Primates are part of the Order Primates, divided into two Suborders, Strepsirrhini and Haplorrhini. The Haplorrhini comprises the infraorders Tarsiiformes (tarsiers) and Simiiformes (apes, monkeys, and humans) (1). About the Simiiformes, these non-human primates are divided into two Parvorders, the Catarrhini, which comprises the species of monkeys known as Primates of the Old World that inhabit regions of Africa and Asia, and the Platyrrhini, which contains the species that inhabit areas of Mexico and the Americas Central and South that are called the New World or Neotropical Primates (2). The Neotropical primates are currently distributed among 152 species (204 species and subspecies), 20 genera, and five families: Cebidae, Aotidae, Pitheciidae, Atelidae, and Callitrichidae (Figure 1) (3).

To the best of our knowledge, in neotropical primates, the reproductive B-mode ultrasound examination has been described in the Aoetidae, in the genus Aotus (Aotus azarai infulatus and A. nancymaae), in the Callitrichidae, genus Callithrix (Callithrix jacchus), Saguinus (Saguinus ursulus and S. fuscicollis) and Leontopithecus (Leontopithecus rosalia, L. chysomelas and L. chrysopygus), and in the Cebidae, in subfamily Cebinae, genus Sapajus (Sapajus apella, S. xanthosternos, and S. robustus), and subfamily Saimiriinae, genus Saimiri (Saimiri bolivensis bolivensis, S. sciureus sciureus) as described in Table 1.

Ultrasonography is a non-invasive imaging technique that provides real-time bidimensional (2D) or three-dimensional/four-dimensional (3D/4D) B-mode images (brightness) (28, 29); generates high-resolution characterization of reproductive organs; and provides precise linear measurements of the ovary, uterus, conceptus, and fetus (30). In association with Doppler ultrasonography, B-mode ultrasonography can also monitor mammalian blood flow to the reproductive tract (31, 32). Color ultrasonography and spectral Doppler ultrasonography can provide qualitative and quantitative data concerning blood flow, respectively (33). Color ultrasonography and spectral Doppler ultrasonography were used to detect hemodynamic changes in the iliac (6), uterine, ovary, and utero-ovarian ligament (UOL) arteries during follicular growth and ovulation, as well as the formation and regression of the corpus luteum (CL) (10, 25). In obstetrics, color ultrasonography and spectral Doppler ultrasonography are crucial for evaluating strategic conceptus vessels by fetal development (25). Nowadays, portable ultrasound equipment provides a vast possibility of modes (2D/3D/4D and color/spectral/power Doppler) and is reliable in animal research institutes, zoos, and field studies (34).

Gynecological and obstetric ultrasonography is essential for establishing the female reproductive anatomical and physiological ovarian and uterine health status (10), gestational diagnosis, and fetal growth monitoring (25). Ultrasound has become an indispensable method for routine management, health assessment, and research on the reproduction of non-human primates (NHPs) in captivity (35). Uterine and ovarian examinations have always been of great concern in gynecology because they can host numerous diseases related to reduced fertility or cause serious damage to the health of females (6, 36). Several techniques, such as ultrasonography, have been used to detect pregnancy in mammals, some of which represent technological advances in practice with captive animals (27). The increasing use of pregnant primates and their fetuses in scientific research makes diagnosing and monitoring pregnancy important, as it enables proper handling of the pregnant female (37). In the Neotropical primates, researchers have recently been developing gynecological (Tables 2, 3) and obstetric (Table 4) ultrasonography data to improve the reproductive management of these animals in captivity.

Considering the New World or Neotropical primates, 42.2% are listed as threatened, according to the International Union for Conservation of Nature (40). A small group of Neotropical (e.g., Aotus sp., Callithrix jacchus, Saguinus sp., Saimiri sp., and Sapajus sp.) primates is an important model for biomedical research because of its applicability. Only for these species are there studies on reproductive physiology and biotechnology that employ gynecological and obstetric ultrasonography as a tool (Tables 2–4). For instance, this paper discusses the state-of-the-art of gynecological and obstetric ultrasonography in the Neotropical primates, species that are models for biomedical research, and some recent studies on species targets for conservation strategies for wild animal populations.

