The relationship between follicular fluid steroid concentration and successful fertilization of human oocytes in vitro

FERTILITY AND STERILITY Copyright © 1984 The American Fertility Society

Vol. 41, No.6, June 1984 Printed in U.8A.

The relationship between follicular fluid steroid concentration and successful fertilization of human oocytes in vitro

William Botero-Ruiz, M.D.* Neri Laufer, M.D. *H Alan H. DeChemey, M.D.§ Mary Lake Polan, M.D., Ph.D.§ Florence P. Haseltine, Ph.D., M.D.§ Harold R. Behrman, Ph.D.* Yale University School of Medicine, New Haven, Connecticut

Follicular fluids (FF) and their matched oocytes were obtained from 64 follicles of 28 women who failed to conceive after in vitro fertilization (NF) and 33 follicles of 8 women who successfully conceived after the procedure. Ovulation was induced with human menopausal gonadotropin, and follicular aspiration was performed 36 hours after human chorionic gonadotropin administration. The concentration of 17f3-estradiol, progesterone, testosterone, dihydrotestosterone, and androsterone was correlated with the morphology of the oocyte-corona-cumulus complex (DCCC), oocyte fertilization, the rate of cleavage, and the incidence of pregnancy after embryo transfer. In both groups of women, FF progesterone was lowest in follicles containing immature OCCCs. However, follicles aspirated from women who conceived after NF which contained intermediate and mature OCCCs had significantly higher FF estradiol levels than similar follicles from women who failed to conceive after the procedure. Fertilized oocytes and 4- to 6-cell stage embryos which were obtained from follicles of pregnant women contained significantly higher FF estradiol levels than fertilized oocytes and similar embryos from nonpregnant women. It appears that higher FF estradiol levels correlate well with successful fertilization and an enhanced cleavage rate of oocytes associated with pregnancy following NF. Fertil Steril 41 :820, 1984

Received November 14, 1983; revised and accepted February 15, 1984. *Section of Reproductive Biology, Department of Obstetrics and Gynecology. t Andrew W. Mellon Foundation Fellow in Reproductive Sciences on leave from the Department of Obstetrics and Gynecology, Hadassah University Hospital, Jerusalem, Israel. tReprint requests: Neri Laufer, M.D.,. Department of Obstetrics and Gynecology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510. §Section of Reproductive Endocrinology, Department ofObstetrics and Gynecology.

Since the first report of in vitro fertilization (lVF) of human oocytes, l the methodology of fertilization and culture of human embryos has been simplified and, to a large extent, standardized. 2 Although fertilization and cleavage rates have improved substantially,3 pregnancy rates in different programs are in the range of 15% to 25% even when multiple embryos are transferred into the uterine cavity.4 A major determinant of the success of IVF is the choice of optimal follicles containing oocytes capable of producing normal

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pregnancies after fertilization and culture in vitro. When used as a marker for follicular maturation, the steroid content of human follicular fluid (FF) in normal cycles correlated well with follicular growth and the morphology of the oocyte. Estradiol (E 2) and progesterone (P) concentrations were found to progressively increase with follicular growth, while those of androgens decreased. 5 . 7 Oocytes obtained from large estrogenic and nonatretic follicles were healthy and able to mature in vitro, while those derived from small follicles containing high concentrations of androgens were degenerate and necrotic. 6 A major improvement in pregnancy rates of IVF programs has been achieved by inducing the growth of multiple follicles resulting in multiple embryo transfers with either clomiphene3 or human menopausal gonadotropin (hMG).8 In clomiphene-induced cycles, a close correlation was found between high FF P and E2 concentrations and the ability of oocytes to fertilize in vitro.9 In these cycles, follicles containing high concentrations of E2 were the sole source of oocytes associated with pregnancy in IVF patients.lO In hMGinduced cycles, asynchronous follicular development and a large variation in FF steroid content were observed. l l However, the association between the hormonal milieu in hMG-stimulated follicles and the success of oocytes fertilized in vitro was not reported. The present study was undertaken in order to evaluate the relationship between FF E 2, P, and androgen concentrations in FF and to compare then with the morphology, fertilization, and successful implantation of human ova fertilized in vitro obtained from hMG/human chorionic gonadotropin (hCG)-induced cycles.

