OOCYTE RECOVERY FROM THE HUMAN OV ARY*

Transcription

1 FERTILITY AND STERILITY Copyright by The Williams & Wilkins Co. Vol. 23. No. 10. October 1972 Printed in U.S.A. OOCYTE RECOVERY FROM THE HUMAN OV ARY* LARRY L. MORGENSTERN, M.D.,t AND PIERRE SOUPART, M.D., PH.D. Department of Obstetrics and Gynecology, United States Army Tripier General Hospital, APO San Francisco, California 96438, and Department of Obstetrics and Gynecology, Vanderbilt University School of. Medicine, Nashville, Tennessee Recent advances in the in vitro culture of human ova have opened opportunities for experimental investigation of ovum maturation, fertilization, embryonic development, and capacitation of spermatozoa, as well as epididymal spermatozoa maturation studies in the human not possible a few years ago. However, a method is needed whereby a plentiful supply of normal human ova can be made available for study. Methods of recovery of the unfertilized ovum from the excised and the in situ human oviduct have proven to be difficult and productive of inadequate numbers of ova for in vitro culture study. 1 While a recently described technic for obtaining ova from the human uterus by transcervical flushing 2 is simple and nonsurgical, unfertilized ova recoverable by this method still fall far short of the required numbers, and the age of uterine ova is too advanced for studies of in vitro maturation, fertilization, or capacitation. Attempting to bypass the almost insuperable problems inherent in the recovery of ova from the female genital tract, other workers devised methods of oocyte recovery from excised ovarian tissue removed in the process of gynecologic surgery for various clinical conditions. The basic methodology used involved puncturing the follicle in its native site and Received May 24, * Supported by the United States Army Medical Research and Development Command and by United States Public Health Service. Contract NIH and Grant HD t Present address: Department of Obstetrics and Gynecology, University of New Mexico School of Medicine, Albuquerque, New Mexico searching out the released oocyte, but one study mentions oocyte recovery after mincing the excised ovary3 and another worker dissected the intact follicle free from the ovarian tissue prior to puncture. 4 The actual technic used ranged from simple incision with a surgical blades to aspiration of large follicles with syringe and needle 6 or glass pipet and mouth suction,3 while one report mentions puncturing all visible follicles but does not give technical details. 7 Although technically simple and productive of relatively large numbers of oocytes, recovery from the excised ovary has the objectionable feature that oophorectomy usually implies ovarian disease and/or ovarian senility and the resultant oocytes are in an immature form. Recognizing the desirability of obtaining oocytes which had completed much of the maturation process in vivo, Steptoe and Edwards8 developed a laparoscopic method whereby preovulatory oocytes could be recovered from the in situ ovary after treatment of the patient with purified human menopausal and human chorionic gonadotropin. Actual recovery of the oocyte through the laparoscope involved follicular aspiration with either a syringe and needle or a specially developed aspiration apparatus,8 the two technics giving similar oocyte recovery rates of 31.8% and 32.4%, respectively. The laparoscopic approach to the ovary makes recovery of preovulatory oocytes after previous gonadotropin treatment more feasible in that a major operation is not required and the process can be re- 751