The use of pregnant primates and their fetuses in scientific research has remained constant over the last few years. However, one of the major drawbacks of NHPs is that they can be extremely difficult and even dangerous to handle. Manual and/or chemical restraint is necessary and desirable to protect both the investigator and animal. According to Unwin et al. (49), manual restraint should not be considered for any NHP species weighing >5 kg or for any species not used to be handled. In addition, some parameters, such as physiological status (pregnant or not), size, and age, should be considered when deciding whether to anesthetize. For instance, during pregnancy, it is necessary to be careful with manual and/or chemical restraints, as they can cause pregnancy loss. In owl monkeys, anesthetic drugs and capture methods used to monitor pregnancy using ultrasound act as chemical and environmental factors that result in abortions (4). Thus, some small primates (marmosets and owl and squirrel monkeys) can be trained to be handled and, hence, scanned without sedation. Training them to cooperate voluntarily using positive reinforcement training techniques is one means of significantly reducing the adverse impact of physical stress and anesthetic drug. These actions are important for monitoring and understanding the primate gestational physiology (39).

Reproductive applications of ultrasound in NHP breeding colonies can provide an efficient method for pregnancy detection, fetal monitoring during gestation, and routine assessment of breeding females (50). Evaluating the efficacy of ART is crucial, and ultrasound is the method of choice for female cycle monitoring, early pregnancy diagnosis, and conceptus development study (11). Ultrasonography has been used to study gestation in C. jacchus (11–13), Saguinus fuscicollis (20), Aotus azarae infulatus (4, 39), Aotus nancymaae (9), Leontopithecus rosalia, Leontopithecus chrysomelas, Leontopithecus chrysopygus (27), Saimiri sp. (21), Sapajus sp. (Cebus apella) (23), and S. apella (25). With increased studies on NHP pregnancy failures (37) and experimental Zika virus studies (51), adequately describing normal and abnormal conceptus growth and fetal development is crucial.

Serial ultrasonographic sessions were conducted to identify early ultrasonographic signs of gestation (EUSG) and developing ultrasonographic signs of gestation (DUSG). EUSG was considered if the endometrial thickening and UV increased (4) (Figure 5A). Evidence of the gestational sac (GS) and visualization of the embryonic bottom were considered DUSG (4) (Figure 5B). The diameters of the GS (CCD, DVD, and TD) or latero-lateral longitudinal and latero-lateral transversal diameters are the mean parameters for monitoring conceptus growth before fetal morphogenesis (4, 20, 23, 25, 39) as showed in Table 4.

According to Monteiro et al. (39) and Miranda et al. (25), fetal development parameters can be useful for monitoring pregnancy, such as following the crown–rump length (CRL) on the longitudinal section of the fetus, which is obtained from the top of the cranium to the base of the tail (Figure 6A). The biparietal diameter (BPD) and occipitofrontal diameter (OFD) were measured in the thalamic plane, determined by the ambient cistern appearance at the rear of the head (Figure 6B). The BPD was measured by placing calipers from the external surface of the proximal cranial table to the distal internal surface. The OFD was obtained by measuring the distance from one external fetal cranial margin to the other margin perpendicular to the BPD. The head circumference and head area were measured by delineating the fetal cranium's hyperechoic outline in the same scan used for the BPD and OFD. The abdominal circumference and abdominal area were measured by placing the probe around the external portion of the hyperechogenic border of the fetal abdomen (Figure 7A). A transverse scan perpendicular to the vertebral axis was obtained from the fetal abdomen. This scan visualizes the stomach and the umbilical portion of the portal vein in the liver. Color Doppler mode can be used to easily identify the umbilical portion of the portal vein (Figure 7A). The femoral length (FL) was measured from the distal and proximal diaphysis extremities (Figure 7B).

In a study of prenatal development in capuchin monkeys, ultrasonography was used (23). The authors observed the embryonic development and the beginning of fetal development. In six animals, pregnancy was diagnosed between 15 and 27 days of gestation. The GS was visible at a mean of 23.7 ± 2.8days. Embryonic development was evaluated using the largest SG and CRL. Fetal development was evaluated using the CRL, BPD, thoracic height, and FL. The authors concluded that ultrasonography is a useful diagnostic method for determining and assessing the gestational age in this species. The capuchin monkey follows the general embryonic development plan of mammals, and its fetal development is chronologically similar to that observed in other primates (Macaca fascicularis, Macaca mulatta, and Papio anubis), with similar gestational periods (52, 53).

