INTRODUCTION

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THE VARIOUS PROCESSES COMPRISING mammalian pregnancy and parturition evolved within the omnipresent context of the normal gravitational forces on Earth, thus raising the question of whether pregnancy and birth can be successfully sustained in the absence of gravity. In the only previous spaceflight in which pregnant mammals were exposed to microgravity, rats in late stages of pregnancy were flown on the 4.5-day Cosmos-1514 mission in 1983 (9). After this brief flight, four of five dams gave birth to viable litters. Parturition was not observed systematically in the Cosmos study. Also, it is not known how longer flights might affect physiological or behavioral responses of pregnant and parturient females and the process of birth. The most common effects of spaceflight, namely, headward fluid shifts, alterations in bone and calcium metabolism, and muscular deconditioning (5, 8, 10), may provide formidable obstacles to sustaining the gravid state in space and impede the ability of mothers to give birth.

Of particular concern are the potential effects of spaceflight muscle deconditioning on the musculature of pregnant dams in the days preceding parturition. For example, expulsion of the conceptus may be compromised because of deconditioning of the transverse abdominus, an antigravity muscle in tetrapods (6).

Much is known about parturition in the Norway rat (Rattus norvegicus) (4, 14, 15). At the time of birth, the female rat's behavior is centered on the processes of delivery and the products of birth, namely, the fetuses, placentas, and birth fluids (8). The entire process begins just a few hours before birth with the transition from infrequent, low-amplitude uterine contractions to regular, more intense contractions. This shift signals the onset of labor (4, 15). Direct measurements of intrauterine pressure in rats suggest that, near parturition, the rat fetus is exposed to contractions approaching 20 mmHg (7). We previously described and quantified labor and delivery in the rat using 24-h time-lapse videography (15). Several behaviorally distinct types of uterine contractions can be observed during labor. During a lordosis contraction, the dam lies on her ventrum and elongates her body, often arching her back and elevating her outstretched hindlimbs off the ground. More than 70 lordosis contractions may be observed during a typical birth, at intervals less than 35 s apart during the last hour of labor. Lordosis contractions predominate before the birth of the first pup and are believed to transport the conceptus into the lower birth canal. Vertical contractions, observed just before the birth of a pup, consist of a series of rapid, bilateral abdominal lifts. Rat dams typically deliver from 8 to 12 pups over a period of 40-140 min. Elements of maternal behavior, such as licking and retrieving, emerge when the first pup is expelled from the womb. At birth, the mother licks the neonate, removing its birth membranes, thereby helping to initiate postnatal breathing (13). The onset of nursing occurs soon after the last pup is born.

In the present experiment, we tested hypotheses that mammalian pregnancy and parturition can survive exposure to sustained periods of spaceflight. On the basis of the brief Cosmos-1514 mission, we predicted that pregnant rats flown on longer (11 and 9 day) missions would also complete their 22-day pregnancies and undergo vaginal deliveries but that parturition would not be successful for all rat dams. The longer period of spaceflight exposure relative to the Cosmos mission was predicted to increase fetal losses and reduce the number of live births. We predicted specific effects on labor contractions at the time of parturition, mediated via spaceflight-induced changes in uterine contractile proteins (3) and abdominal musculature (6). We also hypothesized that labor contractions would be less effective after spaceflight exposure, possibly lengthening the birth process. We also predicted postflight behavioral changes such as reduced appetite and lethargy and thus quantified the dams' postflight feeding, drinking, and locomotion. Characteristic maternal responses to the young during parturition were analyzed to test the hypothesis that patterns of maternal care would be disrupted after spaceflight.

