DOI 10.1515/hmbci-2013-0002      Horm Mol Biol Clin Invest 2013; 13(2): 19–40

Joaquín Cortés-Prieto*, Maria Lina Vicente-Hernanz, Ana Cortés-García, Jerry Keller, Rosario Cintas del Rio and José Luis Brita-Paja Segoviano

Hormone replacement therapy: evolution of body mass index, bone mineral density, and lipid profile Abstract: One hundred and eighty-five female never-hormone users with a mean age of 50.71 (SD = 5.58) years upon initiation of treatment were studied before instauration and during treatment. The profile of patients subjected to the study included body mass index (BMI), bone mineral density (BMD), and lipid profile (LP). In our population, pretreatment values were within the normal range, which is why a hormone replacement therapy (HRT), conjugated equine estrogen plus medroxyprogesterone acetate, was administered to a healthy population of women for 9.82 ± 5.42 years and follow-up comprised 1815.84 womanyears. To study the effects of treatment on the investigated clinical subjects, we resorted to comparative analyses of pretreatment and posttreatment mean values: descriptive and comparative hypothesis, followed by a repeated measurements design to investigate the evolution on three levels according to age groups just before the initiation of the treatment and on the other three levels during the treatment with a minimum follow-up of 9 years. On the basis of the numerical results, we were able to conclude that BMD decreased significantly (p = 0.00) well above the values found on curves corresponding to these age groups. The total cholesterol decreased significantly at 2–3  years interval (p = 0.04). The rest of the LP factors remained within a safe margin. Therefore, long-term HRT may be considered initially as a beneficial alternative in the treated study population. Keywords: body mass index; bone mineral density; hormone replacement therapy; lipid profile; long HRT. *Corresponding author: Prof. Joaquín Cortés-Prieto, c/ Monasterios de Suso y Yuso 67, D, 4°, 28049 Madrid, Spain, Phone: +34-91-750-8026, Cellular: 610576283, E-mail: [email protected] Joaquín Cortés-Prieto, Ana Cortés-García and Jerry Keller: Gynecology Department of Medical Specialities, Medical School, University of Alcala, Alcala, Spain Maria Lina Vicente-Hernanz, Rosario Cintas del Rio and José Luis Brita-Paja Segoviano: Department of Statistics and Operational Research III, School of Statistics, Complutense University of Madrid, Madrid, Spain

Introduction In our previous paper [1] on hormone replacement therapy (HRT), we have analyzed in depth the observed main clinical events accompanying the described treatment [2–7]. In particular, we focused our attention on subjects with a breast cancer diagnosis or those who presented with a cardiovascular event (CV) during the study, without excluding other minor clinical events. Notwithstanding, in clinical terms, other parameters such as body mass index (BMI), bone mineral density (BMD), and the lipid profile (LP) are known to influence the results of this type of research. For that reason, we have included in our study the corresponding evolutions and interpretations of the results that were gathered throughout the treatment. The completion of a long-duration treatment and a thorough analysis of the results gave us important information about the possible effects of these parameters during hormone administration. The study on the overall body response induced by the medication can be assessed, at least partially, from the ensuing BMI data, and with regard to fractures, whether spontaneous or not, the total response can be effectively analyzed by monitoring BMD. Lastly, LP variations, which may be related at least partially to the incidence of CV events, hypertension, and thrombophlebitis, are also helpful in overall analysis. Combined continuous estrogen-progestin replacement therapy, in our case, conjugated equine estrogen (CEE) always in combination with the opposed progestin medroxyprogesterone acetate (MPA), was prescribed throughout the prolonged oral administration. Browsing through the specialized literature, we can often find comments about the benefits and risks of HRT in relation to clinical parameters, as indicated in the preceding paragraph. When reviewing the literature and trying to focus on results corresponding to the employed HRT treatment, we logically sought specific data but these are very scarce, apart from the fact that in most publications the follow-up periods are quite short with a predominance of year-long studies and an equally limited number of studied subjects.

20      Cortés-Prieto et al.: Hormone replacement therapy The exclusion of some of the consulted bibliographic references does not mean a lack of confidence in the published materials but merely refers to an observation that they do not include specific values that cannot be compared objectively against our findings. Hence, the aim of our study is to contribute to the most relevant literature by reporting the evolution of our clinical results derived from women orally treated with CEE plus MPA over a followup of 1815.84 woman-years. All the women subjected to the study were adamantly advised at the beginning and during the follow-up about the importance of diet, physical exercise, and self-determination. The collaboration between two teams, one from the gynecology department and another from the statistics department, allowed to maximize the analysis of the studied material on the basis of the obtained results, and its outcome is presented in this article for the general consideration of all concerned.

Materials and methods Population One hundred and eighty-five female never-hormone users who were prescribed HRT in our private clinic practice were studied before and during treatment, recording family and personal histories, gynecological and breast examinations, BMI, BMD, and LP. Clinical events occurring throughout the follow-up were duly scrutinized. The follow-up lasted from October 1991 to July 2010. The major general characteristics of the studied population were as follows: menarche in 184 women at an average age of 12.66 (SD = 1.40) years, with a range from 9 to 16 years, with one primary amenorrhea; pregnancies in 146 (78.91%) and none in 39; and with lactation in 126 (68.10%). In the initial reference publication [1], one can find exhaustive information dealing with the subject, which for the sake of clarity needs to be considered; however, at present, it spares repetition of details and concepts. Conventional or mandatory gynecological and breast examinations (regular X-ray and nuclear magnetic resonance) were carried out before and during treatment, as well as BMI, BMD, and LP measurements. BMI was measured during scheduled consultations, most of the time before the BMD, irrespective of the type of system used for this assessment, while performing blood sampling for the measurements of LP before the appointment. BMI is a simple index of weight-for-height that is commonly used to estimate underweight, overweight, and obesity in adults and is influenced by diet and exercise. It is defined as the weight in kilograms divided by the square of the height in meters (kg/m2). This definition was agreed on as the most operational during the Symposium of International Congress on Physical Activity, Fitness and Health, held in Toronto in 1992. The above measurements were evaluated two or more times, considering their means as valid values. The height measurements were done using the same procedure in all women; that is, barefoot;

