In vitro fertilization as a diagnostic and therapeutic tool in a patient with partial 17,20-desmolase deficiency

Transcription

1 FERTILITY AND STERILITY Vol. 55, No.5, May 1991 Copyright If) 1991 The American Fertility Society Printed on acid-free paper in U.S.A. In vitro fertilization as a diagnostic and therapeutic tool in a patient with partial 17,20-desmolase deficiency Antonio Pellicer, M.D.* Fernando Miro, Ph.D. Marcos Sampaio, M.D. Emilio Gomez, Ph.D. Fernando M. Bonilla-Musoles, M.D. Instituto Valenciano de Infertilidad and Department of Obstetrics and Gynecology, Valencia University School of Medicine, Valencia, Spain Objective: To present a case with 17,20-desmolase activity deficiency in which in vitro fertilization (IVF) served not only as a therapeutic approach but also as a diagnostic tool for the specificity of the enzymatic deficiency. Design: IVF in the patient under study compared with a control group. All women treated with pure follicle-stimulating hormone (FSH). Setting: IVF program at the Instituto Valenciano de Infertilidad. Patients, Participants: A patient with primary amenorrhea, who was the subject under study, and seven normally cycling control patients undergoing IVF in the same series. Interventions: IVF, steroidogenesis in vitro of granulosa-luteal cell obtained at ovum pick-up. Main Outcome Measure(s): Oocyte fertilization and embryo cleavage. Serum and follicular fluid (FF) levels of estradiol (E 2), progesterone (P), testosterone (T), androstendione (A), 17ahydroxyprogesterone (17 -OHP). In vitro accumulation of E2 and P. Results: Ovulation induction with FSH was successful in achieving follicular development despite low circulating E 2 Fertilization and cleavage rates were similar to the control subjects. The patient developed ovarian hyperstimulation. The lack of 17,20-desmolase activity was detected by normal P levels in serum and FF, high 17-0HP, and low T, A, and E2 levels in serum and FF. Granulosaluteal cell cultures in the presence of T restored normal E2 and P production in response to gonadotropins. Conclusions: In patients with 17,20-desmolase deficiency, follicular development, oocyte maturation, and fertilization can take place in a low estrogenic environment. Fertil Steril 55:970, 1991 Patients with enzymatic deficiencies other than 21-hydroxylase are very uncommon. Particularly rare is 17,20-desmolase defect. Unlike other enzymatic deficiencies that affect the synthesis of adrenal corticosteroids and androgens, lack of 17,20-desmolase activity would only affect androgen and subsequent estrogen formation. Because 17,20-desmolase and 17a-hydroxylase activity in adrenal cortex and gonads are catalyzed by the polypeptide complex cytochrome P a\ activity lacking in both Received August 13, 1990; revised and accepted January 30, * Reprint requests: Antonio Pellicer, M.D., Instituto Valenciano de Infertilidad, Guardia Civil 23, Valencia-46020, Spain. usually is coincidental. However, there are some reports of specific defects in a single enzymatic activity.2,3 The 17,20-desmolase deficiency syndrome is a homozygotic genetic disease that can be easily detected in n~wborn males because of ambiguous external genitalia.2 Diagnosis of females with the enzymatic defect is extremely rare in the neonatal period because it would be obviously asymptomatic at this age. 4 However, primary amenorrhea and sexual infantilism due to negligible estrogen production would make the detection easier during the pubertal period. The diagnosis is based on elevated basal levels of C-21 steroids and accumulation of precursor C-19 steroids after adrenal and ovarian stimulation Pellicer et al. IVF in 17,20-desmolase deficiency Fertility and Sterility

2 Follicular development is controlled by endocrine, paracrine, and autocrine hormones in mammals. Despite recent studies that suggest that peptide hormones and growth factors play a major role in ovarian physiology, estradiol (E 2 ) is still a critical hormone responsible for regulation of ovarian function in conjunction with follicle-stimulating hormone (FSH).5,6 In addition, the intrafollicular steroid environment may be essential for appropriate oocyte maturation and fertilization in in vitro fertilization (IVF).7 Thus, follicular and oocyte maturation should be theoretically impaired in patients with 17,20-desmolase deficiency because of the estrogenic abnormality. There is, however, evidence in a patient with 17 (X-hydroxylase deficiency of normal IVF and early embryo development. 