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Effect of induced uterine retroversion on bone mass in rats
European Journal of Obstetrics & Gynecology and Reproductive Biology 83 (1999) 101–104
Original Article
Effect of induced uterine retroversion on bone mass in rats b ´ ´ a , Manuel Revilla b , Emma R. Hernandez ´ Ana Lopez-Castejon , Luis F. Villa b , Horacio Rico b , *, ´ Cortes ´ a Joaquın
b
a Department of Medical Specialties, University of Alcala´ , Madrid, Spain Department of Medicine, University of Alcala´ de Henares, 28801 Madrid, Spain
Received 23 July 1998; received in revised form 24 October 1998; accepted 19 November 1998
Abstract Objective: To evaluate the effect of surgical uterine retroversion on bone mass in rats. Study Design: Forty-five female Wistar rats were assigned randomly to three groups: 15 unmanipulated rats, 15 rats that underwent uterine retroversion, and 15 rats that underwent sham uterine retroversion (exposure of the uterus to air followed by closure of the abdominal cavity). Sixty days later the rats were killed and their femurs were dissected. Femurs were weighed and measured, and femoral bone mineral content (BMC) and bone mineral density (BMD) were determined by dual energy X-ray absorptiometry. Results: In the group of rats that underwent uterine retroversion, BMC, BMD, and BMC corrected for final body weight were significantly lower (P,0.001) than in the unmanipulated control and sham uterine retroversion groups. Conclusion: Our findings indicate that uterine retroversion induced a loss of bone mass. We could not determine the mechanism of bone loss; in our opinion, these problem merits further investigations, which currently occupy our interest. 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Uterine retroversion; Bone loss; Femur mass loss
1. Introduction The changes in bone mass that accompany menopause are well known [1]; bone mass is lost quickly in the early years of menopause, then more slowly [2]. Similar changes in bone mass occur after ovariectomy [3], and less intense changes have been described in perimenopause [4] and in women being treated with GnRH agonists [5]. These observations suggest that bone mass is dependent on gonadal state in women. It has been reported recently that women who undergo hysterectomy without ovariectomy also experience a loss of bone mass [6], which suggests the involvement of a uterine-dependent mechanism in the bone loss. This may lead one to consider whether uterine retroversion, a relatively common gynecologic disorder that produces disturbances in the uterus and adnexa [7], could be *Corresponding author. Tel.: 134-1-8854552; fax: 134-1-8854526
associated with changes in bone mass. The potential effect of uterine retroversion on bone mass has not been studied previously, so we evaluated the loss of bone mass after surgical uterine retroversion in rats. Rats are recognized as a suitable model for postovariectomy and osteoporosis because changes in bone mass in rats can be extrapolated to human beings [8].
2. Materials and methods A sample of 45 female Wistar rats, all 100 days-old, with mean weight of 250 g, were randomized into three groups based on their body weight. The rat with greatest and smallest weight were assigned each time alternatively to one group, so at the end of the randomization process the mean body weight of each group was comparable. The control group did not undergo any manipulation, one group underwent uterine retroversion, and the third group under-
0301-2115 / 99 / $ – see front matter 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S0301-2115( 98 )00304-2
102
´ ´ et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 83 (1999) 101 – 104 A. Lopez-Castejon
went sham retroversion, in which the uterus was exposed to air followed by closure of the abdomen. Sample size was calculated in a pilot study after determining the variability of densitometric measurements. The standard deviation (SD) of bone mineral density (BMD) was 10 and the hypothesized difference between groups was 15 units. Alpha risk was 0.05 and beta risk 0.20 for a two-sided control. The number of animals in each group was established according to the formula n c 5n e 52(Za 1 Zb )2 s 2 /D2 , where n c 5number of animals in the control group; n e 5number of animals in the experimental group; s 5SD; D5difference to be detected. All the rats were fed type A04 feed (Panlab, Barcelone, Spain), containing 7.1 g / kg calcium and 5 g / kg phosphorus, which had a calorie value of 3100 kcal / kg. Water was given ad libitum. The rats were maintained for the 60 days that the experiment lasted in the animal laboratory of the University of Alcala´ de