erect with the back of the subject against the meter bar; weight evenly distributed over both feet with tightly clenched heels and knees; toes separated in 60° angle; back straight; arms relaxed along the body; head, shoulder blades, buttocks, and heels in contact with the vertical plane; horizontal Frankfort plane as an imaginary line inferior to the orbital crest in the same horizontal plane as the ear canal. The measuring lip of the meter is lowered gently, slightly brushing the hair and maintaining contact with head’s occipital region and relative to the length of the measuring bar located immediately behind. The readings on the measuring scale were taken to the nearest 0.5 cm. In continuation, the subjects were weighed on the scale that forms the part of the measuring equipment. BMI was measured in 155 women at the beginning of the treatment from the total of 185 women included in the study. The mean values obtained during this procedure were 24.68 (SD = 3.59) kg/m2 in 59.89% of the cases (normal range-underweight) and >24.68  kg/m2 (overweight) in 40.14% of the cases. BMD was evaluated by means of osteosonography and the mean ultrasound velocity method through the distal metaphyses of the proximal phalanx of the second through the fifth finger of the non-dominant side (bone digital measurement; DBM Sonic 1200, Igea, Italy). Sound velocity is defined as the transmission capability of ultrasound from a transmitter to a receiver through the bone tissue, i.e., Ad-SOS (amplitude-dependent speed of sound, in m/s), through the proximal phalanx. The measured value was estimated on a curve plotted by the machine manufacturers with maximum, minimum, and average values, all of which decrease with advancing age. We used the t-score to assess the deviation that exists in the Ad-SOS (m/s) relative to a pattern of young postmenopausal women (usually 20–30 years before the most probability of integrity of ovarian function). The z-score was used in women > 70 years of age. For this calculation, the t-score is the individual value found less the average pattern of young women, divided by the standard deviation value (pattern of normality: 2151 ± 39 m/s). The z-score is the value of the studied individual minus the mean value of normal women of the same age, divided by the standard deviation of the value of normal women of the same age. There are other techniques for measuring BMD: (a) using ultrasound that penetrates across the calcaneus, and (b) analyzing the lumbar spine (L2–L4) as measured by dual-energy X-ray absorptiometry, then evaluating the t-score: –2.5 considered osteoporosis; between –1.0 and –2.5 diagnosed as osteopenia. We opted to discard these last techniques and only consider the measurement obtained using DBM Sonic 1200. The measurements obtained using the (a) and (b) techniques were found insignificant; thus, we excluded them from this study despite the available equations for converting the values obtained with these techniques into the finger measurements corresponding to the ultrasound method. Pretreatment values for BMD measured with the ultrasound finger method, DBM Sonic 1200, obtained from 58 of the studied 185 women had yielded a mean of 2097.87 (SD = 100.22) Ad-SOS (m/s). LP measurements were done following an animal fat-free meal in the preceding 48 h, and 12 h fast before blood extraction. The measurements included total cholesterol (T Chol), high-density lipoprotein cholesterol (HDL Chol), low-density lipoprotein cholesterol (LDL Chol), and triglycerides (TG), expressed in mg/dL. The atherogenic index (AI) was taken as the ratio between T Chol and HDL Chol. The mean pretreatment values of the various considered entities are reported in Table 1.

Cortés-Prieto et al.: Hormone replacement therapy      21

Table 1 Pretreatment values of the investigated entities. Entities

Valids

BMI BMD T Chol AI LDL Chol/HDL Chol index TG

155 58 163 156 156 163

Mean (SD) 24.68 (3.59) kg/m2 2097.87 (100.22) Ad-SOS m/s 224.75 (44.86) mg/dL 3.40 (1.04) mg/dL 2.20 (0.93) mg/dL 95.36 (41.21) mg/dL

of repeated measurements, which we called “repeated evolutionary control”. Whenever the sphericity conditions and equal variancecovariance matrices could be satisfied, we performed the corresponding univariate F statistic based on Snedecor. In cases where such hypotheses were not acceptable, we resorted to contrasts based on multivariate statistics (Traza de Pillai, Wilks’ lambda, and Hotelling’s trace).

Results Treatment All the women included in the study were prescribed HRT. They had never been treated with hormone replacement of estrogens and they were in an appropriate health condition for initiating HRT administration. The study group of 185 women was treated continuously with a combination of CEE+MPA throughout a long-term oral administration. CEE was always administered at a dosage of 0.625 mg/day and together with the opposed MPA. This progestin was administered from 1991 until 1995 at a dosage of 2.5 mg/day, and from 1995 at 5  mg/day in accordance with the recommendation at the American Fertility Society Meeting in Seattle. CEE and MPA were always administered together, with the exception of two cases with previous breast cancer surgeries where only MPA was prescribed. Treatment was administered according to two patterns: (a) perimenopausal women from day 1 to 25 of the cycle, and (b) postmenopausal women from Monday to Friday. No further treatment was prescribed nor specific calcium supplements or other medications known to affect the skeleton were administrated. In addition to individual explanations of the effects of hormone treatment, all women were provided with exhaustive information about the importance of diet (low-fat dietary patterns), as well as physical exercise and self-determination.

Statistical analysis Statistical analysis was performed using SPSS 20.0. To reach conclusions from our studied data, we first performed a comparative analysis of pretreatment and posttreatment values for each entity included in our study. We used descriptive and hypothesis test methods for overall statistical analysis. We performed final assessment including final risk when it was relevant, as well as the assessment differential against the final difference vs. the pretreatment values or in the case of BMD, the differences between measured and estimated values according to the curve with respect to pretreatment values. By not reporting previously on all of the above in a precise manner and concerning the parameters’ evolution throughout treatment, we have decided to employ other methods that could provide more information about the outcome of the treatment, as now there are enough data to draw broader statistical conclusions. Therefore, in view of all the above, we chose consecutive periods – regarding the total duration of treatment – that could be compared with each other in their development and also depending on the age at the onset of treatment. That is, we designed a model

The total duration of the follow-up was calculated on the basis of the period ranging from the respective initiation of treatment to the last general follow-up checkups (July 2010). Cases with temporary stops were included in the overall duration of follow-up by subtracting these stops to obtain the net treatment duration (Table 2). The mean treatment duration was 9.82 ± 5.42 years for all of the women (0.11–18.53 years). According to the literature, and in most publications, comparisons of the results deal with only 1 year of administration of HRT, whereas we have followed through for much longer periods. Furthermore, we present our results in the form of an evolutionary study conducted on each of the entities included in the investigation. It is also important to stress that there were no documented deaths during the entire follow-up period from October 1991 to July 2010 (1815.84 woman-years) despite the severity of the pathologies found in several cases.

