Human menopausal gonadotropinlhuman chorionic gonadotropin follicular maturation for oocyte aspiration: Phase I, 1981

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1 FERTLTY AND STERLTY Copyright 1983 The Amerian Fertility Soiety Vol. 39, No.2, February 1983 Printed in U.8A. Human menopausal gonadotropinlhuman horioni gonadotropin folliular maturation for ooyte aspiration: Phase, 1981 Jairo E. Garia, M.D.*t Georgeanna Seegar Jones, M.D.* Anibal A. Aosta, M.D. * George Wright, Jr., Ph.D.=!: Eastern Virginia Medial Shool, Norfolk, Virginia Thirty-one human menopausal gonadotropin and human horioni gonadotropin (hmg/heg) ovulation indution yles from 25 normally ovulating patients who applied to a program for the Vital nitiation of Pregnany (VP) are disussed. Three different ategories of serum estradiol (E:d response were found. Serum E2 and progesterone responses were inversely related to the amount of hmg, indiating a patient sensitivity rather than a dosage relationship. Luteinizing hormone levels were suppressed by gonadotropins. Daily evaluation of vaginal smear, ervial muus, serum E2 determinations, and pelvi ultrasound are neessary for an optimum ovulation indution with gonadotropins. Two suessful pregnanies are reported. Fertil Steril39:167, 1983 Pregnanies through in vitro fertilization (lvf) were first aomplished after ooyte aspiration during spontaneous yles. Edwards et al. l reported no suess using menopausal gonadotropin, in spite of the ourrene of pregnanies after ovulation indution in anovulatory patients in many enters.2 Following an experiene in 1980, with 41 natural yles and no pregnanies, it was deided to attempt to stimulate folliular development with human menopausal gonadotropin (hmg) and ontrol ovulation by human horioni Reeived May 13, 1982; revised and aepted Otober 12, *Department of Obstetris and Gyneology. treprint requests: Jairo E. Garia, M.D., Department of Obstetris and Gyneology, Eastern Virginia Medial Shool, 304 Medial Tower, Norfolk, Virginia *Department of Mirobiology and mmunology and the mmunology Program. gonadotropin (hcg). Gonadotropins were hosen, rather than lomiphene itrate, beause they are physiologi hormones, and in our experiene folliular response seems to be more dependable following pituitary gonadotropin stimulation than after lomiphene itrate. The suessful use of lomiphene itrate for folliular stimulation and ovulation indution with hcg has sine been reported by Trounson et al. 3 Sine almost no information was found in the literature onerning hmghcg ovulation indution in normal yling women, an arbitrary protool was adopted, based on personal experiene with dosages used in anovulatory women and on our estimation of when the dominant follile was seleted. Although the main purpose of this protool was to reruit more than one fertilizable ooyte and thereby inrease the suess of folliular aspiration, fertilization, and embryo transfer through VF, the use of physiologi hormones provided Vol. 39, No.2, February 1983 Garia et a. hmglhcg for folliular maturation: Phase 167

2 ; l j:'! information about the varying response to pituitary hormones and its onsequene in the normal menstrual yle. The hormonal findings in relation to ooyte maturation and VF are reported in the first 31 yles from 25 patients in whom were used folliular stimulation and ontrolled ovulation by hmg/hcg. Two suessful pregnanies are reported in this series. MATERALS AND METHODS n 31 yles from 25 regularly menstruating and ovulating patients (measured by basal body temperature [BBT] hart) who applied to a program for the Vital nitiation of Pregnany (VP) at the Eastern Virginia Medial Shool between January 1 and July 31, 1981 (Phase, 1981), ovulation was indued with hmg (Pergonal, Serono Laboratories, n., Braintree, MA) and hcg. All patients had normal, fertile husbands as judged by the semen analysis and postoital testing. ndution was arried out in one yle in 20 patients, in two yles in 4 patients, and in three yles in 1 patient. An arbitrary dosage of hmg, two ampules intramusularly (1M) daily for 3 days, ontaining 75 U follile-stimulating hormone (FSH) and 75 U luteinizing hormone (LH), and redued to one ampule for 3 days, was administered between 8:00 AM. and 10:00 AM., starting on the 1st, 3rd, or 5th day of the menstrual yle, depending upon the yle length < 25 days, 25 to 35 days, and> 35 days, respetively. This arbitrary dosage