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a Reproductive Biology Research, Indian Institute of Chemical Biology, Jadavpur, Calcutta 700 032, India
b Institute of Reproductive Medicine, DD 18/5/1, Salt Lake City, Calcutta 700 064, India
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult female Sprague-Dawley rats procured from the random-bred colony of our institute's animal house were maintained under standard temperature (23 ± 1°C) and light (12L:12D) conditions with food and water ad libitum. All rats were allowed to acclimatize in the experimental cage for 15 days, during which they were monitored daily for estrous cycles by vaginal lavage. Rats exhibiting 3–4 consecutive regular cycles (4 or 5 days) were selected for the study.
Surgical Implantation of Endometrial Tissue
Endometrial tissue was implanted by surgical technique as described by Vernon and Wilson [9]. Briefly, rats on the morning of proestrus were subjected to light ether anesthesia. Under aseptic conditions, the abdominal cavity was entered through a small midventral incision and the mesentery was exposed. One uterine horn was removed and dissected along its longitudinal axis. Pieces of uterine tissue (1–2 mm2; 4.2 ± 0.6 mg) were prepared and thoroughly rinsed with Ham's F-10 nutrient medium equilibrated at 37°C. For each rat, 5 of the uterine explants were attached to the mesentery adjacent to blood vessels with a single tie of nylon suture; the serosal layer of the square was placed in direct apposition to the peritoneal surface, and the endometrial surface faced the lumen of the peritoneal cavity. To prepare sham-operated controls, rats were subjected to unilateral hysterectomy followed by attachment of 2-mm wedges of fat, removed from the utero-ovarian ligament, at 5 sites on the mesentery. After each surgical procedure the abdominal cavity was irrigated with 1 ml of sterile saline. The abdominal incision was then closed by sutures. The animals were allowed to recuperate for 1 wk and then assigned to one of the groups detailed below. In all groups, if not otherwise stated, observations were made during three specific phases of the study: Days 20–30 (phase I), 50–70 (phase II), and 90–100 (phase III) posttransplantation.
Experiment 1: Assessment of Endometrial Growth in the Peritoneum and Its Effect on ReproductiveCycle and Ovulation
Rats were monitored daily for reproductive cytology until autopsy. To examine the effect of PEI on ovulation, rats were killed at noon of estrus. The oviduct and uterine horns were separated from the ovaries and flushed with 0.9% saline under a dissecting microscope; after addition of hyaluronidase, they were examined for the presence or absence of eggs, and if present, eggs were counted. Anovulation was defined as the absence of ovum in the oviduct or uterine horns. After autopsy, the peritoneal cavities of all rats were systematically examined for the presence of endometrial-like tissue. If present, endometrial implants were surgically dissected out from the adherent tissue, weighed, and processed for histological examination.
Experiment 2: Effect of PEI on Mating Rate,Pregnancy, and Pseudopregnancy
To examine the influence of PEI on reproductive potential, after confirmation of pelvic endometrial growth by laparotomy, rats were subjected to induction of pseudopregnancy during various phases after surgery. Pseudopregnancy was induced either by copulomimetic stimulation with a glass rod at 08–00 h of estrus (Day 1 of pseudopregnancy) or by overnight exposure of proestrous females to vasectomized males. Continuous diestrous phase for at least 8 consecutive days was used to confirm pseudopregnancy. The length of pseudopregnancy was considered to be the number of days between cervical stimulation and appearance of the next proestrus. The animals were killed on the day of termination of pseudopregnancy (proestrus), when the condition of the endometriotic implants was assessed. Another group of implanted rats (phase I, phase II, and phase III) were co-caged with males of proven fertility in the ratio 2:1 in the afternoon of their proestrus. The next morning, mating was confirmed by the presence of spermatozoa in the vaginal lavage. The mating rate (number of rats with sperm-positive vaginal lavage per 100 rats exposed to breeder male) was recorded. The rats were autopsied 21 days after mating, when the presence and absence of fetuses were noted for determination of wastage of pregnancy. A subset of mated rats from the Day 50 to 70 posttransplant group were autopsied on Day 7, when the number of implantation sites and functional corpora lutea in these animals was counted. Another subset of rats from the same group were laparotomized under ether anesthesia on Day 7, when the numbers of implantation sites were counted. The incision was closed by sutures, and the rats were maintained under postoperative care until Day 21, when they were autopsied. The presence and absence of fetuses were noted and the number of fetuses was recorded.