The data presented are derived from two spaceflight missions jointly sponsored by National Aeronautics and Space Administration (NASA) and National Institutes of Health (NIH) and are called the Rodent 1 (NIH.R1) and Rodent 2 (NIH.R2) missions. Ten rat dams were flown on each mission, launched at the approximate midpoint of pregnancy [gestational day (GD) 9 for NIH.R1 and GD11 for NIH.R2] and landed close to the time of parturition (GD20 of the rat's 22-day pregnancy). The mission lengths were 11 and 9 days, respectively. The dams on each flight were treated similarly. Continuous postflight video surveillance of both NIH.R1 and NIH.R2 rat dams, including time-lapse recordings of labor and delivery, permitted us to replicate the parturition analyses. This was particularly important because the NIH.R1 study involved performing a unilateral hysterectomy on each dam soon after recovery on GD20, the major difference between the two flights. This was done so that both fetal and neonatal samples could be obtained from each of the NIH.R1 subjects (1).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Subjects. Forty nulliparous, pregnant Sprague-Dawley rats (Taconic Farms, Germantown, NY) weighing between 165 and 205 g were used. The time-bred dams were shipped to Kennedy Space Center (KSC) on GD2 (spermatozoa positive = GD1). Animals were housed in a room with controlled lighting (6 AM to 6 PM) and temperature (~22°C). Pregnant rats were housed individually in standard vivarium cages (47 cm × 26 cm × 21 cm) with corncob bedding material. Rat chow and water were available ad libitum. All animal procedures adhered to NASA guidelines and the NIH Guide for the Care and Use of Laboratory Animals. [DHHS Publication No. (NIH) 85-23, Revised 1985, Office of Science and Health Reports, Bethesda, MD 20892].

Treatment of dams. Two treatment groups were used in these experiments. For each study, 10 dams were housed in groups of five in flight animal modules (described in Surgical laparotomy of the NIH.R1 and NIH.R2 dams, below) and exposed to launch, spaceflight, and landing (flight group). Synchronous control dams (n = 10) were treated identically to flight animals but were not exposed to launch, landing, or spaceflight. These animals were run at the same gestational ages as the flight group but with a 24-h delay relative to the flight group to allow time for downlinking from the shuttle of the previous day's environmental conditions. In this way, environmental parameters (i.e., temperature, humidity, and exposure to augmented lighting during video recording) onboard the shuttle could be mimicked for the synchronous control group. The preflight (12:12 h) light-dark cycle was maintained in the housing for both the flight and ground groups. Before flight, dams were carefully matched according to weight across flight and synchronous control conditions.

Maternal surgeries. All experimental dams sustained two surgical procedures during the study (described below). On GD7, surgical laparotomy was performed to confirm pregnancy and establish the number of implantation sites. Dams were selected for inclusion in the study only if a minimum of five embryos populated each of their paired uterine horns. On GD20, immediately after recovery from the space shuttle, the NIH.R1 dams (but not the NIH.R2 dams) were given a unilateral hysterectomy under anesthesia, yielding for immediate analysis of fetuses from all 10 dams (i.e., n = 10). The same dams then recovered from anesthesia, completed gestation, and underwent vaginal delivery of pups from the remaining uterine horn, thereby providing neonates for postnatal analyses (n = 10). The NIH.R2 dams were either observed until birth (n = 6) or dissected on recovery (n = 4). Only the dams that underwent parturition are discussed in this report.

Surgical laparotomy of the NIH.R1 and NIH.R2 dams. Laparotomy was conducted on flight and synchronous control dams under aseptic conditions on GD7, the earliest day on which implantation sites (decidual swellings) can be reliably visualized. This procedure is described elsewhere (1). Briefly, the dam was anesthetized with isoflurane (IsoFlo, Abbott Labs, North Chicago, IL) vapor using a nonrebreathing rodent anesthesia unit (Viking Products, Medford Lakes, NJ). The fur overlying the abdomen of the anesthetized rat was shaved, the skin was cleansed with antiseptic and alcohol, a veterinary opthalmic ointment was applied, and an antibiotic and analgesic mixture [Microcillin, Anthony Products, Arcadia, CA (10,000 IU) and butorphanol tartrate, Fort Dodge Labs, Fort Dodge, IA (10 mg/kg)] was given by subcutaneous injection. An incision was made, beginning ~2 cm cranial to the pubis and extending cranially 2-3 cm. Each uterine horn was gently grasped between decidual swellings and gently externalized for close visual inspection and counts of implantation site, which were recorded. The uteruses were carefully reinserted into the abdominal cavity, after which interrupted sutures were used to close the peritoneum and muscle layer. The overlying skin was then closed with 9-mm wound clips. The entire procedure lasted 10 min.

Unilateral hysterectomy of the NIH.R1 dams. The purpose of the unilateral hysterectomy was to provide a sample of fetuses soon after return from spaceflight (see Ref. 1 for further background and discussion of the rationale). Within 3 h after recovery from spaceflight, the first flight dam was anesthetized. Anesthesia and surgical preparations were identical to those described for the laparotomy procedure on GD7. The uterine horns were exteriorized by extending the midventral abdominal incision. The uterine horn removed was alternated across rats in each treatment group. The horn was ligated cranially and caudally with braided silk (20, Ethicon, Somerville, NJ) and then excised. The incision was closed by following the procedures used for laparotomy.

