The effect of laparoscopic follicular aspiration on ovarian steroidogenesis during the early preimplantation period*

FERTILITY AND STERILITY Copyright © 1986 The American Fertility Society

Vol. 45, No.2, February 1986 Printed in U.SA.

The .effect of laparoscopic follicular aspiration on ovarian steroidogenesis during the early preimplantation period*

Joyce Vargyas, M.D. Oscar Kletzky, M.D. Richard P. Marrs, M.D. t Department of Obstetrics and Gynecology, University of Southern California School of:Medicine, Women's Hospital, Los Angeles, California

Seventeen patients were .studied for the first 72 hours of the luteal phase after spontaneous ovulation or follicle aspiration. Nine patients (group I) underwent follicle aspiration after clomiphene citrate (Ce) administration, three women were studied after CC for ovulation induction (group II), and five spontaneous ovulating subjects served as controls (group.III). Serum progesterone (P) concentrations were significantly elevated in stimulated cycles with or without follicle aspiration, compared with subjects. Moreover, when aspirated subjects were compared with nonaspirated stimulated subjects, a significant difference in P concentration was identified. It can be concluded that with follicle aspiration, P decreases can be observed, but with multiple follicle development, inadequate P levels are nat seen in these stimulated subjects. On the contrary, with such elevated P levels, endometrial advancement may playa role in poor implantation success with human in vitro fertilization procedures. Fertil SteriI45:221, 1986

. Despite the many advances made in. research surrounding the process of in vitro fertilization (lVF) , the clinical procedure remains limited, with reported pregnancy rates ranging from 15% to 25%.1-3 The cause(s) of this inefficiency is unknown but may be secondary to an unreceptive endometrium. induced by the IVF. process itself. Some of the factors affecting the endometrium, and thus implantation of the embryo, may be related to the· use of ovarian stimulatory drugs, general anesthesia during the laparoscopic aspi-

Received May 20, 1985; revised and accepted October 24, .1985. *Presented in part at the Annual Meeting of the Society for Gynecologic Investigation, March 17, 1983, Washington, D.C. tReprint. requests: Richard P. Marrs, M.D., Department of Obstetrics and Gynecology, Women's Hospital, 1240 North Mission Road, Los Angeles, California 90033.

ration of the follicles, or possibly the removal of granulosa cells during follicle aspiration. An abnormal luteal phase has been reported after aspiration of preovulatory follicles during spontaneous cycles in normally .cycling monkeys.4 The monkeys had either an inadequate secretion of progesterone (P) for the. first 7 or 8 days after the aspiration with subsequent recovery or a P deficiency throughout the cycle. 4 In humans, follicle aspiration resulted in decreased P secretion and a shortened luteal phase. These studies were done in unstimulated cycles with only a single dominant follicle present; steroid secretion was measured daily, with emphasis on midluteal phase levels. 5 The purpose of this study was to investigate cycles in which clomiphene citrate (CC) was used for. ovarian stimulation before IVF and embryo replacement (lVF"ER). Frequent sampling for the

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evaluation of hormonal parameters was performed for the first 72 hours after follicular aspiration to assess the possible effects of general anesthesia and/or follicle aspiration on the early preimplantation period. MATERIALS AND METHODS

Seventeen women participated in the study and were divided into three groups. Group I comprised nine ovulatory patients who were selected for the IVF program on the basis of irreparable tubal disease. These patients received 150 mg CC/day on cycle days 5 through 9. The cycles were monitored with ultrasound and hormonal parameters as previously described. 1 When the estrogen levels reached a plateau and the follicle size (measured by ultrasound) was> 18 mm in diameter, 4000 IU of human chorionic gonadotropin (hCG) was administered. Laparoscopy was performed 36 hours after hCG injection and follicle aspiration was performed with a 14-gauge Teflon-lined 20-cm needle connected to constant suction at 100-mm negative pressure. Once follicle collapse was visualized, the needle was used gently to curette the entire follicle surface and was then removed. No follicle flushing was performed. Patients were admitted to the Clinical Research Center (CRC) after a short postanesthetic recovery period. Blood samples were obtained 30 to 60 minutes before aspiration and every 6 hours thereafter for 72 hours, with the use of a 19-9auge butterfly heparin lock in the antecubital vein. Group II consisted of three ovulatory women who were undergoing timing for washed intrauterine insemination. Similar to the patients in group I, these patients received 150 mg CC/day on cycle days 5 through 9, and 4000 IU hCG was administered when the follicle diameter was> 18 mm. Thirty-six hours after hCG injection, intrauterine insemination with the husband's washed sperm was performed. 6 After insemination, these patients were admitted to the CRG and followed the same protocol as group I patients. Group III consisted of five normally cycling women who received no medication and served as controls. The timing of their ovulation and subsequent serum sampling was monitored by daily ovarian ultrasound beginning on day 10 of the spontaneous cycle. When ovulation appeared to be imminent by virtue of size of the dominant follicle, frequent blood sampling was performed. The patients were admitted to the CRC and un222

Vargyas et al.