Experiment 3: Effect of PEI on Hormone Concentrations Preceding Ovulation
The effect of PEI on reproductive hormones was assessed only during the middle phase of the study, i.e., 50–70 days posttransplantation. The rats were anesthetized between 1600 and 1700 h on the day of proestrus, and blood was collected by heart puncture. The serum procured after centrifugation was stored at -20°C until analysis by RIA for LH, estradiol (E2), and progesterone (P4).
RIA was performed using the previously validated method of Ray et al. [10]. LH concentration was determined by double-antibody RIA using the method and immunoreagents supplied by Dr. A.F. Parlow of the NHPP, NIDDK in Torrance, California. The values were expressed in terms of NIDDK-r-LH-RP-3. E2 and P4 were assayed using kits purchased from ICN Biomedicals, Inc. (Costa Mesa, CA). As specified in the kit literature, P4 antisera had the highest cross-reactivity (2.5%) with 17-hydroxyprogesterone, and E2 antisera had 6.2% and 1.4% cross-reactivities with estrone and estriol, respectively. The assay sensitivities (95% confidence limits of buffer control), as determined in our laboratory, were 0.6 ng/ml LH, 10 pg/ml E2, and 0.2 ng/ml P4. The coefficients of variation within the LH, E2, and P4 assays were 6%, 8.5%, and 5.5%, respectively. The respective interassay coefficients of variation of LH, E2, and P4 were 11%, 12.5%, and 9%.
Statistical Analysis
Values were expressed as means ± SD. Rates of mating, ovulation, pregnancy, and pseudopregnancy were compared using chi-square test. Student's t-test was used to compare mean fetus number and hormone levels. Differences were adjudged significant at a level of p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Normal cyclical function was resumed by the third week after surgery in both the sham-operated control and experimental rats as assessed by vaginal cytology of the characteristic 4- or 5-day estrous cycles that continued until the final phase of the study.
Ectopic Growth of Endometrial Tissue
The ectopic endometrial tissue developed into highly vascularized nodes in 87% of the recipient rats at 3–5 sites. The implants had grown gradually from 4.2 ± 0.6 mg (mean ± SD) at the time of transplantation to 10.3 ± 4.2 mg, 34.5 ± 14.4 mg, and 29.8 ± 13.0 mg, respectively, during the three consecutive phases of the study. Most of the implanted sites in both sham-operated and experimental rats were surrounded by semi-adherent fat deposits, while the ovary-oviduct unit remained largely free from adhesions. Rats that failed to show appreciable growth of endometrial implants at any of the five sites of transplantation, or developed infection at the transplantation site (as assessed by the presence of pus in thickly encapsulated large spherical mass), or had severe adhesions in areas surrounding the ovary were discarded from the study.
Histological examination of the uterine explants showed the presence of stromal and endometrial epithelium and endometrial-like glands.
Ovulation (Table 1)
During the first 20–30 days posttransplantation, no alteration in ovulation rate was evident under the influence of PEI. Ovulated eggs were present in the fallopian tubes of all the animals in the sham-operated as well as experimental groups. However, during the 50–70 days postsurgery, 43% (6 of 14) of the experimental rats failed to ovulate; this contrasted with 100% (8 of 8) ovulation in sham-operated rats. At 90–100 days after transplantation, the rate of ovulation was partially restored. Anovulation in PEI rats was recorded to be 20% (2 of 10), while no failure was observed in the sham-operated control group.
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Experiment 2: Mating Receptivity (Table 1)
All sham-operated rats in each of the three phases of the study conceived after a single mating as evidenced by presence of viable intrauterine fetuses on Day 21 of pregnancy. In the experimental group, significant loss of mating receptivity was evidenced during the 50–70 days posttransplantation. During this phase, 44% (12 of 27) of the rats exhibited no sperm in vaginal smears on the day after overnight exposure to a breeder male. During the first and third phase of the study, mating failed on 14% (1 of 7) and 13% (1 of 8) of occasions, respectively.
