components, i.e., endometriosis lesion and adhesion in the pelvis. In most patients with endometriosis, adhesion became worse in proportion to the severity of the endometriosis lesion. However, in some patients with endometriosis who are in the higher stages of the revised AFS, there were severe adhesions in pelvic organs associated with minimal or mild endometriosis lesions, especially in the ovaries. In these patients, the disturbance of folliculogenesis might be minimal, and the good follicles can be developed in the IVF cycle. The existence of both adhesions and endometriosis lesions in the pelvic organs clearly affects the fecundity of the patients. Therefore, the revised AFS classification is a reasonable classification to evaluate fertility achieved through natural sexual intercourse. In the IVF treatment, however, adhesion is not a major factor influencing the fecundity of patients because oocytes are retrieved and introduced to the insemination place mechanically. Therefore, the quality of oocytes and follicles is one of the most essential factors to establish good outcomes in IVF treatments. Thus, in the ovaries, which indicate the presence of endometriosis, have a great influence on folliculogenesis through apoptosis in GCs; the existence of chocolate cysts should be determined, and endometriosis in the ovaries should be treated before IVF is performed to obtain good-quality oocytes and consequently to establish a high pregnancy rate in patients with endometriosis who are undergoing IVF. References 1. Mahadevan MM, Trounson AO, Leeton JF. The relationship of tubal blockage, infertility of unknown cause, suspected male infertility, and endometriosis to success of in vitro fertilization and embryo transfer. Fertil Steril 1983;40: Matson PL, Yovich JL. 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