hum. reprod.-1999-pengdi-970-5

7/23/2019 Hum. Reprod.-1999-Pengdi-970-5

1/6

Human Reproduction vol.14 no.4 pp.970975, 1999

The effect of a levonorgestrel-releasing intrauterinedevice on human endometrial oestrogen andprogesterone receptors after one year of use

Zhu Pengdi, Liu Xiaoqun, Luo Hongzhi, Gu Zhao,Cheng Jie, Xu Ruhua, Lian Shizhu, Wu Shangchun,Wang Jiedong1

National Research Institute for Family Planning, Beijing, 100081,China

1To whom correspondence should be addressed

Thirty-four women bearing a levonorgestrel-releasing

intrauterine device, 20 g/day (LNG-IUD-20), for 1215

months were recruited. Endometrial biopsies were collected

during the late proliferative phase of the cycle (on cycle

days 1012) before (control) and after the use of the IUDfor 12 months, and assayed for oestrogen receptors (ER)

and progesterone receptors (PR). An immunohistochemical

technique with the peroxidaseantiperoxidase detection

system (PAP method) was employed. D75 and JZB39 were

the primary antibodies for ER and PR respectively. The

immunostaining semiquantitative analysis was performed

with a computerized microscope image processor, and

expressed as grey value. Both endometrial ER and PR

populations were significantly lower after insertion of the

IUD (P < 0.01) than in control biopsies. The intensity of

nuclear staining and the percentage of positively stained

cells for ER and PR in women with LNG-IUD were

each about 50% of those in control biopsies. The results

suggested that LNG released locally from the IUD has a

depressive action on the ER and PR, which may contribute

to the contraceptive effectiveness of this type of IUD and

also to the possible causes of LNG-IUD-induced irregular

bleeding and amenorrhoea.

Key words: endometrium/immunohistochemistry/levonorges-

trel-releasing intrauterine device/oestrogen receptor/progester-

one receptor

IntroductionA levonorgestrel-releasing intrauterine device (LNG-IUD)

developed in Finland has been in wide clinical use for more

than 15 years. Although its clinical performance, including

long duration of action, low pregnancy rate, reduced menstrual

blood loss and relief of symptoms of dysmenorrhoea is

encouraging, some side effects such as intermenstrual spotting

and amenorrhoea still cause problems for the users. Many

studies have suggested that the mode of action and side effects

of this IUD were, at least in part, based on the changes in

endometrial function and morphology that are produced by

the steroid liberated from the IUD (Nilsson et al., 1984;

970 European Society of Human Reproduction and Embryology

Barbosa etal., 1995; Gu etal., 1995; Luukkainen and Toivonen,

1995; Xiao et al., 1995).

The endometrium is the target of oestrogen and progesterone.

The steroid hormone may not be directly responsible for the

endometrial functional and morphological changes, as the

hormonal actions not only coincide with the plasma hormone

concentrations but also relate closely to the numbers of

endometrial oestrogen receptors (ER) and progesterone recep-

tors (PR). Some studies have demonstrated the effect of

progestin-releasing intrauterine devices on steroid receptor

expression in the endometrium (Janne and Ylostalo, 1980; Lu,

1991). These studies used radiochemical techniques to identifytotal ER and PR populations in whole endometrium, and did

not permit identification of the receptors in particular cell

types. In the present study we used immunohistochemistry to

describe the cellular localization of ER and PR in the endomet-

rium before and after insertion of a LNG-IUD. A semiquantit-

ative analysis was also employed with a computerized

microscope image processor, which was intended to render

results more objective, the main aim of the study being to

help in our understanding of the mechanism of the action and

side effects of the LNG-IUD.

Materials and methodsSubject selection

Thirty-four healthy women, aged between 23 and 35 years, with

proven fertility and regular menstrual periods were recruited for the

study. The women had no known systematic or gynaecological

disorder, had not received any hormonal therapy, steroid contraceptives

or intrauterine device for at least three cycles, and had no history of

abortion or pregnancy during the last 6 months before the start of

the study. After having obtained written informed consent from each

woman, a levonorgestrel-releasing intrauterine device (LNG-IUD)

was inserted into each subject for a period of 1215 months. The

study was approved by the Ethical Committee of the National

Research Institute for Family Planning, Beijing.

Intrauterine devices

The levonorgestrel-releasing IUD were provided by Leiras Company

(Turku, Finland). Each device contains 46 mg levonorgestrel, and

releases approximately 20 g/day. The effective lifetime of the IUD

is expected to be 7 years.