Comparison of pretreatment and posttreatment values Table 3 presents the posttreatment values of clinical variables analyzed for their comparison with the pretreatment values (Table 1). Table 4 presents the percentage distributions before and after treatment of the analyzed clinical entities. As far as BMI is concerned, the underweight and normal weight levels have experienced a slight decrease, whereas overweight and obese levels have experienced an increase. As far as BMD is concerned, one can observe a large increase of low level and a very patent decrease of the normal level. Finally, observing the behavior of the LP, it can be seen that Table 2 Duration of follow-up (woman-years). Groups

All women

Cases

Total duration

Temporary stop

Net treatment duration

185

1815.84

31.67

1784.17

22      Cortés-Prieto et al.: Hormone replacement therapy

the T Chol plus AI and LDL Chol/HDL Chol plus TG had improved with treatment, as there was an increase in the percentage of patients with normal levels of the three measurements, while decreasing in that which corresponds to the risk level. Table 5 shows the results of the tests that were carried out in order to detect significant differences in the mean levels of the considered variables as measured before and after treatment. The results achieved in contrasts led to the following conclusions: 1. There were significant differences (at any level of those usually considered, p = 0.00) between the average levels measured before and after BMI treatment. An increase of the average value of this index with p = 0.0005 can be accepted. Figures 1 and 2 present results that are consistent with these findings. Figure 1 represents the final assessment of BMI reflecting treatment duration in years and its correspondence with BMI. In its pretreatment distribution, we can observe 59.89% of underweight women with BMI < 25  kg/m2 and 40.11% overweight women (BMI > 25 kg/m2). In each graph, whenever included, the numbers correspond to the age of each woman at the time of

Table 3 Posttreatment values. Entities BMI BMD T Chol AI LDL Chol/HDL Chol index TG

Valids 155 58 165 156 156 163

Mean, SD 25.33 (3.88) kg/m2 2079.5 (72.64) Ad-SOS, m/s 215.47 (34.53) mg/dL 3.28 (0.85) mg/dL 1.99 (0.72) mg/dL 98.69 (39.69) mg/dL

measurement. Taken as being the final assessment, both coordinates refer to the final measurement. The distribution reflected in Figure 1 respects the intervals of the World Health Organization (WHO), whose classification is reproduced below in order to inform about this topic, as frequently the limits shown in the world literature are not always the same. The international classification of adult underweight, overweight, and obesity according to BMI is as follows: BMI principal cut-off points, kg/m2

Classification

< 18.50 18.50–24.99   ≥  25.00   ≥  30.00 30.00–34.99 35.00–39.99   ≥  40.00

Underweight Normal range Overweight Obese Obese class I Obese class II Obese class III Source: Adapted from WHO, 1995, 2000, and 2004.

2. There were significant differences (at any habitually considered level, p < 0.01) between the average levels measured before and after BMD treatment. We accept a reduction of the average value assigned to this index with a p = 0.00. Figures 3–5 show the results for BMD. Consequently, Figure 3 represents final values of BMD plotted on the normal evolution curve of this variable with age. However, in Figure 4, one can observe a decrease in the BMD value, which as indicated is statistically significant. In the follow-up represented in Figure 5, one can observe that when a given patient followed a more intense exercise regimen, as advised during the checkups, the effect translated in the maintenance of

Table 4 Percentage distribution of clinical profiles before and after treatment.

BMI, kg/m2

BMD, AD-SoS, m/s

T Chol, mg/dL AI, mg/dL LDL Chol/HDL Chol, mg/dL TG, mg/dL

Underweight ( < 18.5) Normal (20–25) Overweight (25–30) Obese ( > 30) Low ( < 2112) Normal (2112–2190) High ( > 2190) Normal ( < 240) High ( > 240) Normal ( < 4.50) Risk ( > 4.50) Normal ( < 3.22) Risk ( > 3.22) Normal ( < 190) High ( > 190)

Pretreatment, %

Posttreatment, %

6.78 53.11 30.51 9.60 48.51 42.54 8.96 60.20 39.80 84.28 15.72 86.16 13.84 96.91 3.09

5.16 49.68 33.55 11.61 70.14 21.53 8.33 75.80 24.20 91.03 8.97 92.95 7.05 97.50 2.50

Cortés-Prieto et al.: Hormone replacement therapy      23

the difference between both values of the variable INDEX 1. INDEX 1 is calculated as the observed densitometry minus the densitometry below the curve, where “observed densitometry ” is the value of the last BMD measurement on a patient, i.e., the final value of BMD, and the densitometry below the curve value corresponds to the subject’s age on the normal curve and at the time of testing. The result obtained for INDEX 1, after performing adequate contrasting, shows the existence of significant differences between the average value of this index and 0 (at any habitually considered level, p = 0.00). That is, we accept that the last densitometry value of the given subject is higher than the average value for her age, according to the normal curve, p = 0.0005 (Figure 6). 3. With respect to T Chol, we accept the existence of significant differences between average levels before and after treatment with a p = 0.001. We accept the reduction in the average level of T Chol, after treatment, with a p = 0.0005 (see Figures 7 and 8). 4. As far as AI (T Chol/HDL Chol index), the three employed contrasts did not yield significantly higher values in the p-value domain; thus, there is no statistical evidence for the existence of significant differences in the average level of this index before and after treatment (see Figures 9 and 10).

Table 5 Contrasts of differences of mean values before and after treatment. p-Value Entities BMI BMD T Chol AI LDL Chol/HDL Chol TG

Sign test

Signed rank test

t-Testa

0 0.001 0.018 0.798 0.148 0.036

0 0 0.002 0.149 0.014 0.045

0 0 0.001 0.142 0.008 0.277

a

The t-test was used for related samples, although the studied variables do not correspond to a normal distribution, as the sample size is large enough to guarantee results.

the observed values, which nevertheless suffered a rapid decline. This was due to the pronounced drop in physical activity, as the studied subject had to care for her mother who was admitted to the hospital for serious health problems. BMD analysis deserves special consideration. Observing graphical representation of the value of this posttreatment entity on the normal value curve as based on age, Figure 3 shows that most patients exceed, in posttreatment, the value that corresponds to their normal age curve. Consequently, we evaluated

Index 40 38 36 34 32

BMI

30 28 26 24 22 20 18 16 14 -1

0

1

2

3

4

5

6

7

8

9

10

11

Treatment duration in years

Figure 1 Final assessment of BMI.

12

13

14

15

16

17

18

19

24      Cortés-Prieto et al.: Hormone replacement therapy

10.00

Difference BMI final vs. pretreatment

7.50

5.00

2.50

0

-2.50

-1

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

Treatment duration in years

Figure 2 Differential assessment of BMI.

