COLLECTION OF HUMAN OOCYTES AT LAPAROSCOPY AND LAPAROTOMY

Transcription

1 FERTILITY AND STERILITY Copyright 1974 The American Fertility Society Vol. 25, No. 12, December 1974 Printed in U.s.A. COLLECTION OF HUMAN OOCYTES AT LAPAROSCOPY AND LAPAROTOMY ALEXANDER LOPATA,* M.B., B.S., PH.D., IAN W. H. JOHNSTON, M.B., B.S., F.RC.O.G., JOHN F. LEETON, M.B., B.S., F.R.C.O.G., DOROTA MUCHNICKI, B.S., B.Sc., JOHN Mc. TALBOT, M.B., B.S., M.RC.O.G., AND CARL WOOD, M.B., B.S., F.RC.O.G. Department of Obstetrics and Gynaecology, Monash University, Queen V ictoriahospital, Melbourne, Victoria, 3000, Australia, and Sterility Clinic and Department of Pathology, Royal Women's Hospital, Carlton, Victoria, 3053, Australia Meiotic maturation, fertilization, and growth in vitro of human follicular oocytes have been studied in media of various complexities supplemented with sera and other ingredients. HI Despite these studies, little is known about the pattern of metabolism of human oocytes and zygotes at various stages of their development. The paucity of studies in this area is in part due to the inadequate numbers of oocytes available. Thus, the purpose of this study was to determine whether ovarian follicular aspiration at laparoscopies would provide sufficient numbers of oocytes to permit adequate in vitro studies, including preliminary testing of culture media for in vitro fertilization and meiotic chromosome preparation. Steptoe and Edwards l2 used a laparoscopic method for collecting oocytes from the ovaries of women who could not conceive due to pelvic or fallopian tube pathology or because of oligospermic husbands. These women were treated with human postmenopausal and chorionic gonadotropins. About 30 hours after the chorionic gonadotropin injection, laparoscopy was performed; follicles ranging from 0.25 to 3.0 cm were aspirated either with a syringe and needle or with an aspiration device. All other workers have gained access to the ovaries at major surgical procedures for gynecologic pathology or tuballigations.1-7,lo.11,13 Since laparoscopy Received January 4, *Present address: Department of Biology, University of California, Los Angeles, California is now frequently used for sterilization of normal women, follicular aspiration during this procedure should provide a method of collecting oocytes during the normal menstrual cycle. Moreover, follicular aspiration carried out during diagnostic laparoscopy for infertility enables examination of the patient's oocytes and follicular fluid, thus broadening the scope of the investigation. In the present study the laparoscopic method of collecting human oocytes was compared with two different methods of oocyte recovery used at laparotomies. MATERIALS AND METHODS Collection of oocytes at laparoscopy. Most of the patients requiring elective laparoscopy were married women who were being investigated due to their inability to conceive. The other patients required laparoscopy for sterilization by tubal diathermy or as a diagnostic procedure for various gynecologic conditions. An attempt was made to perform all laparoscopies during the preovulatory stage of the menstrual cycle; this was determined by the length of the cycle and the dates of the last period. Patients taking oral contraceptives were not included in these studies. Clomiphene or gonadotropins were not administered prior to surgery. After dilatation and curettage, a metal vacuum cannula was attached to the cervix to facilitate manipulation of the uterus during the laparoscopy. Approxi-

2 Vol. 25, No. 12 COLLECTION OF OOCYTES 1031 ~ I FIG. 1. Arrangement of instruments for aspirating ovarian follicles at laparoscopy. The needle used for aspirating follicles is passed through the anterior abdominal wall midway between the laparoscope and the Palmer forceps. Aspirated follicular fluid collects in the test tube trap placed on the surgical drapes. mately 2.5 liters of carbon dioxide was introduced into the peritoneal cavity using a Semm pneumoperitoneum apparatus. The laparoscope used was a Hopkins forward oblique telescope, wide angle 5.B mm (Storz), incorporating fiber optic light transmission. The instrument was introduced through a small stab incision immediately below the umbilicus; once in position the sheath of the laparoscope was used to deliver a reduced supply of CO 2 to maintain the pneumoperitoneum. A