Henares. Their living conditions (12 h light and 12 h darkness, mean room temperature 228C, and 50% environmental humidity) and diet conformed to current guidelines for care and use of the animals, approved by the local institution and European Union. To create the uterine retroversion, animals were anesthetized intraperitoneally with ketamine hydrochloride (10 mg / kg) and acepromazine (3 mg / kg). The abdomen was shaved and the skin was cleaned with 70% ethanol followed by povidone solution. A longitudinal midline incision was made in the umbilical region to expose the rectus abdominous muscle and abdominal cavity. The urinary bladder was drawn forward and uterine horns were identified. Each hemiuterus was fixed to the presacral fascia with 0000 Vicryl sutures (Ethican, Somerville, NJ). The abdominal musculature was sutured and the skin was closed with staples. The uterine retroversion was always performed by the same person (ALC). The manipulated rats were controlled to observe the development of intestinal function alterations and infection or dehiscence of surgical sutures. A vaginal cytology was performed on each rat 1 week before the termination of the experiment. On day 60 of the experiment, rats were anesthetized with sodium thiopental 4 mg / 100 g body weight and killed
by sectioning the aorta. Blood was collected and stored at 220C for estradiol determination. Estradiol was measured in the samples using a 1230 Arcus fluorimeter (LKB, Turku, Finland). All samples were measured in the same assay to eliminate interassay variation. In our laboratory these biochemical determinations have intra- and interassay coefficient of variation of less than 6% referred to a concentration of 25 pg / ml. The femurs of the rats were removed and disarticulated. Femurs were placed in an 858C water bath to remove soft tissue. Femur length was measured with a calliper, femur weight was measured on a precision scale, and bone densitometry was carried out with techniques for small animals [9] using a Norland X-R 26 instrument (Norland Co., Fort Atkinson, WI) to determine femoral bone mineral content (BMC) and BMD. The coefficient of variation of BMC and BMD found in 25 measurements made in five different femurs at an interval of 2 weeks were both 0.4%. Bone mineral content was corrected for final body weight to obtain the BMC5weight ratio in milligrams per gram. The femur length, weight, BMC, BMC5weight ratio, and BMD were analyzed as continuous variables and the groups (unmanipulated controls, sham uterine retroversion, and true uterine retroversion) as nominal variables using analysis of variance with post hocs Newman-Keuls multiple comparison test in relation to the nominal variables. Simple linear regression analysis was done for the final body weight and femoral BMC of each separate group of rats to determine the influence of body weight on bone mass. The length and weight of the dissected femurs were also examined by simple regression analysis. All statistical analyses were done on a Macintosh computer (Apple Computer, Cupertino, CA) running Statview 4.02 (Abacus Concepts, Berkeley, CA).
3. Results Our findings are summarized in Table 1. The weight of the femur and femoral BMC, BMD, and BMC5weight ratio were significantly lower in the uterine retroversion group than in the other two groups.
Table 1 Body weight and femoral measurements Controls Initial body weight (g) Final body weight (g) Femur length (mm) Femur weight (mg) BMC (mg) BMC / W (mg / g) BMD (mg / cm 2 )
Unmanipulated retroversion n515
Uterine retroversion n515
Sham
237619 288635 34.360.8 768641 404693 1.4060.08 151611
249626 290639 34.260.9 693687 342642 1.1760.08 11767
246620 300620 33.960.9 751676 407691 1.3560.08 149610
n515
Data are presented as mean6standard deviation. BMC5bone mineral content; BMC / W5bone mineral content5weight ratio; BMD5bone mineral density.
´ ´ et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 83 (1999) 101 – 104 A. Lopez-Castejon
There were significant correlations between final body weight and femoral BMC (r50.71, P,0.001 in unmanipulated controls; r50.65, P,0.005 in sham uterine retroversion; and r50.58, P,0.05 in true uterine retroversion) and significant correlations between femur length and femur weight (r50.81, P,0.001 in unmanipulated controls, r5 0.75, P,0.005 in sham uterine retroversion; and r50.67, P,0.05 in true uterine retroversion). No differences were observed in the levels of estradiol in the three groups of rats (21.266.7, 19.866.1, and 20.365.9 pg / ml in the control, sham, and uterine retroversion groups, respectively), nor in the vaginal cytology performed 1 week before the termination of the experiment (data not shown).
