Introduction

Ectopic pregnancy, which occurs when an embryo implants outside of the uterus, affects approximately 2% of pregnancies in the United States annually, and in more than 90% of cases, the ectopic embryo implants in the fallopian tube (i.e., tubal ectopic pregnancy)1,2. The current treatments for ectopic pregnancy include expectant management, methotrexate administration, salpingectomy, and salpingostomy3,4,5,6,7. The aim of the current treatments is to protect the pregnant woman from the potentially lethal consequences of ongoing embryo growth and placental invasion through the thin fallopian tube, which could lead to tubal rupture and hemorrhage. However, these treatment options, for obvious reasons, do not allow for the continuation of the pregnancy4,7,8.

Prevalence is high, unfortunately, among populations who may desire to continue a pregnancy, but there is no option for women who wish to retain a viable ectopic embryo. The available literature pertaining to any procedure involving the relocation of an embryo in the case of ectopic pregnancy is scarce, and generally vague9,10,11,12, providing little clarity on the realistic feasibility of relocating an ectopic embryo into the uterus, allowing for normal gestational development.

Investigating a potential surgical procedure with the intention of allowing for the continuation of a desired ectopic pregnancy, while also protecting the mother, provides a significant scientific and ethical challenge. Scientifically, the significant changes that occur during pregnancy, at both the micro (e.g., molecular, cellular, endocrinological, etc.) and macro (e.g., organ, anatomical, physical, etc.) levels, are immensely complex. While our understanding has exponentially expanded in recent years, even contributing to extending the limits of fetal viability, the reality remains that significant further investigation remains necessary. From an ethical perspective, challenges are present in relation to protecting the woman’s life, the potential desire for the continuation of the pregnancy, and preserving fertility (e.g.,13). Additionally, ethical concerns arise in relation to the dangers associated with potential abuses that may result from human experimentation (e.g.,14,15). Thus, a common first step in approaching such situations often requires preclinical studies (e.g., animal models)16,17.

Pre-clinical experimentation involving animal models (e.g., rat models) has its limitations and it is understood that no animal model can sufficiently or perfectly explain human physiology, behavior, or pathology (including lack of direct translation from the animal to human level). Nevertheless, it is well established that the similarities that are present allow for significant foundational investigation with potential implications and reasonable, cautious extrapolation to the human level.

Specifically related to our investigation, differences exist between humans and rats in relation to pregnancy and uterine anatomy (e.g., uterine structure, eventual divergence in the source of progesterone synthesis, implantation process, etc.; see Limitations). However, there are also important similarities including placentation type, in which both rats and humans undergo hemochorial placentation (i.e., maternal blood comes in direct contact with the fetal chorion)18,19, the initial hormonal maintenance of pregnancy20,21, and the combined influence of hormones and the presence of embryos on the distention of the uterus during pregnancy22,23,24,25,26. These similarities allow for the rat to be utilized as a feasible first-step model to investigate pregnancy and pregnancy-related questions27,28.

Given (1) the prevalence of ectopic pregnancies, (2) the absence of treatment options that allow for the continuation of the pregnancy, despite the desire in some women for such an option, (3) the lack of sufficient investigation into the potential relocation of an ectopic embryo into the uterus, (4) the scientific, medical, and ethical challenges surrounding such an investigation, and (5) the availability of animal models which allow for the investigation of pregnancy-related questions with cautious extrapolation to human pregnancy, this study sought to initiate an investigation into the potential design of a surgical procedure that could serve as a putative foundation that stimulates further research into the possibility of an ectopic embryo relocation into the uterus at the clinical level.

Based on the abnormal placement of the placenta in ectopic pregnancies, the model was developed with a theoretical procedure designed for human use in mind, which would rely on placing the entire implantation site (including part of the fallopian tube) inside of the uterus. The present animal model relies on previous literature in humans, which relates that:

  1. (1)

    While the location of implantation is abnormal in ectopic pregnancies, there also appear to be significant similarities (e.g., in vascularization, trophoblastic flow) between ectopic and intrauterine pregnancies, and the placenta in ectopic pregnancies appears to have a relatively normal development until a certain point, due to the differences between the tubal and uterine anatomy29,30,31,32.

  2. (2)

    A transmural incision can be made in the uterus during pregnancy (e.g., during fetal surgery, etc.). In such situations, it is necessary to closely monitor the mother and fetus for the remainder of the pregnancy, and given an incision was made in the contractile portion of the uterus, delivery would necessitate a Cesarean section (e.g.,33,34).

  3. (3)

    One purported approach to transplanting ectopic embryos11 involved making a transmural incision in the uterus and moving the embryo and part of the fallopian tube into the endometrial cavity through that incision, after debulking tubal tissue not immediately adjacent to the site of implantation, and the blood supply to that area.

Moving a residually vascularized fallopian tube containing the gestational sac into the uterus through a transmural incision would likely eventually lead to cessation of reliable blood flow to the tubal tissue. However, such a procedure may permit the embryo to follow the natural progression of tubal ectopic pregnancy (i.e., growth through the fallopian tube) and allow for ongoing placental growth into the hormonally-ready endometrium deep to the residual fallopian tube. While the capacity for the placenta to grow into the endometrium in this context is currently unknown, it would be dependent on whether hemochorial placentas can transition from blood supply from one area of the reproductive tract to another.