3 In the present study, we have characterized a patient with partial 17,20-desmolase deficiency during an IVF cycle. MATERIALS AND METHODS Patients and Ovarian Stimulation Protocols A 31-year-old woman was referred to our institution from a private office. She was first studied at age 17 with primary amenorrhea. At this time she was diagnosed as having female phenotype and 46,XX karyotype. Thyroid function was normal and basal FSH and luteinizing hormone (LH) were 1.2 and 0.1 miu/ml, respectively. A LH-releasing hormone stimulation test increased the levels of both gonadotropins to 4.5 and 5.9 miu/ml, respectively, within 60 minutes. The sense of smell was not affected. She was diagnosed as having hypogonadotropic hypogonadism and was maintained on hormone replacement therapy until she desired to conceive at age 26. At that time, she had two canceled stimulation cycles with human menopausal gonadotropin because of low «100 pg/ml) serum E 2 levels despite normal follicular development by ultrasound scans. During our infertility work-up she showed normal hysterosalpingogram, and the husband provided us with two normal semen analyses. Follicle-stimulating hormone, LH, prolactin, and cortisol levels were 1.5 miu/ml, 1.7 miu/ml, 9.6 ng/ml, and 4.5 ttg/dl, respectively. After discussing the therapeutic possibilities with the couple, we decided to perform an IVF cycle. Ovarian stimulation with four ampules per day of pure FSH (Fertinorm; Serono Laboratories, Madrid, Spain) began after withdrawal bleeding with progestagens and a negative ultra- sound scan. Stimulation was maintained during 11 days until the ultrasonographic criteria for human chorionic gonadotropin (hcg) administration (10,000 IU; Profasi, Serono) were reached. Our routine criteria for hcg injection include the presence of two or more follicles ~ 1. 7 cm in greatest diameter, and serum E2 > 800 pg/ml. In this particular case, however, E2 values were not taken into account because of previous experience with her cycles. The control group consisted of seven women undergoing IVF in the same series (April 1 to June 30, 1990) and treated with a similar stimulation protocol. Three ampules per day FSH were administered to these patients over a period of 5 days. Beginning day 6, FSH was administered on an individual basis according to the serum E2 and transvaginal ovarian ultrasound scans until the criteria for hcg injection were reached. Oocyte retrieval was scheduled 35 hours after hcg administration. The IVF procedure has been previously described. 8 Three to four embryos were transferred into the uterus, whereas the others were cryopreserved with 1,2-propanediol and sucrose as the cryoprotectants. 9 In a second cycle, the patient in question underwent a steroid replacement cycle according to the protocol previously described 10 using natural progesterone (P) (intramuscular in oil, 25 mg/ml). This cycle served for the transfer of frozen and thawed embryos. Hormones, Drugs, and Reagents Human FSH (hfsh, NIDDK, AFP-4161B, 3,500 IU/mg; National Institutes of Health, Bethesda, MD) and hcg (Pregnyl, >3,000 IU/mg; Organon Co., Oss, Holland) were diluted in M-199 medium (biomerieux, Charbonnieres les Bains, France). Fetal calf serum (FCS) and L-glutamine were obtained from Gibco Co. (Paisley, Scotland, United Kingdom). Testosterone (T), Percoll, and trypan blue were purchased from Sigma Chemical Company (St. Louis, MO). Gentamycin (Schering Corp., Boston, MA) and amphoterizine B (Squibb Ind. Farmaceutica, S.A.; Barcelona, Spain) were added as antibiotics to the culture medium. Ham's F-lO medium was obtained from Seromed (Berlin, Germany). Cell Preparation and Culture All visible follicles in women included in the present study were harvested by ultrasound-guided vaginal aspiration. Oocytes were identified under the dissecting microscope and isolated for further in- Vol. 55, No.5, May 1991 Pellicer et al. IVF in 17,20-desmolase deficiency 971

3 semination and culture. The remaining follicular contents were centrifuged at 1,500 rpm for 10 minutes and the supernatants stored for follicular fluid (FF) hormonal determinations. Cells from five follicles of the patient under study were collected and combined for further culture. Cells were washed twice in 2 ml Ham's F-lO medium supplemented with 20% FCS and centrifuged at 1,500 rpm for 10 minutes. Subsequently, cells were layered onto 5 ml 50% Percoll columns and centrifuged at 500 rpm for 30 minutes to pellet red cells. A purified granulosaluteal cell preparation was aspirated from the interface, washed, resuspended, and counted in a hemocytometer. Viability in all cases was >95%, as assessed by trypan blue staining. All cell cultures were performed in triplicate at a density of 50,000 cells per well in tissue culture dishes (Nunclon; Nunc Delta, Kamstrup, Denmark). Cells were incubated in 1 ml M-199 supplemented with 10-7 M T, 20% FCS, and 2 mm L-glutamine in 5% CO2 in air atmosphere at 37 C. Cells were cultured in the absence or presence of 1 IV hfsh and 1 IV hcg, and maintained 48 hours. The