Sample collection

An endometrial biopsy was taken from the anterior or posterior wall

of the mid uterus on cycle days 1012, just before insertion of the

IUD, and served as the control representing the proliferative phase

of the normal menstrual cycle. One year later, immediately after

removal of the IUD on cycle days 1012, a second biopsy was taken.

In women with amenorrhoea who bore the IUD, the device was


2/6

Effect of levonorgestrel IUD on steroid receptors

removed on any day after 12 months of use, and a biopsy taken at

the time of removal. No biopsy could be obtained in 16 cases because

of endometrial atrophy.

Specimen preparation

A small portion of the endometrial specimen was fixed in Bouins

solution and paraffin sections were stained with haematoxylin and

eosin (H&E) for morphology and dating. The major portion of the

specimen was rinsed immediately in cold normal saline, frozen inliquid nitrogen, and stored at 70C until analysed.

Immunohistochemistry

Monoclonal antibodies D75 to human ER and JZB39 to human PR

were the generous gifts of Dr G.L.Greene, University of Chicago. A

peroxidaseantiperoxidase method was employed (Wanget al., 1992),

the frozen serial sections of 5 m thickness being fixed in picric

acidparaformaldehyde for 15 min, then immersed in 0.5% hydrogen

peroxide solution for 15 min to block endogenous peroxidase activity.

After washing the sections in phosphate-buffered saline (PBS),

primary antibodies (rat monoclonal antibody to human ER, rat

monoclonal antibody to human PR respectively), secondary (bridge)

antibody (rabbit anti-rat IgG), and rat peroxidaseantiperoxidase

complex were applied to sections sequentially. Between each step thesection was rinsed in PBS. The formation of an immunoprecipitate

in the section was demonstrated with 3,3-diaminobenzidine (Serva,

Heidelberg, Germany). One of the serial sections in each case was

treated with normal rat IgG instead of the primary antibody to

serve as a negative control. A section from receptor-positive tissue,

processed using the same procedure, served as a positive control in

each assay. No sections were counterstained, as this procedure is

thought to reduce the sensitivity of the image analysis system in

terms of recording the integrated optical density of the nuclei.

Analysis of endometrial immunoreactivity

In order to assess the effect of LNG-IUD (in terms of ER and PR),

two types of scoring system were used.

Subjective score: the immunostaining intensity of all tissue sectionswas scored on a five-point scale, where 0 no staining, 1 mild,

2 moderate, 3 high and 4 intensive staining. The recorded

immunostaining score was based on the intensity of staining expressed

by the majority of cells in each section. In brief, 310 areas were

chosen randomly in each section according to the size of the sample.

In each area (at 200 magnification), the percentage of stained cells

of each scale was estimated by eye. The score of each area was

obtained as (IIP), where I is the intensity of staining from 04 and

P is the corresponding percentage. The mean value of all areas gives

the score of the section. All samples were evaluated by the same

observer to rule out interobserver variability.

Objective score: the immunostaining semiquantitative analysis was

performed with a computerized microscope image processor. The

method was described previously by Zhuet al. (1995). Sections wereanalysed in blind fashion (Olympus BH light microscope, 200

magnification). In each case, about 30 positive and negative nuclei

at the level of stroma and glandular epithelium respectively, were

evaluated and expressed as a grey value. The only selection criterion

was the absence of superimposition of the nuclei to obtain the real

optical density. The background was measured at the same time for

each cell in the area immediately outside the cell, in order to eliminate

batch or area differences in staining intensity. The score of each cell

was expressed as background minus nuclear values.

Data management and statistical analyses

The grey values of the endometrial cells (stromal and glandular cells)

in each subject were measured with minicomputer, VAXII, SAS-

971

language Program. Statistical analysis was performed, results were

evaluated with a t-test, and significant differences between the pre-

and post-insertion data were calculated.

Results

Histologically, the cellular components of the endometrium

could be clearly subdivided into glands and surrounding stromacells comprising the vasculature. Samples taken before IUD

insertion showed the morphology of the proliferative phase

(Figure 1a) according to the criteria of Noyes et al. (1950)

and Johannisson et al. (1987). Samples from the IUD users

displayed a pattern of uniform suppression (Figure 2a), the

glands being scarce in number and very small. The stroma

showed pseudodecidual features.

