Arias-stella 현상의 전자현미경적 연구

Abstract

[한글]

일반적으로 융모조직의 체내 잔존시 자궁내막의 상피세포에 나타나는 특이한 변화로 알려진 Arias-Stella 현상은 임신이나 융모상피계통의 질환시에 여러 빈도로 나타나고 이의 출현은 많은 산과적 질환의 감별 진단에 도움을 주며 탈락세포검사상 또는 조직학적 진단상 악성세포와의 감별에 유의해야 한다고 알려져 있다(Arias-Stella, 1954 및 1972).

그러나 아직도 그 미세구조가 분명히 밝혀지지 안고있으며, 발생기원에 관해서도 홀몬상승으로 인하여 변화된 활동성 분비세포라는 설과(Arias-Stella, 1955**2, Birch 및 Colli

ns, 1961), 홀몬저하로 인한 퇴행성 변화라는 설이 (Fienberg 및 Lloyd, 1974)서로 엇갈린 주장을 하고 있어 분명하지 않다.

이에 본 연구에서는 인체 Arias-Stella 현상의 조직화학적 특성과 그 미세구조를 관찰함과 아울러 백서에 여러 홀몬을 혼합투여하여 가능한한 인체 Arias-Stella 현상과 유사한 변화를 유발시켜서 그 조직화학적 특징 및 미세구조등을 관찰하여 인체의 그것과 비교

연구하므로써 Arias-Stella 현상의 미세구조를 밝혀내고 아울러 그 발생기전의 이해에 기여하고져 다음과 같이 검색하였다.

연구재료 및 방법

본 연구는 인체 자궁내막에 대한 검새과 동물실험으로 나누어 시행하였다.

인체에 대한 검색은 원주기독병원 산부인과에서 소파술로 얻은 총 101례의 자궁내막을 대상으로 하여 광학현미경과 전자현미경으로 관찰하였다. 조직의 일부는 10% formalin에 고정한 후 통상의 방법에 의하여 H-E, PAS, dPAS, methylgreen pyronin 및 oil red O 염

색등을 시행하여 광학현미경으로 검경하였고 일부의 조직은 4% glutaraldehyde 고정 후 1% osmium tetroxide에 이중 고정하여 통상적 방법으로 Hitachi HU-11E형 전자현미경으로 관찰하였다.

실험적으로 Arias-Stella 현상의 발생기전을 추구하기 위하여 체중 200gm 내요ㅣ의 자성 백서를 다음과 같이 구분하여 처치하였다.

제Ⅰ군: 임신군

제Ⅱ군: HCG 및 estrogen 투여군

제Ⅲ군: HCG, estrogen 및 progesterone 투여군

제Ⅳ군: HCG 및 progesterone 투여군

제Ⅴ군: 거세후 HCG 및 estrogen 투여군

제Ⅵ군: 거세후 HCG, estrogen 및 progesterone 투여군

제Ⅶ군: 거세후 estrogen 및 progesterone 투여군

제Ⅷ군: 정상 및 거세 대조군

실험동물에 대한 검색은 임신 또는 투약 중 3,7,10,15 및 20일에 ehter 마취하에 도살, 개복하여 육안적 검색을 마친 후 자궁내막에 대하여 인체에 대한 것과 같은 방법에 의거하여 광학 및 전자현미경적 검색을 시행하였다.

연구성적 및 결론

이상과 같이 연구하여 다음의 성적과 결론을 얻었다.

Electron Microscopic study of the Arias-Stella Reaction

Jin Kook Choe, M.D.

Department of Medical Science, The Graduate School, Yonsei University

(Directed by Professors: Dong Sik Kim, M.D. and Yoo Bock Lee, M.D.)

