Echography of the Cervix and Uterus during the Proliferative and Secretory Phases of the Menstrual Cycle in Bonnet Monkeys (Macaca radiata)

Abstract

We undertook the present study to investigate the echographic characteristics of the uterus and cervix of female bonnet monkeys (Macaca radiata) during the proliferative and secretory phases of the menstrual cycle. The cervix was tortuous in shape and measured 2.74 ± 0.30 cm (mean ± SD) in width by 3.10 ± 0.32 cm in length. The cervical lumen contained 2 or 3 colliculi, which projected from the cervical canal. The echogenicity of cervix varied during proliferative and secretory phases. The uterus was pyriform in shape (2.46 ± 0.28 cm × 1.45 ± 0.19 cm) and consisted of serosa, myometrium, and endometrium. The endometrium generated a triple-line pattern; the outer and central lines were hyperechogenic, whereas the inner line was hypoechogenic. The endometrium was significantly thicker during the secretory phase (0.69 ± 0.12 cm) than during the proliferative phase (0.43 ± 0.15 cm). Knowledge of the echogenic changes in the female reproductive organs of bonnet monkeys during a regular menstrual cycle may facilitate understanding of other physiologic and pathophysiologic changes.

Ultrasound imaging is a noninvasive, atraumatic, and simple method to assess various organs in humans and nonhuman primates.¹¹ Ultrasonography has been used to detect fetal growth, endometrial thickness, and endometriosis in rhesus macaques (Macaca mulatta),^17,20,26 and color Doppler ultrasonography has used for characterizing uteroplacental hemodynamics and assessing fetal and placental blood flow in that species.^22,29 In cynomolgus macaques (Macaca fascicularis), ultrasound techniques have been applied in transfundal uterine sperm recovery, follicular fluid aspiration, assessment of endometrial changes during the menstrual cycle, evaluation of folliculogenesis, and ovulation induction studies.^5,33-35 Other primates species wherein ultrasound techniques have been used are vervet monkeys (Cercopithecus aethiops),³¹ owl monkeys (Aotus nancymaae),³⁰ and common marmosets (Callithrix jacchus).^9,12

Bonnet macaques (Macaca radiata) have been used as an animal model for research on contraceptive agents,^6,23,24 endometrial biology,^18,27,28 and assisted reproductive technologies.¹⁰ Although bonnet macaques bear close similarity with rhesus macaques, ultrasound profile of cervix and uterus in these species has not yet been characterized. We undertook the present study to evaluate the echographic profile of the cervix and uterus in regularly cycling bonnet macaques in the proliferative (days 6 through 13 after overt bleeding) and secretory phases (days 16 through 21) of menstrual cycle. Knowledge of the normal ultrasonographic profiles of the female reproductive organs may facilitate the identification of pathophysiological changes during natural or stimulated cycles in bonnet monkeys.

Materials and Methods

Animals and husbandry practices.

Adult female bonnet macaques (Macaca radiate; n = 15) showing normal menstrual cycles underwent abdominal ultrasonography during their late proliferative (days 6 through 13) and midsecretory (days 16 through 21) phases. Bonnet monkeys used in the present study were wild-caught from a forest in southern India. Monkeys were housed individually and fed with fresh seasonal vegetables, fruits, ground nuts, bangal grams, and so forth. Water was provided ad libitum. Housing conditions were a 12:12-h light:dark cycle, with a temperature of 24 to 28 °C and relative humidity of 30% to 70%. Previous reports from our laboratory have shown that the length of menstrual cycle in bonnet monkeys is 25 to 30 d.^6,24 Menstrual cyclicity was monitored for at least 2 cycles by daily vaginal swab examination at 1100. The day of visible vaginal bleeding was considered as day 1, and ultrasonography was performed on days 6 through 13 (proliferative phase) and 16 through 21 (secretory phases). The ultrasound protocol was approved by the Institutional Animal Ethics Committee, and ultrasound examinations were performed during routine health monitoring of bonnet monkeys in the colony. To ensure that the monkey's urinary bladders should be full during ultrasound examination, water bowls were removed during the evening prior to the exam and replaced the morning of the ultrasonography. The animal facility at the institute is registered with the Committee for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Social Justice and Empowerment, Government of India (registration no. 78/1999/CPCSEA).

Gynecologic ultrasound examination.

