Uterine Dysfunction in Biglycan and Decorin Deficient Mice Leads to Dystocia during Parturition

Cesarean birth rates are rising. Uterine dysfunction, the exact mechanism of which is unknown, is a common indication for Cesarean delivery. Biglycan and decorin are two small leucine-rich proteoglycans expressed in the extracellular matrix of reproductive tissues and muscle. Mice deficient in biglycan display a mild muscular dystrophy, and, along with mice deficient in decorin, are models of Ehlers-Danlos Syndrome, a connective tissue anomaly associated with uterine rupture. As a variant of Ehlers-Danlos Syndrome is caused by a genetic mutation resulting in abnormal biglycan and decorin secretion, we hypothesized that biglycan and decorin play a role in uterine function. Thus, we assessed wild-type, biglycan, decorin and double knockout pregnancies for timing of birth and uterine function. Uteri were harvested at embryonic days 12, 15 and 18. Nonpregnant uterine samples of the same genotypes were assessed for tissue failure rate and spontaneous and oxytocin-induced contractility. We discovered that biglycan/decorin mixed double-knockout dams displayed dystocia, were at increased risk of delayed labor onset, and showed increased tissue failure in a predominantly decorin-dependent manner. In vitro spontaneous uterine contractile amplitude and oxytocin-induced contractile force were decreased in all biglycan and decorin knockout genotypes compared to wild-type. Notably, we found no significant compensation between biglycan and decorin using quantitative real time PCR or immunohistochemistry. We conclude that the biglycan/decorin mixed double knockout mouse is a model of dystocia and delayed labor onset. Moreover, decorin is necessary for uterine function in a dose-dependent manner, while biglycan exhibits partial compensatory mechanisms in vivo. Thus, this model is poised for use as a model for testing novel targets for preventive or therapeutic manipulation of uterine dysfunction

Lack of a significant compensatory mechanism at the gene level between biglycan and decorin in the knockout mice.

<p>Levels of biglycan mRNA in wild-type and <i>Bgn+/+Dcn−/−</i> mouse uterus, as well as decorin mRNA in wild-type and <i>Bgn−/−Dcn+/+</i> mouse uterus were determined using qPCR at progressive gestational ages. Biglycan and decorin are not developmentally regulated in the pregnant uterus, nor do they compensate for each other at the transcript level in the <i>Bgn−/−Dcn+/+</i> or the <i>Bgn+/+Dcn−/−</i> (<i>P</i> = 0.271 and <i>P</i> = 0.351 respectively). Two-way ANOVA Holm-Sidak. n = 4−6 samples from 4–6 pregnant dams per condition. E = embryonic day. Error bars = SD.</p

Altered TGF-β expression in biglycan knockout mice.

<p><b>A:</b> TGF-β expression was evaluated in uterine samples of pregnant dams at E18. Western blotting was performed on wild-type, <i>Bgn−/−Dcn+/+</i> and <i>Bgn+/+Dcn−/−</i> uteri. GAPDH was used as an internal standard. <b>B:</b> TGF-β is decreased in the absence of biglycan. Digitally scanned and densitometrically analyzed Western blots from three experiments from three individual mouse uteri are expressed as a ratio of TGF-β over GAPDH and normalized to the wild-type ratio. Student's t-test. <i>P</i> = 0.004 for biglycan knockout; <i>P</i> = 0.46 for decorin knockout. Error bars = SD.</p

<i>Bgn+/−Dcn−/−</i> dams display an increase in length of gestation (delayed onset of parturition after embryonic day 21) compared to all other genotypes.

<p>***Chi square test: <i>P</i><0.001.</p><p><i>Bgn</i> = biglycan. <i>Dcn</i> = decorin.</p

Abnormal rate of dystocia in biglycan/decorin knockout mice.

<p><b>A:</b> Percentage of births displaying dystocia per mouse genotype. Biglycan and decorin are necessary for contractile activity leading to birth in a dose dependent manner. <i>Bgn−/−Dcn+/+</i> females are not at increased risk of dystocia, while mice lacking one or both decorin alleles are at increased risk of dystocia. <i>P</i><0.001. Chi-square test. <i>Bgn+/+Dcn+/+</i> n = 13; <i>Bgn−/−Dcn+/+</i> n = 33; <i>Bgn+/+Dcn−/−</i> n = 11; <i>Bgn+/−Dcn+/−</i> n = 14; <i>Bgn+/−Dcn−/−</i> n = 20; <i>Bgn−/−Dcn+/−</i> n = 30; <i>Bgn−/−Dcn−/−</i> n = 7. Bgn = biglycan; Dcn = decorin. <b>B:</b> The percentage of dams displaying dystocia increases with decreasing number of maternal total SLRP (<i>P</i><0.001) and decorin (<i>P</i><0.001) alleles, but is independent of biglycan alleles (<i>P</i> = 0.348). Chi-square test. 4 SLRP alleles n = 13; 2 SLRP alleles n = 58; 1 SLRP allele n = 50; 0 SLRP alleles n = 7. SLRP = small leucine rich proteoglycan.</p

Abnormal uterine contractile force in response to oxytocin.

<p>Uterine contractile force development was examined after exposure to increasing doses of oxytocin. The wild-type displays a stepwise increase in contractile force on exposure to oxytocin. The absence of biglycan, decorin, or both results in attenuation of this response. Data is normalized to the average force of spontaneous contraction for each genotype. Baseline represents relaxation period after activation by K-PSS. <i>P</i> = 0.005. Two-way ANOVA Holm-Sidak. <i>Bgn+/+Dcn+/+</i> n = 4; <i>Bgn−/−Dcn+/+</i> C3H n = 5; <i>Bgn−/−Dcn+/+</i> C3H/C57BL n = 3; <i>Bgn+/+Dcn−/−</i> n = 5; <i>Bgn−/−Dcn−/−</i> n = 3. <i>Bgn</i> = biglycan. <i>Dcn</i> = decorin. Error bars = SD.</p

Abnormal uterine contractions in biglycan/decorin knockout mice.

<p><b>A:</b> Representative contractile force tracings of spontaneous isometric uterine contractions display phenotypic differences per genotype. The wild-type displays regular, phasic contractions. In contrast, the absence of biglycan, decorin, or both, leads to decreased amplitude. <b>B:</b> Quantification of isometric uterine contractile amplitudes. The waveform amplitude is decreased in all knockouts compared to the wild-type. <i>P</i> = 0.012. One way ANOVA Holm-Sidak. Error bars = SD. <i>Bgn</i> = biglycan. <i>Dcn</i> = decorin.</p