5. For the LDL Chol/HDL Chol index, the sign test accepts the equality of average levels to any significance level, the signed rank test does it for a level of 1%, and the t-test rejects this equality to any significance level with p = 0.008; therefore, the reduced average levels of this index after treatment is accepted with a p = 0.004 (see Figures 11 and 12). 6. As for the TG variable, we can reject the average level equality to a significance level of 5% in the sign test and the signed rank Wilcoxon test. However, the t

contrast accepts equality with p = 0.277 (see Figures 13 and 14).

Analysis of the evolution of the patients treated with HRT in the clinical entities as investigated for a period of 9–10 years The first problem we encounter is that as it is an empirical study, there are no available precise data that would

BMD, AD-SoS(m/s)

2300

2200

2100

2000

1900

1800 40

50

60 Age

Figure 3 Final assessment of BMD.

70

80

Cortés-Prieto et al.: Hormone replacement therapy      25

250

200

Difference BMD final vs. pretreatment

150

100

50

0

-50

-100

-150

-200 -1

0

1

2

3

4

5

6

7

8

9

10 11

12

13 14

15

16 17

18

19

20

21

Treatment duration in years

Figure 4 Differential assessment of BMD.

be helpful while dealing with this study. Thus, among the patients who had undergone regular follow-up for at least 9 years of treatment, a random sample was selected so that the maximum permissible error in estimates has remained around 12% in the worst situation of maximum variance and a significance level of 5%. With these patients in mind, we studied the evolution of the parameters investigated during treatment, using a repeated-measures design that considered an intersubject factor and an intra-subject factor. The first refers to the years of treatment with four levels: control of variable evolution in the study that presents the patient at the beginning of treatment for selected valid cases, and in situ, during the treatment durations of 2–3, 5–7, and 9–10 years. The inter-subject factor considers three age levels for the initiation of treatment: < 48 years, between 48 and 52 years, and > 52 years. In each clinical entity, the selected number of women is logically the same in order to correctly compare the initial evolutive control over the different indicated treatment durations. This number of samples will be the proper one, displayed for each entity and according to the selected corresponding female patients. For the BMD

analysis, we considered the last three levels, as we did not have evolution control average value as in the case of other values. The results obtained for each of the variables are presented as follows.

Analysis of the evolution of BMI In case of BMI, upon carrying out the contrasts of average equalities for the factors in the study, the following results were obtained (Table 6): – There was no significant interaction between treatment duration and age factors at the start, p = 0.96. – There were significant differences between the mean BMI values for the four levels of treatment duration, p = 0.018. – No significant differences in the mean BMI values for different age levels at start of treatment p = 0.14 were observed. Therefore, it seems logical to analyze the evolution of BMI throughout the years of treatment. In Table 7, we present

26      Cortés-Prieto et al.: Hormone replacement therapy

Figure 5 Individual ultraosteosonography.

the results of the comparison of (means) averages in the four studied periods. In view of the results in Table 7, the following can be concluded: – There was a significant increase in the value of BMI between the average found in the evolutive control and that obtained after 9–10 years of treatment duration.

–

–

There was a significant increase in the value of BMI between the average after 2–3 years of treatment and that obtained after 9–10 years. BMI values were higher than normal, although the age of the patients should be taken into account when assessing the pattern of normality.

Cortés-Prieto et al.: Hormone replacement therapy      27

350

280

210

Index 1

140

70

0

-70

-140 0

1

2

3

5

4

6

7

8

9

10

11

12

13

14

15

16

17

18

Treatment duration in years

Figure 6 Final assessment of INDEX 1.

350

Total cholesterol, mg/dL

300

250

200

150

100 -1

0

1

2

3

4

5

6

7

8

9

10

11

12

Treatment duration in years

Figure 7 Final assessment of T Chol.

13

14

15

16

17

18

19

20

21

28      Cortés-Prieto et al.: Hormone replacement therapy

150

Difference final total cholesterol vs. pretreatment

100

50

0

-50

-100

-150

-1

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

16

17

19

20

Treatment duration in years

Figure 8 Differential assessment of T Chol.

Risk 7

6

AI

5

4

3

2

1 -1

0

1

2

3

4

5

6

7

8

9

10

11

Treatment duration in years

Figure 9 Final risk assessment of the AI: T Chol/HDL Chol.

12

13

14

15

18

19

21

Cortés-Prieto et al.: Hormone replacement therapy      29

3

Difference final AI vs. pretreatment

2

1

0

-1

-2

-3 -1

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

Treatment duration in years

Figure 10 Differential assessment of the AI.

Analysis of the evolution of BMD

within the normal range for age (see Figure 3), two studies were carried out: 1. With the BMD values of patients 2. With the INDEX 1 values, evaluated for each of the patients who followed the treatment for 9–10 years

In view of the results obtained in the previous section for BMD, where it was found that, while there is a decreasing chronological trend in this variable, most of the values are

Figure 16 graphically represents the evolution of the patients in the considered period, on the normal age curve, and justifies the second analysis.

Figure 15 shows the evolution of BMI during the years of treatment.

Risk 5

Index of LDL Cholesterol/HDL cholesterol

5 4 4 3 3 2 2 1 1 0 -1

0

1

2

3

4

5

6

7

8

9

10

11

12

Treatment duration in years

Figure 11 Final risk assessment of the LDL Chol/HDL Chol index.

13

14

15

16

17

18

19

30      Cortés-Prieto et al.: Hormone replacement therapy

Difference LDL cholesterol/HDL cholesterol index, final vs. pretreatment

3

2

1

0

-1

-2

-3

-4 -1

0

1

2

3

4

5

6

7 8 9 10 11 12 Treatment duration in years

13

14

15

16

17

18

19

Figure 12 Differential assessment of the LDL Chol/HDL Chol index.

Analysis of BMD

3.

There were significant differences in the BMD value according to age at the onset of treatment, p = 0.005.

From the results associated with the BMD values of the selected patients, first 1. It can be concluded that the effect of the interaction between treatment duration and age at the start was not significant, p = 0.066. 2. It is accepted that the BMD value was not equal after 2–3 years, after 5–7 years, and after 9–10  years of treatment duration, p = 0.0000.

Table 8 shows the mean BMD values in selected periods. A decrease of this value can be observed (Table 9). The decline is significant between levels 2–3 and 5–7 of the treatment duration factor, and also between levels 2–3 and 9–10. However, the decrease is not significant between the 5–7 and 9–10 levels. Regarding the age at the onset of treatment, we obtained the following results.

250

TG/mg/dL

200

150

100

50

0 -1

0

1

2

3

4

5

6

7

8

9

10

11

12

Treatment duration in years

Figure 13 Final assessment of TG.

