Vital initiation of pregnancy (VIP) using human menopausal gonadotropin and human chorionic gonadotropin ovulation induction: Phase I—1981

FERTILITY AND STERILITY Copyright © 1983 The American Fertility Society

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Vol. 39, No.6, June 1983 Printed in U.SA.

Vital initiation of pregnancy (VIP) using human menopausal gonadotropin and human chorionic gonadotropin ovulation induction: Phase 1-1981

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J. W. Edward Wortham, Jr., Ph.D.*t+ Lucinda L. Veeck, M.L.T. (A.S.C.P.)* Jeannine Witmyer, B.A.*t Howard W. Jones, Jr., M.D.* Eastern Virginia Medical School and Old Dominion University, Norfolk, Virginia

Laparoscopies for oocyte aspiration in 31 cycles were performed on 25 patients receiving human menopausal gonadotropin and human chorionic gonadotropin. Sixty oocytes were aspirated, of which 48 were considered preovulatory. Ninety-seven percent (58 of 60) of the oocytes were found in the original aspirate, and the remaining oocytes were found in either the first or second follicle wash. The fertilization rate per preovulatory oocyte was 33% (16 of 48), whereas on a per cycle basis it was 39% (12 of 31). A total of 15 conceptuses (2-cell = 5; 3-cell = 3; 4-cell = 7) were transferred to 12 patients, and two pregnancies were established. These pregnancies were established by transfers of 3-cell and 4-cell conceptuses at approximately 47 hours after insemination. Both pregnancies resulted in term deliveries of normal infants. Fertil Steril39:785, 1983

The establishment of pregnancies by in vitro fertilization (IVF) and embryo transfer has been reported by several investigative groupS.I-3 These groups have all reported term deliveries with programs of IVF. Edwards et aLl achieved their pregnancies and subsequent term deliveries by IVF of oocytes recovered from spontaneous cycles. Similarly, Lopata et al.,2 at the University of Melbourne in Australia, reported the birth of the world's third baby by IVF of an oocyte recovered

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Received June 28, 1982; revised and accepted February 17, 1983. *Department of Obstetrics and Gynecology, Eastern Virginia Medical School. tDepartment of Biological Sciences, Andrology Laboratory, Old Dominion University. :j:Present address and reprint requests: J. W. Edward Wortham, Jr., Ph.D., Department of Obstetrics and Gynecology, University of Oklahoma, Tulsa Medical College, 2808 South Sheridan Road, Tulsa, Oklahoma 74129. Vol. 39, No.6, June 1983

during a spontaneous cycle. Other pregnancies have been established since these by IVF of 00cytes from stimulated cycles using clomiphene citrate and human chorionic gonadotropin (hCG). Wood et al.,3 at Monash University in Melbourne, Australia, have reported on the clinical assessment of nine pregnancies resulting from oocytes recovered after clomiphene/hCG stimulation. This report describes the results of the vital initiation of pregnancy (VIP) IVF program at Eastern Virginia Medical School from January 1 to July 31, 1981, referred to as VIP Phase I, 1981. These results include oocyte and embryo data from 31 laparoscopies performed on 25 patients receiving human menopausal gonadotropin (hMG) and hCG. The laboratory results associated with the two pregnancies, which resulted in term deliveries of healthy infants, will be described in detail. Clinical information on these pregnancies, including the details of gonadotropin stimulation, has been discussed elsewhere. 4 Wortham et a!. Vital initiation of pregnancy-phase I

785

MATERIALS AND METHODS

CO 2 in air, at 37° C, and 97% to 98% relative humidity.