In 1954 Arias-Stella described a peculiar atypism of the endometrial glandular

epithelium in cases associated with uterine abortion, hydatidiform mole,

chorionepithelioma, syncytial endometritis and ectopic pregnancy. this atypism has

subsequently come to bear his name; the Arias-Stella reaction (ASR). Also ASR has

been reported in caseof intrauterine pregnancy (Arias-Stella, 1959) and

occasionally in cases treated with oral contraceptive pills(Azzopardi and Zayid,

1967) or clomiphene citrate (Bernhardt et al., 1966).

The ASR, which occurs focally in glandular and/or surface epithelium of the

endometrium, consistsof enlargement of the gland cell, loss of cellular polarity,

lack of orientation of the nucleus, nuclear hypertrophy with hyperchromasia and the

presence of a voluminous vacuolated and foamy cytoplasm (Arias-Stella, 1954).

A wide variation in the incidence of ASR has been reported: 2.9% (Roach et al.,

1960) to 100% (Frederiksen, 1959) in ectopic pregnancy; 0-81.3% in intrauterine

abortion or pregnancy (Roach et al., 1960; Silverberg, 1972); and 24.3-34.6% in

diseases of chorionic tissue (Arias-Stella, 1954; Roach et al., 1960). However, the

presence of the ASR has been known to be helpful in differential diagnosis of

diseases of pregnancy and of chorionic tissue (Arias-Stella, 1972).

Despite the well known characteristics and the clinical significance of ASR, only

a few invesetigators have studied this lesion by the electron microscope. De Brux

and Ancla(1964) observed a single case of ASR with electron microscope and they

described that the atypia consisted of "clear and dark cells" and the

characteristic "clear cell" did not contain glycogen. Recently, Thrasher and

Richart (1972) studied this lesion in 8 cases and stated that the ASR cells were in

an active metabolic state since they contained abundant glycogen and rough

endoplasmic reticulum in the cytoplasm.

There are many opinions as to the genesis and causes of ASR. It was previously

thought to be secondary to an inflammatory reaction (Deelman, 1933). Arias-Stella

(1954 and 1972) and many other authors believed that it was a hormonally induced,

metabolically active secretory cells of endometrial gland due to the presence of

chorionic tissue. Arias-Stella demonstrated an ASR-like lesion in rats by

administration of gonadotropin with estrogen in normal animals and by a large

amount of estrogen and progesterone in the castrated rats (1955). In contrast to

the above, some investigators claimed that ASR is an involutional, regressive and

degenerative change which is related to fetal death or trophoblastic disorders

(Lloyd and Fienberg, 1965).

As mentioned above, the ultrastructural nature and pathogenesis of the ASr are

still very controversial among investigators.

This present study examined a large number of cases of ASR by histological,

histochemical and electron microscopic methods, to study the cellular

ultrastructure. It investigated the pathogenesis of ASR by comparing ASR-like

lesions in rats, which lesions were induced by the administration of various

combinations of hormones.

Materials and Methods

The study was divided into two parts: first the examination of human endometrial

specimens and second the animal experiments.

1. Examination of human material

One hundred and one human endometrial tissues removed by curette were obtained

from the Department of Obstetrics and Gynecology in Wonjoo Union Christian

Hospital. Twenty eight of the tissues came from patients with intrauterine

pregnancy, 10 had ectopic pregnancy, 1 had hydatidiform mole, 49 had a normal

menstrual cycle and 13 patients had other diseases of which specimens were

discarded. A portion of each specimen was fixed in 4% glutaraldehyde, double-fixed

in 1% phosphate buffered osmium tetroxide(pH 7.4), prepared as usual for electron

microscopy, and examined by the Hitachi HU-11E electron microscope. The remainder

of each specimen was fixed in 10% neutral formain solution. The paraffin embedded

sections were stained by hematoxylin and eosin, periodic acid-Schiff reaction with

or without diastase treatment (PAS and dPAS) for glycogen and glycoprotein, and

methylgreen pyronin for ribonucleic acid. A frozen section of formalin fixed tissue

was stained by oil red O to detect lipid.

