Preoperative treatment with oestradiol in women scheduled for vaginal operation for genital prolapse. A randomised, double-blind trial

Objective — To disclose a clinical and histopathological effect of local low-dose oestradiol treatment on the vagina. Design — A randomised, double-blind trial. Setting — Two gynaecological departments at University Hospitals. Subjects — Forty-eight postmenopausal women scheduled for surgery because of genital prolapse. Intervention — 25μg oestradiol or placebo, administered as vaginal pessaries daily, 3 weeks prior to surgery. Main outcome measures — Cytological, histological and clinical changes of the vaginal mucosa. Results — The thickness of the vaginal wall increased as did the oestrogenic index. No clinical effect was seen apart from decreased incidence of recurrent cystitis postoperatively. Conclusions — Preoperative oestrogen treatment has been shown to reduce the incidence of recurrent cystitis and may be needed for stimulation of vaginal mucosa; the short-term clinical effect is not convincing, however.     

Effects of oestradiol administration via different routes on the lipid profile in women with bilateral oophorectomy

To determine the influence of various oestrogenenic administrations on lipid response, 63 women with total abdominal hysterectomy and bilateral anexectomy were studied before and 6 and 12 months after receiving 17β-oestradiol by different means. The effect on the levels of lipids and lipoproteins of the 2 mg/24 h administration of oestradiol valerate was compared with 1.5 mg/day of percutaneous 17β-oestradiol and 0.05 mg/day of transdermic oestradiol. The treatments were given continuously over a year. The oestradiol valerate produced a statistically significant increase (P < 0.05) of the HDL-C levels both after 6 and 12 months (10.6% vs. 11.6%). A significant increase was also observed (P < 0.05) in the Apo Al levels during the treatment (18 and 25%). On the other hand, unfavorable side effects with oestradiol were not produced, either percutaneous or transdermic, on lipid plasmatic or lipoprotein levels. These data show the benefit of oral oestrogenic therapy and the maintenance of the lipid profile in percutaneous and transdermic therapies in oophorectomized women.     

Combined oestrogen-progestin replacement therapy prevents atherosclerosis in postmenopausal women

The incidence of atherosclerotic plaques in the aorta and in the carotid and iliac arteries was investigated using high resolution B-mode ultrasonography in 40 postmenopausal women using sequentially combined oestradiol valerate 2 mg—levonorgestrel 0.25 mg replacement therapy. In addition, serum lipid, lipoprotein and apolipoprotein levels were measured. Similar studies were performed for 40 postmenopausal women using oestradiol valerate alone and for 40 women who had never used hormone replacement therapy (HRT). Ultrasonograhpically observed atherosclerotic plaques were less common (P = 0.011) in oestradiol valerate—levonorgestrel users than in women without HRT. More than three atherosclerotic plaques were observed in 62% of the women without HRT but only in 30% of the women using combined therapy (P = 0.003). The total number of plaques did not differ between the two hormone using groups. The mean serum triglyceride, total cholesterol and apolipoprotein B levels were lowest in oestradiol valerate — levonorgestrel users when compared with those of women with oestradiol valerate monotherapy and those of women without HRT. Combined oestradiol valerate — levonorgestrel replacement therapy, like oestradiol valerate monotherapy, prevents the progression of atherosclerosis in postmenopausal women. The lipid patterns were not adversely affected by the addition of the androgenic levonorgestrel.     

Ethanolic extracts of black cohosh (Actaea racemosa) inhibit growth and oestradiol synthesis from oestrone sulphate in breast cancer cells

Extracts of black cohosh (Actaea racemosa) and soy are used as ‘natural’ alternatives to conventional hormone replacement therapy (HRT) and there is some evidence that soy may protect against breast cancer by inhibiting the production of active oestrogens. This study compares the action of ethanolic extracts of black cohosh (BCE) and genistein on growth and enzyme activity in MCF-7 and MDA-MB-123 breast cancer cells. BCE inhibited growth at the two highest doses tested, i.e. 50 and 100 μg/ml, whilst genistein stimulated growth in the oestrogen receptor positive (ER⁺) MCF-7 cells, but at high doses it inhibited growth in both cell lines. BCE did not affect the conversion of androstenedione to oestradiol and only the highest doses (50 and 100 μg/ml) significantly inhibited the conversion of oestrone to oestradiol in MDA cells. In contrast, BCE induced a dose-dependent inhibition of the conversion of oestrone sulphate to oestradiol in both cell lines, whilst in human granulosa lutein (GL) cells enzyme activity was only inhibited at the highest dose of BCE. Genistein had no significant effect on enzyme activity in breast cancer cells and like BCE only the highest doses (10 and 50 μM) inhibited enzyme activity in human GL cells. In vivo genistein may have growth stimulatory effects on breast tissue but BCE not only inhibits growth but inhibits the conversion of oestrone sulphate to active oestradiol, considered by some, to be the preferred pathway of oestradiol synthesis in breast tissue.     