2 752 MORGENSTERN AND SOUPART Vol. 2'3 peated, if necessary, on the same patient. Problems reported with the laparoscopic approach include limited access to the ovary and consequent difficulty in aspiration of the follicles if pelvic adhesions are present. Also, the clinical indications for laparoscopy (investigation of infertile married couples, pelvic pain, etc.) prevent classifying the oocytes as the product of "normal" donors. Recovery of viable oocytes from normal ovaries in quantities sufficient for meaningful investigative study requires a technic that: (a) is adaptable to both vaginal and abdominal exposure of the ovary; (b) can be used with safety and ease on the in situ ovary; (c) permits recovery of oocytes from small as well as large follicles; and (d) allows rapid recovery and identification of the oocyte in its native follicular fluid with a minimum of manipulation and trauma. This study describes results obtained with an oocyte recovery unit (ORU) which can be used on the in situ ovary independent of the surgical approach and is adaptable to all sizes of follicles. MATERIALS AND METHODS Oocyte Recovery Unit. The aspiration of oocytes from the follicles of in situ and excised ovaries was performed with an oocyte recovery unit (ORU) designed by one of the authors (L.L.M.). The assembled ORU is shown in Fig. 1 and consists of six components: (1) a 25- or 27 -gauge cartridge type double-end dental needle on a plastic base (Monoject Style #400, long); (2) a 20-gauge hypodermic needle bent into a gentle curve not greater than 45 0 and inserted through the plastic base of the dental needle; (3) a neoprene glass cartridge fitment diaphragm (West Company, Phoenixville, Penn., size No.5) through which the short end of the dental needle and the tip of the hypodermic needle are passed; (4) an 8.75 x 38 mm. silicone coated glass vial; (5) a 20-inch plastic extension tube; (6) a 10 ml. plastic syringe. All components are disposable and can be autoclaved. With the tip of the dental needle in the follicle, withdrawal of the syringe plunger creates a gentle vacuum in the vial via the hypodermic needle and the contents of the follicle are aspirated into the collection vial under low pressure. For exceptionally large follicles, modifications of the basic ORU included use of standard 2- ml. Vacutainer tubes, double-end 20- and 21-gauge needles and use of a vacuum pump instead of a syringe. As the latter modifications were found to result in more trauma to the follicle, the basic ORU depicted in Fig. 1 was utilized in the large majority of cases. Surgical Aspiration of Follicles. Follicle aspiration from the in situ ovary was carried out in the course of elective vaginal and abdominal operations for surgical sterilization and minor gynecologic pathology. The average delay in the surgical procedure was 5 min. Follicle aspiration after exposure of the ovaries by the vaginal approach is illustrated in Figs. 2 and 3. After removal of the uterus, a Babcock clamp adjacent to the lower pole of the ovary permitted easy manipulation, good exposure of the surface, and served to sta- FIG. 1. Assembled ovum recovery unit (ORU). Details of the component parts described in text.

3 October OOCYTE RECOVERY FROM HUMAN OVARY FIG. 2. Approach to follicle aspiration with the ORU in the course of elective surgical sterilization by the vaginal approach. Vaginal hysterectomy has been completed. The left ovary is exposed and stabilized by a Babcock clamp adjacent to one pole of the ovary. The follicle about to be aspirated is visible as a dark spot to the right of the ORU needle tip. FIG. 3. Completion of follicle aspiration with the ORU in the course of elective surgical sterilization by the vaginal approach. Vaginal hysterectomy has been completed. The left ovary is stabilized by means of a Babcock clamp adjacent to one pole of the ovary. A tonsil artery forceps in the left hand is being used as an extension for guiding the needle of the ORU. Aspiration of the follicle has been completed by an assistant out of the operative field. The follicular contents are visible in the collection vial of the ORU. FIG. 4. Follicle aspiration with the ORU in the course of elective surgical sterilization by the abdominal approach. The ovary is completely exposed subsequent to tubal ligation and permits access to all identifiable follicles. A follicle has just been aspirated and the contents are visible in the collection vial of the ORU. bilize the ovary during aspiration. After noting the number and size of the follicles present, the needle point of the ORU is introduced into a follicle (Fig. 2) with the bore of the needle oriented toward the base of the follicle. Tonsil artery forceps on the ORU were frequently useful as an extension for guiding the needle into the follicle. The follicular contents are then aspirated into the collecting vial by an assistant operating the syringe distant from the operative field (Fig. 3). Aspiration is terminated as soon as collapse of the follicle is noted and/or a drop of gross blood appears at the end of the dental needle in the collection vial. Aspiration of follicles after exposure of the ovaries by the abdominal route (e.g., tubal ligation) followed a similar procedure and is illustrated in Fig. 4. Aspiration of follicles from excised ovaries was performed with the ORU in the laboratory. RESULTS In 76 patients undergoing elective surgical procedures permitting exposure of the ovaries, 17 (22.4%) resulted in removal

