**A Novel Concept of Fundus-Ovary-Salpinx-Para-Aorta Implantation Promoting Unit during Human Embryo Implantation**

Hiroshi Fujiwara, Yoshihiko Araki, Shigeru Saito, Kazuhiko Imakawa, Satoru Kyo, Minoru Shigeta, Masahide Shiotani, Akihito Horie and Takahide Mori

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60634

### **Abstract**

Human embryo implantation is mainly regulated by the endocrine system. Since the ovary, fallopian tube, and fundus can directly communicate through the mesosalpinx and ovarian ligament, the local concentration of progesterone in the pathway of the developing embryo is considered to be higher than in systemic blood circulation. The immune system promotes embryo implantation by stimulating progesterone production of the ovary and by inducing endometrial differentiation. The recognition of the developing embryo in the fallopian tube by the immune system is achieved through the para-aortic lymph nodes. On the basis of the above evidence, the autologous immune cells activated in vitro were demonstrated to improve clinical pregnancy rates in patients with repeated implantation failures. In addition, the autonomic nerve system that innervates the fundus, the ovary, and the fallopian tube from the para-aortic region is proposed to regulate the environment of the pathway of the developing embryo. From these findings, we suppose that a unique unilateral functional unit to promote human embryo implantation exists in the pathway of the developing embryo including the para-aortic regions and propose naming this novel functional unit the Fundus-Ovary-Salpinx-Para-aorta Implantation Promoting unit (FOSPa-IP unit).

**Keywords:** embryo, FOSPa-IP unit, implantation

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **1. Introduction**

In humans, the corpus luteum, which is formed from the ovulated follicle, produces proges‐ terone that induces adequate endometrial differentiation for embryo implantation. During pregnancy, the embryo trophoblast cells secrete human chorionic gonadotropin (HCG) that stimulates the maternal corpus luteum to sustain progesterone production. In turn, it acts on the endometrium to maintain embryo implantation in the uterus. Thus, human embryo implantation is mainly regulated by the endocrine system.

In addition to this endocrine system, we have demonstrated that the immune system is involved in the process of promoting embryo implantation by stimulating progesterone production of the ovary and by inducing endometrial differentiation [1]. It is also suggested that recognition of the developing embryo in the fallopian tube by the immune system is achieved through the para-aortic lymph nodes from a very early stage of pregnancy. The intrauterine administration of autologous immune cells that was activated by HCG in vitro was demonstrated to improve embryo implantation rates in patients with repeated failure of in vitro fertilization and embryo transfer treatment [2].

These lines of evidence led us to conceive a novel concept that there is a unique unilateral functional unit to promote human embryo implantation among the fundus, the ovary, the fallopian tube, and the para-aortic regions. In this chapter, we propose naming this novel functional unit as the Fundus-Ovary-Salpinx-Para-aorta Implantation Promoting unit (FOSPa-IP unit) and describe its estimated characteristics.

### **2. The implantation pathway of the developing embryo**

In humans, the ovulated oocyte is picked up by the fimbria of the fallopian tube and then the fertilized oocyte is transferred to the uterine cavity through the fallopian tube, causing embryogenesis to proceed toward the blastocyst stage (Figure 1). Recently, it has been widely accepted that a synchronized dialog between the developing embryo and the temporally and coordinately differentiated maternal endometriumis is necessary for successful embryo implantation [3]. Accordingly, the adequate preparation of endometrial receptivity as well as the quality of the embryo affects the success of the outcome of in vitro fertilization-embryo transfer (IVF-ET) therapy.

To support the significance of this period, the phenomenon of delayed implantation is well known in rodents and it has also been reported in humans [4]. Furthermore, in cows, this process continues for at least a few days and bovine early embryos become elongated during the pre-implantation period. Consequently, it can be speculated that embryonal signals locally induce further endometrial differentiation and/or an environment suitable for subsequent embryo implantation. We previously proposed that the Eph-ephrin system, which can induce a repulsive force between the epithelial cell layers, contributes to maintaining these crosstalk phases [5]. Several other systems may be involved in the molecular mechanisms of regulation of embryo-maternal crosstalk [4].

A Novel Concept of Fundus-Ovary-Salpinx-Para-aorta Implantation Promoting Unit during Human Embryo… http://dx.doi.org/10.5772/60634 79

**1. Introduction**

78 New Discoveries in Embryology

In humans, the corpus luteum, which is formed from the ovulated follicle, produces proges‐ terone that induces adequate endometrial differentiation for embryo implantation. During pregnancy, the embryo trophoblast cells secrete human chorionic gonadotropin (HCG) that stimulates the maternal corpus luteum to sustain progesterone production. In turn, it acts on the endometrium to maintain embryo implantation in the uterus. Thus, human embryo

In addition to this endocrine system, we have demonstrated that the immune system is involved in the process of promoting embryo implantation by stimulating progesterone production of the ovary and by inducing endometrial differentiation [1]. It is also suggested that recognition of the developing embryo in the fallopian tube by the immune system is achieved through the para-aortic lymph nodes from a very early stage of pregnancy. The intrauterine administration of autologous immune cells that was activated by HCG in vitro was demonstrated to improve embryo implantation rates in patients with repeated failure of

These lines of evidence led us to conceive a novel concept that there is a unique unilateral functional unit to promote human embryo implantation among the fundus, the ovary, the fallopian tube, and the para-aortic regions. In this chapter, we propose naming this novel functional unit as the Fundus-Ovary-Salpinx-Para-aorta Implantation Promoting unit (FOSPa-

In humans, the ovulated oocyte is picked up by the fimbria of the fallopian tube and then the fertilized oocyte is transferred to the uterine cavity through the fallopian tube, causing embryogenesis to proceed toward the blastocyst stage (Figure 1). Recently, it has been widely accepted that a synchronized dialog between the developing embryo and the temporally and coordinately differentiated maternal endometriumis is necessary for successful embryo implantation [3]. Accordingly, the adequate preparation of endometrial receptivity as well as the quality of the embryo affects the success of the outcome of in vitro fertilization-embryo

To support the significance of this period, the phenomenon of delayed implantation is well known in rodents and it has also been reported in humans [4]. Furthermore, in cows, this process continues for at least a few days and bovine early embryos become elongated during the pre-implantation period. Consequently, it can be speculated that embryonal signals locally induce further endometrial differentiation and/or an environment suitable for subsequent embryo implantation. We previously proposed that the Eph-ephrin system, which can induce a repulsive force between the epithelial cell layers, contributes to maintaining these crosstalk phases [5]. Several other systems may be involved in the molecular mechanisms of regulation

implantation is mainly regulated by the endocrine system.

in vitro fertilization and embryo transfer treatment [2].

IP unit) and describe its estimated characteristics.

transfer (IVF-ET) therapy.

of embryo-maternal crosstalk [4].

**2. The implantation pathway of the developing embryo**

**Figure 1.** The implantation pathway of the developing embryo In humans, the ovulated oocyte is picked up by the fimbria of the fallopian tube and then the fertilized oocyte is transferred to the uterine cavity through the fallopian tube, causing embryogenesis to proceed toward the blastocyst stage.

In contrast to the majority of mammals with uterus bicornate bicollis, in humans, women have a single fused uterus derived from bilateral paramesonephric (Müllerian) ducts. From this perspective, the uterine fundus is a structure specific to primates among mammals. Shiotani et al. showed that the human uterus possesses a latent fluid-retaining space along the trans‐ versely communicating line (TCL) between the bilateral utero-tubal angles on the fundus [6]. To build on and confirm their findings, when we injected a small amount of contrast dye (70 µl) into the upper portion of the cavity, the dye spontaneously migrated toward the ceiling of the cavity, spread bilaterally to the utero-tubal angles, and formed along cylindrical space (TCL space) that was gently expanded by the dye (Figure 2A).This space communicated directly with the bilateral fallopian tubes (Figure 2B). When we observed the uterine cavity from a sagittal perspective using a surgically resected uterus, the TCL space was macroscopically manifested by innate tissue pressure of the muscle layer (Figures 3A and B). In contrast, macroscopic TCL space was not formed in the uterus with diffusion and firm enlargement by adenomyosis lesions (Figure 3C).

From these findings, we speculate that the main site of crosstalk between the human embryo and maternal tissues before implantation is the upper site of the uterine cavity in the fundus, that is, the TCL space. In support of this theory, Minami et al. reported that gestational sacs of patients in the early stage of spontaneous normal pregnancy were mainly observed on the right or left side of the upper third of the uterine cavity. They also reported that patients with gestational sacs in the upper region had a significantly lower miscarriage rate than those in the middle and lower regions, proposing that the endometrium at the uterine fundus, especially near the utero-tubal junction, is suitable for human blastocyst implantation under physiological conditions [7].

**Figure 2.** TCL space detected by hysterosalpingography A and B: The human uterus possesses a latent fluid-remaining space along the transversely communicating line (TCL, arrows) between the bilateral utero-tubal angles at the top of the cavity in the fundus. A: When a small amount of contrast dye was injected into the upper portion of the cavity, the dye spontaneously migrated toward the ceiling of the cavity, spread bilaterally to the utero-tubal angles, and formed along cylindrical space (TCL space) that was gently expanded by the dye. B: By subsequent conventional hysterosal‐ pingography, this space communicated directly with the bilateral fallopian tubes.

**Figure 3.** Macroscopic observation of TCL space A: When the uterine cavity was observed from a sagittal perspective using a surgically resected uterus due to carcinoma in situ lesion in the cervix, the TCL space was macroscopically manifested by innate tissue pressure of the muscle layer. B: A magnified figure of the square area of A. C: A uterine specimen that was resected due to adenomyosis. Macroscopic TCL space was not formed in the uterus with diffusion and firm enlargement by adenomyosis lesions.

### **3. The endocrine network around the implantation pathway**

### **3.1. Local concentration of progesterone in the implantation pathway**

the middle and lower regions, proposing that the endometrium at the uterine fundus, especially near the utero-tubal junction, is suitable for human blastocyst implantation under

**Figure 2.** TCL space detected by hysterosalpingography A and B: The human uterus possesses a latent fluid-remaining space along the transversely communicating line (TCL, arrows) between the bilateral utero-tubal angles at the top of the cavity in the fundus. A: When a small amount of contrast dye was injected into the upper portion of the cavity, the dye spontaneously migrated toward the ceiling of the cavity, spread bilaterally to the utero-tubal angles, and formed along cylindrical space (TCL space) that was gently expanded by the dye. B: By subsequent conventional hysterosal‐

**Figure 3.** Macroscopic observation of TCL space A: When the uterine cavity was observed from a sagittal perspective using a surgically resected uterus due to carcinoma in situ lesion in the cervix, the TCL space was macroscopically manifested by innate tissue pressure of the muscle layer. B: A magnified figure of the square area of A. C: A uterine specimen that was resected due to adenomyosis. Macroscopic TCL space was not formed in the uterus with diffusion

pingography, this space communicated directly with the bilateral fallopian tubes.

and firm enlargement by adenomyosis lesions.

physiological conditions [7].

80 New Discoveries in Embryology

Corpus luteum in the ovary is the main endocrine organ that produces progesterone. It has been widely accepted that progesterone prepares the uterus for embryo implantation, induces endometrial differentiation and decreases the contractility of uterine smooth muscle cells. Human endometrial decidualization is also induced by progesterone. The ovary is anatomi‐ cally connected with the ipsilateral fallopian tube and the corneal region of the uterus. The vascular network is present among the fallopian tube as well as the ovary through the mesosalpinx and ovarian ligament. Therefore, it is speculated that the local concentration of progesterone is very high in the ipsilateral fallopian tube adjacent to the ovary that has an ovulated follicle.

Clinically, vaginal administration of progesterone is usually performed for luteal support for infertile patients receiving in vitro fertilization therapy. After vaginal administration, the uterine tissue concentration of progesterone has been found to exceed more than 10 fold the levels achieved by systemic administration. To explain this phenomenon, the "first uterine pass effect," that is, direct preferential vagina-to-uterus transport, was proposed [8]. By drug delivery analysis using tritiated progesterone, Bulletti et al. obtained experimen‐ tal data to support this hypothesis [9]. Consequently, similar to the direct preferential vagina-to-uterus transport system, estrogen and progesterone produced by the corpus luteum in the ovary can be delivered to the corneal region of the uterus by a direct ovaryto-uterus transport system.