4. Comment Our results indicate that femoral bone mass was lower 2 months after surgery in rats that underwent uterine retroversion compared with the unmanipulated control and sham uterine retroversion groups. Specifically, femoral BMC and BMD determined by dual-energy X-ray absorptiometry, BMC5weight ratio, and femur weight were significantly lower. The rat model used is considered adequate for extrapolating changes in bone mass to humans [8]. Among the advantages of using rats are [10]: (1) studies can be carried out under standardized conditions and are highly reproducible; (2) studies are relatively inexpensive; (3) studies can be carried out quickly; (4) rats have lamellar bone and trabecular bone remodelling similar to humans; (5) the skeletal anatomy of rats is similar to humans (periosteum is lacking in the femoral neck), and (6) ovariectomy and natural menopause in rats induces changes similar to those seen in women with age and menopause [10]. Another important aspect is the similar response to treatment of bone in rats and human beings, as reported by Abe et al. [11]. This means that changes in bone mass in rats can be extrapolated to human beings. Dual-energy X-ray absorptiometry is recognized as a useful and exact densitometric technique for small animal studies [9,12]. The low coefficient of variation that we found (0.4%) demonstrates its precision and validates it as a suitable technique for bone mass studies [12]. Using dual-energy X-ray absorptiometry we found that uterine retroversion in normal rats induced a significant loss in bone mass within 2 months, a time interval equivalent to 5 years of a woman’s life [13] The correlation between final body weight and femoral BMC, also observed in our earlier study [9], confirmed the importance of body weight on bone mass in rats, which also occurs in humans [14]. This reaffirms the usefulness of rats as an experimental model because the changes in bone mass that they experience are similar to those observed in humans. The final weight of the rats did not change, perhaps due
103
to the lack of postoperative complications. Body weight is the main determinant of bone mass in humans [14]. In rats, body weight correlates significantly with BMC [9]. In our study, the BMC5weight ratio (femoral BMC corrected for final body weight to eliminate the influence of weight) was lower in the rats that underwent uterine retroversion than in the unmanipulated control and sham uterine retroversion groups, which confirms that uterine retroversion induced a loss of bone mass. It is difficult to imagine how uterine retroversion led to the loss of bone mass observed in this study, but it probably was related to the intimate relation between uterus and ovaries [15]. The uterine retroversion technique produces ovarian and vascular disturbances that could have contributed. For instance, it has been observed that simple hysterectomy without ovariectomy induces early menopause [16]. The fact that oestradiol levels were not different in the three groups may indicate that ovary failure was not the cause of the observed bone mass loss in the rats of our study. Nevertheless, additional experiments are necessary to discard definitely a loss of ovarian function, given the limitations of quantitative determination of oestrogens such as uncertainty about cross reacting steroids or widely varying ranges of serum levels. We cannot compare our findings to those of other authors because, to our knowledge based on extensive review of the literature of the last 15 years quoted in Medline, no other studies have been published on this topic. However, it is clear that uterine retroversion produced a 15% loss in bone mass compared with unmanipulated controls. The frequency of uterine retroversion in women is fairly high. The evaluation of how this potential loss of bone mass might affect the eventual development of osteoporosis, the intrinsic relation between uterine retroversion and bone loss, and the mechanisms of bone loss are problems that must be resolved and currently occupy our interest. Additional studies are needed to ascertain the mechanisms and specificity of the observed changes; as are an increased in circulating cytokines due to trauma and / or a temporary arrest of ovary hormone production, among possible others, and whether they are extrapolable to what happens in humans.
References ´ ´ [1] Rico H, Hernandez ER, Seco C, Villa LF, Fernandez S. Quantitative peripheral computed tomodensitometric study of cortical and trabecular bone mass in relation with menopause. Maturitas 1994;18:183–9. ´ [2] Rico H, Revilla M, Villa LF, Lopez-Alonso A. The relation of total body bone mineral (TBBM) to anthropometric variables in postmenopausal women, and contributions of age and years since menopause to TBBM loss. Clin Rheumatol 1993;12:475–8. [3] Orimo H, Shiraki M, Inoue S. Estrogen and bone. Osteoporosis Int 1993;3(suppl 1):S153–6. ´ ´ [4] Hernandez ER, Seco-Durban C, Revilla M, Gonzalez-Riola J, Rico
104
[5]
[6]
[7] [8] [9]
[10]
´ ´ et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 83 (1999) 101 – 104 A. Lopez-Castejon H. Evaluation of bone density with peripheral quantitative computed tomography in healthy premenopausal, perimenopausal, and postmenopausal women. Age Ageing 1995;24:447–50. Rico H, Arnanz F, Revilla M, Perera S, Iritia M, Villa LF, et al. Total and regional bone mineral content in women treated with GnRH agonists. Calcif Tissue Int 1993;52:354–7. Watson NR, Studd JWW, Garnett T, Savvas M, Milligan P. Bone loss after hysterectomy with ovarian conservation. Obstet Gynecol 1995;86:72–7. ´ ´ Bret AJ, Bardiaux M. Les deviations uterines. Encycl Med Chir Gynecol 1980;250:1–12. Jee WSS. Animal models in the prevention and treatment of osteopenia: Forward. Bone 1995;17(4 Suppl):113S–4S. ´ Rico H, Amo C, Revilla M, Arribas I, Gonzalez-Riola J, Villa LF, et al. Etidronate versus Clodronate in the prevention of postovariectomy bone loss. An experimental study in rats. Clin Exp Rheumatol 1994;12:301–4. Mosekilde L. Assessing bone quality-animal models in preclinical osteoporosis research. Bone 1995;17(4 Suppl):343S–52S.