4 754 MORGENSTERN AND SOUPART Vol. 23 of at least one ovary but ovarian conservation was practiced in 59 (77.6%) patients. Of the 76 patients involved in this study, 71 were found to have follicles present on at least one exposed ovary. Table 1 shows the number of patients yielding at least one oocyte according to the status of the ovary at the time of follicular aspiration and the type of surgical approach to the in situ ovary. Oocytes were recovered from the ovaries of 73.2% of 71 patients having follicles on at least one exposed ovary. Table 2 analyzes the oocyte recovery rate as a function of the number of follicles aspirated, the status of the ovary at the time of follicular aspiration, and the type of surgical approach to the in situ ovary. The recovery of 157 oocytes required the aspiration of 513 follicles for an over-all recovery rate of 30.6%. A comparison of the recovery rate from excised ovaries (40.6%) and in situ ovaries (25.9%) indicates that the aspiration of follicles from excised ovaries is more productive of oocytes than the aspiration of follicles from ovaries in situ. However, the similarity of recovery rates from the vaginal and abdominal approach to the in situ ovary, 24.1% and 27.8%, respectively, suggests that the method of exposure of the in situ ovary is of little consequence in successful oocyte recovery. Table 3 compares the results of different investigative teams with the ORU technic in terms of the number of patients serving as successful oocyte donors. Table 4 compares the results of the two research teams in terms of the number of follicles aspirated and the oocyte recovery rate using the ORU technic. Table 3 shows the vaginal approach to be more common in the TripIer series than in the Vanderbilt series (71.4% and 40.0% of the operations, respectively) and reflects the common practice at TripIer General Hospital of utilizing vaginal hysterectomy as a means of elective surgical sterilization. Likewise, the higher percentage of oophorectomies performed in the Vanderbilt series as compared with the TripIer series (37.5% and 10.3% of the operations, respectively) reflects the ovarian conservation expected in the latter institution with surgical sterilization of young women having normal reproductive function. However, in spite of these obvious differences in the clinical populations as well as the surgical approach and management of the two teams, it is apparent from Table 4 that the over-all recovery rates, 26.1% and 35.9%, are not widely divergent. Table 5 analyzes the relationship between age of the patient, the number of follicles observed, and the number of oocytes recovered. Unless the donor was over 40 years of age, age did not seem to influence either the number of follicles available for aspiration or the oocyte recovery rate. TABLE 1. Number of Patients Yielding at Least One Oocyte According to the Status of the Ovary at the Time of Follicular Aspiration and the Type of Surgical Approach to the in Situ Ovary In situ ovary Vaginal approach Abdom- inal ap- proach Excised ovary Grand No. of patients No. of patients yielding oocyte. Patients yielding 57.6% 86.4% 69.1% 87.5% 73.2% oocytes TABLE 2. Oocyte Recovery as a Function of Number of Follicles Aspirated, Ovarian Status at the Time of Follicular Aspiration, and the Type of Surgical Approach to the in Situ Ovary In situ ovary Vaginal approach Abdom- inal ap- proach Excised ovary Grand Follicle. aspirated Oocytes recovered Recovery rate 24.1% 27.8% 25.9% 40.6% 30.6%

