Effects of quingestanol acetate on the histology, histochemistry, and ultrastructure of the human endometrium

Effects of quingestanol acetate on the histology, histochemistry, and ultrastructure 1"\f +hi"\ h1 lt"V'\''U""' /"H"'rll"\ml"\+viiii""V"V lllc; IIUIIIQII c;IIUVII t;ll lUI II

VI

CHARLES E. FLOWERS, JR., M.D. WALTER H. WILBORN, PH.D. JACQUELINE ENGER, B.S. Birmingham and Mobile, Alabama

Quingestanol acetate is a potent progestogen which significantly alters the histology and histochemistry of the endometrium when studied by light and electron microscopy. The steroid did not alter transport mechanisms across the cell surfaces but, probably because of competition with estrogen for binding sites, altered sequences in carbohydrate and protein synthesis. It caused the development of platelet aggregates within the endometrial vessels and degeneration of endometrial cells through the entire menstrual cycle. The effectiveness of quingestanol acetate as a "mini-pill" is related to its alteration of the biology of the endometrial cells as well as its suppression of FSH and LH.

T H E c A T A s T R o P H 1 c problems associated with the geometric rise in world population have stimulated the development of many compounds capable of acting as oral contran·ptives. One of these newer compounds is quinge,tanol acetate. This steroid has an encouraging chemical formula: it has the addition of a five-carbon ring attached to the oxygen at the 3 position of norethisterone acetate (Fig. 1). The pharmacologic activity of any proc-:esrin is increased by adding side chains ai

the 3 and 17 positions; thus, the activity of the potent norethindrone acetate should be enhanced and be capable of use with estrogen as a combination product or as a "minipill.'' In 1966 Martinez-Manautou and his associates 1 reportPd the use of the first "minipill," chlormadinone acetate (0.5 mg. daily) 11ithot1t estrogen as a contraceptive. This new principle for oral contraceptives was cntllllsiastically embraced h\' manv clinicians :tnd invPstigators. Since the progestin w<~s tt;;Pd alone. it 11:ts belin·ed that there would

Frmn the Department of Obstetrics ,-;,,.}

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in Birmingham School of Medicine, and the Department of Anatomy, Uniz,ersity of South Alabama School of Medicine.

. llld unfurtuna!t' :--ide dTt>cts that \\'en· possiblv related to the estrogen in combination products. It \\as felt that estrogen was most likely rPsponsible for altered coagulation factors that resulted in an increasPd incidence of pulmonary embolism. thrombophlebitis, and ccrebroyascular :tccidents. ! t \\'as also thought that estrogen was the primary cause of such side effects as weight gain, headachP, fluid retention, nausea. breast pain, and chloasma. Unfortunately, "every sword has two

Supported by a grant from Warner-Lambert Pharm. Co. Presented at the Ninety-seventh Annual Meeting of the American Gynecological Society, Hot Springs, Virginia, May 23-25, 1974. Reprint requests: Dr. Charles E. Flozcers, Jr., Department of Obstetrics and Gynecology, University of Alabama Medical, Center, University Station, Birmingham, Alabama 35294.

589

590

Flowers, Wilborn, and Enger

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November 1, 1974 Obstet. Gynecol.

J.

0

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OCCH, CH~ -- C ~CH

Fig. 1. Steroid formulas of quingestanol acetate and norethindrone acetate.

Table I. Endometrial biopsies* Control Early proliferative phase (days 5-7) Middle proliferative phase (days S-1 0) Late proliferative phase (days 11-14) Early secretory phase (days 15-18) Middle secretory phase (days 19-23) Late secretory phase Beginning regression (days 24-25) Regression with ischemia (days 26-28) Totals

Quingestanol acetate

2

7 6

3 4

4 15

3 5 2

2 3

2 2

39

21

*Grand total, 60; age range, 22 to 46 years.

edges." It was soon evident that the "minipills" without estrogen had an increased incidence of breakthrough bleeding and a higher pregnancy rate when compared to sequential or combination products. However, it was felt that if a sufficiently potent progestogen could be developed, there would be an acceptable incidence of pregnancy and a reduced incidence of breakthrough bleeding, since the progestin could possibly cause sufficient endometrial regression to offset the absence of estrogen. The "mini-pills" have an interesting and important effect upon the hypothalamic pituitary axis, ovary, cervix, and endometrium. Moghissi and associates 2 - 4 have clearly delineated the effects upon these structures of norethindrone (0.3 mg.), norgestrei (0.075 mg.), and quingestanol acetate (0.3 mg.) daily. These three compounds had similar effects upon follicle-stimulating hormone (FSH) and luteinizing hormone (LH). They all reduced the midcycle serum FSH from ap~--------~~~~ ... ~1

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24. The LH surge was similarly reduced to about one-sixth of control values. The reduc-

tions in midcycle FSH and LH had the expected effect upon the corpus luteum, causing a significant reduction in estrogen and approximately a 50 per cent fall in serum progesterone. Sperm penetration of the cervical mucus was slowed because of the continuous progestogen dosage. Apparently, therefore, the microdose pills cause a significant suppression in hypothalamic-pituitary-ovarian function, cause a reduction in corpus luteum activity despite ovulation, but have a favorable contraceptive effect upon sperm penetration of the cervical mucus. This report concerns the effects of quingestanol acetate on the histology, histochemistry, and ultrastructure of the endometrium. t-Aoterials and methods

Ten patients were used for this study. Table I shows the number of endometrial biopsies taken and the stage of the menstrual cycle in which they were obtained. Of the 60 biopsies, 39 were taken before the patients were given qumgestanol acetate; the remaining 21 were taken after the patients had received the steroid for three months (Table I).