Kuederling and Heistermann (20) studied the gestation of S. fuscicollis (142–150 days), comparatively, by ultrasound and plasmatic progesterone dosage, and verified that ultrasonographic examination was suitable for earlier for pregnancy diagnosis. After conception, the endometrial surfaces separated and formed a lumen, which was detected between 16 and 18 days of gestation (17.2 ± 1.0 days), when uterine cavity had a small circular hypoechoic structure in the middle of the endometrial line, a safe indication of gestational sac and pregnancy. The amniotic sac was visualized between the fourth and 5th weeks, at ~32–52 days of gestation. One week before the estimated time of conception, low progesterone levels were detected, with mean values of 4.7 ± 1.6 ng/mL. A marked postconception increase was observed, resulting in a mean level of 107.1 ± 46.0 ng/mL around day 24 of gestation. After that, the progesterone levels decreased to 71.6 ± 30.3 ng/mL and remained at this plateau until ~2 months before delivery, when a second significant increase occurred. Peaks of 278.5 ± 67.3 ng/mL were found 1 month before delivery, declining to levels close to preconception levels (7.4 ± 7.8 ng/mL) within a week after delivery.

The mean gestational and fetal echo-biometry parameters evaluated in neotropical primates are shown in Table 4. The serial evaluation gestational sac was described only for Sapajus sp. (23, 25), Saguinus fuscicollis (20), and Aotus azarai infulatus (39). In the same way, for the fetal echo-biometry, the biparietal diameter is the mean parameter for monitoring fetal development in neotropical primates. In Callithrix jacchus, the BPD was the best parameter to relate with the gestational age, showing that the diagnosis of a small BPD for gestational age results in fetal death 7 days after birth (13). However, other parameters important to diagnosing intrauterine growth restriction, such as AC and HC, are described just for Sapajus sp. and Aotus sp. It is important to notice that a small AC for gestational age may reflect a reduction in the size of the liver or other intra-abdominal organs, and HC reflects the head size and brain growth (54). Microcephaly, for example, is diagnosed when the HC is below the mean for gestational age (55). In neotropical primates, studies about HC related to gestational age still need to be completed. Recently, craniofacial malformation was diagnosed by B-mode ultrasound measurement of HC, OFD, and BPD in Saimiri collinsi experimentally infected with ZIKV in the second trimester of gestation (51).

Fetal echo-biometry ultrasound monitoring during the gestational period may be suitable for predicting the whelping time using regression analysis formulas (Table 5). Nonetheless, considering the lack of studies on fetal echobiometry in neotropical primates, the use of ultrasound to predict whelping time is limited to Aotus nancymae (9), Leontoptecus sp. (27), and Sapajus sp. (23, 25).

Fetal wellbeing is usually monitored based on the fetal heartbeat (23). Relationships between the fetal size, fetal wellbeing, and characteristics of blood flow in the uterine artery (UA) and umbilical artery evaluated by triplex Doppler showed suitability for evaluating fetal viability using 2D B-mode ultrasonography (25). Among the Doppler indices, the pulsatility index (PI) and resistivity index (RI) are commonly used for obstetric applications. As described for humans, in capuchin monkeys (Sapajus sp.), an early diastolic notch is ordinarily present in the UA wave flow until the second trimester of gestation. The early diastolic notch disappears concurrently with significant decreases in the RI and PI of the UA (25).

In summary, this review shows the importance of ultrasound for understanding reproductive physiology in neotropical primates. However, it also arises that studies on ultrasonography applied to non-human primate reproduction are almost restricted to species used in biomedical research. Even in the biomedical research primates species, there species, there is a lack of studies about ovulation predicted day, early diagnosis of gestation, echo-biometry parameters to determine gestational age, fetal vitality, and whelping time, as well as studies involving Doppler parameters for ovarian, uterine, and umbilical arteries. It is important to notice that all these aspects are essential for the development of reproductive programs related to the conservation of these non-human primates, and in biomedical research such as studies on the effect of the Zika virus on the fetal development of both human and non-human primates.

SD, FM, and DL contributed to conception and design of the study and wrote the first draft of the manuscript. DL organized the database. FM wrote sections of the manuscript and contributed with figures. All authors contributed to manuscript revision, read, and approved the submitted version.