Using slice computed tomography (CT) scanning and vascular casting, it was demonstrat‐ ed that both the intramuscular uterine artery and the ovarian artery had a typical ovari‐ an branch connected as an arterial arch, that is, the ovarian artery-to-uterine artery anastomoses [10]. Importantly, these ovarian artery-to-uterine artery anastomoses are located in the mesosalpinx region. In mammals, the anatomical structure among the uteroovarian vein and the ovarian artery is considered to be important to regulate the countercurrent system of exchange from the uterus to the ovary and back again. In humans, the utero-ovarian vein forms a plexus around the ovarian artery. Therefore, it has been suggested that counter-current transfer facilitates local communication between the ovary, the fallopian tube, and the uterus [11]. Later, it was also reported that serum levels of estradiol and progesterone in the uterine vessels were more than 2–4 times higher than those in the systemic circulation, demonstrating the preferential transport of sex steroids produced in the ovary to the uterus [12]. Interestingly, the same group also suggested that the main blood supply to the uterine corneal region from uterine and ovarian arteries is shifted following ovulation [13]. This suggests the possibility that progesterone regulates counter-current blood flows in the mesosalpinx. Consequently, the local concentration of progesterone along the implantation pathway of the human embryo is speculated to be considerably high during its developmental process (Figure 4).

**Figure 4.** The local concentration of progesterone along the implantation pathway The local concentration of progester‐ one along the implantation pathway of the human embryo is speculated to be considerably high during its develop‐ mental process.

### **3.2. Hormonal regulation of the contractility around the implantation pathway by ovarian steroid hormones**

Strict regulation of contractility in the uterus and the fallopian tube is essential for various reproductive functions including expulsion of menstrual debris, sperm transport, and adequate embryo placement during implantation [14]. More than half a century ago, the precise profiles of contractile activity of the non-pregnant uterus throughout the menstrual cycle were reported using an intrauterine pressure recording system [15, 16]. Recently, it has become possible for uterine contractility to be directly and non-invasively assessed using ultrasound scans and ultrafast magnetic resonance imaging techniques [17]. Accordingly, the inherent contractility of the uterus is classified into two patterns: a focal and sporadic bulging of the myometrium and a rhythmic and subtle stripping movement in the subendometrial myometrium, known as uterine peristalsis. Using these direct and non-invasive techniques, the precise profiles of several wavelike activity patterns throughout the menstrual cycle have been thoroughly analyzed, and it has been widely accepted that ovarian steroid hormones regulate contractions of the non-pregnant uterus. Clinically, uterine contractility has been demonstrated to influence the human embryo implantation process in both spontaneous cycles and assisted reproduction [18, 19].

Waves from fundus to cervix are dominant in the follicular phase, but diminish after ovulation. In contrast, waves from cervix to fundus were observed in the late follicular and luteal phases [20, 21], supporting implantation of the embryo at the upper region of the uterine cavity [22]. During the luteal phase, the movement of the upper fundal region is relatively quiescent facilitating embryo implantation [23]. By the sequential administration of estradiol (days 1– 28) and progesterone (days 14–28), waves from fundus to cervix were induced by estradiol, but were immediately diminished after the administration of progesterone, whereas waves from cervix to fundus were observed in both the estradiol-only and the estradiol and proges‐ terone combined phases [18]. It was also reported that there is horizontal movement at the fundus, often to and fro, not unidirectional [24]. Importantly, this horizontal movement can theoretically induce the migration of pre-implanted embryo back to the fallopian tubes along the TCL through the fluid by endometrial secretion (Figure 5).

**Figure 4.** The local concentration of progesterone along the implantation pathway The local concentration of progester‐ one along the implantation pathway of the human embryo is speculated to be considerably high during its develop‐

**3.2. Hormonal regulation of the contractility around the implantation pathway by ovarian**

Strict regulation of contractility in the uterus and the fallopian tube is essential for various reproductive functions including expulsion of menstrual debris, sperm transport, and adequate embryo placement during implantation [14]. More than half a century ago, the precise profiles of contractile activity of the non-pregnant uterus throughout the menstrual cycle were reported using an intrauterine pressure recording system [15, 16]. Recently, it has become possible for uterine contractility to be directly and non-invasively assessed using ultrasound scans and ultrafast magnetic resonance imaging techniques [17]. Accordingly, the inherent contractility of the uterus is classified into two patterns: a focal and sporadic bulging of the myometrium and a rhythmic and subtle stripping movement in the subendometrial myometrium, known as uterine peristalsis. Using these direct and non-invasive techniques, the precise profiles of several wavelike activity patterns throughout the menstrual cycle have been thoroughly analyzed, and it has been widely accepted that ovarian steroid hormones regulate contractions of the non-pregnant uterus. Clinically, uterine contractility has been demonstrated to influence the human embryo implantation process in both spontaneous cycles

mental process.

**steroid hormones**

82 New Discoveries in Embryology

and assisted reproduction [18, 19].

**Figure 5.** Hormonal regulation of the contractility around the implantation pathway by ovarian steroid hormones. Ovarian steroid hormones regulate contractions of the uterus. Waves from cervix to fundus were observed in the late follicular and luteal phases. There is also horizontal movement at the fundus, often to and fro, not unidirectional, which can theoretically induce the migration of pre-implantation embryo back to the fallopian tubes along the TCL.

Recently, it has been reported that a new population of c-kit-positive cells, interstitial Cajallike cells, now called telocytes, had been found on the borders of smooth muscle bundles in human myometrium. These cells resemble interstitial cells of Cajal in the gut, which generate electrical slow waves to control peristalsis [14]. Telocytes in the myometrium are doublepositive for c-kit and CD34, and have very long cellular extensions called telopodes that release extracellular vesicles, sending macromolecular signals to neighboring cells. It was proposed that they modulate spontaneous contractions of the non-pregnant human uterus, through a tyrosine-kinase-independent signaling pathway [25, 26]. Although the precise effects of ovarian steroid hormones on telocyte functions remain unclear, immunoreactive estrogen and progesterone receptors localized at the nucleus level of uterine telocytes suggested their involvement in the hormonal regulation of uterine contractility [27].

### **3.3. Hormonal regulation of immune environment around the implantation pathway by ovarian steroid hormones**

During the implantation process, the semi-allogeneic embryo is not rejected by the maternal immune system. The mechanisms regarding how the fetus is tolerated by the maternal immune system are still not well understood. It is generally accepted that ovarian sex steroids regulate the function and population of endometrial and/or decidual immune cells in the uterus [28] and these immune–endocrine interactions contribute to fetal survival within the maternal uterus, suppressing adverse maternal immune responses and promoting immunotolerance pathways [29].

Progesterone regulates immune function by producing mediators such as the progesteroneinduced blocking factor that induces Th2-dominant cytokine production [30] and glycodelin A that protects the embryo from maternal immune attack by inhibiting the proliferation of T cells and B cells and the activity of natural killer cells, or by deleting the monocytes [31]. The physiological effects of progesterone are mediated by its specific nuclear progesterone receptor (PR) that activates transcription factors. Nuclear PR has two main isoforms: PR-A and PR-B. PR-B acts as an activator of progesterone-responsive genes, while PR-A can inhibit the activity of PR-B. Using nuclear PR knockout mice, it has been shown that progesterone antagonizes estrogen-induced recruitment of macrophages and neutrophils into the uterus [32]. Recently, it has been demonstrated that progesterone at a relatively high concentration also acts on cells by a non-genomic mechanism through progesterone binding membrane proteins such as progesterone membrane components 1 and 2, and the membrane progestin receptors [33, 34]. Considering a local high concentration of progesterone, these non-genomic mechanisms may operate in the implantation pathway.

CD56(high+)CD16(–) uterine natural killer cells are the predominant population in the decidual tissues during the late secretory phase of the menstrual cycle and early pregnancy. They may be derived from natural killer cell progenitors and/or peripheral natural killer cells and are considered to contribute to the remodeling of maternal uterine vasculature in inter‐ action with extravillous trophoblasts [29, 35]. Although the level of the expression of PR on uterine natural killer cells is very low [36], the progesterone-induced endometrial environment is an important factor for the in situ proliferation or differentiation of uterine natural killer cells in human endometrium, inducing reprogramming of their chemokine receptor profiles [37, 38]. Progesterone is also reported to reduce the antigen-presenting capacity of dendritic cells, monocytes, and macrophages and induce the recruitment of regulatory T (Treg) cells, contributing to local accumulation of pregnancy-protective cells [29]. These lines of evidence

suggest the relationship between the endocrine and immune systems for establishing the embryo implantation environment.

### **4. The autonomic nerve network around the implantation pathway**

extracellular vesicles, sending macromolecular signals to neighboring cells. It was proposed that they modulate spontaneous contractions of the non-pregnant human uterus, through a tyrosine-kinase-independent signaling pathway [25, 26]. Although the precise effects of ovarian steroid hormones on telocyte functions remain unclear, immunoreactive estrogen and progesterone receptors localized at the nucleus level of uterine telocytes suggested their

**3.3. Hormonal regulation of immune environment around the implantation pathway by**

During the implantation process, the semi-allogeneic embryo is not rejected by the maternal immune system. The mechanisms regarding how the fetus is tolerated by the maternal immune system are still not well understood. It is generally accepted that ovarian sex steroids regulate the function and population of endometrial and/or decidual immune cells in the uterus [28] and these immune–endocrine interactions contribute to fetal survival within the maternal uterus, suppressing adverse maternal immune responses and promoting immunotolerance

Progesterone regulates immune function by producing mediators such as the progesteroneinduced blocking factor that induces Th2-dominant cytokine production [30] and glycodelin A that protects the embryo from maternal immune attack by inhibiting the proliferation of T cells and B cells and the activity of natural killer cells, or by deleting the monocytes [31]. The physiological effects of progesterone are mediated by its specific nuclear progesterone receptor (PR) that activates transcription factors. Nuclear PR has two main isoforms: PR-A and PR-B. PR-B acts as an activator of progesterone-responsive genes, while PR-A can inhibit the activity of PR-B. Using nuclear PR knockout mice, it has been shown that progesterone antagonizes estrogen-induced recruitment of macrophages and neutrophils into the uterus [32]. Recently, it has been demonstrated that progesterone at a relatively high concentration also acts on cells by a non-genomic mechanism through progesterone binding membrane proteins such as progesterone membrane components 1 and 2, and the membrane progestin receptors [33, 34]. Considering a local high concentration of progesterone, these non-genomic mechanisms may

CD56(high+)CD16(–) uterine natural killer cells are the predominant population in the decidual tissues during the late secretory phase of the menstrual cycle and early pregnancy. They may be derived from natural killer cell progenitors and/or peripheral natural killer cells and are considered to contribute to the remodeling of maternal uterine vasculature in inter‐ action with extravillous trophoblasts [29, 35]. Although the level of the expression of PR on uterine natural killer cells is very low [36], the progesterone-induced endometrial environment is an important factor for the in situ proliferation or differentiation of uterine natural killer cells in human endometrium, inducing reprogramming of their chemokine receptor profiles [37, 38]. Progesterone is also reported to reduce the antigen-presenting capacity of dendritic cells, monocytes, and macrophages and induce the recruitment of regulatory T (Treg) cells, contributing to local accumulation of pregnancy-protective cells [29]. These lines of evidence

involvement in the hormonal regulation of uterine contractility [27].

**ovarian steroid hormones**

84 New Discoveries in Embryology

operate in the implantation pathway.

pathways [29].

In pigs, it was demonstrated that the oviduct is innervated by various efferent autonomic neurons such as the inferior mesenteric ganglion, ovarian ganglion, and celiac-superior mesenteric ganglion complex, forming discrete "oviductal centers" and implying that these nerve fibers regulate oviductal blood flow, non-vascular smooth muscle contraction, trans‐ mission of sensory information, and epithelial secretion [39, 40]. In monkeys, noradrenaline of the sympathetic nerves innervating the smooth musculature of the oviduct was demon‐ strated to change cyclically during the menstrual cycle, suggesting that the system of adre‐ nergic nerves in the primate oviduct is under the control of endogenous estrogen and progesterone [41]. It was also reported that estrogen and progesterone affect not only the noradrenaline content of adrenergic nerves in the uterus and oviduct but also the turnover of noradrenaline, the activity of the enzymes that synthesize it, and the release of noradrenaline from nerve terminals [42]. By electron microscopic examination, non-vascular adrenergic nerves were found in smooth muscle bundles of human fallopian tube and electrical field stimulation of adrenergic nerves in the isthmic smooth muscle induced an alpha-receptormediated contractile response [43]. In rats, the sympathetic nerve fibers of the upper part of the uterus arise from the ovarian plexus nerve that mainly originates from neurons of the suprarenal ganglia and of the T10 to L3 ganglia of the paravertebral sympathetic chain, whereas most of the sympathetic innervation of the lower uterus arises from neurons of the paravertebral ganglia T13 to S2, principally at the L2–L4 levels, suggesting that regulation of myometrial activity by the sympathetic nerve system is functionally different between the oviduct and the cervical ends of the uterus [44].

