How many fetuses are in the uterine horns of the rat

To evaluate our animal model, vaginal smears were collected daily between and AM for estrous cycle determination. Adult SD rats were anesthetized Supplementary Figure S1E , first panel , and the uterine horns were exposed through an abdominal incision.

The uterus of rat was opened along the side of nonmesometrium, and uterine walls comprise three layers—the serosa, the myometrium and the endometrium, with the myometrium consisting of circular smooth muscle near the lumen and subserosal longitudinal smooth muscle Supplementary Figure S1E , second panel. A segment of circular smooth muscle and endometrium approximately 1. Immediately after resection surgery, the resected surface was pressed with hemostatic gauze for hemostasis, and the damaged area was confirmed to be thin.

In a self-correlative study, rat uterine side-by-side comparisons were observed for diversity restoration effects between the cell sheet group and the control group.

The cell sheets show strong adhesion, of which we take advantage to complete the transplant operation. In the cell sheet transplant group the left uterine horn , triple-layer ADSCs sheet were implanted to replace the segmental uterine excised tissue Supplementary Figure S1F , and the uterine surface wound with the ADSC sheets remained exposed for 30 min to ensure engraftment.

A mm culture dish covered the wound to prevent from water loss. The control group the right uterine horn had uterine lesions that were left untreated Supplementary Figure S1F and the sham group rats received abdominal incisions with the uterine horns intact.

The uterine wound was closed by absorbable sutures and abdominal incision was closed by absorbable sutures. All rats received penicillin by intramuscular injection twice a day for three days. At 21, 30, and 60 days after surgery, all the uterine horns were tested by histological and immunofluorescence stain Supplementary Figure S1F.

Additionally, all slides were incubated for 15 min with 4, 6-diamidinophenylindole DAPI, Santa Cruz for nuclear staining. Slides were observed with a confocal laser scanning microscope Leica, Nussloch, Germany. Staining results were analyzed using an optical microscope Leica, Nussloch, Germany. Uterine function was assessed by determining whether the regenerating uterine horn was capable of implanting a fertilized ovum and providing adequate nourishment to the developing fetus.

Vaginal plugs were tracked every day at AM to confirm whether mating had occurred. Animals were euthanized A number of fetuses at scar site were calculated by independent sample t-tests between control and cell sheet group.

Fisher's exact test was performed for comparison of pregnancy rate at scar site in cell sheet group versus control group. Phenotype and multidifferentiation of rat ADSCs.

Scaffold-free ADSC sheets represent a new technology to regenerate injured or damaged tissues. After 10—14 days of cultivation in inducing medium, ADSCs sheets were harvested from 6-well plates in vitro. The first panel in Figure 2A shows the surface of a sheet that detached at the bottom with the edge cocked, while the second panel in Figure 2A was floating ADSCs sheet.

The intact ADSC sheets were harvested and reshaped to the rectangle. Based on scanning electron microscopy examination, ADSCs sheets were compact overlayer films. C SEM images of ADSCs sheet formation and distinct secretory granules with the appearance of spherical vesicles on cell sheet surfaces red arrow. Abundant endometrial glands were observed in the endometrial stromal layer Figure 3A , left panel.

Masson's trichrome staining was performed on the uterine horns to characterize the extent of the muscle layers, which were composed of smooth muscle cells in circular and longitudinal patterns in the nonoperative area, leaving behind only the longitudinal muscle layer in the operative area Figure 3A , right panel.

Immunofluorescence staining with an anti-Ck antibody revealed Ck-positive epithelial cells on the normal uterine adluminal surface and the endometrial gland in the endometrial stromal layer white arrow.

No simple columnar epithelial cells were found on the endometrial surface of the resected tissue, and no endometrial glands were observed either Figure 3B , first panel. Vm-positive stromal cells were distributed in region under the simple columnar epithelial cells white arrow. Conversely, we did not find Vm-positive stromal cells in the resected area of the endometrium Figure 3B , middle panel. Sm-positive muscle bundles were labeled with green fluorescence following immunofluorescence staining.