OOCYTE RETRIEVAL AND CULTURE

The protocol for stimulation was that of Jones et a1. 4 A 12-gauge needle measuring 2.16 mm in inside diameter and 43 cm in length was used for oocyte aspiration. The needle was attached to an Argyle DeLee (Sherwood Medical Industry, St. Louis, MO) suction catheter with mucus trap (20 ml) by a segment of 10 French catheter tubing supplied with the trap.5 The follicular fluid containing the oocyte was aspirated into the trap, which contained 2 ml of Dulbecco's phosphatebuffered saline (#450-1300 supplemented with 0.1 gm/l calcium chloride, GIBCO Laboratories, Grand Island, NY) which had previously been adjusted to a pH of 7.35 and osmolarity of 285 mOsmlkg. The system was then flushed with an additional 2 ml of Dulbecco's phosphate-buffered saline. The follicular fluid/phosphate-buffered saline mixture was immediately transferred from the operating room to the adjacent culture laboratory for examination in tissue culture dishes (#3002, Falcon Plastics, Oxnard, CA). The volume and color of the fluid were noted and the granulosa cells evaluated for maturity. Mature oocytes were usually located quickly by the presence of an expanded cumulus mass, which was often visible macroscopically. Both a Nikon MS (Nikon Inc., Garden City, NY) inverted microscope and an American Optical (Buffalo, NY) dissecting microscope were used for scanning. The inverted microscope provided high-resolution viewing and photomicrographic capabilities, and the dissecting microscope permitted fast scanning and facilitated oocyte identification and transfer. Once located, the oocyte was briefly examined to assess its general morphologic features and maturity, and 35-mm black and white photomicrographs were often taken for future reference. The oocyte was then quickly transferred into 3 ml of insemination medium (1M, described below) in an organ culture dish (#3037, Falcon), which had been incubated in 5% CO 2 in air at 37° C for approximately 1 hour. If the follicular fluid was heavily contaminated with blood, the oocyte was washed briefly in a separate portion of 1M prior to incubation. We then incubated the oocyte for 5 hours prior to insemination in order to allow for equilibration and completion of maturation. All phases of incubation were carried out under conditions of 5% 786

Wortham et a1. Vital initiation of pregnancy-phase I

INSEMINATION

The patient's husband collected a semen sample by masturbation and delivered it to the laboratory approximately 90 to 120 minutes prior to insemination. The sample was allowed to liquefy at room temperature, a process usually taking 10 to 30 minutes. After liquefaction, a semen analysis was performed, including count, motility, and general morphologic characteristics, to confirm previous assay findings. Upon completion of the analysis, the remainder of the sample was diluted with 15 ml of 1M and centrifuged at 427 x g for 10 minutes, and the supernatant was discarded. This washing procedure was repeated in 10 ml of 1M, and the remaining sperm pellet was resuspended in 3 to 5 ml of 1M. At that time, a second count and motility analysis were performed. Motile sperm were added to the insemination dish containing the oocyte at a concentration of 0.8 x 106/ml, or 2.4 million motile sperm in the 3 ml of 1M. Periods of insemination ranged from 5 to 8 hours, after which time the oocytes were transferred to growth medium (GM, described below), which had been incubated and equilibrated during the insemination period. Because pronuclei were typically not seen after so short a period,

Figure 1 Typical aspirated oocytes and membrana granulosa cells at various stages of maturation. (a), Preovulatory oocyte/corona radiata (e)/cumulus (eu) complex (x 40). (b), Immature oocyte displaying lack of cumulus and compact corona (e) (x 100). (e), Atretic oocyte displaying lack of cumulus, lack of corona degenerative ooplasm (0), and zona pellucida (zp) ( x 100). (d), Cells of the membrana granulosa characteristic of a mature follicle. Note the intercellular spacing and roundness ofthe cells ( x 40). (e), Compact granulosa cells associated with an immature follicle (x 40). (fl, Small clump of degenerative cells associated with an atretic follicle ( x 40).