was then readjusted aording to the patient's response. The hmg administration was monitored aording to daily observation of the karyopyknoti index of the ells of the vagina, ervial muus, daily serum LH determination, and real-time ultrasound of the ovaries to detet folliular development. hmg was disontinued with the appearane of a biologi shift, defined as (1) 30% pyknoti ells; (2) a volume of ervial muus> 0.2 ml; (3) a spinnbarkeit > 10 m; (4) good quality of the muus based on its lear aspet, lak of ells, and a 4 + ferning pattern; and (5) dilatation of the external os of the uterine ervix. Administration of 10,000 units of hcg 1M was given 2 or 3 days after the biologi shift, depending upon when the largest follile reahed 18 mm in diameter, as measured by ultrasound. n 29 yles, we inreased blood sample frequeny to every 4 hours, after the 1st day of the shift, in an effort to detet the LH surge. Laparosopy and folliular aspiration were arried out under general anesthesia, 36 to 38 hours from the time of hcg administration, using the tehnique reported elsewhere. 4 Daily blood samples were taken between 8:00 A.M. and 9:00 AM. for serum radioimmunoassay (RA) LH and estradiol (E2) determinations (Pantex, Santa Monia, CA). An aliquot of patient serum was extrated with a 3:2 mixture of ethylaetate and hexane and evaporated to dryness in a 50 C waterbath under nitrogen. The dried extrat was reonstituted with a phosphate buffer supplied in the kit. After 30 minutes at 37 C an RA was performed on the reonstituted extrats, with 1251 as a trae. Sample, trae, and antibody were allowed to sit at 37 C for about 30 minutes before the addition of a seond antibody. The tubes were entrifuged, and the pellets were ounted in a gamma ounter. The minimum onentration of E2 detetable by the Pantex RA was 10 pg/ml. The intraassay and interassay for both the low and high E2 ontrols was between 10% and 12%. LH in the serum was assessed daily at 8:00 A.M. with the use of an RA kit (Amersham, Arlington Heights, L). A modified protool, hanging the inubation period from between 16 and 24 hours to 3 hours, allowed the shortening of the assay time to 5 hours and permitted serum spe imens to be proessed and LH onentrations alulated on a daily basis. Although lower zero binding ourred as expeted with this modifiation (approximately 65% of that observed in the overnight assay), the two protools nevertheless maintained a lose orrelation (r = 0.97). Blood for serum progesterone (P) RA was obtained every 8 hours, beginning on the day of hcg injetion (day - 1) until aspiration (day + 1) and every other day thereafter during the luteal phase (Progesterone Test Set, Wien Laboratories, Suasunna, NJ). Daily ultrasound determinations were made with the use of an ADR Setor Sanner Model 2140 (ADR Ultrasound, Tempe, AZ). The ultrasonographer was always present at laparosopy. RESULTS Laparosopy and folliular aspiration for ooyte retrieval were performed in 31 yles. During this time, only one patient aborted her yle and menstruated on day 14, making it neessary to pass the yle. This has never ourred during subse- 168 Garia et al. hmg/heg for folliular maturation: Phase Fertility and Sterility

3 use Table 1. Serum E2 Response, hmg Dosage, and hmglhcg nterval in 31 Ovulation ndution Cyles from 25 Patients VP Phase, 1981 No. E. response yles hmgampules Low" ± 1.3 b Normal ± 1.4 High ± 1.2 alnadequate stimulation: four patients. bstandard deviation. hmghcg interval hr 71.2 ± 16.2 b 73.4" ± ± 9.9 quent stimulated yles. Tehnial failure preluded ooyte aspiration in one patient. Sixty 00- ytes were aspirated from 113 folliles in 30 yles; there were 48 preovulatory and 12 immature, or degenerated, ooytes. The omplete details of the harateristis of these ooytes are the subjet of another paper. 5 n none of the 16 yles adequately studied did an LH surge our before hcg administration. The serum LH levels during the folliular phase of all patients were low, and this phase of the study will be reported separately. 