As expected, no circular fibers were found in the muscle layer. Only Sm-positive longitudinal muscle bundles were observed in the resected area yellow arrow. Conversely, Sm-positive circular and longitudinal fibers were observed in normal areas white arrow Figure 3B , below panel. Based on these results, the endometrial epithelial layer, basal layer, and circular fibers were successfully ablated in the uterine damage model, laying the foundation for the regeneration of damaged uteri.

Experimental procedure for the rat uterine regeneration model. A Schematic illustration showed the experimental procedure. B Uterine horns were exposed, and half of the uterus, approximately 1. Triple-layer ADSCs sheets were transplanted onto the damaged inner uterine surface, and the uterine wound was left open for 30 min after transplantation to ensure engraftment.

C and D To assess the uterine damage model, uteri were resected, and specimens were subjected to histological analysis after surgery. Black arrows and blue arrows indicate endometrial glands and the circular muscle layer, respectively. D Immunofluorescence staining of specimen components using antibodies against Ck, Vm, and Sm.

White arrows show the endometrial epithelial layer first panel , endometrial basal layer middle panel , and circular muscle layer, respectively last panel. Yellow arrows indicate the presence only of the remaining longitudinal muscle layer in the regeneration model.

Dotted lines indicate repair sites. Gross examination showed that luminal stenosis occurred in the surgical regions of the uteri in the control group at 30 days Figure 4C , and adhesions or pathological stenosis formed at 60 days Figure 4E. However, luminal stenosis and scars were not observed in the cell sheet group Figure 4D and F , and the uterine anatomy was similar to the sham group Figure 4A and B.

Representative images show that more blood vessels were present in the cell sheet group at both 30 and 60 days after surgery Figure 4D and F , while the control group exhibited pale serosa Figure 4C and E.

Gross view and histological evidence for uterine horn regeneration among the groups. The appearance of surgical regions indicated superior vascularization for the repair of segmental defects in the rat uterus in the cell sheet group compared with the control group.

J, K Statistical analysis of endometrial thickness revealed a thicker endometrium in the cell sheet group than in the control group; however, the endometrium was thinner than that of the sham group. Black arrows indicate endometrial glands in the basal layer. As shown in Figure 4H , the cell sheet group exhibited greater numbers of endometrial glands in the endometrial stromal layer and more apparent neovascularization 30 days after surgery.

There were significant differences in endometrial thickness between the cell sheet group At 60 days, the specimens in the cell sheet group appeared near-normal and exhibited more angiogenesis. No epithelial-like cells were observed in uterine centers in the control group, but columnar epithelium lining was present in the cell sheet group Figure 4H. Endometrial thickness in the cell sheet group According to Masson's trichrome staining on postoperative day 30, uterine tissue in the sham group was present in the muscle layer, which consists of internal-circular muscle and external-longitudinal muscle Figure 4I.

At 30 days, positively stained smooth muscle cells turned light red and were arranged in a fascicular pattern in the cell sheet group but not the control group Figure 4I.

At 60 days, we observed the orderly arrangement of smooth muscle cell fascicles at near-normal levels in the cell sheet group. However, the endometrium in the control group exhibited fibrous tissue hyperplasia and collagen deposition and eventually formed scars Figure 4I. We assessed epithelial regeneration using an anti-Ck antibody.

Ck-positive epithelial cells covered the surface of the excised uterine lumen after 30 days in the cell sheet group. In contrast, few epithelial cells were found at excision sites in the control group Figure 5A. The lumenal surface of the uterus was a simple columnar epithelium, which became more organized and exhibited secretory glands after 60 days in the cell sheet group but lacked epithelial cells and secretory glands in the control group Figure 5A.

D CD31 expression, indicative of neovascularization in the regenerated endometrium, was detected at 30 and 60 days in the sham group, control group, and cell sheet group. Each experiment was repeated four times. Uterine specimens from each group were tested for Vm expression by performing immunofluorescence microscopy to investigate stromal cell regeneration.