Accordingly, the influence of the sympathetic nerve response on the female genital tract should be considered clinically. In fact, it was demonstrated that mock embryo transfer stimulation (injection of 20 µl of ultrasound contrast agent alone) evoked uterine peristalsis that could cause embryo migration and extrude the transferred embryo with fluid [45]. The density of nerve fibers in the oviduct isthmus in women with hydrosalpinx was revealed to be low compared with that in women without hydrosalpinx, suggesting the involvement of autono‐ mous nerve system in the mechanism of hydrosalpinx-associated infertility [46]. A recent study showed that transcutaneous electrical acupoint stimulation significantly improved the clinical outcome of embryo transfer [47].

### **5. The immune network around the implantation pathway**

### **5.1. Circulating immune cell contribution to embryo implantation**

Mammals are a unique group of species in terms of accepting embryos within the maternal uterus (embryonal parasitic strategy). In this respect, maternal recognition of the developing embryo in the genital tract is a very important process to prepare a favorable maternal environment for subsequent embryo implantation. In humans, HCG secreted by the implant‐ ing embryo stimulates the corpus luteum of pregnancy to produce progesterone, maintaining embryo implantation in the uterus. Previously, we found that the immune system also contributes to this process and proposed that "corpus luteum function is maintained not only by HCG (endocrine system), but also by lymphocytes (immune system)" [48]. In mouse, implantation experiments, intravenous or intrauterine administration of splenocytes derived from early pregnant mice induced endometrial differentiation and successful implantation in pseudopregnant recipient mice [49, 50]. On the basis of these results, we proposed a new concept that "The immune system recognizes some information on the presence of the developing embryo around the implantation period and, thereafter, circulating immune cells transmit this information to the ovary and the uterus through blood circulation to induce adequate differentiation of pregnancy CL and endometrium to facilitate embryo implanta‐ tion." Furthermore, we found that peripheral blood mononuclear cells (PBMC) promoted endometrial receptivity in vitro, while HCG affected PBMC function not through authentic HCG receptor, but sugar chain receptors, which is a primitive mechanism in the immune system [51, 52]. These experimental facts led us to pay attention to sugar chain moieties as candidate key structures of embryonal signals to the maternal immune system. These findings also suggest the important roles of circulating immune cells in embryo implantation from a very early stage [53].

### **5.2. Direct and functional communication between para-aortic lymph nodes and the implantation pathway of the developing embryo**

What is the main immune organ for the first recognition of the developing embryo in the implantation pathway? From insight obtained from gynecologic oncology, para-aortic lymph nodes are classified as regional lymph nodes in patients with uterine corpus cancer. When we used a fluorescent indocyanine green to confirm the sentinel lymph nodes from the fundus lesion, rapid drainage into para-aortic lymph nodes, especially around the proximal site of the branch of the inferior mesenteric artery, from the uterine fundus through the suspensory ligament of the ovary and the meso-oviductal space, was initially detected using a PDA camera system (Figure 6). Theoretically, this supports the presence of direct communication between para-aortic lymph nodes and the implantation pathway of the developing embryo through the immune system.

Recently, Treg cells have been shown to facilitate maternal immune tolerance of the semiallo‐ geneic conceptus and proposed to play a crucial role in embryo implantation and fetal development. During the pre-implantation period, factors in the seminal fluid delivered at coitus cause expansion of a CD4(+)CD25(+) putative Treg cell population in the para-aortic lymph nodes [54]. They were also reported to be rapidly recruited to para-aortic lymph nodes and activated in the first days after embryo implantation [55]. In mouse, implantation experi‐ ments, splenocytes derived from early pregnant mice (post-ovulation day 4) when the embryo had not yet attached to the endometrium could induce endometrial differentiation and successful implantation in the early stage of pseudopregnant recipient mice that had been

A Novel Concept of Fundus-Ovary-Salpinx-Para-aorta Implantation Promoting Unit during Human Embryo… http://dx.doi.org/10.5772/60634 87

embryo in the genital tract is a very important process to prepare a favorable maternal environment for subsequent embryo implantation. In humans, HCG secreted by the implant‐ ing embryo stimulates the corpus luteum of pregnancy to produce progesterone, maintaining embryo implantation in the uterus. Previously, we found that the immune system also contributes to this process and proposed that "corpus luteum function is maintained not only by HCG (endocrine system), but also by lymphocytes (immune system)" [48]. In mouse, implantation experiments, intravenous or intrauterine administration of splenocytes derived from early pregnant mice induced endometrial differentiation and successful implantation in pseudopregnant recipient mice [49, 50]. On the basis of these results, we proposed a new concept that "The immune system recognizes some information on the presence of the developing embryo around the implantation period and, thereafter, circulating immune cells transmit this information to the ovary and the uterus through blood circulation to induce adequate differentiation of pregnancy CL and endometrium to facilitate embryo implanta‐ tion." Furthermore, we found that peripheral blood mononuclear cells (PBMC) promoted endometrial receptivity in vitro, while HCG affected PBMC function not through authentic HCG receptor, but sugar chain receptors, which is a primitive mechanism in the immune system [51, 52]. These experimental facts led us to pay attention to sugar chain moieties as candidate key structures of embryonal signals to the maternal immune system. These findings also suggest the important roles of circulating immune cells in embryo implantation from a

**5.2. Direct and functional communication between para-aortic lymph nodes and the**

What is the main immune organ for the first recognition of the developing embryo in the implantation pathway? From insight obtained from gynecologic oncology, para-aortic lymph nodes are classified as regional lymph nodes in patients with uterine corpus cancer. When we used a fluorescent indocyanine green to confirm the sentinel lymph nodes from the fundus lesion, rapid drainage into para-aortic lymph nodes, especially around the proximal site of the branch of the inferior mesenteric artery, from the uterine fundus through the suspensory ligament of the ovary and the meso-oviductal space, was initially detected using a PDA camera system (Figure 6). Theoretically, this supports the presence of direct communication between para-aortic lymph nodes and the implantation pathway of the developing embryo through the

Recently, Treg cells have been shown to facilitate maternal immune tolerance of the semiallo‐ geneic conceptus and proposed to play a crucial role in embryo implantation and fetal development. During the pre-implantation period, factors in the seminal fluid delivered at coitus cause expansion of a CD4(+)CD25(+) putative Treg cell population in the para-aortic lymph nodes [54]. They were also reported to be rapidly recruited to para-aortic lymph nodes and activated in the first days after embryo implantation [55]. In mouse, implantation experi‐ ments, splenocytes derived from early pregnant mice (post-ovulation day 4) when the embryo had not yet attached to the endometrium could induce endometrial differentiation and successful implantation in the early stage of pseudopregnant recipient mice that had been

very early stage [53].

86 New Discoveries in Embryology

immune system.

**implantation pathway of the developing embryo**

**Figure 6.** Direct communication between para-aortic lymph nodes and the embryo through the immune system. When fluorescent indocyanine green was injected into the uterine fundus that was affected by endometrial cancer cells in or‐ der to confirm the sentinel lymph nodes from the fundus lesion, rapid drainage into para-aortic lymph nodes, especial‐ ly around the proximal site of the branch of the inferior mesenteric artery was detected by the PDA camera system. A: The retroperitoneal para-aortic region was opened. B: A magnified figure of the square area of A. Indocyanine greenpositive para-aortic lymph nodes and an afferent lymph vessel (arrowhead) were clearly detected. C: A figure from after lymph node dissection. D: Para-aortic lymph nodes communicated by vessels and nerves (arrows). Pa-LN, paraaortic lymph nodes; Ao, aorta; IVC, inferior vena cava; IMA, inferior mesenteric artery; l-RV, left renal vein.

mated with vasectomized male mice, indicating that functional change of peripheral immune cells has already occurred before embryo implantation [49, 50]. Importantly, since the immune system of pseudopregnant recipient mice mated with vasectomized male mice was already sensitized with seminal plasma component of seminal fluid, the changes in splenocyte function were induced by the presence of developing embryos [56]. In addition, it was reported that functional changes in the endometrium could be induced in pregnant mice even when the uterotubal transition sites were ligated and entry of the developing embryos into the uterine cavity was inhibited [57], indicating that the developing embryo in the fallopian tube can influence maternal endometrial differentiation. Collectively, it is speculated that mothers can recognize the developing embryo during this early phase through the para-aortic lymph nodes.

The human para-aortic lymph nodes are rich in not only vascular but also automatic nerve networks (Figure 6D). Importantly, all primary and secondary immune organs receive substantial sympathetic innervation from sympathetic post-ganglionic neurons. This sympa‐ thetic nervous system either enhances or inhibits the activity of both acquired and adaptive immune systems [58]. Adrenergic nerve fibers were found following both afferent and efferent blood vessels as well as T areas, supporting a regulatory role of the sympathetic nervous system in human lymph nodes [59]. Intriguingly, amputation of autonomic nerves innervating the uterus was reported to cause on-time implantation failure in rats, increasing the population of uterine mast cells and facilitating the release of histamine by mast cells [60]. These findings support the concept that the neuro-immune network plays an important role in embryo implantation.

### **6. Fundus-Ovary-Salpinx-Para-aorta Implantation Promoting Unit (FOSPa-IP unit)**

On the basis of the above evidence, we suppose that there is a unique unilateral functional unit to promote human embryo implantation among the fundus, the ovary, the fallopian tube, and the para-aortic regions (Figure 7). From an anatomical viewpoint, we here propose naming this novel functional unit as the Fundus-Ovary-Salpinx-Para-aorta Implantation Promoting Unit, that is, the FOSPa-IP unit. This functional unit seems to be co-operatively regulated by the endocrine, immune, and nerve systems.

Recently, increasing attention has been paid to patients with repeated implantation failures under IVF-ET treatment. It should be noted that the process of maternal recognition by the immune system in the FOSPa-IP unit is largely skipped in the treatment of IVF-ET. Considering the intrinsic function of the FOSPa-IP unit, we developed a novel therapy for patients with repeated implantation failures to complement the functions of the unit. Concretely speaking, PBMC are isolated from patients and incubated for two days with HCG in order to activate them. Thereafter, activated PBMC are administered into the uterine cavity to induce adequate endometrial differentiation. Three days later, blastocysts are transferred into the uterine cavity. A Novel Concept of Fundus-Ovary-Salpinx-Para-aorta Implantation Promoting Unit during Human Embryo… http://dx.doi.org/10.5772/60634 89

**Figure 7.** Proposal ofthe FOSPa-IP unit. We propose a unique unilateral functional unit that promotes human embryo implantation among the fundus, the ovary, the fallopian tube, and the para-aortic regions. This functional unit seems to be co-operatively regulated by the endocrine, immune, and nerve systems and can be named the Fundus-Ovary-Salpinx-Paraaorta Implantation Promoting Unit, that is, FOSPa-IP unit.

We applied this treatment to patients with 4 or more repeated failures in IVF therapy and it effectively improved the clinical pregnancy and implantation rates [2, 61].