A gynecologic ultrasound evaluation of the cervix and uterus was performed in B mode by using high-resolution linear curved transducers (L12-5 MHz and C5-2 MHz probes, HDXE11 ultrasound system, Philips, Andover, MA). The shape, echogenic texture, position, and dimensions of the cervix and uterus were studied during the late proliferative and midsecretory phases of the menstrual cycle. Monkeys were sedated with ketamine (10 mg/kg body weight) and placed in a supine position. The region was disinfected (Savlon, Johnson and Johnson, Mumbai, India), and the hair was shaved from 2/3 of the abdominal cavity, to the pubic symphysis. Ultrasound gel (RTrace, RWave Cardio Products, Mumbai, India) was applied to the shaved area. Initially the entire reproductive tract was examined by using the C5-2MHz transducer. The transducer was placed sagittally on the lower midline of the pelvic–abdominal region to locate the vagina, cervix, and uterus. The linear L12-5 MHz transducer then was used for individual examination of the cervix and uterus. The uterus was localized in the lower 2/3 of the abdominal area. The cervix was localized above the pubic symphysis. The craniocaudal and dorsoventral diameters of the cervix, uterus, and endometrium were measured from sagittal scans, and the transverse diameters of these organs were obtained from transverse scans.¹⁶ Four observations were made for each monkey during both the proliferative and secretory phases of the menstrual cycle. The cervix was measured from the internal os to external os (craniocaudal diameter) and from the dorsal colliculi to ventral colliculi (dorsoventral diameter). The uterus was measured from the outer body of uterus (fundus) to the internal os of the cervix to obtain the craniocaudal and dorsoventral diameters (extending from the dorsal to the ventral side of uterus). The endometrium appeared as a triple-line pattern consisting of a prominent outer and central hyperechogenic lines and an inner hypoechogenic or black region on sagittal and transverse views. The central hypoechogenic region of the endometrium was measured on sagittal scans as the craniocaudal (longitudinal) diameter. Endometrial thickness (dorsoventral diameter) was determined by measuring the distance between the outermost interfaces of both sides of the echogenic interface adjacent to myometrium. The color Doppler mode was used to assess the vascularity of the uterus during the proliferative and secretory phases of menstrual cycle.

Statistical analysis.

Measurements of cervix and uterus obtained during ultrasonography are expressed as mean ± 1 SD. Endometrial thickness during the proliferative and secretory phases was compared by using the Student t test(Prism 5, GraphPad Software, San Diego, CA).

Results

In the present study, the cervix and uterus of 15 bonnet monkeys (average weight, 5.1 ± 1.3 kg) with regular menstrual cycles were evaluated by ultrasonography during the late proliferative (days 6 through 13) and midsecretory (days 16 through 21) phases. We used 2 different transducers for this purpose. The C5-2 MHz probe can scan the entire reproductive system for localization of the vagina, cervix, and uterus. The L12-3 MHz transducer is a high-resolution linear probe that allows excellent evaluation of the echogenic texture of the cervix, uterus, and endometrium.

Cervix.

Both probes were used to assess the entire cervical canal ultrasonographically (Figures 1 through 3). Table 1 summarizes the cervical measurements. The cervix was broader (P < 0.05) than the uterus. The cervical canal contained several turns, giving rise to a tortuous shape. The 3 collicular structures were attached by broad bases to the cervical walls. A large prominent ventral colliculus and the intermediately sized cranial dorsal colliculus were readily identified as discrete structures near the ectocervix. The third, caudal dorsal colliculus merged with the fibrous dorsal lip of the external os of the cervix (Figure 1 A and B). The ventral and cranial dorsal collicululi were demarcated easily on sagittal and transverse scans during the proliferative phase (Figure 2 A and B). During the secretory phase of the menstrual cycle, the ventral and cranial dorsal colliculi were hyperechoic and appeared merged on sagittal and transverse scans (Figure 3 A and B). In addition, the echogenicity and texture of the cervix changed during the proliferative and secretory phases of the menstrual cycle. The walls and cervical canal were distended and hypoechoic during the proliferative phase (Figure 2 A and B); this appearance likely was due to excessive cervical secretions. The cervix was hyperechoic and the cervical canal was collapsed during the secretory phase (Figure 3 A and B).

Figure 1.

An ultrasound image of the reproductive organs of bonnet monkeys obtained by using the C5-2 probe during (A) late proliferative and (B) midsecretory phases of the menstrual cycle, demonstrating the echogenicity of the uterus and cervix during the proliferative (hypoechoic) and midsecretory (hyperechoic) phases. The cervical canal appears open and filled with mucus during the proliferative phase compared with secretory phase. C, cervix; CC, cervical canal; CrDC, cranial dorsal colliculus; CuDC, caudal dorsal colliculus; Is, isthmus; U, uterus; UC, uterine cavity; V, vagina; VC, ventral colliculus.

