Introduction

Pelvic floor disorders are a major women’s health issue with significant negative effects on quality of life. The etiology of pelvic organ prolapse is multifactorial, however, vaginal delivery has been consistently shown to be a risk factor. This disorder occurs gradually and is most commonly seen in aging women who have delivered ≥1 infants vaginally. The overarching hypothesis is that injury to the integrity of the nerve muscle units of the pelvic floor initiates this process, leading to a reduction in the muscle’s ability to oppose intraabdominal forces. It also has been thought to result from direct damage to the muscles or associated connective tissues during parturition. Cesarean deliveries (CD) have been proposed as being a protective factor.

The squirrel monkey is an excellent animal model developing dystocia during parturition requiring CD and changes in the pelvic support structures resulting in prolapse. Little is known about the specific effects of pregnancy alone on the pelvic floor muscles. We have previously identified changes in the pelvic floor associated with pregnancy and parturition in the squirrel monkey model. The coccygeus (COC) muscle, within the posterior pelvis in squirrel monkeys, is correlated to the pubo-COC (levator ani [LA]), anteriorly located in human beings, as they both undergo significant strain, stretch, and injury during parturition. The monkeys have a fixed pubic symphysis so the pelvic floor has to widen toward the posterior pelvic rim, to accommodate the fused fetal skull producing forces that would inherently affect the COC. We examined animals prepregnancy, immediately prior to delivery, and a few days postpartum. Both pregnancy and delivery affected the COC muscle volume, but there was no consistent change in either the obturator internus (OI) or LA muscles following pregnancy or parturition. The study, however, was performed on multiparous females.

To determine if parturition-related changes in pelvic floor muscle volume and contrast changes could be prevented or diminished by CD we conducted a randomized controlled trial of scheduled primary CD on nulliparous females.

Materials and Methods

Squirrel monkeys were obtained from the National Squirrel Monkey Breeding and Research Resource in Bastrop, TX. Sixteen were housed at the Bastrop facility and 4 at the Scott and White Healthcare animal facility in Temple, TX. Both facilities are Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International accredited and institutional animal care and use committees with oversight for each facility reviewed and approved the project prior to initiation. Squirrel monkey breeding activity is seasonal with ovulatory cycles occurring from December through April in Texas even as animals are housed inside in light- and temperature-controlled rooms. Gestation lasts about 152 days. All females were evaluated with magnetic resonance imaging (MRI) prior to pregnancy. Once conception was confirmed, the pregnancies were dated and monitored using ultrasound. Spontaneous delivery was estimated to occur >147 days of gestation, so CD was planned for 144-147 days of gestation. All CD were performed by the veterinarian (J.C.R.) involved with the study, who is experienced with this procedure in squirrel monkeys. All pregnant animals within the study were randomized by computer-generated sequence number so that 10 females were assigned to spontaneous vaginal delivery and 10 assigned to scheduled CD. Assignments were made as pregnancies were confirmed and dated.

MRI was performed of the pelvic floor from L7-C4 using the validated procedure, previously described by Kramer et al and modified by Bracken et al. Using 3-dimensional fast low-angle shot MRI sequences, 224 sagittal images were acquired. A neonatal blood pressure cuff was placed around the animal’s abdomen to provide transabdominal pressure. A dynamic series (using 2-dimensional fast low-angle shot) of images was obtained during 25 seconds while inflating the cuff with a volume of air (40 mL) to reproduce prolapse as evaluated by perineal exam, if present, or voiding of bladder contents as described by Pierce et al. Twenty sagittal images along the midline were obtained to evaluate the position of the bladder neck relative to bony landmarks. A bony reference line was created from the anterior aspect of the pubic symphysis to the anterior aspect of the first tail vertebra. The position of the bladder neck relative to this reference line in millimeters was measured as an objective description of the degree of support of this pelvic structure. The females that had undergone CD did not have a dynamic series performed in the immediate postpartum exam. Instead, the position of bladder neck was measured in sagittal images without abdominal pressure. These are reported for completeness, but not used in analysis. As a monitor of sedation status, animals were visually observed for motion of limbs during the entire MRI process, which typically lasted <15 minutes. The high-resolution gradient echo sagittal views were reformatted as 224 axial views to obtain muscle volume and contrast intensity measurements. Animals were returned to the facility and monitored during recovery of sedation. All females were examined during the breeding season, prior to conception for their first MRI, then between 1-5 days after delivery and finally at 3 months postpartum.

The Digital Imaging and Communications in Medicine (DICOM) image files were processed using software (3D-Doctor; Able Software Corp, Lexington, MA) for review, manipulation, measurement, and analysis. The left and right sides of the OI, LA, and COC muscles were traced by authors (E.S.C., F.M.L., W.I.L., and T.J.K.) who were blinded to mode of delivery of the animal subjects. Each tracing was completed twice by different examiners and the averages used for analysis. The volumes were calculated by the software algorithm from the surfaces and thicknesses of the image series. The average volume for each paired muscle group is reported. For all of the muscles, high-contrast areas were traced separately. Recording and analysis of images using 3D-Doctor software has been established in our laboratory with good interobserver correlations for pelvic floor muscle volume measures. The pelvic outlet diameter is the distance between the inferior lateral margins of the obturator foramina at the level of the pubic arch. This measure was made in an axial MRI as reported previously for this species.