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Qu!ngestano! acetate on endometrium

Each biopsy was divided into smaller pieces in order that several investigative procedures might be performed on each biopsy. A portion of the biopsy was fixed in cold (5° C.) 10 per cent calcium fonnol, dehydrated in alcohol, cleared in xylene, embedded in paraffin. and sectioned at 6 rc. Some of these sections were stained with hematoxylin and <>osm (H&E), others with periodic acid Schiff (PAS), and tht- remaining ,,·ith alcian blue (AB) at pH 2.5 or 0.5. Slides stained \\·ith H&E sPrved as the basis for dating the biopsy according to tlw criteria of Noyes, Hertig, and Rock" and as a common reference point to which other findings, both histochen1ical and ultrastructural, could be related. Sections stained with the PAS technique of McManus" and Lillie' were used to demonstrate glycogen and neutral mucopolysaccharides. The PAS method depends on the selective oxidation by periodic acid which acts by breaking the carbon bonds of adjacent 1,2- glycols, such as those in glycogen, and converting them to aldehydes. The aldehydes then react with Schiff's reagent to produce a rc>ddish color. Since incllbation of tissue sections in diastase removes ,glycogen. it \\as possiblP to determine thE' site of glycogen in the tissue by comparing diastase-treated sections '' i th untreated sections. Sections were stained by the alcian blue techniquP to demonstrate arid mucopolysaccharides i acid mucosubstancesJ which in thr endometrium are belie\Td to provide a barrier against infection, coat the ovum and sperm. aid in the implantation of the fertilized oyum. ;md act as a transport ag-ent for desqu;11nated cdb and menstrual debris.' f"\YO

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mucins and sulfotmH in,, han· been described and hoth classes are bclicYed to he glycoprotcins. It has bcPn found that ~ilomucins stain '' ith alcian blue at pH 2.:l \\hik sulfomucins c:tn be idcntif-if'd hy ~t;~ining· \Yith ~tlcian blue at pH 0.5."· '" A portion of each biopsy ,,·as frozt·n at 20° C. and sections were cut with a cryostat at 10 I' in thickness. SonH' of thP sections wPre incubated at ::l7° C. as outlined for the dPmonstration of sites of succinate dchydro-

591

genase activity.' 1 Enzyme activity \\as absent in control sections which were incubated without substrate or in a mixture containing equal concentrations of sodium succinate and sodium malonate. Additional frozen sections were incubated at 37° C. to demonstrate acid phosphatase activity (at pH 5.0) and others were incubated at 37° C. to show sites of alkaline phosphatase activity (at pH 9.0). Both phosphatase procedures were carried out according to the method of Gomori, '" with sodium beta-glycerophosphate as the substrate and lead nitrate as the capturing agent for the cleaved phosphate groups. Sites of lead phosphate were visualized by the use of arn.n1onium sulfide which reacted to form lead or cobalt sulfide, respectively. No enzyme activity occurred when sections were incubated without substrate. The phosphatase enzymes were also demonstrated in frozen sections \vith the napthol-AS phosphate azo-coupling procedure and the results cornparPd to those obtained by the Gomori. 1 " Techniques for demonstrating activities of thP three enzymes studied in frozen sections, succinate dehydrog-enase, acid phosphatase, and alkaline phosphatase, are found in standard tt'xtbooks of histochemistry and ha\'e been used for several years in our laboratory with reproducible results.,,_,,, These particular enzymes wert' selected for study because of their imporLmt rok in normal endometrial function. Succinate d(·hydrogcnasc, an enzyme loc;~ tPd in mitochondria, is part of the succinate oxidase system \\ hich contains the cytochromes and other components necessary to :~t:L the c·rL'Ylnc \\·ith ox,·uen lti 'rhe stinnda\()J'\' f'fJ'Fct<; of e-.trddiol and the absence of .IllY dTcct of progesterone nn this enzyme systern in lmrnan t'ndometrium are well docunwnted and prm·ick a basis for comparing \ar1ou~

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Since

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biopsy does not provide sufficient tissue for biochemical determination of enzyme acti...-ity \vith methods that are commonly used. histochemicd methods are suitable alternatives. They also ha\T tlw advantage nf shO\\·ing cdlubr sites of enznrw ;ICtivitv as well as

592

November I, !974 Am.]. Obstct. Gynccol.

Flowers, Wilborn, and Enger

Fig. 2. Middle proliferative endometrium. A and B, Control. (x120.) C and D, (x480. )

sites of low, moderate, high, and intense activity. Acid phosphatase is a hydrolytic enzyme associated with lysosomes. 1" Functions of this enzyme include degradation and digestion of secretory products and tissue debris. 2 (' Alkaline phosphatase is associated with the apical plasma membranes of endometrial epithelial cells of the glands and the uterine cavity. It appears to be involved in the processes of growth and differentiation of new cells and is also associated with membrane transport. 21 It is also associated with vascular endothelium, where its role is commonly accepted to be that of expediting the transport of substances across the blood vessels. As mPntinnPcl l';o,rliPr P:-trh hinnsv

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vided into several small pieces, some of which were used for histology and histochemistry.

The remammg pieces were cut into 1 mm. cubes and fixed for 4 hours in cold ( 0 to 4° C.) glutaraldehyde prepared m Clark's 22 buffer (pH 7.4). Following this initial fixation in glutaraldehyde, the tissues were postfixed for 2 hours in cold 2 per cent osmium tetroxide which was also prepared in Clark's buffer (pH 7.4). Buffers other than Clark's, such as Millonig's 23 and collidine, were also used, but they did not provide as good overall tissue preservation as did Clark's. 24 It should .be pointed out, however, that Millonig's buffer was superior to any other buffer in respect to glycogen preservation. Following fixation, regardless of the buffer employed, tissues were handled in the same mannpr Tho hY!D~ ~;~~~-!~ V-'~~- dchydr:tted through ethyl alcohols and then in propylene oxide before it was embedded m Dow epoxy

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Quingestanol acetate on endometrium

593

Fig. 3. A, Early secretory endometrium with subnuclear vacuoles in the control glands. B, Absence of vacuoles in quingestanol acetate glands. (X480.)

With the use of an ultramicrotome, a I {t section of the plastic-embedded material was cut with glass or diamond knives, stained with 1 per cent toluidine blue, and examined with the light microscope. Examination with the light microscope of sections from the same blocks, from which thinner sections were to be cut for electron microscopy, provided a general over-all view of the tissue and made it easier to correlate light microscopic and ultrastructural findings . The 1 /'plastic st'ction also showed the finer cytological details of the endometrial constituents much more vividly than did the thicker paraffin sections. For electron microscopy, ultrathin sections (60 to 90 mp.) were cut on glass or diamond knives with a Porter-Blum Ultramicrotome (MT-2B), mounted on 200 or 300 mesh naked copper grids, and double stained, first '>Vith uranyl acetate and then with lead citrate. Tht' stained sections were exam ined with a Philips EM-301 electron resin. ~ "

llllCfOSCOpe.