Video recording of labor and birth. Within several hours of both recovery and unilateral hysterectomy (NIH.R1) or recovery (NIH.R2), the dams were housed singly in Plexiglas observation cages (12.5 cm × 8.5 cm × 9.25 cm) lined with corn cob bedding and placed in a vivarium. Food bars were placed on the cage floor, and Lixit spouts were positioned near the base of the cage to facilitate the mothers' access to food and water. Daily records of food bar consumption, water intake, and body weight were maintained for the remainder of the study. The dams were videotaped continuously beginning soon after recovery until the completion of parturition and the onset of nursing. A mirror was angled at the rear of each observation cage to permit camera views from both the front and rear of the cage. Cages were positioned four per camera view. Red lighting was illuminated during the dark phase to enable 24-h video data collection (12:1 record-to-playback ratio).

Data analysis. Video data were analyzed by trained scorers during real-time playback of the videotapes time locked to a computerized event-scoring program (13). Briefly, the amount of time dams spent feeding, drinking, or ambulating was quantified with the use of this system. Interrater reliability (IRR) for these measures was R² > 0.99. The number and duration of labor contractions, number of neonates born, placentophagia (ingestion of placenta), the total duration of birth, maternal care (licking and handling of neonates), and the onset of nursing were also encoded from the video recordings (IRR was R² > 0.98). Individual data were expressed as litter means and analyzed with the use of ANOVA, t-tests, or simple regression.

	RESULTS

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NIH.R1 and NIH.R2 dams at recovery. Because of inclement weather, the shuttle carrying the NIH.R1 payload landed at the Hugh Dryden Flight Research Facility (HDFRF) alternate landing site in California. Within 3 h of landing, the rats were delivered to the payload receiving facility. The dams were then carefully unloaded from the AEMs, given a health examination, and weighed. Dam body weight gains at shuttle load and unload were identical in the flight and synchronous control groups (percent change from GD9 to GD20 as follows: flight = 45.7 ± 2.0 and synchronous control = 42.4 ± 1.7%; not significant). All of the dams were deemed to be in good condition. Unilateral hysterectomy was performed on the flight group dams without complication. Over the next several hours, the dams showed characteristic signs of recovery from general anesthesia and surgery.

Readaptation of flight dams to 1 G. In contrast to the NIH.R1 dams, which received postflight surgery (i.e., unilateral hysterectomy), data from the NIH.R2 dams provide an unbiased perspective of the effects of spaceflight on pregnant mothers' behavioral readaptation to 1 G. These data are shown in Fig. 1. Results of the time-lapse analyses are presented across three consecutive 12-h time intervals beginning with the dark phase of the circadian cycle on GD20 [corresponding to recovery (R) + 12 h (R + 12)] and ending 36 h later (at R + 48), coincident with the onset of the light phase of the cycle on GD22. This analysis revealed that flight dams ambulated less than did synchronous control dams [Fig. 1A; gravity F(1,10) = 14.5, P < 0.01; Newman-Keuls test, P > 0.05] but only during the dark phase of the circadian cycle [time interval F(2,20) = 12.5, P < 0.001; gravity × time interval F(2,20) = 7.5; Newman-Keuls, P > 0.05]. During the light phase of the cycle (R + 24 and R + 36), a floor effect was observed that obscured potential group differences: both flight and synchronous control dams locomoted for less than 5% of the observation interval during the lights-on period. Despite the reduced activity of the flight dams during the dark phase of the cycle, the amount of time dams spent eating and drinking was equivalent to that of synchronous dams (Fig. 1B; gravity F < 1), and the typical circadian rhythm of feeding behavior was observed [time interval F(2,20) = 29.3; P < 0.0001].

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Behavioral readaptation of National Institutes of Health (NIH) Rodent 2 (NIH.R2) flight and synchronous control dams to 1 G (n = 6 per condition). Locomotion (top) and eating and drinking (bottom) across consecutive 12-h dark-light-dark periods beginning at 6 PM on gestational day (GD) 20 [recovery (R) + 12h (R + 12)], at 6 AM on GD21 (R + 24), and at 6 PM on GD21 (R + 36). Locomotion differed across flight and synchronous dams during the dark phase of the circadian cycle (*P < 0.05); eating and drinking were identical across the groups.