5 October 1972 OOCYTE RECOVERY FROM HUMAN OVARY 755 TABLE 3. A Comparison of the Use of the ORU Technic by Two Different Investigative Teams in Terms of Number of Patients Serving as Successful Oocyte Donors Vaginal approach In situ ovary Abdominal approach Excised ovary Grand total Tripier series No. of patients No. of patients yielding oocytes Patients yielding oocytes 56.0% 90.0% 65.7% 100% 69.2% Vanderbilt series No. of patients No. of patients yielding oocytes Patients yielding oocytes 62.5% 83.3% 75.0% 83.3% 78.1% TABLE 4. A Comparison of the Use of the ORU Technic By Two Different Investigative Teams in Terms of the Number of Follicles Aspirated and the Oocyte Recovery Rate Ab dom- inal ap proach Vagi nal ap- proach In situ ovary Excised ovary Grand TripIer Series Follicles aspirated Docytes recovered Recovery rate 20.5% 26.2% 22.7% 36.9% 26.1% Vanderbilt Series Follicles aspirated Oocytes recovered Recovery rate 38.7% 28.3% 30.6% 43.0% 35.9% TABLE 5. Relationship between Age, Number of Follicles Identified, and Oocyte Recovery Age group Follicles Oocytes No. of Mean/ No. of Mean/ patients patients patients patients Grand total 75* * One patient was excluded because of absence of a follicle count. Table 6 is a tentative distribution of freshly recovered oocytes based on evaluation of their quality at the time of recovery using the dissecting and inverted phase microscope. Approximately 37% of the oocytes were obviously degenerate and would have virtually.no value as objects of study in culture. The remaining 63% are classified as likely to mature in an in vitro culture environment. While preselection for cell culture studies based on direct observation is only approximate at best, the results do indicate that the ORU technic is capable of supplying a large number of oocytes of good quality. The ORU technic seemed to result in little actual trauma to the oocyte. Only 6 (3.8%) of the 157 oocytes recovered showed anatomical damage to the zona pellucida that could be related to the recovery method. In most cases, corona radiata cells and cumulus obscured the zona damage, perforation not becoming apparent until examination of the oocytes after incubation in the fertilization medium. 9 That is, the perivitelline space was noted to be tightly packed with spermatozoa and rolling of the ovum eventually exposed the zona perforation. No problems with ovarian bleeding were encountered at the operation as a consequence of the follicular aspiration of in situ ovaries. A total of 55 patients were subjected to in situ follicle aspiration and none has developed hemorrhage or infection by 6 weeks postoperation. DISCUSSION This report describes a technic for oocyte recovery that takes advantage of

6 756 MORGENSTERN AND SOUPART Vol. 23 TABLE 6. Classification of Oocyte Quality Based on Observation at the Time of Recovery by the ORU Technic Using the Dissecting and Inverted Microscope Type of oocyte Cumulus mass Corona radiata cells Degenerate None None or few Nonovulatory None Several compact layers Preovulatory Undetermined Extensive, sticky, Loosley applied silvery and loosely applied Estimated Vitellus potential No. of oocytes for maturation Nonhomogeneous and/or Nil 52 (37.4) shrinkage within zona pellucida When details observable, Good 72 (51.8) germinal vesicle generally observed When details observable, Excellent 13 (9.4) germinal vesicle may be seen 2 (1.4) % 139 (100) elective gynecologic surgery in which the ovaries are exposed and the follicles can be directly aspirated. The probability that a given patient will serve as a successful oocyte donor (i.e., yield at least one'oocyte) seems to be proportional to the degree to which the ovarian surface is ex" posed and the extent to which manipula~ tion of the ovary is possible. Thus, the chances that an individual patient will yield an oocyte are greater if an ovary is excised during the operative procedure (87.5%) or the in situ ovary is approached by the abdominal route (86.4%). The smaller percentage of successful donors, when the in situ ovary is approached by the vaginal route (57.6%), is most likely related to limited access to the distal ovarian pole for purposes of follicle aspiration. For example, if the only follicle available on the ovary is at the upper pole and cannot be aspirated, the patient will represent an unsuccessful donor. On the other hand, a solitary follicle on an ovary exposed by the abdominal route, or on the excised ovary, can be utilized regardless of its location and the case will be counted as a successful oocyte donor experience. Although the abdominal approach to the in situ ovary was advantageous if only one or a limited number of follicles were available for aspiration, the actual number of oocytes recovered was independent of the route by which the in situ ovary was exposed. The; results show similar recovery rates from the in situ ovary approached by the vaginal (24.1%) and the abdominal route (27.8%). However, a comparison of the over-all recovery rate from in situ ovaries (25.9%) with the yield from excised ovaries (40.6%) indicated that, in the long run, the aspiration of follicles from the excised ovary was more productive ofoocytes than aspiration from follicles of the in situ ovary. The most likely explanation for the greater yield from the excised ovaries concerns the use of ovary bisection and transillumination to expose deep follicles and better de lin - eate follicular size and boundaries. In the present study, only 22.4% of the surgical procedures involved removal of the ovaries with subsequent oocyte recovery in the permissive environment of the laboratory. On the other hand, 77.6% of the surgical procedures required ovarian conservation and oocyte recovery was achieved from the in situ ovary with all the precautions and technical restrictions imposed by a surgical procedure. Thus, the lesser yield of oocytes from the in situ ovary as compared with the ex-