Therefore, to this end, while an actual ectopic pregnancy was not utilized in this rat model, we exploited the bicornuate nature of the rat uterus in order to explore the potential for an embryo in one part of the reproductive tract (native uterine horn) to develop normally after being physically enveloped by another part of the reproductive tract (contralateral uterine horn).

Methods

Subjects

Female Long-Evans rats (n = 24) were purchased from Hilltop Lab Animals (Scottdale, PA, USA). All animal protocols were approved by the Franciscan University Institutional Animal Care and Use Committee (Approval Numbers: 2018-02 & 2022-01) and adhere to the Guide for the Care and Use of Laboratory Animals published by the USPHS. The study is reported in accordance with the ARRIVE guidelines. Animals were housed on Aspen shavings (Nepco®) and were single-housed (beginning at 7 weeks of age), positioned in a way that they could see, hear and smell other animals of the same species, under a 12/12 hour light/dark cycle (Lights on 2.15am) and controlled temperature and humidity (20–26 C, 30–70% relative humidity), with ad libitum access to standard laboratory chow (RMH 1800, LabDiet) and water.

Experimental procedures & groups

In order to track the health35 of the single-housed female rats, rat weight and food consumption were measured daily beginning at 8 weeks of age. Additionally, vaginal impedance measurements were also initiated at 8 weeks of age, measured daily, using an Electronic Vaginal-Estrous Cycle-Monitor (MK-11, Stoelting, Wood Dale, IL, USA), in order to determine and track estrus36. At 10 weeks of age, female rats were bred with male rats of the same stock (purchased from Hilltop Lab Animals, Scottdale, PA, USA), with Day 0 (D0) representing the day of breeding. Signs of mating were recorded after the removal of the male.

Animals were randomly assigned to one of two groups: Embryo Relocation (ER) or Normal Pregnancy (NP). Those in the ER group (n = 12) underwent the embryo relocation surgery detailed below, while the NP group (n = 12) carried a normal pregnancy with no surgical intervention. Within each group, 8 rats were allowed to carry the pregnancy to term and deliver, while 4 rats had their uteri collected on D20 or D21 of gestation for histological investigation.

Drugs and chemicals

Meloxicam (5 mg/ml; Loxicom®, Norbrook Laboratories Limited) and Isoflurane, USP (Piramal Critical Care) were purchased from Med-Vet International. All other chemicals were of the highest grade commercially available.

Anesthesia for surgery, ultrasound and tissue collection

Rats were anesthetized in the induction chamber using 5% isoflurane in oxygen, followed by 2–3.5% in the rebreather nosecone, using the SomnoSuite® Low Flow Anesthesia System (Kent Scientific Corporation, Torrington, CT, USA). The hind limb compression reflex was tested periodically to confirm proper anesthesia.

Surgical procedure

Rats in the ER group underwent surgery on D7 of gestation (by which time implantation is reported to be complete37). Briefly, the procedure (see Figs. 1A-H and 2A-I) involved the relocation of one or two embryos and the uterine horn immediately surrounding them into the contralateral uterine horn (simulating the movement of a human fallopian tube containing an ectopic embryo into the uterus). The selection of the receiving horn and embryo(s) to be transplanted was determined at the beginning of surgery, once the pregnant uterus was visualized, but prior to beginning the embryo relocation. This selection was based on optimal distribution and placement of the embryo(s) in the uterine horns.

Fig. 1
figure 1

Diagram representative of surgical procedure for embryo relocation in a rat model using a bicornuate uterus. (A) Both uterine horns and ovaries were visualized. Green horizontal arrows indicate ovaries and black vertical arrow indicates the corpus and cervix. Bulges in the uterine horns reflect the presence of gestational sacs with maroon crescents indicating placentas. Diagonal lines extending downwards from the uterus reflect mesometrial fat (not included in all images, despite being present in situ). (B) The number of gestational sacs to be relocated was determined and both the uterine horn and mesometrial vessels were ligated superior to the gestational sacs to be relocated (green arrows), as well as inferior to the ovary (not indicated). (C) The ligated portion of the uterine horn (red star) was then excised, preserving the ovary, and the portion of the uterine horn containing the gestational sacs to be relocated was disconnected from as much mesometrial fat as possible (dotted line). The ovary on the original side of the relocated gestational sacs is omitted from future images, but remained in situ. (D) The blood supply from the uterine artery on the cervical side was retained. (E) A longitudinal incision was made along the opposite uterine horn and the contents emptied, creating a “flap”. (F) The gestational sacs to be relocated were rotated so that the placenta(s) were aligned with the mesometrial vessels of the receiving horn. (G & H) Cyanoacrylate surgical glue was used to affix the mesometrial surface of the uterine horn containing the gestational sacs to be relocated to the inner surface of the flap. The flap was brought down and around the gestational sacs and affixed with additional surgical glue to enclose the relocated gestational sacs with the receiving horn. Following the procedure, the cervix remains patent (green arrow).