of serum E2 and by an increase in follicular diameter measured by ultrasonography (Siemens PHO-Sonic Scanner, Santa Clara, CA) starting on day 8. hCG (Pregnyl, Organon Ltd., Oss, The Netherlands), 10,000 IU, was administered intramuscularly when follicular size reached at least 1.6 cm with E2 levels> 400 pg/ml. Laparoscopy was performed 36 hours later, and the contents oflarge follicles (> 1.5 cm in diameter) were aspirated. Oocytes were identified and immediately classified under the microscope, according to the morphologic appearance of the oocyte-coronacumulus complex (OCCC). FF was centrifuged at 1000 x g, and cell-free supernatant was kept frozen until assayed. IN VITRO FERTILIZATION PROCEDURE

Three main types of OCCC were identified: immature OCCC, with a tight corona and cumulus; intermediate OCCC, with a dispersed cumulus but only a partly dispersed corona; and mature OCCC, with advanced dispersal of both the cumulus and the corona (Fig. 1). Intermediate and mature complexes were incubated for 6 to 8 hours, and immature complexes were incubated for 24 hours before insemination, as reported previously.4 Culture of the OCCCs was carried out in a modified Ham's F-I0 medium, which was prepared weekly from a stock solution. 4 The insemination medium was supplemented with 10% of the individual woman's heat-inactivated serum. Sixteen to 18 hours after insemination, the ova were moved to growth medium, which was similarly prepared to contain 20% serum. Oocytes and embryos were cultured in I-ml medium culture a.

MATERIALS AND METHODS

Ninety-seven follicles were aspirated in 36 women who underwent an IVF procedure at YaleNew Haven Hospital because of irreparable tubal damage. All patients were under 37 years of age, with normal ovulatory cycles. Ovulation was induced with 3 ampules of hMG (Pergonal, Serono Laboratories, Inc., Randolph, MA), 75 IU of follicle-stimulating hormone, and 75 IU ofluteinizing hormone (LH) per ampule from day 3 of the cycle for 5 days. The dose was increased in a stepwise manner by administration of 3 to 4 ampules/day for another 2 to 3 days, depending on the individual response, which was monitored by daily levels Vol. 41, No.6, June 1984

Figure 1 Morphology of three main types of OCCCs: immature (1m), with a tight and compact corona/cumulus mass; intermediate (In), with a dispersed cumulus but a tight corona; mature (Ma), with a complete dispersal of both cumulus and corona complexes (original magnification, x 150; Hofman Interference Optics). Botero-Ruiz et al. Follicular fluid steroids in IVF

821

Table 1. Effect of aeee Maturation on Fertilization Status Nonpregnant

Pregnant

Maturation stage

Total Fertilized Fertil· OCCCs OCCCs ized n

n

Immature Intermediate Mature

9 38 17

8 33 11

88 87 65

Total

64

52

81

Immature Intermediate Mature

3 17 13

3 15 12

100 87 92

Total

33

30

90

%

dishes (#3037, Falcon Plastics, La Jolla, CA) at 37.5° C in sealed jars after gassing with 5% CO2, 5% O2, and 90% N 2. Inseminated oocytes were incubated for 38 to 40 hours, at which time cell cleavage was documented, and all fertilized 00cytes were transferred into the uterus. STEROID RADIOIMMUNOASSAY

Estradiol was measured by diluting 50-J.LI aliquots of FF (1:40) in buffer. Fifty microliters was extracted in 5 ml of ether and reconstituted after evaporation in 0.5 ml of buffer and assayed as described previously. 12 For assay of P, 10 J.LI of FF was extracted in 8 ml of petroleum ether, the extract was evaporated to dryness, the residue reconstituted in 1 ml of buffer, and 10-J.LI aliquots were assayed as described previously.13 Two hundred microliters ofFF was extracted in 5 ml of ether and reconstituted in 0.2 ml chloroform in order to measure testosterone (T) and dihydrotestosterone (DHT). The samples were chromatographed prior to radioimmunoassay as previously

described. 14 Androsterone levels were measured after extraction of 100 J.LI of FF with 3 x 3 ml of ether and reconstitution in 0.2 ml chloroform. Separation of androsterone was carried out by column chromatography using heptane/chI oroform/ethanol (50:50:1) saturated with water as solvent. Radioimmunoassay was performed as previously described. 15 STATISTICAL ANALYSIS Co~parisons between groups were made using Student's two-tailed t-test.