Palmer diathermy biopsy forceps was then introduced in the midline through its own trocar and cannula at a point approximately 3 cm above the symphysis pubis. The laparoscope and forceps were thus separated by the greatest possible distance; which allowed for ease of independent manipulation and minimal visual interference. A 20 gauge needle with 45 bevel (24 cm long with its own 15 gauge sheath) was then introduced in the midline half way between the laparoscope and the Palmer forceps (Fig. 1). This needle was connected by a no. 2 gauge translucent vinyl tubing to a glass test tube (inner diameter, 10 mm; capacity, 5 ml) by a metal inlet passing through a rubber stopper which was used to seal the tube. A metal outlet in the stopper was connected in tum by heavy gauge rubber tubing to a Twin-O-Vac suction jar, a venturi vacuum system (Fig. 2) activated by the piped O2 or air supply. The test tube acted as a collection trap for fluid aspirated from follicles when suction was generated in the system. The vacuum gauge of the venturi system was set at 200 mm of

3 1032 LOPATAET AL December 1974 Pf'ED AIR LAPAROSCOPY TO LAPAROTOMY ON -Cff Control FO..UCULAR FLUID TRAP GAUGE FIG. 2. A diagrammatic representation of the equipment used for aspirating ovarian follicles at laparoscopy and laparotomy. mercury, which was found to be optimal for oocyte collection at laparoscopies. Thereafter, suction at the preset vacuum was activated-as required by the operator-by an "on-off' foot valve. An assistant held the uterus in full anteversion by using the metal vacuum cannula attached to the cervix; this permitted manipulation of the ovaries which were grasped by the Palmer forceps. The follicles appeared as slightly raised grey areas on the surface of the unstimulated ovary and those over 5 mm in diameter could readily be aspirated. When the ovary had been grasped and rotated so that the follicle to be aspirated was visible, the forceps handle was then passed to the assistant who maintained this position. The vacuum was activated immediately before a follicle was penetrated with a gentle but firm pressure on the needle (the aim being to penetrate the follicle without damaging its inner wall, thus avoiding bleeding). It was necessary to start the vacuum before penetrating the follicle to prevent loss of follicular fluid. A follicle being aspirated collapsed rapidly; the needle was then withdrawn and the vacuum turned off. The test tube now containing the follicular fluid was removed, and the procedure was repeated using a clean tube for each follicle. If the collection system became soiled with blood the needle was withdrawn from its sheath and a small amount of normal saline was sucked through the system to clean it. A little bleeding occurred at the puncture sites but diathermy was not necessary at any of these points at the conclusion of the collection. The instruments were removed except for the laparoscope cannula which was used to empty the peritoneum of CO2, The cannula was then removed and the two larger skin punctures were closed with a single clip which was removed 24 hours later. The puncture made by the aspirating needle required no closure. Collection of oocytes at laparotomy. Before the follicular aspiration equipment used at laparoscopies was developed, a basically similar aspiration device (Fig. 2) was used for collecting oocytes during laparotomies performed for fallopian tube division. Whenever possible, the operation was planned to take place shortly before the patient was expected to ovulate. This was timed from basal body temperature records kept by the patient; the operation was scheduled for the day of the menstrual cycle which preceded the expected thermal shift. The patients in this group had not taken oral contraceptives for at least two menstrual cycles prior to surgery and had not been treated with clomiphene or gonadotropins. At laparotomy, all ovarian follicles over 5 mm in diameter were aspirated using

