RECOVERY OF MIDCYCLE HUMAN FOLLICULAR OOCYTES: CORRELATION OF THEIR MORPHOLOGY WITH ENDOMETRIAL AND FOLLICULAR HISTOLOGY

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1 r FERTILITY AND STERILITY Copyright ~ 1978 The American Fertility Society Vol. 29,.5, May 1978 Printed in U.S.A. RECOVERY OF MIDCYCLE HUMAN FOLLICULAR OOCYTES: CORRELATION OF THEIR MORPHOLOGY WITH ENDOMETRIAL AND FOLLICULAR HISTOLOGY DONALD EARL MOORE, M.D.* ROBERT S. THOMPSON, PH.D.t ROBERT ISRAEL, M.D. Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Southern California School of Medicine, Los Angeles County Medical Center, Los Angeles, California The ovarian follicles of 23 of 33 women undergoing elective pelvic laparotomy during menstrual cycle days 11 to 17 were aspirated by a low-vacuum device. The study compared the ovulatory status of women in midcycle with the health of the oocytes obtained, and determined the frequency of recovery of mature oocytes without the preadministration of exogenous hormones. Maturing or mature oocytes were obtained from 22% and nonmature oocytes from 48% of the 23 women. Sixty per cent of the women in each of the maturing or mature and nonmature groups were postovulatory as judged by evaluation of the endometrial and ovarian tissue recovered. Furthermore, 20% of the maturing or mature oocytes and 65% of the nonmature oocytes showed a moderately heavy amount of cytoplasmic degeneration, which was independent of ovulatory status. Therefore, it is concluded that, because of the relatively low rate of return of oocytes, the frequent postovulatory status of patients in midcycle, and uncertainty about the viability of the oocytes so obtained, women not stimulated by exogenous hormones are ill-suited for recovery of maturing or mature follicular oocytes for in vitro fertilization studies. Fallopian tube destruction accounts for a large proportion of infertility. Despite improved surgical techniques, intrauterine pregnancy following repair of severe tubal disease is infrequent. Oocyte retrieval, in vitro fertilization, and embryo transfer offer an alternate means of treatment in instances of irrevocable tubal infertility. Much successful animal and human research has been conducted in this field. 1-4 Recovery of preovulatory, and potentially mature, oocytes has been enhanced by the administration of clomiphene citrate or human menopausal gonadotropin to patients The following study was. designed to Received October 27,1977; accepted vember 21, *Present address and address for reprint requests: Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, RH-20, University of Washington School ofmedicine, Seattle, Wash tpresent address: California Primate Research Center, University of California, Davis, Calif provide supportive physiologic and histologic correlates for oocyte recovery by assessing follicular and oocyte development in the midcycle human ovary not exposed to exogenous hormones. MATERIALS AND METHODS To facilitate recovery of mature ova, 37 women undergoing elective pelvic laparotomy between menstrual cycle days 11 and 17 were selected. All but one woman had proven fertility, all had regular menstrual cycles, and they ranged in age from 20 to 50 years (average, 36.2 years). Basal body temperatures were not recorded and no attempt was made to induce ovulation. Of the 37 women undergoing laparotomy, a syringe with a 20- or 25-gauge needle was used for aspiration in 4 women. ova were obtained. In addition, no aspiration was attempted in 10 other women, of whom 7 had no discernible follicles and 3 had only corpora lutea on the

2 Vol. 29,.5 RECOVERY OF MIDCYCLE HUMAN FOLUCULAR OOCYTES 519 FIG. 1. The low-pressure device used to aspirate human follicles. Left to right: aspiration needle, control clamp, collection tube with sterile medium, baftle, and vacuum tube. ovarian surface. Therefore, these 14 women were eliminated from the study. A low-vacuum aspiration device similar to the one described by Berger et a1. 7 was used to evacuate follicles in the remaining 23 women (Fig. 1). The operating end of the device consisted of a 1~ inch, blunted, 20cgauge needle. The.needle was connected to a polyethylene catheter leading to a 15-ml blood-collection tube containing 1 mlof sterile culture medium (Ham's. FlO or F12). A second polyethylene catheter connected the collection tube to the vacuum source, which consisted of a 15-ml vacuum blood-collection tube and a 125- or 250-ml Erlenmeyer flask baffie (located between the vacuum tube and collection vessel). A small Kelly clamp between the aspiration needle and collection tube served to control the system and to prevent entry of blood into the collection tube. When possible, each follicle was aspirated into a separate collection tube so that the morphology and stage of maturation of any recovered ovum could be compared with the histology of its respective follicle. The contents of all aspirated follicles were examined