6 Three types of hormonal response were seen in a retrospetive analysis of ovulation indution with gonadotropins in relation to serum E2 values: (1) a low response, with E2 values < 300 pg/ml at the time hmg was disontinued; (2) a normal response, with E2 values between 300 and 600 pg/ml when hmg was disontinued; and (3) a high response, with E2 values> 600 pg/ml when hmg was disontinued. The relation of these E2 values and disontinuation of hmg is seen in relation to the biologi shift in Table 1. There were seven yles in the low-response group, ten in the normal-response group, and ten in the high-response group. There were four patients whose stimulation was inadequate. The serum E2 profiles taken every 4 hours at the time of hcg administration in these three different groups are shown in Figure 1. The biologi shift was the main indiator for the disontinuation of hmg during this phase. Ultrasound served a seondary or omplementary role, exept in those patients who had a positive history of onization of the ervix. The arbitrary hmghcg protool was adjusted in relation to the number of ampules of the drug per day and the total dosage of hmg aording to the individual patient response. The low E2 responders reeived an average of 11.2 ± 1.3 ampules, the normal E2 responders 10.4 ± 1.4 am- pules, and the high E2 responders 9.8 ± 1.2 ampules. The time of administration of heg was determined to a great extent in this series by the results of ultrasound examination. By protool this was to be at 6:00 P.M. on the day the folliular diameter reahed 18 mm. The interval between the last hmg dose and the hcg dose was 71.2 ± 16.2, 73.4 ± 16.6, and 65.2 ± 9.9 hours, respetively (Table 1). n some patients this interval was inreased up to 108 hours in an effort to obtain larger folliles by ultrasound. This was frequently not pratial, and by ultrasound the follile ranged between 15 and 17 mm during this phase when hcg was given. Serum P levels were also obtained on the 4- hour blood samples taken at the time of hcg administration. n the group of high responders, levels were statistially elevated above the low and normal responders. Following hcg administration, P inreased to a maximum value of 0.94, 1.41, and 5.1, respetively, in these three groups between 12 and 20 hours after hcg administration and delined thereafter (Fig. 2). At the time of aspiration, P values were 0.76, 1.1, and ng/ml, for the low, normal, and high E2 responders, respetively (Table 2). At the time of aspiration, two unusual phenomena were seen in this series: (1) darkness of the granulosa ells in the folliles aspirated from seven yles and (2) fragmentation of ten preovulatory ooytes. These findings ourred when the P levels were elevated beyond the normal values, when the interval between disontinuing hmg and hcg administration was inreased signifiantly above the usual, or in one instane when the preinubation time before fertilization was prolonged beyond permissible limits. n three patients, the first two phenomena ourred onomitantly (Tables 3 and 4). Sixteen of 48 preovulatory ooytes were fertilized and leaved: 9 single, 2 twin, and 1 triplet oneptuses were transferred transervially to the endometrial avities of 12 patients. Two pregnanies resulted from single transfers. A normal female was delivered Deember 28, 1981, and a normal male infant Marh 30, DSCUSSON There is almost no information on hmghcg ovulation indution in normal yling women. Although Edwards et al. l initially used gonadotro- Vol. 39, No.2, February 1983 Garia et al. hmglhcg for folliular maturation: Phase 169

4 'i'i E 200 ":.; f) 100 Low nadequate...,, HCG Aspiration 50 A o E... R 'i'i.n 300 E :.; " f) B _normal Menstrual Cyle Day o.1 Menstrual Cyle Day Figure 1 (A), Morning (8:00 A.M.) serum E2 values in 11 low E2 responders. On review of the ooyte maturation of these yles, seven patients were adequately stimulated (- - -) and four. were inadequately stimulated (--). Although the pattern is similar, the values of the inadequately stimulated patients are onsistently lower. (B), Morning (8:00 A.M.) serum E2 levels in ten patients with a normal E2 response to hmglhcg stimulation. Both low E2 responders and normal E2 responders show a ontinued E2 rise at 8:00 A.M. after 10,000 units hcg at 6:30 P.M. the previous evening. Day 0 at 8:00 A.M., over 12 hours after hcg administration, is equivalent to the day of the LH surge in the unstimulated yle. (e), Morning (8:00 A.M.) serum E2 values in ten high E2 responders to hmglhcg stimulation. E2 has begun to fall, perhaps indiating folliular maturation and oupany of LH reeptors prior to hcg administration. No further rise is seen in the E2 values at 8:00 A.M. after hcg, day O. pins to indue ovulation for VF, their efforts were unsuessful, and aspiration of unstimulated yles were