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Corpus luteum function after follicle aspiration for oocyte retrieval*
FERTILITY AND 8TEruiJTY Copyright c 1981 The American Fertility Society
Vol. 36, No. 5; November 1981 Print£d in U.S.A.
CORPUS LUTEUM FUNCTION AFTER FOLLICLE ASPIRATION FOR OOCYTE RETRIEVAL*
JAIRO GARCIA, M.D. GEORGEANNA S. JONES, M.D. ANffiAL A. ACOSTA, M.D.t GEORGE .L. WRIGHT, JR., PH.D. Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, Virginia 23508
Follicle aspiration for in vitro fertilization is associated with a statistical disruption of the luteal phase. The severity of the disruption seems to be in relation to the vigorousness and the number of aspirations and therefore the number of granulosa .cells that are dislodged from the membrana granulosa layer. Although the statistical ·importance of this disruption from a biologic point of view. does not seem to be significant, as measured by the length of the luteal phase, an analysis of individual cases must be made in order to determine the frequency with which a biologically significant luteal defect may be produced. At the present time, it seems that the fewer the granulosa cells removed at aspiration, the less the luteal disruption will be. The effect of the surgical trauma and anesthesia that should be constant throughout the series is discounted as an important factor in inducing luteal dysfunction. Fertil Steril 36:565, 1981
Attempts to use in vitro fertilization as a tubal bypass procedure in two medical centers have shown that embryo transfer itself seems to be the step that accounts for the majority of failures. 1 It has been suggested that interference with corpus luteum function due to disruption of the previous dominant follicle may cause corpus luteum insufficiency and account for the low success rate of embryo transfer. 2 Several etiologic factors have been mentioned in the literature that may be responsible for corpus luteum insufficiency. 3 Among these are two specific factors that may play a role in in vitro fertilization; one is surgical stress associated with hyperprolactinemia, 4 and the other, in the subhuman primate, is disruption
of the corpus luteum by follicle aspiration. 5 It therefore seemed that evaluation of corpus luteurn function after follicle aspiration for in vitro fertilization in the human was .of some importance.6 This investigation was initiated during an in vitro fertilization program in order to determine (1) the effect of surgical anesthesia and (2) the effect of aspiration of the dominant follicle in the periovulatory period of the normal menstrual cycle on subsequent corpus luteum development and function. MATERIALS AND METHODS
Patient Selection Thirty-two patients from the Vital Initiation of Pregnancy Program at the Eastern Virginia Medical School underwent hormonal monitoring of menstrual cycle before and after oocyte retrieval attempts. A total of 85 menstrual cycles were reviewed, and 71 were found to be adequate for endocrine analysis. A total of 30 cycles was inves-
Received April15, 1981; revised and accepted July 23, 1981. *Presented at· the Thirty~Seventh Annual Meeting of the American Fertility Society, March 14 to 18, 1981, Atlanta, ·Georgia. tReprint requests: Dr. Anibal A. Acosta, Department of Obstetrics and Gynecology, DePaul Hospital, 150 Kingsley Lane, Norfolk, Virginia 23505.
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GARCIA ET AL.
tigated before any attempt of egg retrieval was done. This group seemed to have normal menstrual cycles characterized by length and hi phasic basal body temperature charts and is designated as group A. There were 15 cycles within this group in which an endometrial biopsy was taken for further evaluation of corpus luteum function. These were designated as group B. All endometrial biopsies were examined and dated by the same observer (J. Donald Woodruff, Department of Obstetrics and Gynecology, Johns Hopkins Hospital). Eleven of these 15 cycles also had estradiol serum values. Forty-one menstrual cycles of these same patients were reinvestigated during an oocyte retrieval attempt by laparoscopy. These cycles were retrospectively classified into four different categories according to the degree of maturation of the oocyte recovered, failure to retrieve an oocyte, finding of an already ruptured follicle (corpus luteum), and whether stimulation or supplementation during the luteal phase was used or avoided. There were eight cycles in which successful follicle aspiration was associated with mature oocyte retrieval and subsequent fertilization (group C). After embryo transfer these eight patients received either supplementation of corpus luteum function by progesterone suppositories 25 mg twice daily (seven cycles) or corpus luteum stimulation with human chorionic gonadotropin at 2500 IU intramuscularly every other day up to day 10 (one cycle). Group D comprised 8 cycles from patients with successful follicle aspiration and retrieval of a mature oocyte, which nevertheless failed to fertilize, and therefore no embryo transfer was accomplished and no supplementation of corpus luteum function was attempted. Group E consisted of nine cycles in patients with follicle aspiration in which no oocyte was retrieved. Group F consisted of nine cycles in patients in whom a recent corpus luteum was found at the time of operation that was nevertheless aspirated in an unsuccessful attempt to retrieve the oocyte. A total of seven cycles were excluded from consideration. Five of these were so excluded because the oocyte recovered was immediately transferred to the uterus following an in vivo protocol to be reported separately. Two cycles were not considered because cystic structures were found in the ovaries rather than mature follicles. No cycle in group D, E, or F was supplemented during the luteal phase.
Clinical Monitoring Extensive monitoring of the menstrual cycle was carried out in each of the patients. These included investigation of clinical parameters (basal body temperature (BBT) chart, daily cervical mucus examinations, and karyopyknotic index investigations) and endocrine evaluations daily and every 4 hours in the periovulatory period, including serum LH, serum FSH, Sjlrum estradiol, and serum progesterone. Additionally daily ultrasound scanning for follicle growth monitoring was done.