samples were stored in aliquots at -20 C for subsequent analysis. The normal range of steroid production in culture medium was assessed in 10 normal ovulatory patients undergoing IVF in whom similar experiments were performed for different reasons. Hormonal Analyses Several steroid hormones were measured in serum, FF, and culture media using commercially available kits. Progesterone, E2, androstendione (A), and T were evaluated by radioimmunoassay (RIA; Diagnostic Products Corporation, Los Angeles, CA). The interassay and intra-assay variability for P, at a concentration of 0.6 ng/ml, were 5.8% and 7.2%, respectively. Estradiol had an interassay variability of 5.5% and intra-assay of 5.6% at a concentration < 40 pg/ml. Androstendione, at a concentration of 0.5 ng/ml, showed 8.6% interassay and 5% intraassay variability and T, 9.2% interassay and 10.4% intra-assay, at a concentration of 0.5 ng/ml. 17a Hydroxyprogesterone (17 -OHP) was determined by a immunosystem using enzymatic reactions (Sibar diagnostici, Perugia, Italy). The interassay and intra-assay variability for 17 -OHP at a concentration of 5 pg/ml were 8.3% and 12%, respectively. Correlation with RIA performed on the same samples was y = 0.95x (r = 0.972, P < 0.001). Progesterone and E2 in culture medium were measured in triplicate incubation mixtures after appropriate dilutions whenever necessary. Statistical Analysis Data are expressed as means ± SEM. For mean comparisons ANOVA and the Student's t-test were employed. When the ANOV A test showed statistical differences, the Duncan test was used. Significance was defined as P < RESULTS Table 1 shows some data from the IVF cycle of the subject under study as well as the patients from the control group. In addition, serum steroid levels the day of oocyte recovery are represented, except for E2 in which peak values 1 day before retrieval were recorded. The number of FSH ampules and days necessary to reach an optimal response was higher for the patient under study; she developed a greater number of follicles, however, and as a consequence the number of oocytes retrieved was higher. Even though 11 oocytes had normal fertilization and cleavage, fertilization rate was slightly lower than the controls. Four embryos were transferred approximately 48 hours after insemination and the remaining embryos were cryopreserved. The luteal phase was supported with intramuscular P in oil (50 mg/d). She developed hyperstimulation with> 12 cm large ovaries, moderate ascites, 43% hematocrit, and normal electrolytes. Hyperstimulation was successfully treated with bed rest and analytical and clinical control. In a subsequent cycle, the patient underwent an artificial cycle with steroids, and four surviving embryos were replaced into the uterus after thawing. This embryo transfer also failed to reach implantation. Table 1 Ovarian Stimulation, IVF Results, and Serum Steroid Levels in the Seven Control Patients Compared With the Patient Under Study Controls b Patient under study Age 32.3 ± FSH (ampules) 25.0 ± Days of stiil!-ulation 10.1 ± Follicles 7.0 ± Oocytes 5.6 ± Fertilization rate (%) E2 peak (ng/ml) 2,160 ± A (ng/ml) 6.4 ± T (ng/ml) 1.1 ± HP (ng/ml) 0.7 ± P (ng/ml) 1.8 ± Blood was drawn the day of oocyte recovery except for E2 in which peak levels 1 day before retrieval are represented. b Values are means ± SEM. 972 Pellicer et al. IVF in 17,20-desmolase deficiency Fertility and Sterility

4 Steroid levels in serum are also represented in Table 1. Although P levels in the subject under study were in the range of the controls, 17 -OHP was> 10 times higher. The levels of A, T, and E2 were 10 to 20 times lower in the patient in question than in the controls, suggesting a blockage at the level of the 17,20-desmolase enzyme activity. This suspicion was confirmed by analyzing the FF steroid contents of three individual follicles in each of the control patients and five follicles in our patient. A significant (P < 0.05) increase in 17-0HP was observed in the subject under study in comparison with the control women, whereas the levels of P in FF were no different between the patient and controls. Also, a significant decrease in the accumulation of T (P < 0.005), A (P < 0.001), and E2 (P < 0.005) was detected in the patient's FF compared with the controls (Table 2). The specificity ofthe enzymatic defect was further tested by culturing the granulosa-luteal cells obtained at ovum pick-up in the subject under study (Fig. 1). Progesterone accumulation after 48 hours in basal conditions was in the range of normal patients. Basal E2 production, however, was very low in comparison with normal individuals. When the cells were cultured in the presence of T, the enzymatic deficiency was overcome and E2 was accumulated in amounts similar to those of