human chorionic gonadotropin for in vitro fertilization*

Vol. 41, No.1, January 1984 Printed in U.8A.

FERTILITY AND STERILITY Copyright 0 1984 The American Fertility Society

Advanced endometrial maturation after ovulation induction with human menopausal gonadotropinlhuman chorionic gonadotropin for in vitro fertilization*

Jairo E. Garcia, M.D.t:J: Anibal A. Acosta, M.D. t Jeng-Gwang Hsiu, M.D.§ Howard W. Jones, Jr., M.D.t Eastern Virginia Medical School, Norfolk, Virginia

Endometrial biopsy was performed between the first and third luteal phase day in 22 normally cycling patients following human menopausal gonadotropin and human chorionic gonadotropin ovulation induction for in vitro fertilization but in whom embryo transfer was not accomplished. Eleven patients showed an "advanced" pattern and 10 an "in-phase" endometrium according to the Noyes criteria. A significant difference in serum progesterone levels on days 16 and 18 was found in these two groups. Serum progesterone levels were significantly higher by day 18 if pregnancy was established. In in vitro fertilization and embryo transfer the embryo arrives 24 to 48 hours earlier than in natural conception in the endometrial cavity. Therefore, the "advanced" endometrium may have some benefit for embryo implantation. Fertil Steril 41 :31, 1984

Much progress has been made during the first lustrum of success in in vitro fertilization and embryo transfer OVF-ET) in human beings. There has been a great proliferation of in vitro centers around the world, ovulation induction in normal women has been refined, and the fertilization rate has improved along with the ET rate. Nevertheless, implantation and pregnancy rates

Received May 2, 1983; revised and accepted August 30, 1983. *Presented at the Thirty-Ninth Annual Meeting of The American Fertility Society, April 16 to 20, 1983, San Francisco, California. tDepartments of Obstetrics and Gynecology and Reproductive Endocrinology. :j:Reprint requests: Jairo E. Garcia, M.D., Assistant Professor, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, Virginia 23507. §Department of Pathology. Vol. 41, No.1, January 1984

are still far from desirable. The reason remains enigmatic. The observation of higher serum progesterone (P) levels in the early luteal phase of cycles stimulated by human menopausal gonadotropin (hMG) and human chorionic gonadotropin (hCG) than in spontaneous ovulatory cycles caught our attention. Therefore, early luteal phase endometrial biopsies were performed in some hMG/hCG-stimulated patients and correlated with serum P levels. The purpose of this report was to study these relationships in more depth. MATERIALS AND METHODS

Early luteal phase endometrial biopsies were obtained from 22 patients who participated in a program oflVF-ET at the Eastern Virginia Medical School during 1982. All patients had been admitted into the program according to the cri-

Garcia et aI. Endometrial biopsy after ovulation induction for IVF

31

teria previously reported. 1 Ovulation was stimulated with hMGlhCG according toa protocol published elsewhere. 2 Laparoscopy and IVF were carried out as previously described. 3 ETtook place on the second day after laparoscopy. An endometrial biopsy was obtained in patients whose ET was not accomplished because of an oocyte retrieval failure or inaccessible ovaries, technical failure at aspiration, early ovulation, or lack· of fertilization related to oligospermia. The biopsy specimen was obtained from the lateral wall of the endometrial cavity using a Novak curette. The specimen was promptly fixed in formalin and stained with hematoxylin and eosin. The biopsy was carried out the day of laparoscopy in 2 instances, the following day in 3 cases, and 2 days after laparoscopy in 17 patients. The dating of the endometrium was classified according to the Noyescriteria. 4 To avoid confusion, the days of the biopsy were designated as the 14th menstrual cycle day when the biopsy was obtained on the day of aspiration (equivalent to the day of ovulation) and day 15, 16,and so on when the biopsy was taken 24, 48, or more hours later. The slides were reviewed independently and blindly by all three authors and by a pathologist qualified in dating endometrial biopsies. Serum P levels were determined by radioimmunoassay following a procedure previously described. 5 Determinations were carried out first on the aspiration day and then every other day thereafter throughout the luteal phase in most of the patients. Nevertheless, for the purpose of this report, only those values from the day offollicular aspiration to the 18th menstrual cycle day will be considered. The serumP levels of the biopsy group were correlated with the serum P levels of 18 patients who had successfully conceived after hMGlhCG ovulation induction, follicular aspiration, IVF, and ET, and with the P values from 116 cycles of patients whose ovulation was successfully stimulated but in whom no pregnancy resulted after ET. In\all patients who underwentET, the luteal phase was supplemented with daily P in oil, 12.5 mg intramuscularly,on a routine basis, after blood was drawn for P determination, to prevent any P deficiency subsequent to the removal of granulosa cells at follicular aspiration, but none of the patients from which an endometrial biopsy was obtained was exposed to exogenous P. Student's one-tailed t-test was used for statistical analysis. 32

Table 1. Serum P Levels at the Time of the Endometrial Biopsy Day of endometrial biopsy

No. of __~~____~~______~~___ MeanP

cycles

14"

2 3 1 4 5 3 2 1

Proliferative

15

16

level nglml

Proliferative Proliferative Secretory 16 Secretory 17 Secretory 17-18 Secretory 18 Secretory 19-20

1.3 2.2 1.3 5.2 8.3 16.2 14.7 20.8

aDay 14: follicular aspiration day.