with a dissection microscope, and any ovum found in the aspirate was fixed in 2.5% glutaraldehyde, postfixed in 1% osmium tetroxide, dehydrated, embedded in Epon 812, and serially sectioned for high-resolution light microscopy.8 The criteria for ovum maturation (nonmature, and maturing or mature classification) are those described by Zamboni et a1.8 Aspiration was performed in situ in 10 of the 23 women. The uterus and ovaries were extirpated from the other 13 women, and aspiration of the surface follicles was performed within 3 hours of ovariectomy. All tissues were subsequently fixed in formalin, and representative areas of the endometrium and ovary were trimmed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. In 9 of the 13 women, areas of aspirated follicle walls.were dissected and individually fixed in formalin or Technicon FU48 so as to compare the oocyte with the histology of its respective follicle. All tissues were stained with hematoxylin and eosin after sectioning, and the endometrium was dated' according to the criteria of yes et a1. 9 Corpora lutea were distinguished from follicles by gross appearance and/or by the criteria of Corner. 10 The degree of follicular atresia was characterized by the following criteria: pyknosis of nuclei, foamy-appearing cytoplasm, extremely palelooking cytoplasm, and the presence of isolated cell nuclei lacking cytoplasmic components. Serum samples were obtained from 9 of the 23 patients at the time of surgery. Luteinizing hormone (LH) and progesterone levels were measured

3 520 May 1978 MOOREETAL. TABLE 1. Relationship between Stage of Meiotic Maturation, Oocyte and Follicular Cell Degeneration, and Stage of the Menstrual Cycle Patient Stage of meiosis Metaphase II Prophase I Metaphase I Meta-anaphase I Metaphase I Cytoplasmic vacuolization Moderately heavy Profile deformity Follicle cell degeneration Corpus luteum Secretory endometrium Cycle day Moderate Moderate in the sera of these patients by established procedures in our laboratory. 11 By these studies, any LH surge could be identified and progesterone levels suggesting prior ovulation (above 3 ng/ml) could be ascertained Sixty-one follicles ranging in size from 4 to 25 mm were aspirated from 23 women, for an average of 2.7 follicles/woman. Of the 61 follicles, 14 were aspirated from the in situ ovaries of 10 women (1.4 follicles/woman); 47 follicles were evacuated from the extirpated ovaries of 13 women (3.6 follicles/woman). Ova were successfully recovered from 70% of the patients studied (16 of 23). Seven ova were obtained from five of the ten women (50%) who underwent in situ ovarian aspiration, and all seven ova displayed prominent nuclei by light microscopy and were therefore classified as nonmature. On the other hand, 18 ova were obtained from the aspirates of 11 of the 13 women (85%) who had undergone ovariectomy. Of the 18 ova, 13 were judged nonmature and 5 were maturing or mature on the basis of microscopic identification of chromosomes and/or polar bodies. A description of the general microscopic appearance of the five maturing or mature ova is presented in Table 1. One ovum was found to be in first prophase, two ova were in metaphase I, one ovum was in meta-anaphase I, and one ovum was fully mature in the metaphase II configuration. The general cytologic appearance of nonmature and maturing or mature ova is displayed in Figures 2 to 5. Both ova in metaphase I were obtained from women (patients 10 and 23) presumed to be preovulatory on the day of surgery according to serum LH levels of 133 and 88 miu/ml (LH surge levels), serum progesterone levels of 0.5 and 0.7 ng/ml, late proliferative endometria, and no evidence of corpora lutea. In both ova, the cytoplasm showed slight vacuolization with minimal profile deformity. Although one ovum showed no follicular atresia; in the other ovum approximately one-third of the surrounding follicular cells showed signs of atresia. The remaining three maturing or mature ova were recovered from women who were considered postovulatory on the day of surgery as confirmed by the presence of recent corpora lutea. The ovum in metaphase II (mature) and the surrounding follicular cells were in excellent condition; the ovum in prophase I showed moderately heavy signs of degeneration as evidenced by cytoplasmic vacuolization and organelle clumping, whereas the ovum in meta-anaphase I showed only slight cyto- 2. This nonmature follicular oocyte has not resumed meiosis and possesses a nucleus with associated chromatin. A slight amount of cytoplasmic vacuolization is evident in the oocyte (toluidine blue-stained, l-llm thick section, x800). FIG. 3. This follicular oocyte in prophase I demonstrates the process of chromatin condensation and nuclear membrane dissolution characteristic of this meiotic phase. The oocyte shows a moderately heavy amount of vacuolization (toluidine blue-stained, l-llm thick section, x800). RESULTS FIG.