normal patients. In the presence of substrate, E2 production was stimulated by FSH and heg, demonstrating a normal aromatase activity of these cells. DISCUSSION The results of the present study suggest that women with 17,20-desmolase enzymatic deficiency can undergo IVF as a therapeutic approach. However, because no pregnancy has been reported, the o C B B+T FSH+T hcg+t Figure 1 In vitro E2 and P accumulation after 48 hours in granulosa-luteal cells obtained at ovum pick-up in the patient under study. Measurements have been made in basal (B) conditions and in the presence of T, either without stimulus (B + T) or after stimulation with FSH (1 IU/mL) and hcg (1 IU/mL). The normal range of basal steroid production (C) in culture medium was assessed in 10 normal ovulatory patients undergoing IVF. present findings have to be completed with the analysis of the endometrial response to altered serum steroid levels. If an abnormal pattern is confirmed, cryopreservation of zygotes and subsequent replacement in an artificial cycle should be the method of choice. Our patient underwent such a cycle when the frozen and thawed embryos were used, but unfortunately she did not become pregnant. The lack of enzymatic activity was demonstrated measuring different steroids in serum and FF. In Table 2 Follicular Fluid Steroid Levels in the Seven Control Patients Compared With the Patient Under Study" Patient P 17-0HP T A E2 (ng/ml) (ng/ml) (ng/ml) (ng/ml) (ng/ml) 1 8,162 ± 5, ± 4 12 ± 2 76 ± 11 2,080 ± ,654 ± 4, ± 8 20 ± ± 77 1,416 ± ,258 ± 2, ± ± ± 76 1,872 ± ,158 ± 4, ± ± ± 25 1,697 ± ,575 ± 3, ± 3 20 ± ± ± ,726 ± 8, ± 4 15 ± ± ± ,456 ± 5, ± 5 16 ± 4 56 ± 7 1,692 ± 503 Patient under study 15,657 ± 1, ± ± ± ± 6.2 Probability <0.05 <0.005 <0.001 <0.005 " Each value in the controls represents the means ± SEM of three individual follicles. A total of five follicles selected at random were analyzed from the patient under study. Vol. 55, No.5, May 1991 Pellicer et al. IVF in 17,20-desmolase deficiency 973

5 addition, granulosa-cell cultures precisely showed the enzymatic blockage in the steroidogenic pathway by restoring normal E2 accumulation with the addition of appropriate substrate, despite the fact that granulosa cells theoretically contain low levels of 17,20-desmolase, which is mainly located in the thecay,12 The use of homologous tissue to demonstrate such abnormalities has already been reported. Zachman et al. 13 obtained testicular slices from two male first cousins with the clinical picture of 17,20- desmolase deficiency. Incubation of this tissue with C-21 precursors showed deficient conversion to T. To our knowledge, this is the first time that ovarian cells have been used to identify the enzymatic defect. Moreover, the cells have been obtained during ovum pick-up, confirming the use ofivf and related technology not only as therapeutic, but also as a diagnostic approach. Enzymatic 17,20-desmolase and 17a-hydroxylase activities in adrenal cortex and gonads are catalyzed by the cytochrome P a complex localized in the microsomes.1 When a lack of activity is detected, it usually involves both enzymatic activities as a consequence of a mutation, sometimes affecting just a single amino acid.14 This anomaly is typical of consanguineous families. 15 Interestingly enough, the defect in our patient was specific for 17,20-desmolase. We believe that future studies using Southern and Northern blots may detect a new mutation at the molecular level that only affects one of the catalytic activities. We feel that these studies will help to clarify the molecular structure of the enzyme, and also to treat the disease. The study of women with such deficiencies may be also useful in understanding ovarian physiology in humans. The fact that multiple follicular development was achieved by administering FSH in the presence of 10% of aromatase activity suggests that FSH is the principal trophic factor able to mediate most intrafollicular events necessary to achieve follicular and oocyte maturation in the presence of very low E2 levels. These data put into perspective the critical importance of FSH versus a lesser autocrine and paracrine role of E2 in follicular development. Perhaps the physiological role of E2 is limited to control the pituitary response by the feedback mechanisms, but other nonsteroidal intraovarian regulators orchestrated by FSH are also key to normal follicular growth. A good example may be plasminogen activators. Their secretion by granulosa cells is FSH-dependent.16 By blocking their action with serine proteases inhibitors we were able to impair follicular development and ovulation in the rat.17 However, it has to be kept in mind that pharmacological doses of FSH were employed in the patient under study, and therefore the effect of FSH