Immunoreactivity of endometrial ER and PR in control

samples

As shown in Figure 1b and c, both ER and PR were localized

in the nuclei of epithelial and stromal cells. Luminal and

glandular epithelial cells showed strong staining in nearly all

of their nuclei, and stromal cells were strongly stained in most

nuclei. The glandular cells were slightly more heavily stained,

the percentage of stained cells being greater than that in

stromal cells. The intensity and percentage of cell nuclei

staining for PR were greater than those for ER (Figure 1c;

Table I). Many of the nuclei contained a small focal area,

interpreted as a nucleolus, which was unstained. In contrast to

the densely stained nuclei, the cytoplasm and intercellular

spaces were unstained. Endometrial vascular smooth muscle

cell nuclei and endothelial cell nuclei were consistently

unstained. Results obtained from the two scoring systems were

parallel (Table I). The percentages of stained cells for ER andPR were not shown.

Immunoreactivity of endometrial ER and PR in LNG-IUD

users

By using a series of adjacent sections, it was shown that

ER and PR immunostaining remained in the nuclei of the

endometrial cells, as before. There was a substantial reduction

(P 0.01) in both the intensity of nuclear staining and the

proportion of cells stained for ER and PR (Figure 2bd) in

comparison with the control (Figure 1bd). The immuno-

staining in most cases was almost eliminated, with only

occasional isolated cells showing a variable amount of staining.

However, these occasional isolated cells were quite strongly

stained and contrasted sharply with the minimal staining in

adjacent cells. The nuclear staining intensity and percentage

of stained cells in the gland and stroma were similar (P0.05),

but the stromal cells were slightly more intensely stained and

the percentage of stained cells was higher than that of the

glandular cells only for ER. During this post-insertion period,

the intensity and percentage of cell nuclear staining for PR

remained slightly greater than that for ER as in the pre-

insertion period, though no significant difference was found

(Table I). The endometrial vascular cells remained unstained,

as in the controls.


3/6

Z.Pengdi et al.

Figure 1. Light micrographs of endometrial sections for the immunoreactivity of oestrogen receptors (ER) and progesterone receptors (PR)in IUD users. Before insertion, at cycle day 12. ( a) A haematoxylin and eosin-stained section shows a normal late proliferative phaseendometrial pattern. Epithelial cells and the stromal cells in the upper functionalis exhibit strong immunoreactivity for ER ( b) and PR (c)with the specific nuclear staining using D75 and B39 as primary antibodies (PAP method). No specific cytoplasmic staining was observed.No immunoreactivity for PR or ER was detected in the vascular endothelial cell nuclei and smooth muscle cell nuclei. ( d) Background for

PR (ER not shown) in the control section. Scale bars 100 m.

Discussion

A number of studies have defined the location and distribution

of ER and PR in the endometrium (Lessyet al., 1988; Snijders

et al., 1992; Bergqvist et al., 1994). The expression of ER and

PR in the endometrium changes cyclically, although some

differences exist among the reports. In general, ER and PR

are located in the nuclei and the positive rate and degree of

expression peak during the mid cycle and then decrease.

Progestins downregulate this expression (Natrajanet al., 1982).

In this study, using an immunohistochemical method, we

showed that the staining for endometrial ER and PR in LNG-

IUD users was rather similar to the pattern of the mid to

late secretory phase of the normal menstrual cycle reported

previously (Lessy et al., 1988; Snijders et al., 1992; Bergqvist

et al., 1993). There was a substantial reduction in both the

intensity of the nuclear staining and the proportion of the cells

stained for ER and PR in comparison with controls. The

released LNG caused the downregulation of both ER and PR,

which is in agreement with other studies as the populations

of both endometrial oestrogen and progesterone cytoplasmic

receptors were reduced significantly after insertion of a LNG-

IUD (Janne and Ylostalo, 1980; Lu, 1991).

However, the LNG which is released locally from the IUD

972

does not mimic the effect of physiological progesterone on the

endometrium. The endometrium of LNG-IUD users displayed a

pattern of uniform depression. In the IUD users, the endometrial

steroid receptor population was uniformly decreased independ-

ent of the cell types, while in the normal cycle it was cell-

specific, there being a major difference between epithelial and

stromal cells. Interestingly, Critchley et al. (1993) reported

that the immunoreactive PR population remained high in the

endometrium of Norplant users, whereas the population of ER

was relatively low. These findings are significantly different

from those of the present study, and may suggest that the

effect of LNG released locally on the endometrium is different

from that of circulating LNG. Moreover, Lau et al. (1996)

showed an increase in immunoreactive PR population, but a

reduction in PR mRNA levels in Norplant endometrium, while

in the normal menstrual cycle the protein is consistent with the

mRNA. It would be interesting to investigate the endometrial

expression of isoforms of ER and PR in both LNG-IUD users

and Norplant users.