13

14

15

16

17

18

19

Cortés-Prieto et al.: Hormone replacement therapy      31

150

Difference final TG vs. pretreatment

100

50

0

-50

-100

-150 -1

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

Treatment duration in years

Figure 14 Differential assessment of TG.

In Table 10, we present the averages of the BMD values obtained at the end of treatment, depending on the age of the patients at the onset of treatment. It can be seen that the greatest average BMD value is reached when the age at the start of treatment falls between 48 and 52 years. According to the results presented in Table 11, the difference between BMD values is only statistically Table 6 Means and CI of BMI, according to duration of treatment. Duration

Pretreatment 2–3 years 5–7 years 9–10 years

Average BMI, kg/m2 24.67 24.78 24.94 25.29

95% CI Lower limit

Upper limit

23.89 23.95 24.16 24.38

25.45 25.62 25.72 26.20

significant among the age group comprising subjects 48 and 52 years old, and older than 52 years. Figure 17 illustrates in the combined manner the above results. Analysis of BMD evolution according to INDEX 1: The results obtained for INDEX 1, after performing the appropriate contrasts, showed the existence of significant differences between the average value of the index and 0 (at any habitually considered level, p = 0.00). That is, it is accepted that the last densitometry value of the subject is higher than the average value for her age, according to the normal curve, p = 0.0005.

BMI

Table 7 Comparison of mean BMI according to duration of treatment. Duration (I)

Pretreatment

2–3 years 5–7 years a

Duration (J)

2–3 years 5–7 years 9–10 years 5–7 years 9–10 years 9–10 years

Difference of BMI average (I–J)

p-Value

–0.11 –0.26 –0.61a –0.15 –0.50a –0.35

1.000 1.000 0.044 1.000 0.037 0.364

The mean difference is significant at the 0.05 level.

Estimated marginal means

25.40

25.20

25.00

24.80 Pretreatment

2–3 5–7 Treatment duration in years

9–10

Figure 15 Evolution of the mean values of BMI according to duration of treatment (9–10 years).

32      Cortés-Prieto et al.: Hormone replacement therapy

2300

2200

BMD

2100

2000

1900

1800 40

45

50

55 Age

60

65

70

Figure 16 Evolution of BMD in patients treated for 9–10 years.

1.

2.

3.

INDEX 1 analysis yielded the following results: The effect of the interaction between treatment duration and age at the start of the treatment was not significant, p = 0.584. It is accepted that the value of INDEX 1 was not the same after 2–3 years, after 5–7 years, and after 9–10 years of treatment, p < 0.001. As far as the age factor at the start is concerned, there were significant differences in the INDEX 1 value for different levels of the factor, p = 0.001.

Analysis of INDEX 1 evolution, depending on the length of treatment, yielded the following results. Table 8 Means and CI of the BMD, according to duration of treatment. Duration, years

Average length

2–3 5–7 9–10

2094.63 2074.88 2067.27

Table 10 Means and CI of the BMD by age at the start of treatment. 95% CI

Lower limit

Upper limit

2074.99 2056.29 2049.74

2114.26 2093.48 2084.80

Table 9 Comparison of mean BMD according to treatment duration. Duration, years (a)

(b)

2–3 2–3 5–7

5–7 9–10 9–10

The results from Table 12 indicate that as the years of treatment duration increase, the difference between the average value of BMD and the average normal value for the patients’ age also increase. There were significant differences between INDEX 1 values in the sense that with increasing treatment duration, the value of INDEX 1 increased (see Table 13). The difference between the first two levels of the duration factor is not significant. Nevertheless, it is significant between levels 1 and 3, and between levels 2 and 3. On the basis of the results from Table 14, we can conclude that the greatest increase in INDEX 1 occurs when initiating treatment at ages between 48 and 52 years.

BMD mean difference between (a) and (b)

p-Value

19.74a 27.35a 7.61

0.007 0.000 0.600

Age, years

< 48 48–52 > 52

Average

2084.333 2111.944 2040.519

95% CI Lower limit

Upper limit

2054.63 2082.24 2010.81

2114.03 2141.64 2070.21

Table 11 Comparison of mean BMD for age at baseline. (I) Age, years

(J) Age, years

Age average difference (I–J)

p-Value

< 48 < 48 48–52

48–52 > 52 > 52

–27.61 43.81 71.42a

0.578 0.124 0.004

Cortés-Prieto et al.: Hormone replacement therapy      33

Age at treatment begining in years 2140

Below 48

Estimated marginal means for BMD

Between 48 and 52 Upper 52

2120

2100

2080

2060

2040

2020 2–3

5–7 Treatment duration in years

9–10

Figure 17 Evolution of the mean values of BMD, according to the duration (9–10 years) and age at the start of treatment.

In Table 15, we compare the averages of INDEX 1 values, as compounded for different age levels at the start of the treatment. We therefore obtained the following conclusions: – INDEX 1 increased significantly in the second age group and the third with respect to the first. – No significant differences existed between the averages of INDEX 1 values from the third age group with respect to the second. Figure 18 contains graphical explanations together with the above-mentioned results.

As a summary of the two studies related to BMD, we conclude that – BMD decreased significantly over the treatment duration, although this reduction was influenced by the increase in the patients’ age. – However, the BMD value was significantly superior to the value that would correspond to the patients’ age according to the normal curve of this variable. – INDEX 1 increased accordingly to the length of treatment. The difference was statistically significant between duration levels 2–3 and 9–10, and between levels 5–7 and 9–10.

Table 14 Means and CI of INDEX 1 by age at the start of treatment.

Table 12 Means and CI of INDEX 1, according to duration of treatment.

Age, years Duration, years

Average length

Lower limit

Upper limit

2–3 5–7 9–10

36.44 50.48 69.19

17.80 32.36 51.25

55.08 68.61 87.12

Table 13 Comparison of average INDEX 1 values according to treatment duration.

< 48 48–52 > 52

Average

3.591 81.212 71.317

95% CI Lower limit

Upper limit

–25.52 52.09 42.20

32.70 110.32 100.43

Table 15 Comparison of the means of INDEX 1 by age at baseline.

(I) Duration, years

(J) Duration, years

Difference of INDEX 1 averages (I-J)

p-Value

(I) Age, years

(J) Age, years

Difference of averages (I-J)

p-Value

2–3 2–3 5–7

5–7 9–10 9–10

–14.04 –32.74a –18.70a

0.061 < 0.001 0.008

< 48 < 48 48–52

48–52 > 52 > 52

–77.62a –67.72a 9.89

< 0.001 0.002 0.632

34      Cortés-Prieto et al.: Hormone replacement therapy

Age at treatment begining in years

100

Below 48 80 Between 48 and 52 Estimated marginal means

Upper 52 60

40

20

0

-20 2–3

5–7

9–10

Treatment duration in years

Figure 18 Evolution of INDEX 1, according to the duration (9–10 years) and age at the start of treatment.

