The baboon model (Papio hamadryas) of fetal loss: Maternal weight, age, reproductive history and pregnancy outcome

Abstract

Background

Several risk factors are associated with the incidence of human stillbirths. The prevention of stillbirths in women is a pressing clinical problem.

Methods

We reviewed 402 pathology records of fetal loss occurring in a large baboon (Papio spp.) colony during a 15-year period. Clinical histories of 565 female baboons with one or more fetal losses during a 20-year period were analyzed for weight, age, and reproductive history.

Results

Fetal loss was most common at term (35.57%) and preterm (28.61%) and less common in the first half of gestation (11.20%) and post-term (5.22%). Greater maternal weight, older age, history of stillbirth and higher parity were independent predictors for stillbirth. An exponential increase in the incidence of fetal loss was observed beginning at age 14 years in baboons.

Conclusion

Fetal loss and maternal risk factors associated with stillbirths in baboons were similar to those documented in women.

Introduction

Stillbirth in the women is defined as intrauterine fetal death occurring greater than 20 weeks of gestation and represents the majority of perinatal death. In the United States stillbirth occurs in about one in 200 of all births (or 6.4 per 1000 of live births). Approximately 50% of fetuses die of unknown causes. Several risk factors associated with the incidence of human stillbirths include maternal weight, age, race and parity [20]. The prevention of stillbirths is a pressing clinical problem, as evident by the on-going NIH-sponsored Stillbirth Collaborative Research Network [56].

The baboon (Papio hamadryas) is one of many nonhuman primates species used in biomedical research and is a well established model for studying reproductive function [26]. Stillbirths in this species has been reported to occur in 5.9 to 20% of pregnancies and varies depend on facility, housing condition, habitat [3, 5] with influence of seasonality and associated hormonal changes on fetal loss have been investigated intensively in this species [3, 19], the incidence and causes of fetal loss in a large baboon population has not been thoroughly studied [61].

The aims of this study were to evaluate the epidemiology and pathology associated with fetal loss and to analyze maternal risk factors for stillbirth in the baboon model.

Materials and Methods

Animal husbandry

The main breeding colony at the Southwest National Primate Research Center (SNPRC) consists of approximately 3800 baboons housed in either corrals or metal and concrete gang cages that usually contain between 16 and 20 animals. Two study sets were performed respectively: 1) pathology records of all fetal losses that occurred during the 15-year period from 1988 through 2002 (n=402) were analyzed for gross pathology and microscopic findings, main and secondary diagnoses and fetal sex), 2) female baboons with a history of stillbirth, recorded in the SNPRC animal database, were evaluated (n=565) for maternal age, parity, number of stillbirths, and weight. Maternal age was recorded at the time of stillbirth. Maternal weight was recorded at the non-pregnant stage during maximum one year prior to the pregnancy that ended as stillbirth and then calculated as average weight over this time period. Complete data sets (maternal age, weight, and parity) were available in 261 cases. All animal procedures were approved by the SNPRC Institutional Animal Care and Use Committee.

Calculation of gestational age and recording of fetal loss

Clinical and reproductive histories were retrieved from the computerized animal records database (Computerized Animal Management Program, CAMP). When the menstrual cycle records were available, pregnancies were scored as followed: the cessation of sexual cycling (>40 days) without evidence of menstruation (i.e., vaginal bleeding followed by sexual swelling within 1 week) and presence of pregnant color (pink) [27]. The estimated day of conception (first day of pregnancy) was then counted as the day that a female's sexual swelling began to reduce minus two days, as this is common practice for gestational age estimation for all pregnancies in this facility [27]. Sexual swelling has proven to correlate greatly with oocyte development and ovulation in the baboons [60].

Fetal loss was established in early gestation (0-89 days gestational age [dGA]) by a history of females who previously showed pregnant color and signs of vaginal bleeding after that or absence of fetus upon ultrasound examination. In addition to these criteria, at gestational age 90 dGA and above fetal loss was determined either by presence of a fetus (or its remnants) or placental tissue.