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Fig. 2
figure 2

Representative images from embryo relocation surgical procedure and follow-up. Images (A-I) are shown with superior (towards the head) aspect at the bottom left corner and inferior (towards the tail) aspect towards the top right corner. (A) Visualization of uterine horns containing gestational sacs (thick black arrows). (B) Insertion of first suture, superior to the gestational sacs (thick green arrows) to be relocated. (C) Ligated portion of the uterine horn to be excised. (D) Excision of the ligated portion of the uterine horn. (E) Removal of mesometrial fat on remaining portion of the uterine horn containing gestational sacs to be relocated. (F) Portion of the uterine horn containing gestational sacs to be relocated prior to relocation to other horn. (G) Creation of the flap in the receiving uterine horn. Inset indicates the flap once created. (H) Gestational sacs being relocated onto flap. (I) Relocated gestational sacs fully enclosed by flap. (J) Rat in the Embryo Relocation group at full-term (D20/D21), prior to tissue collection. Visualization of uterus surrounding the relocated fetus and mesometrial blood vessels. (K) Representative ultrasound image of fetus from full-term rat in Embryo Relocation group, with the head, spine and rib cage labelled.

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More specifically, once the rat was sufficiently anesthetized, the abdominal area of the rat was closely shaved from the lower pelvic area up to the sternum, and the shaved area was cleaned three times using alcohol, Betadine®, and alcohol. Eye lubricant (Puralube® Vet Ointment (Dechra)) was applied to the eyes of the rat. The rat was placed in a sterile bench-top surgical area and a Press’n Seal® drape38 was placed over the rat. An opening was made in the drape to expose the abdomen of the rat, and a midline vertical incision was then made in the outer skin, followed by another midline vertical incision in the abdominal wall.

Both uterine horns and ovaries were visualized (Figs. 1A and 2A), and the number of gestational sacs to be relocated was determined based on inter-embryo spacing within the uterus that would allow for the appropriate manipulations involved in the embryo relocation procedure. Both the uterine horn and mesometrial vessels were ligated, using 3 − 0 silk suture (MV-684; Med-Vet International, Mettawa, IL, USA), superior to the gestational sacs to be relocated and another inferior to the ovary (Figs. 1B, 2B & C). The ligated portion of the uterine horn was then excised (Fig. 2D), preserving the ovary, and the portion of the uterine horn containing the gestational sacs to be relocated was disconnected from as much mesometrial fat as possible (Figs. 1C, 2E & F). The blood supply from the uterine blood vessels on the cervical side was retained (Fig. 1D). A longitudinal incision was made along the opposite uterine horn and the contents emptied, allowing it to become a “flap” to wrap around the gestational sacs to be relocated (Figs. 1E and 2G). The cut consisted of several diagonal lines in order to maximize usable internal surface area of the flap. The endometrial lining was gently curettaged to ensure removal of all previously implanted sacs and to improve bonding between the flap and the myometrium surrounding the sacs being transplanted. The gestational sacs chosen for relocation were rotated so that the placenta(s) were aligned with the mesometrial vessels of the receiving horn (Figs. 1F and 2H). Cyanoacrylate surgical glue (3 M Vetbond™ Tissue Adhesive) was used to obtain hemostasis and affix the mesometrial surface of the myometrium surrounding the sacs to the exposed inner surface of the uterine flap. The flap was then brought down and around the gestational sacs in the relocated horn with its vasculature and mesometrial fat, and affixed with additional cyanoacrylate surgical glue. The ovary was pulled down (without being compromised) so that the entire length of the flap could be used in enclosing the relocated horn (Figs. 1G-H and 2I).

Following the surgical procedure, sterile body temperature saline was applied to the abdominal cavity for rehydration and adhesion prevention. The abdominal wall was closed using 3 − 0 silk suture (MV-684; Med-Vet International, Mettawa, IL, USA) followed by the outer skin incision using wound clips ensuring that the underlying tissue was not clipped (clips were removed 10–14 days post-surgery). Betadine® was applied to the wound once it was completely clipped (9 mm clips; Ted Pella, Inc., Redding, CA, USA) and then dabbed off. One dose of Meloxicam (1.0–1.5 mg/kg) was administered subcutaneously (as well as every 24 h for two days), and the rat was placed in a recovery cage with clean bedding and a paper towel, with a heating pad under part of the cage in order to keep the rat warm. After an observation period, the rat was returned to routine care and the relocated embryo(s) was/were allowed to continue developing to term (Fig. 2J).

Post-surgical measurements

In the ER group, rat body weight and food consumption continued to be measured following surgery, until either tissue collection (n = 4) or two weeks post-weaning (for the rats allowed to deliver, n = 8). With the exception of the first two days post-surgery, vaginal impedance was also measured until either tissue collection or two weeks post-weaning. Equivalent measurements (i.e., rat body weight, food consumption and vaginal impedance) were also recorded in rats in the NP group.

Ultrasound

Transabdominal ultrasound imaging (Fig. 2K) was performed on all rats (i.e., both ER and NP rats) on either D20 or D21 of pregnancy in order to determine the presence or absence of fetuses and fetal cardiac activity. Ultrasound imaging was conducted under isoflurane anesthesia (see above), using the EDAN U50 VET Ultrasound Machine, using a Linear array transducer (L15-7b) (Universal Diagnostic Solutions, Inc., Vista, CA, USA). Uterine horns were scanned for fetuses, and fetal heart rates (beats per minute, bpm) were recorded from fetuses within each horn, when present. The average fetal heart rate of the offspring of each mother was then calculated.