Laboratory Assays Serum luteinizing hormone (LH) was assessed daily by using a radioimmunoassay (RIA) kit · (Amersham, Arlington Heights, Ill.). The RIA kit protocol was modified from a 16- to 24-hour incubation period to a 3-hour incubation period. This permitted serum specimens to be processed and LH concentrations calculated on a daily basis (total test time approximately 5 hours). Although lower zero binding occurred as expected with the faster modification (approximately 55% of that observed in the overnight assay), a close correlation (r = 0.97) between values with the two protocols was maintained. The precision of the rapid LH assay was determined on 30 within-assay tests (CV = 7.99%) and 18 between-assay tests (CV = 10.8%). Serum FSH was measured by RIA with the double antibody method of Midgley. 7 Serum progesterone was assessed with the use of the progesterone RIA kit from Wien Laboratories (Succasunna, N. J.). The unknown control sera and reagent blanks were all extracted with glass-distilled petroleum ether. An appropriate aliquot was taken from the top ether layer and evaporated to dryness in a 45 to 50° C waterbath with the aid of a nitrogen airstream. The unextracted progesterone standards were also evaporated to dryness. All samples, including the standards, were run in duplicate. The RIA portion of the procedure was run by adding 3 H-progesterone, phosphate buffer, and working progesterone antibody solution consecutively. All tubes were then incubated in 6 to 10° C icebath for 60 minutes. At the end of that time, cold dextran-coated charcoal was added to all tubes to absorb the unbound progesterone. The charcoal was then precipitated by centrifugation and the supernatant transferred to counting vials with scintillation cocktail. After standing in the dark at room tern-
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FOLLICLE ASPIRATION AND CORPUS LUTEUM FUNCTION
perature for at least an hour, the samples were counted in a Beckman model LS-250 scintillation counter. A standard curve for each assay was constructed by plotting the progesterone standards (picogram per tube) against a ratio of CPM of the zero to each unknown. Each progesterone concentration minus the value ofthe blank was converted from picograms per tube to nanograms per milliliter. Within-assay precision (CV = 8.2%) and between-assay precision (CV = 12.0%), based on 18 consecutive assays, were within allowable limits of the RIA procedure. The serum estradiol concentration was determined by RIA with the use of a modification of the procedure proposed by Abraham et al. 8 All endocrine values were plotted and normalized around the LH peak (0 time). Finally, the total length of the secretory phase of the menstrual cycle was determined in each counting from 10 hours after the LH peak to the first day of the menstrual period. During oocyte retrieval attempts the patient was hospitalized immediately before the procedure. Premedication consisted of Demerol (Winthrop, New York, N.Y.), 50 mg, Vistaril (Pfizer, New York, N. Y.), 50 mg, and Robinul (A. H. Robins, Richmond, Va.), 0.2 mg on call to the operating room. A common anesthesia protocol was used for all patients having laparoscopy for oocyte retrieval. n-Tubo-curare, 5 mg intravenously, fentanyl 1.0 to 2.0 ml intravenously, according to the patient's weight, Na-Pentothal (Abbott, North Chicago, Ill.), 250 to 300 mg intravenously, succinylcholine, and L.T.A. topical to the trachea and endotracheal intubation followed by N202 2-5:2 ratio were used as anesthesia, and the patient recovered in the usual manner with the use of oxygen 100% by mask until she was alert and reoriented. The surgical procedure used for oocyte retrieval was a routine type of laparoscopy by the double puncture technique performed by the same group of surgeons. Oocyte retrieval was attempted in all cases by aspiration of the dominant follicle and in some instances other secondary follicles or a recent corpus luteum with a 2.2-mm (inside diameter) needle and syringe with suction by hand pressure. A representative group of follicles was selected for study in an effort to determine the number of granulosa cells retrieved at the time of follicle aspiration. The unpaired Student's t-test was used to analyze the differences between progesterone and es-
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Control Groups
FIG. 1. Serum progesterone values in 30 normal control cycles (group A). No aspiration was performed in these cycles. A subset of 15 cycles is included (group B) in which endometrial biopsy showed a normal, in-phase pattern, further confirming the normality of the cycle.
tradiol levels on specific cycle days. In order to test the differences between the progesterone values and the estradiol values between groups throughout the entire cycle, an analysis of variance with covariance was used. The day of the cycle and the square of the day of the cycle were used as covariants in the analysis in order to eliminate the effect of position in the cycle on the overall progesterone or estradiol levels. RESULTS
Progesterone In analyzing progesterone levels in the control group of 30 cycles a comparison was made be-
Group
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FIG. 2. Serum progesterone values (-- -) in cycles in which a mature oocyte was aspirated from the follicle and an embryo was transferred to the uterus subsequently (group C). These cycles were all supplemented with progesterone, 12.5 mg in oil intramuscularly daily, or 25 mg progesterone vaginal suppositories twice daily after day + 4 with the exception of one cycle, which was stimulated with 2500 IU ofhCG daily, days +5, +7, +9, and +11. These values are plotted against the nonaspirated control cycles (-). There was a statistically significant prolongation of the cycle and no statistical significant difference in the progesterone secretion. These cycles were associated with the least vigorous aspirations and the fewest numbers of follicle washes.
, li
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GARCIA ET AL.
November 1981
Group F
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FIG. 3. Serum progesterone values(---) in cycles in which a mature oocyte was aspirated from the follicle but no embryo transfer was possible, (group D). These cycles are plotted against the nonaspirated control cycles (-). There is a small but statistically significant difference in the progesterone serum values between the aspirated and the unaspirated cycles.
tween the values in the 15 cycles of patients with endometrial biopsies taken and in the 15 cycles without biopsies. No significant differences were found by computer analysis (results not shown). Since group B had a normal luteal phase as judged by properly timed and dated endometrial biopsies, it was assumed that the entire group was homogeneous. Therefore the total number of 30 cycles in group A was used as the final control series. The similarity between the entire group and the subgroup, group B, is seen in Figure 1. When the values of group C, represented by cycles with mature follicles, as indicated by the recovery of a mature oocyte that fertilized, were compared with those of the control cycle (Fig. 2), with the use of analysis of variance with covariance, no significant difference between the groups
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FIG. 5. Serum progesterone values (-- -) in cycles in which the follicle was found to have ovulated but was nevertheless aspirated through the recently ruptured stoma. These cycles are plotted against the nonaspirated control cycles (-). The cycles in group F, in which follicles had discharged their content of granulosa cells into the peritoneal cavity and then were further traumatized with mechanical aspiration, showed the most significant decrease in the progesterone values.
was found. In this group progesterone substitution or human chorionic gonadotropin (hCG) stimulation was used for support of embryo implantation, perhaps accounting, at least in part, for the prolongation of this cycle. When group D was plotted against the control group (Fig. 3) and analysis of variance with covariance was performed, a significantly lower progesterone level (P = 0.025) was demonstrated. The only difference between the cycles in group C and group D was the absence of corpus luteum supplementation or stimulation. When the curves of group E cycles, in which no oocyte was retrieved (Fig. 4) and F cycles, associated with ruptured follicles and corpus luteum formation (Fig. 5), were plotted against the control cycles, again, a significantly low progesterone level was demonstrated (P = 0.022 and P = 0.001, respectively).