Observations Histology. Certain striking changes in histology. carbohydrate histochemistry, and ultr:lstructure w<>re noted in the endometrial gla nds of women taking quingestanol acetate. On the other hand. the epithelium of the ute rine cavity and the stromal cells did not show any consistent effect. Regardless of the phase of the cycle, glands of control endometrium in sections stained

with H&E contrasted markedly with those seen in endometrial biopsies of patients taking quingestanol acetate. Even low-power views of the sections showed that the glands were fewer and smaller in the biopsies of patients on the progestin (Fig. 2) . Examination at higher magnification of glands in the proliferative phase showed much more mitotic activity in the control glands. Moreover, by the twelfth day of the cycle, glands of the treated patients showed considerable eosinophilic secretion in their apices. Subnuclear vacuoles of glycogen developed in r.ontrol glands during the early secretory phase but failed to develop in glands of treated patients (Fig. 3). During the late secretory phase, vacuolated areas appeared in the supranuclear cytoplasm of cells of the control glands. Such foci were shown by the PAS technique to be rich in glycogen. Secretion subsequently appeared in the glandular lumina and then the ';crn·tory process subsided Tn rontrast, such <:m orderly secretory process was seldom observed in gla nds of patients receiving quingestanol acetate: most of the secretory material remained in the glandular apices until the glands began fragmentation. This process characteristically began much earlier in glands of treated patients (Fig. 4 ). Histochemistry. By the late proliferative phase, cells of control glands contained a striking a mount of diastase-labile glycogen (Fig. 5 ) . The material encircled the supranu-

594

Flowers, Wilborn, and Enger

N uvember I, 1974 Am,

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Obstl't. (;yncco1.

Fig. 4. Late secretory endometrium. A and B, Control. (X120.) Quingestanol acetate. C and D, (X480.)

clear region and was present as scattered droplets in the subnuclear region. All subnuclear droplets and most of the PAS-positive material in the supranuclear cytoplasm dissolved when sections were treated with diastase. This indicated that the subnuclear vacuoles contained glycogen and that glycogen represented the predominant material in the supranuclear cytoplasm. It also illustrated that the supranudear cytoplasm contained a carbohydrate other than glycogen which was PAS-positive and diastase resistant. The chemical nature of the latter is not known but it would seem to represent a proteinaceous secretory material bound to a -r

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Celis of endometrial glands influenced by quingestanol acetate in the late proliferative

phase also contained a considerable amount of PAS-positive material (Fig. 5). As in control glands, diastase removed the subnuclear foci along with most of the apical material, but the enzyme removed far less of the supranuclear material than it did in control glands (Fig. 5, Band D). This finding confirms the impression gained from studying numerous H&E sections, many of which showed an eosinophilic apical zone that corresponded in position to the PAS-positive, diastase-resistant material. The material was evidently secreted during the secretory phase. Alteration of the secretory process occurred during the early proliferative phase and .,.,, uvc ~.-u11ecteu uUiiug the remainder of the cycle. Aithough more glycogen and secretory material occupied the lumina of some

Voilllllt' 1:!0 :--.rumht>r :-,

Quingestanol acetate on endometrium

595

Fig. 5. Late proliferative endometrium showing PAS-positive material indudin)l; the luminal surface with droplets near the base. A, Control; B, c~ntrol diastase treated; quingestanol acetate: D, quingestanol act> tate diastase trt>ated. ( X480.)

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glands under the influence of quingestanol acetate, this condition obviously resulted from early breakdown of glands and not from acceleration of the normal secretory mechanisms (Fig. 6). In addition to the alteration in PAS-positiw· material in the glands, blood vessels of the endometrium of patients receiving quingcstanol acetate contained platelet aggregates \vhich \verc Pr\S positive and diastao;e resistant (Fig. 7 .1. Quingt·sLrr,ol <•CTt:< tc did nut appe:u· to alter the cyclic \·aria tions in acid mucosubstances. Sulfornucins predominated during the proliferati\e phase and carboxylrnucins in the secretory phase. Platelet aggregates in blood vessels of tlH· steroid-treated patients \H're alcian blue-positive at pH 2.5 and, as characteristic of platelets, alcian blue-negative at pH 0.5. Alcian blue staining at pH 2.5 has been shown by neuraminidase digestion to be due to sialic acid which adheres to the microvilli at the surface of the platelet.

Frozen sections incubated for the histochemical demonstration of succinate dehydrogenase, alkaline phosphatase, and acid phosphatase were meticulously studied and evaluated for any possible alterations that might be related to the contraceptive. None were found except for the cyclic and individual \ariations noted by Dallenbach-Hcllweg"'' dnd Schrnidt-~vfatthiesen.~~ The observation that quingestanol acetate did not alter sucnn:tte dehydrngenasf' activity was in agreement >vith the reports of Villec"· '' for other progestogens. This finding was a !so supported by electron micrographs which showed that mitochondria \\ere normal ultrastructurallv in cells of tlw glands of treated patients. Histochemical observations indicated that they were functioning normally. .:\'onnal alkaline phosphatase activity in the glands under the influence of quingestanol acetate suggested that the transport ;1bility of the epithelial cells was not seriously

596

November l, 1974 Am. ]. Obstet. Gynecol.