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evidence of fertilization was not obtained in all cases. Despite the insemination of all oocytes regardless of type (Fig. 1a to c), data on the fertilization rate could not be established for this series of patients. Subsequent observations were made at 40 to 46 hours after insemination, at which time the oocytes were examined for cleavage, and the patients were scheduled for embryo transfer if cleavage had occurred. Noncleaving oocytes were fixed for electron-microscopic evaluation. CULTURE MEDIUM

The stock culture medium was prepared each week from liquid Ham's F-10 (#320-1550, GIBCO Laboratories), supplemented with calcium lactate, 0.25 gm/l (#4208, Mallinckrodt Laboratories, St. Louis, MO), penicillin, 100 Dlml, and streptomycin, 100 /-lg/ml (#600-5140, GIBCO Laboratories). The osmolarity was then adjusted to 285 to 295 mOsm/kg. INSEMINATION MEDIUM

1M was made by adding bovine serum albumin (#A4503, Sigma, St. Louis, MO) to the Ham's F-10 stock solution at a concentration of 5 mg/ml. The 1M was then adjusted to a pH of 7.4 to 7.45, filter-sterilized, and stored at 4° C until use. GROWTH MEDIUM

GM was made by adding 1.5 volumes of heat-inactivated human fetal cord serum to 8.5 volumes of Ham's F-10 stock solution. The pH of the GM was adjusted to 7.3 to 7.35 and filter-sterilized. All culture media were tested for their ability to support the development of mouse embryos fertilized in vivo. GRANULOSA CELLS

The aspirated cells of the membrana granulosa in the follicular fluid were routinely evaluated for maturity. Correspondence between the maturity of the granulosa cells and oocyte maturity was generally good, although there were occasions noted when the maturity of the oocyte appeared to lag behind that of the granulosa cells. Mature granulosa cells displayed an increased cytoplasmic/nuclear ratio, as compared with immature cells. In addition, mature granulosa cells appeared in loosely aggregated clumps or sheets with discrete intercellular spaces, while immature cells appeared as compact cellular masses Vol. 39, No.6, June 1983

lacking definite intercellular spacing (Fig. 1d to f). Sheets of very mature cells often closely re-

sembled an expanded cumulus mass. OOCYTE CLASSIFICATION

Oocytes were primarily classified on the basis of two criteria: the presence or absence ofa cumulus mass and the maturity of associated granulosa cells. An oocyte was classified as preovulatory if it was associated with an expanded cumulus mass (Fig. 1a). The size of the cumulus varied from extremely small to massive. Identification of mature granulosa cells in the corresponding follicular fluid also warranted assignment to the preovulatory category. The morphologic characteristics of the corona radiata were not used in classifying preovulatory oocytes, although the degree of expansion seems to reflect maturation. Generally, an oocyte was classified as immature if it was not associated with an expanded cumulus mass and if it possessed a compact or scanty corona (Fig. 1b). Granulosa cells from follicles with immature oocytes were usually immature, but occasionally they showed some enlargement and intercellular spacing characteristic of mature cells. Atretic oocytes displayed a complete lack of corona cells and cumulus and often exhibited degenerative features within the ooplasm or zona pellucida (Fig. 1c). Atretic oocytes were derived from follicles with varying types of granulosa cells. It was sometimes difficult to distinguish between degenerative and very immature cells by observation under the inverted microscope. Often, follicles from which atretic oocytes were derived displayed a complete absence of any cells. Such follicles were considered to be cystic.

RESULTS Laparoscopies were performed in 31 cycles on 25 patients after hMG and hCG stimulation. A total of 60 oocytes was recovered from 113 aspirated follicles. Oocytes were recovered from all but one patient. Fifty-eight (97%) of the oocytes were recovered in the first aspirate, while the remainder were recovered in either the first or second follicle wash. Forty-eight (80%) of the 00cytes were considered preovulatory, 9 (15%) immature, and 3 (5%) atretic (Table 1). Each oocyte considered preovulatory was embedded ina cumulus mass, and each corresponding follicular fluid contained mature granulosa Wortham et al. Vital initiation of pregnancy-phase I

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Table 1. Cleavage of in Vitro Inseminated Preovulatory and Nonovulatory Human Oocytes Classification

No. of oocytes inseminated

No. of oocytes cleaved (%)

No. not cleaved (%)

48

16 (33) 0(0) 0(0)

32 (67) 9 (100) 3 (100)