GroupE
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FIG. 4. Serum progesterone values(---) in cycles in which no oocyte was retrieved from the follicle (group E). The most vigorous aspirations and the greatest number of follicle washes were performed in these cycles. There was a statistically significant decrease in the amount of progesterone in the aspirated cycles, as compared with the unaspirated control cycles.
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FIG. 6. Daily serum estradiol levels during the luteal phase and 4-hour estradiol levels during the periovulatory and ovulatory phase in 11 of 30 control cycles in which no aspiration was accomplished.
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FOLLICLE ASPIRATION AND CORPUS LUTEUM FUNCTION
Group C
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FIG. 7. Serum estradiol values plotted as described in Figure 6 for patients in group C (---),described in Figure 2, against control cycles seen in Figure 6 (-). Although there are many fluctuations, there is no statistical difference in the group C estradiol values and those of the control cycles. Interestingly, these patients received progesterone supplementation but no estradiol supplementation.
In summary, supplementation with progesterone suppositories or stimulation of corpus luteum with hCG in cycles with successful egg fertilization and transfer resulted in progesterone levels similar to those in the control group. In patients receiving neither stimulation nor supplementation following follicle aspiration,_ significantly lower progesterone levels were recorded.
Estradiol Estradiol values, obtained in 11 cycles out of 30 in the control group, were plotted and analyzed in a similar manner (Fig. 6). No significant difference from control cycles to the cycles of group C (Fig. 7) were found, although no estradiol supplementation had been used during these cycles. In group D, again, no significant difference was found from the control group A (Fig. 8). Groups E
FIG. 9. Serum estradiol levels (-- -) in group E as described in Figure 4, plotted against the control cycles (-). These estradiol values showed a statistically significant decrease as compared with estradiol findings in the control cycles.
and F (Figs. 9 and 10) showed a highly significant reduction of estradiol levels from those seen in the control cycles (P = 0.001 and P = 0.003, respectively).
Duration of the Luteal Phase Length of the secretory phase was measured from 10 hours after the LH peak to the time menstrual bleeding began. Twenty-nine cycles were available for analysis and a mean of 12.3 days (standard deviation (SD) = 3.5) was found, with variation ranging from 12 to 16 days. Group C showed a mean cycle variation of 19.6 days (SD = 3.9) with variation ranging from 14 to 26 days. This is obviously considered statistically significant (P = 0.001), and the luteal phase was clearly prolonged in many of these cycles. The biologic significance of this will be discussed. Group F
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FIG. 8. Serum estradiol levels in patients in group D (-- -) described in Figure 3, again plotted against the control cycles (-). As in the progesterone-supplemented group, there was no evidence of an estrogen insufficiency, contrary to the results with progesterone serum assays in group D.
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FIG. 10. Estradiol values(---) in group F as described in Figure 5, again plotted against clinical values in Figure 6. There was a statistically significant lowering of the estradiol values in these patients, who had ovulated prior to follicle aspiration but had additional aspiration through the stoma of the follicle. Both group E and group F showed similar decreases in estradiol and progesterone values.
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GARCIA ET AL.
Group D shows a mean of 15.1 days (SD = 1.45), the shortest luteal phase being 13 days and the longest being 18 days. This also represents a significant increase in duration of the luteal phase (P = 0.030). Group E shows a mean cycle luteal phase of 13.6 days (SD = 0.8), with a total duration fluctuating between 13 and 15 days. This is not significantly different from the control groups. Group F has a mean luteal phase duration of 13.7 days (SD = 1.59) with extremes of 11 and 16 days for the ovulatory phase. Again these figures are not significantly different from those of the control group.
Granulosa Cell Aspiration The number of viable granulosa cells was available in 30 cases (Table 1). These data cannot be assumed to be exactly comparable, because the time interval between follicle aspiration and cell count was variable, and the number of viable cells may differ with this time interval. Nevertheless, the average number of granulosa cells aspirated was 3 x 106 , the lowest being 6000 and the highest being 12 million. The diameter of the follicle was considered in 19 aspirates. In 10 observations the follicle measured 18 mm or more. The average number of viable cells counted was 4. 72 x 106 , the lowest count being 102,000 and the highest 12 million. In 9 cases the follicle was reported as being 17 mm or less in diameter. The average count of viable granulosa cells recovered was 2.11 x 106 , the lowest count being 6000 and the highest 6,320,000. DISCUSSION
The results of statistical analysis of the total surgical procedures of oocyte aspiration for in vitro fertilization lead to the conclusion that there is a disturbance of luteal function. The most severe disruption of function in both progesterone and estrogen steroidogenesis occurred in those cycles in which the most vigorous and repetitive TABLE 1. Number of Viable Granulosa Cells per Milliliter
Retrieved at the Time of Follicle Aspiration No. of cases
30 11 10 9
Diameter of follicle
Average no. of cells
3,008,140 (6000--12,000,000) Not stated ;a.18mm .;;; 17mm
4,720,200 (102,000--12,000,000) 2,116,111 (6000--6,320,000)
November 1981 aspirations were carried out. Cycles of group F, those in which the ovulation had occurred prior to the procedure and a leaking follicle was present, showed the most severe luteal defects (Fig. 5). These follicles had already spilled their contents, including a number of luteinized granulosa cells, into the peritoneal cavity. In an effort to retrieve an oocyte these recent corpora lutea were subjected to further mechanical insult with aspiration and removal of additional granulosa cells from the membrana granulosa. The next greatest statistical disruption of the luteal function occurred in group E, cycles in which no oocyte was obtained (Fig. 4). In this group multiple washings of the follicle were carried out in an effort to secure an oocyte. The least disruption of the luteal function was found in group D, in which a mature follicle was recovered, but because of failure to fertilize no embryo was transferred, and therefore no supplementation of the luteal phase was given (Fig. 3). The follicles in group D corresponded completely to follicles in group C as far as aspiration procedures were concerned. However, in group C, the oocyte was fertilized and the embryo transferred. Supplementation of the luteal phase with progesterone was carried out in eight cycles, and stimulation of corpus luteum function with hCG in one. There was no statistical evidence of disruption of luteal function in this group of cycles (Fig. 2). It is presumed that this is because the luteal phase was supplemented, because otherwise these patients clinically resembled those described in group D. This suggestion of a progression of severity in disruption of the luteal phase according to the vigor and number of follicular aspirations lends support to the theory that the mechanical removal of granulosa cells plays the most important part in the luteal disruption, while surgical stress and anesthesia are unimportant or minor, because these variables were similar in all groups of cycles. Although general anesthesia per se has been shown to be associated with hyperprolactinemia and a decrease corpus luteum function after surgery,4 Edwards and Steptoe and the Melbourne group in Australia have demonstrated that although their patients did have hyperprolactinemia, they had relatively little disruption of the luteal function. We therefore did not think it necessary to repeat prolactin assays. Some disruption of estradiol steroidogenesis was also statistically demonstrated in the two most vigorously and frequently aspirated groups