Flovlers, VVi!bcrn, and Enger

Fig. 6. Late secretory endometrium. A, PAS-positive material in the cellular apices; B, PASpositive debris in the lumen of a degenerating gland.

endometrial glands. This finding was of value in identifying glands that were near the end of their life span or already in the process of breaking down. In biopsies of treated patients it was not unusual to find a few highly acid phosphatase-positive, degenerating glands throughout the menstrual cycle, although they were more numerous during the second half of the cycle. Degeneration of these glands and blood vessels which surround them could explain the breakthrough bleeding before the appearance of true menstrual flow. Electron microscopy

Fig. 7. Late secretory endometrium showing platelet aggregates in blood vessels. (x480.)

affected. This idea was also supported by electron micrographs which showed that the fine· structure of the cell membranes was perfectly·normal. Lysosomal acid phosphatase activity peak.1

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Cells of control glands showed considerable ultrastructural variation during the proliferative phase, but it was obvious that the variation was due to cells in different stages of maturation, rather than to diverse cell types. Fig. 8 shows the salient features of the supranuclear cytoplasm of the younger population of gland cells, including a few foci of glycogen-rich areas, some strands of granular endoplasmic reticulum with little or no cisternal distention, a few free ribosomes and Lunufilaments, flattened Golgi membranes, several pleomorphic (round, ovoid, or eion-

Volume 120 :-.lumber 5

Quingestanol acetate on endometrium

L

-

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.

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Fig. 8. Control endometrium, early. proliferatiye phase. Portions o{ three "young" gland cells and characteristics of their supranuclear cytoplasm are shown. Nucleus (N) , Golgi membranes (G), microvilli (MV), and the glandular lumen (L) are depicted. Mitochondria ( M ) vary in appearance, some being cut in longitudinal section and o thers in cross section. A few strands of granular endoplasmic reticulum (ER) and areas rich in glycogen (gly) are present. Glycogenrich areas have a pale, granular apppearance and differ from the numerous, electron-dense ribosomes scattered through out the cytoplasm. Electron-lucent vesicles (v ) can be identified beneath the microvilli . ( X24,750 .)

597

598

Flowers, Wilborn, and Enger

Fig. 9. Control endometrium, early proliferative phase. A basal cell illustrated. Basal cells do not extend to the free surface and are numerous free ribosomes (arrows ). A gland cell lies on each side " - ..--·-•'-'-' u v •u Lut ua,al cell by an intercelluar space (ics) that is (x24,750.)

November I , 1974 Am. j . Obstct. Gynccol.

and its nucleus (N) are further characterized by of thP. has;ol rP.ll hPin~ prominent at some si tes.

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Quingestanol acetate on endometrium

Fig. 10. Quingestanol acetate endometrium, late proliferative phase. A dark, degenerating cell (de) with an irregular nucleus ( N) and dilated perinuclear cisternae ( pnc) is shown near the renter of the micrograph next to the lumen ( L) that is lined by minovilli. Th£' cell at the right of the micrographs , which extends from the base to the lumen of the gland, also appea rs to be degenerating. A small infranuclear area of glycogen (gl)') is in o ne of the nondegenerating cells. (X 1!,25Q.)

599

600

Flowers, Wilborn, and Enger

Nove mbe r I, 19i4 Am .

J. O bstct. Gy nccol.

Fig. II. Quingesta nol aceta te endometrium , la te prolifera tive phase. Portions of two basal cells

are sho wn , o ne at th e lower left margin of th e micrographs and th e o ther a t the lower right margin. The n ucle us ( N ) of each basal cell is surrounded by electron-dense cytoplasm which cont a ins free ribosom es a nd much gra nula r end oplasmic retic ulum ( ER ) with dilated cisternae. The gla nd ce ll in the ce nter of th e micr ogra ph with th e p romin e nt nucleus ( N) also contains eo nsidera ble granular endoplasmic. re ticulum ( ER ) , but th e cisternae are nut as dil atP.n as in : __ : ... ... : ,, ;; _, ;-~ ;c.u,uuu.:s, '" well as hl am ents (/ ) , a re es pecia lly abundant in the gland cell, but g lycogen is a bsent in this particula r sec tion . (X24,750. )

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Quingestanol acetate on endometrium

Fig. 12. Quingestanol acetate endometrium, middle proliferative phase. Portions of three gland cells and characteristics of their supranuclear cytoplasm are shown. Golgi membranes (G) are somewhat dilated. Small vesicles (v) and multivesicular bodies (mbj can be seen in the vicinity of the Gol,gi. Multivesicular bodies are formed in the Golgi regio n by the coalescence of small vesicles and then move apically for expulsion into the lumen ( L). Microvilli project into the lumen and appear somewhat longer and more filamentous than those of control gland cells of the corresponding phase. Many small secretory granules ( sg) of intermediate electron density are also present. Less glycogen ( gly) is present than in control glands, but filaments (/) are more widely distributed. (x24,750 )

601

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November 1, 1974 Am. j. Obstet. Gynccol.

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Fig. 13. Quingestanol acetate endometrium, early secretory phase. The infranuclear region of a gland is depicted. Unlike the situation in cells of control glands, only a few small areas of glycogen ( gly) are present. Mitochondria, endoplasmic reticulum ( ER), and ribosomes (arrows) are shown. (X24,750.)

gated) mitochonana, and occasional l!p!O droplets. The apical surface bore microvilli. Cilia were sparse except on the "clear" cells. A few vesicles of secretory material were present but few of them appeared to be entering the glandular lumina. The older popu11 -

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the proliferative phase progressed and differed from the younger population in that

they showed greater evidence of secretory activity. Their granular endoplasmic reticulum and Go!gi membranes were distended, and secretory material was present in the glandular lumina (Fig. 8). The basal region of gland cells, both young aw.l ulJ, in the proliferative phase was far less specialized than the apical region. There was, however, an additional cell type at the

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Quingestanol acetate on endometrium

603

Fig. 14. Quingestanol acetate endometrium, early secretory phase. Apices of gland cells are shown. Degeneration is evident. Mitochondria ( M) have disrupted cristae. Dense bodies resembling lysosomes (l) are present. Cellular apices are irregular and project into the lumen (L). (X24,750.)

base of the gland . Such cell t ypes did not reach the glandular lumina and were designated basal cells (Fig. 9) . The basal region of the young gland cell was characterized by numerous free ribosomes, a few mitochon-

dria, and scattered tonofilarnents and microtubules. Both of the latter were often associated with small glycogen-rich areas. Older gland cells differed mainly in that they contained larger foci of glycogen. They became

604

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November 1, 1974 Am.]. Obstet. Gynecol.