[11] Abe T, Chow JWM, Lean JM, Chambers TJ. Estrogen does not restore bone lost after ovariectomy in the rat. J Bone Miner Res 1993;8:831–8. [12] Yamauchi H, Kushida K, Yamazaki K, Inoue T. Assessment of spine bone mineral density in ovariectomized rats using DXA. J Bone Miner Res 1995;10:1033–9. [13] Thompson DD, Simmons HA, Pirie CM, Ke HZ. FDA guidelines and animal models for osteoporosis. Bone 1995;17(Suppl):125S– 33S. [14] Rico H, Revilla M, Villa LF, Alvarez de Buergo M, Ruiz-Contreras D. Determinants of total and regional bone mineral content and density in postpubertal normal women. Metabolism 1994;43:263–6. [15] Anderson LL, Musah AI, Uterine control of ovarian function. In: Wynn RM, Jollie WP, editors. Biology of the uterus, 2nd ed. New York: Plenum Publishing Co., 1989:505–558. [16] Siddle N, Sarrel P, Whitehead MI. The effect of hysterectomy on the age at ovarian failure: Identification of a subgroup of women with premature loss of ovarian function and literature review. Fertil Steril 1987;47:94–100.
Original Article
Effect of induced uterine retroversion on bone mass in rats b ´ ´ a , Manuel Revilla b , Emma R. Hernandez ´ Ana Lopez-Castejon , Luis F. Villa b , Horacio Rico b , *, ´ Cortes ´ a Joaquın
b
a Department of Medical Specialties, University of Alcala´ , Madrid, Spain Department of Medicine, University of Alcala´ de Henares, 28801 Madrid, Spain
Received 23 July 1998; received in revised form 24 October 1998; accepted 19 November 1998
Abstract Objective: To evaluate the effect of surgical uterine retroversion on bone mass in rats. Study Design: Forty-five female Wistar rats were assigned randomly to three groups: 15 unmanipulated rats, 15 rats that underwent uterine retroversion, and 15 rats that underwent sham uterine retroversion (exposure of the uterus to air followed by closure of the abdominal cavity). Sixty days later the rats were killed and their femurs were dissected. Femurs were weighed and measured, and femoral bone mineral content (BMC) and bone mineral density (BMD) were determined by dual energy X-ray absorptiometry. Results: In the group of rats that underwent uterine retroversion, BMC, BMD, and BMC corrected for final body weight were significantly lower (P,0.001) than in the unmanipulated control and sham uterine retroversion groups. Conclusion: Our findings indicate that uterine retroversion induced a loss of bone mass. We could not determine the mechanism of bone loss; in our opinion, these problem merits further investigations, which currently occupy our interest. 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Uterine retroversion; Bone loss; Femur mass loss
1. Introduction The changes in bone mass that accompany menopause are well known [1]; bone mass is lost quickly in the early years of menopause, then more slowly [2]. Similar changes in bone mass occur after ovariectomy [3], and less intense changes have been described in perimenopause [4] and in women being treated with GnRH agonists [5]. These observations suggest that bone mass is dependent on gonadal state in women. It has been reported recently that women who undergo hysterectomy without ovariectomy also experience a loss of bone mass [6], which suggests the involvement of a uterine-dependent mechanism in the bone loss. This may lead one to consider whether uterine retroversion, a relatively common gynecologic disorder that produces disturbances in the uterus and adnexa [7], could be *Corresponding author. Tel.: 134-1-8854552; fax: 134-1-8854526
associated with changes in bone mass. The potential effect of uterine retroversion on bone mass has not been studied previously, so we evaluated the loss of bone mass after surgical uterine retroversion in rats. Rats are recognized as a suitable model for postovariectomy and osteoporosis because changes in bone mass in rats can be extrapolated to human beings [8].