advised. Previous suessful results with ovulation indution in anovulatory patients enouraged us to attempt hmg stimulation for VF. The seletion of an arbitrary dose of 150 units of FSH and 150 units of LH daily for 3 days (two ampules of Pergonal) and reduing it to 75 units of FSH and 75 units of LH for 3 days was based on experiene with hypopituitary, anovula- tory patients. The time of administration of gonadotropins in relation to the menstrual day was based on the estimate that the dominant follile is seleted not prior to day 7 or 8. t was rapidly established that day 3 in women with a 28-day yle and day 5 in women with a 35 ± 3-day yle were suffiiently early to reruit more than one dominant follile in most patients. When hmg was started later, it was impossible to reruit additional folliles. 170 Garia et a. hmglhcg for folliular maturation: Phase Fertility and Sterility

5 E 5 " '"., r:: 4!., '" "- 3 2 ow - _ inodequate 7 6 E 5 " '"., 4! ;; '" "- HCG Aspiration!! j......:l high _normal HCG - ;' \ \ \ Aspiration 8:00 20:00 24:00 8:00 16:00 24:00 8: :00 24:00 8:00 16:00 24:00 8:00 Menstrual Cyle Doy Menstrual Cyle Day Figure 2 Serum P values by RA immediately prior to hcg administration and through aspiration. Again we see the levels divided aording to the patient's E2 response to hmg/hcg stimulation. Among the low responders who were inadequately stimulated, the P values are below the normal level. The values of the low responders approximate those ofthe normal E2 responders. The high responders show values prior to hcg administration that are sometimes within the normal luteal range, probably representing exessive or premature oupany of LH reeptors related to the fixed ratio of FSH to LH administered. During this series, the patients were monitored by linial evaluation, serum LH determination, and ultrasound examination of the developing follile. Serum samples were also obtained for E2 and P assays. These were evaluated retrospetively. As expeted, the linial parameters were found to be more responsive in the hmghcg-indued yles than in the unstimulated yles; the maturation index was higher, and the quality and quantity of muus greater. nitially, all patients reeived two ampules of hmg daily for 3 days. This was dereased to one ampule daily for 3 days, depending upon the biologi linial estrogen response and the folliular size at ultrasound examination. n several patients the dose of one ampule daily was prolonged as neessary until the estimated ideal response was ahieved. No LH surge ourred during the interval of hmg indution, regardless of the length of treatment or the amount of hmg reeived by anyone patient, as suggested elsewhere. 7 LH determinations are therefore of no value in monitoring hmg-stimulated yles and have sine been disontinued. hcg was administered usually 1, 2, or 3 days after hmg was disontinued and oasionally was delayed as long as 5 days beause the protool alled for administration ofhcg when the folliu- lar diameter of the largest follile was 18 mm in diameter. n the following paragraphs the problems with the initial arbitrary protool will be disussed. n an effort to determine the ideal protool for hmghcg stimulation and ooyte aspiration, the 31 yles stimulated and aspirated were analyzed in relation to the quality of the ooytes obtained and the linial monitoring, using the serum E2 determinations that were retrospetively obtained, the linial parameters, and the ultrasound data. This analysis revealed three different patient responses, as judged by the E2 serum levels in relation to the biologi estrogen response. Preovulatory ooytes that beame fertilized were obtained in all three types of response, provided the response was reognized and hmg stopped aordingly. The first group, the low es- Table 2. Serum P Levels at the Time of Laparosopy-VP Phase 1,1981 Low E2 response Normal E2 response High E2 response astandard deviation. P level nglml 0.76 ± O.4 a 1.1 ± ± 2.0 Range Vol. 39, No.2, February 1983 Garia et a. hmglhcg for folliular maturation: Phase 171