Fig. 15. Control endometrium, middle secretory phase. Apical portions of gland cells are shown.

Much glycogen ( gly) is present in the apical projection that extends into the lumen ( L) . Microvilli line the luminal surface. Golgi membranes (G ) a nd mitochondria ( M ) are abundant. ..:>tu ew r y VttilLlt!ti ! V) , granular endoplasmic reticulum ( ~H. ) , and nbosomes are present. (X24,750.)

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Quingestanol acetate on endometrium

Fig. 16. Quingestanol acetate endometrium, middle secretory phase. The apex of the gland and its lumen ( L) are depicted . The cell at the right margin of the micrograph appears relatively normal. Its nucleus (N), Golgi membranes (G), mitochondria, granular endoplasmic reticulum , a nd free ribosomes are shown. Glycogen is absent. A degenerating cell (de) with dilated cytoplasmic spaces is present. The nucleus ( N) of another degenerating cell is shown at the lower left margin of the micrograph. ( x24, 7 50. )

605

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November I, 19i4 Am. ]. Obstct. Gynccol.

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Fig. 17. Quinges tanol acetate endometrium, late secretory phase. The base of the gland is depicted. Above the ill-defined basal lumina (BL), portions of two degenera ting cells (d e) are shown. Large cytoplasmic spaces lie below the nucleus (N) of each cell. The intercellular space (ics) is quite large and filled with leaflets of plasma membranes from adjacent cells. (X24,750.)

most numerous during the later proliferative phase. Quingestanol acetate-stimulated gland cells of the proliferative phase differed strikingly, both basally and apically, from cells f'f rnntrnl

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Perhaps the most distinctive feature of these glands was the presence of degenerating cells (Fig. 10) . The cytoplasm of such cells was

very electron dense and the nuclei were irregular with marginal chromatin and dilated perinuclear cisternae. Basal cells of glands under the influence of quingestanol acetate also differed from those of control glands ~Fi;. 8 ) in th~t they \~· e: r t; clialac tefizeU by electron-dense cytoplasm and abundant granular endoplasmic reticulum with dilated cisternae . It appeared as if the basal cells had

Volume 120 Number 5

been triggered prematurely to differentiate into secretory cells. This is indicated by their highly dewloped endoplasmic reticulum (Fig. 11). Because of the pn:sence of a progestin early in the cycle, there was greater cytoplasmic density due to the increase in granular endoplasmic reticulum. free ribosomes, and tonoftlaments. It was difficult. therefore, to characterize cells as young or old in progestinstimulated glands of the proliferative phase. It was apparent that the progestogen prevented the usual sequence of events leading to normal development and differentiation. The accelerated production of ribosomes and granular endoplasmic reticulum, especially in the basal region, provided the cytological machinery for the synthesis of the tonofiliments and accounted for their excess. A few areas of infranuclear glycogen were present in cells of the glands of treated patients, hut they were less frequent and smaller than in the control glands (Fig. 10). During the mid and later proliferative phase, the apical cytoplasm of gland cells showed a greater quantity of secretory granules than the supranuclear cytoplasm of the control glands (Fig. 12) . Many of the granules were multivesicular bodies which could be seen forming in the vicinity of Golgi memhranPs and then migrating toward the apical plasma membrane to be expelled by reverse pinocytosis into the glandular lumina. The other granules in the apical cytoplasm approximated the multivesicular bodies in electron density. It should be recalled that the granules corresponded in position to the eosinophilic zone seen in H&E sections and LU

the lase-n:sista!Jl ,cone ob-

served with the PAS-diastase technique (Fig. 5. D). Electron micrographs, as did the photomicrographs, showed that the granules disappeared in the secretory phase. Besides granules, glycogen-rich areas were occasionally present in the cellular apices, but as a rule such areas were less frequent than in apices of control glands. In contrast, tonofilaments were more numerous than in control glands. During the early secretory phase. large lakes of glycogen developed in the infranu-

Quingestonol acetate on endometrium

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clear region of many cells of control glands. The apical cytoplasm was rich in mitochondria, which ~hawed wide variation in size. Profiles of granular endoplasmic reticulum were especially numcreous in the apical region and thPir distended cisternae contained finely fibrillar and granular material. Several electron-lucent yesicles and occasional glycogen-rich areas also occupied the supranuclear cytoplasm. The apical surface of the cell, however, was rather smooth, except for microvilli. and showed little evidence of discharging secretion into the glandular lumina. Nuclei of many cells developed the nucleolar channel system during this phase. This structure has been lucidly described and its appearance is dependent on estrogen priming followed by progesterone stimulation. 28 The appearance of the nucleolar channel system seems to be dependent upon an exact ratio between estrogen and progesterone. Infranuclear lakes of glycogen did not develop in the gland cells of patients receiving quingestanol acetate during the secretory phase. Fig. 13 shows the maximal extent of subnuclear glycogen development in these cells. Although several small foci of glycogen appeared, they failed to coalesce into the large lakes characteristic of control gland cells and this, in turn, accounted for the anovulatory appearance of the endometrium when studied in H&E sections. Free ribosomes, often in the form of rosets, short segments of granular endoplasmic reticulum, and mitochondria were fairly numerous in the subnuclear cytoplasm. Thus, the cytological machinery was available for t-!;lycogen svnthesis. but the process seemed to be inLibited by sornr enzyrn;1tic n1ech~nistr1 opc-r
608