Pathological evaluation and pathological diagnosis

Gross examination of fetuses/placentas was generally performed within 12-18 hours of delivery. In several cases, the time between birth and necropsy was difficult to estimate because of logistic variations in time of the delivery. The primary criteria for intracranial trauma were hemorrhage in the brain and meninges, luxation and fracture of the bones of the skull and distortion of the face and head. Diagnosis of stillbirth was confirmed by negative floating test and microscopic lung evaluation. In all 402 cases of available pathology records histological evaluation was performed.

Statistical analysis

Baboons with fetal loss were divided into two major groups based on the definition of stillbirth in the human population [14, 56]. These two groups were: early pregnancy loss (first half of gestation, 0-89 dGA) and stillbirths (second half of gestation, 90dGA and above). The stillbirth group was comprised of three subgroups: 1) preterm stillbirths (90-163 dGA), 2) stillbirths at term (164-185 dGA), and 3) post-term stillbirths (186 dGA and above). These stillbirth subgroups were formed based on the published data for full-term pregnancy in the baboons at 175±11days of gestation [3, 39]. We used a Poisson regression for prediction of stillbirth number, adjusted by maternal age, weight, and parity. Poisson regression models are generalized linear models with a natural logarithm as the (canonical) link function. The Poisson distribution for the dependent variable is limited to positive values, and has a variance equal to its mean. Thus, in populations in which events are rare, the distribution is highly skewed to the right; as the mean of events rises, the distribution is increasingly normal. Fetal weight comparison was performed using one-way ANOVA. Values were considered to be statistically significant with p<0.05.

Results

The number of births in SPNRC colony ranged from 379 to 689 per year, with a total of 8,336 for the 15-year period. Recorded fetal loss during this same period ranged from 15-49 per year, with a total of 402 losses (4.82 % of all births). We established accurate gestational ages in 323 (80.34%) cases: 45 (11.20%) cases of fetal loss were in the first half of gestation, 115 (28.61%) were preterm, 143 (35.57%) were at term, and 21 (5.22%) were post-term (Table 1). The placenta was available for examination in 35 (8.71%) cases and 351 (87.31%) fetuses were necropsied. The etiology of stillbirth was undetermined in 93.5% of early fetal loss, 65.79% of preterm stillbirths, and 57.14% of post-term stillbirths. In pregnancies at term, the most common cause of fetal death was intracranial trauma or intracranial and intraabdomial hemorrhages (52.45%). Stillbirths of undetermined etiology comprised 25.87% of all diagnoses in this group. Table 2 lists the distribution of cause of death across different gestational ages.

Table 1. Number of the stillbirths and distribution of main pathological diagnosis.

Gestational age	Total number of stillbirths/abortions registered	No. evaluated		Main pathological diagnosisa
		Fetuses	Placentas	Relative disproportion of fetal size and maternal pelvis	Undetermined etiology	Placental and umbilical cord pathology	Intrapartum trauma or intracranial heamorrhage	Pathology of fetusb	Maternal conditions
0-89	45 (11.2%)	12 (3.0%)	2 (0.5%)	NDa	42 (93.3%)	2 (4.4%)	1 (2.2%)	NA	NA
90-163	114 (28.4%)	103 (25.6%)	13 (3.2%)	ND	75 (65.8%)	7 (6.1%)	22 (19.3%)	7 (6.1%)	4 (3.5%)
164-185	143 (35.5%)	143 (35.2%)	10 (2.5%)	7 (4.9%)	37 (25.9%)	6 (4.2%)	75 (52.4%)	11 (7.7%)	7 (4.9%)
186 and more	21 (5.2%)	21 (5.2%)	2 (0.5)	ND	12 (57.1%)	1 (4.8%)	6 (28.6%)	2 (9.5%)	NA
Unknown gestational age	79 (19.7%)	73 (18.2)	8 (2.0%)	ND	52 (65.8%)	1 (1.3%)	25 (31.6%)	ND	1 (1.3%)
Sum	402 (100%)	351 (87.3%)	35 (8.7%)		219 (54.3%)	17 (4.2%)	128 (31.8%)	20 (5.0%)	12 (3.0%)

^aPercentage was calculated from total incidence of stillbirths at particular gestational age.