Tissue collection

Following the ultrasound (see above) on either D20 or D21 of pregnancy, rats undergoing tissue collection in either the ER or NP group were deeply anesthetized using isoflurane, and the uteri collected for gross anatomical examination and histological investigation (see below), and stored in 10% Neutral Buffered Formalin (Azer Scientific, Inc., Morgantown, PA, USA). Following the tissue collection, rats were euthanized via a bilateral thoracotomy and exsanguination.

Gross anatomy and histological examination of placentas

All placentas resulting from surviving fetuses at D20 or D21 of gestation in the ER group (specifically those that underwent tissue collection; see above) (n = 4; one per rat) were dissected from the collected uteri, retaining the section of uterine horn to which the placenta was attached. Regarding the NP group, the two placentas closest to the cervix (i.e., one placenta from each uterine horn) were dissected (n = 8; two per rat), again retaining the section of the uterine horn to which the placenta was attached.

The fetuses associated with the dissected placentas were also removed and disconnected from the placentas, and the following measurements were made: fetal weight (g), fetal width (mm) and fetal length (mm) (Fig. 3A). Additionally, prior to obtaining placental slices for histological analysis, both the major and minor axes were measured (both mm) (Fig. 3B).

Fig. 3
figure 3

Gross anatomical measurements (A & B) and representative images of histological analysis from both Normal Pregnancy (C & D) and Embryo Relocation (E & F) groups. (A) Measurement of fetal width (left) and fetal length (right). (B) Measurement of placental major and minor axes. Green lines represent placental slices for histological investigation. (C & E) Measurement of the specific placental layers at 10x. (D & F) Measurement of basal shoulder, measured from the apex of the labyrinth zone to the furthest point of the basal zone (including giant cells) at 40x. LZ = labyrinth zone, BZ = basal zone, MT = maternal tissue.

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Histological sections (75 μm slices) were then obtained from all placentas described above, mounted, and stained with Hematoxylin and Eosin39 to allow for histological determination of the various placental zones.

Three tissue slices (identified at the location of the umbilical cord insertion, as well as one from either side of the cord insertion) from each placenta were analyzed/assessed using a microscope for the thickness (mm) of the following placental zones (Fig. 3C & E): labyrinth zone, basal zone, and maternal tissue (decidua basalis and metrial gland combined). Additionally, the height of the basal zone was measured at both corners of each placenta (referred to as the basal shoulder, Fig. 3D & F), measured using two parallel lines, one at the apex of the labyrinth zone and the other at the furthest point of the basal zone (including giant cells). The height was then measured as the height/distance between those two parallel lines. All measurements were made by one investigator and independently confirmed by at least one other investigator.

Final follow-up

In adulthood (approximately 17 weeks of age; ER group offspring: M = 17.5 weeks of age, SEM = 0.3; NP group offspring: M = 17.3, SEM = 0.4), the ER-group offspring (those resulting from the embryo relocation procedure) and appropriate sex-matched controls born of the normal pregnancies (NP-group offspring) were weighed.

Statistical analysis

Data analysis was conducted using SigmaPlot version 14.0 (Systat Software, Inc.). Independent measures t-test (two-tailed) was utilized to compare differences in (1) average day of delivery between the ER and NP groups, (2) average fetal heart rate between the ER and NP offspring, (3) average rat body weight of offspring delivered following the embryo relocation procedure and a normal pregnancy, and (4) fetal weight, length and width, placental major and minor axes, basal shoulder height, and total placental thickness between the ER and NP groups. Additionally, independent measures t-test (two-tailed) was also utilized to compare the average rat weight between the ER and NP groups for the combined data of D1 to D7 of gestation (days prior to and including day of surgery). Moreover, one-way repeated measures ANOVA was utilized to assess the rat body weight of the mothers within both the ER and NP groups across gestational days. Similar tests (t-test; one-way RM-ANOVA) were also conducted for food consumption. A two-way independent measures ANOVA was utilized to assess differences in pre-breeding and post-weaning vaginal impedance both within and across the ER and NP groups. Given that rats (n = 4/group) that received tissue collection at the end of gestation did not receive post-weaning vaginal impedance measurements, in the analysis, post-weaning impedance included the eight rats per group that were allowed to deliver, while pre-breeding impedance included all 12 rats per group. A two-way repeated measures ANOVA with one factor repetition (placental zone) was utilized to compare differences in specific placental zone thickness, as well as the percentage thickness of the specific placental zones relative to the total placental thickness, both between and within the ER and NP groups. Differences were considered significant at p < 0.05 for all analyses. While not considered as significant, p-values within the range of 0.05 < p < 0.1 are indicated as tendencies towards significance.

Results

Of the eight rats that underwent the embryo relocation procedure and were allowed to carry to full-term and deliver, one rat had one embryo relocated, while seven rats had two embryos relocated, giving a total of 15 total relocated embryos (see Table 1). Pertaining to the four rats undergoing tissue collection following the embryo relocation procedure, three rats had one embryo relocated, while one had two embryos relocated, giving a total of five relocated embryos (see Table 1). Median anesthetic time was 47 min (interquartile range [IQR] 43–49 min) and surgical time 39 min (IQR 37–41 min).

Table 1 Outcomes of offspring of rats in the embryo Relocation group.
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Embryo relocation procedure outcomes

The information regarding number of embryos relocated, number of offspring with cardiac activity detected via ultrasound, number of pups delivered and number of offspring that survived to adulthood (approximately 17 weeks of age) for each of the specific rats that underwent the embryo relocation procedure are outlined in Table 1.