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11crc FspFcially numerous. The lysosomps were of special interest since they could accntltJt for the intense acid phosphatase activity se('l1 in degenerating cells with the light microscope (Fig. 14). I )ming· the mid-secretor>· phase. glycogen mcrFased in the apices and dt>c-reased in the bases of control gland cells. The cell apices wt>re irregular or domf'-shapcd and could be sef'n breaking off to enter the glandular lumina. Mitochondria were abundant, Golgi mt>mbranes extensive, and the cristernae of granular endoplasmic reticulum less distended than in the cariy secretory phast> (Fig. 15). Small foci of glycogen persisted in the base of gland cells of treated patients during the mid-st>cretory phase and the basal region resembles that seen in the early secretory phase. Many cells were in the process of degt>neration; others attempted to secrete during this phase and had rather long apical projections which extended into the lumina. Such projections contained little glycogen. Degenerating cells 11·ere also present during this period and often lay adjacent to cells of near normal appearance (Fig. 16). In the late secretory phase, interceliuiar spaces were prominently distended at the base of control gland cells and distended to a lesser degree at their apex. Leaflets of the lateral plasma membranes projected into the intercellular spaces. Except for the clefts, the basal region was unimpressive. Glycogen was sparse or absent. The basal region was unirnpressin· except for a few mitochondria ~orncs

and ribosorr1es. SC'cretion \Vas still present in

the lumen and glycogen in the cell apex. Mitochondrial cristae lacked sharpness and Golgi membranes set>med to be fragmenting, indicating that the cells \\ere near thP end of their life span. In the latP secretory phase, cells of glands influenced by the progestin yaried greatly in appearance. Some, as in the previous phases of the cycle, were undt>rgoing degener;ltion and lay adjacent to prominent inter... 11..1

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explanation was apparent as to why some of the Ct'lls were unresponsivE' and thl'ir eyentual fate was uncertain (Fig. 17) . Comment

The endometrium is a mirror that reflects to the most minute degree the slightest variation in estrogen and progesterone stimulation. Mort'over, the endometrium is programmed to receive only estrogen during the proliferativt> phasP and have progesteronE' product' a secretory effect under the proliferative t>ndometrium framework. Quingestanol acetate suppresses FSH and LH and thus reduces the amount of cirrulating- estrogen and progesterone. But more important, this potent artificial progestin is introduced into the entire life cycle of the endometrium which is programmed to be stimulated by estrogen alont' during the proliferatiYe phase. There thus occur competition for binding sites and altered sequences in carbohydrate and protein synthesis. Quingestanol acetate seemed to influence many aspects of protein synthesis, produced an irregularity in the sequence of the secretory process, and caused the gland cells to produce a carbohydrate otht'r than glycogen. The proliferative endometrium of patients receiving this steroid had fewer and smaller glands. Glycogen appeared in these glands during the early proliferative phase and was readily apparent by mid-cycle; but despite ovulation, therE' was a failure to produce normal subnuciear vacuolation. Quingestanol acetate-stimulated glands sho,ved no derHonstrable ~1ltcration in alka>= line phosphatase, succinate dehydrogenase. acid phosphatase, and affinity for alcian blue at pH 2.5 and 0.5. These findings are interpreted as meaning that this progestogen did not alter the transport mechanism across cell surfaces. Many cells showt>d ahundant tonofilalllents and microtubules which resulted from an increase in ribosomes and granular endoplasmic reticulum. These findings indicated that the Iw·clla11isms \\ere present for glycogen synthesis and transport; however, there was a reduction in the actual amount of glyco-

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protein in the cells and the transport systems within the cells wen· altered. Platelet aggregates ·were seen in the en dometrial vessrls during the late proliferative and early secretory phase of patirnts receiving quingestanol acetate. There is little question that these PAS-positive structures were platelets, since they did not contain the acid phosphatase of granulocytes and \\·ere positive for alcian bluC:'. There were no platelet aggregates found in the vessels of 39 control patients despite the many sections which werr made. It is not known what relationship these platelet aggregates bear to thromboembolic phenomena which are occasionally seen in worrwn taking oral contraceptives,"" but this finding and similar descriptions by others demand further study. This study possibly explains breakthrough blt>eding which is often seen in patients taking oral contraceptives. Quingestanol acetate was associated with the degeneration of endome-

Quingestanol acetate on endometrium

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trial gland and vessel cells throughout the entire menstrual cycle. This was evident in both light and electron rmcroscopy. It is possible that this progestogen altered protein synthesis and caused cells to die prematurely, but most likely the explanation lies in the failure of the cells to undergo orderly regression and digestion through a lysosomal mechanism during menstruation. I .ysosomal activity within the endometrial cells requires an orderly production of adequate amounts of estrogen and progesterone. Quingestanol acetate alters the production of these steroids. Thus, endometrial cells and thr cells of blood vessels were breaking down throughout the entire menstrual cycle rather than during menstruation. It is apparent that quingestanol acetate has as profound rffect upon the endometrium as it has upon the hypothalarnic-pituitaryovarian axis.

REFERENCES

I. Martinez-Manautou, J., Cortez, V., Giner, J., Aznar, R .• Casasola, J., and Rudel, H. W.: Fertil. Steril. 17:49. 1966. :2. Moghissi, K. S., and M<~rks, C.: FertiL SteriL 22: 424, 1971 :'. Moghissi, K. S.: Fntil. Stcril. 22: 739, 1972. L Moghissi, K. S., Syner, F. N., and McBride, L. C.: Obstt>t. Gynecol. 41: 585, 1973. 5. Noyes, R. W., Hertig, A. T., and Rock, J.: Fertil. Steril. I: :1, 1950. 6. McManus, J. F. A.: Nature 158: 202, 1946. 7. Lillie, R. W.: J. Lab. Clin. Med. 32: 910. 1947. 8. Hester, L. L. Kclktt. W. W., Spicn, S. M .• Williamson. J. 0., and Pratt-Thomas, H. R.: AM. J. OnsTJ:T. GY!\FCOI.. 106: 11 H, 1970. q. Mowry, R. ·w.: Ann. N Y. :\cad. Sci. 106: +U~.

]II

11.

12 1:l.

H. 15.

l'lbi.

Leppi, T. j., and Spicer. S. S.: Am. J. .'\nat. 118: S:l:,, 1966. Nachlas, M. M .. Tsou, K. C., OnesouLa, E .. Cheng, C. S., and Seligman. A. M.: J. Histochem. Cytochem. 5: ~:!0, 1957. C:omori. G.: Microscopic Histochemistry. Chica,go, 1952. Cniversity of Chicago Press. Burston<", M. S.: Enzyme Histochemistry and Its Application in tlw Study of Neoplasms, :--.lew York, 1962. Academic Press, Inc. Lillie, R. D.: Histopathologic Technique and Practical Histochemistry, Nc~w York, 1965, The Blakiston Company. Pearse, A. C. E.: Histochemistry Theoretical

16.