RESULTS Ova from 64 follicles of 28 women in whom IVF did not result in a pregnancy and 33 follicles of 8 women who conceived after the procedure showed an overall fertilization rate of > 80%, which was not significantly different between OCCCs at different maturity (Table 1). In both groups of women, immature OCCCs were derived from follicles which contained significantly lower levels of P (P < 0.02) and androsterone (P < 0.03) than follicles from which intermediate and mature OCCCs were derived (Table 2). Intermediate OCCCs of both groups contained significantly (P < 0.01) higher FF T concentrations than those from which a mature OCCC was recovered (Table 2). Intermediate and mature OCCCs obtained from follicles of pregnant women contained significantly higher levels of FF E2 than similar OCCCs aspirated from follicles of women who failed to conceive after IVF (Table 2). Fertilized oocytes in both groups of women were obtained from follicles containing higher levels of E2 than follicles that

Table 2. Relationship Between FF Levels of E 2 , P, and Androgens and Maturation of Human Corona/cumulus maturation

aeee (Mean

± SE)

E.

P

T

DHT

TIlJlml

TIlJlml

TIlJlml

TIlJlml

TIlJlml

2.82 ± 0.47 4.10 ± 0.49 b 2.59 ± 0.35 b

7.95 ± 1.1 c 12.07 ± 0.75 c 10.88 ± 1.35

5.62 ± 0.64d 12.43 ± 1.34d 9.88 ± 1.35d

2.7 ± 0.31 3.91 ± 0.518 2.30 ± 0.218

NM NM NM

6.80 ± 0.57 h 10.1 ± 1.36h 11.2 ± 1.49h

Nonpregnant

Immature (n = 9) Intermediate (n = 38) Mature (n = 17)

Pregnant

Immature (n = 3) 566 ± 13W 2357 ± 867 Intermediate (n = 17) 1051 ± 168eJ 6179 ± 1125",i 722 ± 87{ Mature (n = 13) 4833 ± 986

435 ± 102 503 ± 44 e 477 ± 65'

3834 ± 698 a 6238 ± 746 a 5729 ± 925

Androsterone

NM, not measured in pregnant women. Groups with identical letters differed significantly. < 0,05. c,b,e,gp < 0.01. dp < 0.01, immature versus intermediate and mature. hp < 0.03, immature versus intermediate and mature.

a,i,{Jp

822

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Fertility and Sterility

Table 3. Relationship Between FF Levels of E2 , P, and Androgens and the Success Rate of Fertilization of Human Oocytes (Mean ± SE)

Oocyte fertilization

Ea

p

T

DHT

Androsterone

nglml

nglml

nglml

nglml

nglml

6259 ± 623 6447 ± 1028

3.70 ± 0.39 3.50 ± 0.58

11.0 ± 0.64 11.2 ± 1.60

11.74 ± 1.21 9.32 ± 1.04

4830 ± 793 6556 ± 1004

3.68 ± 0.42 3.53 ± 0.77

NM NM

12.4 ± 1.40 13.8 ± 3.33

Nonpregnant

528 ± 37",b Fertilized (n = 52) Not fertilized (n = 12) 352 ± 37",c

Pregnant

Fertilized (n = 30) Not fertilized (n = 3)

804 ± 71 b 967 ± 41 C

NM, not measured in pregnant women. Groups with identical letters differed significantly. "P < 0.01. b,cp < 0.001.

yielded unfertilized oocytes. Furthermore, both fertilized and unfertilized oocytes of pregnant women contained higher FF E2 levels than their counterparts obtained from follicles of nonpregnant women (Table 3). FF levels of P, T, and androsterone did not differ between fertilized and unfertilized oocytes. At the time of transfer (38 to 40 hours after insemination), 44 of 52 fertilized oocytes cleaved in the nonpregnant group, 19 achieved a 2- to 3-cell stage, and 25 achieved a 4to 6-cell stage. In this group, FF T levels were significantly higher (P < 0.05) in follicles from which 2- to 3-cell embryos were obtained (Table 4). In the pregnant group, 27 of 30 fertilized 00cytes cleaved, of which 11 were at the 2- to 3-cell stage and 16 were at the 4- to 6-cell stage. FF from 16 follicles yielding embryos at the 4- to 6-cell stage from women who subsequently conceived were found to have a 60% increase in E2 concentration when compared with FF of25 similar embryos not associated with pregnancy following IVF (Table 4). DISCUSSION