•
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FOLLICLE ASPIRATION AND CORPUS LUTEUM FUNCTION
of follicles (groups E and F) (Figs. 9 and 10). It is assumed that the decrease in estradiol values is a reflection of the decrease of steroid substrate progesterone. This assumption seems to be strengthened by the finding that the E 2 values were within normal limits in those cycles in which progesterone supplementation or stimulation with hCG of corpus luteum function occurred (Fig. 7). In our series the average figure of aspirated viable granulosa cells was 3 x 106 . It seems that this figure varies with the diameter of the dominant follicle and undoubtedly represents a sizable number of cells. Whether these cells are dislodged by aspiration or are already floating in the follicular fluid cannot be determined with any accuracy. Nevertheless, since luteinized granulosa cells are found in the cul-de-sac, in the peritoneal fluid after ovulation, and since sheets of luteinized granulosa cells apparently from the membrana granulosa are found frequently in the follicular wash specimens, both sources of dislodged granulosa cells are undoubtedly present. The number of active granulosa cells in the mature human follicle has been estimated to be approximately 50 x 106 •9 Thus, the number of granulosa cells removed at aspiration, particularly when multiple procedures are done, is theoretically of some concern. In subhuman primates progesterone insufficiency has been shown in the early part of the postovulatory phase following follicle aspiration,5 but the individual pattern of corpus luteurn disruption in our patients did not seem to follow this same curve. Although the luteal function can be shown to be impaired after follicle aspiration in relation to controlled cycles in the same individuals, these mathematical differences may not be biologically significant. This possibility is borne out by the fact that in cycles E and F, in which both progesterone and estradiol were statistically lower than in controlled cycles, there was no association with a shortened secretory phase. The mean duration of the luteal phase in the controlled cycles was 12.3 days, with variations ranging between 12 and 16 days. This was 13.6 days, with a fluctuation between 13 and 15 days, and 13.7 days, with an extreme of 11 to 16 days in groups E and F, respectively. In group C, cycles that were supplemented with progesterone, there was a statistically significant prolongation of the luteal phase. The statistical mean of 19.6 days is highly significant, especially since it can be seen from the spread that some patients had a normal 14-day span in spite of supplementation, while others were greatly prolonged, one being 26
571
days, that is, almost 2 weeks past the expected time of the period. This is of particular interest, because in the study of supplemented cycles both in infertility patients with either normal corpus luteum function or inadequate corpus luteum function, 10• 11 no such prolongation was seen, and the longest delay was 4 days in an inadequate luteal phase cycle. In many years of supplementing the luteal phase with progesterone, such pseudopregnancy reactions had never before occurred. In none of these cycles, however, was it possible with the serum available to document any significant changes in ~-hCG levels. We have nevertheless asked whether this could pos--;., sibly represent a failure of the implanted embryo, which was matured in vitro, to develop a trophoblastic hormone function. If this were the case, such a fundamental defect would probably be associated with other defects incompatible with embryo development. It is important at this time to state that this study relates only to the overall statistics. Study of the relation of individual cases to these statistics is required for an understanding of the biologic significance of the various procedures. Thus, as indicated above, the analysis of the individual patients' records in group C with prolonged cycles and those with 14-day cycles will perhaps help us to understand the biologic significance and to evaluate the possible role of progesterone supplementation. Likewise, although the mean values of progesterone in the control cycles, compared with the mean values in groups E and F, appear to represent relatively insignificant differences (Figs. 4 and 5), when the progesterone curves for individual patients are plotted against these curves and against the normal curves, some are strikingly deficient and are associated with relatively short luteal cycles. Thus, individual analysis may lend further support, or discredit, to the theory that the vigorousness or repetitiousness, or both,_ of follicle aspiration is associated with more severe defects in corpus luteum function.
Acknowledgments. The authors would like to express their thanks to Connie Halforty, Becky Spear, and Diane Brussel, Dr. George Wright's technicians, for technical assistance in LH and progesterone determinations; to all of the nursing personnel in labor and delivery at Norfolk General Hospital for their unselfish cooperation, and to Mrs. Cathy Bannon for typing the manuscript.
GARCIA ET AL.
572 REFERENCES
1. Steptoe PC, Edwards RG, Purdy JM: Clinical aspects ?f pregnancies established with cleaving embryos grown m vitro. Br J Obstet Gynaecol 87:757, 1980 2. Edwards RG, Steptoe PC, Fowler RE, Baillie J: Observations on preovulatory human ovarian follicles and their aspirates. Br. J Obstet Gynaecol 87:769, 1980 3. Wentz AC: Treatment of luteal phase defects. J Reprod Med 18:159, 1979 4. Soules MR, Sutton GP, Hammond ChB, Haney AF: Endocrine changes at operation under general anesthesia: reproductive hormone fluctuations in young women. Fertil Steril 33:364, 1980 5. Hodgen GD: Personal communication 6. Lopata A: Successes and failures in human in vitro fertilization. Nature 288:642, 1980
d' !
I
November 1981 7. Midgley AR: Radioimmunoassay of human follicle stimulating hormone. J Clin Endocrinol Metab 27:295, 1967 8. Abraham GE, Swerdloff R, Tulchinsky D, Odell WD: Radioimmunoassay of plasma progesterone. J Clin Endocrinol Metab 32:619, 1971 9. McNatty KP, Smith DM, Makris A, Sathanond R, Ryan KJ: The microenvironment of the human Graafian follicle: interrelationship among the steroid levels in antral fluid, the population of granulosa cells and the status of the oocyte in vivo and in vitro. J Clin Endocrinol Metab 49:851, 1979 10. Jones GS, Aksel S, Wentz AC: Serum progesterone values in the luteal phase defects: effect of chorionic gonadotropin. Obstet Gynecol44:26, 1974 11. Aksel S, Jones GES: Effect of progesterone and 17 -hydroxyprogesterone caproate on normal corpus luteum function. Am J Obstet Gynecol118:466, 1974
Vol. 36, No. 5; November 1981 Print£d in U.S.A.