17. lB.

19.

21)

~,.

:22. :!3.

~t.

25. 26. 27.

and Applied, Boston. 1960, Little, Brown & Company. Singer, T. P., Kenney. E. B., and Massey, V.: In Gaebler, 0. H., editor: Enzymes: Units of Biological Structure and Function, Pd. 9, Nt>w York, 1956, Academic Press, Inc., pp. ~17-~32. Villec, C. A.: Cancer Res. 17: 507, 1957. Villt'e, C. A.: In Young, W. C., and Corner, G. W., editors: Sex and Internal Secretions, Bal timorP, 1961, The Williams & Wilkins Company, pp. 6~3-665. DeDuevt>, C.: In deReuck, A. V. S., and CarnPron, M. P., editor:<: Lysomcs, Boston, 1963, Little;>, Brown & ':>n1pany, pp. 1-35. Swift, H .. and Hruban, Z.: Fed. Proc. 23: !010, 196·1 hnutseh,, J. C., de :\cl'i. J. C., t:llny, J C .. and Georgt', 0. T.: Obstct. & GynecoL 21: 423, 1963. Clark, S. L., Jr.: Am. J. Anat. 112: I, 1963. Millonig, G.: En Fifth Internationl Congress for Electron Microscopy, New York, 1962, Academic Press, Inc .. voL 2. p. 8. Bennett, H. S. and Luft, J. H.: J. Biophys. Biochcm. Cytol. 6: 113, 1959. Winborn, \V. B.: Stain Techno!. 40: 227. 1965. Dallenbach-Hellweg, G.: Histopathology of the Endometriurll, New York, 1971, SpringerVerlag. Schmidt-Matthiesen, H.: The Normal Human

610

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November 1, 1974 Am. j. Oh>tt•t. Gynerol.

Endometrium, New York, 1963, Blakiston Division, McGraw-Hill Book Co., Inc., pp. 1 ~5-207.

28. Terzakis, ]. A.: J. Cell. Bioi. 27: 293, 1965. 29. Irey, N. S., Marion, W. C., and Taylor, H. B.: Arch. Pathol. 89: 1, 1970.

Discussion DR. RALPH M. WYNN, Chicago, Illinois. Using

these organelles contain a variety of enzymes; nor should alkaline phosphatase necessarily be equated with unimpaired membrane transport. Finally, any discussion of acid phosphatase should distinguish the lysosome-bound from the ergastoplasm-associated enzyme, for the former may be biologically inert. Do the authors believe that quingestanol is a direct labilizer of lysosomal membranes or does it cause disruption of the lysosome through production of ischemia via an effect on blood vessels? Recent data suggest that lysosomal phospholipase is critical in the synthesis of prostaglandins. Thus the effect of steroid hormones on lysosomal labilization might be mediated via production of prostaglandin. Dr. Flowers and colleagues have begun an interesting investigation of a subject of clinical and theoretical significance. It is most likely to stimulate further correlative biochemical and ultrastructural studies. DR. SAUL B. GusnERG, New York, New York. I am almost embarrassed to ask Dr. Flowers this question, but he sort of "threw away" a statement that this compound competed with the binding site for estradiol. This would be a very important finding if it were substantiated. I would like to know what is the evidence for that. DR. EDWARD C. HuGHEs, Syracuse, New York. I am interested in Dr. Flowers' using a form of progesterone that is practically acting determinally upon the subnuclear structures and enzymes of the endometrium than what we have found in our laboratory. We have kept endometria both in vitro and in vivo, using the organ culture method of determining various enzymes involved in the metabolism of glucogen and mucopolysacharrides. His findings are a little bit contrary to the effects of other progesterone compounds which we have found, particularly in organ culture. I think one of the most interesting and unique characteristics of the endometrium is its ability to synthesize carbohydrates, mostly in the form of glycogen, which it does on a daily basis. And it reaches its peak accumulation on about the sixteenth to eighteenth day with about 800 to 1,000 mg. per I 00 mi. But this, of course, is in preparatiOn to rece1ve the tertilized ovum . .Failing conception, the endometrium must get rid of the amount of glycogen it has stored in the

standard histologic, histochemical, and electron microscopic techniques, Dr. Flowers and colleagues have confirmed the abundant morphologic data relevant to the normal and hormonally modified human endometrial cycles. They have, I believe, added one interesting observation, namely, the absence of nucleolar channel systems and giant mitochondria even under the influence of a compound that presumably does not inhibit ovulation. In our earlier ultrastructural studies of conventional combined and sequential agents we were unable to attribute the absence of these specialized organelles specifically to anovulation, as opposed to a deviation from the optimal ratio of estrogenic to progestational substances. The structures to which I refer are shown in Figs. I and 2. Unfortunately, the authors do not depict nucleolar channel systems in their controls nor do they mention giant mitochondria. Were these structures, in fact, found in the controls as expected? The data are valid but the possibility of their overinterpretation remains. Caution is required in the attribution to a purely progestational compound of the capacity to stimulate, as indicated by an increase in free ribosomes and granular endoplasmic reticulum, an endometrium unprimed by estrogen; but quingestanol, a 3-enol ether of a ~ 4 -3-ketosteroid, has potent intrinsic estrogenic properties. During a cycle of treatment with this drug, furthermore, ovarian production of estrogen, albeit diminished, continues. The aggregates of platelets and the possibility of thromboembolism may thus reflect intrinsic estrogenic activity. Even the progestin-only "mini-pill" is therefore not free of this potential hazard The enzyme-histochemical data should be interpreted with particular caution. The authors describe no changes, detectable by optical microscopy of the treated endometria, in the distribution of three enzymes, despite the lowered le\'eJ., of both estrogen and progesterone. Electron microscopic localization of these enzymes would aid greatly in the differentiation of artifacts of intracellular distribution from sites of production. . .,,~ ,,u'"u ·"'JUlt'> m progress;' 111e delnunstratwn of succinate dt'hydrogenase is not an adequate index of normal mitochondrial function, for

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Quingestanol acetate on endometrium

611

Fig. 1. Arrow points to nucleolar channel system in early postovulatory endometrium.