4 Vol. 25, No. 12 COLLECTION OF OOCYTES 1033 a suction device. The suction device consisted of a 20 gauge needle, about 10 cm in length, connected to a translucent vinyl tube which emptied into a 5 ml test tube through a metal inlet (20 gauge bore) in the rubber stopper sealing the tube (Fig. 2). Another needle passing through the stopper was joined by its Luer adaptor to a short length of rubber tubing which in turn was connected to the surgical suction tubing via a three-way plastic connector (Portex). Suction through the needle used for aspirating follicles was produced by covering the free opening in the three-way connector; some control of suction strength could be achieved by varying the size of this opening with a finger tip. After a follicle was aspirated, the test tube containing the fluid was replaced by another tube to establish a fresh collection trap. The follicular fluid samples were later examined for the presence of oocytes using a dissecting microscope. In some patients follicles were aspirated in one ovary and a wedge of tissue containing several follicles was excised from the other. The wedge was transferred to the laboratory in warm saline or tissue culture medium. All follicles clearly visible with the aid of a dissecting microscope were incised with a no. 11 scalpel blade; in most cases the contained oocytes could be released into the medium under observation. Oocytes were classified as morphologically normal if: the shapes of their vitelline membranes and the enclosed ooplasm were regular and fully occupied the space within the zona pellucida, and if they were surrounded by several layers of corona and cumulus cells. Oocytes with incomplete coverings, with no surrounding cells, or with irregularities in the vitelline membrane or fragmenting ooplasm were classified as atretic. RESULTS Table 1 summarizes the menstrual cycle data of patients in the laparoscopy series and shows that at least 32 of the 45 women were preovulatory at the time of the operation. This was judged by the presence of a proliferative endometrium and the absence of a visible corpus luteum. Eight patients in this group were postovulatory. This was judged by the presence of a secretory endometrium and the presence of a corpus luteum. In the laparotomy group, six patients were preovulatory and 17 were postovulatory (Table 1). Of the patients that ovulated, most had a corpus luteum in an advanced stage of development, judging by the size and well developed yellow convolutions. Only three of the patients in this group probably ovulated less than a day before the operation; their ruptured follicles had the appearance of a 1 to 2 cm cyst filled with fresh blood. Oocyte recoveries at laparoscopy and laparotomy are compared in Table 2. The total number of oocytes recovered in each TABLE 1. The Stage of the Menstrual Cycle of Patients in the Laparoscopy and Laparotomy Groups Day of menstrual cycle Length of menstrual cycle Stage of No. of menstrual cycle patients Average Range Average Range Laparscopy Preovulatory Postovulatory Undetermined Laparotomy Preovulatory Postovulatory Undetermined

5 ~ LOPATAET AL December 1974 TABLE 2. Recovery of Human Oocytes From Their Follicles at Laparoscopy and Laparotomy No. of Total no. of % of follicles No. of oocytes per patient Method of No. of follicles oocytes yielding oocyte collection patients aspirated recovered oocytes Average Range Laparoscopy Suction vacuum ( mm Hg) Suction vacuum (200 mm Hg) Total group Laparotomy Aspirated from in situ ovaries Punctured follicles in ovarian wedges 11 (18)a Total group 25b Grand total anumber of ovarian wedges used in parenthesis. bin four patients, follicles were aspirated from one in situ ovary and a wedge was removed from the other. group was almost the same, although 20 additional patients were used for the laparoscopy. Therefore, a larger average number of oocytes was recovered per patient at laparotomies. This difference was due mainly to the better oocyte recoveries from ovarian wedges (Table 2). If the aspiration of oocytes from in situ ovaries is compared, the average number of oocytes recovered per patient was approximately the same at both procedures providing that a suction vacuum of 200 mm of mercury was used at laparoscopies. Moreover, under these conditions a greater percentage of follicles yielded oocytes at laparoscopies than at laparotomies. Overall, 498 follicles were aspirated and 217 oocytes were collected; the average recovery rate was about three oocytes per patient. TABLE 3. Recovery of Human Oocytes From Their Follicles in Relation to Age Average no. Average no. No. of Total no. of follicles of oocytes Age group No. of follicles of oocytes aspirated recovered (years) patients aspirated