The outcomes of the relocated embryos in the eight rats that underwent the surgical procedure and were allowed to carry to full-term and deliver are as follows: From the 15 relocated embryos, all 15 embryos (100% of those relocated) were detected via ultrasound with cardiac activity at the end of gestation. 14 of the offspring (93% of those relocated and detected with cardiac activity via ultrasound) were delivered vaginally. Of these 14 offspring that were delivered, 12 (80% of the original 15 relocated embryos; 86% of the 14 offspring delivered) survived until adulthood (approximately 17 weeks of age), with seven being male (58%) and five being female (42%). The other two offspring that were delivered (but did not survive until 17 weeks of age) died prior to weaning.

Regarding the rats that underwent surgery and had tissue collection at full-term rather than being allowed to deliver, four of the five relocated embryos (80%) were detected on ultrasound with cardiac activity at the end of gestation (prior to tissue collection).

In regard to the fetuses that died, in the rats that underwent the surgical procedure for embryo relocation and were allowed to deliver, pup demise post-delivery was observed in two rats. Our notes indicate that, in one case (Rat #6 in Table 1), the low number of gestational sacs on the flap side led to the transferred sacs being wrapped more tightly than in the other surgeries. This may be the cause of the pre-birth demise of one of the fetuses in this rat (Rat #6). This rat delivered one fetus and did appear to possibly be nursing. However, its growth appeared stunted relative to other pups of the same age, and while it had developed light fur/coloring, it remained significantly smaller. In the other case where a pup was delivered (Rat #8 in Table 1), we do not know what the reason for the demise was. There were no surgical complications and cardiac activity was observed and within normal range (279 bpm) on the ultrasound on D20, and its color appeared normal at birth.

In the one rat (Rat #10 in Table 1), in the ER group that had tissue collection performed, that underwent surgery to relocate two embryos, where cardiac activity was only observed and recorded from one pup during ultrasound, we noted in our surgical notes that one sac on the relocated side was not completely enclosed due to difficulty associated with the distance between sac 1 and 2. This is most likely the cause for the demise of the embryo. The death of this embryo appears to have taken place early in the process as it was also not observed during the ultrasound nor was there any evidence of it present at tissue collection.

Average day of delivery

Analysis indicated no significant difference (t(14) = 1.72, p > 0.05) between the average day of delivery of the ER group (M = 23.4 days of gestation, SEM = 0.3) relative to the NP group (M = 22.9 days of gestation, SEM = 0.1).

Rat body weight and food consumption (dams)

Pertaining to the rat body weight (g) and food consumption (g/100 g of rat weight) (both indicators of overall health35) of the mothers, analysis indicated no significant difference between the NP and ER groups in the combined data of D1-D7 (the days prior to and including the day of surgery for the ER group) for both rat weight (t(166) = -1.05, p > 0.05) and food consumption (t(160) = -0.25, p > 0.05).

Regarding rat body weight of the mothers during gestation (D1-D21) (Fig. 4), analysis indicated a significant effect across gestational days in both the ER (F(20, 214) = 238.81, p < 0.001) and NP (F(20, 218) = 631.49, p < 0.001) groups. In relation to the NP group, as expected, rat weight increased steadily from D1 through to the end of gestation (D21). Regarding the ER group, consistent with the surgical procedure, post-hoc analysis indicated that there was a significant decrease (p < 0.001) in average rat weight only on D8 (day immediately following surgery) relative to D7 (day of surgery). Rats then resumed gaining weight with the weight becoming significantly higher than D7 beginning on D13 through the rest of gestation (all p < 0.001).

Fig. 4
figure 4

Percentage rat body weight change across gestation in both experimental groups (Embryo Relocation and Normal Pregnancy). Percentage weight change calculated relative to day 1 (D1) of gestation. NP = Normal Pregnancy group, ER = Embryo Relocation group. *** p < 0.001 relative to D7 of gestation (day of surgery) for the ER group.

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Pertaining to average food consumption, analysis revealed an overall significant effect across gestational days within both the ER (F(20, 195) = 4.36, p < 0.001) and NP (F(20, 218) = 2.77, p < 0.001) groups. While the overall analysis indicated a significant effect across days in the NP group, the post-hoc test indicated that, with the exception of D21 (p < 0.05), there was no significant difference (all p > 0.05) across all days relative to D7. Within the ER group, similar to rat body weight, food consumption was also significantly lower relative to D7 of gestation only on the day immediately following the surgery (D8) (p < 0.001). All other days post D8 (and pre-D7) were not significantly different from D7 (p > 0.05).

Vaginal impedance

Analysis of vaginal impedance (Fig. 5) indicated a significant difference across time (pre-breeding vs. post-weaning; F(1, 635) = 6.47, p < 0.05), but not group (ER vs. NP; F(1, 635) = 0.02, p > 0.05) or the interaction of time and group (F(1, 635) = 0.90, p > 0.05). Post-hoc analysis revealed no significant difference (both p > 0.05) between the ER and NP groups in average vaginal impedance both pre-breeding (ER: M = 3.6, SEM = 0.2; NP: M = 3.4, SEM = 0.2) and post-weaning (ER: M = 2.8, SEM = 0.2; NP: M = 3.0, SEM = 0.3).