Preovulatory Immature Atretic

9 3

cells. A typical preovulatory oocyte-cumulus mass can be seen in Figure 1a. The preovulatory oocyte was usually spherical and surrounded by a few layers of attendant corona cells. The oocyte/ corona complex was usually located toward the periphery of the cumulus mass. The cumulus is said to be expanded because the cells are separated from one another by the intercellular deposition of hyaluronic acid. 6 Although highly variable in size and shape, cumulus masses could be as large as 0.5 cm in diameter. The morphologic characteristics of the corona radiata appear to reflect oocyte maturity; however, the corona was often difficult to evaluate morphologically because of the density and amount of cumulus. Certainly, the expansion of the corona was varied among the oocytes, but we were uncertain of the significance of this observation with regard to maturation. Forty-five of the 48 (94%) preovulatory oocytes possessed an expanded corona, 1 had a compact corona, and 2 had no recognizable corona, although all had an expanded cumulus and were associated with mature granulosa cells in the follicular fluid. Conversely, the coronas of the immature oocytes (Fig. 1b) were all compact, even though some of these oocytes were associated with mature granulosa cells. None of the atretic oocytes (Fig. 1c) displayed corona, cumulus, or mature granulosa cells. Evaluation of the characteristics of the oocytes recovered during a particular cycle showed that 20 (65%) of the laparoscopies produced only preovulatory oocytes, 3 (10%) produced only immature oocytes, and 1 (3%) produced a single atretic oocyte. In six (19%) additional cycles, at least one preovulatory oocyte was recovered, although immature or atretic oocytes were recovered as well. In one cycle (3%), no oocytes were recovered. Five of the 25 patients underwent multiple treatment cycles in this series (Table 2). Of these, one patient underwent three laparoscopies, and two or three preovulatory oocytes were obtained each time. Two cycles resulted in oocyte cleavage and transfer, and the patient subsequently became pregnant after a fourth laparoscopy. This 788

Wortham et al.

Vital initiation of pregnancy-phase I

pregnancy will be addressed in a later paper dealing with the next series of cycles (VIP Phase 11). The other four patients had two treatment cycles each. In two of these patients, preovulatory 00cytes were recovered during each cycle, but only one of these patients had a subsequent transfer. In another patient, immature oocytes were obtained during the first cycle and three preovulatory oocytes during the second cycle, but all failed to cleave. In the last patient, an atretic oocyte was recovered during the first cycle and immature oocytes during the second cycle, but no apparent fertilization or cleavage resulted. The characteristics and the fates of these 00cytes are reported in Table 3. The only developing conceptuses resulted from fertilization of preovulatory oocytes. Sixteen of the 48 (33%) preovulatory oocytes cleaved, and the conceptuses were transferred. All of the oocytes that cleaved possessed at least some form of expanded cumulus and expanded corona, but so did 29 of the 32 preovulatory oocytes that did not divide. The three preovulatory oocytes with a nonexpanded corona all failed to cleave. None of the immature or atretic oocytes produced cleaving embryos under the culturing techniques used in phase I. Fragmentation of oocytes was occasionally seen. In review of the associated endocrine findings, this seems to be a problem related to hMG/ hCG stimulation or response to stimulation. 7 This phenomenon was usually accompanied by dark-appearing granulosa cells in the follicular fluid. When these dark cells were present, there was no associated preovulatory oocyte that cleaved. Fragmenting oocytes were considered to be postmature and non cleaving in respect to reporting data. The individual fragments appeared to be larger than those described by Sundstrom and co-workers. 8 Table 2. Variation in Oocytes Recovered During Multiple

Treatment Cycles Treatment cycles II

Patient I Patient II Patient III Patient IV Patient V

III

2 preovulatoryU 2 preovulatoryU 3 preovulatory 1 atretic 2 immature 3 preovulatory 2 preovulatoryU 1 preovulatory 1 immature 1 preovulatory 1 preovulatory 2 immature 1 immature 1 "tretic

UEmbryo transfer was carried out during this treatment cycle.