Fig. 2. Arrow points to giant mitochondrion normally associated with early postovulatory endometrium. Glycogen (G) is abundant.

glandular epithelium, and it goes through another exciting and extraordinary physiologic effect. In other words, the glandular epithelium is gorged with glycogen, and it must eliminate it in order to proceed to the next cycle. The process of elimination is just as important as the process of synthesis. We have studied glycogen synthetase and glycogen phosphorylase, two of each kind. Glycogen synthetase, of course, builds up carbohydrase to the deposition of hydrogen and estrogen. It reaches its peak on the seventeenth day of the cycle. Glycogen phosphorylase, the two, in time reach their peak activity on the twenty-fourth day of the cycle. And they are definitely under the influence of progesterone as proved in organ culrure. The progesterones that Dr. Flowers has nsed in observing these histologic sections have ~·ll entirely differeut effect upuu thi,. I would lih to try this and evaluate what happens to the activity of the phosphorylases. Amazingly enough, in abnormal endometrium, hyperplasia and carcinoma, the glycogen synthetase is very high, but the phosphorylases are very low, meaning that the cell does not probably get rid of the glycogen as it should and has a constant storage for some period of time. The phosphatas<'s, L and S phosphatases, are interesting enzymes. They, too, are under the influence of progesterone, and they rise markedly, particularly in organ culture after the use of

practically all forms of progesterone except the one Dr. Flowers has used, which we have not used. The action of the phosphatases, both alkaline and acid, are particularly on cell membranes, where they break down phosphates and phospholipids. It is important to have a cell membrane that can excrete and get rid of the stored glycogen. And without this action, of course, the cell retains more glycogen. So I am interested to see what Dr. Flowers finds with this progestin material because without adequate progesterone, we may have a failure of the cell to metabolize, to breakdown glycogen as it should. DR. FRITZ K. BELLER, Muenster, Germany. Dr Flowers provided some very interesting data on the endometrium. 1 ne observation regarding clusters in the treatment group impressed me. You call them platelet aggregates not being present in the control group. Did you try to identify these aggregates besides your staining technique? If these clusters were indeed platelet aggregates, it would be an interesting attempt to administer to these patients aspirin or any other platelet aggregate inhibitor. Did you try this modification of treatment? DR. E. S. E. HAFEZ, Detroit, Michigan. Scanning electron micrographs of human endometrium untreated show two types of cells-the "secretory" cells with microvilli and ciliated cells with kinocilia. With a higher magnification, the cili-

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Am. J. Obstet. Gynecol.

ated cells appear in clusters and the microvilli are among them. Before secretion of the secretory material, there is rupture of the cell mem· brane and the release of the secretory material and remnants of microvilli. These are the degen· erating cells that Dr. Flowers was talking about. They appear also i'n the untreated endometrium. Ciliated cells during the secretory phase are erect, but during the premenstrual phase they become "droopy." These findings are summarized as follows: The secretory material is formed, coalesced, stored, released, and distributed in the lumen with the aid of the cilia, which beat toward the cervix. DR. FLOWERs (Closing). I appreciate the comments of Drs. Wynn and Hughes, who are international authorities. Dr. Wynn has delineated the endometrium's ultrastructure and Dr. Hughes and his group have placed into perspective its complicated enzyme systems. We did find the delicate nucleolar channel system in our control endometrium but it was absent in patients receiving quingestanol acetate. These channels are extraordinarily delicate and apparently require a precise balance of progesterone and estrogen to properly develop. The artifical progestin upset this balance. We are sure that we are observing platelet aggregates within the endometrial vessels. These findings are disturbing. However, there is no apparent intimal injury to the vessels, as described by lrey, in patients dying from pulmonary emboli while receiving oral contraceptives. It is appropriate for Dr. Wynn to question the statement that quingestanol acetate does not alter transport mechanism across the cell surface. This was a histochemical and ultrastructure study of the endometrium. We could thus not quantitate the levels of succinate dehydrogenase or alkaline phosphatase within the cells. Since progestin altered the morphology of some of the cells and in others caused premature aging, it would seem that very accurate quantitative measure· ments of enzymes might indicate that the progestin did alter membrane transport. However, utilizing the techniques that we have described, there was no such evidence. We believe that the lysosomes contain a num· ber of hydrolytic enzymes and play an important and fundamental role in endometrial digestion and menstruation. The lysosomes are manufactnrpr1

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the influence of balanced levels of estrogen and progesterone. The destruction of their membranes and the release of the potent digestive hydrolytic enzymes are apparently due to hypoxia secondary to vascular collapse which is associated with decrease in the ovarian steriods. However, some of the Golgi, under the influence of quingestanol acetate, produced lysosomes with defective lysosomal membranes. It was asked "was there any alteration in cervical epithelium in patients receiving quingestanol acetate?" There were no dysplasias in the 125 patients who were subjects of the clinical trials. Our continuing studies of all patients with cervical dysplasia have not indicated that oral contraceptives play a significant role. Cervical dysplasia and carcinoma in situ are associated with so many cultural and socioeconomic variables, it is difficult to incriminate one single factor. Dr. Hughes is a far better interpreter of the enzymatic alterations associated with endometrial hyperplasia than I. However, I feel that endometrial hyperplasia results from continued growth of endometrial cells beyond their physiologic life span. In the prolonged absence of ovulation, there is irregular sloughing of some of the endometrial cells due to periodic decline in estrogen. Since progesterone is absent, there is minimal lysosomal production and there is no orderly digestion of the endometrium by the lysosomal enzymes. The continued growth stimulus to the endometrium of some patients may markedly alter RNA, DNA, and enzymatic activity. We did not chemically determine binding sites, but I believe this is the best biologic explanation for our findings. Endometrial cells have been programmed for millions of years to respond to estrogen during the proliferative phase and progesterone during the secretory phase. One might say the endometrium cells were confused when two active steroids stimulated them at a time when only one was expected. Our electron microscopic pictures and our studies with microcapsules lead us to agree with Dr. Hafez that the glandular cells and superficial cell villi are capable of moving glycoproteins and particles of various size with great ease. We did not appreciate the platelet aggregates until the clinical studies were terminated. Thus, we did not administer aspirin.