Fig. 5
figure 5

Comparison of average vaginal impedance measurements (kΩ) between experimental groups (Embryo Relocation and Normal Pregnancy) pre-breeding and post-weaning. NP = Normal Pregnancy group, ER = Embryo Relocation group; Pre = Pre-breeding impedance, Post = Post-weaning impedance.

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In regard to potential changes in vaginal impedance between pre-breeding and post-weaning within the individual groups, analysis indicated that within the ER group, the post-weaning impedance was significantly lower (p < 0.05) than that of the pre-breeding impedance. There was no significant difference (p > 0.05) between pre-breeding and post-weaning impedance in the NP group.

Fetal heart rates

Regarding fetal heart rate (bpm) detected via ultrasound at the end of gestation, analysis indicated that the average fetal heart rate of offspring from the ER group (M = 240, SEM = 6) was significantly higher (t(22) = 3.60, p < 0.01) than that of the NP group (M = 211, SEM = 5). The range of fetal heart rates of the offspring for the ER group was 200–279 bpm (Median = 235), while that of the NP group was 161–264 bpm (Median = 210). These heart rates are all within the normal heart rate range.

Uterine weight at end of gestation

As would be expected, average uterine weight (g) following tissue collection at the end of gestation was significantly higher (t(6) = -6.24, p < 0.001) in the NP group (M = 60.5, SEM = 7.8) relative to the ER group (M = 10.5, SEM = 2.0). This weight included both pregnant horns in the NP group (i.e., a normal pregnancy), but only 1–2 fetuses enveloped by the flap in the ER group.

Rat body weight (offspring)

Analysis indicated no significant difference (t(22) = 0.13, p > 0.05) between the average body weight (g) at adulthood between offspring (sexes combined) born following the embryo relocation procedure (ER group offspring; M = 361.7, SEM = 24.3) and a normal pregnancy (NP group offspring; M = 356.5, SEM = 30.3).

Gross anatomical fetal and placental measurements

Data pertaining to the average, standard error of the mean and t-statistic for fetal weight (g), length and width, as well as placental major and minor axes (mm) are shown in Table 2 for both the ER and NP groups. While there were no significant differences in these parameters between the ER and NP groups, the placental major and minor axes showed a tendency towards significance (p = 0.092 and 0.074, respectively).

Table 2 Placental and fetal measurements.
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Histological measurements

Total placental thickness

In relation to total placental thickness, as measured by the sum of the labyrinth zone, basal zone and maternal tissue thickness (mm) (Fig. 6 inset), analysis revealed that placentas of the ER group (M = 6.16, SEM = 0.22) were significantly larger (t(34) = 2.179, p < 0.05) than those of the NP group (M = 5.66, SEM = 0.12).

Fig. 6
figure 6

Placental zone thickness across groups as a percentage of total placental thickness. Comparison between Normal Pregnancy and Embryo Relocation groups: *p < 0.05, **p < 0.01, ***p < 0.001. Inset: Placental zone thickness (mm) across groups. LZ = labyrinth zone, BZ = basal zone, MT = maternal tissue; NP = Normal Pregnancy group, ER = Embryo Relocation group.

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Thickness of placental zones

Regarding the thickness (mm) of the measured placental zones (labyrinth zone, LZ; basal zone, BZ; maternal tissue, MT; Fig. 6 inset), analysis indicated a significant difference across group (ER vs. NP; F(1, 68) = 4.75, p < 0.05), placental zone (F(2, 68) = 167.58, p < 0.001), and the interaction between group and placental zone (F(2, 68) = 12.51, p < 0.001). Post-hoc analysis revealed that the average thickness (mm) of both the labyrinth zone (ER: M = 2.94, SEM = 0.12; NP: M = 2.42, SEM = 0.06; p < 0.001) and the basal zone (ER: M = 1.17, SEM = 0.13; NP: M = 0.81, SEM = 0.04; p < 0.01) was significantly higher in the ER group relative to the NP group. In regard to the maternal tissue (ER: M = 2.05, SEM = 0.14; NP: M = 2.43, SEM = 0.10), the thickness of the ER group was significantly lower (p < 0.01) than that of the NP group.

Similarly, in relation to the percentage thickness (%) of the various placental zones relative to the total/overall thickness of the placenta (Fig. 6), analysis indicated a significant difference across placental zones (F(2, 68) = 168.02, p < 0.001) and the interaction of group and placental zone (F(2, 68) = 13.21, p < 0.001), but not across group (F(1, 68) = 1.24, p > 0.05). In this regard, post-hoc analysis revealed that the percentage thickness of both the labyrinth zone (ER: M = 48.2, SEM = 2.3; NP: M = 42.9, SEM = 1.0; p < 0.01) and basal zone (ER: M = 18.8, SEM = 1.8; NP: M = 14.4, SEM = 0.8; p < 0.05) were significantly larger in the ER group relative to the NP group. Conversely, the average thickness of the maternal tissue (ER: M = 33.0, SEM = 1.4; NP: M = 42.7, SEM = 1.1; p < 0.001) was significantly higher in the NP group.

Relating to the actual thickness (mm) and the percentage thickness relative to the total placental thickness, within both the ER and NP groups, both the labyrinth zone and the maternal tissue were significantly thicker than the basal zone (all p < 0.001). Additionally, in the NP group, there was no significant difference (both p > 0.05) between the labyrinth zone and the maternal tissue in relation to both the actual thickness and percentage thickness. However, in the ER group, the labyrinth zone was significantly thicker and constituted a significantly higher percentage of the total thickness than the maternal tissue (both p < 0.001).