^bPathology of fetus includes fetal infection, fetal malformation, hydrops, and growth retardation.

^cND, not done.

Table 2. Distribution of main pathological diagnoses among the groups of different gestational ages (dGA – days of gestation).

	dGA
	0-89	90-163	164-185	186 and over	Unknown gestational age
Trauma and hemorrhages	1 (2.2%)	22 (19.3%)	75 (52.4%)	6 (28.6%)	20 (25.3%)
Isolated intracranial trauma or intracranial hemorrhage	1 (2.2%)	12 (10.5%)	33 (23.1%)	2 (9.5%)	10 (12.7%)
Liver trauma	0	1 (0.9%)	1 (0.7%)	0	1 (1.3%)
Isolated intraabdominal hemorrhage	0	0	12 (8.4%)	0	3 (3.8%)
Combined intracranial trauma and intraabdominal hemorrhage		3 (2.6%)	22 (15.4%)	3 (14.3%)	5 (6.3%)
Chest cavity trauma	0	0	2 (1.4%)	0	0
Diagnosed dystocia	0	0	19 (13.3%)	0	3 (3.8%)
Breech presentation	0	0	1 (0.7%)	1 (4.8%)	1 (1.3%)
Face presentation	0	0	4 (2.8%)	0	1 (1.3%)
Macrosomia and intrauterine growth retardation (IUGR).	0	2 IUGR (1.80%)	4 IUGR 3 fetuses with macrosomia (4.90%)	1 IUGR (4.80%)	1 IUGR (1.30%)
Placental pathology	2 (4.4%)	7 (6.1%)	8 (5.6%)	0	1 (1.3%)
Placentitis	2 (4.4%)	5 (4.3%)	3 (2.1%)	0	1 (1.3%)
Abruptio	0	1 (0.9%)	3 (2.1%)	0	1 (1.3%)
Maternal pathology	4 (8.8%)	4 (3.5%)	7 (4.9%)	0	1 (1.3%)
Shock	0	0	2 (1.4%)	0	0
Diabetes	1 (2.2%)	1 (0.9%)	4 (2.8%)	0	0
Uterine rupture	0	0	3 (2.1%)	0	0
Fetal pathology	2 (4.4%)	7 (6.1%)	11 (7.7%)	0	0
Pneumonia	0	2 (1.8%)	4 (2.8%)	0	0
Malformation	0	0	2 (1.4%)	0	0
Hydrops and hydrocephalus	2 (4.4%)	0	2 (1.4%)	0	0
Twins	0	1 (0.9%)	0	0	0
Severe growth retardation	0	0	0	0	0
Other fetal infection	0	2 (1.8%)	1 (0.7%)	0	0
Total number of fetal loss in the subgroups	45	114	143	21	79

In the group of stillbirths at term intracranial trauma and intracranial hemorrhage (n=33; 23.08%) were registered more often compared to abdominal hemorrhage (n=12; 8.39%), combined intracranial / abdominal hemorrhage (n=22; 15.38 %), liver trauma (n=1; 0.70%), and whole body trauma (n=1; 0.70%). Birth weight in the intracranial trauma and hemorrhages subgroup was not different than in other subgroups of pathological diagnoses (maternal, placental and fetal pathology) and the population of live-born fetuses, but it was higher than birth weight in cases of stillbirth of undetermined etiology (Fig. 1). Fetal infection with unknown etiology was diagnosed in 2 fetuses in this group, 2 fetuses had malformations, 4 were small for their gestational age (one had hydrocephalus), 2 had lung pathology (atelectasis and hyalinosis) and one fetus had confirmed encephalomyocarditis virus infection (EMCV). Five cases (6.41%) of infections in the stillborn term fetuses and placentas were of unknown etiologies. Maternal pathology included maternal diabetes, hemorrhagic shock, uterine rupture, and histoplasmosis. Three cases of placental abruption, three cases of placentitis, one case of massive placental necrosis associated with hydrops fetalis, and one case of placenta previa were diagnosed. One animal had intrauterine death of twins of undetermined cause. The functional discrepancies between fetal and maternal pelvic size involved face presentation (4 cases), breech presentation (1 case), and transverse position of fetus (1 case).