In sham-operated control rats, all sperm-positive matings during the three phases of the study led to successful pregnancy. In contrast, in PEI rats, 17% (1 of 6), 34% (5 of 15), and 29% (2 of 7) of pregnancies, respectively, during the three phases of the study terminated prematurely (Table 1). On Day 7 postcoitus, number of implantation sites/functional corpora lutea was statistically comparable between the sham-operated control (4.7 ± 1.3/5.4 ± 1.7) and the PEI rats (4.4 ± 1.7/5.3 ± 1.8) (Table 2). In the sham control group, the mean number of viable fetuses on Day 21 of pregnancy (4.3 ± 0.7) did not differ significantly from the mean number of implantation sites noted on Day 7 (4.7 ± 1.3). In the PEI rats, however, the number of viable fetuses decreased from 4.3 ± 1.3 on Day 7 to 1.9 ± 1.4 on Day 21.
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Pseudopregnancy (Table 3)
All sham-operated rats responded to the copulomimetic stimulation to exhibit a mean 11 ± 2 days of pseudopregnancy. In contrast, 43% (3 of 7), 100% (12 of 12), and 37% (3 of 8) rats with ectopic endometrial implants did not respond to the cervical stimulation, respectively, during the three phases of the study. However, during the maximum effective phase (50–70 days posttransplantation) when cervical stimulus was given in the form of sterile mating, 46% (6 of 13) of experimental rats exhibited the development and maintenance of pseudopregnancy (11 ± 3 days).
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Experiment 3: Effect on Preovulatory Hormone Levels (Table 4)
The mean serum E2 concentration (pg/ml) of PEI rats (53.1 ± 33.0) did not differ significantly from that of sham control rats (72.1 ± 14.1). However, E2 levels in 48% (10 of 21) of the PEI rats were below the lowest value (53.6 pg/ml) observed in the sham control group. The evening proestrous levels of P4 (ng/ml; control: 27.8 ± 7.7 vs. PEI: 15.9 ± 9.6) and LH (ng/ml; control: 13.3 ± 3.9 vs. PEI: 7.2 ± 4.1) were significantly lower (p < 0.01) in the PEI rats.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The essential prerequisites in the process of ovulation are a complex sequence of hormonal events. These include a timed preovulatory rise of threshold level of E2 followed by an ovulatory LH peak and subsequent release of P4. In the rat, preovulatory E2 reaches its peak level in the afternoon of proestrus. Release of LH for ovulation occurs under the positive feedback influence of E2 during the afternoon on the same day [14]. When measuring on the afternoon of proestrus, we observed a tendency toward lower levels of E2 and significantly reduced serum LH in the rats with PEI. The mean E2 levels did not differ significantly; however, individual E2 levels in ~50% of the PEI rats were lower than the lowest concentration of E2 observed in the sham control group. This implies that at least a sizable population of PEI rats suffered from hypoestrogenic ovarian function. Possibly these rats represent the subpopulation that exhibited anovulation or loss of mating receptivity. Lower preovulatory LH level was possibly due to lower level of preovulatory E2, and failure of ovulation was a secondary consequence to this altered hormonal milieu.
In a female rat with the proper hormonal milieu, mating receptivity appears in the afternoon or early evening of proestrus and persists until the morning of estrus [14]. Exposure of a receptive female to a reproductively proven male generally results in pregnancy, whereas copulomimetic stimulation or exposure to a sterile male leads to the development of pseudopregnancy. We observed that cervical stimulation in the morning of estrus failed to induce pseudopregnancy in PEI rats, but ~50% of the PEI rats developed pseudopregnancy after exposure to vasectomized males. Taken together, these observations may be interpreted to mean that approximately 50% of the PEI rats completely lost their receptivity, while in the remainder, either the span of receptivity was shortened or the rats became less responsive.