2. Materials and methods A sample of 45 female Wistar rats, all 100 days-old, with mean weight of 250 g, were randomized into three groups based on their body weight. The rat with greatest and smallest weight were assigned each time alternatively to one group, so at the end of the randomization process the mean body weight of each group was comparable. The control group did not undergo any manipulation, one group underwent uterine retroversion, and the third group under-
0301-2115 / 99 / $ – see front matter 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S0301-2115( 98 )00304-2
102
´ ´ et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 83 (1999) 101 – 104 A. Lopez-Castejon
went sham retroversion, in which the uterus was exposed to air followed by closure of the abdomen. Sample size was calculated in a pilot study after determining the variability of densitometric measurements. The standard deviation (SD) of bone mineral density (BMD) was 10 and the hypothesized difference between groups was 15 units. Alpha risk was 0.05 and beta risk 0.20 for a two-sided control. The number of animals in each group was established according to the formula n c 5n e 52(Za 1 Zb )2 s 2 /D2 , where n c 5number of animals in the control group; n e 5number of animals in the experimental group; s 5SD; D5difference to be detected. All the rats were fed type A04 feed (Panlab, Barcelone, Spain), containing 7.1 g / kg calcium and 5 g / kg phosphorus, which had a calorie value of 3100 kcal / kg. Water was given ad libitum. The rats were maintained for the 60 days that the experiment lasted in the animal laboratory of the University of Alcala´ de Henares. Their living conditions (12 h light and 12 h darkness, mean room temperature 228C, and 50% environmental humidity) and diet conformed to current guidelines for care and use of the animals, approved by the local institution and European Union. To create the uterine retroversion, animals were anesthetized intraperitoneally with ketamine hydrochloride (10 mg / kg) and acepromazine (3 mg / kg). The abdomen was shaved and the skin was cleaned with 70% ethanol followed by povidone solution. A longitudinal midline incision was made in the umbilical region to expose the rectus abdominous muscle and abdominal cavity. The urinary bladder was drawn forward and uterine horns were identified. Each hemiuterus was fixed to the presacral fascia with 0000 Vicryl sutures (Ethican, Somerville, NJ). The abdominal musculature was sutured and the skin was closed with staples. The uterine retroversion was always performed by the same person (ALC). The manipulated rats were controlled to observe the development of intestinal function alterations and infection or dehiscence of surgical sutures. A vaginal cytology was performed on each rat 1 week before the termination of the experiment. On day 60 of the experiment, rats were anesthetized with sodium thiopental 4 mg / 100 g body weight and killed
by sectioning the aorta. Blood was collected and stored at 220C for estradiol determination. Estradiol was measured in the samples using a 1230 Arcus fluorimeter (LKB, Turku, Finland). All samples were measured in the same assay to eliminate interassay variation. In our laboratory these biochemical determinations have intra- and interassay coefficient of variation of less than 6% referred to a concentration of 25 pg / ml. The femurs of the rats were removed and disarticulated. Femurs were placed in an 858C water bath to remove soft tissue. Femur length was measured with a calliper, femur weight was measured on a precision scale, and bone densitometry was carried out with techniques for small animals [9] using a Norland X-R 26 instrument (Norland Co., Fort Atkinson, WI) to determine femoral bone mineral content (BMC) and BMD. The coefficient of variation of BMC and BMD found in 25 measurements made in five different femurs at an interval of 2 weeks were both 0.4%. Bone mineral content was corrected for final body weight to obtain the BMC5weight ratio in milligrams per gram. The femur length, weight, BMC, BMC5weight ratio, and BMD were analyzed as continuous variables and the groups (unmanipulated controls, sham uterine retroversion, and true uterine retroversion) as nominal variables using analysis of variance with post hocs Newman-Keuls multiple comparison test in relation to the nominal variables. Simple linear regression analysis was done for the final body weight and femoral BMC of each separate group of rats to determine the influence of body weight on bone mass. The length and weight of the dissected femurs were also examined by simple regression analysis. All statistical analyses were done on a Macintosh computer (Apple Computer, Cupertino, CA) running Statview 4.02 (Abacus Concepts, Berkeley, CA).