Basal shoulder height

Pertaining to the basal shoulder height (measured as described in Sect. 2.9), analysis indicated that the height of the ER group (M = 1.34, SEM = 0.05) was significantly larger (t(34) = 4.414, p < 0.001) than that of the NP group (M = 0.99, SEM = 0.05).

Discussion

A rudimentary model of embryo relocation from one position in the reproductive tract to another is described. The model demonstrates a successful trans-uterine embryo relocation in an animal model with hemochorial placentation. It is important that it is understood at the outset that this model does not replicate the relocation of ectopic embryos (i.e., it does not involve an actual ectopic pregnancy), but rather provides a model for the relocation of an embryo(s), with potential implications for ectopic pregnancy in humans following further investigation in higher animals. Briefly, the model involved the relocation of a section of one uterine horn containing 1–2 embryos (with both the uterine horn and vasculature disconnected completely anteriorly, but connected posteriorly on the cervical end) to the contralateral vacated horn. The contralateral uterine horn was incised, the embryonic contents cleared and the uterine horn wrapped around the relocated uterine horn section containing the living embryo(s). It is anticipated that a comparable procedure in humans will involve the translocation of the fallopian tube into the uterus, as outlined above (Fig. 7).

Fig. 7
figure 7

Diagram of potential human surgical equivalent. (A) Human female internal genitalia with green arrow indicating an ovary. Black arrow indicates ectopic embryo in fallopian tube. (B) Green lines indicate potential locations of incisions on the fallopian tube, as well as in the uterus. (C) Indicates the rotation of the section of the fallopian tube containing the ectopic embryo. (D) Indicates the uterus sutured closed, with the section of fallopian tube containing the ectopic embryo inside of the uterus. Inset. Represents the ectopic embryo within the uterus.

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Our findings indicate that, in the dams that underwent the embryo relocation procedure and were allowed to deliver, 100% of the relocated embryos were alive, as indicated by heart rate detected on ultrasound, at the end of gestation. All relocated embryos, except one, were delivered vaginally, indicating that the cervix remained patent and the ability to deliver vaginally was not compromised by the surgical procedure. Furthermore, the average day of delivery was not affected by the embryo relocation procedure relative to those with a normal pregnancy. Additionally, 80% of the relocated embryos (86% of the embryos delivered) survived until adulthood.

Of note, our findings suggest that the surgical procedure did not result in undue stress on the mothers, as the rats lost weight and showed a significant decrease in food consumption (as would be expected following a major surgical intervention and the removal of a part of one uterine horn), both indicators of health, only on the day immediately following surgery. The rats then proceeded to gain weight throughout the rest of gestation and return to normal food consumption. Pertaining to fertility, in addition to rats in the Embryo Relocation group having the same average vaginal impedance as those in the Normal Pregnancy group prior to breeding, there was no significant difference in average vaginal impedance post-weaning, between the two groups following either the embryo relocation procedure or a normal pregnancy. Unlike the rats that carried a normal pregnancy, those that underwent the embryo relocation procedure experienced a significant decrease in vaginal impedance over time (post-weaning relative to pre-breeding).

These findings pertaining to vaginal impedance potentially suggest that while the fertility of the rats that underwent the embryo relocation procedure showed some impact, fertility was still present at a level that was comparable to the rats that carried a normal pregnancy, to the extent that there was no significant difference between the two groups in impedance following the weaning of the pups. This could potentially be due to both ovaries remaining intact following the embryo relocation procedure. At the human level, previous literature has suggested that following treatment for an ectopic pregnancy (e.g., salpingectomy), the health of the mother, including the health of the contralateral fallopian tube (i.e., the tube that did not have the ectopic pregnancy), plays a fundamental role in their future fertility40,41.

Regarding the well-being of the offspring resulting from the embryo relocation procedure, our findings appear to suggest that the development of the offspring resulting from the procedure was relatively unaffected by the procedure. Specifically, in relation to fetal heart rate, despite the significant difference in average fetal heart rates of the Embryo Relocation versus Normal Pregnancy group, both averages appear to be within the range of fetal heart rates, obtained utilizing a variety of methods, reported in previous literature42,43,44. Moreover, in adulthood, there was no significant difference in the average body weight of rats born following the embryo relocation procedure relative to a normal pregnancy. This is further corroborated by the gross anatomical measurements of the fetuses at the end of gestation following tissue collection, which indicated no significant difference in fetal weight, length or width between those resulting from the embryo relocation procedure relative to a normal pregnancy.

Pertaining to the histological investigation of the placentas at the end of gestation following either a normal pregnancy or the embryo relocation procedure, our results indicated that, while the placental major and minor axes were not significantly different between the two groups (but indicated a tendency towards significance), the total placental thickness (as measured by the sum of the labyrinth zone, basal zone and maternal tissue thickness) was significantly greater in the Embryo Relocation group relative to the Normal Pregnancy group. The increased thickness in the placentas of the Embryo Relocation group appears to be the result of thicker layers on the fetal side of the placenta (labyrinth and basal zones), as opposed to the maternal placental tissue, which was significantly smaller in the Embryo Relocation group.