recovered per patient per patient Table 3 shows the numbers of follicles aspirated and oocytes recovered from patients of different age groups (20 to 44 years). No statistically significant difference was found between the age groups for the mean number of follicles aspirated and that of oocytes recovered from the ovaries. The number of women over 40 years old was too small for statistical analysis of the results. The dimensions of ovarian follicles aspirated during the periovulatory stage of the menstrual cycle are shown in Table 4. The size of a follicle was established by measuring the volume of follicular fluid aspirated and extrapolating the diameter from a graph relating volume with the diameter of a sphere. This procedure Diameter of follicles (mm) TABLE 4. Recovery of Human Oocytes in Relation to the Size of the Follicles Aspirated at Laparoscopy No. of follicles aspirated No. of oocytes recovered % of follicles State of oocytes yielding oocytes Normal Degenerated

6 Vol. 25, No. 12 COLLECTION OF OOCYTES 1035 TABLE 5. The Size of Follicles Present in the Ovaries of Infertile and Fertile Patients Type of patient No. of patients 5-7 Primary infertility Secondary infertility Fertile 7 18 Diameter of follicles (mm) assumed that the total volume of follicular fluid was aspirated and that the follicles were spherical. Follicles 7 to 9 mm in diameter were aspirated most frequently and yielded the largest number of oocytes. About one third of all follicles aspirated were 10 mm or more in diameter. The smallest follicles contained the greatest proportion of atretic oocytes. On the other hand, the highest percentage of morphologically normal oocytes was recovered from follicles larger than 7 mm. in diameter (Table 4). No significant difference was found between the mean size of follicles present at the surface of the ovaries in the age groups studied. The mean follicular diameter was 9.4 mm at 20 to 24 years, 8.9 mm at 25 to 29 years, 8.6 mm at 30 to 34 years, and 8.5 mm at 35 to 39 years. The ovaries of infertile patients contained a considerably higher proportion (18.5%) offollicles over 11 mm in diameter compared with that (7.0%) of the fertile patients (Table 5). The mean follicular diameter was 9.1 mm in infertile and 8.0 mm in fertile patients; this was statistically significant (P<0.05). The state of oocytes recovered from the ovaries of primary and secondary infertility patients was compared with that of oocytes aspirated from fertile women (Table 6). Analysis of these using the X 2 test showed that a significantly greater proportion of morphologically normal oocytes was obtained from the fertile group compared with the infertile group of patients. DISCUSSION This report describes an effective method of collecting oocytes at laparoscopies performed during the periovulatory stage of the menstrual cycle. When ovarian follicles were aspirated using a suction vacuum of 200 mm of mercury, oocytes were obtained from 45.6% of 217 follicles, a recovery rate considerably higher than that reported by other workers However, in an earlier series of laparoscopies, oocytes were obtained from only 20.6% of aspirated follicles when suction vacuums ranging from 120 to 180 mm of mercury were used. Steptoe and Edwards 12 performed laparoscopic recoveries of oocytes in patients whose ovaries were stimulated with gonadotropins. They recovered oocytes from 31.8% offollicles aspirated with a syringe TABLE 6. Comparison of the Number and State ofoocytes Recovered at the Periovulatory Stage of the Menstrual Cycle from the Infertile Group with Those Obtained from the Total Group of Fertile Women State of oocytes Mean Total day of No. of no. of Normal Degenerated Patient group No. of menstrual follicles oocytes patients cycle aspirated recovered No. % No. % X' Combined infertile group Combined fertile group "P< "