4 Vol. 29,.5 RECOVERY OF MIDCYCLE HUMAN FOLLICULAR OOCYTES 521 granulosa cells. The corona radiata and adherent layers of granulosa cells around the 20 nonmature oocytesshowed some signs of degeneration in 16 of 20 cases (80%). In 40% of cases (8 of 20) degeneration of the corona and granulosa ranged from moderate to heavy. The relationship of these signs of oocyte and/or follicular degeneration to the potential viability of ova in culture is not known with certainty. DISCUSSION FIG. 4. This follicular oocyte has reached the metaphase I stage of meiosis; some of the condensed chromosomes are seen. This oocyte did not possess a polar body, the latter being a feature of the second metaphase stage of meiotic maturation (toluidine blue-stained, I-JLm thick section, x800). plasmic vacuolization. Of the surrounding follicular cells, only those around the ovum in metaanaphase I showed moderate signs of degeneration; the cells around the other ovum showed no degeneration. Of the 20 nonmature oocytes recovered, 7 showed no or slight signs of atresia, 9 showed moderate signs, and 4 showed severe signs. In instances where nonmature oocytes were obtained from women who underwent total abdominal hysterectomylbilateral salpingo-oophorectomy, 60% (6 of 10) were postovulatory as evidenced by the presence of a recent corpus luteum and/or a secretory endometrium. Biopsies of specific follicles in two preovulatory women, from which healthyappearing nonmature oocytes were aspirated, showed no signs of atresia. Specific follicular biopsies in four postovulatory women, from which nonmature oocytes with various stages of atresia were found, did show signs of atresia in the FIG. 5. A fully mature human follicular oocyte in metaphase II of meiosis. te the fluted appearance of the first polar body and some of the second metaphase chromosomes (arrow) in the adjacent oocyte cytoplasm (toluidine bluestained, I-JLm thick section, x800). An attempt was made to obtain oocytes from women in midcycle for two main reasons: (1) to test the feasibility of obtaining maturing or mature oocytes from non-hormonally primed women and (2) to test the operation of a lowvacuum aspiration collecting device. The results obtained with the device are in general agreement with those of Steptoe in that, compared with syringe aspiration, the collecting device obtained clearer fluid and improved the rate of oocyte recovery.s In the present study, aspiration of ovarian follicles with a syringe yielded no ova, whereas with the device ova were recovered from 41% of the follicles aspirated and from 70% of the women who had visible ovarian follicles. The aspiration device was inexpensive, required only readily available materials, was easy to use, afforded good control because of its low pressure, and required no connection to an external suction source. Five maturing or mature oocytes were obtained from sixty-one follicles for a recovery rate of 8%. These ova were recovered from 5 of the 23 women in whom aspiration was attempted, for an individual recovery rate of 22%. This is one of the higher rates reported in the literature for normally cycling women without hormonal priming. This high rate may be attributed to the midcycle range of all of the patients. Several investigators have obtained human oocytes in all phases of the cycle without hormonal stimulation. Sanyal et al. 13 reported only 3.3% mature or maturing oocytes of 152 recovered by dissection from 58 ovaries, compared with 20% of 25 oocytes reported in the present study. All of the maturing or mature oocytes recovered by Sanyal et al. 13 were obtained from women undergoing oophorectomy during the late proliferative phase of the cycle. Of the 145 women whom Shea et al. 14 studied, mature oocytes were obtained from only 3 (2.1%), compared with the 22% rate (5 of 23) reported in this study. Morgenstern and Sou-