3. Results Our findings are summarized in Table 1. The weight of the femur and femoral BMC, BMD, and BMC5weight ratio were significantly lower in the uterine retroversion group than in the other two groups.
Table 1 Body weight and femoral measurements Controls Initial body weight (g) Final body weight (g) Femur length (mm) Femur weight (mg) BMC (mg) BMC / W (mg / g) BMD (mg / cm 2 )
Unmanipulated retroversion n515
Uterine retroversion n515
Sham
237619 288635 34.360.8 768641 404693 1.4060.08 151611
249626 290639 34.260.9 693687 342642 1.1760.08 11767
246620 300620 33.960.9 751676 407691 1.3560.08 149610
n515
Data are presented as mean6standard deviation. BMC5bone mineral content; BMC / W5bone mineral content5weight ratio; BMD5bone mineral density.
´ ´ et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 83 (1999) 101 – 104 A. Lopez-Castejon
There were significant correlations between final body weight and femoral BMC (r50.71, P,0.001 in unmanipulated controls; r50.65, P,0.005 in sham uterine retroversion; and r50.58, P,0.05 in true uterine retroversion) and significant correlations between femur length and femur weight (r50.81, P,0.001 in unmanipulated controls, r5 0.75, P,0.005 in sham uterine retroversion; and r50.67, P,0.05 in true uterine retroversion). No differences were observed in the levels of estradiol in the three groups of rats (21.266.7, 19.866.1, and 20.365.9 pg / ml in the control, sham, and uterine retroversion groups, respectively), nor in the vaginal cytology performed 1 week before the termination of the experiment (data not shown).
4. Comment Our results indicate that femoral bone mass was lower 2 months after surgery in rats that underwent uterine retroversion compared with the unmanipulated control and sham uterine retroversion groups. Specifically, femoral BMC and BMD determined by dual-energy X-ray absorptiometry, BMC5weight ratio, and femur weight were significantly lower. The rat model used is considered adequate for extrapolating changes in bone mass to humans [8]. Among the advantages of using rats are [10]: (1) studies can be carried out under standardized conditions and are highly reproducible; (2) studies are relatively inexpensive; (3) studies can be carried out quickly; (4) rats have lamellar bone and trabecular bone remodelling similar to humans; (5) the skeletal anatomy of rats is similar to humans (periosteum is lacking in the femoral neck), and (6) ovariectomy and natural menopause in rats induces changes similar to those seen in women with age and menopause [10]. Another important aspect is the similar response to treatment of bone in rats and human beings, as reported by Abe et al. [11]. This means that changes in bone mass in rats can be extrapolated to human beings. Dual-energy X-ray absorptiometry is recognized as a useful and exact densitometric technique for small animal studies [9,12]. The low coefficient of variation that we found (0.4%) demonstrates its precision and validates it as a suitable technique for bone mass studies [12]. Using dual-energy X-ray absorptiometry we found that uterine retroversion in normal rats induced a significant loss in bone mass within 2 months, a time interval equivalent to 5 years of a woman’s life [13] The correlation between final body weight and femoral BMC, also observed in our earlier study [9], confirmed the importance of body weight on bone mass in rats, which also occurs in humans [14]. This reaffirms the usefulness of rats as an experimental model because the changes in bone mass that they experience are similar to those observed in humans. The final weight of the rats did not change, perhaps due
103
to the lack of postoperative complications. Body weight is the main determinant of bone mass in humans [14]. In rats, body weight correlates significantly with BMC [9]. In our study, the BMC5weight ratio (femoral BMC corrected for final body weight to eliminate the influence of weight) was lower in the rats that underwent uterine retroversion than in the unmanipulated control and sham uterine retroversion groups, which confirms that uterine retroversion induced a loss of bone mass. It is difficult to imagine how uterine retroversion led to the loss of bone mass observed in this study, but it probably was related to the intimate relation between uterus and ovaries [15]. The uterine retroversion technique produces ovarian and vascular disturbances that could have contributed. For instance, it has been observed that simple hysterectomy without ovariectomy induces early menopause [16]. The fact that oestradiol levels were not different in the three groups may indicate that ovary failure was not the cause of the observed bone mass loss in the rats of our study. Nevertheless, additional experiments are necessary to discard definitely a loss of ovarian function, given the limitations of quantitative determination of oestrogens such as uncertainty about cross reacting steroids or widely varying ranges of serum levels. We cannot compare our findings to those of other authors because, to our knowledge based on extensive review of the literature of the last 15 years quoted in Medline, no other studies have been published on this topic. However, it is clear that uterine retroversion produced a 15% loss in bone mass compared with unmanipulated controls. The frequency of uterine retroversion in women is fairly high. The evaluation of how this potential loss of bone mass might affect the eventual development of osteoporosis, the intrinsic relation between uterine retroversion and bone loss, and the mechanisms of bone loss are problems that must be resolved and currently occupy our interest. Additional studies are needed to ascertain the mechanisms and specificity of the observed changes; as are an increased in circulating cytokines due to trauma and / or a temporary arrest of ovary hormone production, among possible others, and whether they are extrapolable to what happens in humans.