7 1036 LOPATA ET AL December 1974 and from 32.4% with an aspiration device, but they did not report the suction vacuums that were used. Morgenstern and Soupart13 used an oocyte recovery unit for aspirating the follicles of ovaries exposed at elective abdominal and vaginal operations. Their overall oocyte recovery rate from in situ ovaries was 25.9%. However, since these authors also did not indicate the suction vacuums used, nor the stage of the menstrual cycle at which oocyte collections were performed, it is uncertain whether the higher oocyte recovery rate reported in the present study is a function of stronger suction vacuums or other factors. In initial studies the vacuum generated by a surgical sucker was used for aspirating follicles of in situ ovaries at laparotomies. It was found that oocytes recovered in this way were partially denuded of corona cells and had vitelline membrane deformities. The vacuum used during these early studies was more than twice that finally employed. Reducing the vacuum arbitrarily provided morphologically normal oocytes and a recovery rate of 30.2%; this is similar to that reported by others.12,13 Morphologic damage could not be detected in oocytes recovered at a vacuum of 200 mm of mercury. When such suction was used for aspirating preovulatory follicles of infertile patients treated with clomiphene and human chorionic gonadotropin, the recovered oocytes were surrounded by a mass of cumulus cells embedded in a delicate ~ viscous material and their zona pellucida and vitelline membrane appeared to be morphologically normal (in preparation). Moreover, a number of these oocytes cleaved following in vitro insemination and some developed to the eight-cell stage. 14 Human oocytes reach a mature size in follicles that have developed to 1 mm in diameter. 15 It has been suggested, however, that further growth of these small follicles is probably necessary for them to attain the capacity to ovulate The diameter of ovulatory follicles in human ovaries has been reported to be from 2 to 4 mm19 and up to 20 mm.20 In the present study follicles ranging from 7 to 9 mm in diameter were most frequently encountered at the surface of ovaries near the expected time of ovulation. It was also found that follicles over 7 mm in diameter yielded by far the greatest proportion of morphologically normal oocytes, whereas the smaller follicles yielded mainly atretic oocytes. These findings are consistent with the observations that in subhuman primate ovaries most of the follicles in the new crop that begins to develop during the luteal phase of the preceding cycle undergo degeneration at various stages of their development and the few whose oocytes and granulosa cells escape atretic changes continue to develop to the ovulatory stage. 18 The mean number of ovarian follicles that could be readily aspirated and the mean number of oocytes recovered did not vary significantly for age groups ranging from 20 to 40 years. Similar results were obtained by other investigators. 13,21 In addition, the present study showed that the average size of follicles, present at the ovarian surface during the periovulatory stage of the menstrual cycle, did not vary significantly in the age groups studied. Comparison of the size of follicles present at the surface of ovaries showed that infertile patients contained a much higher percentage of follicles over 11 mm in diameter than did fertile patients. Follicles up to 21 mm in size were found in the infertile group; all follicles in the fertile group were less than 15 mm in diameter. This difference in the population of ovarian follicles accounted for the significantly greater mean follicular diameter in the infertile group. The ultimate size of ovarian follicles is probably determined by several mechanisms. These include: the levels of gonadotropins and sex steroids in the target tissues which control their growth,1m an

8 Vol. 25, No. 12 COLLECTION OF OOCYTES 1037 ' interaction between the oocyte and follicular cells,22 and a process of atresia and involution of non ovulatory follicles.23 Thus the large follicles found in infertile patients and in particular the polycystic ovaries often associated with infertility may result from an impairment of one or more of these follicular control mechanisms. Significantly more atretic oocytes were recovered from the infertile than from the fertile patients. This difference reflects the state of oocytes in small ovarian follicles since only a few were recovered from follicles over 11 mm in diameter. The reason for the low oocyte recovery rate from large follicles of unstimulated ovaries is unknown. Hertig24 postulated that if ovulation is delayed the oocyte within the ovulatory follicle has an increasing chance of being abnormal after fertilization. It could also be argued, however, that ovulation was delayed because an