Laparoscopic follicular aspiration

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derwent the same blood collection protocol as the other two groups when by ultrasound the dominant follicle disappeared. All samples were assayed for estradiol (E 2), prolactin (PRL), and l3-subunit luteinizing hormone (I3-LH) by radioimmunoassay.7 All blood samples were centrifuged and the plasma separated and kept at - 20°C until assayed. To minimize the variation of results, we measured all samples of each woman in the same assay. Statistical analysis was performed by use of Student's t-test where a ppropria te.

RESULTS

E 2, P, and I3-LH concentrations in the stimulated aspirated cycles (group I) are demonstrated in Figure 1. At the time of laparoscopy (time 0) the E2 levels were 424 ± 98 pg/ml (mean ± standard error of the mean [SEM]). A nadir was reached (132 ± 14.6 pg/ml) 24 hours after laparoscopy and steadily increased thereafter to reach a level of 278 ± 33 pg/ml at 72 hours after laparoscopy. The mean serum P level at laparoscopy was 2.6 ± 0.3 ng/ml and steadily rose in a fairly linear fashion to reach a mean of 15.3 ± 1.0 ng/ml Fertility and Sterility

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at 72 hours. ~-LH concentrations remained low and unaltered throughout the study interval. The E 2 , P, and ~-LH values for the stimulated unaspirated cycles (group II) are illustrated in Figure 2. Insemination was performed at time 0, which was 36 hours after hCG administration. The mean E2 concentration at time 0 was 206 ± 110 pg/ml and increased to 620 ± 240 pg/ml at 72 hours after insemination. The mean P value was 2.B ± 0.2 ng/ml at insemination and increased to 22.9 ± 2.B ng/ml 72 hours later. ~-LH remained low and unaltered throughout the study interval. E2 concentrations at time 0 and ± 72 hours were not significantly different in groups I and II. Figure 3 illustrates the hormonal values in the unstimulated control group (group III). At the time of follicle disappearance (time 0), the mean E2 concentration was 379 ± lOB pg/ml. Seventytwo hours after expected ovulation, the mean E2 level was 109 ± 33 pg/ml. The mean P value in this group at expected ovulation was 2.4 ± 0.4 ng/ml; 72 hours after ovulation it increased to B.3 ± O.B ng/ml. ~-LH concentrations reflect the midcycle LH increase in this group. The E2 concentration at 72 hours was significantly lower than Vol. 45, No.2, February 1986

in groups I and II (P < 0.05, P < 0.01, respectively). Figure 4 compares the PRL level in the early luteal phase of all three groups. The serum PRL level was significantly elevated at 6 hours in stimulated aspirated and unstimulated unaspirated cycles, compared with the stimulated unaspirated cycles, but was similar at other time points (P < 0.005) . Figure 5 demonstrates the serum P concentrations in all three groups. P values were signifi" cantly elevated in the stimulated aspirated group and the stimulated unaspirated group, compared with the unstimulated unaspirated group from 0 to 72 hours (P < 0.001). Moreover, the P values were elevated in the stimulated unaspirated group, compared with the stimulated aspirated group at 6, 30, 54, and 72 hours (P < 0.005, P < 0.05, P < 0.05, and P < 0.002, respectively). There was a slight elevation of P in the stimulated aspirated group at 36 and 42 hours, but this was not significant. In the group I patients, the P levels remained elevated at 1 week after ER (30.9 ng/ml ± 4.6 ng/mI) and at 2 weeks after ER (17.3 ± 6.3 ng/ mI). In all nine patients, mature oocytes capable of fertilization were obtained at aspiration. The mean number of follicles per patient was 3.2 ± 0.5, and the mean number of eggs aspirated and fertilized was 2.2 ± 0.5 and 1.3 ± 0.4, respectively. The mean number of embryos transferred per patient was 1.3 ± 0.03. Two of the nine pa,~------=~~-------------,500

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Figure 3 Serum E 2 , P, and I3-LH coconcentrations in unstimulated unaspirated cycles during the first 72 hours after follicle disappearance, determined by ovarian ultrasound. Hormonal concentrations are represented by the mean ± SEM.