7 October 1972 OOCYTE RECOVERY FROM HUMAN OVARY 757 cised ovary is offset by the fact that the opportunity for aspiration of follicles from in situ ovaries was 3.5 times more frequent than with excised ovaries. The usefulness of the ORU technic as a means of supplying human oocytes for laboratory study was especially apparent in recovery from the in situ ovary. The safety of in situ recovery is indicated by the absence of both operative and postoperative complications that could be attributed to the method. The over-all recovery rate of 30.6% is comparable to that reported by Steptoe and Edwards 8 for laparoscopic recovery with syringe and needle (31.8%) and a special aspiration apparatus (32.4%). A comparison of the experience of two different investigative teams at different institutions shows similar oocyte recovery rates and indicates that the ORU technic is capable of reproducible results. Also, variation in the surgical technic of the operator is not a major factor in determining the over-all recovery rate. Age of the patient did not seem to significantly influence either the potential or actual recovery rate. Approximately 63% of the oocytes recovered by the method were of such quality as to permit utilization in in vitro culture studies. 9 Oocyte damage incident to the recovery technic is low (3.8%). The ORU technic described is a safe method whereby the investigator can obtain a sufficient supply of oocytes for systematic in vitro studies on human preimplantation phenomenon. The oocytes can be recovered "at random" from untreated ovum donors undergoing surgery and unscheduled with respect to their menstrual cycle. The oocytes can also be recovered following preoperative treatment designed to impose some control over the menstrual cycle and oocyte maturation. 8, 9 The usefulness of the technic for in situ oocyte recovery by the vaginal approach makes it possible for women with normal reproductive function but undergoing vaginal hysterectomy as a sterilization procedure to serve as normal donors of oocytes. SUMMARY A simple method is described for recovery of oocytes from the in situ ovary exposed in the course of vaginal and abdominal surgery. The in situ recovery rate of 25.9% was not significantly influenced by age of the patient, surgical approach to the in situ ovary or variations in the operative technic of different surgeons. An oocyte yield of 40.6% was obtained using the same technic on excised ovaries for an over-all recovery rate of 30.6%. Approximately 63% of the oocytes were of adequate quality for in vitro culture studies and only 3.8% showed evidence of mechanical damage. Application of the technics to the in situ ovary was shown to be safe and successful thus providing a method whereby women with normal reproductive function undergoing elective sterilization can serve as oocyte donors. Acknowledgment. The authors wish to acknowledge the cooperation of the resident physicians and operating room staff at TripIer General Hospital and Vanderbilt University Hospital, as well as. the secretarial skills of Mrs. Barbara Tsuchiya. REFERENCES 1. CLEWE, T. H., MORGENSTERN, L. L., NOYES, R. W., BONNEY, W. A., BURRUS, S. B., AND DEFEO, V. J. Searches for ova in the human uterus and tubes. Amer J Obstet Gynec 109:313, CROXATTO, H. B., FUENTEALBA, B., DIAZ, S., PAS TENE, L., AND TATUM, H. J. A simple nonsurgical technique to obtain unimplanted eggs from human uteri. Amer J Obstet Gynec 112:662, JACOBSON, C. B., SITES, J. G., AND ARIAs-BERNAL, L. F. In vitro maturation and fertilization of human follicular oocytes. Int J Fertil 15:103, EDWARDS, R. G. Maturation in vitro of human ovarian oocytes. Lancet ii:926, SEITZ, H. M., ROCHA, G., BRACKETT, B. G., AND MASTROIANNI, L. Cleavage of human ova in vitro. Fertil Steril 22:255, SHETTLES, L. B. Human blastocyst grown in vitro

8 758 MORGENSTERN AND SOUPART Vol. 23 in ovulation cervical mucus. Nature (London) 229: 343, KENNEDY, J. F., AND DONAHUE, R. P. Human oocytes: Maturation in chemically defined media. Science 164:1292, STEPTOE, P. G., AND EDWARDS, R. G. Laparoscopic recovery of pre-ovulatory human oocytes after priming of ovaries with gonadotropins. Lancet i: 683, SOUPART, P., AND MORGENSTERN, L. L. Human sperm capacitation and in vitro fertilization. In Preparation.