In this study, the endometrial blood vessels were not stained

in either controls or after insertion of the LNG-IUD. However,

conflicting data have been obtained regarding this issue. Lessy

et al. (1988) demonstrated no immunostaining at all, whereas


4/6


Figure 2. Light micrographs of endometrial sections stained for the immunoreactivity of ER and PR in IUD users. Post-insertion; cycle day12. (a) A haematoxylin and eosin-stained section showing typical uniform suppression of endometrium. A substantial reduction in both theintensity of nuclear staining and the proportion of cells stained for ER ( b) and PR (c) in comparison with the control. Specific cytoplasmicstaining was not observed. The endometrial endothelial cells and vascular myometrial cells remained unstained for ER and PR, as in thecontrol. (d) Background for PR (ER not shown) was shown in the control section by using normal non-immunized rat immunoglobulin in

place of monoclonal anti-receptor antibody, B39.

others reported the presence of both ER and PR in vascular

smooth muscle cells of endometrial blood vessels (Perrot-

Applanatet al., 1988, 1994). A earlier study in this laboratory

reported immunostaining for PR in the endothelial cells from

human decidua (Wang et al., 1992), while Perrot-Applanat

et al. (1988, 1994) took tissue at the end of the luteal phase

or during pregnancy. It is possible that endometrial blood

vessels express the receptors when endometrium becomes

decidualized. The endometrium of LNG-IUD users also showed

a pseudodecidual pattern, but this was uniformly and heavily

depressed, including the vessels which were small with a thin

wall that was only single-layered in most cases. In addition,

combining results of the present study with those reported

previously (Lessy et al., 1988; Snijders et al., 1992; Critchley

et al., 1993; Bergqvist et al., 1994; Lau et al., 1996), it is

likely that locally released LNG affects the endometrium in a

different manner from other circulating progestins. However,

Rogers et al. (1996) demonstrated highly varied ER and PR

immunoreactivity in the vascular smooth muscle cells of

endometrial blood vessels, ranging from 0% to 85%, with a

tendency towards greater immunoreactivity in the late secretory

phase. We could not find an adequate explanation for the

difference between our results and those of Rogers et al.

973

(1996), who identified receptors as being present in blood

vessels throughout the menstrual cycle, albeit with high vari-

ability. However, the fact that the antibodies used in these

studies were different might provide an explanation.

Many studies suggested that the changes in endometrial

function and morphology are caused mainly by the local

effect of progestin on the endometrium (Nilsson et al., 1984;

Luukkainen and Toivonen, 1995). Previous studies of ovarian

function in LNG-IUD users have suggested that the majority

of women studied had ovulatory cycles (Nilsson et al., 1984;

Barbosaet al., 1995; Xiaoet al., 1995). The intrauterine release

of levonorgestrel results in high local tissue concentration in

the endometrium (Nilsson et al., 1982). Oral administration of

a dose 10-fold higher than the daily dose administered with

LNG-IUD resulted in endometrial concentrations of levonor-

gestrel which were similar to those in adjacent tissues, and

the effects on the endometrium were correspondingly much

weaker than with locally released LNG (Nilsson et al., 1982).

Many LNG-IUD usersthough none of the LNG intracervical

device usersdeveloped amenorrhoea, which confirms the

local effect of LNG-IUD (Kurumaki et al., 1984).

Several studies have indicated that endometrial morphology

in women bearing a LNG-releasing IUD shows profound and


5/6

Z.Pengdi et al.