6 Table 3. nterval hmg/hcg in Patients with Dark Granulosa Cells and/or Fragmented Ooytes Compared with Those with Similar ntervals but with Normal Ooytes hmglhcg interval Patients -:==;: ,:=- trogen responders, were those patients who had serum E2 levels of < 300 pg/ml, in spite of 3 days of biologi estrogen response or "shift." Preovulatory ooytes were obtained when hmg was disontinued on the third day of the "biologi shift." The normal estrogen responders were those patients with a serum E2 of 300 pg/ml or above at the time of the "linial estrogen shift." Preovulatory ooytes were obtained in this group when hmg was disontinued at the time ofthe biologi estrogen shift and when E2 was 300 pg/ml or above. Patients with a high E2 response were those whose serum E2 level was above 600 pg/ml prior to the ourrene of the biologi shift. Preovulatory ooytes were obtained in this group of patients ifhmg was disontinued on the day that E2 reahed 600 pg/ml or above, regardless of the absene of biologi shift. Although E2 values reahed levels of over 1000 pg/ml in patients with a high E2 response, no patient showed linial hyperstimulation. Aording to the initial protool, hcg should have been administered when the diameter of the largest follile reahed 18 mm by ultrasound examination. This proved to be unrealisti. The ultrasound diameter when hmg was disontinued was usually between 12 and 14 mm, and when hcg was given, the diameter was usually between 15 and 17 mm. Thus, the diameter of the follile measured by ultrasound in hmglhcgstimulated yles seemed smaller than that in the unstimulated yle. This finding was substantiated by the amount of folliular fluid aspirated from preovulatory folliles in whih preovulatory 00- ytes that were obtained fertilized and leaved normally. This finding may be attributed to the inhibition of endogenous LH and the exposure of a number of folliles to a fixed amount of exogenous LH, thus influening their growth. n the blood samples taken every 4 hours at the time of hcg administration, the patterns refleted the three different response groups, with mean peak E2 values ourring between 12 and 20 hours after hcg administration, or 16 to 24 hours prior to aspiration for an ooyte. During the final 24 hours, E2 values dereased but at aspiration remained higher than prior to hcg administration. Four patients in whom hmg dosage was insuffiient showed an E2 profile below the normal range in the low-response group. Serum P was also evaluated in these blood samples, and three different patterns were again orrelated losely with the E2 response. n the low and normal responders, P values were in the range reported in spontaneous menstrual yles. 8 n the high E2 responders P was three times higher than in the other groups. t was not orrelated with the numbers of folliles aspirated, and it was also inversely proportionate to the hmg dosage. Therefore, both the E2 and P responses represent hypersensitivity in the patient or the ovarian follile response to hmg, rather than hyperstimulation. n the unstimulated yle the degree of maturity (luteinization) of the granulosa ells was one of the best indiators of the maturation of the follile, whih in turn orrelated well with the quality and maturation of the ooyte. Thus, in a follile whih at aspiration showed well-utein ized granulosa ells, reovery of a preovulatory ooyte with an expanded umulus and a radiant orona was expeted. n hmglhcg-stimulated yles this orrelation was not as exat. The expansion of the orona and the umulus was undoubtedly affeted by hcg, and not until the umulus was removed by the sperm at fertilization ould one always be sure whether one was atually dealing with a preovulatory ooyte. The details of these ooyte findings are reorded in another paper. Table 4. P in Patients with Aspirations Showing Fragmented Ooytes and Dark Granulosa Cells Patients At hcgtime At laparosopy ng/ml ng/ml Dark granulosa ± 1.0 b 4.7 ± 2.0 ells Fragmented ± ± 2.2 ytes Nonnalooytesa ± ± 0.2 acontrol, patients with similar hcg to aspiration intervals, but nonnal ooytes. bstandard deviation. i 172 Garia et al. hmg/hcg for folliular maturation: Phase Fertility and Sterility