Vm-positive stromal cells in excised sections were detected by the presence of a bright green fluorescent signal in the basal layer of the endometrium in the cell sheet group Figure 5B. Although Vm expression was also observed in the same region in the control group, it was present at a lower frequency, and the endometrium was thinner than that in the cell sheet group Figure 5B. At 60 days, greater numbers of endometrial glands were observed in the endometrial stromal layer in the cell sheet group, and the fluorescence intensities of Vm-positive stromal cells were enhanced compared with those at 30 days; however, the control group still exhibited lower frequencies of Vm-positive cells Figure 5B.

We used a panel of monoclonal rabbit anti-rat antibodies raised against smooth muscle actin Sm to explore smooth muscle regeneration at sites of injury. At 30 days, new circular muscle bundles formed a thin successive layer beneath the endometrium in the cell sheet group. However, muscle bundles were absent at injured sites in the control group with the exception of anastomosis sites Figure 5C. AOD in the cell sheet group 0.

At 60 days, some collagen deposition and scar tissue formation was observed in the control group, but no obvious circular muscle bundles had formed. The smooth muscle tissue converged to form thick muscle layers in the cell sheet group that were denser and arranged. However, at the site of anastomosis, only circular fibers were observed, rather than circular and longitudinal fibers Figure 5C. The AOD in the cell sheet group 0. Thus, cell sheets promoted the regeneration of injured muscle bundles, and this outcome became increasingly evident over time.

Neovascularization is one of significant standards to evaluate the regeneration of the injured uterine regions.

At 30 days after the implantation of cell sheet grafts, blood vessel density within the cell sheet group was higher, with neovascularization located near the site of injury Figure 5D. Blood vessel density in injured uterine regions in cell sheets group At 60 days, the blood vessel was increased in all groups compared with 30 days, however, the cell sheet group Twenty-one days later, ADSCs fused with host cells and prompted local cells migration to the wound edges. Notably, clusters of cells labeled with red fluorescence were primarily distributed in the basal layer of the regenerated endometrium Figure 6A.

These typical images illustrate the differentiation of ADSC sheets during development in the uterine microenvironment. The location and differentiation of ADSCs in the newly regenerated uterine endometrium 21 days after cell sheet transplantation as determined by immunofluorescence staining.

Programmed cell death apoptosis is the physiological process by which excess or dysfunctional cells are eliminated during development or normal tissue homeostasis, and nowhere is this process more dramatic than in the reproductive system.

Apoptosis occurs cyclically in human nonpregnant endometrium; throughout pregnancy in the decidua, placental, and amnion epithelial cells; in cervical smooth muscle cells SMCs at later pregnancy; and in mammary glands during weaning [ 7 — 12 ].

Two different pathways of apoptosis activation exist: the extrinsic pathway involving Fas ligand or tumor necrosis factor alpha activation of specific death receptors and the intrinsic pathway activated by cellular stressors such as hypoxia, DNA damage, or ionizing radiation. Importantly, caspases in higher organisms have acquired functions in cellular processes other than apoptosis, including involvement in cell survival, cell cycle [ 14 ], and normal differentiation [ 15 , 16 ].

Fernando et al. In addition, activation of CASP3 has been correlated with reduced myocyte contractility in cardiac [ 18 ], skeletal [ 19 ], and smooth muscle [ 20 ]. CASP3 is transiently activated at midgestation in the rat and mouse myometrium, but this activation is not associated with biochemical or morphological features of apoptosis [ 1 , 21 ]. Jeyasuria et al. Though the mechanisms leading to CASP3 activation remain unclear, it coincides with the transition between the phases of myometrial hyperplasia and hypertrophy, which occurs around Gestational Day 14 in rats.

We have suggested that the myocyte differentiation that induces the transition of phenotypes is related to acute stretching of the uterine wall by the growing conceptus [ 1 ].

The goal of this study was to investigate the mechanisms underlying caspase activation associated with differentiation of the myometrium from a state of hyperplasia to hypertrophy. We hypothesize that activation of this pathway is induced by myocyte hypoxia resulting from an acute mechanical stretch on the uterine wall. Thus, our specific aims were to 1 determine whether the myometrium is exposed to hypoxia during this period of pregnancy and to determine the contribution of mechanical stretch exerted on caspase activation at different time points throughout gestation 2 physiologically as a result of embryo growth or 3 artificially through implantation of an expandable material within the empty horn of unilaterally pregnant rats.