Bleeding pattern and climacteric symptoms during different sequential combined HRT regimens in current use

Four sequential combined oestrogen and progestogen regimens were compared in terms of bleeding pattern and relief of climacteric symptoms. Treatment was with either 2 mg 17β-oestradiol with I mg norethisterone acetate [E₂ + NEta]; 2 mg oestradiol valerate with 75 μg levonorgestrel [E₂V + Lng]; 2 mg oestradiol valerate with 10 mg medroxyprogesterone acetate [E₂V + Mpa]; or 1.5 mg 17β-oestradiol with 150 βg desogestrel [E₂ + DG]. A placebo-controlled study lasting 12–24 months was completed by 143 healthy early postmenopausal women. Bleeding lengths were not substantially different; in all regimens the majority of women were bleeding for 3–6 days. Bleeding onset showed differences when related to the 11th day of progestogen addition; in the regimen with E₂V + LNG, 21% of the women were bleeding before the 11th day of progestogen addition 26% on, and 53% after that day. In the regimen with E₂V + MPA, 56% of the women were bleeding before the 11th day, 28% on, and 17% after that day, whereas in the regimen with E₂ + DG, 15% of the women were bleeding before the 11th day, 5% on, and 80% after that day. All regimens reduced climacteric symptoms to the same extent. Breast tenderness occurred in all the regimens, except in the E₂ + DG. Conclusively, the differences between the responses to treatment were not conspicuous. However, our data indicate that one regimen (E₂ + DG) resulted in optimal bleeding control, optimal effect on climacteric symptoms, and no production of breast tenderness.     

Progestogen addition during oestrogen replacement therapy--effects on vasomotor symptoms and mood

Vasomotor symptoms and mood changes were followed in 2 groups of post-menopausal women receiving cyclic replacement therapy with 3 mg/day of percutaneous oestradiol-17 beta alone or in sequential combination (days 11-21) with lynestrenol 5 mg/day. During 6 mth of therapy, daily symptom ratings were performed using a visual analogue scale technique. Serum levels of oestradiol were similar in both groups but sequential oestrogen/progestogen treatment was more efficient in suppressing vasomotor symptoms and serum gonadotropins. However, all negative mood symptoms were also more pronounced in this group. Progestogen addition may enhance the therapeutic effect on hot flushes and sweats but, at least in individual women cause unfavourable effects on mental status, mood and well-being.     

Evaluation of irritation and sensitisation of two 50 μg/day oestrogen patches

Objectives: To comparatively assess the irritation and sensitisation of the Estradot^® transdermal oestrogen patch, in healthy postmenopausal women, using the Menorest^® transdermal oestrogen patch, as a comparator. Methods: In an open-label, single-centre, randomised, active-treatment, within-patient controlled study, 208 healthy postmenopausal women, age range 40–70 years, received and completed simultaneous treatment with a 5 cm² (50 μg/day) oestradiol patch (Estradot) and a 14.5 cm² (50 μg/day) oestradiol patch (Menorest). The treatment was given for 72 h, then 96 h, for eight successive applications during an induction phase, and for 72 h during a challenge phase. There was a 14-day resting period between phases. Skin irritation (measured by erythema on a scale of 0–4), topical sensitisation, patch adherence and local skin reaction, were assessed and recorded immediately before or after removal of each patch, as appropriate. Results: Most patients experienced a significant difference in irritation with Menorest than with Estradot (P<0.0001) at the end of the induction phase. Patch loss was also significantly higher for Menorest as compared to Estradot (P=0.0253) at the end of the induction phase. Most incidences of erythema were classified as slight (score of 1), and there was no significant difference in the percentage of topical sensitisation, or in the incidence of local skin reactions between Estradot and Menorest. Patch loss was low for both systems. Conclusions: Estradot demonstrates reduced skin irritation, superior adhesion and a lower rate of patch loss compared to Menorest.     