–

The best results were obtained, in all cases, for patients that begin treatment between 48 and 52 years.

Evolution of T Chol Here again, the only significant differences were obtained among the average values of T Chol for the four levels of treatment duration, p = 0.02. Consequently, we can conclude that the evolution of T Chol is not dependent on the interaction between two variables under study. Likewise, it does not depend on the age at the start of the treatment. From the analysis on the evolution of T Chol and depending on the years of treatment, the following results were obtained.

Table 16 represents the average values of T Chol in the four studied periods. Therefore, it can be observed that there was a decrease in the value of this variable with respect to the relative values of control in any studied duration levels. On the basis of the results from Table 17, we can conclude that – T Chol remained below the 240 limit value – T Chol decreased at the considered periods with respect to the considered evolutive control values, although the decrease was significant when the duration is 2–3 years. In Figure 19, we graphically represent the evolution of the average T Chol value in the studied periods. Table 17 Comparison of T Chol mean according to treatment duration.

Table 16 Means and CI of T Chol, according to duration of treatment. Duration

Pretreatment 2–3 years 5–7 years 9–10 years

Average T Chol, mg/dL 235.39 218.09 228.23 222.52

95% CI Lower limit

Upper limit

222.72 206.19 218.44 211.95

248.06 229.99 238.02 233.09

Duration (I)

Duration (J)

Difference of T Chol averages (I-J)

p-Value

Pretreatment

2–3 years 5–7 years 9–10 years 5–7 years 9–10 years 9–10 years

17.30a 7.15 12.87 –10.14 –4.43 5.71

0.043 1.000 0.225 0.381 1.000 1.000

2–3 years 5–7 years a

The mean difference is significant at 0.05 level.

Cortés-Prieto et al.: Hormone replacement therapy      35

Total cholesterol/HDL cholesterol

Total cholesterol 3.40

Estimated marginal means

Estimated marginal means

235

230

225

220

3.30

3.20

3.10

3.00 Pretreatment

2–3 5–7 Treatment duration in years

9–10

Pretreatment

2–3 5–7 Treatment duration in years

9–10

Figure 19 Evolution of the mean values of T Chol according to the duration of treatment (9–10 years).

Figure 20 Evolution of the average values of AI according to the duration of treatment (9–10 years).

Evolution of T Chol/HDL Chol index

2.

In the case of T Chol/HDL Chol or AI, we could not observe any significant interaction among factors, p = 0.13. Likewise, no significant differences between the levels of treatment duration, p = 0.054, or between age levels at the start, p = 0.34, were observed. The average of T Chol/HDL Chol index values in the four treatment duration levels are presented in Table 18. With regard to AI, it can be concluded that – There was a slight increase in the rate of T Chol/HDL Chol, which was not statistically significant. – The value of this index remained within the normal limits, i.e., < 4.5.

3.

In Figure 20, we graphically represent the evolution of T Chol/HDL Chol throughout the treatment. Evolution of LDL Chol/HDL Chol index The results for this index are as follows: 1. There was no significant interaction between treatment duration and age at the start factors, p = 0.47.

There were no significant differences among different age levels at the onset of treatment, p = 0.88. There were significant differences among the average LDL Chol/HDL Chol index values for the four levels of treatment duration, p = 0.03.

The average LDL Chol/HDL Chol index values in the four studied periods are shown in Table 19. In Table 19, we can clearly see a decrease in the value of this index from the evolution control value until 2–3  years of treatment. In successive periods, the evolution control value increases. On the basis of the results found in Table 20, we can conclude that – The value of the LDL Chol/HDL Chol index, after 9–10  years of treatment duration, increased significantly with respect to the value that was present after 2–3 years of treatment duration. – There were no other significant changes. – The index value was maintained within normal limits, < 3.22. Figure 21 illustrates the evolution of the average of the LDL Chol/HDL Chol index during the years of treatment.

Table 18 Means and CI of T Chol/HDL Chol, according to treatment duration.

Table 19 Means and CI of LDL Chol/HDL Chol, according to treatment duration.

Duration

Duration

Pretreatment 2–3 years 5–7 years 9–10 years

Average AI

3.01 3.09 3.30 3.36

95% CI Lower limit

Upper limit

2.81 2.85 3.05 3.10

3.21 3.34 3.56 3.62

Pretreatment 2–3 years 5–7 years 9–10 years

Average LDL Chol/ HDL Chol index 2.41 2.11 2.38 2.50

95% CI Lower limit

Upper limit

2.13 1.895 2.09 2.22

2.68 2.33 2.67 2.79

36      Cortés-Prieto et al.: Hormone replacement therapy Table 20 Comparison of mean LDL Chol/HDL Chol according to treatment duration.

Table 21 Means and CI of TG, according to duration of treatment. Duration

Duration (I)

Duration (J)

Difference in averages LDL/HDL (I-J)

p-Value

Pretreatment

2–3 years 5–7 years 9–10 years 5–7 years 9–10 years

0.29 0.02 –0.09 –0.27 –0.39a

0.244 1.000 1.000 0.135 0.020

2–3 years

Average TG, mg/dL

Pretreatment 2–3 years 5–7 years 9–10 years

95% CI Lower limit

Upper limit

85.48 83.04 83.76 88.08

110.28 100.80 106.70 113.42

97.88 91.92 95.23 100.75

Analysis of TG

Discussion

In order to carry out contrast calculations on equal terms, we resorted to multivariate contrasts because Mauchly’s test indicates an extreme situation, p = 0.056, to fulfill the assumption of sphericity. The results reached with such contrasts are as follows: 1. No significant interaction between treatment and age at the start factors, p = 0.12, were observed. 2. Likewise, there were no significant differences between the average TG values for four levels of treatment duration, p = 0.32, or for different age levels at the start, p = 0.65 (Table 21).