CORPUS LUTEUM FUNCTION AFTER FOLLICLE ASPIRATION FOR OOCYTE RETRIEVAL*
JAIRO GARCIA, M.D. GEORGEANNA S. JONES, M.D. ANffiAL A. ACOSTA, M.D.t GEORGE .L. WRIGHT, JR., PH.D. Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, Virginia 23508
Follicle aspiration for in vitro fertilization is associated with a statistical disruption of the luteal phase. The severity of the disruption seems to be in relation to the vigorousness and the number of aspirations and therefore the number of granulosa .cells that are dislodged from the membrana granulosa layer. Although the statistical ·importance of this disruption from a biologic point of view. does not seem to be significant, as measured by the length of the luteal phase, an analysis of individual cases must be made in order to determine the frequency with which a biologically significant luteal defect may be produced. At the present time, it seems that the fewer the granulosa cells removed at aspiration, the less the luteal disruption will be. The effect of the surgical trauma and anesthesia that should be constant throughout the series is discounted as an important factor in inducing luteal dysfunction. Fertil Steril 36:565, 1981
Attempts to use in vitro fertilization as a tubal bypass procedure in two medical centers have shown that embryo transfer itself seems to be the step that accounts for the majority of failures. 1 It has been suggested that interference with corpus luteum function due to disruption of the previous dominant follicle may cause corpus luteum insufficiency and account for the low success rate of embryo transfer. 2 Several etiologic factors have been mentioned in the literature that may be responsible for corpus luteum insufficiency. 3 Among these are two specific factors that may play a role in in vitro fertilization; one is surgical stress associated with hyperprolactinemia, 4 and the other, in the subhuman primate, is disruption
of the corpus luteum by follicle aspiration. 5 It therefore seemed that evaluation of corpus luteurn function after follicle aspiration for in vitro fertilization in the human was .of some importance.6 This investigation was initiated during an in vitro fertilization program in order to determine (1) the effect of surgical anesthesia and (2) the effect of aspiration of the dominant follicle in the periovulatory period of the normal menstrual cycle on subsequent corpus luteum development and function. MATERIALS AND METHODS
Patient Selection Thirty-two patients from the Vital Initiation of Pregnancy Program at the Eastern Virginia Medical School underwent hormonal monitoring of menstrual cycle before and after oocyte retrieval attempts. A total of 85 menstrual cycles were reviewed, and 71 were found to be adequate for endocrine analysis. A total of 30 cycles was inves-
Received April15, 1981; revised and accepted July 23, 1981. *Presented at· the Thirty~Seventh Annual Meeting of the American Fertility Society, March 14 to 18, 1981, Atlanta, ·Georgia. tReprint requests: Dr. Anibal A. Acosta, Department of Obstetrics and Gynecology, DePaul Hospital, 150 Kingsley Lane, Norfolk, Virginia 23505.
565
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November 1981
GARCIA ET AL.
tigated before any attempt of egg retrieval was done. This group seemed to have normal menstrual cycles characterized by length and hi phasic basal body temperature charts and is designated as group A. There were 15 cycles within this group in which an endometrial biopsy was taken for further evaluation of corpus luteum function. These were designated as group B. All endometrial biopsies were examined and dated by the same observer (J. Donald Woodruff, Department of Obstetrics and Gynecology, Johns Hopkins Hospital). Eleven of these 15 cycles also had estradiol serum values. Forty-one menstrual cycles of these same patients were reinvestigated during an oocyte retrieval attempt by laparoscopy. These cycles were retrospectively classified into four different categories according to the degree of maturation of the oocyte recovered, failure to retrieve an oocyte, finding of an already ruptured follicle (corpus luteum), and whether stimulation or supplementation during the luteal phase was used or avoided. There were eight cycles in which successful follicle aspiration was associated with mature oocyte retrieval and subsequent fertilization (group C). After embryo transfer these eight patients received either supplementation of corpus luteum function by progesterone suppositories 25 mg twice daily (seven cycles) or corpus luteum stimulation with human chorionic gonadotropin at 2500 IU intramuscularly every other day up to day 10 (one cycle). Group D comprised 8 cycles from patients with successful follicle aspiration and retrieval of a mature oocyte, which nevertheless failed to fertilize, and therefore no embryo transfer was accomplished and no supplementation of corpus luteum function was attempted. Group E consisted of nine cycles in patients with follicle aspiration in which no oocyte was retrieved. Group F consisted of nine cycles in patients in whom a recent corpus luteum was found at the time of operation that was nevertheless aspirated in an unsuccessful attempt to retrieve the oocyte. A total of seven cycles were excluded from consideration. Five of these were so excluded because the oocyte recovered was immediately transferred to the uterus following an in vivo protocol to be reported separately. Two cycles were not considered because cystic structures were found in the ovaries rather than mature follicles. No cycle in group D, E, or F was supplemented during the luteal phase.
Clinical Monitoring Extensive monitoring of the menstrual cycle was carried out in each of the patients. These included investigation of clinical parameters (basal body temperature (BBT) chart, daily cervical mucus examinations, and karyopyknotic index investigations) and endocrine evaluations daily and every 4 hours in the periovulatory period, including serum LH, serum FSH, Sjlrum estradiol, and serum progesterone. Additionally daily ultrasound scanning for follicle growth monitoring was done.