Haemodynamic and hormonal effects of short-term oestradiol treatment in postmenopausal women

Haemodynamic changes during a 3-wk treatment with oestradiol valerianate (2 mg/day orally) were studied in 12 postmenopausal women by isotope ¹¹³In^m radiocardiography.

Systolic blood pressure measured in the supine position decreased during oestradiol treatment by 3% (P < 0.05) and the diastolic blood pressure decreased by 4% (P < 0.01). The heart rate decreased by 15% (P < 0.001).

Blood volume increased during oestrogen treatment by 5% (P < 0.05) whereas cardiac output decreased by 9% (P < 0.05). Stroke volume increased by 13% (P < 0.001) due to concomitant decrease in heart rate.

Changes in plasma oestrone and oestradiol concentrations during oestradiol valerianate substitution showed a positive correlation with the changes of blood volume.     

Skin contamination by oestradiol gel--a remarkable source of error in plasma oestradiol measurements during percutaneous hormone replacement therapy

Objectives: To study the consequence of skin contamination by oestradiol gel on circulating plasma oestradiol levels. Methods: We studied ten healthy, hysterectomized postmenopausal women who had used percutaneous oestradiol gel for at least 2 years. After wash-out period percutaneous dose of 1.5 mg 17β-oestradiol was administered once a day in the evening. The gel was applied with a bare or gloved hand to an arm or thigh according to the schedule. Blood samples for assay of plasma oestradiol concentrations were collected from both cubital veins 12 h after gel administration, at baseline and every time after using the gel, for 2 weeks. Results: Plasma oestradiol concentrations were significantly higher in the gel-contaminated samples: in the cubital vein of the gel-applying arm and in the cubital vein of the forearm on which the gel had been spread. Conclusions: Skin contamination by topical 17β-oestradiol can distort plasma oestradiol measurements by giving much higher oestradiol concentrations than in reality there are in the systemic circulation. This has an important meaning when tailoring individual oestrogen therapy.     

Clinical evaluation, dose-finding and acceptability of AERODIOL, the pulsed estrogen therapy for treatment of climacteric symptoms

S21400 (AERODIOL^®) is a new intranasal formulation of 17β-estradiol. It provides a pulsed estrogen therapy that ensures sufficient estrogenisation of tissues to treat estrogen deficiency symptoms, particularly those of the menopause. This multicentric study was designed to determine dose-range, efficacy and acceptability of S21400. One hundred and thirty four women were allocated a daily dose of 100–900 μg for 12 weeks. The doses of 100, 600 and 900 μg were given in two daily administrations, the doses of 200, 300 and 450 μg were given in one and two daily administrations. Oral progestogen was added the last 10–14 days of each cycle of estrogen therapy in all non-hysterectornized women. S21400 showed a dose-effect relationship and provided adequate estrogenisation in more than 80% of patients receiving a dose ranging from 200 to 600 μg daily. Hormonal impregnation was judged sufficient in 23% of women receiving the lowest dose (100 μg). It was often considered excessive for daily doses of 900 μg (36%). After 12 weeks of treatment, efficacy was similar whether the total daily dose was given in one or two administrations. Treatment was well tolerated and accepted, with only minor nasal events (prickling, sneezing). It was perceived by 92% of patients as good or excellent and 81% chose to continue the nasal treatment when it was offered to them. An initial dose of 300 μg per day provides an optimal efficacy/tolerability ratio.

In summary, the pulsed estrogen therapy with AERODIOL^® in one daily administration offers a safe, well accepted and highly effective treatment to alleviate climacteric symptoms. It can be adapted easily to ensure optimal clinical efficacy.     

Study on the Effect of Calf Thymic Peptides Preparation (Tp) on Human B' Lymphocytes

Extensive experimental evidence has shown that thymic hormones (or factors) affect and regulate the differentiation and function of T lymphocytes. However, little is known about the action, if any, of thymic hormones on B lymphocytes. This paper reports the results of an investigation of the effect of a calf thymic peptide preparation (TP) prepared in our laboratory, on the proliferation and differentiation of human B lymphocytes.