### **7. Conclusion**

mated with vasectomized male mice, indicating that functional change of peripheral immune cells has already occurred before embryo implantation [49, 50]. Importantly, since the immune system of pseudopregnant recipient mice mated with vasectomized male mice was already sensitized with seminal plasma component of seminal fluid, the changes in splenocyte function were induced by the presence of developing embryos [56]. In addition, it was reported that functional changes in the endometrium could be induced in pregnant mice even when the uterotubal transition sites were ligated and entry of the developing embryos into the uterine cavity was inhibited [57], indicating that the developing embryo in the fallopian tube can influence maternal endometrial differentiation. Collectively, it is speculated that mothers can recognize the developing embryo during this early phase through the para-aortic lymph

The human para-aortic lymph nodes are rich in not only vascular but also automatic nerve networks (Figure 6D). Importantly, all primary and secondary immune organs receive substantial sympathetic innervation from sympathetic post-ganglionic neurons. This sympa‐ thetic nervous system either enhances or inhibits the activity of both acquired and adaptive immune systems [58]. Adrenergic nerve fibers were found following both afferent and efferent blood vessels as well as T areas, supporting a regulatory role of the sympathetic nervous system in human lymph nodes [59]. Intriguingly, amputation of autonomic nerves innervating the uterus was reported to cause on-time implantation failure in rats, increasing the population of uterine mast cells and facilitating the release of histamine by mast cells [60]. These findings support the concept that the neuro-immune network plays an important role in embryo

**6. Fundus-Ovary-Salpinx-Para-aorta Implantation Promoting Unit**

On the basis of the above evidence, we suppose that there is a unique unilateral functional unit to promote human embryo implantation among the fundus, the ovary, the fallopian tube, and the para-aortic regions (Figure 7). From an anatomical viewpoint, we here propose naming this novel functional unit as the Fundus-Ovary-Salpinx-Para-aorta Implantation Promoting Unit, that is, the FOSPa-IP unit. This functional unit seems to be co-operatively regulated by

Recently, increasing attention has been paid to patients with repeated implantation failures under IVF-ET treatment. It should be noted that the process of maternal recognition by the immune system in the FOSPa-IP unit is largely skipped in the treatment of IVF-ET. Considering the intrinsic function of the FOSPa-IP unit, we developed a novel therapy for patients with repeated implantation failures to complement the functions of the unit. Concretely speaking, PBMC are isolated from patients and incubated for two days with HCG in order to activate them. Thereafter, activated PBMC are administered into the uterine cavity to induce adequate endometrial differentiation. Three days later, blastocysts are transferred into the uterine cavity.

nodes.

88 New Discoveries in Embryology

implantation.

**(FOSPa-IP unit)**

the endocrine, immune, and nerve systems.

In humans, the TCL space at the top of the uterine cavity in the fundus may be the main site of crosstalk between embryo and mother before implantation. Including this space, we propose the presence of a unique unilateral functional unit, named the FOSPa-IP unit, among the fundus, the ovary, the fallopian tube, and the para-aortic regions, which promotes human embryo implantation. The local concentration of progesterone along the implantation pathway of the human embryo is considered high, regulating uterine contractility and influencing human embryo implantation. On the other hand, the immuneendocrine interactions along the implantation pathway of the embryo generate an environ‐ ment that promotes embryo implantation, contributing to fetal survival within the maternal uterus, suppressing adverse maternal immune responses and promoting immunotoler‐ ance pathways. In addition, circulating immune cells were shown to contribute to em‐ bryo implantation in a very early stage, probably after being activated in para-aortic lymph nodes. Furthermore, the influence of the sympathetic nerve response on the female genital

tract has been clinically noticed, based on the concept that the neuro-immune network plays an important role in embryo implantation. Considering the intrinsic function of the FOSPa-IP unit, we developed a novel therapy for patients with repeated implantation failures to complement functions of this unit. Further understanding of reproductive organs from the viewpoint of the FOSPa-IP unit is expected to contribute to the development of new therapies, especially in the field of reproductive medicine.

### **Author details**

Hiroshi Fujiwara1\*, Yoshihiko Araki2 , Shigeru Saito3 , Kazuhiko Imakawa4 , Satoru Kyo5 , Minoru Shigeta6 , Masahide Shiotani7 , Akihito Horie8 and Takahide Mori9

\*Address all correspondence to: fuji@kuhp.kyoto-u.ac.jp

1 Department of Obstetrics and Gynecology, Kanazawa University Graduate School of Medical Science, Japan

2 Institute for Environmental and Gender-specific Medicine, Juntendo University Graduate School of Medicine, Japan

3 Department of Obstetrics and Gynecology, Faculty of Medicine, University of Toyama, Toyama, Japan

4 Laboratory of Animal Breeding and Reproduction, Veterinary Medical Sciences, Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan

5 Department of Obstetrics and Gynecology, Faculty of Medicine, Shimane University, Japan

6 Advanced Fertility Center of Fuchu Nozomi, Osaka, Japan

7 Hanabusa Women's Clinic, Kobe, Japan

8 Department of Gynecology and Obstetrics, Kyoto University Graduate School of Medicine, Japan

9 Academia for Repro-Regenerative Medicine, Tokyo, Japan

### **References**

[1] Fujiwara H: Do circulating blood cells contribute to maternal tissue remodeling and embryo-maternal cross-talk around the implantation period? Mol Hum Reprod. 2009;15:335-43. doi: 10.1093/molehr/gap027.

[2] Yoshioka S, Fujiwara H, Nakayama T, Kosaka K, Mori T, Fujii S: Intrauterine admin‐ istration of autologous peripheral blood mononuclear cells promotes implantation rates in patients with repeated failure of IVF-embryo transfer. Hum Reprod. 2006;21:3290-4.

tract has been clinically noticed, based on the concept that the neuro-immune network plays an important role in embryo implantation. Considering the intrinsic function of the FOSPa-IP unit, we developed a novel therapy for patients with repeated implantation failures to complement functions of this unit. Further understanding of reproductive organs from the viewpoint of the FOSPa-IP unit is expected to contribute to the development of new

, Shigeru Saito3

, Akihito Horie8

1 Department of Obstetrics and Gynecology, Kanazawa University Graduate School of

2 Institute for Environmental and Gender-specific Medicine, Juntendo University Graduate

3 Department of Obstetrics and Gynecology, Faculty of Medicine, University of Toyama,

4 Laboratory of Animal Breeding and Reproduction, Veterinary Medical Sciences, Graduate

5 Department of Obstetrics and Gynecology, Faculty of Medicine, Shimane University, Japan

8 Department of Gynecology and Obstetrics, Kyoto University Graduate School of Medicine,

[1] Fujiwara H: Do circulating blood cells contribute to maternal tissue remodeling and embryo-maternal cross-talk around the implantation period? Mol Hum Reprod.

, Kazuhiko Imakawa4

and Takahide Mori9

, Satoru Kyo5

,

therapies, especially in the field of reproductive medicine.

, Masahide Shiotani7

\*Address all correspondence to: fuji@kuhp.kyoto-u.ac.jp

School of Agricultural and Life Sciences, University of Tokyo, Japan

6 Advanced Fertility Center of Fuchu Nozomi, Osaka, Japan

9 Academia for Repro-Regenerative Medicine, Tokyo, Japan

2009;15:335-43. doi: 10.1093/molehr/gap027.

7 Hanabusa Women's Clinic, Kobe, Japan

**Author details**

90 New Discoveries in Embryology

Minoru Shigeta6

Toyama, Japan

Japan

**References**

Medical Science, Japan

School of Medicine, Japan

Hiroshi Fujiwara1\*, Yoshihiko Araki2


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[43] Sporrong B, Helm G, Owman C, Sjöberg NO, Walles B: Electron microscopic and pharmacologic evidence for a functional adrenergic innervation of the smooth mus‐

[44] Houdeau E, Rousseau A, Meusnier C, Prud'Homme MJ, Rousseau JP: Sympathetic innervation of the upper and lower regions of the uterus and cervix in the rat have

[45] Zhu L, Xiao L, Che HS, Li YP, Liao JT: Uterine peristalsis exerts control over fluid migration after mock embryo transfer. Hum Reprod. 2014;29:279-85. doi: 10.1093/

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[47] Zhang R, Feng XJ, Guan Q, Cui W, Zheng Y, Sun W, Han JS: Increase of success rate for women undergoing embryo transfer by transcutaneous electrical acupoint stimu‐ lation: a prospective randomized placebo-controlled study. Fertil Steril.

[48] Hashii K, Fujiwara H, Yoshioka S, Kataoka N, Yamada S, Hirano T, Mori T, Fujii S, Maeda M: Peripheral blood mononuclear cells stimulate progesterone production by luteal cells derived from pregnant and non-pregnant women: possible involvement of interleukin-4 and interleukin-10 in corpus luteum function and differentiation.

[49] Takabatake K, Fujiwara H, Goto Y, Nakayama T, Higuchi T, Maeda M, Mori T: Intra‐ venous administration of splenocytes in early pregnancy changes the implantation

[50] Takabatake K, Fujiwara H, Goto Y, Nakayama T, Higuchi T, Fujita J, Maeda M, Mori T: Splenocytes in early pregnancy promote embryo implantation by regulating endo‐

[51] Egawa H, Fujiwara H, Hirano T, Nakayama T, Higuchi T, Tatsumi K, Mori T, Fujii S: Peripheral blood mononuclear cells in early pregnancy promote invasion of human

[52] Kosaka K, Fujiwara H, Tatsumi K, Yoshioka S, Sato Y, Egawa H, Higuchi T, Nakaya‐ ma T, Ueda M, Maeda M, Fujii S: Human chorionic gonadotropin (HCG) activates monocytes to produce interleukin-8 via a different pathway from luteinizing hor‐

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### **Human Embryology**

Shigehito Yamada, Mark Hill and Tetsuya Takakuwa

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61453

### **Abstract**

The study of human embryology has a very long history. Modern embryology owes its initial development to the key embryo collections that began in the 19th century. The first large collection was that of Carnegie, and this was followed later by the major 7 collections. The second role of the Carnegie collection was for researchers to establish a defined set of Carnegie stages based on embryo morphological features. Today, embryos are imaged three-dimensionally (3D) by a range of imaging modalities including, magnetic resonance microscopy (MRM), episcopic fluorescence image capture (EFIC), phase-contrast X-ray computed tomography (pCT), and optical projection tomography (OPT). Historically, embryo serial images were reconstructed using wax-plate and model techniques. The above new 3D imaging techniques now allow 3D computer reconstructions, analysis, and even 3D printing. This chapter will describe how the classical embryology collections and techniques have developed into today's imaging and analysis techniques, giving new insights to human embryonic development.

**Keywords:** Human Embryo, Embryo Collection, Developmental Stages, Imaging, 3D reconstruction, 3D printer

### **1. Introduction**

Human embryology in the 19th century began by using human embryo samples derived from maternal deaths, abortion, or surgery. Nothing has been changed in the 21st century, because animal experimental biology developed in the 20th century could not and should not apply to human embryology on its ethical aspect. However, human embryology has progressed little during the last 100 years, with only recently some limited molecular studies on small numbers of human material. In contrast, recent studies using both nondestructive and destructive

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

imaging techniques on existing collections have allowed many morphological measurements of these embryos using these novel imaging techniques.

Here we summarize the historic collections of embryos used in the study of human develop‐ ment, explain the criteria used for developmental staging, show sectioned and reconstructed images from newer three-dimensional (3D) imaging in high resolution, and discuss the future directions for the analyses of the human embryo.

### **2. Human embryo collections**

During the history of human embryology the establishment and study of key human embryo collections has greatly contributed to our current understanding. In this section we briefly summarize the history of some of these collections, such as the Carnegie Collection, the Kyoto Collection, the Blechschmidt Collection, and the Madrid Collection (Table 1). More online information can be found on existing historic human collections (http://tiny.cc/ Human\_Embryo\_Collections). The human embryo collections shown in Table 1, along with other collections, form part of the Digital Embryology Consortium (http://human-embryolo‐ gy.org), formed to electronically preserve and make available for research and education these irreplaceable historic collections.

Not included in this chapter will be descriptions of the smaller, less described human embryo collections, species comparative embryo collections, or collections that are of nonembryonic material, such as placenta. An example of one of the best and largest historic comparative embryo collections is the embryological collection of the Natural History Museum in Berlin, which includes many other species in the combined collections of Hubrecht, Hill, Dohrn, Bolk, and Kückenthal. An example of a mainly human placenta and early implanted uterus is the Hamilton-Boyd Collection in Cambridge. More recently, there are smaller collections of embryos used mainly for molecular studies, such as the Human Developmental Studies Network (HuDSeN) in Newcastle and London. Note that many anatomy departments hold their own small collections of human material that are not covered here.

A key factor in understanding the developmental morphological changes is the possession of human embryo samples at sequential developmental stages. The following are the major historic collections used in most research and textbook publications that have aided our understanding of human development.