Laboratory Assays Serum luteinizing hormone (LH) was assessed daily by using a radioimmunoassay (RIA) kit · (Amersham, Arlington Heights, Ill.). The RIA kit protocol was modified from a 16- to 24-hour incubation period to a 3-hour incubation period. This permitted serum specimens to be processed and LH concentrations calculated on a daily basis (total test time approximately 5 hours). Although lower zero binding occurred as expected with the faster modification (approximately 55% of that observed in the overnight assay), a close correlation (r = 0.97) between values with the two protocols was maintained. The precision of the rapid LH assay was determined on 30 within-assay tests (CV = 7.99%) and 18 between-assay tests (CV = 10.8%). Serum FSH was measured by RIA with the double antibody method of Midgley. 7 Serum progesterone was assessed with the use of the progesterone RIA kit from Wien Laboratories (Succasunna, N. J.). The unknown control sera and reagent blanks were all extracted with glass-distilled petroleum ether. An appropriate aliquot was taken from the top ether layer and evaporated to dryness in a 45 to 50° C waterbath with the aid of a nitrogen airstream. The unextracted progesterone standards were also evaporated to dryness. All samples, including the standards, were run in duplicate. The RIA portion of the procedure was run by adding 3 H-progesterone, phosphate buffer, and working progesterone antibody solution consecutively. All tubes were then incubated in 6 to 10° C icebath for 60 minutes. At the end of that time, cold dextran-coated charcoal was added to all tubes to absorb the unbound progesterone. The charcoal was then precipitated by centrifugation and the supernatant transferred to counting vials with scintillation cocktail. After standing in the dark at room tern-
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FOLLICLE ASPIRATION AND CORPUS LUTEUM FUNCTION
perature for at least an hour, the samples were counted in a Beckman model LS-250 scintillation counter. A standard curve for each assay was constructed by plotting the progesterone standards (picogram per tube) against a ratio of CPM of the zero to each unknown. Each progesterone concentration minus the value ofthe blank was converted from picograms per tube to nanograms per milliliter. Within-assay precision (CV = 8.2%) and between-assay precision (CV = 12.0%), based on 18 consecutive assays, were within allowable limits of the RIA procedure. The serum estradiol concentration was determined by RIA with the use of a modification of the procedure proposed by Abraham et al. 8 All endocrine values were plotted and normalized around the LH peak (0 time). Finally, the total length of the secretory phase of the menstrual cycle was determined in each counting from 10 hours after the LH peak to the first day of the menstrual period. During oocyte retrieval attempts the patient was hospitalized immediately before the procedure. Premedication consisted of Demerol (Winthrop, New York, N.Y.), 50 mg, Vistaril (Pfizer, New York, N. Y.), 50 mg, and Robinul (A. H. Robins, Richmond, Va.), 0.2 mg on call to the operating room. A common anesthesia protocol was used for all patients having laparoscopy for oocyte retrieval. n-Tubo-curare, 5 mg intravenously, fentanyl 1.0 to 2.0 ml intravenously, according to the patient's weight, Na-Pentothal (Abbott, North Chicago, Ill.), 250 to 300 mg intravenously, succinylcholine, and L.T.A. topical to the trachea and endotracheal intubation followed by N202 2-5:2 ratio were used as anesthesia, and the patient recovered in the usual manner with the use of oxygen 100% by mask until she was alert and reoriented. The surgical procedure used for oocyte retrieval was a routine type of laparoscopy by the double puncture technique performed by the same group of surgeons. Oocyte retrieval was attempted in all cases by aspiration of the dominant follicle and in some instances other secondary follicles or a recent corpus luteum with a 2.2-mm (inside diameter) needle and syringe with suction by hand pressure. A representative group of follicles was selected for study in an effort to determine the number of granulosa cells retrieved at the time of follicle aspiration. The unpaired Student's t-test was used to analyze the differences between progesterone and es-
567
Control Groups
FIG. 1. Serum progesterone values in 30 normal control cycles (group A). No aspiration was performed in these cycles. A subset of 15 cycles is included (group B) in which endometrial biopsy showed a normal, in-phase pattern, further confirming the normality of the cycle.
tradiol levels on specific cycle days. In order to test the differences between the progesterone values and the estradiol values between groups throughout the entire cycle, an analysis of variance with covariance was used. The day of the cycle and the square of the day of the cycle were used as covariants in the analysis in order to eliminate the effect of position in the cycle on the overall progesterone or estradiol levels. RESULTS
Progesterone In analyzing progesterone levels in the control group of 30 cycles a comparison was made be-
Group
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FIG. 2. Serum progesterone values (-- -) in cycles in which a mature oocyte was aspirated from the follicle and an embryo was transferred to the uterus subsequently (group C). These cycles were all supplemented with progesterone, 12.5 mg in oil intramuscularly daily, or 25 mg progesterone vaginal suppositories twice daily after day + 4 with the exception of one cycle, which was stimulated with 2500 IU ofhCG daily, days +5, +7, +9, and +11. These values are plotted against the nonaspirated control cycles (-). There was a statistically significant prolongation of the cycle and no statistical significant difference in the progesterone secretion. These cycles were associated with the least vigorous aspirations and the fewest numbers of follicle washes.
, li
568
GARCIA ET AL.
November 1981
Group F
Group D
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FIG. 3. Serum progesterone values(---) in cycles in which a mature oocyte was aspirated from the follicle but no embryo transfer was possible, (group D). These cycles are plotted against the nonaspirated control cycles (-). There is a small but statistically significant difference in the progesterone serum values between the aspirated and the unaspirated cycles.
tween the values in the 15 cycles of patients with endometrial biopsies taken and in the 15 cycles without biopsies. No significant differences were found by computer analysis (results not shown). Since group B had a normal luteal phase as judged by properly timed and dated endometrial biopsies, it was assumed that the entire group was homogeneous. Therefore the total number of 30 cycles in group A was used as the final control series. The similarity between the entire group and the subgroup, group B, is seen in Figure 1. When the values of group C, represented by cycles with mature follicles, as indicated by the recovery of a mature oocyte that fertilized, were compared with those of the control cycle (Fig. 2), with the use of analysis of variance with covariance, no significant difference between the groups
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FIG. 5. Serum progesterone values (-- -) in cycles in which the follicle was found to have ovulated but was nevertheless aspirated through the recently ruptured stoma. These cycles are plotted against the nonaspirated control cycles (-). The cycles in group F, in which follicles had discharged their content of granulosa cells into the peritoneal cavity and then were further traumatized with mechanical aspiration, showed the most significant decrease in the progesterone values.
was found. In this group progesterone substitution or human chorionic gonadotropin (hCG) stimulation was used for support of embryo implantation, perhaps accounting, at least in part, for the prolongation of this cycle. When group D was plotted against the control group (Fig. 3) and analysis of variance with covariance was performed, a significantly lower progesterone level (P = 0.025) was demonstrated. The only difference between the cycles in group C and group D was the absence of corpus luteum supplementation or stimulation. When the curves of group E cycles, in which no oocyte was retrieved (Fig. 4) and F cycles, associated with ruptured follicles and corpus luteum formation (Fig. 5), were plotted against the control cycles, again, a significantly low progesterone level was demonstrated (P = 0.022 and P = 0.001, respectively).