Figure 1.

Fetal weight distribution in control population of baboons (vaginal deliveries (CTR, n=6)), and stillbirths at term (due to intracranial trauma/hemorrhages (Intracr T/H, n=75), maternal, fetal and placental pathology (MFP, n=26) and stillbirths of undetermined etiology (UN, n= 37). “a” is statistically significant different from “b”. Data presented as mean ± SEM.

In the group of post-term stillbirths 1 fetus was small for its gestational age, 1 had congenital heart failure, and a true umbilical cord knot was diagnosed in 1 case.

In preterm stillbirths there were 7 cases of placental pathology: 5 were cases of placentitis (one positive culture for two microorganisms: Klebsiella pneumonia and Aeromonas hydrophila), one case with probable placenta abrupta was identified and one case with placental infarction and hemorrhage. In diagnosed cases of fetal pathology, 2 were small for the gestational age, and 3 had signs of infection (pneumonia and EMCV). The total number of cases with intrauterine infection was 8 (6.8% of all fetuses examined at this gestational age). When the cause of death could be determined, maternal conditions that led to stillbirth were maternal diabetes with ketoacidosis and maternal trauma.

Of the 565 animals recorded with a history of pregnancy loss during all years of observation at SNPRC, data analysis revealed that 45 (7.96%) animals had no previous offspring; 159 (28.1%) had 1 to 4; 321 (56.8%) had 5 to 10; and 40 (7.1%) had more than 11 offspring. Of those animals with a history of pregnancy loss 21.2% had one stillbirth/abortion in the clinical history, 14.2% - two; 12.2% - three, and 44.3%- 4 or more stillbirths/abortions. The average age of the baboons at the time of stillbirth/abortion was 11.2±4.5 years, and weight was 17.3±3.0 kg (data presented as mean±SEM). Each factor studied (maternal age, weight and parity) showed a significant and independent prediction for the incidence of fetal loss (Table 3, Fig. 2). Exponential increase in the incidence of fetal loss was observed beginning at age 14 in this group (Fig. 3).

Table 3.

Independent variables that explain the incidence of stillbirths in captive baboons.

Variable	Coefficient (SE)a	p-Value	95% Confidence Interval
Number of offspring	0.118 (0.012)	<0.001	0.094, 0.142
Maternal weight (kg)	0.019 (0.010)	0.051	-0.001, 0.038
Age (years)	0.077 (0.008)	<0.001	0.061, 0.092
Constant	-0.665 (0.189)	<0.001	-1.036, -0.294

^aCoefficients were calculated for a general lineal model using Poisson process (n=565).

Figure 2.

Predicted fetal loss number using a Poisson process adjusted by the total offspring number. The graphic estimates numbers of stillbiths/abortions for a 10- and 15-year-old baboon weighing 10, 15, or 20 kg. As weight increases, the number of negative pregnancy outcome increases (n=565).

Figure 3.

Poisson process adjusted by maternal age, weight, and the number of offspring. Around 14 years of age the predicted number of stillbirths/abortions increases exponentially (n=261).

Discussion

Incidence of fetal loss in nonhuman primates and humans

The incidence of fetal loss in the SNPRC baboon colony remains within the range published by Hendrickx in 1966 [28] for the same facility (2.4-11.2%). The stillbirth rate in our study (35/1,000 live births) is higher than has been reported for human population (6.4/1000 live births). This difference is primarily due to pregnancy loss caused by intracranial trauma/hemorrhages (56.7%) that were much more frequent than in the human population (0-1%). Our data concerning distribution of fetal loss among a captive baboon population contrast with those of Packer et al. [45] who reported an equal distribution of stillbirths across the trimesters in a population of wild baboons. These differences may be explained by the data arrangement in the two studies. We did not use a trimester approach in our study as did Packer et al. [45]; rather, we divided the groups by gestational age according to the definition of stillbirth. The observation that peak pregnancy loss is approximately at 175 days (term stillbirths) in our study agrees with the data published for wild baboons [3].