Table 1.

Cervical, uterine, and endometrial measurements (cm; mean ± SD) during the late proliferative and midsecretory phases of the menstrual cycle of bonnet monkeys

	Late proliferative	Midsecretory
Cervix
Craniocaudal diameter	2.74 ± 0.30	2.69 ± 0.24
Dorsoventral diameter	3.10 ± 0.32a	2.88 ± 0.37b
Transverse diameter	2.86 ± 0.28	2.62 ± 0.27
Uterus
Craniocaudal diameter	2.46 ± 0.28	2.41 ± 0.32
Dorsoventral diameter	1.45 ± 0.19a	1.39 ± 0.24b
Transverse diameter	1.42 ± 0.37	1.52 ± 0.42
Endometrium
Craniocaudal diameter	1.20 ± 0.23	1.34 ± 0.42
Dorsoventral diameter	0.43 ± 0.15c	0.69 ± 0.12c
Transverse diameter	0.56 ± 0.85d	0.79 ± 0.14d

Data were obtained by using the L12-3 probe during sagittal and transverse scans.

Values indicated by the same lowercase letter differ significantly (P < 0.05, Student t test).

Figure 2.

An ultrasound image of the cervix of bonnet monkeys during the late proliferative phase of the menstrual cycle, obtained by using the L12-3 probe. (A) Sagittal view. (B) Transverse view. The cervical canal appears open and contains mucus, and colliculi are separated and distinctly visible. CC, cervical canal; CrDC, cranial dorsal colliculus; CW, cervical wall; VC, ventral colliculus.

Figure 3.

An ultrasound image of the cervix of bonnet monkeys during the midsecretory phase of the menstrual cycle, obtained by using the L12-3 probe. (A) Sagittal view. (B) Transverse view. The cervical canal appears collapsed, and colliculi are merged. CC, cervical canal; CrDC, cranial dorsal colliculus; CuDC, caudal dorsal colliculus; VC, ventral colliculus.

Uterus.

The uterus was located centrally in the abdominal cavity, below the urinary bladder. On sagittal scans, the uterus measured 2.5 ± 0.28 cm × 1.45 ± 0.20 cm (length × width) and appeared pyriform in shape (Figure 4 A); on transverse scans, the uterus was oval in shape (Figure 4 B). The myometrium was homogeneously echodense. The inner layer of myometrium was moderately echogenic, but the junctional zones of the endometrium were well demarcated and smooth in outline (Figure 4 A and B). Color Doppler ultrasonography revealed the location of the uterine artery on the left, at the junction of the cervix and uterine corpus (Figure 5 A). Blood flow and vascularity in the uterus were increased during the proliferative phase (Figure 5 A) compared with the secretory phase (Figure 5 B).

Figure 4.

An ultrasound image of the uterus and endometrium of bonnet monkeys during the late proliferative phase of the menstrual cycle, obtained by using the L12-3 probe. (A) Sagittal view. (B) Transverse view. The uterus appears in a typical triple-line pattern consisting of a prominent outer and centrally hyperechogenic line (endometrium) and an inner, hypoechogenic or black region (uterine cavity) separated by homogeneously grayish myometrium. E, endometrium; M, myometrium; U, uterus; UB, urinary bladder; UC, uterine cavity.

Figure 5.

A sagittal view of a color Doppler ultrasound image of the uterus of bonnet monkeys during the (A) late proliferative and (B) midsecretory phases of the menstrual cycle, obtained by using the L12-3 probe. The uterus demonstrated increased vasculature and blood flow during the proliferative phase compared with the secretory phase. E, endometrium; IA, iliac artery; M, myometrium; U, uterus; UA, uterine artery.

Endometrium.

On sagittal and longitudinal views, the uterine endometrium had a triple-line pattern, with 2 prominent, outer and central hyperechogenic lines and an inner hypoechogenic or black region (Figures 4 and 6). During the proliferative phase, the endometrium appeared more echogenic than was the myometrium and developed a multilayered appearance, in which an echogenic basal layer and hypoechoic inner functional layer were separated by a thin echogenic median layer of uterine cavity (Figure 4 A and B). The echogenicity of the endometrium increased during the secretory phase as the multilayer pattern disappeared (Figure 6 A and B). Endometrium thickness was measured from echogenic border to echogenic border, across the endometrial cavity, on a sagittal midline image. The endometrium was significantly (P < 0.005) thicker during the secretory phase (6.9 ± 0.8mm) as compared with during the proliferative phase (4.3 ± 1.1 mm; Table 1).