RESULTS Twenty-one of 22 biopsy specimens were adequate for analysis. One patient was excluded from the study because ovulation had occurred prior to laparoscopy, and only one blood sample was available for analysis; furthermore, the biopsy specimen was of poor quality for dating. The biopsy specimens obtained on day 14 (two cases) and day 15 (three cases) showed proliferative endometrium. Eleven of 16 patients who had an endometrial biopsy performed 2 days after aspiration had endometria which were dated 1, 2, or up to 3 days later ("advanced" endometrial biopsy group), in contrast to 5 other patients whose endometria were dated "in phase" ("in-phase" endometrial biopsy group). There was an increase in the mean serumP levels in those patients whose endometria were advanced, compared with patients whose endometria were "in phase" on the day of the biopsy (Table 1). There was no statistically significant difference between the serum P levels on days 14 and 16 between the "advanced" endometrial biopsy group and those patients who underwent ET (pregnant or nonpregnant). Also, there was no significant difference between the serum P levels in the "advanced" endometrial biopsy group and nonpregnant patients on the 18th cycle day, but there was a statistically significant difference between those two groups and the pregnant group. There was also a statistically significant difference between the serum P levels of the "advanced" endometrial biopsy group and the P levels of the "in-phase" endometrial biopsy group, primarily on the 16th and 18th cycle days (Table 2). The serum P profile for all groups is shown in Figure 1. The shadowed area represents the mean

Garcia et al. Endometrial biopsy after ovulation induction for IVF

Fertility and Sterility

Table 2. Serum P (ng/ml ± 1 Standard Error of the Mean) During the Early Luteal Phase Menstrual cycle day Group

No. of cycles

Pregnant Nonpregnant "Advanced" endometrial biopsy "In-phase" endometrial biopsy

18 116 11 10

14

2.1 ± 2.7·± 3.0.± 0.9 ±

17

16

12.0 12.4 12.7 4.9

0.3 0.5 0.8 0.1

P = .0.104

± 2.3a ± l.2a ± 1.7b ± 1.3b

18

:33.1 22.2 21.7 11.7

22.6 ± 16.4 ± 3.2a 7:6 a

NA" 9.8 ± 1.7

P = 0.003

± ± ± ±

5.4a 'l.4a 2~9b

1.8b

P = 0.009

ap'supplementation 12.5 mg intramuscularly. bStatistically significant difference. ~ot available.

± 1 standard error of the mean (SEM) of the pregnant group from the 14th to the 18th menstrual cycle day. Serum P levels of the nonpregnantand "advanced" endometrial biopsy groups were within the same range on days 14 and 16 .. By day 17, the P levels fell but were still within the same SEM. But by day 18, the P levels were statistically significantly lower than in the pregnant group. The serum P levels in the "in-phase" endometrial biopsy group were significantly lower than the P levels of the above group, although they still remained within physiologic levels.

On the other hand, it was observed that in patients who underwent IVF-ET the P levels were higher. subsequent to transfer if pregnancy was established, but at the present time it is impossible to determine whether this is due to an unknown factor or signal that promotes the func-

35

30 ___ PregnantNon PregnantAdvanced Endo. Bx ~--o In Phase Endo. Bx ...-

0--0

DISCUSSION

criteria4

~

The Noyes of-dating endometrialbiopsies has been useful for diagnosis and evaluation of fertility problems, for example, luteal phase defect. The criteria were based on histopathologic analysis of the endometrium, expected menstrual date, and presumptive ovulation by basal temperature chart. Depending upon these parameters, accuracy was observed within 1 day. Since 1950, when the pilot work was published, better laboratory techniques for hormone determination (luteinizing hormone, follicle-stimulating hormone, estradiol, and P) and diagnostic procedures such as laparoscopy and ultrasound have allowed us to pinpoint precisely the time of ovulation in spontaneous menstrual cycles. Therefore, more accurate dating of the endometrium is feasible. Early during hMGIhCG ovulation induction in the normal cycle for IVF it was noticed that P levels were ,higher than in spontaneous cycles. This was probably related to better follicular development, or at least to a higher number of folliclesor a higher degree ofluteinization, regardless of the trauma suffered by the follicle at the time of oocyte retrieval by laparoscopy.5-7 Nevertheless, it was observed that in some patients serum P remains at "physiologic levels." Vol. 41, No.1, January 1984

25

CJI

c::

.,c::

..:.,e

20 Aspiration

~

.