The higher percentage of intracranial trauma and hemorrhages in nonhuman primates could be explained by differences in the birth mechanism in human and non-human primates, e.g., less degree of head rotation, possible natural delivery with face presentation, and absence of social contact at birth (assistance at birth) in baboons [50].

Infection as a cause of pregnancy loss in baboons and humans

Combined incidences of intrauterine infection as indicated by placentitis and fetal infection were very similar in the term and preterm stillbirth (6.8%) groups in this study. The incidence of infections as a cause of stillbirths in this baboon population was lower than those published for human populations (10-25%) [9, 23, 43]. This observation is remarkable, since most of the infectious agents causing stillbirths in humans [23] are also found in the baboon population with few exceptions (Table 4): Chlamydia trachomatis, varicella zoster virus, parvovirus (B19), Western equine encephalitis and Brucella spp. For example, Ureaplasma urealyticum and Mycoplasma hominis, which cause intrauterine infection in women [49], were detected in baboons in previous studies [10]. However, their role in spontaneous intrauterine infection in baboons is unknown. Only Ureaplasma urealyticum has been experimentally evaluated to determine its effects on fetal baboons [62]. Klebsiella spp. were reported to be natural infectious agents in baboons, but our study is the first to associate this agent with placentitis in Papio sp. Herpesvirus papio 2 (HVP2) and cytomegalovirus (CMV) infections among baboons are high (almost the same prevalence as in human population), with 95% seropositivity in the colony [6, 17]. Both herpes virus and CMV have been reported to be associated with pregnancy loss in human studies [12, 48].

Table 4.

Comparative incidences of infections in baboon and human populations associated with fetal loss in human population (selected publications).

Infectious agent	Human population	References	Baboon population	References
Spirochetes
Treponema pallidum	2.4 cases per 1,000,000 population	11	Naturally occurring cases. 65-76% seropositivity in some areas of Africa	21
Protozoa
Toxoplasma gondii	15-80% of women of childbearing age	36	32% of population	41
Trypanosoma cruzi	0.6% of population of pregnant women in some regions in United States	15	9.4-22.5% of whole population	2
Plasmodium falciparum	25.3-30.5% slides positive in endemic areas	57	Not reported	N/A
Viruses
Rubella	0.13:100,000 per year among adult population	47	Experimental infection	28
Cytomegalovirus	antibodies present in 40-100% of adult population	14	antibodies present in more than 95% of population	6
Herpes simplex 2	40% of the population of young women	8	93.5% of population seropositive for Herpesvirus papio 2 (HVP2)	17
Varicella zoster virus	0.05% of pregnant women	7	Not reported	N/A
Lymphocytic choriomeningitis	0.3-5.4% seropositivity among adult population	46	3 of 22 captive baboons were seropositive	38
Parvovirus (B19)	1-5% of pregnant women	18	Not reported	N/A
Bacterial and mycoplasmal infections
Chlamydia trachomatis	Present in 25.4% of vaginal cultures of the women with preterm deliveries	10	Experimental infection	35
Klebsiella pneumoniae	Present in 41.7% of vaginal cultures of women with preterm deliveries	10	Bacteria were isolated from oral swabs of captive baboons	37
Ureaplasma urealyticium	Present in cervix or vagina of 40-80% of sexually mature asymptomatic women	58	10% of baboon population	62
Escherichia coli	Present in 30.8% of vaginal cultures of women preterm deliveries	10	Pathogenic and atypic E. coli was isolated in 20 from 208 captive baboons (9.6%)	37
Mycoplasma hominis	Present in cervix or vagina of 21-53% of sexually mature asymptomatic women	58	Detected by PCR in 29% of baboon population	55