**Table 1.** Comparison among major human embryo collections

### **2.1. Carnegie collection**

imaging techniques on existing collections have allowed many morphological measurements

Here we summarize the historic collections of embryos used in the study of human develop‐ ment, explain the criteria used for developmental staging, show sectioned and reconstructed images from newer three-dimensional (3D) imaging in high resolution, and discuss the future

During the history of human embryology the establishment and study of key human embryo collections has greatly contributed to our current understanding. In this section we briefly summarize the history of some of these collections, such as the Carnegie Collection, the Kyoto Collection, the Blechschmidt Collection, and the Madrid Collection (Table 1). More online information can be found on existing historic human collections (http://tiny.cc/ Human\_Embryo\_Collections). The human embryo collections shown in Table 1, along with other collections, form part of the Digital Embryology Consortium (http://human-embryolo‐ gy.org), formed to electronically preserve and make available for research and education these

Not included in this chapter will be descriptions of the smaller, less described human embryo collections, species comparative embryo collections, or collections that are of nonembryonic material, such as placenta. An example of one of the best and largest historic comparative embryo collections is the embryological collection of the Natural History Museum in Berlin, which includes many other species in the combined collections of Hubrecht, Hill, Dohrn, Bolk, and Kückenthal. An example of a mainly human placenta and early implanted uterus is the Hamilton-Boyd Collection in Cambridge. More recently, there are smaller collections of embryos used mainly for molecular studies, such as the Human Developmental Studies Network (HuDSeN) in Newcastle and London. Note that many anatomy departments hold

A key factor in understanding the developmental morphological changes is the possession of human embryo samples at sequential developmental stages. The following are the major historic collections used in most research and textbook publications that have aided our

> About 10,000 Human fixed specimens and histology

> > histology

About 120 Human histology 1950s

1887

1961

**Collection Place Number Characteristics Establishment**

Madrid Madrid, Spain 100+ Human histology 1935

Kyoto Kyoto, Japan About 44,000 Human fixed specimens and

their own small collections of human material that are not covered here.

of these embryos using these novel imaging techniques.

directions for the analyses of the human embryo.

**2. Human embryo collections**

98 New Discoveries in Embryology

irreplaceable historic collections.

understanding of human development.

Germany

**Table 1.** Comparison among major human embryo collections

Carnegie Washington DC, USA

Blechschmidt Göttingen,

Franklin P. Mall (1862–1917) began his human embryo at Johns Hopkins University in the early 1900s; these formed the beginnings of the Carnegie collection. He and Franz Keibel (1861–1929) used these embryos in their textbook *Manual of Human Embryology* [1, 2] and also in the Carnegie Institution of Washington Series *Contributions to Embryology* beginning in 1915. Organizing some of these human embryos form the first 8 weeks into a developmental sequence formed the basis of their "23 Carnegie Stages" staging criteria (see Figure 7), described in detail later in this chapter. The same staging criteria have been subsequently applied in the organizing of the other major human embryo collections. These stages will be described in detail from the Kyoto Collection later in this chapter. Reconstructions from histological sections of the collection embryos were the basis of the larger Carnegie models (Figure 1) and this technique has also been used in the development of other collection models, as in the Blechschmidt Collection.

Franklin P. Mall received his medical degree at the University of Michigan in 1883. He traveled to Germany to receive a clinical training, where he met the German embryologist Wilhelm His (1831–1904). This initiated his interest in studying human embryology, and he began collecting human embryos in 1887. His collection had reached several hundreds of specimens by the time he returned to the Anatomy department of the Johns Hopkins School of Medicine in Baltimore, Maryland. He received a Carnegie research grant in 1914 and became the first director of the Department of Embryology at the Carnegie Institution of Washington, in Baltimore, MD. The embryo collection grew at a rate of about 400 specimens a year, donated by clinicians and researchers, and the number of samples reached over 8,000 by the early 1940s. Researchers at the institute then began the complex task of organizing these embryos into a developmental sequence. Note that size alone was a difficult criterion due to the variable effects of fixation shrinkage. The solution was a "staging" system, developed by Mall, based instead on devel‐ opmental ape embryo morphological features. Internal features were identified histologically from embryos that were serially sectioned, and also formed the basis of hundreds of 3D models and 700 wax-based reconstructions.

During Mall's era, several department members became renowned scientists. George L. Streeter (1873–1948) and Franz J. Keibel were also both former students of the important German embryologist Wilhelm His; Osborne O. Heard worked as an embryo modeler; and James D. Didusch as a scientific illustrator. Mall documented his research in a series of papers compiled in the *Contributions to Embryology* of the Carnegie Institution of Washington, published from 1915 to 1966. These articles even today are considered the core findings for studying human embryology. Mall unexpectedly died in 1917 and was replaced as director by Streeter. Streeter was then the first to define the 23 Carnegie Stages currently used to classify the developmental stages of the human embryo (see Table 2). The collection continued to grow by hundreds of specimens every year and included rare, very young normal specimens. At the time, induced abortions were illegal in the United States and miscarriages usually resulted from embryo abnormalities.

Streeter retired in 1940 and George W. Corner (1889–1981]) became the third departmental director. Corner was a former Johns Hopkins researcher who studied the menstrual cycle and identified the ovarian hormone progesterone. During his direction until 1956, many advances in human reproductive physiology were made and embryology research continued but came to an end with the succeeding director. Relocation of the collection began in 1973 to the University of California at Davis Medical School and was completed in 1976. Ronan O'Rahilly was the new director of the collection for the next 15 years, publishing many studies, often with Fabiola Müller, on human embryonic development. At the retirement of the director in 1991 the collection was relocated again to its current location at the Walter Reed Army Medical Center in Washington, D.C., forming part of the Human Developmental Anatomy Center 20 historic embryology collections and remains available for researcher study. In 2014, prelimi‐ nary work began with the current curator on establishing a partnership with the Digital Embryology Consortium to eventually digitize, preserve, and make more widely available this collection. Further details of the embryo collection can be found in earlier publications [3, 4] as well as on the web (http://tiny.cc/HDAC\_Collections), see also (http://tiny.cc/Carne‐ gie\_Collection).

**Figure 1.** Carnegie models located at the Carnegie Collection. (Embryos shown in the bottom left-hand corner were laminated from individual layers and then painted.)

### **2.2. Harvard collection**

identified the ovarian hormone progesterone. During his direction until 1956, many advances in human reproductive physiology were made and embryology research continued but came to an end with the succeeding director. Relocation of the collection began in 1973 to the University of California at Davis Medical School and was completed in 1976. Ronan O'Rahilly was the new director of the collection for the next 15 years, publishing many studies, often with Fabiola Müller, on human embryonic development. At the retirement of the director in 1991 the collection was relocated again to its current location at the Walter Reed Army Medical Center in Washington, D.C., forming part of the Human Developmental Anatomy Center 20 historic embryology collections and remains available for researcher study. In 2014, prelimi‐ nary work began with the current curator on establishing a partnership with the Digital Embryology Consortium to eventually digitize, preserve, and make more widely available this collection. Further details of the embryo collection can be found in earlier publications [3, 4] as well as on the web (http://tiny.cc/HDAC\_Collections), see also (http://tiny.cc/Carne‐

**Figure 1.** Carnegie models located at the Carnegie Collection. (Embryos shown in the bottom left-hand corner were

laminated from individual layers and then painted.)

gie\_Collection).

100 New Discoveries in Embryology

Originally collected by Charles Minot (1852–1914), sometimes referred to as the Minot Collection, it now forms part of the larger Carnegie Collection. By 1905, the collection consisted of 937 histologically sectioned embryos from human and other species (Figure 2).

**Figure 2.** Harvard Collection histology slide No. 839 E, showing 10 micron serial sections from human embryo (No. 318) 13.6 mm in length.

### **2.3. Blechschmidt collection (University of Göttingen, Germany)**

The Blechschmidt Collection is located in the Department of Anatomy and Embryology, Center of Anatomy, University of Göttingen. The University of Göttingen was founded in 1737, and has a long history in research that includes producing 45 Nobel Prize winners.

The human embryo collection is named after Erich Blechschmidt (1904–1992), who directed the Anatomical Institute from 1942 until 1973, and consists of two parts: firstly, a large histology collection of serial sections and, secondly, a model collection based upon these sections.

The histology collection is made up of about 120 human embryos that have been cut in a range of anatomical planes into some 200,000 serial sections. In 1972, some of the embryo serial section sets were temporarily incorporated into the Carnegie Collection and assigned Carnegie Nos. 10315 to 10434. These embryos have since been returned to their original home at the University of Göttingen.

The model collection (Figure 3) "Human embryologische Dokumentations sammlung Blechschmidt" forms a permanent exhibition housed at the Centre of Anatomy and consists of 64 large models, generated from 1946 to 1979. The models are available for viewing upon request and are arranged in perspex cases that allow each model to be observed from all directions. The models range from selected parts or systems of a specific embryo to whole embryos in surface view. In addition, parts of the embryos have been selectively removed or "windows" generated to observe internal system structures including: circulatory, respiratory, gastrointestinal, neural, and the musculoskeletal system.

The modeling method from the histological material used a technique based upon Blechsch‐ midt's own method, described below. Each model illustrates whole embryo surfaces, some organic systems (including a circulatory organ, respiratory organs, a digestive organ, central nerve, and the skeletal system) in precision, in addition to the right-side out.

The embryo collection has probably the largest number of excellently preserved specimens of the latter half of the embryonic period (covering weeks 5–8 post conception). Detailed documentation on individual specimens of the collection is sparse and some of the specimens are also depicted as color drawings in Blechschmidt [5]. The high quality and standard of the histology material was achieved by a combination of a "state-of-the-art" embryo collection gynecological practice (mechanical curettage or hysterectomy) from operations including termination of pregnancy and development of a special fixation procedure. As a result, the quality of paraffin histological sections mounted on large glass microscope slides is unsur‐ passed and reveals valuable morphological detail of early organ development in the human embryo.

**Figure 3.** The Blechschmidt models and histology slides (photo by Saki Ueno).

Like many historic collections, even with optimal storage conditions, the slide histology has gradually deteriorated with evaporation of cover glass glue and bleaching of histological stains. Secondly, the large glass microscope slides are delicate and easily damaged during use. Both these issues highlight the pressing need for generating a "digital copy" of these historic collections.

"windows" generated to observe internal system structures including: circulatory, respiratory,

The modeling method from the histological material used a technique based upon Blechsch‐ midt's own method, described below. Each model illustrates whole embryo surfaces, some organic systems (including a circulatory organ, respiratory organs, a digestive organ, central

The embryo collection has probably the largest number of excellently preserved specimens of the latter half of the embryonic period (covering weeks 5–8 post conception). Detailed documentation on individual specimens of the collection is sparse and some of the specimens are also depicted as color drawings in Blechschmidt [5]. The high quality and standard of the histology material was achieved by a combination of a "state-of-the-art" embryo collection gynecological practice (mechanical curettage or hysterectomy) from operations including termination of pregnancy and development of a special fixation procedure. As a result, the quality of paraffin histological sections mounted on large glass microscope slides is unsur‐ passed and reveals valuable morphological detail of early organ development in the human

nerve, and the skeletal system) in precision, in addition to the right-side out.

gastrointestinal, neural, and the musculoskeletal system.

**Figure 3.** The Blechschmidt models and histology slides (photo by Saki Ueno).

embryo.

102 New Discoveries in Embryology

Photomicrographs of individual histological sections from several specimens were included in Blechschmidt's embryology textbook [5]. At that time, the only way to preserve for posterity morphological information contained in these specimens consisted in building large-scale polymer plastic reconstruction models. These models were made from camera-lucida draw‐ ings at an intermediate magnification of regularly spaced histological sections [6]. Using the same series of serial sections several times over, Blechschmidt made reconstructions of the surface anatomy and the morphology of several organ systems of the same embryo, thereby enabling direct comparison of topographical characteristics and their dynamic changes during development, even though the cellular detail detectable at high magnification remained unexplored with this method. Currently, the way to preserve the collection in its current condition lies with the scanning and digital preservation of the histological material with the Digital Embryology Consortium.

**Figure 4.** The Orts-Llorca Madrid Collection. Slides of serially sectioned embryos are stored in individual box sets. (Photo by Mark Hill)

### **2.4. Madrid institute of embryology human embryo collection**

The human embryo histology collection was started in 1935 by the Spanish embryologist Francisco Orts-Llorca (1905–1993) and is located at the Embryology Institute of Complutense University of Madrid [7]. The collection consists of histological serial sections of more than 100 human embryos in thousands of serial sections covering the embryonic and fetal periods (Figure 4). The collection includes both normal and abnormal embryos. The sectioning is in a number of different anatomical planes and includes both normal and abnormal embryonic material. The collection has unfortunately suffered from the rigors of time, handling by many researchers, and fading of histological stains. The collection though still contains many very useful and unexplored embryos of a broad range of stages of development and the current head of department Professor José F. Rodríguez-Vázquez is determined to return this collec‐ tion to a better condition and preserve this valuable research collection.