Table I. The immunoreactivity score of endometrial cells for oestrogen andprogesterone receptors (arbitrary units) before and after insertion ofLNG-IUD-20. Values are mean SD

Index Stroma cells Epithelial cells

Scorea Grey valueb Scorea Grey valueb

Oestrogen receptor

Before 2.97 0.17 14.45 1.80 2.94 0.24 15.25 1.12(n 34) (n 34) (n 34) (n 34)

After 1.69 0.48* 8.09 1.35* 1.43 0.79* 8.02 1.97*(n 16) (n 16) (n 7) (n 7)

Progesterone receptor

Before 3.62 0.49 17.55 0.97 3.74 0.45 17.98 1.20(n 34) (n 34) (n 34) (n 34)

After 1.43 0.51* 8.66 1.81* 1.80 0.79* 9.80 3.73*(n 14) (n 14) (n 10) (n 10)

*P 0.01 versus control.aSubjective quantification.bObjective grey value.

uniform suppression which is independent of the stage of the

menstrual cycle (Zhu et al., 1989; Gu et al., 1995). Locally

released levonorgestrel causes atrophy of the endometrium,

such that the glands are scarce and the stroma shows pseudo-

decidual features. This effect on the endometrium is seen as

early as 1 month after insertion, and is maintained as long as

levonorgestrel is released. The mechanism is likely to be a

complex process (Hsueh et al., 1976), and many factors may

be involved. For example, the decidualized cells produce

insulin-like growth factor binding protein-1, which can block

insulin-like growth factor I that is thought to be a mediator of

oestrogen-induced mitotic effects (Pekonenet al., 1992). From

the present data, we might suggest that the continuous highconcentration of locally released LNG inhibits the expression

of ER, which in turn makes the endometrium insensitive to

circulating oestradiol and provokes an anti-proliferative effect

during the use of LNG-IUD. Thus, endometrial proliferative

activity is totally arrested, and this is thought to be the main

contraceptive action of the IUD.

Taken together, the results of the present study indicate that

there is a statistically significant decrease of ER and PR in

the endometrium of LNG-IUD users, when compared with

controls, and confirms that progesterone downregulates its

receptor. However, the decrease of ER and PR populations in

LNG-IUD users was somewhat uniform, and not cell-specific

a situation which is different from that in the normal menstrual

cycle (Lessyet al., 1988; Snijderset al., 1992; Bergqvistet al.,

1994). In addition, the immunoreactivity of ER and PR in the

LNG-IUD endometrium is significantly different from that in

the Norplant endometrium (Critchley et al., 1993; Lau et al.,

1996), though it is unclear why such differences exist. However,

the data acquired in the present study show that 16 out of 34

subjects failed to produce a tissue samplea proportion similar

to that seen in Norplant users (Hadisputra et al., 1996). As,

therefore, data are unavailable from these patients, the samples

that were collected may not be truly representative, and so

care must be taken when interpreting our results.

974

Acknowledgements

The authors are highly appreciative of the gifts of LNG-IUD andanti-receptor monoclonal antibodies from Prof. Luukkainen of LeirasCompany, Turku, Finland, and Dr G.L.Greene of Ban May Institute,University of Chicago, USA. We are also very grateful to Dr ElisabethJohannisson, LACQ, Geneva for scientific advice and revision of themanuscript. The present study was supported by WHO SpecialProgramme of Research, Development and Research Training in

Human Reproduction, Geneva, Switzerland.

References

Barbosa, I., Olsson. S.E., Odlind, V. et al. (1995) Ovarian function after sevenyears use of a levonorgestrel IUD. Adv. Contracept.,11, 8595.

Bergqvist, A. and Ferno, M. (1993) Oestrogen and progesterone receptors inendometriotic tissue and endometrium: comparison of different cycle phasesand ages. Hum. Reprod.,8 , 22112217.

Critchley, H.O.D., Bailey, D.A., Au, C.L. et al. (1993) Immunohistochemicalsex steroid receptor distribution in endometrium from long-term subdermallevonorgestrel users and during the normal menstrual cycle. Hum. Reprod.,8, 16321639.

Gu, Z., Zhu, P.D., Luo, H.Z. et al. (1995) A morphometric study on theendometrial activity of women before and after one year with LNG-IUDin situ. Contraception, 52, 5761.

Hadisaputra, W., Affandi, B., Witjaksono, J. and Rogers, P.A.W. (1996)Endometrial biopsy collection from women receiving Norplant. Hum.Reprod., 11 (suppl. 2), 3134.

Hsueh, A.J., Peck, E.J., Jr and Clark, J.H. (1976) Progesterone antagonism ofthe estrogen receptor and estrogen-induced uterine growth. Nature, 254,337338.

Janne, O. and Ylostalo, P. (1980) Endometrial estrogen and progestin receptorsin women bearing a progesterone-releasing intrauterine device.Contraception , 22, 1923.

Johannisson, E., Landgren, B.M., Rohr, H.P. and Diczfalusy, E. (1987)Endometrial morphology and peripheral hormone levels in women withregular menstrual cycles. Fertil. Steril., 48 , 401408.