The discrepancies in distribution of intrauterine infections in baboon and human pregnancy loss could be explained by absence of placental material for evaluation at necropsy for fetal loss occurring in the first half of gestation and a restricted number of placentas available for evaluation in the second half of gestation in our study baboons. In the population of wild baboons Beehner et al. [3] observed, most fetal loss was associated with fetal, and not placental, abnormalities as indicated by a decreasing estradiol, and not progesterone, level prior to stillbirths. We could not rule out that a natural placental barrier to infection is present in nonhuman primates. The relatively high incidence (4 cases) of pregnancy loss associated with ECMV in our study could be explained by an outbreak of this viral infection at SNPRC during the period of evaluation [32].

Maternal age, weight, and parity

The analysis of this baboon population found that an increased risk of a negative pregnancy outcome was associated with a higher number of offspring. Moreover, our observation that a history of stillbirths is correlated with a greater risk of a subsequent stillbirth parallels epidemiological studies in the human population [53].

The World Health Organization (WHO) has declared obesity to be one of the top 10 adverse health risk conditions in the world and one of the top 5 in developed nations (www.who.int/nut/obs.htm.). Between 20 and 34% of women of reproductive age are obese [9]. Maternal obesity is associated with a 5-fold increase in risk of stillbirth with placental dysfunction, an increased risk of pregnancy and delivery complications (gestational diabetes, pre-eclampsia, macrosomia, shoulder dystocia, higher rates of cesarean sections, and infections), neural tube defects, and fetal mortality in the human population [44]. In our study, increased maternal weight was associated with an increased incidence of fetal loss. Baboons spontaneously develop obesity and diabetes, and they have been used extensively as models of these conditions [13]. However, there are no previous reports of diabetes during gestation or development of gestational diabetes in this species. Our records demonstrate the presence of two documented cases (one with maternal ketoacidosis) of diabetes as the cause of fetal death. In the human population, the prevalence of gestational diabetes is 5-10% [51]. The low percentage of diabetes in our study baboons may be due, in part, to lack of maternal evaluation during pregnancy.

Mean perimenopausal age (based on menstrual cycle regularity) is 18.89 years of age in this baboon colony [40]. In our study, an increase in fetal loss was seen beginning at 14 years of age. In the observation of baboons in the wild, other authors have observed either a decrease in reproduction, and a higher rate of miscarriages beginning at 21 years of age [45], or no increase in the rate of fetal loss with age in this species [59]. Our data are consistent with data from the human population, where onset of decreased fecundity takes place before perimenopausal changes associated with irregularities of the menstrual cycle occur [24]. In humans, the association of advanced maternal age with an increased rate of chromosomal aneuploidy has been recognized for decades [25, 34]. Ten of 34 fertilized human ova recovered during the first 17 days of development were found to be abnormal [29, 30]. A high rate of chromosomal abnormalities has been found in oocytes (see reviews by Jacobs [34] and Hunt and Hassold [33]), in pre-implantation embryos [1, 4], and in embryonic/fetal or extra-embryonic tissues after spontaneous abortion [22]. The analysis of meiosis or abnormal live births in nonhuman primates has been sparse. The only study of aneuploidy in nonhuman primate oocytes involved five younger and seven older animals in which 4 out of 30 oocytes were aneuploid [54]. There are 15 cases of autosomal trisomies reported in nonhuman primates [16, 31, 42].

Conclusion

The data presented here have shown that the causes of fetal loss are similar in human and baboon populations. The incidence of fetal infection is lower and intracranial trauma/hemorrhages higher compared to those observed in humans. We present for the first time a documented case of Klebsiella infection associated with placentitis and two cases of maternal diabetes during pregnancy in the baboons. As seen in women, age, parity, and history of fetal loss are independent predictors of fetal loss in baboons, suggesting similar pathological mechanisms behind this phenomenon.