Implantation

All obstetricians should be aware of the basic reproductive bio- logical processes re-
quired for women to successfully achieve pregnancy. Several abnormalities can affect
each of these and lead to infertility or pregnancy loss. In most women, sponta- neous,
cyclical ovulation at 25- to 35-day intervals continues during almost 40 years between
menarche and menopause. Without contraception, there are approximately 400 oppor-
tunities for pregnancy, which may occur with intercourse on any of 1200 days—the day
of ovulation and its two preceding days. This narrow window for fertilization is con-
trolled by tightly regulated production of ovarian steroids. Moreover, these hormones
promote optimal endometrial regeneration after menstruation in preparation for the next
implantation window.

Should fertilization occur, events that begin after initial blastocyst implantation onto the
endometrium and con- tinue through to parturition result from a unique interaction be-
tween fetal trophoblasts and maternal endometrium- decidua. The ability of mother and
fetus to coexist as two

distinct immunological systems results from endocrine, para- crine, and immunological
modification of fetal and maternal tissues in a manner not seen elsewhere. The placenta
mediates a unique fetal–maternal communication system, which cre- ates a hormonal en-
vironment that initially maintains preg- nancy and eventually initiates events leading to
parturition. The following sections address the physiology of the ovarian- endometrial
cycle, implantation, placenta, and fetal mem- branes, as well as specialized endocrine ar-
rangements between fetus and mother.
THE OVARIAN–ENDOMETRIAL CYCLE
Predictable, regular, cyclical, and spontaneous ovulatory menstrual cycles are regulated
by complex interactions of the hypothalamic-pituitary axis, ovaries, and genital tract
(Fig. 5-1). The average cycle duration is approximately 28 days, with a range of 25 to 32
days. The hormonal sequence leading to ovulation directs this cycle. Concurrently, cycli-
cal changes in endometrial histology are faithfully reproduced. Rock and Bartlett (1937)
first suggested that endometrial histological features were sufficiently characteristic to
permit cycle “dating.” In this scheme, the follicular-proliferative phase and the postovu-
latory luteal-secretory phase are cus- tomarily divided into early and late stages.

The Ovarian Cycle

Follicular or Preovulatory Ovarian Phase
The human ovary contains 2 million oocytes at birth, and approximately 400,000 folli-
cles are present at puberty onset (Baker, 1963). The remaining follicles are depleted at a
rate of approximately 1000 follicles per month until age 35, when this rate accelerates
(Faddy, 1992). Only 400 follicles are normally released during female reproductive life.
Therefore, more than 99.9 percent of follicles undergo atresia through a process of cell
death termed apoptosis (Gougeon, 1996; Kaipia, 1997).

Follicular development consists of several stages, which include the gonadotropin-inde-

pendent recruitment of pri- mordial follicles from the resting pool and their growth to the
antral stage. This appears to be controlled by locally produced growth factors. Two
members of the transforming growth factor-β family—growth differentiation factor 9
(GDF9) and bone morphogenetic protein 15 (BMP-15)—regulate granulosa cell prolifer-
ation and differentiation as primary follicles grow (Trombly, 2009; Yan, 2001). They
also stabi- lize and expand the cumulus oocyte complex in the oviduct

(Hreinsson, 2002). These factors are produced by oocytes, sug- gesting that the early
steps in follicular development are, in part, oocyte controlled. As antral follicles develop,
surround- ing stromal cells are recruited, by a yet-to-be-defined mecha- nism, to become
thecal cells.

Although not required for early follicular maturation, follicle-stimulating hormone

(FSH) is required for further development of large antral follicles (Hillier, 2001). During
each ovarian cycle, a group of antral follicles, known as a cohort, begins a phase of
semisynchronous growth based on their maturation state during the FSH rise in the late
luteal phase of the previous cycle. This FSH rise leading to follicle development is called
the selection window of the ovarian cycle (Macklon, 2001). Only the follicles progress-
ing to this stage develop the capacity to produce estrogen.

During the follicular phase, estro- gen levels rise in parallel to growth of a dominant fol-
licle and to the increase in its number of granulosa cells (see Fig. 5-1). These cells are
the exclusive site of FSH receptor expression. The rise of circulating FSH levels during
the late luteal phase of the previous cycle stimulates an increase in FSH receptors and
subsequently, the ability of cytochrome P450 aromatase within granulosa cells to convert
androstene- dione into estradiol. The requirement for thecal cells, which respond to
luteinizing hormone (LH), and granu- losa cells, which respond to FSH, rep- resents the
two-gonadotropin, two-cell hypothesis for estrogen biosynthesis (Short, 1962). As
shown in Figure 5-2, FSH induces aromatase and expansion of the antrum of growing
follicles. The follicle within the cohort that is most responsive to FSH is likely to be the
first to produce estradiol and initiate expression of LH receptors.