Our results demonstrate a close association between OCCC morphology and FF steroid content. Follicles from which immature OCCCs were obtained contained 60% less P and DHT and 80%

less androsterone than follicles from which intermediate and mature OCCCs were derived. FF E2 levels did not differ between the three types of OCCCs aspirated from nonpregnant women but were significantly lower in follicles containing immature OCCCs obtained from women who subsequently became pregnant. In the normal ovulatory cycle prior to the LH surge, growth of the follicle was associated with a progressive and rapid increase in FF E2 concentrations, whereas FF P levels remained relatively low and increased in small increments. 5 , 6 Following the LH surge, a change in FF steroid content was measured, with FF E2 levels declining as ovulation approached, whereas those of FF P increased dramatically.7 In carefully monitored cycles during which follicles were aspirated, the same trend was seen: FF P concentrations increased, and FF E2 decreased as the interval between the beginning of the LH surge to aspiration was increased, i.e., as luteinization progressed. 16 Similar to the sequence of events in the normal cycle, we found that follicular maturation as evidenced by the morphology of the OCCC correlated well with the degree of follicular luteinization reflected in FF P levels. The process of follicular maturation in follicles from which immature OCCCs were obtained was probably intercepted by aspiration prior to adequate exposure to hCG.

Table 4. Relationship Between FF Levels of E 2 , P, and Androgens and the Cleavage Rate of Human Ova" Ova cleavage

Ea nglml

= 19) = 25)

Nonpregnant

2-3 cells (n 4--5 cells (n

Prel{llant

2-3 cells (n = 11) 4-6 cells (n = 16)

p nglml

515 ± 55 535 ± 49 b

6134 ± 1290 6325 ± 681

601 ± 52 861 ± 110b

8939 ± 3672 5777 ± 851

T nglml

4.9 ± 0.83 b 3.0 ± 0.37 b 3.65 ± 0.71 3.41 ± 0.35

DHT

Androsterone

nglml

nglml

13.25 ± 1.31 9.78 ± 0.62

13.61 ± 2.66 10.78 ± 1.27

NMc NM

12.81 ± 1.85 11.73 ± 1.17

"Mean ± standard error. bp < 0.05. cNM, not measured in pregnant women. Vol. 41, No.6, June 1984

Botero-Ruiz et aI. Follicular fluid steroids in IVF

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This is reflected in low FF P concentrations and the inability of these OCCCs to fertilize without a prolonged period of continuous in vitro maturation.3, 17 Inadequate exposure of immatUl'e OCCCs to hCG is further reflected by their markedly reduced capacity to produce P in culture when compared with mature OCCCS. 18 The morphologic immaturity of these OCCCs and the inadequate luteinization of their respective follicles are probably not due to inaccessibility of hCG, because follicles from which immature OCCCs were obtained were found to contain levels of LH and hCG similar to those from which intermediate and mature OCCCs were derived. 19 It may be assumed, therefore, that hCG reaches these follicles before they attain a full complement of LH receptors, and they are, therefore, unable to respond maximally to hCG stimulation. In contrast to the normal cycle in which follicular size correlated well with both FF steroid content and oocyte morphology in hMG-induced cycles, asynchronous development may occur: all three types of OCCC can be found in the same woman in a given cycle, and the largest follicles present at the time of aspiration (> 1.5 cm in diameter) are not necessarily the most estrogenic or contain the most mature OCCCs. These observations corroborate previous findings by Fowler et a1. 11 in hMGinduced cycles as well as those of Carson et a1. 10 in clomiphene-induced cycles. These investigators found a wide variation in FF steroid content between large follicles of the same patient. Higher FF E2 concentrations were shown in this study to be associated with successful IVF, a higher cleavage rate, and subsequent implantation. These findings in hMG-induced cycles are similar to previous observations in clomipheneinduced cycles where significantly higher follicular concentrations ofE 2 were associated with successful fertilization 9,l0 and pregnancy following embryo transfer. Our observations provide further direct evidence supporting the assumption that the most estrogenic follicles are the major source of fertilizable oocytes capable of more rapid cleavage and emphasize the important role of E2 in mammalian oocyte cytoplasmic maturation. 20 It should be noted, however, that multiple embryo transfer is associated with a higher pregnancy rate. 3, 8 Since pregnant women in this series had more embryos transferred than nonpregnant women (mean, 3.7 versus 1.8 oocytes/ woman), it may be concluded that the association 824