5 522 MOOREETAL. May 1978 part15 reported 13 "preovulatory" oocytes recovered from 76 women, or 17.1%. If one desires to fertilize oocytes without preculturing and without exogenous hormonal stimulation, the 22% recovery rate in this study is low. By contrast, Steptoe and Edwards, 5 in a report concerned with ovarian stimulation, indicated that 23 to 35 of a total of 68 oocytes that they evaluated-cytologically from a larger pool were probably maturing or mature (34% to 51%), compared with only 5 of 25 (20%) in our study without exogenous hormones. Many of the women in our study were postovulatory as evidenced by the presence of a recent. corpus luteum and/or a secretory endometrium. Three of the five women (60%) from whom maturing or mature oocytes were obtained had corpora lutea, indicating prior ovulation. Likewise, 60% of the women from whom nonmature oocytes were recovered were also postovulatory. In contrast, Steptoe and Edwards 5 reported that a corpus luteum was observed in only one woman (1.7%) after 59 courses of human menopausal gonadotropin/human chorionic gonadotropin priming. In the present study, 1 of the 5 maturing or mature ova (20%) and 13 of the 20 nonmature ova (65%) showed moderate to severe signs of cytoplasmic degeneration. The granulosa cells surrounding 20% of the maturing or mature ova (1 of 5) showed moderate signs of degeneration, while the granulosa around 40% of the nonmature oocytes (8 of 20) showed moderate to heavy signs of atresia. In toto, only 40% (2 of 5) ofthe maturing or mature and only 20% (4 of 20) of the nonmature oocytes revealed no or slight degeneration in the cytoplasm of the ovum or in the surrounding follicular cells. Although one cannot be certain that cytoplasmic vacuolization and/or follicular cell atresia imply a poor chance for fertilization and embryonic development, the postovulatory status of many of the women in this study and the low yield of oocytes obtained per woman indicate that the use of exogenous hormonal stimulation for follicular growth and timing of laparoscopic retrieval of oocytes is needed to ensure that a relatively large population of maturing or mature oocytes is available for recovery. Acknowledgment. The authors would like to acknowledge the assistance of D. Moyer, M.D., in dating the endometrial, follicular, and corpora lutea tissue. REFERENCES 1. Rock J, Menkin MF: In vitro fertilization and cleavage of human ovarian eggs. Science 100:105, Menkin MF, Rock J: In vitro fertilization and cleavage of human ovarian eggs. Am J Obstet Gynecol 55:440, Edwards RG: Studies on human conception. Am J Obstet Gynecol 117:587, Wood C, Talbot M, McMaster R, Erhardt A, Leeton J, Lawson R, Dooley M, Paterson P, Blackwell J: Treatment of tubal infertility by artificial fertilization. In Obstetrics and Gynecology Year Book. Chicago, Year Book Medical Publishers, 1975, p Steptoe PC, Edwards RG: Laparoscopic recovery of preovulatory human oocytes after priming of ovaries with gonadotrophins. Lancet 2:683, Edwards RG, Steptoe PC: Control of human ovulation, fertilization and implantation. Proc R Soc Med 67:30, Berger MJ, Smith DM, Taymor ML, Thompson RS: Laparoscopic recovery of mature human oocytes. Fertil Steril 26:513, Zamboni L, Thompson RS, Smith DM: Fine morpholgoy of human oocyte maturation in vitro. BioI Reprod 7:425, yes RW, Hertig AT, Rock J: Dating the endometrial biopsy. Fertil Steril1:3, Corner GW: The histological dating of the human corpus luteum of menstruation. Am J Anat 98:377, Mishell DR, Nakamura RM, Crosignani PG, Stone S, Kharma K, Nagata Y, Thorneycroft IH: Serum gonadotrophin and steroid patterns during the normal menstrual cycle. Am J Obstet Gynecol 111:60, Israel R, Mishell DR, Stone SC, Thorneycroft IH, Moyer DL: Single luteal phase serum progesterone assay as an indicator of ovulation. Am J Obstet Gynecol 112:1043, Sanyal MK, Taymor ML, Berger MJ: Cytologic features of oocytes in the adult human ovary. Fertil Steril 27:501, Shea BF, Baker RD, Latour JPA: Human follicularoocytes and their maturation in vitro. Fertil Steril26:1075, Morgenstern LL, Soupart P: Oocyte recovery from the human ovary. Fertil Steril 23:751, 1972