Fertility and Sterility

Table 3. Relationship of Granulosa Cells, Cumulus, and Corona Cells at the Time of Aspiration to the Success of Cleavage Oocytes Preovulatory Cleaved Not cleaved

No.(%) 48 16 (33)

16 Mature

32 (67)

32 Mature

Immature Cleaved Not cleaved

9 0(0) 9 (100)

Atretic Cleaved Not cleaved

3 0(0) 3 (100)

)

1 i

Granulosa

Cumulus size 12 4 25 7

Corona

Moderate/massive Small Moderate/massive Small

16 Expanded 29 Expanded 1 Compact 2 Absent

5 Mature 3 Immature 1 Undetermined

9 Absent

9 Compact

3 Immature

3 Absent

3 Absent

I

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Because observations of cleavage were limited, it was difficult to evaluate the growth rates of developing conceptuses. In this series, a total of eight 4-cell, three 3-cell, and four 2-cell conceptuses were transferred (Fig. 2). Observations of seven conceptuses in the 2-cell stage were made between 42 and 48 hours after insemination. Four were still in the 2-cell stage when transferred at 48 hours after insemination. Of the three that did progress, two had developed into 3-cell and 4-cell conceptuses by transfer at 48 hours and one into the 3-cell stage by 47 hours. This last one produced pregnancy A (Table 4). Eight 4-cell conceptuses were transferred, one (conceptus 10, Fig. 2) resulting in pregnancy B (Table 4). Three of the 4-cell conceptuses were growing progressively at the time of transfer (Fig. 2, conceptuses 3, 4, and 9b), but none of these produced a pregnancy. Some apparent asynchrony was observed between cleavage rates of individual conceptuses from patients who had more than one embryo. In one patient, both conceptuses were in the 2-cell stage at first observation (44 hours after insemination), and only one progressed to the 3-cell stage by transfer at 48 hours (Fig. 2, conceptus 2a). In the second patient, both a 2-cell and a 3-cell conceptus were observed at 44 hours after insemination. Three hours later, the 2-cell conceptus had not changed, but the 3-cell conceptus had progressed to the 4-cell stage (Fig. 2, conceptus 9b). In the third patient, one 2-cell and two 4-cell conceptuses were seen at 43 hours after insemination, and their status had not changed by the time oftransfer at 46 hours (Fig. 2, conceptuses 12a to c). In all, 15 conceptuses were transferred to 12 patients and resulted in two pregnancies. The Vol. 39, No.6, June 1983

pregnancies were established by the transfer of a 3-cell conceptus at 47 hours after insemination and a 4-cell conceptus at 46 hours after insemination (Table 4). Both pregnancies have ended successfully in term deliveries.

DISCUSSION

The Norfolk IVF program, while similar in many ways to the programs in Melbourne and the United Kingdom, has some unique approaches of its own. The stimulation of patients has been with hMG rather than with clomiphene citrate. The technique for aspiration of oocytes has utilized a large-diameter needle (inside diameter, 2.16 mm) and manual suction, as compared with a small-diameter needle (inside diameter, 1.4 mm) and wall

......

..... u

40

. .. Hours

..

50

A,8,e represenl mullipJe oocyte/patients

Figure 2 The single points represent the cell stage of the embryos at both the initial evaluation and at the time of transfer. The dotted lines indicate a change in the status of the embryos occurring between the initial observation and transfer.

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Table 4. Comparative Characteristics of Follicles, Oocytes, Fertilization, and Conceptus Development in Patients Achieving Pregnancy Characteristics Follicles and oocytes Interval between anesthesia and aspiration (min) Follicular fluid volume (per ml) Estimated follicle size (large, medium, or small) Follicle wall transparency (transparent or opaque) Follicle wall thickness (thin or thick) Granulosa cells (mature or immature) Oocyte classification (preovulatory, immature,or atretic) Cumulus (massive, moderate, small, or absent) Corona (expanded, slightly expanded, compact, or absent) Fertilization and Development Ejaculate concentration (total no. of sperm) Motility (%) Incubation period prior to insemination (hr) No. of motile sperm used for insemination (per ml) Period of insemination (hr) Interval between insemination and examination (hr) No. of blastomeres at examination (NC, not cleaved) Age of conceptus at transfer