7 The separation of the appearane of the preovulatory ooyte and the luteinization of the granulosa ells with the maturation of the ooyte were observed during VP Phase in two interesting phenomena that had not been seen during aspiration of ooytes in the unstimulated yle: (1) darkness of the granulosa ells and (2) fragmentation of the ooytes. Darkness of the granulosa ells in light mirosopy is equated to inlusion of lipids in the ells-therefore, exesive luteinization. The former ourred in seven yles, and the latter in ten ooytes. Five of seven patients were in the high E2 responder group, and the other two in the low and normal responder groups. n all seven yles, however, the hmg/hcg interval was inreased, as observed in Table 3. Although an inreased hmghcg interval was observed in another five patients, the quality of the ooytes in these five yles were preovulatory, and no darkness of the granulosa ells or fragmentation of ooytes ourred. P levels at hcg administration and aspiration time were signifiantly higher in those yles showing dark granulosa ells and fragmented ooytes than in those five yles having preovulatory ooytes and no dark granulosa ells (Table 4). This finding might be the expression of ooyte overexposure to hmg (FSH/LH) prior to hcg. Suh an exposure may result in postmature ooytes. The P values at the time of hcg administration were higher in these yles than in those in whih fertilization and leavage of ooytes ourred, indiating that FSH exposure had been suffiient to indue LH reeptors on the granulosa ells in exess of those usually present just prior to ovulation. This finding might be the result of the fixed FSH:LH ratio and the failure of the spontaneous LH surge, allowing the ooyte to be trapped and thus overexposed. n summation, the administration of pituitary gonadotropins to normally ovulating women, in an effort to enter into the normal physiologi hain of events and, thus, selet two or three rather than one dominant follile with normally fertilizable ooytes for aspiration in VF, an be suessfully aomplished. During this effort several interesting observations were made. First, it was onfirmed that during exogenous administrtion of gonadotropins, endogenous LH is suppressed and the "sensitization"ofthe pituitary by E2 either does not our or does not result in an LH surge. Therefore, no spontaneous ovulation will be expeted. This may result in entrapment of the ooyte and postmaturity. Eah individual patient appears to have a speifi sensitivity to hmg administration. Preovulatory ooyte aspiration was only suessfully aomplished when these individual responses were reognized. n low responders it was neessary to observe a biologi E2 shift for 2 days, and hmg was disontinued the morning of the 3rd day of the shift, regardless of the E2 values at that time. n the normal E2 response group, hmg was disontinued when E2 reahed 300 pg/ml if the linial parameters had shifted. f no shift had ourred, the hmg dose was ontinued for one more day, at whih time all patients had shown a shift. n the high E2 responders, hmg was disontinued the day E2 reah 600 pg/ml, regardless of the linial parameters. hcg was given between 50 and 60 hours after the last hmg administration in all patients, regardless of their response. Although ultrasound was helpful in onfirming the size of the follile and the number of folliles and their loation, it ould not be used as a definitive measure for either stopping hmg or giving hcg. Aknowledgments. The authors would like to express their thanks to Connie Holforty, Beki Spear, Diane Brassil, Sheri Fowlkes, Dr. Wright's tehniians, for tehnial assistane in LH, E 2, and P determinations; to Mrs. Doris Gentilini, R.N., and to all of the nursing personnel in Labor and Delivery at the Norfolk General Hospital for their unselfish ooperation; and to Mrs. Linda Lynh for typing the manusript. REFERENCES 1. Edwards RG, Steptoe P, Purdy JM: Establishing full term human pregnanies using leaving embryos grown in vitro. Br J Obstet Gynaeol 87:737, Shwartz M, J ewelewiz R: The use of gonadotropins for indution of ovulation. Fertil Steril 35:3, Trounson AO, Leeton JF, Wood D, Webb J, Wood J: Pregnanies in humans by fertilization in vitro and embryo transfer in the ontrolled ovulatory yle. Siene 212:681, Jones HW Jr, Aosta AA, Garia J: A tehnique for the aspiration of ooytes from human ovarian folliles. Fertil Steril 37:26, Wortham E, Veek L, WitmyerJ: Vital initiation ofpregnany using hmghcg indution-a 7 months' review. Unpublished data 6. Ferraretti A, Garia JE, Aosta A, Jones GS: Serum LH during ovulation indution for in vitro fertilization in normally menstruating women. Unpublished data 7. Shoemaker J, Wentz AC, Jones GES, Dubin NH, Sapp KC: Stimulation of folliular growth with "Pure" FSH in patients with anovulation and elevated LH levels. Obstet Gyneol 51:270, Garia JE, Jones GS, Wright GL Jr: Predition of the time of ovulation. Fertil Steril 36:308, 1981 Vol. 39, No.2, February 1983 Garia et al. hmglhcg for folliular maturation: Phase 173