Wistar rats Charles River Co. Louis, MO and water ad libitum. Female virgin rats were mated with male rats. Day 1 of gestation was designated as the day a vaginal plug was observed.

Under general anesthesia, virgin female rats underwent tubal ligation through a flank incision to ensure that they subsequently became pregnant in only one horn [ 23 ]. Animals were allowed to recover from surgery for at least 7 days before mating.

Pregnant rat myometrial samples from nongravid empty and gravid horns were collected at noon on Gestational Days 6, 12, 13, 14, 15, 19, and 23 Labor , and 1 day postpartum four animals per time point. Labor samples were collected during active labor once the animals had delivered at least one pup. After pregnancy was confirmed, unilaterally ligated rats were randomized into six groups.

The rats were euthanized on day 14 24 h after the tubal insertion surgery. On day 13, rats underwent insertion of an expandable tube, 2 mm in diameter and 3 cm in length, into the nongravid uterine horn through midline abdominal incision intra-uterine expandable tube, [IUET].

Two different expandable materials were used for stretch experiments: 1 laminaria MedGyn Products Inc. The rats were euthanized 2, 14, and 24 h after the tubal insertion surgery four to six animals per time point. IUET stretches the uterine horn approximately 3-fold in both diameter and length as compared to the unstretched empty horn and becomes comparable in size to the gravid horn. On Day 5 after mating, animals were anesthetized with isofluorane, and an empty uterine horn was exposed via an abdominal incision.

The empty horn was injected intraluminally from the cervical end with 0. The cervix was ligated with silk thread to prevent oil leakage. Deciduomas and myometrium from the empty horn were collected separately on Gestational Day 6, 8, 12, 14, and The experiment was repeated twice. Animals were euthanized by carbon dioxide inhalation. The protein extraction was done as described in [ 23 ].

To minimize intra-animal variation, each whole frozen myometrial tissue sample was pulverized in liquid nitrogen with a mortar and pestle for protein extraction. These segments were further cross-sectioned or sectioned longitudinally. Total protein was extracted from frozen tissues using RIPA lysis buffer, resolved using SDS polyacrylamide gel, transferred onto polyvinylidene fluoride membrane, and probed with anti-cleaved CASP3 antisera as described earlier in detail [ 1 ].

The intensity of and kDa bands of cleaved CASP3 was quantified by densitometry, and their protein levels were normalized to the calponin 1 CNN1 protein levels 34 kDa and expressed as relative optical density ROD units.

To compare the level of CASP3 activation in paired myometrial samples from each unilaterally pregnant animal, data were expressed as the percentage of cleaved CASP3 in the empty horn relative to the gravid horn: the ROD for empty horn was divided by the ROD for the gravid horn and multiplied by The fixed myometrial tissues were gradually dehydrated in ethanol and embedded in paraffin. Paraffin sections were deparaffinized and rehydrated.

After quenching in 0. All sections were incubated with primary antibodies overnight. For the negative controls, normal rabbit or normal mouse serum ABC kit was used at the same concentration as primary antibodies, and sections were also incubated with secondary antibodies in the absence of primary antibodies. Secondary antibodies used for detection were biotinylated anti-mouse or biotinylated anti-rabbit ; ABC kit; Vector Laboratories.

Counterstaining with Harris Hematoxylin Sigma, St. A minimum of five fields were examined for each gestational day and uterine horn for each set of tissue, and representative tissue sections were photographed with a Sony DXC MD 3CCD color video camera Sony Ltd. Protein adducts of reductively activated pimonidazole are effective immunogens for the production of antibodies and are easily detected by immunohistochemistry.

Early pregnant Gestational Day 6 , midpregnant Day 14 , and late pregnant Day 20 Wistar rats were randomly assigned for i. Uterus, liver, kidney, and brain tissue samples were collected 1 h after injection and fixed in formalin. Liver and kidney have cells with pO2 below 10 mmHg positive control but brain does not negative control.