GroupE
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FIG. 4. Serum progesterone values(---) in cycles in which no oocyte was retrieved from the follicle (group E). The most vigorous aspirations and the greatest number of follicle washes were performed in these cycles. There was a statistically significant decrease in the amount of progesterone in the aspirated cycles, as compared with the unaspirated control cycles.
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FIG. 6. Daily serum estradiol levels during the luteal phase and 4-hour estradiol levels during the periovulatory and ovulatory phase in 11 of 30 control cycles in which no aspiration was accomplished.
!'
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FOLLICLE ASPIRATION AND CORPUS LUTEUM FUNCTION
Group C
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569
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FIG. 7. Serum estradiol values plotted as described in Figure 6 for patients in group C (---),described in Figure 2, against control cycles seen in Figure 6 (-). Although there are many fluctuations, there is no statistical difference in the group C estradiol values and those of the control cycles. Interestingly, these patients received progesterone supplementation but no estradiol supplementation.
In summary, supplementation with progesterone suppositories or stimulation of corpus luteum with hCG in cycles with successful egg fertilization and transfer resulted in progesterone levels similar to those in the control group. In patients receiving neither stimulation nor supplementation following follicle aspiration,_ significantly lower progesterone levels were recorded.
Estradiol Estradiol values, obtained in 11 cycles out of 30 in the control group, were plotted and analyzed in a similar manner (Fig. 6). No significant difference from control cycles to the cycles of group C (Fig. 7) were found, although no estradiol supplementation had been used during these cycles. In group D, again, no significant difference was found from the control group A (Fig. 8). Groups E
FIG. 9. Serum estradiol levels (-- -) in group E as described in Figure 4, plotted against the control cycles (-). These estradiol values showed a statistically significant decrease as compared with estradiol findings in the control cycles.
and F (Figs. 9 and 10) showed a highly significant reduction of estradiol levels from those seen in the control cycles (P = 0.001 and P = 0.003, respectively).
Duration of the Luteal Phase Length of the secretory phase was measured from 10 hours after the LH peak to the time menstrual bleeding began. Twenty-nine cycles were available for analysis and a mean of 12.3 days (standard deviation (SD) = 3.5) was found, with variation ranging from 12 to 16 days. Group C showed a mean cycle variation of 19.6 days (SD = 3.9) with variation ranging from 14 to 26 days. This is obviously considered statistically significant (P = 0.001), and the luteal phase was clearly prolonged in many of these cycles. The biologic significance of this will be discussed. Group F
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FIG. 8. Serum estradiol levels in patients in group D (-- -) described in Figure 3, again plotted against the control cycles (-). As in the progesterone-supplemented group, there was no evidence of an estrogen insufficiency, contrary to the results with progesterone serum assays in group D.
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FIG. 10. Estradiol values(---) in group F as described in Figure 5, again plotted against clinical values in Figure 6. There was a statistically significant lowering of the estradiol values in these patients, who had ovulated prior to follicle aspiration but had additional aspiration through the stoma of the follicle. Both group E and group F showed similar decreases in estradiol and progesterone values.
570
GARCIA ET AL.
Group D shows a mean of 15.1 days (SD = 1.45), the shortest luteal phase being 13 days and the longest being 18 days. This also represents a significant increase in duration of the luteal phase (P = 0.030). Group E shows a mean cycle luteal phase of 13.6 days (SD = 0.8), with a total duration fluctuating between 13 and 15 days. This is not significantly different from the control groups. Group F has a mean luteal phase duration of 13.7 days (SD = 1.59) with extremes of 11 and 16 days for the ovulatory phase. Again these figures are not significantly different from those of the control group.
Granulosa Cell Aspiration The number of viable granulosa cells was available in 30 cases (Table 1). These data cannot be assumed to be exactly comparable, because the time interval between follicle aspiration and cell count was variable, and the number of viable cells may differ with this time interval. Nevertheless, the average number of granulosa cells aspirated was 3 x 106 , the lowest being 6000 and the highest being 12 million. The diameter of the follicle was considered in 19 aspirates. In 10 observations the follicle measured 18 mm or more. The average number of viable cells counted was 4. 72 x 106 , the lowest count being 102,000 and the highest 12 million. In 9 cases the follicle was reported as being 17 mm or less in diameter. The average count of viable granulosa cells recovered was 2.11 x 106 , the lowest count being 6000 and the highest 6,320,000. DISCUSSION
The results of statistical analysis of the total surgical procedures of oocyte aspiration for in vitro fertilization lead to the conclusion that there is a disturbance of luteal function. The most severe disruption of function in both progesterone and estrogen steroidogenesis occurred in those cycles in which the most vigorous and repetitive TABLE 1. Number of Viable Granulosa Cells per Milliliter
Retrieved at the Time of Follicle Aspiration No. of cases
30 11 10 9
Diameter of follicle
Average no. of cells
3,008,140 (6000--12,000,000) Not stated ;a.18mm .;;; 17mm
4,720,200 (102,000--12,000,000) 2,116,111 (6000--6,320,000)
November 1981 aspirations were carried out. Cycles of group F, those in which the ovulation had occurred prior to the procedure and a leaking follicle was present, showed the most severe luteal defects (Fig. 5). These follicles had already spilled their contents, including a number of luteinized granulosa cells, into the peritoneal cavity. In an effort to retrieve an oocyte these recent corpora lutea were subjected to further mechanical insult with aspiration and removal of additional granulosa cells from the membrana granulosa. The next greatest statistical disruption of the luteal function occurred in group E, cycles in which no oocyte was obtained (Fig. 4). In this group multiple washings of the follicle were carried out in an effort to secure an oocyte. The least disruption of the luteal function was found in group D, in which a mature follicle was recovered, but because of failure to fertilize no embryo was transferred, and therefore no supplementation of the luteal phase was given (Fig. 3). The follicles in group D corresponded completely to follicles in group C as far as aspiration procedures were concerned. However, in group C, the oocyte was fertilized and the embryo transferred. Supplementation of the luteal phase with progesterone was carried out in eight cycles, and stimulation of corpus luteum function with hCG in one. There was no statistical evidence of disruption of luteal function in this group of cycles (Fig. 2). It is presumed that this is because the luteal phase was supplemented, because otherwise these patients clinically resembled those described in group D. This suggestion of a progression of severity in disruption of the luteal phase according to the vigor and number of follicular aspirations lends support to the theory that the mechanical removal of granulosa cells plays the most important part in the luteal disruption, while surgical stress and anesthesia are unimportant or minor, because these variables were similar in all groups of cycles. Although general anesthesia per se has been shown to be associated with hyperprolactinemia and a decrease corpus luteum function after surgery,4 Edwards and Steptoe and the Melbourne group in Australia have demonstrated that although their patients did have hyperprolactinemia, they had relatively little disruption of the luteal function. We therefore did not think it necessary to repeat prolactin assays. Some disruption of estradiol steroidogenesis was also statistically demonstrated in the two most vigorously and frequently aspirated groups