Analysis of variance with repeated measures design was utilized for parametric measures of muscle volumes and percentage of contrast (Statistica software; StatSoft, Tulsa, OK). Post hoc testing of means was performed using Duncan test with P < .05 indicating significance. Power analysis of 0.80 was calculated to detect a 24% difference in COC muscle volume ( P < .05).

Materials and Methods

Squirrel monkeys were obtained from the National Squirrel Monkey Breeding and Research Resource in Bastrop, TX. Sixteen were housed at the Bastrop facility and 4 at the Scott and White Healthcare animal facility in Temple, TX. Both facilities are Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International accredited and institutional animal care and use committees with oversight for each facility reviewed and approved the project prior to initiation. Squirrel monkey breeding activity is seasonal with ovulatory cycles occurring from December through April in Texas even as animals are housed inside in light- and temperature-controlled rooms. Gestation lasts about 152 days. All females were evaluated with magnetic resonance imaging (MRI) prior to pregnancy. Once conception was confirmed, the pregnancies were dated and monitored using ultrasound. Spontaneous delivery was estimated to occur >147 days of gestation, so CD was planned for 144-147 days of gestation. All CD were performed by the veterinarian (J.C.R.) involved with the study, who is experienced with this procedure in squirrel monkeys. All pregnant animals within the study were randomized by computer-generated sequence number so that 10 females were assigned to spontaneous vaginal delivery and 10 assigned to scheduled CD. Assignments were made as pregnancies were confirmed and dated.

MRI was performed of the pelvic floor from L7-C4 using the validated procedure, previously described by Kramer et al and modified by Bracken et al. Using 3-dimensional fast low-angle shot MRI sequences, 224 sagittal images were acquired. A neonatal blood pressure cuff was placed around the animal’s abdomen to provide transabdominal pressure. A dynamic series (using 2-dimensional fast low-angle shot) of images was obtained during 25 seconds while inflating the cuff with a volume of air (40 mL) to reproduce prolapse as evaluated by perineal exam, if present, or voiding of bladder contents as described by Pierce et al. Twenty sagittal images along the midline were obtained to evaluate the position of the bladder neck relative to bony landmarks. A bony reference line was created from the anterior aspect of the pubic symphysis to the anterior aspect of the first tail vertebra. The position of the bladder neck relative to this reference line in millimeters was measured as an objective description of the degree of support of this pelvic structure. The females that had undergone CD did not have a dynamic series performed in the immediate postpartum exam. Instead, the position of bladder neck was measured in sagittal images without abdominal pressure. These are reported for completeness, but not used in analysis. As a monitor of sedation status, animals were visually observed for motion of limbs during the entire MRI process, which typically lasted <15 minutes. The high-resolution gradient echo sagittal views were reformatted as 224 axial views to obtain muscle volume and contrast intensity measurements. Animals were returned to the facility and monitored during recovery of sedation. All females were examined during the breeding season, prior to conception for their first MRI, then between 1-5 days after delivery and finally at 3 months postpartum.

The Digital Imaging and Communications in Medicine (DICOM) image files were processed using software (3D-Doctor; Able Software Corp, Lexington, MA) for review, manipulation, measurement, and analysis. The left and right sides of the OI, LA, and COC muscles were traced by authors (E.S.C., F.M.L., W.I.L., and T.J.K.) who were blinded to mode of delivery of the animal subjects. Each tracing was completed twice by different examiners and the averages used for analysis. The volumes were calculated by the software algorithm from the surfaces and thicknesses of the image series. The average volume for each paired muscle group is reported. For all of the muscles, high-contrast areas were traced separately. Recording and analysis of images using 3D-Doctor software has been established in our laboratory with good interobserver correlations for pelvic floor muscle volume measures. The pelvic outlet diameter is the distance between the inferior lateral margins of the obturator foramina at the level of the pubic arch. This measure was made in an axial MRI as reported previously for this species.

Analysis of variance with repeated measures design was utilized for parametric measures of muscle volumes and percentage of contrast (Statistica software; StatSoft, Tulsa, OK). Post hoc testing of means was performed using Duncan test with P < .05 indicating significance. Power analysis of 0.80 was calculated to detect a 24% difference in COC muscle volume ( P < .05).

Results

Two groups of 10 females were randomized to either spontaneous vaginal delivery or scheduled primary CD upon diagnosis of pregnancy with serial ultrasound. The baseline evaluations of both groups were similar ( Table 1 ) for age, body size, and pelvic floor measurements including muscle volumes, bladder neck position with abdominal pressure, and width of the bony pelvic outlet. No vaginal lacerations were found on exam and no detected anal sphincter tears observed on MRI. Pregnancy outcomes were similar ( Table 2 ) except that animals undergoing scheduled CD were delivered about 3 days earlier than those delivering spontaneously. There was an impression that the first CD animal may have had the beginnings of labor, however the head was not engaged. Perinatal mortality was equal in both groups. The first 2 infants delivered by CD developed respiratory depression related to maternal anesthesia. The anesthetic regimen was modified and no other complications were noted. One of the females in the spontaneous vaginal delivery group had perinatal fetal demise related to a spontaneous preterm delivery at 139 days, about 2 weeks premature. The other perinatal mortality event was related to protracted labor due to an abnormal presentation resulting in dystocia. This infant died in utero, but was delivered by CD. The female was included in the analyses as a vaginal delivery based on the intention-to-treat principle.

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