Rat tissues were immunostained with mouse anti-pimonidazole primary antibodies MAB-1; ; Chemicon. Two animals were used for each gestational day. Densitometric analysis of immunoblots was performed with the aid of Scion Image software version 4. Statistical analysis of immunoblots was performed with SigmaStat version 2. Data from immunoblot analysis of cleaved CASP3 protein expression in the unilaterally pregnant model were subjected to a two-way ANOVA followed by pairwise multiple comparison procedures Student-Newman-Keuls method to determine differences between groups.

Many years ago, Reynolds [ 22 ] described uterine conversion as a unique change in embryo shape at midgestation from spheroid to ellipsoid that was suggested to be associated with 1 circulatory stasis of the maternal blood flow, 2 transient ischemia in the stretched myometrium, and 3 a hypoxic response in the tissue.

Therefore, we investigated the occurrence of myometrial hypoxia throughout gestation in pregnant rats at early Day 6 , mid- Day 14 , and late gestation Day 20 using immunohistochemical techniques Fig.

As shown in Figure 1 , A and B, in decidua Gestational Day 6 , multiple glands were stained positive for both markers; however, only a small number of immunopositive SMCs were detected in the early Day 6 or late Day 20 pregnant myometrium. Importantly, both markers were expressed more intensively in the circular muscle layer of myometrium as compared to the longitudinal layer.

Rat kidney stained positive for pimonidazole hydrochloride, whereas rat brain was negative Supplemental Fig. S1 , available online at www. It is a reasonable hypothesis that uterine hypoxia may consequently activate the intrinsic apoptotic pathway. Immunohistochemical localization of hypoxia in pregnant rat myometrium.

Both markers were expressed more intensively in the circular muscle layer of myometrium CL as compared to the longitudinal layer LL. Note the lack of staining after incubation of Day 14 myometrial tissue with nonspecific mouse IgGs G , H.

For each day of gestation, tissues were collected from three different animals. We hypothesized that myometrial hypoxia was caused by mechanical stretch of the uterine walls by the growing fetus es , placenta, and amniotic fluids.

In order to prove this assumption, we studied the effect of gravidity on caspase cascade activation using the rat model of unilateral pregnancy. Since Western immunoblot analysis in bilaterally pregnant rats demonstrated a transient induction of active CASP3 in myometrium between Gestational Day 12 and Day 15 [ 1 ], particular attention was given to midgestation. The results in Figure 2 show that cleaved CASP3 protein levels were very low in the empty horn of unilaterally pregnant rats. At late gestation and during labor, cleaved CASP3 expression was reduced to a barely detectable level in both uterine horns.

Expression of cleaved CASP3 protein in the myometrium of unilaterally pregnant rats during gestation and postpartum. B Densitometric analysis illustrating CASP3 protein expression levels in empty white bars and gravid black bars horns normalized to calponin protein and expressed as ROD.

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Unfortunately, few rodent investigations have examined whether intrauterine position influences fetal body weight at term, thereby contributing to study variability. The number of feti in a litter is negatively correlated with birth weight Romero et al. Romero and colleagues reported an inverse relationship between fetal weight and litter size in Sprague Dawley SD rats, and emphasized that if this relationship is not taken into account when toxicity on fetal weight is analyzed there is a potential to mask a decrease in fetal weight due to litter size reduction Romero et al.

Importantly, the number of feti deposited within the right and left uterine horns, respectively, not total litter size, determines the influence of the intrauterine position effect; therefore, experimental outcomes should be evaluated on a per-horn basis Raz et al.

Lastly, maternal weight may be an important consideration, as smaller dams may have less energy reserve per fetus to produce feti of a similar weight compared to a larger female Thame et al. The impact of environmental exposure during pregnancy affecting intrauterine positional growth has received little attention.

Our model of maternal exposure to titanium dioxide nanoparticle nano-TiO 2 aerosols during gestation has identified impairments in gestational health Stapleton et al. Utilizing this model, we have previously reported effects on fetal reabsorption and placental and fetal weight depending on the window s of gestational exposure Stapleton et al.