Kurumaki, H., Toivonen, J., Lahteenmaki, P. and Luukkainen, T. (1984)Pituitary and ovarian function and clinical performance during the use of alevonorgestrel-releasing intracervical contraceptive device. Contraception,29, 3143.

Lau, T.M., Witjaksono, J. and Rogers, P.A.W. (1996) Progesterone receptor

in Norplant endometrium. Hum. Reprod., 11 (suppl. 2), 9094.Lessy, B.A., Killam, A.P., Metzger, D.A. et al. (1988) Immunohistochemical

analysis of human uterine estrogen and progesterone receptors throughoutthe menstrual cycle. J. Clin. Endocrinol. Metab., 67 , 334340.

Lu, S.M. (1991) Effect of levonorgestrel intrauterine device on humanendometrial estrogen and progesterone receptors. Chin. J. Obstet. Gynecol.,26, 293294.

Luukkainen, T. and Toivonen, J. (1995) Levonorgestrel-releasing IUD as amethod of contraception with therapeutic properties. Contraception, 52,269276.

Natrajan, P.K., Muldoon, T.G., Greenblatt, R.B. and Mahesh, V.B. (1982) Theeffect of progestins on estrogen and progesterone receptors in the humanendometrium. J. Reprod. Med., 27 , 277230.

Nilsson, C.G., Hankkamaa, M., Vierola, H. and Luukkainen, T. (1982) Tissueconcentrations of levonorgestrel in women using a levonorgestrel-releasingIUD. Clin. Endocrinol. (Oxford), 17 , 529536.

Nilsson, C.G., Lahteenmaki, P. and Luukkainen, T. (1984) Ovarian function inamenorrhoeic and menstruating users of levonorgestrel-releasing intrauterinedevices.Fertil. Steril., 41 , 5255.

Noyes, R.W., Hertig, A.T. and Rock, J. (1950) Dating the endometrial biopsy.Fertil. Steril.,1 , 325.

Pekonen, F., Nyman, T., Lahteenmaki, P. et al. (1992) Intrauterine progestininduces continuous insulin-like growth factor-binding protein production inthe human endometrium. J. Clin. Endocrinol. Metab.,75 , 660664.

Perrot-Applanat, M., Groyer-Picard, M.T., Garcia, E. et al. (1988) Immuno-cytochemical demonstration of oestrogen and progesterone receptors inmuscle cells of uterine arteries in rabbits and humans. Endocrinology,123 ,15111519.

Perrot-Applanat, M., Deng, M., Fernandez, H. et a l. (1994) Immuno-histochemical localisation of estradiol and progesterone receptors in humanuterus throughout pregnancy: expression in endometrial blood vessels.

J. Clin. Endocrinol. Metab., 78, 216224.


6/6


Rogers, P.A.W., Lederman, F., Kooy, J. et al. (1996) Endometrial vascularsmooth muscle oestrogen and progesterone receptor distribution in womenwith and without menorrhagia. Hum. Reprod., 11, 20032008.

Snijders, M.P.M.L., de Goeij, A.F.P.M., Debets-Te Baerts, M.J.C. et al. (1992)Immunocytochemical analysis of oestrogen and progesterone receptors inthe human uterus throughout the menstrual cycle and after menopause.

J. Reprod. Fertil., 94, 363371.

Wang, J.D., Fu, Y., Zhu, P.et al. (1992) Immunohistochemical localization ofprogesterone receptor in human decidua of early pregnancy. Hum. Reprod.,7, 123127.

Xiao, B.L., Zeng, T., Wu, S.C. et al. (1995) Effect of levonorgestrel-releasingintrauterine device on hormonal profile and menstrual pattern after longterm use. Contraception, 51, 319365.

Zhu, P., Luo, H., Xu, R. et al. (1989) The effect of intrauterine device,stainless steel ring, the copper T220, and releasing levonorgestrel, on thebleeding profile and the morphological structure of the human endometrium:a comparative study of three IUDs. A morphometric study of 96 cases.Contraception , 40, 425438.

Zhu, P.D., Liu, X.Q., Wang, J.D. et al. (1995) Analysis of endometrial FactorVIII activity in normal proliferative phase by digital image processingsystem. Contraception, 52, 2327.

Received on April 27, 1998; accepted on December 23, 1998

975

hum. reprod.-1999-pengdi-970-5

Documents