Our current contribution focuses on the pathological events in a population of female never-hormone users treated continuously with a combination of CEE plus MPA, administrated orally as a long-term preventive therapy. According to the American Society for Reproductive Medicine [8], individualized evaluation of the risk/benefit profile by a well-informed clinician is clearly the most prudent course to follow. Evidently, regular advice on diet, physical exercise, and self-determination in each patient, associated with the hormonal effects, benefited the final results. That is why the global results must not be considered exclusively as being due to the action of CEE plus MPA during HRT. In the review of the literature from the last 20 years, selecting studies on HRT modalities to achieve the type of treatment used by us (CEE plus MPA) reduced the number of available references concerning the viable parameters, such as BMI, BMD, and LP, for studying the outcome of the treatment employed in our study. Most significantly, out of nearly a hundred publications found for each of these modalities, those in which our proposed criterion

Therefore, on the basis of the above results, we can conclude that – The average value of the variable TG showed a decrease from its evolutive control value until it was measured after a period of 2–3 years of treatment. In subsequent periods, the value increased, although not statistically significantly. Figure 22 graphically illustrates the above results.

Triglycerides

2.60

102

2.50

100 Estimated marginal means

Estimated marginal means

LDL cholesterol/HDL cholesterol

2.40

2.30

2.20

2.10

98 96 94 92 90

Pretreatment

2–3 5–7 Treatment duration in years

9–10

Figure 21 Evolution of the mean values of the LDL Chol/HDL Chol index according to treatment duration (9–10 years).

Pretreatment

2–3 5–7 Treatment duration in years

9–10

Figure 22 Evolution of the mean values of TG according to treatment duration (9–10 years).

Cortés-Prieto et al.: Hormone replacement therapy      37

was used do not in fact reach a dozen, in all reviewed publications, and failed to supply more or less specific data concerning the aforementioned parameters. Moreover, as already stated, the available references mostly refer to short periods of follow-up and deal with equally small populations. The longer duration of our follow-up and the greater number of the studied population will undoubtedly help alleviate the shortage of references and hopefully situate our contribution within the world literature as worthy of consideration. While reviewing the vast number of publications devoted in general to the action of HRT on distinct clinical entities, one finds an obvious controversy that also appears while considering in concrete terms the exclusive action of CEE plus MPA in relation to parameters regarding the clinical examinations under consideration. Even more, the number of such publications appears to be less patent, whereas the majority of them are merely monographic in nature. We do accept that, at least partially, the evolutive data of BMI can shed light on the nutritional status and the possible global action of the administrated medication by which the percentual BMI distribution values may have the maximum initial interest. With the same logic, one could assume that the LP and the response expressed as CV events, hypertension, and thrombophlebitis are of greater interest. Evidently, for the WHO, BMI values are age independent and the same is true for both sexes. It is not our intention to dispute these claims; however, we ought to stress that the patients’ height can variate with advancing years and may thus alter the interpretation of results as calculated for height. To prevent these distorting readings, other skeleton measurement methods were proposed. For information purposes only, let us comment on the measurement of knee height, which remains stable throughout the adult life and leads to consider this measurement as the one that comes closest to the value of actual height (measured with leg bent at a 90° angle between the distance of the heel to the top of the kneecap). In 2007, Thorneycroft et  al. [9] published an article concerning 822 randomized, double-blind, placebocontrolled women, where the substudy of CEE plus MPA combination does not show weight gain or body composition changes. Vigorous exercise (EXER program) increased by 3.5% the lumbar spine measurement of BMD vs. 1.5% with a more flexible exercise (HOME program) in 28 women > 75 years old with bone fragility treated for 9 months with CEE plus MPA [10]. The normal chronological decrease of BMD may vary in some cases, slightly increase, or at least remain at the

previous levels as a possible benefit from the exercise and the response to HRT. In some publications [5, 11–16], this is considered a favorable action of the CEE plus MPA treatment on BMD, whereas for Ortiz-Nuñez et al. [17] increased BMD exhibited no statistically significant difference in treatment for up to 4 years. In an analysis carried out on reviewed publications dealing with the action of CEE plus MPA on BMD, we point out that in the Women’s Health Initiative study [5], the authors hint that in their clinical opinion combined therapy may be appropriate for certain women. They also indicate that more studies with appropriate statistical power are needed to show fracture prevention efficacy. Grey et  al. [11] communicate a comparative study of 79 osteoporotic women, of which 23 hysterectomized women were treated with 0.625 mg CEE daily and 50 non-hysterectomized women were continuously treated with 0.625 mg CEE daily combined with 5 mg MPA daily. After 1 year of therapy, the spinal BMD increased by 6.6% [95% confidence interval (95% CI) 5.6–7.6, p < 0.0001 vs. baseline] in the combined CEE plus MPA group compared with 4.0% (CI 2.4 –5.7, p < 0.001 vs. baseline) in the unopposed estrogen group (difference between means, 2.6%; 95% CI 0.8–4.4, p < 0.01), concluding that in postmenopausal osteoporotic women 1 year of continuous combined CEE/MPA therapy is associated with a 65% greater increment in spinal BMD than observed in response to unopposed estrogen. Hence, the prescription of such a therapy should be considered also in osteoporotic postmenopausal women who have undergone hysterectomy, in order to maximize the skeletal protection provided by HRT. In a comparative study [12] of 875 postmenopausal women with a follow-up of 36 months, a therapy with an active regimen and different doses of CEE plus MPA had a positive effect on BMD, while with placebo there was a 1.8% decrease, most notably in the spine measurement, reaching 5% vs. the hip measurement, which only increased by 1.7%. When it comes to measurements, BMD measurements are more demonstrative at the hip level. Similar results were subsequently published [13], with 9-month follow-up and in 67 women who were 75  years old, which also showed a decrease in BMD with placebo administration, 4.3% increase of BMD measured in the spine, and only 1.7% increase in hip measurements. We ought to underline that comparative BMD measurements should be carried out at 12-month intervals in order to ensure similar climatic conditions. Very similar readings appear [14] in the review on 16,608 postmenopausal women, with BMD measurements after 3 years of CEE plus MPA treatment showing a 3.7% increase in spine and only a 0.14% increase with placebo (p < 001), and presenting 733