Botero-Huiz et aI. Follicular fluid steroids in NF

ofFF E2 with oocytes of good quality is only one of multiple factors contributing to the success of IVF. In tpe normal ovulatory cycle, FF androgen concentrations of healthy preovulatory follicles fall as ovulation approaches, while atretic follicles are characterized by androgen accumulation. 6, 21 The exact role of androgens in follicular function and oocyte maturation is not fully understood, but recent studies22 have suggested that androgen may be necessary for full development of granulosa cells, and androgen enhancement of P secretion by human granulosa cells has been demonstrated. In the present studies, FF T levels were significantly lower than those observed in FF prior to the LH surge. 6 , 21 However, these concentrations are comparable to those found in the normal cycle 30 to 38 hours following the onset of the LH surge 23 , 24 and similar to those found in clomiphene/hCG FF.24 T levels were low in FF from which immature and mature OCCCs were obtained and highest in FF from which intermediate OCCCs were derived. Follicles from which an immature OCCC was aspirated appear similar to healthy nonovulatory follicles which contain low concentrations of both FF P and FF T.21 It seems possible that if these OCCCs had not been artificially aspirated, spontaneous ovulation of their respective follicles would not have occurred, resulting in entrapped oocytes in anovulatory luteinized follicles. 25 The fact that T levels were found to be higher in FF from which intermediate OCCCs were obtained may be a further reflection of asynchronous intrafollicular maturation. LH and hCG stimulate T production by theca cells of antral follicles; but with progressive maturation and accumulation of LH receptors on theca and granulosa cells, the former lose their capacity to respond to the preovulatory surge of LH, and androgen production by these cells falls. On the other hand, granulosa cells luteinize and respond to the same stimulus by P production. 26 It is suggested that follicles that yield an intermediate OCCC were intercepted at a point in differentiation where part of the theca was still relatively immature and responded to the ovulatory dose of hCG by T production, whereas the granulosa cells had matured adequately and were capable of producing P in quantities similar to those observed in follicles containing a mature OCCC. Elevated FF T levels were also found to be associated with a lower rate of embryo development in Fertility and Sterility

nonpregnant women. This observation reflects the fact that - 70% of the fertilized oocytes developing into 2- to 3-cell embryos, after 40 hours of culture, were derived from intermediate OCCCs, which were shown to contain higher FF T levels. The association between FF T levels and the cleavage rate in human oocytes has not been reported before, and the mechanism by which T may affect the cleavage rate is unknown. However, increasing androgenization of the intrafollicular environment has been shown to interfere with the resumption of the first meiotic division. 6 It is possible that FF T concentrations may have either a direct or an indirect effect on the capacity of oocytes to undergo cleavage subsequent to fertilization at a rate comparable to that of oocytes exposed to lower concentrations of T. The fact that FF T levels did not differ between embryos at the 2- to 3-cell stage and 4- to 6-cell stage ofpregnant and nonpregnant women may alternatively suggest that the cleavage rate of oocytes derived from highly estrogenic FF is not affected by FF T concentrations. The presence of DHT in FF has been reported previously6, 20 and was not found to inhibit maturation or to induce mouse oocyte degeneration. 27 The levels ofDHT reported herein are significantly lower than those reported in follicles in the normal cycle. 6, 21 These differences probably stem from the different population studied, i.e., small antral follicles versus large preovulatory follicles in the present study. The presence of androsterone in human FF has not been previously reported. This metabolite is a major nonaromatizable end-product of androstenedione and T metabolism in the rat ovary.28 FF androsterone levels were found to be lower in FF of immature OCCCs and comparable in concentration to those of FF DHT. The exact source and relative importance of this hormone in the human follicular milieu remain to be elucidated. It may be concluded that our present observations on the steroid content ofFF from hMGIhCGstimulated cycles show a close association between FF P levels and the morphology of the OCCC. In addition, they emphasize the important role of FF E2 in the intrafollicular environment by demonstrating the association between high FF E2 levels and successful IVF and subsequent pregnancy. FF T levels were found to be associated with a decreased rate of oocyte cleavage and either may be an indirect marker of oocyte quality or may directly affect metabolic processes in Vol. 41, No.6, June 1984