PregnancyB

Pregnancy A Oocyte 1 15

Oocyte 2

31

26

4.0

Oocyte 3

8.0

Oocyte 1 24

Oocyte 2 28

2.2

1.0

Oocyte 3 37

5.0

4.2

L

L

S

M

L

L

Tr

Tr

Op

Tr

Tr

Op

Tn

Tn

Tk

Tn

Tn

Tn

Mat

Mat

Mat

Mat

Mat

Mat

Preov

Preov

Immat

Preov

Preov

Preov

Mod

Mass

Abs

Mod

Mod

Sm

Exp

Exp

Comp

Exp

Exp

Comp

129 x 106

?

5.5 0.8 x 106 7 44

64% 5.5 0.8

X

106

7 44

NC

2

5.5 0.8

X

106

52% 5

5 0.8

X

7 44

9 45

NC

NC

106

0.8

X

5

106

9 45

0.8

X

106

9 45

4

NC

47

46.5

3

4

0.15

0.15

Term delivery: healthy female

Term delivery: healthy male

(hr)

No. of blastomeres at transfer Volume of culture medium used for transfer (ml) Status of pregnancy

suction. The use of organ culture dishes instead of culture tubes or Petri dishes containing microdrops for fertilization and embryo growth has facilitated viewing and transfer of developing embryos. The use of an inverted microscope has also allowed for critical embryo evaluation without the necessity of transferring the embryo out of its own medium, as is required with culture tubes. The use of both an inverted microscope and dissecting microscope for identification, transfer, and oocyte evaluation provides for rapid location and critical oocyte and granulosa cell assessment. Rapid evaluation of granulosa cells gives the laboratory personnel an immediate estimate of the type of oocyte to be expected. While the preovulatory oocyte is easily identifiable because of its 790

Wortham et aI. Vital initiation of pregnancy-phase I

cumulus mass, nonovulatory oocytes require close scrutiny during search and seem more likely to be found in the follicle wash. Although preovulatory oocytes can be identified by morphologic criteria, the fate of these apparent preovulatory oocytes recovered after hMG/ hCG stimulation is less certain. Preovulatory 00cytes were recovered in 26 (84%) of the laparoscopies, but only 12 (46%) of these cycles produced cleaving conceptuses. It is difficult with our limited observations to explain these data; however, it is obvious that the morphologic criteria for judging preovulatory oocytes do not allow for fine distinction between the stages of oocyte maturation 01' overmaturation. This may be additionally complicated by the exogenous gonadotropin stimulaFertility and Sterility

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tion in relation to our dependence on the expanded cumulus as a key criterion for oocyte classification. The practical need of this approach is obvious, but the cumulus expansion is probably related to hormonal or chemical stimuli and may vary independently with oocyte maturation. Eppig9 • 10 has demonstrated that mouse cumuli oophori may expand in response to follicle-stimulating hormone and that this response may be enhanced by prostaglandin estradiol. hMG injections make available exogenous follicle-stimulating hormone and luteinizing hormone to the system and could cause premature luteinization of the granulosa cells and cumulus expansion, whereas hCG is a patent luteinizing hormone stimulus with inherent follicle-stimulating hormone activity. These factors may well explain the occasional advanced maturity of the granulosa cells over that of the oocyte. Further evidence is that supplementary oocyte incubation prior to insemination allowing for additional oocyte maturation appears to be beneficial, even when the oocyte seems to be preovulatory by morphologic criteria. Hormonal levels within the follicular fluid have been shown to be a better indicator of oocyte maturation than the morphologic features. Wramsby et al. l l have demonstrated a close correlation between high progesterone and high estradiol concentrations in follicular fluids and the ability of the associated oocytes to fertilize. It should be noted, however, that these levels can also be changed by exogenous hormonal stimulation. As difficult as it may seem to determine the state of individual preovulatory oocytes, it would seem even more difficult to determine the normality of developing embryos. The state of development of the conceptus at transfer appears not to be the determining factor for establishing a pregnancy. Pregnancies have been established in various clinics by transferring stages ranging from 2-cell embryos to blastocysts. 12 It has been thought that the fastest growing embryos have been the most successful in establishing pregnancies,13 but our data indicate that the mere ability to cleave rapidly does not ensure implantation. Pregnancy A was established by one of the slower growing embryos. The morphologic variations in cleaving embryos have been very striking, even in embryos that produced pregnancies. The 16 embryos that cleaved in this series displayed differences in blastomere size and shape and many times appeared to have extra minor cytoplasmic fragments or small blebs. Other investigators Vol. 39, No.6, June 1983