38      Cortés-Prieto et al.: Hormone replacement therapy

fractures during the active regimen (8.6%) and 896 with placebo (11.1%) (hazard ratio 0.76, 95% CI 0.69–0.83). The review of Pinkerton and Dalkin [15] has been of great use to us for evaluating osteoporosis treatment with different agents; however, unfortunately, there are no large trials of an adequate veracity to determine the fracture prevention efficacy. We cannot deny the possible relation between BMD and bone fractures, as all being traumatic in nature, after a detailed anamnesis, it is impossible to refute that poor skeletal quality may play a role in fractures. According to the data from Lindsay et  al. [16], HRT significantly increased BMD by approximately 2%–3% during 2 years of therapy in women who had been in menopause for 4 years and up to those who were 40–65 years old. Despite these publications in favor of 0.625 mg CEE daily combined with 2.5 mg MPA daily for increasing BMD, for other authors [17] as noted above, the increased BMD showed no statistically significant differences in treatments of up to 4 years. The action of CEE plus MPA on the LP was favorable according to several publications [2,  18,  19]. Reviewing these publications, we ought to stress that with regard to cholesterol and lipoproteins, we can highlight the ample meta-analysis review carried out by Salpeter et al. [2], who find, as in similar studies, beneficial effects of HRT, including increases in HDL Chol and a reduction in all other (LDL Chol and TG). The administration of CEE combined with MPA, studied at the end of 1 year of treatment of about 345 postmenopausal women, demonstrated a protective effect, more specifically reaching a 17.5% increase in HDL Chol in non-overweight women (BMI < 25 kg/m2) compared with overweight women in whom the increase was only 10.4% (p = 0.015), whereas decreases in LDL Chol are not statistically significant [18]. In another study dealing with TG [19], 99 postmenopausal women were randomly treated with 0.625 mg CEE plus 2.5 mg MPA every other day (50 women) or every day (49 women) for 1 year. In women whose BMI was 25 kg/m2 or higher, TG levels during daily treatment increased by 26.8%, while TG levels during every-otherday treatment decreased by 12.3%. There was a significant (p < 0.05) difference between the percentage changes in TG during the daily and every-other-day treatments. In women whose BMI was < 25 kg/m2, TG levels during daily treatment increased by 21.7%, whereas TG levels during every-other-day treatment did not change. TG levels during daily treatment increased more in overweight and obese postmenopausal women. These data require confirmation in order to assess whether giving the combination treatment every other day, in which TG decreased by 12.3%, could be a more convenient method of administration than the combined daily administration.

Table 22 Lipid profile of Spanish women > 40 years old, in mg/dL. Lipid profile

DRECE study

Private laboratorya

≈220 ≈60 ≈130 ≈100 ≈3.67 ≈2.17

< 240 > 50 (  ≤  35)   ≤  150 ( > 180) < 190 < 4.5 < 3.22

T Chol HDL Chol LDL Chol TG AI LDL Chol/HDL Chol a

High-risk values in parentheses.

Blood tests prescribed to all women submitted to the current study were carried out in the officially recognized, prestigious private laboratory, whose pretreatment values are shown in the preceding charts (see Table 1). The LP for Spanish women > 40  years old included in DRECE Study Group [20] are listed in the Table 22. We think that as always in biology, the strict evaluation of datum with absolute value has more transcendence over its relation with the existing, preceding, and subsequent trends, as well as their separation or proximity with respect to normality. Numerical analysis of our material and the mean values of pretreatment BMI, BMD, and LP were already shown in Table 1. Comparing these values with those published in the literature shows that our population treated with HRT is basically sound because these values fall mostly within the normality. To study the possible evolutive effect of HRT according to the treatment duration and also as a function of age at the onset of the treatment, we resorted to a design of repeated measurements, known as evolutive control, which required a selection of patients treated for at least 9 years. The most significant numerical results throughout the treatment were as follows: – For BMI, there was a statistically significant increase with respect to the initial drop at 2–3 years of treatment (p = 0.037) (Figure 15). – BMD decreased significantly (p = 0.00) (Figure 16). The decrease was influenced by the increasing age of patients. As shown in Figure 5, exercise influences positively and because of that it can maintain the reached values, instead of following a chronological and habitual decline. Considering the INDEX 1 value for BMD – the value of the difference in the observed densitometry and the densitometry below curve – such index increases as the treatment lengthens, the difference being statistically significant between the duration levels of 2–3 and 9–10 years and between the duration levels of 5–7 and 9–10  years (Figure 18).

Cortés-Prieto et al.: Hormone replacement therapy      39

–

–

–

–

T Chol remained below the limit value, 240 mg/dL (Figure 7). T Chol decreased at the considered time periods with respect to the value of the corresponding evolutive controls, while becoming statistically significant (p = 0.04) when the duration is 2–3  years (Figure 19). Most of the average AI values were below 0, thereby inferring that the selected population maintained basically healthy eating habits (Figure 9). This index increased discretely but was not statistically nor significantly important beyond its normal range of 4.5 (Figure 20), which is why the increase has no clinical significance. Most of the LDL Chol/HDL Chol index mean values were below the risk limit, < 3.22 (Figure 11). This index increased significantly (p = 0.02) with respect to the initial drop at 2–3 years of treatment but stayed within normal; therefore, such an increase is not clinically relevant (Figure 21). Most of the TG mean values were found below the risk of < 190 mg/dL (Figure 13) whose significance can be identified with the preceding risk indices. The average TG value provides a graphical representation that is similar to the previous index, again without straying off the normal readings; hence, their changes have no clinical significance (Figure 22).

Specifically, changes in the mean values of BMD found during the treatment decreased but remained higher than the values to which they ought to correspond (Figures 3 and 17). This is also true for T Chol (Figures 7 and 19) and other integrating LP values, and therefore without clinical significance, which indicate a possible beneficial HRT action. Naturally, more research that could endorse our findings is required.

Comments One hundred and eighty-five postmenopausal women were treated with CEE and MPA, complemented by advice

on diet, physical exercise, and self-determination. The mean duration of treatment was 9.82 ± 5.42  years and the follow-up comprised 1815.84 woman-years. The pretreatment values for BMI, BMD, and LP were within the normal limits at the onset of HRT that was administered to a healthy population. To study the possible effects of HRT treatment throughout the investigated clinical entities, we first performed comparative analysis of pretreatment and posttreatment values using descriptive methods and contrast hypotheses and then analyzed the evolution through a repeated measurements method, considering three levels according to age at the onset and three levels during the minimum 9  years of follow-up of selected patients. From the abundant numerical data on the clinical evolution during treatment, we can conclude that – BMD decreased significantly (p = 0.00) but maintained superior values than the corresponding values according to the normal evolutive curve for age. Physical exercise has a positive influence. – T Chol decreased mostly after 2–3  years into the treatment (p = 0.04). – Most of the AI values indicate that the studied population maintained basically healthy eating habits. – Most of the LDL Chol/HDL Chol index mean values remained below the risk index. – Most of the TG mean values were below the risk limit. Consequently, the values that we found for BMD and LP in our studied population, subjected to long-term HRT, indicate a possible beneficial effect. Naturally, these relevant findings require more studies about the possible benefit of long-term HRT. Acknowledgments: Recognition is due to the two teams for their close and sustained collaboration, as well as to Mrs. Olga Boltova for her dedicated transcription of the text and figures.

Received January 25, 2013; accepted February 18, 2013; previously published online March 13, 2013

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