Figure 6.

An ultrasound image of the uterus and endometrium of bonnet monkeys during the midsecretory phase of the menstrual cycle, obtained by using the L12-3 probe. (A) Sagittal view. (B) Transverse view. The uterus appears as homogeneously echogenic, with absence of the characteristic triple-line pattern. E, endometrium; M, myometrium; UC, uterine cavity.

Discussion

South Indian bonnet monkeys are closely similar to rhesus macaques. Previous reports from our laboratory showed that the length of the menstrual cycle in bonnet monkeys is 25 to 30 d.^6,24 Menstrual cyclicity reflects cyclic changes in the levels of reproductive hormones, such as estrogen and progesterone. Endometrial proliferation, ovarian follicular development, cervical mucus secretions are associated with the menstrual cycle phase in bonnet monkeys.¹⁴ The gross anatomy of cervix uteri has been compared among nonhuman primates. The cervical canal of bonnet and rhesus macaques have several sharp turns due to the protrusion of colliculi, giving rise to the characteristic tortuous shape to the cervix.⁸ Vaginal hysterography of bonnet monkeys and contrast films of excised uteri have shown the similar cervical morphology in the bonnet and rhesus macaques.²³ Our ultrasound observations are in agreement with previous investigations in bonnet monkeys. The exact role of these colliculi is not known. However, microscopically, colliculi consist of columnar epithelium, and they support mucus secretion during the estrogen-dominant proliferative phase of the menstrual cycle.²¹ In addition, the cervical colliculi may act as a sperm reservoir and, alternatively, as a barrier to prevent excessive spermatozoa from reaching the site of fertilization. Motile sperm have been recovered from the cervix of humans,⁷ cattle,¹⁵ and marmosets,⁸ 2 to 5 d after insemination. The echogenicity, texture, and mucus secretion of the cervix change during the proliferative and secretory phases of menstrual cycle in macaques and humans. The quantity of cervical mucus secretion increases proportionately with plasma estrogen concentration in bonnet monkeys¹⁴ and humans.¹³ We noted that the cervical walls of bonnet monkeys are hypoechoic during the proliferative phase, suggesting actively secretion into the lumen of the cervix. In addition, we found that the cervical canal was open and completely echoic, indicating hydrated mucus accumulation. Compared with rhesus macaques, bonnet monkeys secrete very high amounts of cervical mucus.²¹ Under the influence of estrogen, the cervical canal widens due to accumulation of cervical secretions in dogs,³² cats,¹ and humans.⁴

The endometrium of bonnet monkeys proliferates during the proliferative phase (days 6 through 13) and differentiates during the secretory phase (days 16 through 21) of the menstrual cycle. During the proliferative phase and as a result of estrogen signaling, the endometrium develops glands, blood vessels, and stroma.³¹ Ultrasound examination of the endometrium during the proliferative phase was found it to be echogenic relative to the myometrium. A typical multilayer endometrium consists of an echogenic basal layer and hypoechoic inner functional layer, which are separated by a thin echogenic median layer arising from the luminal content. In rhesus macaques, the reflectivity (echogenicity) of the endometrium varies according to the stage of the menstrual cycle; the maximal thickness of the endometrium was 8 mm during the secretory phase.¹⁷ We similarly observed highly echogenic endometrium during the proliferative phase of the menstrual cycle and maximal endometrial thickness (0.69 ± 0.12 cm) during the secretory phase. Color Doppler imaging showed increased vascularity and blood flow during the proliferative phase compared with the secretory phase. Endometrial echogenicity and thickness are associated with pregnancy outcomes in humans.^2,25 A nonhomogeneous hyperechoic (rather than a triple line) pattern of the endometrium before pregnancy is associated with lower pregnancy rates.² Several studies in humans have suggested a correlation between endometrial thickness and endometrial receptivity during autologous,³⁶ oocyte donor,³ and in vitro fertilization cycles.¹⁹

The present study reports the echogenic changes of the cervix and uterus of bonnet monkeys during the proliferative and secretory phases of the menstrual cycle. This information may help in the delineation of pathologic conditions in these organs and the selective enrolment of macaques for cervical and endometrial research. In addition, our findings likely will facilitate the evaluation of female reproductive organs during gonadotropin stimulation and the development of appropriate nonhuman primate models for reproductive disorders.