Labor in flight dams. Dams from both flights began labor at the expected time (on GD22 and GD23) with three exceptions: two NIH.R1 flight dams and one synchronous dam did not show signs of impending parturition by 1500 on GD23. In accordance with predetermined project requirements, the neonates of these dams were delivered by cesarean section. There was no corresponding requirement for NIH.R2; however, cesarean delivery was performed on one dam from the flight because she appeared to be in distress during labor. Video failure caused data from two additional NIH.R1 control animals to be lost. We present complete data from eight flight dams and seven synchronous control dams for NIH.R1 and from five flight dams and six synchronous control dams for NIH.R2.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Top: behavioral expression of a lordosis contraction in the parturient rat dam. Bottom: number of lordosis contractions observed in NIH Rodent 1 (NIH.R1) (n = 15) and NIH.R2 (n = 12) flight (left) and synchronous control (right) dams. Observations antedated the birth of the first pup by 6 h and continued until the birth of the last pup. The number of lordosis contractions observed in flight and synchronous dams differed from one another (*P < 0.05). Note: NIH.R1 dams underwent unilateral hysterectomy before parturition.

Birth. The dams successfully delivered strong and viable pups. Table 1 shows the number of decidual swellings, the numbers of neonates born, neonatal birth weights, total duration of birth, ingestion of placentas (placentophagia), and maternal behavior during parturition, as measured by licking and retrieving of neonates. In contrast to our initial prediction, morbidity was very low. For NIH.R1, 4 of 60 pups born to flight dams and 1 of 58 pups born to synchronous dams were found dead on the day of birth. For each of the other measures, identical results were obtained for flight and synchronous conditions. The results of the two flights were highly consistent with each other.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The pregnant spaceflight dams returned to Earth in good condition. For NIH.R1, weight gain of dams during flight, particularly important to the fetuses developing in utero, was comparable to the weight gain of ground control dams. This finding is consistent with normal Earth gravity body weight gains seen in adult male rats during 14-day spaceflight missions (14). NIH.R2 flight dams gained about 5% less than synchronous controls, but differences were not observed in any other measure of maternal, fetal, or neonatal outcome after flight. Body weights of dams in the flight and synchronous control conditions were identical at launch for both flights. Because the NIH.R2 mission was 2 days less in duration than the NIH.R1 mission, one possibility is that initial postflight weight loss was fully regained in flight on the longer 11-day mission (NIH.R1) but not achieved by 9 days (NIH.R2). Additional studies are needed to characterize profiles of body mass change in pregnant animals following launch, on orbit, and on recovery from space.

The major finding of our analyses is that flight dams had uncomplicated, successful vaginal deliveries. Parturition occurred at the appropriate gestational time. Number and size of the litters were equivalent to those of controls. Because we had noted during preflight laparotomy the number of implantation sites in the uterine horns of each dam on the 7th day of pregnancy, we were also able to determine that fetal loss, i.e., the difference between the number of implantations and number of pups born to each dam, was equivalent between groups. These findings were seen in both the NIH.R1 and NIH.R2 flight groups. The correspondence between these two data sets is striking particularly because the NIH.R2 dams were not surgically manipulated before collection of observational data.

Readaptation of flight dams to 1 G. The NIH.R2 dams provided the first systematic and continuous observational data ever collected on the postflight readaptation of rats to 1 G. The flight dams were generally less active than the synchronous control dams, as indicated by reduced ambulation during the dark (active) phase of the circadian cycle. There were differences in time spent eating and drinking between the flight dams and the synchronous controls, and both groups followed characteristic circadian fluctuations. The postflight reduction in the locomotor activity of dams is analogous to that of pregnant dams undergoing adaptation from the normal 1 G on Earth to 1.5-G hypergravity (14).

Labor after spaceflight. Labor contractions were affected by spaceflight during pregnancy. The NIH.R2 dams did not receive the abdominal surgery shortly before labor; therefore, their data are not confounded in any way. Nevertheless, the pattern of results from the two spaceflights was strikingly clear and reliable.

Birth and maternal care of neonates after spaceflight. Although more contractions may have been required for parturition in the flight group, this difference did not affect the duration or temporal patterns of birth. The flight dams appeared to be competent mothers. Maternal licking and handling of neonates during parturition and the consumption of birth fluids and membranes (i.e., placentophagia) were indistinguishable in flight and synchronous control dams. Within 3 h of birth, mammary tissue was visually inspected and mammary gland metabolic activity was analyzed (11). These studies indicated that the dams' were physiologically prepared for lactation.