2. Experimental meterial

Female albino rats weighing around 200 gm. were used in the experiment divided

and treated as follows;

Group Ⅰ: Pregnant animals

Group Ⅱ: Normal animals receiving human chorionic gonadotropin (HCG) and

estrogen

Group Ⅲ: Normal animals receiving HCG, estrogen and progesterone

Group Ⅳ: Normal aniamls receiving HCG and progesterone

Group Ⅴ: Castrated animals receiving HCG and estrogen

Group Ⅵ: Castrated animals receiving HCG, estrogen and progesterone

Group Ⅶ: Castrated animals receiving estrogen and progesterone

Group Ⅷ: Normal and castrated controls

Each group consisted of 15 animals except Group Ⅷ which was composed of 10

normal and 10 castrated control animals.

The HCG(Gonadotropin, Upjohn) in a dosage of 100 I.U. daily per animal was

injected subcutaneously. Estrogen (Progynon-DP, Korean Schering) and progesterone

(Proluton, Korean Schering) were injected intraperitoneally in olive oil solution.

The respective dosage was 35 γ and 2 mg. daily per animal.

Under the ether anesthesisa 3 animals from each group were sacrificed after 3, 7,

10, 15, and 20 days of treatment and endometrial tissues were examined by the light

and electron microscopes as the same way as the human tissues.

Results ans Summary

The histochemical and ultrastructural characteristics of human ASR were studied.

In addition, the histochemical and ultrastructural nature of the ASR-like lesion

induced by various hormonal combinations in albino rats was studied.

The results of this study are summarized as follows:

1. The ASR changes were noted in the human endometrium in 17 of 28 intrauterine

pregnancies (60.7%), in 6 of 10 ectopic pregnancies (60%) and in 1 case of

hydatidiform mole. However, the atypism was not detected from the various stages in

the normal menstrual cycle.

2. The clearness of foamy appearance of the ASR cell was due to its abundant

glycogen and to the small amount of mucopolysaccharides which were demonstrated by

PAS and dPAS methods. In adddition, methylgreen pyronin stain revealed that the

cytoplasm of the ASR cell contained many concentrated RNA granules in its

supranuclear position. However, lipid was not regulary observed by the oil red O

stain.

3. The characteristic ultrastructural features of the ASR cell included

supranuclear parallel arrays of rough endoplasmic reticulum, an abundant collection

of glycogen, diffusely scattered small elongated mitochondria and abundant

microvilli on the luminal surface. These features were interpreted as an evidence

that ASR cells were active secretory cells rather than degenerative or involutional

cells.

4. In animal experiment to detect which hormone plasyed a major role in the

pathogenesis of ASR, the specific changes (ASR-like lesion) which were

intraepithelial cysts, were noted along the surface epithelium. These change were

especially prominent in Group Ⅱ (normal animals receiving HCG and estrogen) and

Group Ⅲ (normal animals receiving HCG, estrogen and progesterone).

The lesion was characterized by focal epithelial proliferation, intracytoplasmic

vacuolation, dintraepithelial cyst formation, surrounding focal balloon cells and

an occasional enlarged nuclei. The content of vacuoles and cysts was an amorphous

material and/or hyaline droplets which were PAS positive even after diastase

treatment. The cytoplasm of the component cells was strongly positive for

methylgreen pyronin stain. However, no lipid was noted by the oil red 0 stain.

5. As seen through the electron microscope the cells of the lesion showed well

developed microvilli and contained and abundant rough endoplasmic reticulum and

large intracytoplasmic vacuoles. These vacuoles were filled with an abundant

secretion, an amorphous material with a moderate electron density similar to that

of the uterine lumen. This material was considered as mucopolysaccharides.

In summary, the Arias-Stella reaction which seemed to be metabolically active

cells was noted in the states associated with an elevated gonadotropic hormone

level which might be responsible for the pathogenesis of the Arias-Stella

sreaction. The author postulates that gonadotropic hormone produces an Arias-Stella

reaction through the stimulation of the ovary to secrete sex hormones, especially

estrogen.