Therefore, the purpose of this study was to develop a stepwise method of analysis to organize and normalize fetal growth to more accurately assess FGR in our model. We evaluated fetal weight in a gravid SD rat model under control conditions and after maternal exposure to nano-TiO 2 aerosols by position, maternal GD 20 weight, and number of feti per uterine horn. To ascertain whether exposure caused FGR in a particular uterine location and if the timing of the exposure generated a different outcome, the analysis was conducted with data from four different exposure scenarios: single exposure on gestational day GD 4, GD 12, or GD 17, and repeat exposures occurring between GD 4 and GD This challenges the traditional methods and serves useful for future investigations of fetal growth.

Animals arrived on GD 2 or GD 3. Control animals were exposed to filtered air via whole-body inhalation. Primary particle size was determined to be 21 nm with a mean surface area of The nano-TiO 2 preparation and aerosol exposure has been detailed from one other study previously published Fournier et al. Briefly, animals were administered whole-body inhalation exposures in a custom rodent inhalation facility IEStechno, Morgantown, WV.

Aerosols were collected on a nm PTFE membrane filter for gravimetric sampling to confirm concentration. The exposures were carried out for 4-hr on single days i. Average aerosol concentrations were measured at 9. On GD 20 animals were anesthetized via isoflurane inhalation i. Surgical scissors were used to create a Y-shaped incision through the abdomen to expose the uterus.

The left and right gravid uterine horns were identified, removed, and individually pinned to a dissecting dish positioned with the ovary to the left and the vaginal end to the right. Using a surgical scissor, the uterine muscle was cut lengthwise to reveal placentas, amniotic sacs, and feti. The fetus from the ovary-most end was designated as fetus number one Figure 1. Moving toward the vaginal end, each fetus was numbered, removed from its amniotic sac, and weighed.

Associated placentas were weighed, and wet weights were recorded. Figure 1. Schematic of rodent uterine horn anatomy from the ventral aspect and acronym key. Feti are numbered within each horn from the ovary end OE as fetus one to the cervical end CE as fetus six. Anatomy of major uterine and ovarian arteries and associated arcuate and radial artery branching is displayed.

The inclusion criteria in this analysis required a uterine horn to have at least five feti. Data from horns that met inclusion criteria were organized by intrauterine position [i. Table 1. Reported number of litters and uterine horns excluded from analysis and older analyses of fetal weight demonstrating per dam and per treatment group averages along with percent reduction in fetal weight.

Existing literature has shown that maternal horn size and litter size can influence the intrauterine position effect in the Sprague Dawley rat Raz et al. To correct for differences in maternal weight and litter size, each fetal weight was normalized to the number of feti within that horn by the following equation:.

Placental efficiency, a ratio of fetal weight to placental weight, is often used as a proxy measurement of placental function in human and laboratory animal studies Hayward et al. Placental efficiency was calculated by dividing the raw fetal weight by its respective raw placental weight that were measured on GD A Grubb's test was employed to remove any outliers from the data set before further analysis.

Student's t -test was then used to compare fetal weights using the traditional approach. Fetal weights were also analyzed by arranging and normalizing according to our developed protocol, the individual intrauterine positions from each group were compared between the right and left horn using a two-way ANOVA and Sidak's multiple comparisons test.

The nano-TiO 2 exposed group was compared with controls by position for the right and left horn using a two-way ANOVA and Sidak's multiple comparison test.

Placental weights and placental efficiencies, calculated as a ratio of fetal weight to placental weight, were evaluated with a similar approach using raw unnormalized values. Effect sizes were calculated and included as Supplementary Table 1. Statistical analysis was conducted with GraphPad Prism 8. Fetal body weights were compared using the traditional approach of averaging fetal weight by litter and then by treatment group Table 1.

No significant differences and thus no FGR was found using this approach. Fetal weights were compared using our developed approach by organizing data by intrauterine position and normalizing by maternal weight at necropsy GD 20 and number of feti per horn.

In controls, average of total fetal weights from the left horn 0. Figure 2. Fetal weight normalized by maternal weight on GD 20 and number of feti per uterine horn. A Normalized fetal weights comparing the right and left horn within each exposure timing condition. This analysis identifies differing fetal growth between the uterine horns within the same experimental condition. B Normalized fetal weight of the right and left horns for each exposure group compared to control.