### **2.5. Hinrichsen collection (Bochum specimens)**

Klaus V. Hinrichsen was a pupil of Blechschmidt and had the chair of Anatomy and Embry‐ ology at the Ruhr University Bochum in 1970. Many excellent specimens were collected by Hinrichsen's team between 1969 and 1994 and are now housed in the Department of Anatomy and Molecular Embryology at the Ruhr-Universität Bochum, Germany. The total number of the Hinrichsen Collection reached 70, and details of many of these specimens were published in Hinrichsen's textbook on human embryology [8] and in many original publications [9]. The reconstructions have not been attempted from these specimens and many specimens have likewise remained unexplored, to date.

### **2.6. Kyoto collection**

Hideo Nishimura began this collection in 1961 and currently has over 44,000 human embryo specimens. It was further developed and managed by Kohei Shiota for a long period and is currently managed by Shigehito Yamada and all professors in the Department of Anatomy at Kyoto University School of Medicine.

Under the Maternity Protection Law of Japan, induced abortions were legal and in a great majority of cases pregnancies were terminated for social reasons during the first trimester. These provided Nishimura the beginning of the Kyoto collection. In 1975, he formed the Congenital Anomaly Research Center and the collection had now reached over 36,000 specimens. Currently, this collection is the largest in the world with over 45,000 specimens (Figure 5) and provides a key resource for international embryology researchers.

An important characteristic of the collection is inclusion of both normal and many abnormal embryos with severe malformations [10], including holoprosencephaly. Holoprosencephaly (HPE) is a rare newborn anomaly (1/10,000-20,000) occurring more frequently (1/250 or more) in the embryo, being the most common structural malformation of the human embryonic forebrain due to abnormal midline cleavage of the prosencephalon into cerebral hemispheres. This in turn leads to the characteristically abnormal facial development. [11]. Note that the estimation of embryonic frequency may be lower than the actual prevalence, as milder forms of holoprosencephaly also exist, but are more difficult to diagnose [12, 13].

Another unique feature of the Kyoto Collection is the associated maternal epidemiological data and detailed clinical information on the pregnancies that were collected with each specimen. The epidemiological data has been used for statistical analysis to determine potential causative links between maternal factors and congenital anomalies [14].

**2.4. Madrid institute of embryology human embryo collection**

tion to a better condition and preserve this valuable research collection.

**2.5. Hinrichsen collection (Bochum specimens)**

likewise remained unexplored, to date.

Kyoto University School of Medicine.

**2.6. Kyoto collection**

104 New Discoveries in Embryology

The human embryo histology collection was started in 1935 by the Spanish embryologist Francisco Orts-Llorca (1905–1993) and is located at the Embryology Institute of Complutense University of Madrid [7]. The collection consists of histological serial sections of more than 100 human embryos in thousands of serial sections covering the embryonic and fetal periods (Figure 4). The collection includes both normal and abnormal embryos. The sectioning is in a number of different anatomical planes and includes both normal and abnormal embryonic material. The collection has unfortunately suffered from the rigors of time, handling by many researchers, and fading of histological stains. The collection though still contains many very useful and unexplored embryos of a broad range of stages of development and the current head of department Professor José F. Rodríguez-Vázquez is determined to return this collec‐

Klaus V. Hinrichsen was a pupil of Blechschmidt and had the chair of Anatomy and Embry‐ ology at the Ruhr University Bochum in 1970. Many excellent specimens were collected by Hinrichsen's team between 1969 and 1994 and are now housed in the Department of Anatomy and Molecular Embryology at the Ruhr-Universität Bochum, Germany. The total number of the Hinrichsen Collection reached 70, and details of many of these specimens were published in Hinrichsen's textbook on human embryology [8] and in many original publications [9]. The reconstructions have not been attempted from these specimens and many specimens have

Hideo Nishimura began this collection in 1961 and currently has over 44,000 human embryo specimens. It was further developed and managed by Kohei Shiota for a long period and is currently managed by Shigehito Yamada and all professors in the Department of Anatomy at

Under the Maternity Protection Law of Japan, induced abortions were legal and in a great majority of cases pregnancies were terminated for social reasons during the first trimester. These provided Nishimura the beginning of the Kyoto collection. In 1975, he formed the Congenital Anomaly Research Center and the collection had now reached over 36,000 specimens. Currently, this collection is the largest in the world with over 45,000 specimens

An important characteristic of the collection is inclusion of both normal and many abnormal embryos with severe malformations [10], including holoprosencephaly. Holoprosencephaly (HPE) is a rare newborn anomaly (1/10,000-20,000) occurring more frequently (1/250 or more) in the embryo, being the most common structural malformation of the human embryonic forebrain due to abnormal midline cleavage of the prosencephalon into cerebral hemispheres. This in turn leads to the characteristically abnormal facial development. [11]. Note that the estimation of embryonic frequency may be lower than the actual prevalence, as milder forms

(Figure 5) and provides a key resource for international embryology researchers.

of holoprosencephaly also exist, but are more difficult to diagnose [12, 13].

The collection has more recently been analyzed using several new advanced imaging tech‐ nologies that allow 3D embryo imaging and subsequent generation of digital models. Firstly, magnetic resonance microscopy (MRM, see 4.1 in this chapter) of embryos has been carried out [15-18] and analyzed morphologically using 3D reconstruction [19-21]. Secondly, episcopic fluorescence image capture (EFIC) and phase-contrast X-ray computed tomography (pCT) techniques have also been applied to these embryos (18, 22, see 4.2 and 4.3 in this chapter). The current curator, Shigehito Yamada, has now commenced the lengthy process of digitizing all histological sections within this collection and is also a contributing partner in the new digital consortium. The Kyoto Collection is currently one of the largest and best catalogued human embryo collections, containing approximately equal numbers of both normal and abnormal specimens. The collection is also divided into whole wet specimens (see sub-heading 4.4 OPT) as well as about 1,000 serially, histological sectioned embryos (see 5.3, computer reconstruc‐ tions). More recently, the current curator has digitized and made available online sections from some of the normal embryos in the collection (http://atlas.cac.med.kyoto-u.ac.jp).

**Figure 5.** Kyoto Collection of human embryos. (Image shows embryo storage, fixed wet whole embryos, histological collection, and digitization process.)

### **2.7. Hubrecht collection**

Ambrosius A.W. Hubrecht (1853–1915) was a Dutch embryologist who held a chair in comparative embryology at the University of Utrecht from 1910 and founded the "Institut Inteniational d'Embryologie" in 1911. This huge collection of comparative embryonic material from 600 vertebrate species consists of 3,000 wet specimens and 80,000 histological sections from many species including human [23]. There is also a significant collection of photographic material and documentation available. This collection along with the Hill Collection and other German collections forms the Embryological Collection at the Museum für Naturkunde in Berlin and is currently curated by Peter Giere (Figure 6). The collection is made available for researchers upon request. (http://tiny.cc/MfN\_Berlin\_Embryo)

**Figure 6.** The Embryology Collection photomicroscopy setup at the Museum für Naturkunde. With permission, collec‐ tion slides can be photographed and used for research purposes. (Photo by Mark Hill)

### **2.8. HUDSEN collection**

The Human Developmental Studies Network (HuDSeN) atlas is based on 12 optical projection tomography (OPT) models covering the range of Carnegie stage 12–23 [24]. The Human Developmental Biology Resource (http://www.hdbr.org/) was established in 1999 in line with the ethical guidelines laid out in the Polkinghorne Report. There are also histological sections (hematoxylin and eosin stained) from human embryos covering these stages of development.

### **3. Human embryonic development**

Classification into developmental stages is necessary to accurately describe prenatal growth. Embryonic staging of animals was introduced at the end of the 19th century [25], and was first applied to human embryology by Mall [26]. At first, human embryos were classified based on their length like "3-mm stage," but this approach was quickly obsolete because there are individual variations between each embryo. Subsequently, Streeter developed a 23-stage developmental scheme of human embryos in the 1940s called developmental "Horizons." Finally, stages 1–9 were established by O'Rahilly [1973], stage 10 was summarized by Heuser and Corner in 1957 from Streeter's note [27], and stages 11–23 were described in detail by Streeter [28–31].

### **3.1. Carnegie stages**

**2.7. Hubrecht collection**

106 New Discoveries in Embryology

Ambrosius A.W. Hubrecht (1853–1915) was a Dutch embryologist who held a chair in comparative embryology at the University of Utrecht from 1910 and founded the "Institut Inteniational d'Embryologie" in 1911. This huge collection of comparative embryonic material from 600 vertebrate species consists of 3,000 wet specimens and 80,000 histological sections from many species including human [23]. There is also a significant collection of photographic material and documentation available. This collection along with the Hill Collection and other German collections forms the Embryological Collection at the Museum für Naturkunde in Berlin and is currently curated by Peter Giere (Figure 6). The collection is made available for

**Figure 6.** The Embryology Collection photomicroscopy setup at the Museum für Naturkunde. With permission, collec‐

The Human Developmental Studies Network (HuDSeN) atlas is based on 12 optical projection tomography (OPT) models covering the range of Carnegie stage 12–23 [24]. The Human Developmental Biology Resource (http://www.hdbr.org/) was established in 1999 in line with the ethical guidelines laid out in the Polkinghorne Report. There are also histological sections (hematoxylin and eosin stained) from human embryos covering these stages of development.

tion slides can be photographed and used for research purposes. (Photo by Mark Hill)

**2.8. HUDSEN collection**

researchers upon request. (http://tiny.cc/MfN\_Berlin\_Embryo)

The Carnegie stage is commonly known as a staging scheme which remains widely used today. Table 2 shows the relationship between embryonic ages from various researchers and the equivalent Carnegie stages proposed by O'Rahilly and Müller [32]. It is important to note that Streeter's human series included pathological specimens obtained from spontaneous abortion or ectopic implantation.


**Table 2.** Embryonic age (days) based on developmental stages (CS) of human embryos, according to various authors. Streeter [28-31], Nishimura [33, 34], Jirásek [35], and O'Rahilly and Müller [32] show the approximate ovulation age (days); O'Rahilly and Müller [36] show embryonic ages calculated from the greater length of embryo and ultrasound findings

### **3.2. Image and summary of each Carnegie stage (Figure 7)**

Carnegie stage 1: Zygote

1 day after fertilization, cell size 120–150 µm in diameter.

At fertilization, the oocyte completes meiosis II, forming the female pronucleus. The sperma‐ tozoa nucleus in the oocyte cytoplasm decompresses, forming the male pronuclei. These two pronuclei fuse to form the first diploid cell, the zygote. The first mitosis occurs during the 24 h after zygote formation. The term "conceptus" is now used to describe all the cellular products of the zygote.

Carnegie stage 2: Morula.

1.5–3 days after fertilization, conceptus 0.1–0.2 mm in diameter.

The zygote forms two blastomeres. Mitosis of these blastomeres forms a solid ball of 16 cells, then 32 cells, still enclosed by the zona pellucida. This cleavage stage divides the large zygote cytoplasm into sequentially smaller cells. The term "morula" means berry, referring to the appearance of the solid ball of cells.

Carnegie stage 3: Free blastocyst

4 days after fertilization, conceptus 0.1–0.2 mm in diameter.

Cell division continues after the 32 cell stage occurring more rapidly at the surface and slower in the center cells. This and directional fluid transfer leads to a cavity, the blastocoel, in the conceptus. The surface cells form an outer squamous trophoblast layer linked by both tight and gap junctions. The larger inner cells form the inner cell mass or embryoblast.

Carnegie stage 4: Attaching blastocyst

**3.2. Image and summary of each Carnegie stage (Figure 7)**

1 day after fertilization, cell size 120–150 µm in diameter.

1.5–3 days after fertilization, conceptus 0.1–0.2 mm in diameter.

**Figure 7.** Examples of the Carnegie Collection embryos arranged into the classic Carnegie stages.

Cell division continues after the 32 cell stage occurring more rapidly at the surface and slower in the center cells. This and directional fluid transfer leads to a cavity, the blastocoel, in the

4 days after fertilization, conceptus 0.1–0.2 mm in diameter.

At fertilization, the oocyte completes meiosis II, forming the female pronucleus. The sperma‐ tozoa nucleus in the oocyte cytoplasm decompresses, forming the male pronuclei. These two pronuclei fuse to form the first diploid cell, the zygote. The first mitosis occurs during the 24 h after zygote formation. The term "conceptus" is now used to describe all the cellular products

The zygote forms two blastomeres. Mitosis of these blastomeres forms a solid ball of 16 cells, then 32 cells, still enclosed by the zona pellucida. This cleavage stage divides the large zygote cytoplasm into sequentially smaller cells. The term "morula" means berry, referring to the

Carnegie stage 1: Zygote

108 New Discoveries in Embryology

Carnegie stage 2: Morula.

appearance of the solid ball of cells.