References ´ ´ [1] Rico H, Hernandez ER, Seco C, Villa LF, Fernandez S. Quantitative peripheral computed tomodensitometric study of cortical and trabecular bone mass in relation with menopause. Maturitas 1994;18:183–9. ´ [2] Rico H, Revilla M, Villa LF, Lopez-Alonso A. The relation of total body bone mineral (TBBM) to anthropometric variables in postmenopausal women, and contributions of age and years since menopause to TBBM loss. Clin Rheumatol 1993;12:475–8. [3] Orimo H, Shiraki M, Inoue S. Estrogen and bone. Osteoporosis Int 1993;3(suppl 1):S153–6. ´ ´ [4] Hernandez ER, Seco-Durban C, Revilla M, Gonzalez-Riola J, Rico
104
[5]
[6]
[7] [8] [9]
[10]
´ ´ et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 83 (1999) 101 – 104 A. Lopez-Castejon H. Evaluation of bone density with peripheral quantitative computed tomography in healthy premenopausal, perimenopausal, and postmenopausal women. Age Ageing 1995;24:447–50. Rico H, Arnanz F, Revilla M, Perera S, Iritia M, Villa LF, et al. Total and regional bone mineral content in women treated with GnRH agonists. Calcif Tissue Int 1993;52:354–7. Watson NR, Studd JWW, Garnett T, Savvas M, Milligan P. Bone loss after hysterectomy with ovarian conservation. Obstet Gynecol 1995;86:72–7. ´ ´ Bret AJ, Bardiaux M. Les deviations uterines. Encycl Med Chir Gynecol 1980;250:1–12. Jee WSS. Animal models in the prevention and treatment of osteopenia: Forward. Bone 1995;17(4 Suppl):113S–4S. ´ Rico H, Amo C, Revilla M, Arribas I, Gonzalez-Riola J, Villa LF, et al. Etidronate versus Clodronate in the prevention of postovariectomy bone loss. An experimental study in rats. Clin Exp Rheumatol 1994;12:301–4. Mosekilde L. Assessing bone quality-animal models in preclinical osteoporosis research. Bone 1995;17(4 Suppl):343S–52S.
[11] Abe T, Chow JWM, Lean JM, Chambers TJ. Estrogen does not restore bone lost after ovariectomy in the rat. J Bone Miner Res 1993;8:831–8. [12] Yamauchi H, Kushida K, Yamazaki K, Inoue T. Assessment of spine bone mineral density in ovariectomized rats using DXA. J Bone Miner Res 1995;10:1033–9. [13] Thompson DD, Simmons HA, Pirie CM, Ke HZ. FDA guidelines and animal models for osteoporosis. Bone 1995;17(Suppl):125S– 33S. [14] Rico H, Revilla M, Villa LF, Alvarez de Buergo M, Ruiz-Contreras D. Determinants of total and regional bone mineral content and density in postpubertal normal women. Metabolism 1994;43:263–6. [15] Anderson LL, Musah AI, Uterine control of ovarian function. In: Wynn RM, Jollie WP, editors. Biology of the uterus, 2nd ed. New York: Plenum Publishing Co., 1989:505–558. [16] Siddle N, Sarrel P, Whitehead MI. The effect of hysterectomy on the age at ovarian failure: Identification of a subgroup of women with premature loss of ovarian function and literature review. Fertil Steril 1987;47:94–100.