Furthermore, in the Embryo Relocation group, unlike the observations in the placentas of the Normal Pregnancy group, as well as previous literature45, the labyrinth zone was significantly larger than the maternal tissue. Previous literature (e.g.,46,47,48,49) indicates the ability for fetal development to continue without significant negative effects despite significant insults, such as decreases in blood perfusion (i.e., reflects the ability for a compensatory mechanism to occur during gestation in the presence of/following a significant and potentially negative change). While significant blood loss during surgery did not occur, it is not unreasonable to assume that some compensatory mechanism may potentially have occurred in the placenta following the embryo relocation procedure due to changes to blood supply and other potential factors, allowing for the apparent normal development of the offspring in our study. Moreover, the compensatory growth of the layers of the fetal side of the placenta is reasonable and plausible given the fundamental roles of the labyrinth and basal zones in providing nutrients to the fetus, the exchange and removal of oxygen/carbon dioxide and waste, as well as the production of hormones to maintain pregnancy45. While the findings of this study cannot be directly extrapolated or applied at the human level, they provide a foundation for the investigation of a methodology that, with further development, could ultimately be of benefit in ectopic pregnancy.

Limitations

It is fundamentally important, at the outset, that it is understood that the results we present do not represent a model of ectopic embryo relocation. The bicornuate (as opposed to simplex in humans) nature of the rat uterus provided the opportunity to investigate the potential feasibility of mimicking the relocation of a “tubal” embryo into the “uterus” through the relocation of a section of a healthy uterine horn carrying one to two viable offspring into a contralateral vacated, healthy uterine horn also previously carrying viable embryos. Thus, the model clearly does not address an extrauterine implantation or account for any potential prior presence of pathophysiology (e.g., acute inflammation, tubal damage, scarring, etc.) that may precipitate an ectopic pregnancy. Moreover, the differences between the rat model and humans need to be taken into consideration. These differences include uterine structure (bicornuate in the rat vs. simplex in humans, e.g.,50), the eventual divergence in the source of progesterone synthesis from the corpus luteum to the placenta in the human relative to the rat20,21, the implantation process (including the process of decidualization;28), placentation, and the presence of placental scars (areas of hemosiderin accumulation) in postpartum rats51.

An additional limitation of this study relates to the measurement of fertility, which we address from the perspective of vaginal impedance, but did not actively test through re-breeding of any of the rats in the ER group. While additional investigation is certainly necessary, the scientific literature does document vaginal impedance as an indicator of estrus, increased sexual receptivity, fecundity, histological changes supportive of fertility and also reports the use of vaginal impedance measurements as an objective method for the purpose of fertility management36,52,53,54,55,56,57. Moreover, while not addressed in this study, given the aim was not to perfect a particular surgical approach or to optimize future pregnancy outcomes, the surgical alterations to rat anatomy involved in this model procedure could, potentially, still permit fertilization and pregnancy in the rat given the cervix is still connected to one uterine horn, oviduct, and ovary. Fertilization and pregnancy should be possible in the as-yet-untested human analogue of the procedure being proposed. Prior to investigations at the human level, this dimension would need to be addressed in a separate investigation. Pertaining to the offspring, our study was limited to the measurement of the size of the rat. As we indicate in “Future Direction”, future research should address developmental milestones (e.g., those addressed in Smirnov and Sitnikova58 if being further addressed in the rat) of the offspring in greater depth. While these limitations remain a reality, this animal model provides an ethical foundation for the investigation of the potential for the translocation of an ectopic pregnancy.

Future direction

Going forward, in addition to the suggestions for future research related to specific limitations mentioned in the previous section, given the intricacies that exist in such a surgical technique, a significant amount of pre-clinical investigation remains necessary. In particular, it is imperative that future research involves a model that more closely mimics human reproductive anatomy and physiology (e.g., in relation to the fallopian tube, uterus, placentation, etc.). Ultimately, a true model of tubal ectopic pregnancy must be sought, given that ectopic pregnancies in the rat tend to primarily be abdominal59 and since rat fallopian tubes are microscopic and do not permit this type of model. Thus, there is a need for larger animal models (e.g., guinea pigs, sheep, baboons, etc.) that would allow for better translation to human pregnancy27,60. Additionally, once this investigation is taken to higher animals that more closely mimic the human condition and treatment, more extensive investigation of the offspring and their development would be appropriate and necessary. Moreover, future research should address in greater depth the dynamics of the relocated embryo, its placenta and the uterus into which it is being relocated. Such work would need to investigate the exact mechanism by which embryos survive, including in relation to the transition of blood supply to the new site. An optimal model for these investigations would permit histological distinctions, if present. Finally, any additional research must be conducted in an ethical framework that maintains a focus on the safety and effects on the maternal patient.

Conclusion

In conclusion, to our knowledge, this is the first preclinical study that initiates an investigation to address a surgical technique with potential implications in the relocation of an embryo in the case of an ectopic pregnancy. In this regard, our findings reflect a successful transuterine relocation of living (non-ectopic) embryos within the same rat, resulting in viable fetuses, uncomplicated vaginal delivery and living offspring to adulthood. It is hoped that these findings will stimulate future investigation, at the preclinical level in higher animals, leading to potential equivalent surgical options for use in humans, including in situations such as ectopic pregnancy.