TP at concentrations higher than 1 μg (protein)/ml inhibited the proliferative responses of human B lymphocytes to the stimulation by Staphylococcus aureus Cowen strain I (SAC) and F(ab')₂ fragments of goat anti-human IgM μ chain specific antibody (anti-μ). TP itself had neither toxic nor mitogenic effect on B cells. TP at concentrations of 60 and 100 μg/ml did not affect the differentiation of B cells driven by SAC and PWM in a reverse PFC assay, but appeared to suppress the production in some individuals of total IgG and IgM in culture supernatants in a PWM system. Preculture of B cells with 60 μg/ml of TP for 40 hrs showed a suppression of the proliferative response to SAC and anti-μ stimulation, suggesting that TP might act on cells directly and persistently for some time. When TP was added to the culture on day 0 or on day 1, a similar decrease of inhibition of B cell proliferation was observed. A decrease in monocytes from 15-17% to 5% did not appreciably influence the suppression of SAC-or anti-μ-induced proliferation of B cells by TP. These preliminary results suggest that a calf thymic peptides preparation might have some direct effect on B lymphocytes.     

Growth hormone and somatomedin C during post-menopausal replacement therapy with oestrogen alone and in combination with an antioestrogen

Serum concentrations of growth hormone (GH) and somatomedin C were monitored in 14 women during post-menopausal replacement therapy with oestrogen alone and in combination with a specific antioestrogen. During 3 mth of treatment with ethinyl oestradiol (10 μg daily), the mean serum GH level rose from 2.8 ± 0.78 mU/l (mean ± S.E.M.) to 6.5 ± 0.39 mU/l, whereas the concentration of somatomedin C fell from 22.4 ± 0.89 to 15.4 ± 0.43 nM. These changes during unopposed oestrogen treatment were clearly reversed by the addition of tamoxifen (20 mg daily), following which GH concentrations fell to pre-treatment levels. It is suggested that oestrogens inhibit somatomedin C production in the liver and that GH secretion may play an important role in regard to certain liver effects induced by oral oestrogen therapy.     

How do androgens affect episodic gonadotrophin secretion in postmenopausal women?

In the absence of any significant ovarian oestrogen secretion, as in post-menopausal women, the hypothalamic-pituitary axis may still be influenced by the androgens which continue to be produced. The episodic secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by post-menopausal women was accordingly assessed following short-term androgen antagonism induced by flutamide, a specific androgen receptor blocker. Blood samples were collected at 10-min intervals for 10 h in nine women before and during flutamide administration (750 mg/day for 6 days) for the determination of gonadotrophin and sex hormone concentrations by radibimmunoassay. On both occasions, 25 μg of gonadotrophin-releasing-hormone (GnRH) was injected intravenously 8 h after initiation of the blood collections. Flutamide administration decreased (P < 0.01 or less) androgen concentrations (testosterone, androstenedione and dehydroepiandrosterone sulphate) in relation to baseline values, but did not alter oestrogen (oestrone and oestradiol) or sex-hormone-binding globulin levels. The LH and FSH pulse characteristics (frequency, amplitude, interpulse interval and transverse mean levels) determined by a cluster algorithm in the gonadotrophin secretory profiles did not differ before and during androgen blockade. By contrast, androgen antagonism increased LH (P < 0.01) and tended to enhance FSH (P = 0.10) FSH release in response to GnRH stimulation. Hence, short-term androgen receptor blockade with flutamide did not greatly affect episodic gonadotrophin secretion. However, the combined evidence of the enhanced gonadotrophin release observed in response to GnRH stimulation and the unchanged gonadotrophin secretion during androgen antagonism suggests that alterations in the magnitude, but not the frequency, of hypothalamic GnRH release had occurred. Even in the presence of substantial serum androgen concentrations, the gonadotrophin pulse rhythm in hypogonadal women consitutes the maximal-rate GnRH-LH release pattern.     