This analysis identifies fetal growth impaired by maternal exposure to nano-TiO 2 during gestation. RMD 0. Figure 3. Fetal weight normalized by maternal weight on GD 20 and number of feti per horn.

Data is analyzed by anatomical uterine position to identify differences between right and left uterine horns in A control and B—E exposure groups. When analyzing fetal body weights, exposure to a single inhalation of nano-TiO 2 during pregnancy had a differential effect on fetal weight by intrauterine location based on the timing of exposure. Dams that were repeatedly exposed had no position that was significantly impacted Figure 3B.

Exposure on GD 12 resulted in no significant differences between intrauterine locations within and between right and left horns Figure 3D. When comparing fetal weights between exposure groups on a per-location basis, exposure on GD 4 had a statistically significant impact on the ROE 0. No significance was found between control and repeated exposed or control and GD 12 exposed groups at any intrauterine location.

Figure 4. This analysis pinpoints intrauterine positions which are susceptible to growth reduction after certain timing of exposure s compared to controls. When analyzing for placental weight under control conditions, there was no significant impact on placental weight within and between the right and left uterine horns in control animals Figure 5A. Placental weights were not impacted within or between intrauterine locations in the right and left uterine horns with repeated or single exposures to nano-TiO 2 Figures 5B—E.

Comparison of raw placental weights at each intrauterine location between each experimental group and the control group showed no significance differences Figure 6. Figure 5. Raw placental weights g. Data is analyzed by anatomical uterine position to identify differences between right and left uterine horns in A control and B—E exposure conditions.

Figure 6. Raw placental weight g at each intrauterine position for control and exposure groups. This analysis pinpoints intrauterine positions which are susceptible to placental weight change after certain timing of exposure s compared to controls.

When analyzing for placental efficiency, the ratio of fetal weight to placental weight, under control conditions, there was no difference in either horn or at any location Figure 7A. Placental efficiency was not impacted within and between intrauterine locations in the right and left uterine horns with repeated Figure 7B or single Figures 7C—E exposures to nano-TiO 2 aerosols. When comparing placental efficiency between each experimental group and the control group no significance was found with the exception of a single exposure on GD 17 resulting in a significant reduction in placental efficiency at the RMD location 5.

Figure 7. Data is analyzed by anatomical uterine position to identify differences between right and left uterine horns in control A and exposure groups B—E.

Figure 8. This analysis pinpoints intrauterine positions which are susceptible to reduced placental efficiency after certain timing of exposure s compared to controls. In this study, data from controls and experimental exposures to nano-TiO 2 were evaluated in a manner that challenges the traditional dogma of litter data analysis.

Herein, we were able to account for intrauterine position, number of feti per horn and maternal weight. However, upon further evaluation using our revised approach we observed that under control conditions, feti implanted in the left uterine horn tended to be smaller in body weight at term compared with feti implanted in the right uterine horn.

These analyses also demonstrated significant FGR in exposed animals compared with controls, separated by uterine horn and intrauterine position, outcomes that were lost with traditional approaches. This imbalance of fetal growth between the uterine horns was exacerbated after either repeated maternal exposure to nano-TiO 2 aerosols during gestation or a single exposure early GD 4 or in late GD 17 pregnancy. The most severe FGR in terms of magnitude of impact and locations affected were apparent from a single maternal exposure on GD 4 in the middle position of the right uterine horn.

The middle position of the right uterine horn was also impacted with respect to other endpoints including increased placental weight, reduced fetal weight, and decreased placental efficiency after a single exposure late in gestation, on GD Interestingly, there was no significant positional impact after repeated maternal nano-TiO 2 exposure compared to control in this cohort.

Overall, this study demonstrates both critical windows of maternal exposure early and late in gestation and the risks associated with anatomical positioning of implanted fetus in the right horn. Moreover, this information was not gleaned using a traditional approach to data analysis, where no impact on fetal weight was detected. Observations from control conditions are in agreement with findings from other studies, in that despite anatomical similarities, the right horn produced larger feti suggesting a more favorable environment than the left Wiebold and Becker, ; Lan et al.

This observation has also held from human studies, where there is a tendency for right ovary ovulation and implantation on the right side of the reproductive tract Kawakami et al.