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tients delivered full-term infants. The estrogen, P, and PRL values in the pregnancy cycles did not differ significantly from the mean levels for this group. DISCUSSION It is apparent from these results that ovarian stimulation significantly changes the hormonal components in the early luteal phase. It is interesting that serum E2 concentrations were similar in all three groups at the time of ovulation or aspiration, but 72 hours later E2 was significantly increased in the two groups receiving stimulation. This may represent the effect ofthe stimulatory drug or the cumulative effect of multiple cor. pora lutea. The variation and lack of pattern seen in the PRL concentrations in these three groups leads us to believe that this hormone is not significantly effected (at least a sustained effect) by either stimulation or operative manipulation. The significant elevation at 6 hours in groups I and III demonstrates the transitory effect of anesthesia and/or stress on PRL. In contrast to previous studies in unstimulated cycles,4,5 serum P was significantly elevated early in the luteal phase in these stimulated aspirated patients. However, the aspiration procedure may have had some affect on P reduction. When the stimulated aspirated cycles were compared with stimulated unaspirated cycles, the latter cycles had a significant increase in P levels, which were elevated over the stimulated aspirated cy224

Vargyas et al. Laparoscopic follicular aspiration

cles (Fig. 5, P < 0.01). This finding would concur with reports!,2 in unstimulated cycles where P levels were decreased if follicle aspiration were performed. Others have reported 4,5 that in the spontaneous cycle, although P deficiency is noted 3 days after aspiration when compared to the same patients in cycles with no aspiration, the P levels seem to recover by the sixth day after aspiration. 4,5 In patients undergoing follicle aspiration and ER, by day 7 after replacement, mean levels were 30.9, thus demonstrating no deficiency. Therefore it appears that follicle aspiration with removal of follicular components (granulosa cells) does interfere with overall P concentration, but with multiple follicle development, resulting in multiple corpora lutea formation, cumulative P secretion is still markedly elevated. Thus, high levels measured in these women possibly represent P secretion from several corpora lutea. Moreover, the P levels during the period immediately after aspiration and preceding ER are high enough to warrant concern over the possibil" ity that these levels may advance the maturation of the endometrium beyond the appropriate stage for implantation before the time of ER. The endometrium was not sampled at 72 hours in this study; however, in a previous study ofthis nature with the use of endometrial biopsy specimens, a statistically significant difference in endometrial dating between groups was demonstrated. 4 Likewise, in a recent report by Garcia et al.,5 endometrial advancement was demonstrated in 11 of 22 endometrial biopsy specimens taken after human SERUM PROGESTERONE CONCENTRATION (MEAN ± SEMI 24 22 20

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Figure 5 Comparison of serum P concentrations (mean ± SEM) among the three groups during the first 72 hours of the luteal phase. Fertility and Sterility

menopausal gonadotropin stimulation before laparoscopic aspiration. This type of histologic advancement is consistent with the significant increase in serum P concentrations demonstrated in this study. The authors 5 concluded that this may be a beneficial effect from the stimulation; however, the advanced endometrium may also provide an adverse environment for implantation because embryos .are replaced at very early developmental stages. 1-3 Because this study addressed only the early luteal function in IVF-ER cycles stimulated with CC control, trials need to be performed to evaluate luteal function after follicular aspiration when human menopausal gonadotropin is used for ovarian stimulation for IVF-ER cycles. However, from the results of this study, exogenous P supplementation in CC-stimulated IVF-ER cycles appears unwarranted. Moreover, concern over vigorous aspiration of follicles for IVF appears to be unwarranted when ovarian stimulatory agents are used.

Vol. 45, No.2, February 1986

REFERENCES 1. Marrs RP, Vargyas JM, Gibbons WE, Saito H, Mishell DR

2. 3. 4.

5.

6.

7.

Jr: Successful pregnancies using a modified technique of human in vitro fertilization and embryo transfer. Am J Obstet GynecoI147:3, 1983 Lopata A: Concepts in human in vitro fertilization and embryo transfer. Fertil Steril 40:289, 1983 Trounson A: Current perspectives of in vitro fertilization and embryo transfer. Clin Reprod Fertil 1:55, 1982 Kreitmann 0, Nixon WE, Hodgen GD: Induced corpus luteum dysfunction after aspiration of the preovulatory follicle in monkeys. Fertil Steril 35:671, 1981 Garcia JE, Acosa AA, Hsiu J-G, Jones HW Jr: Advanced endometrial maturation after ovulation induction with human menopausal gonadotropin/human chorionic gonadotropin for in vitro fertilization. Fertil Steril 41:31, 1984 Marrs RP, Vargyas JM, Saito H, Gibbons WE, Berger T, Mishell DR Jr: Clinical applications of techniques in human in vitro fertilization research. Am J Obstet Gynecol 146:477, 1983 Marrs RP, Kletzky OA, Mishell DR Jr: Functional capacity of the gonadotrophs during pregnancy and the puerperium. Am J Obstet Gynecol 141:658, 1981

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