Vernon and Wilson [9] reported no adverse impact of endometrial implants on the mating receptivity of rats. Barragan et al. [15] did not critically address the issue of mating receptivity, although they observed that some rats with implants did not get pregnant. Our findings on mating receptivity accord well with the findings of Barragan et al. [15] but apparently differ from those of Vernon and Wilson [9]. It may be significant, however, that Vernon and Wilson [9] made their observations at 3 wk posttransplantation, when we also noted no significant effect of the implants on ovulation or mating rate. The pregnancy failure as reported by Barragan et al. [15], and the present observation of loss of mating receptivity, are based on observations during 40 and 50 days posttransplantation, respectively. The differences in the results, therefore, seem to be attributable to the difference in the time after transplantation when the observations were made.
In the rat, the LH surge, in addition to ovulation or in the process of triggering ovulation, causes secretion of P4 in significant quantity immediately prior to or coincident with the onset of sexual behavior [16]. P4 acts on a background of estrogen [17], and sexual behavior in the female rat is thought to be a consequence of the synergistic interaction between E2 and P4 [18]. We observed that P4 level in the afternoon of proestrus was significantly below the normal level. Lower P4 level in conjunction with deficient E2 level in about 50% of the PEI rats possibly represents the cause of impaired receptivity. Depending on the degree of E2 and P4 deficiency, sexual receptivity was either totally lost, whereby mating failure was observed in 44% of the PEI rats, or the span of receptivity was shortened. It is significant in this context that Sharpe et al. [19] also reported that antide, a GnRH antagonist, led to significant deviation from the normal behavior of rats due to suppressed cyclic secretion of P4. However, with respect to E2 and P4 levels, the present results differ from those of Barragan et al. [15], who observed no difference in hormone levels between rats with and without endometrial implants. But it should be emphasized that E2 and P4 concentrations vary during different phases of the estrous cycle. In the present study the hormone concentrations were measured at a specific phase of the cycle (evening of proestrus), but Barragan and coworkers [15] measured the hormones randomly irrespective of the phase of estrous cycle. This difference in the experimental protocol may contribute to the difference in the hormone concentration results.
PEI led to premature termination of pregnancy in 34% of rats, while in the remaining rats the fetal number was significantly reduced. It was evident that fetal wastage occurred during the postimplantation phase of gestation, because mean number of implantation sites on Day 7 was comparable between the control and PEI groups, but on Day 21 of pregnancy the number of fetuses in the PEI group was significantly lower. It is not known how PEI induces postimplantation interception; however, 25–40% of women with endometriosis have been reported to have luteal phase defects [20, 21], and we observed that PEI adversely affects preovulatory luteal function. Pelvic endometrial implant-induced luteal deficiency may possibly be associated with pregnancy wastage.
The feasibility of the experimental induction of endometriosis in female rats has been demonstrated by a number of workers. The fertility effects with respect to type and magnitude, however, vary between the studies [2, 9, 14]. This difference in results is not attributed to the difference in the number of implants [2]. We made our observations at three arbitrarily segmented phases of the study to explore whether the effects of the implants varied at different posttransplantation phases. The differences in the results between the different phases suggest that the period after transplantation indeed has a significant impact on the implant-mediated effects.
The reason that reproductive function is inhibited mostly in phase II remains obscure. Endometriosis-induced alteration in reproductive function possibly involves alteration in the biochemical and immunological environment of the peritoneum [22, 23]. There is evidence that subtle or mild endometrial lesions are biochemically more active than the classical lesion [24]. It is therefore tempting to speculate that implants in different phases may differ with respect to their biochemical potential. It is possible that a certain minimum period was necessary in order for the implants to grow and acquire secretory potential. During this period (phase I) the implants failed to exert any significant reproductive effect. Again, after longer periods (phase III), perhaps the secretory potential of the implants is lost or blunted. The fertility-inhibiting effects of classical endometrial lesions in humans may involve mechanical factors, which possibly play no role in the rat model.
In conclusion, the present investigation shows that transplanted uterine explants in the peritoneum adversely alter the hormonal milieu and affect the reproductive potential of rats. The observation lends support to the view that extramechanical factors may be involved in mild endometriosis-associated infertility. How uterine explants induce these hormonal alterations is not known. Further work is under way to clarify the mechanism underlying the endometriosis-associated hormonal anomaly.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted: November 24, 1998.
Received: February 3, 1998.
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REFERENCES |
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