abnormal oocyte was already present in the follicle. A possible relationship between abnormal oocytes and a greater incidence of atresia in the infertile group needs further investigation. Nevertheless, an abnormal egg syndrome probably should be considered a cause of infertility and habitual abortion when no other cause can be found. SUMMARY Oocytes were collected during the periovulatory stage of the normal menstrual cycle from 45 women undergoing laparoscopy and 25 women having a laparotomy. At laparoscopies, oocytes were recovered by aspirating ovarian follicles with a needle introduced through the anterior abdominal wall. At laparotomies, oocytes were collected by aspirating follicles of the in situ ovaries or by excising an ovarian wedge and releasing oocytes from incised follicles. A larger mean number of oocytes was recovered per patient at laparotomies (4.3) than at laparoscopies (2.4) due to better oocyte recoveries from ovarian wedges. Overall, 498 follicles were aspirated and 217 oocytes collected with an average recovery of approximately three oocytes per patient. There was no statistically significant difference between the mean number of follicles, the mean diameter of follicles, or the mean number of oocytes recovered per patient in age groups ranging from 20 to 40 years. Follicles over 7 mm in diameter yielded the largest proportion of normal oocytes. The ovarian follicles of infertile patients had a significantly greater mean diameter and contained a significantly greater proportion of atretic oocytes than those of fertile patients. REFERENCES 1. Edwards RG: Maturation in vitro of mouse, sheep, cow, pig, rhesus monkey and human ovarian oocytes. Nature (Lond) 208:349, Edwards RG: Maturation in vitro of human ovarian oocytes. Lancet 2:926, Kennedy JF, Donahue RP: Human oocytes: maturation in chemically defined media. Science 164:1292, Suzuki S, lizuka R: Maturation of human ovarian follicular oocytes in vitro. Experientia 26: 640, Klinger HP, Kava HW, Hachamovitch M: Chromosome studies in human female meiosis. Ann NY Acad Sci 171:431, Chandley AC: Culture of mammalian oocytes. J Reprod Fertil [Suppl] 14:1, Hayashi M: Fertilization in vitro using human ova. Proc 7th Int Conf I.P.P.F., Singapore, Excerpta Med Found 1963, p Edwards RG, Bavister BD, Steptoe PC: Early stages offertilization in vitro of human oocytes. Nature (Lond) 221:632, Edwards RG, Steptoe PC, Purdy JM: Fertilization and cleavage in vitro of preovulator human oocytes. Nature (Lond) 227:1307, Jacobson CB, Sites JG, Arias-Bernal LF: In vitro maturation and fertilization of human follicular oocytes. Int J Fertil 15:103, Seitz HM, Rocha G, Brackett BG, et al: Cleavage of human ova in vitro. Fertil Steril 22:255, 1971

9 1038 LOPATAET AL December Steptoe PC, Edwards RG: Laparoscopic recovery of preovulatory human oocytes after priming of ovaries with gonadotropins. Lancet 1:683, Morgenstern LL, Soupart P: Oocyte recovery from the human ovary. Fertil Steril 23:751, de Kretzer D, Dennis P, Hudson B, et al: Transfer of a human zygote. Lancet 2:728, Green SH, Zuckerman S: Quantitative aspects of the growth of the human ovum and follicle. J Anat 85:373, Pincus G: The reproductive processes and their vulnerabilities in mammals. In The Control of Fertility. Edited by G Pincus. New York, Academic Press, 1965, p Rodbard D: Mechanics of ovulation. J Clin Endocrinol Metab 28:849, Speroff L, Vande Wiele RL: Regulation of the human menstrual cycle. Am J Obstet Gynecol 109:234, Hafez ESE: Introduction to comparative reproduction. In Comparative Aspects of Reproductive Failure. Edited by K Bernischke. New York, Springer-Verlag, 1967, p Franchi LL: The ovary. In Scientific Basis of Obstetrics and Gynaecology. Edited by EE Phillipp, J Barnes, M Newton. London, William Heinemann, 1970, p Kennedy JF: Maturation and fertilization of human oocytes in vitro. In Oogenesis. Edited by JD Biggers, A W Schuetz. Baltimore, University Park Press, 1972, p Nalbandov AV: Interaction between oocytes and follicular cells. In Oogenesis. Edited by JD Biggers, A W Schuetz. Baltimore, University Park Press, 1972, p Ingram DL: Atresia. In The Ovary. Vol I Edited by S Zuckerman. New York, Academic, Press, 1962, p Hertig AT: The overall problem in man. In Comparative Aspects of Reproductive Failure. Edited by K Bernischke. New York, Springer Verlag, 1967, p 11