have reported similar variations and do not consider them abnormal or pathologic. s To date, we have no sure method of judging the quality of cleaving embryos other than the establishment of pregnancy. This method is of little scientific value, because many other factors can exert a negative influence on implantation. Nevertheless, with current techniques and proper quality control and careful patient monitoring, a predictable pregnancy rate can be achieved with IVF. There is, therefore, the opportunity to apply this therapeutic method in a variety of ways in an effort to solve previously unresolvable infertility problems. Acknowledgments. The authors wish to express their appreciation to Drs. Georgeanna Jones and Bruce Sandow for their· advice and criticism during the preparation of this manuscript. This work was supported in part by the generosity of individuals who donated private funds.

REFERENCES l. Edwards RG, Steptoe PC, Purdy JM: Establishing full-

2.

3.

4.

5.

6.

7.

8.

9.

10.

term human pregnancies using cleaving embryos grown in vitro. Br J Obstet Gynaecol 87:737, 1980 Lopata A, Johnston IWH, Hoult IJ, Speirs AL: Pregnancy following intrauterine implantation of an embryo obtained by in vitro fertilization of a preovulatory egg. Fertil Steril 33:117, 1980 Wood C, Trounson A, Leeton J, Talbot JMcK, Buttery B, Webb J, Wood J, Jessup D: A clinical assessment of nine pregnancies obtained by in vitro fertilization and embryo transfer. Fertil Steril 35:502, 1981 Jones HW Jr, Jones GS, Andrews MC, Acosta A, Bundren C, Garcia J, Sandow B, Veeck L, Wilkes C, Witmyer J, Wortham JE, Wright G: The program for in vitro fertilization at Norfolk. Fertil Steril 38:14, 1982 Jones HW Jr, Acosta AA, Garcia J: A technique for the aspiration of oocytes from human ovarian follicles. Fertil Steril 37:26, 1982 Edwards RG, Steptoe PC, Fowler RE, Baillie J: Observations on preovulatory human ovarian follicles and their aspirates. Br J Obstet Gynaecol 87:769, 1980 Garcia JE, Jones GS, Acosta AA, Wright G Jr: Human menopausal gonadotropin/human chorionic gonadotropin follicular maturation for oocyte aspiration: Phase I, 1981. Fertil Steril 39:167, 1983 Sundstrom P, Nilsson 0, Liedholm P: Cleavage rate and morphology of early human embryos obtained after artificial fertilization and culture. Acta Obstet Gynecol Scand 60:109, 1981 Eppig JJ: Gonadotropin stimulation of the expansion of cumuli oophori isolated from mice: general conditions for expansion in vitro. J Exp Zool 208:111, 1979 Eppig JJ: Prostaglandin E2 stimulates cumulus expansion and hyaluronic acid synthesis by cumuli oophori isolated from mice. Bioi Reprod 25:191, 1981

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1l. Wramsby H, Kullander S, Liedholm P, Rannevik G, Sundstrom P, Thorell J: The success rate ofin vitro fertilization of human oocytes in relation to the concentrations of different hormones in follicular fluid and peripheral plasma. Fertil Steril 36:448, 1981

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12. Edwards RG, Purdy JM, Steptoe PC, Walters DE: The growth of human preimplantation embryos in vitro. Am J Obstet Gynecol 141:408, 1981 13. Edwards RG: Test-tube babies, 1981. Nature 293:253, 1981

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