[영문]

In 1954 Arias-Stella described a peculiar atypism of the endometrial glandularepithelium in cases associated with uterine abortion, hydatidiform mole,chorionepithelioma, syncytial endometritis and ectopic pregnancy. this atypism has

subsequently come to bear his name; the Arias-Stella reaction (ASR). Also ASR has been reported in caseof intrauterine pregnancy (Arias-Stella, 1959) and occasionally in cases treated with oral contraceptive pills(Azzopardi and Zayid, 1967) or clomiphene citrate (Bernhardt et al., 1966).

The ASR, which occurs focally in glandular and/or surface epithelium of the endometrium, consistsof enlargement of the gland cell, loss of cellular polarity, lack of orientation of the nucleus, nuclear hypertrophy with hyperchromasia and the presence of a voluminous vacuolated and foamy cytoplasm (Arias-Stella, 1954).

A wide variation in the incidence of ASR has been reported: 2.9% (Roach et al., 1960) to 100% (Frederiksen, 1959) in ectopic pregnancy; 0-81.3% in intrauterine abortion or pregnancy (Roach et al., 1960; Silverberg, 1972); and 24.3-34.6% in diseases of chorionic tissue (Arias-Stella, 1954; Roach et al., 1960). However, the presence of the ASR has been known to be helpful in differential diagnosis of diseases of pregnancy and of chorionic tissue (Arias-Stella, 1972).

Despite the well known characteristics and the clinical significance of ASR, only a few invesetigators have studied this lesion by the electron microscope. De Brux and Ancla(1964) observed a single case of ASR with electron microscope and they

described that the atypia consisted of "clear and dark cells" and the characteristic "clear cell" did not contain glycogen. Recently, Thrasher and Richart (1972) studied this lesion in 8 cases and stated that the ASR cells were in an active metabolic state since they contained abundant glycogen and rough endoplasmic reticulum in the cytoplasm.

There are many opinions as to the genesis and causes of ASR. It was previously thought to be secondary to an inflammatory reaction (Deelman, 1933). Arias-Stella (1954 and 1972) and many other authors believed that it was a hormonally induced, metabolically active secretory cells of endometrial gland due to the presence of chorionic tissue. Arias-Stella demonstrated an ASR-like lesion in rats by administration of gonadotropin with estrogen in normal animals and by a large amount of estrogen and progesterone in the castrated rats (1955). In contrast to

the above, some investigators claimed that ASR is an involutional, regressive and degenerative change which is related to fetal death or trophoblastic disorders (Lloyd and Fienberg, 1965).

As mentioned above, the ultrastructural nature and pathogenesis of the ASr are still very controversial among investigators.

This present study examined a large number of cases of ASR by histological, histochemical and electron microscopic methods, to study the cellular ultrastructure. It investigated the pathogenesis of ASR by comparing ASR-like lesions in rats, which lesions were induced by the administration of various combinations of hormones.

Materials and Methods

The study was divided into two parts: first the examination of human endometrial specimens and second the animal experiments.

1. Examination of human material

One hundred and one human endometrial tissues removed by curette were obtained from the Department of Obstetrics and Gynecology in Wonjoo Union Christian Hospital. Twenty eight of the tissues came from patients with intrauterine pregnancy, 10 had ectopic pregnancy, 1 had hydatidiform mole, 49 had a normal

menstrual cycle and 13 patients had other diseases of which specimens were discarded. A portion of each specimen was fixed in 4% glutaraldehyde, double-fixed in 1% phosphate buffered osmium tetroxide(pH 7.4), prepared as usual for electron microscopy, and examined by the Hitachi HU-11E electron microscope. The remainder

of each specimen was fixed in 10% neutral formain solution. The paraffin embedded sections were stained by hematoxylin and eosin, periodic acid-Schiff reaction with or without diastase treatment (PAS and dPAS) for glycogen and glycoprotein, and methylgreen pyronin for ribonucleic acid. A frozen section of formalin fixed tissue was stained by oil red O to detect lipid.