Simultaneous evaluation of basal FSH and oestradiol response to GnRH analogue (F-G-test) allows effective drug regimen selection for IVF

To determine whether preliminary assessment of ovarian reserve by simultaneous evaluation of basal follicle-stimulating hormone (FSH) and oestradiol response to gonadotrophin releasing hormone (GnRH) analogue (F-G-test) can be used to tailor individually the drug regimen for ovarian stimulation, the in-vitro fertilization (IVF) results of 238 patients were retrospectively analysed. Sixty-two women with abnormal response to the test (DeltaE2 <180 pmol/l and/or FSH >9.5 mIU/ml) had commenced buserelin nasal spray in the mid-luteal phase and discontinued it on cycle day 1. Ovarian stimulation was started on cycle day 3 with 375 IU/day of gonadotrophin. Fifty-three patients completed the treatment cycle (group A). A total of 176 women with normal response to the test (DeltaE2 >180 pmol/l and FSH <9.5 mIU/ml) had continued the GnRH analogue throughout the stimulation cycle and a starting dose of 225 IU/day of gonadotrophin was used from cycle day 3. A total of 158 patients completed the treatment cycle (group B). Group A had significantly higher age (34.9 +/- 4.2 versus 33.2 +/- 4.2) (P < 0.05) and basal FSH (9.2 +/- 3.8 versus 7.0 +/- 2.2) (P < 0.05) and required a higher total dose of gonadotrophin. The numbers of oocytes retrieved and embryos transferred were significantly lower. However, fertilization, clinical pregnancies, and implantation rates were similar in both groups. It was concluded that simultaneous evaluation of basal FSH and oestradiol response to GnRH analogue can be useful in identifying subcategories of women with reduced ovarian reserve who may benefit from reduced GnRH analogue administration and a higher starting dose of gonadotrophin.     

Climacteric symptoms according to body weight in women of different socio-economic groups

The possible influence of body weight on the 'climacteric symptoms of 618 selected women in spontaneous post-menopause has been studied based on data collected at a Geriatric Centre and subsequently incorporated into a postal questionnaire. These cases were divided into 3 groups according to the “obesity degree”: (I) <5 kg (53.1% of the cases); (II) between 5 and 15 kg (31.7%); and (III) >15 kg (15.2%). The data were also analysed according to the socio-economic groups to which the women belonged.

Overweight women, compared with. thin women, seemed to suffer less “somatic” symptoms such as hot flushes and perspiration, independently of their socio-economic level. This might well be a consequence of the higher endogenous oestrogen activity.

On the contrary, “psychic” symptoms (anxiety, depression, irritability, crying spells) seemed to be more frequent and severe (a) in the “obesity degree” sub-group III, compared with sub-groups I and II, in the women belonging to the higher socio-economic group, and (b) in the sub-groups I and III, compared with sub-group II, in the women belonging to the lower socio-economic group. Such a difference between the socio-economic groups is possibly due to cultural factors. The effects of endogenous oestrogens in the overweight women seem to be easily overruled by the influence of psychological factors.     

The effects of continuous combined transdermal oestrogen-progestogen treatment on bleeding patterns and the endometrium in postmenopausal women

Fifty postmenopausal women requiring hormone replacement therapy for the treatment of climacteric symptoms were recruited in six centers. All patients received a new combined norethisterone acetate (NETA)/oestradiol (E₂)-TTS, (Estragest TTS, Ciba-Geigy Ltd), delivering 0.25 mg NETA and 50 μg E₂ per day, continuously for 12 calendar months. Bleeding occurred in 38 (76%) of the 50 patients at any time during the 1 year treatment. The percentage of patients without bleeding increased gradually each month, from 24% in the second month to a relatively stable level of 80% in month 7 and thereafter. Twenty-seven patients (54%) did not complete the whole trial period; 15 of which discontinued the treatment within the first few months due to irregular bleeding. In patients who remained in the trial, a clear decrease in the frequency and intensity of the bleeding was observed with time. Bleeding was mostly light or consisted of spotting only. None of the post-trial biopsies showed proliferation or hyperplasia of the endometrium. The treatment resulted in a substantial decrease of climacteric symptoms (Kupperman index) within 4 months and was well tolerated. It was concluded that the continuous NETA/E₂-TTS treatment is an effective and safe alternative for the treatment of climacteric symptoms in selected patients.     