Carnegie stage 3: Free blastocyst

of the zygote.

5–6 days after fertilization, conceptus 0.1–0.2 mm in diameter.

The blastocyst hatches from the zone pellucida, still floating in uterine secretions of the secretory phase of the menstrual cycle. The surface trophoblast cells can now initially adhere to the endometrial epithelium at the site of implantation. The trophoblast cells proliferate and differentiate into two layers. The outer cells fusing to form syncytiotrophoblasts, the inner close remain as single cells, cytotrophoblasts.

Carnegie stage 5: Implanted but previllous

7–12 days after fertilization, conceptus 0.1–0.2 mm in diameter.

This stage was originally divided into three (a, b, and c) substages based on trophoblast differentiation status before outgrowth (villi) appears. 5a is the initial solid trophoblast cell layer; 5b, lacunar trophoblast with the appearance of spaces (lacunae) within the trophoblast layer; 5c, maternal blood-filled lacuna as capillaries and uterine glands are opened into the trophoblast spaces.

Carnegie stage 6: Chorionic villi and primitive streak

13 days after fertilization, conceptus 0.2 mm in size.

Trophoblast cells extend into the maternal uterine stroma (decidua) forming chorionic villi. The extra-embryonic mesoderm arises, lining the conceptus cavity and forming the chorionic cavity. Three separate cavities or extra-embryonic coeloms form outside the embryonic disc: the chorionic, amniotic, and yolk sac cavities. Toward the end of this stage, the primitive streak appears on the embryonic disc; this is the site of gastrulation.

Carnegie stage 7: Notochordal process

16 days after fertilization, embryonic disc 0.4 mm in length

The embryonic disc establishes axes and has an initial central primitive node (Hensen's node, primitive pit) with the primitive streak extending caudally to the disc edge where the con‐ necting stalk will later form. Gastrulation occurs here forming endoderm and mesoderm that spread laterally and rostrally from the primitive streak. Above the primitive node, cranially, the notochordal process develops in the mesodermal layer. The length of this process increases from 0.03 to about 0.3 mm. The embryonic disc increases in size and the amniotic cavity enlarges over the yolk sac.

Carnegie stage 8: Primitive pit, neuenteric canal

18 days after fertilization, embryonic disc 1.0 mm in length

The embryonic disc is pyriform, tapering caudally, and now has cranio-caudal axis, measured from this stage onward by crown-rump length (CRL). The stage shows three key features: the primitive pit, the notochordal canal, and the neurenteric canal. The notochordal canal is marked by the cavity extending from the primitive pit into the notochordal process. The floor of the canal is lost to form a transient passage, neurenteric canal, between the amniotic cavity and the yolk sac. The notochord process will differentiate into the notochord or axial meso‐ derm. The remainder of new mesoderm layer has not yet segmented and is called the preso‐ mitic stage.

Carnegie stage 9: 1–3 pairs of somites

20 days after fertilization, embryo 1.5 mm in crown-rump length (CRL)

The mesoderm either side of the notochord now segments into paired somites. Segmentation of paraxial mesoderm only occurs at the level of the trunk, not the head, and proceeds in a cranial-caudal direction. Note that the sequential appearance of somite pairs can also be used as a criterion to stage the embryo. The embryonic disc resembles a shoe-sole, with the broad neural plate in the ectoderm layer positioned in the cranial region. The mid-line neural plate begins to fold forming a neural groove.

Carnegie stage 10: Neural folds begin to fuse, 4–12 pairs of somites

22 days after fertilization, embryo 1.8 mm in CRL

Somitogenesis continues increasing from 4 to 12 somite pairs. The neural groove continues to fold bringing the neural plate edges together to commence fusing. This fusion occurs in both cranial and caudal directions and at several sites. In the head region, the optic sulcus and first pharyngeal (branchial) arch appear. In the underlying trunk region mesoderm the cardiac tube appears.

Carnegie stage 11: Anterior neuropore closes

24 days after fertilization, 2.5–3 mm in CRL

Somitogenesis continues increasing from 13 to 20 somite pairs. The neural groove has formed an open-ended neural tube, and the upper head end (anterior, cranial or rostral) opening (neuropore) commences to close. Optic evagination is produced at the optic sulcus and the optic ventricle is continuous with that of the forebrain. The cardiac tube has formed a loop, with a sinus venosus region appearing. The second pharyngeal arch is visible. A ventral indentation (stomodeum) is present at the level of the first arch. The floor of the stomodeum forms the oral membrane (buccopharyngeal) that commences to degenerate. Dorsally at the level of the second arch, paired otic placodes fold inward to form the otic vesicles.

Carnegie stage 12: Posterior neuropore closes

28 days after fertilization, 4 mm in CRL

Somitogenesis continues with 21–29 somite pairs. The posterior (caudal) neuropore is starting to close or is closed. Three of the pharyngeal arches are now clearly visible. The upper limb buds appear, initially as lateral swellings at the level of the heart. Internally, the heart inter‐ ventricular septum has begun to form, the liver is present and the lung buds appear.

Carnegie stage 13: Limb buds, optic vesicle

32 days after fertilization, 5 mm in CRL

primitive pit, the notochordal canal, and the neurenteric canal. The notochordal canal is marked by the cavity extending from the primitive pit into the notochordal process. The floor of the canal is lost to form a transient passage, neurenteric canal, between the amniotic cavity and the yolk sac. The notochord process will differentiate into the notochord or axial meso‐ derm. The remainder of new mesoderm layer has not yet segmented and is called the preso‐

The mesoderm either side of the notochord now segments into paired somites. Segmentation of paraxial mesoderm only occurs at the level of the trunk, not the head, and proceeds in a cranial-caudal direction. Note that the sequential appearance of somite pairs can also be used as a criterion to stage the embryo. The embryonic disc resembles a shoe-sole, with the broad neural plate in the ectoderm layer positioned in the cranial region. The mid-line neural plate

Somitogenesis continues increasing from 4 to 12 somite pairs. The neural groove continues to fold bringing the neural plate edges together to commence fusing. This fusion occurs in both cranial and caudal directions and at several sites. In the head region, the optic sulcus and first pharyngeal (branchial) arch appear. In the underlying trunk region mesoderm the cardiac tube

Somitogenesis continues increasing from 13 to 20 somite pairs. The neural groove has formed an open-ended neural tube, and the upper head end (anterior, cranial or rostral) opening (neuropore) commences to close. Optic evagination is produced at the optic sulcus and the optic ventricle is continuous with that of the forebrain. The cardiac tube has formed a loop, with a sinus venosus region appearing. The second pharyngeal arch is visible. A ventral indentation (stomodeum) is present at the level of the first arch. The floor of the stomodeum forms the oral membrane (buccopharyngeal) that commences to degenerate. Dorsally at the

Somitogenesis continues with 21–29 somite pairs. The posterior (caudal) neuropore is starting to close or is closed. Three of the pharyngeal arches are now clearly visible. The upper limb buds appear, initially as lateral swellings at the level of the heart. Internally, the heart inter‐

level of the second arch, paired otic placodes fold inward to form the otic vesicles.

ventricular septum has begun to form, the liver is present and the lung buds appear.

20 days after fertilization, embryo 1.5 mm in crown-rump length (CRL)

Carnegie stage 10: Neural folds begin to fuse, 4–12 pairs of somites

mitic stage.

110 New Discoveries in Embryology

appears.

Carnegie stage 9: 1–3 pairs of somites

begins to fold forming a neural groove.

22 days after fertilization, embryo 1.8 mm in CRL

Carnegie stage 11: Anterior neuropore closes 24 days after fertilization, 2.5–3 mm in CRL

Carnegie stage 12: Posterior neuropore closes

28 days after fertilization, 4 mm in CRL

Carnegie stage 13: Limb buds, optic vesicle

Somitogenesis continues with more than 30 somite pairs. The numbers of somite pairs are now difficult to determine as staging criteria. Both upper and lower limb buds are visible. The optic vesicle is present, and the lens placode begins to differentiate.

Carnegie stage 14: Lens pit and optic cup

34 days after fertilization, 6 mm in CRL

The upper limb buds elongate and become tapering. Upper limb bud features appear about 2 days before the lower limb. The embryo cephalic and cervical flexures are prominent. Within the head, the future cerebral hemispheres and cerebellar plates are visible. On the head surface, the lens pit invaginates into the optic cup but is not yet closed and the otic vesicle endolymphat‐ ic appendage emerges. Within the trunk, pancreatic buds (ventral and dorsal) are present, the mesonephric duct forms the ureteric bud and at its tip is the metanephrogenic blastemal cap.

Carnegie stage 15: Lens vesicles, nasal pit and hand plates

36 days after fertilization, 8 mm in CRL

The upper limb hand plates are now visible. Lens vesicles are closed and covered by the surface ectoderm. The nasal plate invaginates to form a nasal pit. The auricular hillocks on pharyngeal arch 1 and 2 appear. Within the heart, the foramen secundum is present. Lung buds are now branched into lobar buds and the primary urogenital sinus is formed.

Carnegie stage 16: Nasal pit faces ventrally, retinal pigment, foot plate

38 days after fertilization, 10 mm in CRL

The upper limb hand plates are distinct and the foot plate has begun to form. On the trunk between the upper and lower limbs, a distinct mesonephric ridge is visible. On the head, the nasal pits deepen and face ventrally and the eye retinal pigment is visible externally. The nasolacrimal groove begins to form and lies between the frontal and maxillary processes.

Carnegie stage 17: Head relatively larger, nasofrontal groove, finger rays

40 days after fertilization, 11 mm in CRL

The upper limb hand plates have digital rays, and the foot has acquired a rounded digital plate. The head is now larger than previously and the trunk has begun to straighten. On the first and second pharyngeal arches the auricular hillocks are present and the nasolacrimal grooves are distinct.

Carnegie stage 18: Elbows, toe rays, eyelid folds

42 days after fertilization, 13 mm in CRL

The upper limb elbows are discernible and in the hand plates interdigital notches appear. Toe rays are observed in the foot plate. The trunk shape is more cuboidal and both cervical and lumbar flexures are denoted. On the head, eyelid folds appear and auricular hillocks are fusing to form specific parts of the external ear. Ossification commences in some skeletal structures.

Carnegie stage 19: Trunk elongation and straightening

44 days after fertilization, 16 mm in CRL

The upper and lower limbs are parallel, with preaxial borders cranially and postaxial borders caudally. On the head, eyes are now positioned in the front of the face, due to the growth of the brain, and the external ears have their definitive shape. The trunk continues to elongate and straighten. Within the trunk, the intestines have developed and herniated in the umbilical region.

Carnegie stage 20: Longer upper limb bent at elbow

46 days after fertilization, 19 mm in CRL

The upper limbs have increased in length and flexed at the elbows and hand joints. Fingers are curving slightly over the chest. The angle of cervical flexure becomes small, and the direction of the head goes upward. The head has a superficial scalp vascular plexus. The herniated intestines continue to elongate. Embryo spontaneous movements can occur at this stage.

Carnegie stage 21: Fingers are longer, hands approach each other

48 days after fertilization, 21 mm in CRL

The hands are slightly flexed at the wrists and nearly come together over the cardiac promi‐ nence. The head becomes round and the superficial vascular plexus has spread and now surrounds the head. The trunk tail now becomes rudimentary.

Carnegie stage 22: Eyelids and external ear are more developed

50 days after fertilization, 23 mm in CRL

The head vascular plexus is now very distinct. The eyelids have thickened and lie over the eyes. The external ear position is higher on the head and the tragus and antitragus regions are more definite. The trunk tail is almost lost.

Carnegie stage 23: End of embryonic period

52 days after fertilization, 30 mm in CRL

The head is now rounded out and the trunk has elongated to a more mature shape. The limbs have increased in length and the forearm is level or above the level of the shoulder. The head scalp vascular plexus is approaching the vertex of the head. The eyelids and ear auricles become definite. The external genitalia are developed but not sex-differentiated. The trunk tail has now gone.

### **4. Human embryo imaging**

Rapid advances in medical imaging are facilitating the clinical assessment of first-trimester human embryos at increasingly earlier stages. To obtain data on early human development, we have used some micro-imaging modalities such as magnetic resonance microscopy, episcopic fluorescence capture, and phase-contrast X-ray computed tomography. The follow‐ ing sections describe and show the resulting embryo images from each of these imaging techniques.