CJI

o 15

~

E

.,2

10

(/)

5

14

15

16

17

18

Menstrual Cycle Day - Progesterone 12.5 mg. q.d, after day 16

Figure 1 The shadowed area represents the P levels (± 1 SEM) of the pregnant group after hMGIhCG-stimulated cycles during the early luteal phase. Notice how the P levels of the nonpregnant and "advanced" endometrial biopsy groups were within the same range of the pregnant group on the 14th and 16th menstrual cycle days. By day 17, P levels fell but were still within the 1 SEM, but by day 18 P levels were significantly lower (P = 0.009). The P levels in the "in-phase" endometrial biopsy group are significantly lower on days 16 and 18, although they remain within "physiologic" levels.

.Garcia et aI. Endometrial biopsy after ovulation induction for IVF

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--------

~--~~-----~~-----'------'---------":'_-J

Figure 2 Photomicrograph of a 17 -day secretory endometrium obtained from a patient 48 hours following laparoscopy (the day of biopsy is equivalent to the 16th menstrual cycle day). The glands exhibit nuclei pushed to the center of the epithelial cells, cytoplasm above them, and vacuoles at the base (x 100).

Figure 3 Photomicrograph of a 19- to 20-day secretory endometrium obtained from a patient 48 hours following oocyte aspiration (the day of biopsy is equivalent to the 16th menstrual cycle day). Notice the abundant secretion in the gland lumen. Subnuclear vacuoles are now absent. Stromal edema is beginning (x 130).

tioning of the corpus luteum and, therefore, increases the serum P levels. Endometrial biopsies were found advanced in 11 of 16 patients on day 18, as shown in Table 1 and Figures 2 and 3. The serum P levels were within the same range on days 14, 16, and 17 in the pregnant, nonpregnant, and "advanced" endometrial biopsy groups, but it was greater on day 18 if pregnancy ensued, but similar in the nonpregnant and "advanced" endome~rial biopsy groups. Furthermore, in the "in-phase" endometrial biopsy groups, the levels were statistically significantly lower than in the other groups on days 16, 17, and 18 (Table 2). Figure 1 shows clearly how serum P levels were within 1 SEM up to day 16 (ET day) but in the next 24 hours were lower than in the pregnant group. One hypothesis could be that the endometrium was prepared for acceptance of the conceptuses, but an unknown signal was produced-from the conceptuses ?-to reinforce the function of the corpus luteum or at least to keep its function preventing luteolysis. The histologic features and P levels in the "advanced" endometrial biopsy group reinforce our hypothesis, and it is possible that if an embryo had been transferred into them, pregnancy would have been established. According to the study of Croxatto et al.,8 the embryo usually arrives at the endometrial cavity 72 to 96 hours after ovulation. In IVF-ET it occurs - 52 hours after laparoscopy. In other words, the

endometrium is behind in relation to the arrival ofthe conceptus. It is possible that the "advanced" endometrium after hMGIhCG ovulation induction might be beneficial for implantation. Implantation is a very complex matter that is still poorly understood in human reproduction. It requires a combination of factors at the endometrial and embryonic levels.

34

Acknowledgments. Special thanks to all of our patients who, regardless of their disappointment, allowed us to perform endometrial biopsies. We would also like to thank Dr. Charles Wilkes from our Department of Obstetrics and Gynecology house staff for the statistical analysis, and Mrs. Linda Lynch for her secretarial assistance.

REFERENCES 1. Marsh FH, Self DJ: In vitro fertilization: moving from theory to therapy (The Norfolk Clinic). Hastings Cent Rep June 1980, p 5 2. Garcia JE, Jones GS, Acosta AA, Wright G Jr: Human menopausal gonadotropin/human chorionic gonadotropin follicular maturation for oocyte aspiration: Phase II, 1981. Fertil Steril 39:174, 1983 3. Jones HW Jr, Acosta AA, Garcia J: A technique for the aspiration of oocytes from human ovarian follicles. Fertil Steril 37:26, 1982 4. Noyes RW, Hertig AT, Rock J: Dating the endometrial biopsy. Fertil Steril 1:3, 1950 5. Garcia JE, Jones GS, Wright GL Jr: Prediction of the time of ovulation. Fertil Steril 36:308, 1981

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Fertility and Sterility

6. Garcia J, Jones GS, Acosta AA, Wright GL Jr: Corpus luteum function after follicle aspiration for oocyte retrieval. Fertil Steril 36:565, 1981 7. Acosta AA, Garcia JE, Jones GS, Wright GL Jr: Corpus luteum function in patients with hMGlhCG induced ovulation for in vitro fertilization. Presented at the Ortho Symposium on "In Vitro Fertilization: Medical and Ethi-

Vol. 41, No.1, January 1984

cal Aspects" at the Thirty-Eighth Annual Meeting of The American Fertility Society, March 20 to 24, 1982, Las Vegas, Nevada. Unpublished data 8. Croxatto HB, Ortiz ME, Diaz S, Hess R, Balmaceda J, Croxatto HD: Studies on the duration of egg transport by the human oviduct. II. Ovum location at various intervals following LH peak. Am J Obstet Gynecol 132:629, 1978

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