2. Experimental meterial

Female albino rats weighing around 200 gm. were used in the experiment divided and treated as follows;

Group Ⅰ: Pregnant animals

Group Ⅱ: Normal animals receiving human chorionic gonadotropin (HCG) and estrogen

Group Ⅲ: Normal animals receiving HCG, estrogen and progesterone

Group Ⅳ: Normal aniamls receiving HCG and progesterone

Group Ⅴ: Castrated animals receiving HCG and estrogen

Group Ⅵ: Castrated animals receiving HCG, estrogen and progesterone

Group Ⅶ: Castrated animals receiving estrogen and progesterone

Group Ⅷ: Normal and castrated controls

Each group consisted of 15 animals except Group Ⅷ which was composed of 10 normal and 10 castrated control animals.

The HCG(Gonadotropin, Upjohn) in a dosage of 100 I.U. daily per animal was injected subcutaneously. Estrogen (Progynon-DP, Korean Schering) and progesterone (Proluton, Korean Schering) were injected intraperitoneally in olive oil solution.

The respective dosage was 35 γ and 2 mg. daily per animal.

Under the ether anesthesisa 3 animals from each group were sacrificed after 3, 7, 10, 15, and 20 days of treatment and endometrial tissues were examined by the light and electron microscopes as the same way as the human tissues.

Results ans Summary

The histochemical and ultrastructural characteristics of human ASR were studied.

In addition, the histochemical and ultrastructural nature of the ASR-like lesion induced by various hormonal combinations in albino rats was studied.

The results of this study are summarized as follows:

1. The ASR changes were noted in the human endometrium in 17 of 28 intrauterine pregnancies (60.7%), in 6 of 10 ectopic pregnancies (60%) and in 1 case of hydatidiform mole. However, the atypism was not detected from the various stages in

the normal menstrual cycle.

2. The clearness of foamy appearance of the ASR cell was due to its abundant glycogen and to the small amount of mucopolysaccharides which were demonstrated by PAS and dPAS methods. In adddition, methylgreen pyronin stain revealed that the cytoplasm of the ASR cell contained many concentrated RNA granules in its supranuclear position. However, lipid was not regulary observed by the oil red O stain.

3. The characteristic ultrastructural features of the ASR cell included supranuclear parallel arrays of rough endoplasmic reticulum, an abundant collection of glycogen, diffusely scattered small elongated mitochondria and abundant microvilli on the luminal surface. These features were interpreted as an evidence that ASR cells were active secretory cells rather than degenerative or involutional cells.

4. In animal experiment to detect which hormone plasyed a major role in the pathogenesis of ASR, the specific changes (ASR-like lesion) which were intraepithelial cysts, were noted along the surface epithelium. These change were especially prominent in Group Ⅱ (normal animals receiving HCG and estrogen) and

Group Ⅲ (normal animals receiving HCG, estrogen and progesterone).

The lesion was characterized by focal epithelial proliferation, intracytoplasmic vacuolation, dintraepithelial cyst formation, surrounding focal balloon cells and an occasional enlarged nuclei. The content of vacuoles and cysts was an amorphous

material and/or hyaline droplets which were PAS positive even after diastase treatment. The cytoplasm of the component cells was strongly positive for methylgreen pyronin stain. However, no lipid was noted by the oil red 0 stain.

5. As seen through the electron microscope the cells of the lesion showed well developed microvilli and contained and abundant rough endoplasmic reticulum and large intracytoplasmic vacuoles. These vacuoles were filled with an abundant secretion, an amorphous material with a moderate electron density similar to that of the uterine lumen. This material was considered as mucopolysaccharides.

In summary, the Arias-Stella reaction which seemed to be metabolically active cells was noted in the states associated with an elevated gonadotropic hormone level which might be responsible for the pathogenesis of the Arias-Stella sreaction. The author postulates that gonadotropic hormone produces an Arias-Stella reaction through the stimulation of the ovary to secrete sex hormones, especially estrogen.