### **4.1. Magnetic Resonance Microscopy (MRM)**

Carnegie stage 19: Trunk elongation and straightening

Carnegie stage 20: Longer upper limb bent at elbow

Carnegie stage 21: Fingers are longer, hands approach each other

surrounds the head. The trunk tail now becomes rudimentary. Carnegie stage 22: Eyelids and external ear are more developed

The upper and lower limbs are parallel, with preaxial borders cranially and postaxial borders caudally. On the head, eyes are now positioned in the front of the face, due to the growth of the brain, and the external ears have their definitive shape. The trunk continues to elongate and straighten. Within the trunk, the intestines have developed and herniated in the umbilical

The upper limbs have increased in length and flexed at the elbows and hand joints. Fingers are curving slightly over the chest. The angle of cervical flexure becomes small, and the direction of the head goes upward. The head has a superficial scalp vascular plexus. The herniated intestines continue to elongate. Embryo spontaneous movements can occur at this

The hands are slightly flexed at the wrists and nearly come together over the cardiac promi‐ nence. The head becomes round and the superficial vascular plexus has spread and now

The head vascular plexus is now very distinct. The eyelids have thickened and lie over the eyes. The external ear position is higher on the head and the tragus and antitragus regions are

The head is now rounded out and the trunk has elongated to a more mature shape. The limbs have increased in length and the forearm is level or above the level of the shoulder. The head scalp vascular plexus is approaching the vertex of the head. The eyelids and ear auricles become definite. The external genitalia are developed but not sex-differentiated. The trunk tail

Rapid advances in medical imaging are facilitating the clinical assessment of first-trimester human embryos at increasingly earlier stages. To obtain data on early human development,

44 days after fertilization, 16 mm in CRL

46 days after fertilization, 19 mm in CRL

48 days after fertilization, 21 mm in CRL

50 days after fertilization, 23 mm in CRL

more definite. The trunk tail is almost lost. Carnegie stage 23: End of embryonic period

52 days after fertilization, 30 mm in CRL

**4. Human embryo imaging**

has now gone.

region.

112 New Discoveries in Embryology

stage.

Magnetic resonance (MR) imaging is now widely used as a tool for diagnostic medical imaging. In research, when scanning small samples this technique is called magnetic resonance microscopy (MRM). MRM was first applied to studying the human embryo in the 1990s [37, 38], and has now become a very powerful tool for 3D measurement of chemically fixed human embryos [15]. This research technique is still being developed and MRM images in higher resolution have been obtained using human embryos and a range of contrast agents [39]. The images shown in Table 3 were obtained using MRM equipped with a 2.34T magnet [15].

### **4.2. Episcopic Fluorescence Image Capture (EFIC)**

Episcopic fluorescence image capture (EFIC) was devised and developed in the early 2000s [40, 41]. With EFIC imaging, tissue autofluorescence is used to image the whole embryo block face prior to histologically cutting each section. These individual sections can then be viewed or reconstructed into a 3D image [18], Figure 8. This technique has now been applied to staged human embryos from the Kyoto Collection. The first and only human embryo atlas developed from Kyoto embryos using EFIC can be accessed at website in University of Pittsburgh (http:// apps.devbio.pitt.edu/HumanAtlas/; login ID and password are shown in [18]; the atlas also includes MRM images from similar staged embryos.

### **4.3. Phase-Contrast X-ray Computed Tomography (pCT)**

Phase-contrast X-ray computed tomography (pCT) is a relatively newer technique of imaging. In this technique, the X-rays are used as electric waves characterized by amplitude and phase. Conventional X-ray imaging (radiography) is based on absorption-contrast (i.e., amplitude imaging) and represents the mass-density distribution of X-ray inside the sample.

In comparison, pCT uses the phase-shift, occurring when X-rays pass through samples [42]. The phase shift is converted into a change in X-ray intensity that is collected by a currentdetecting device. There are some conversion methods such as interferometry with an X-ray crystal interferometer [42, 43], diffractometry with a perfect analyzer crystal [44-46], a propa‐ gation-based method with a Fresnel pattern [47, 48], and Talbot interferometry with a Talbot grating interferometer [49, 50]. Devices based on this principle have been developed [51, 52], and an image of human embryo at CS 17 obtained using a two-crystal X-ray interferometer (Yoneyama et al., 2011) is featured in Figure 9.

### **4.4. Optical Projection Tomography (OPT)**

Optical projection tomography (OPT) was devised in 2002, using the principle of projection tomography [53, 54]. During the embedding process, the samples are dehydrated and cleared


**Table 3.** Result of MRM scanning using human embryos

with a mixture of benzyl alcohol and benzyl benzoate, allowing the light to pass through the specimen. This technique has also been applied into human embryo [55, 56], and the atlas regarding gene expression in the developing human brain has been established using OPT [24].

**Figure 8.** Comparison between imaging of the same stage embryo using two different techniques of EFIC (left) and MRM (right).

**Figure 9.** Comparison of human embryos (CS 17) imaged by pCT (A,C) and MRM (B,D). Note that Rathke's pouch can be seen in the embryo by pCT (C) but not detected by MRM (D).

### **5. Three-dimensional models and analyses of human embryos**

In the 19th century, human embryo models were made manually based on macroscopic and microscopic observation. Wax plate technique was introduced into embryology in 1887, and the principle was used continuously until the computer era, although the material of the model has been changed from wax to plastics. Computer-assisted reconstruction started at the end of the 20th century. The reconstruction was made from the histological sections at first, followed by reconstruction from 3D image dataset. Recently, as 3D printers become cheaper and widespread, they are being applied in human embryology.

### **5.1. Ziegler models**

with a mixture of benzyl alcohol and benzyl benzoate, allowing the light to pass through the specimen. This technique has also been applied into human embryo [55, 56], and the atlas regarding gene expression in the developing human brain has been established using OPT [24].

**Table 3.** Result of MRM scanning using human embryos

114 New Discoveries in Embryology

**Figure 8.** Comparison between imaging of the same stage embryo using two different techniques of EFIC (left) and

MRM (right).

By the middle of the 19th century, there had already been 2D illustrations of embryos and 3D embryo models were eagerly awaited, due to the difficulty of obtaining embryos, their fragility and size. Louis Auzoux, a French anatomist, made papier-mâché models in his Normandy workshop (Figure 10A). Later Adolf Ziegler started to render hand-shaped models after he returned to the University of Freiburg in 1854, and completed his first model series "The Development of the Frog." His modeling was applied in developmental biology, including human embryos. Adolf Ziegler retired in 1883 and his son Friedrich Ziegler took over the modeling operations. The "Ziegler models," including trout, sea urchin, beetle, frog, chick, and human embryos (Figure 10B) were displayed at the 1893 World's Columbian Exposition in Chicago, and there they attracted much attention.

**Figure 10.** Examples of historic embryo models. Modeling workshop of Louis Auzoux (A), Ziegler human embryo models (B) and Carnegie Laboratory models and Osborne O. Heard (C).

### **5.2. Wax-plate model and its derivatives**

returned to the University of Freiburg in 1854, and completed his first model series "The Development of the Frog." His modeling was applied in developmental biology, including human embryos. Adolf Ziegler retired in 1883 and his son Friedrich Ziegler took over the modeling operations. The "Ziegler models," including trout, sea urchin, beetle, frog, chick, and human embryos (Figure 10B) were displayed at the 1893 World's Columbian Exposition

**Figure 10.** Examples of historic embryo models. Modeling workshop of Louis Auzoux (A), Ziegler human embryo

models (B) and Carnegie Laboratory models and Osborne O. Heard (C).

in Chicago, and there they attracted much attention.

116 New Discoveries in Embryology

In 1865, Wilhelm His Sr. invented the microtome [57], and he applied it to embryology. In 1883, Gustav Born devised the wax plate technique; 3D reconstruction from serial histological sections was made by wax plate [58]. This technique was applied to embryology [59] and later modified in the Carnegie Laboratory in Baltimore [60]. The material of model originally used was mainly wax (Figure 10C), and changed into plastic or its derivatives [61] in the 20th century. These new models were a significant improvement in detail and accuracy over the earlier Ziegler models. The technique was later further developed, with larger scale and detail, by Blechschmidt in his model series (see above, section 2.3].

### **5.3. Computer graphics from serial sections**

Recent advancement in image technology and computer science has made computer-assisted reconstruction of embryonic structures more effectively, and the reconstructed images can be manipulated as desired on the viewing screen. This technique has been applied to human embryos in 1994, using the Madrid Collection [62], and 3D reconstruction of human embryo has also been established using the Kyoto Collection [62]. In the 21st century, the 3D models were colorized and elaborated [63, and see Figure 11]. In combination with the advance of web technique, some attractive web-based human embryo atlases have been constructed using the Kyoto Collection [18], http://apps.devbio.pitt.edu/HumanAtlas/), and the Carnegie Collection has been established and available freely [64], (http://www.ehd.org/virtual-human-embryo/).

### **5.4. 3D printer and scanner**

A 3D printer is a tool for making 3D solid objects from digital data. Stereolithography was a technique developed at the end of the 20th century; in recent years, it has enabled the creation of inexpensive 3D models in engineering, medical and dental fields, as well as the academic area [65] and has been applied to human embryology [66], (see Figure 12).

**Figure 11.** Histological section (left), embryo surface reconstruction (center) and a 3D reconstruction labeling of the gastrointestinal system (right) from a human embryo.

**Figure 12.** Images related with 3D printer and scanner. (A) The brain ventricle of human embryo ranging from CS13 to 23. (B) Solid reconstruction in the Blechschmidt Collection. (C) Drawing derived from B. (D) Image data of B converted by a commercially available 3D scanner (http://cubify.com/en/Products/Sense).

A 3D scanner is a tool for digitizing the surface of an object as data. Several groups are currently investigating its application to human embryology.

### **Acknowledgements**

We would like to thank Ms Chigako Uwabe at the Congenital Anomaly Research Center for technical assistance in handling human embryos; Prof. Katsumi Kose and Dr. Yoshimasa Matsuda at the Institute of Applied Physics at University of Tsukuba for technical help with MR imaging; Dr. Jörg Männer and Prof. Christoph Viebahn at the Department of Anatomy and Embryology, Georg-August-University of Göttingen for their generous cooperation using the Blechschmidt Collection; Prof. Tohoru Takeda at Allied Health Science, Kitasato University and Dr. Akio Yoneyama at Central Research Laboratory, Hitachi Ltd. For pCT scanning; Dr Peter Giere at the Museum für Naturkunde, Berlin for his generous cooperation with access to the Embryological collection. Research was financially supported by JSPS KAKENHI Grant Number #248055, #268044, #24790195, #25461642, 15H05270, and 15K08134 from the Japan Society for the Promotion of Science (JSPS), MEXT KAKENHI Grant Number #24119002 and #26220004 from Grant-in-Aid for Scientific Research on Innovative Areas. Kyoto studies were approved by the Medical Ethics Committee, Kyoto University Graduate School of Medicine (Kyoto, Japan).

### **Author details**

Shigehito Yamada1\*, Mark Hill2 and Tetsuya Takakuwa1


### **References**

**Figure 12.** Images related with 3D printer and scanner. (A) The brain ventricle of human embryo ranging from CS13 to 23. (B) Solid reconstruction in the Blechschmidt Collection. (C) Drawing derived from B. (D) Image data of B converted

A 3D scanner is a tool for digitizing the surface of an object as data. Several groups are currently

We would like to thank Ms Chigako Uwabe at the Congenital Anomaly Research Center for technical assistance in handling human embryos; Prof. Katsumi Kose and Dr. Yoshimasa Matsuda at the Institute of Applied Physics at University of Tsukuba for technical help with MR imaging; Dr. Jörg Männer and Prof. Christoph Viebahn at the Department of Anatomy and Embryology, Georg-August-University of Göttingen for their generous cooperation using the Blechschmidt Collection; Prof. Tohoru Takeda at Allied Health Science, Kitasato University and Dr. Akio Yoneyama at Central Research Laboratory, Hitachi Ltd. For pCT scanning; Dr Peter Giere at the Museum für Naturkunde, Berlin for his generous cooperation with access to the Embryological collection. Research was financially supported by JSPS KAKENHI Grant Number #248055, #268044, #24790195, #25461642, 15H05270, and 15K08134 from the Japan Society for the Promotion of Science (JSPS), MEXT KAKENHI Grant Number #24119002 and

by a commercially available 3D scanner (http://cubify.com/en/Products/Sense).

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**Chapter 6**