on granulosa-luteal cells may not represent the same situation as in the normal menstrual cycle. Some other clinical lessons might be learned from this case. The patient developed moderate to severe hyperstimulation18 after oocyte retrieval despite low E2 levels. The pathogenesis of the ovarian hyperstimulation syndrome is not yet established. The process requires extensive angiogenesis and increased capilar permeability. These properties of E2 are sufficiently demonstrated during pregnancy. However, because the level of E2 was low in this patient, other angiogenic factors may be responsible for the pathogenesis of the hyperstimulation syndrome. Alternatively, the ovarian renin-angiotensin system may playa role in the generation of the hyperstimulation syndrome.19 In fact, a direct correlation has been found between plasma renin activity and the severity of the ovarian hyper stimulation in women.20 Several groups have found a good correlation between FF E21evels and oocyte maturation, fertilization, embryo cleavage, and implantation. The results, however, are contradictory because others have not been able to link successful IVF and cleavage to a certain level of FF P and E2.7 These contradictory results together with the evidence presented here suggest that oocyte maturation is related to a series of follicular events initially independent of steroids. The abnormal intrafollicular milieu present within the ovaries of our patient with 17,20-desmolase deficiency did not affect oocyte maturation as far as our current methods of oocyte and embryo evaluation identify. The presence of degenerated or nonfertilizable oocytes would be expected for a different reason other than an abnormal milieu. It has been widely demonstrated that E2 is responsible for inducing granulosa-cell intercellular communications or gap junctions.21 Because the arrest of the oocyte at the diplotene stage has been linked to the passage of cyclic AMP through the gap junctions into the cytoplasm,22 premature oocytes unable to be penetrated by sperm should be found in these patients. However, FSH also induces gap junctions between cells,23 and only desensitization of its receptors by the preovulatory peak of gonadotropins will be responsible for oocyte maturation.23 As the patient was treated with high doses of FSH, its effect probably overrode the deleterious effect of low E2 on intercellular communication. 974 Pellicer et al. IVF in 17,20-desmolase deficiency Fertility and Sterility

6 The present findings may be interpreted with caution because there was still an overall 10% normal enzymatic activity. Perhaps a complete blockage of the desmolase activity may show a totally different picture with no ovarian response and/or degenerated oocytes. It is also important to emphasize that the enzymatic deficiency was a secondary problem added to the hypogonadotropic hypogonadism. If the enzymatic deficiency would be the only cause of amenorrhea, gonadotropin levels would be found elevated as it has been previously described. 24 REFERENCES 1. Kominami S, Shinzawa K, Takemora S: Purification and some properties of cytochrome P -450 for steroid 17 -alphahydroxilation and C17-20 bond cleavage from guinea pig adrenal microsomes. Biochem Biophys Res Commun 109:916, Kaufman FRG, Costin D, Goebelsmann F, Stanczyk Z, Zachmann M: Male pseudohermaphroditism due to 17,20- desmolase deficiency. 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Am J Obstet Gynecol 158:670, Rabau E, David A, Serr DM, Mashiach S, Lunenfeld B: Human menopausal gonadotropins for anovulation and sterility. Am J Obstet Gynecol 98:92, Lightman A, Palumbo A, DeCherney AH, Naftolin F: The ovarian renin-angiotensin system. Sem Reprod Endocrinol 7:79, Navot D, Margalioth EJ, Laufer N, Birkenfeld A, Relou A, Rosier A, Schenker JG: Direct correlation between plasma renin activity and severity of the ovarian hyperstimulation syndrome. Fertil Steril48:57, Merk FB, Boticelli CR, Albright JT: An intercellular response to estrogen by granulosa cells in the rat ovary: an electron microscope study. Endocrinology 90:992, Dekel N, Lawrence TS, Gilula NB, Beers WH: Modulation of cell-to-cell communication in the cumulus-oocyte complex and the regulation of oocyte maturation by LH. Dev Bioi 86: 356, Pellicer A, Parmer TG, Stoane JM, Behrman HR: Desensitization to follicle-stimulating hormone in cumulus cells is coincident with hormone induction of oocyte maturation in the rat follicle. Mol Cell Endocrinol 64:179, Larrea F, Lisker R, Banuelos R, Bermudez JA, Herrera J, Nunez-Rasilla V, Perez-Palacios G: Hypergonadotrophic hypogonadism in a XX female subject due to 17,20 steroid desmolase deficiency. Acta Endocrinol (Copenh) 103:400, 1983 Vol. 55, No.5, May 1991 Pellicer et al. IVF in 17,20-desmolase deficiency 975