the fertilized oocyte. Pregnancy rates in hMG-induced cycles for IVF may be improved by manipulating ovulation induction in a manner that will result in a large proportion of follicles containing high FF E 2 levels and low concentrations ofFF T. REFERENCES 1. Edwards RG, Steptoe PC: Pregnancies following implan· tation of human embryos grown in culture. Presented at Scientific Meeting, Royal College of Obstetricians and Gynaecologists, January 26, 1979, London 2. Lopata A, Johnston IWH, Hoult IJ, Speirs AL: Pregnancy following intrauterine implantation of an embryo ob· tained by in vitro fertilization of a preovulatory egg. Fer· til Steril 33:117, 1980 3. Trounson AO, Mohr LR, Wood C, Leeton JF: Effect of delayed insemination on in·vitro fertilization, culture and transfer of human embryos. J Reprod Fertil 64:285,1982 4. Laufer N, DeCherney AH, Haseltine FP, Polan ML, Mez· er HC, Dlugi AM, Sweeney D, Nero F, Naftolin F: The use of high-dose human menopausal gonadotropin in an in vitro fertilization program. Fertil Steril 40:734, 1983 5. Sanyal MK, GergerJJ, Thompson IE, Taymor ML, Horne HW: Development of Graafian follicles in adult human ovary. I. Correlation of estrogen and progesterone concentration in antral fluid with growth of follicles. J Clin Endocrinol Metab 38:828, 1974 6. McNatty KP, Smith DM, Makris A, Osathanondh R, Ryan KJ: The microenvironment of the human antral 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 48:851, 1979 7. Bomsel-Helmreich 0, Gougeon A, Thebault A, Saltarelli D, Milgrom E, Frydman R, Papiernik E: Healthy and atretic human follicles in the preovulatory phases: differences in evolution of follicular morphology and steroid content of follicular fluid. J Clin Endocrinol Metab 48:686, 1979 8. 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 9. Wramsby H, Kullander S, Liedholm P, Rannevik G, Sundstrom P, Thorell J: The success rate of in vitro fertilization of human oocytes in relation to the concentrations of different hormones in follicular fluid and peripheral plasma. Fertil Steril 36:448, 1981 10. Carson RS, Trounson AO, Findlay JK: Successful fertilization of human oocytes in vitro: concentration of estradiol-1713, progesterone and androstenedione in the antral fluid of donor follicles. J Clin Endocrinol Metab 55:798, 1982 11. Fowler RE, Edwards RG, Walters DE, Chan STH, Steptoe PC: Steroidogenesis in preovulatory follicles of patients given human menopausal and chorionic gonadotropins as judged by the radioimmunoassay of steroids in follicular fluid. J Endocrinol 77:161, 1978 12. Haning R, Orczyk GP, Buxton VC, Behrman HR: Plasma estradiol, estrone, estriol and estriol glucuronide. In Methods of Hormone Radioimmunoassay, Edited by BM

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21. Brailly S, Gueron A, Milgrom E, Bomsel-Helmreich 0, Papiernik E: Importance of changes in the transformation of progestin into androgen during preovulatory development and atresia of human follicles. In Follicular Maturation and Ovulation, Edited by R Rolland, EV van Hall, SG Hillier, KP McNatty, JS Schoemaker. Amsterdam, Excerpta Medica, 1982, p 180 22. Moon YS: The role of gonadotropins and testosterone in progesterone production by human ovarian granulosa cells. Mol Cell EndocrinoI23:115, 1981 23. Fowler RE, Chan STH, Walters DE, Edwards RG, Steptoe PC: Steroidogenesis in human follicles approaching ovulation as judged from assays of follicular fluid. J Endocrinol 72:259, 1977 24. Testart J, Castanier M, Feinstein MC, Frydman R: Pituitary and steroid hormones in the preovulatory human follicle during spontaneous or stimulated cycles. In Follicular Maturation and Ovulation, Edited by R Rolland, EV van Hall, SG Hillier, KP McNatty, JS Schoemaker. Amsterdam, Excerpta Medica, 1982, p 193 25. Koninckx PR, De-Moor P, Brosens IA: Diagnosis of the luteinized unruptured follicle syndrome by steroid hormone assays on peritoneal fluid. Br J Obstet Gynaecol 87:929, 1980 26. Henderson KM: Gonadotrophic regulation of ovarian activity. Br Med Bull 35:161, 1979 27. Smith OM, Tenney DY: Mouse oocyte maturation in vitro: effects of steroids. BioI Reprod 18:69, 1978 28. Magoffin DA, Erickson GF: Mechanism by which 17~ estradiol inhibits ovarian androgen production in the rat. Endocrinology 108:962, 1981

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