Biological and endocrine aspects of transdermal 17β-oestradiol administration in post-menopausal women

The plasma protein distribution of oestradiol (E₂) and oestrone (E₁) during transdermal E₂ administration (100 μg/24 hr) was studied in 12 post-menopausal women. The E₂ and E₁ levels observed were 43–83 pg/ml and 37–73 pg/ml, respectively. The levels of the free, albumin-bound and sex-hormone-binding globulin (SHBG) bound fractions were in the ranges 1.4–1.9%, 60–65% and 35–45%, respectively, in the case of E₂, and 2.8–3.0%, 80–89% and 15–20%, respectively, in that of E₁. The SHBG levels also remained unaltered. It was concluded that transdermal administration of E₂ at the dosage employed produces a physiological plasma protein distribution of E₂ and E₁ and does not affect liver protein production.     

An evolutionary perspective on the origin and ontogeny of menopause

The “grandmother hypothesis” proposes that menopause evolved because ancestral middle-aged women gained greater reproductive success from investing in extant genetic relatives than from continuing to reproduce [Williams GC. Pleiotropy, natural selection, and the evolution of senescence. Evolution 1957;11:398–411]. Because middle-aged women faced greater risks of maternal death during pregnancy and their offspring's infancy than did younger women, offspring of middle-aged women may not have received the needed level of prolonged maternal investment to survive to reproductive age. I put forward the “absent father hypothesis” proposing that reduced paternal investment linked with increasing maternal age was an additional impetus for the evolution of menopause. Reduced paternal investment was linked with increasing maternal age because men died at a younger age than their mates and because some men were increasingly likely to defect from their mateships as their mates aged. The absent father hypothesis is not an alternative to the grandmother hypothesis but rather a complement. It outlines an additional cost—reduced paternal investment—associated with continued reproduction by ancestral middle-aged women that could have been an additional impetus for the evolution of menopause. After reviewing additional explanations for the origin of menopause (“patriarch hypothesis,” “lifespan-artifact” hypotheses), I close by proposing a novel hypothesis for the ontogeny of menopause. According to the “adaptive onset hypothesis,” the developmental timing of menopause is a conditional reproductive strategy in which a woman's age at onset is influenced by the likelihood that any children she could produce would survive to reproductive age. Twelve variables predicted to be associated with age at onset and evidence that bears upon the predictions is discussed.     

Long-term post-menopausal hormone therapy and serum HDL-C, total cholesterol and triglycerides

Serum high-density lipoprotein cholesterol (HDL cholesterol), total cholesterol and triglyceride concentrations were determined in 158 post-menopausal women following long-term oral hormone replacement therapy. Oestradiol valerate (2 mg/day) was taken by 53 of the women and oestriol succinate (2 mg/day) by 42 others. The duration (means +/- SD) of the oestradiol valerate therapy was 6.4 (+/- 2.9) yr and of the oestriol succinate therapy 6.4 (+/- 2.3) yr. The remaining 63 women received oestradiol valerate (2 mg/day) combined sequentially with norgestrel (0.5 mg/day). The average duration of treatment with this combination was 3.3 (+/- 2.4) yr. The control group comprised 100 post-menopausal women who received no hormone therapy whatsoever. The HDL cholesterol levels in the women receiving oestradiol valerate were higher than those in the controls (P = 0.001) and in the women on oestradiol valerate plus norgestrel therapy (P less than 0.001). The HDL cholesterol levels in the oestriol succinate group did not differ significantly from those in the controls. The women receiving oestradiol valerate in combination with norgestrel had lower serum HDL cholesterol concentrations than the controls (P less than 0.05). Serum total cholesterol and triglyceride concentrations did not differ in either oestrogen group from those in the controls, but were lower in the oestradiol valerate-plus-norgestrel group than in the controls (P less than 0.001). There were no differences in serum total oestrogen, oestrone, oestradiol and oestriol levels between control subjects with normal HDL cholesterol concentrations and those with low concentrations.     

Skinfold thickness and long-term post-menopausal hormone therapy

Skinfold thickness was measured in 130 post-menopausal women treated with long-term hormone therapy. One group of 50 women took oestradiol valerate 2 mg/day for 3 wk out of 7, a second group comprising 19 women received oestriol succinate 2 mg/day and the remaining group of 61 women used oestradiol valerate 2 mg/day combined sequentially with norgestrel 0.5 mg/day. The duration of treatment in these groups was 6.3 ± 0.4, 6.4 ± 0.4 and 3.3 ± 0.3 yr, respectively. The control group was made up of a further 89 post-menopausal women. The skinfold thickness in all the treated groups was significantly greater than that in the controls.