•
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FOLLICLE ASPIRATION AND CORPUS LUTEUM FUNCTION
of follicles (groups E and F) (Figs. 9 and 10). It is assumed that the decrease in estradiol values is a reflection of the decrease of steroid substrate progesterone. This assumption seems to be strengthened by the finding that the E 2 values were within normal limits in those cycles in which progesterone supplementation or stimulation with hCG of corpus luteum function occurred (Fig. 7). In our series the average figure of aspirated viable granulosa cells was 3 x 106 . It seems that this figure varies with the diameter of the dominant follicle and undoubtedly represents a sizable number of cells. Whether these cells are dislodged by aspiration or are already floating in the follicular fluid cannot be determined with any accuracy. Nevertheless, since luteinized granulosa cells are found in the cul-de-sac, in the peritoneal fluid after ovulation, and since sheets of luteinized granulosa cells apparently from the membrana granulosa are found frequently in the follicular wash specimens, both sources of dislodged granulosa cells are undoubtedly present. The number of active granulosa cells in the mature human follicle has been estimated to be approximately 50 x 106 •9 Thus, the number of granulosa cells removed at aspiration, particularly when multiple procedures are done, is theoretically of some concern. In subhuman primates progesterone insufficiency has been shown in the early part of the postovulatory phase following follicle aspiration,5 but the individual pattern of corpus luteurn disruption in our patients did not seem to follow this same curve. Although the luteal function can be shown to be impaired after follicle aspiration in relation to controlled cycles in the same individuals, these mathematical differences may not be biologically significant. This possibility is borne out by the fact that in cycles E and F, in which both progesterone and estradiol were statistically lower than in controlled cycles, there was no association with a shortened secretory phase. The mean duration of the luteal phase in the controlled cycles was 12.3 days, with variations ranging between 12 and 16 days. This was 13.6 days, with a fluctuation between 13 and 15 days, and 13.7 days, with an extreme of 11 to 16 days in groups E and F, respectively. In group C, cycles that were supplemented with progesterone, there was a statistically significant prolongation of the luteal phase. The statistical mean of 19.6 days is highly significant, especially since it can be seen from the spread that some patients had a normal 14-day span in spite of supplementation, while others were greatly prolonged, one being 26
571
days, that is, almost 2 weeks past the expected time of the period. This is of particular interest, because in the study of supplemented cycles both in infertility patients with either normal corpus luteum function or inadequate corpus luteum function, 10• 11 no such prolongation was seen, and the longest delay was 4 days in an inadequate luteal phase cycle. In many years of supplementing the luteal phase with progesterone, such pseudopregnancy reactions had never before occurred. In none of these cycles, however, was it possible with the serum available to document any significant changes in ~-hCG levels. We have nevertheless asked whether this could pos--;., sibly represent a failure of the implanted embryo, which was matured in vitro, to develop a trophoblastic hormone function. If this were the case, such a fundamental defect would probably be associated with other defects incompatible with embryo development. It is important at this time to state that this study relates only to the overall statistics. Study of the relation of individual cases to these statistics is required for an understanding of the biologic significance of the various procedures. Thus, as indicated above, the analysis of the individual patients' records in group C with prolonged cycles and those with 14-day cycles will perhaps help us to understand the biologic significance and to evaluate the possible role of progesterone supplementation. Likewise, although the mean values of progesterone in the control cycles, compared with the mean values in groups E and F, appear to represent relatively insignificant differences (Figs. 4 and 5), when the progesterone curves for individual patients are plotted against these curves and against the normal curves, some are strikingly deficient and are associated with relatively short luteal cycles. Thus, individual analysis may lend further support, or discredit, to the theory that the vigorousness or repetitiousness, or both,_ of follicle aspiration is associated with more severe defects in corpus luteum function.
Acknowledgments. The authors would like to express their thanks to Connie Halforty, Becky Spear, and Diane Brussel, Dr. George Wright's technicians, for technical assistance in LH and progesterone determinations; to all of the nursing personnel in labor and delivery at Norfolk General Hospital for their unselfish cooperation, and to Mrs. Cathy Bannon for typing the manuscript.
GARCIA ET AL.
572 REFERENCES
1. Steptoe PC, Edwards RG, Purdy JM: Clinical aspects ?f pregnancies established with cleaving embryos grown m vitro. Br J Obstet Gynaecol 87:757, 1980 2. Edwards RG, Steptoe PC, Fowler RE, Baillie J: Observations on preovulatory human ovarian follicles and their aspirates. Br. J Obstet Gynaecol 87:769, 1980 3. Wentz AC: Treatment of luteal phase defects. J Reprod Med 18:159, 1979 4. Soules MR, Sutton GP, Hammond ChB, Haney AF: Endocrine changes at operation under general anesthesia: reproductive hormone fluctuations in young women. Fertil Steril 33:364, 1980 5. Hodgen GD: Personal communication 6. Lopata A: Successes and failures in human in vitro fertilization. Nature 288:642, 1980
d' !
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November 1981 7. Midgley AR: Radioimmunoassay of human follicle stimulating hormone. J Clin Endocrinol Metab 27:295, 1967 8. Abraham GE, Swerdloff R, Tulchinsky D, Odell WD: Radioimmunoassay of plasma progesterone. J Clin Endocrinol Metab 32:619, 1971 9. McNatty KP, Smith DM, Makris A, Sathanond R, Ryan KJ: The microenvironment of the human Graafian follicle: interrelationship among the steroid levels in antral fluid, the population of granulosa cells and the status of the oocyte in vivo and in vitro. J Clin Endocrinol Metab 49:851, 1979 10. Jones GS, Aksel S, Wentz AC: Serum progesterone values in the luteal phase defects: effect of chorionic gonadotropin. Obstet Gynecol44:26, 1974 11. Aksel S, Jones GES: Effect of progesterone and 17 -hydroxyprogesterone caproate on normal corpus luteum function. Am J Obstet Gynecol118:466, 1974