After the appearance of LH recep-

tors, the preovulatory granulosa cells
begin to secrete small quantities of
progesterone. The preovulatory pro-
gesterone secretion, although some-
what limited, is believed to exert
positive feedback on the estrogen-
primed pituitary to either cause
or augment LH release. In addi-
tion, during the late follicular phase, LH stimulates thecal cell production of androgens,
particularly androstenedione, which are then transferred to the adjacent follicles where
they are aromatized to estradiol (see Fig. 5-2). During the early follicular phase, granu-
losa cells also produce inhibin B, which can feed back on the pituitary to inhibit FSH re-
lease (Groome, 1996). As the dominant follicle begins to grow, production of estradiol
and the inhibins increases and results in a decline of follicular-phase FSH. This drop in
FSH lev- els is responsible for the failure of other follicles to reach preovulatory status—
the Graafian follicle stage—during any one cycle. Thus, 95 percent of plasma estradiol
produced at this time is secreted by the dominant follicle—the one destined to ovulate.
Concurrently, the contralateral ovary is relatively inactive.

progesterone and prostaglandin production by the cumulus cells, as well as GDF9 and
BMP-15 by the oocyte, activates expression of genes critical to formation of a hyaluro-
nan-rich extracellular matrix by the cumulus complex (Richards, 2007). As seen in Fi-
gure 5-3, during synthesis of this matrix, cumulus cells lose contact with one another and
move outward from the oocyte along the hyaluronan polymer—this process is called ex-
pansion. This results in a 20-fold increase in the complex volume along with an LH-in-
duced remodeling of the ovarian extracellular matrix to allow release of the mature
oocyte and its surrounding cumulus cells through the surface epithelium. Activation of
proteases likely plays a pivotal role in weakening of the follicular basement membrane
and ovulation (Curry, 2006; Ny, 2002).

Luteal or Postovulatory Ovarian Phase

Following ovulation, the corpus luteum develops from the dominant or Graafian follicle
remains in a process referred to as luteinization. The basement membrane separating the
granulosa-lutein and theca-lutein cells breaks down, and by day 2 postovulation, blood
vessels and capillaries invade the granulosa cell layer. The rapid neovascularization of
the once- avascular granulosa may be due to angiogenic factors that include vascular en-
dothelial growth factor (VEGF) and others

Ovulation
The onset of the gonadotropin surge resulting from increas- ing estrogen secretion by
preovulatory follicles is a relatively precise predictor of ovulation. It occurs 34 to 36
hours before ovum release from the follicle (see Fig. 5-1). LH secretion peaks 10 to 12
hours before ovulation and stimulates resump- tion of meiosis in the ovum and release of
the first polar body. Current studies suggest that in response to LH, increased

produced in response to LH by theca-lutein and granulosa- lutein cells (Albrecht, 2003;

Fraser, 2001). During lutein- ization, these cells undergo hypertrophy and increase their
capacity to synthesize hormones.

LH is the primary luteotropic factor responsible for corpus luteum maintenance (Vande
Wiele, 1970). Indeed, LH injec- tions can extend the corpus luteum life span in normal
women by 2 weeks (Segaloff, 1951). In normal cycling women, the corpus luteum is
maintained by low-frequency, high-amplitude LH pulses secreted by gonadotropes in the
anterior pituitary (Filicori, 1986).

The hormone secretion pattern of the corpus luteum differs from that of the follicle (see
Fig. 5-1). The increased capacity of granulosa-lutein cells to produce progesterone is the
result of increased access to considerably more steroidogenic precursors through blood-
borne low-density lipoprotein (LDL)-derived cholesterol as depicted in Figure 5-2 (Carr,
1981a). The impor- tant role for LDL in progesterone biosynthesis is supported by the
observation that women with extremely low LDL choles- terol levels exhibit minimal
progesterone secretion during the luteal phase (Illingworth, 1982). In addition, high-den-
sity lipo- protein (HDL) may contribute to progesterone production in granulosa-lutein
cells (Ragoobir, 2002).

Estrogen levels follow a more complex pattern of secretion. Specifically, just after ovu-
lation, estrogen levels decrease fol- lowed by a secondary rise that reaches a peak pro-
duction of 0.25 mg/day of 17β-estradiol at the midluteal phase. Toward the end of the
luteal phase, there is a secondary decline in estra- diol production.

Ovarian progesterone production peaks at 25 to 50 mg/day during the midluteal phase.

With pregnancy, the corpus luteum continues progesterone production in response to
embryonic human chorionic gonadotropin (hCG), which binds to the same receptor as
LH (see Fig. 5-2).

The human corpus luteum is a transient endocrine organ that, in the absence of preg-
nancy, will rapidly regress 9 to 11 days after ovulation via apoptotic cell death (Vask-
ivuo, 2002). The mechanisms that control luteolysis remain unclear. However, in part, it
results from decreased levels of circulat- ing LH in the late luteal phase and decreased
LH sensitiv- ity of luteal cells (Duncan, 1996; Filicori, 1986). The role of other factors is
less clear, however, prostaglandin F2α (PGF2α) appears to be luteolytic in nonhuman pri-
mates (Auletta, 1987; Wentz, 1973). The endocrine effects, consisting of a dramatic drop
in circulating estradiol and progesterone levels, are critical for follicular development
and ovulation during the next ovarian cycle. In addition, corpus luteum regres- sion and
decline in circulating steroid concentrations signal the endometrium to initiate molecular
events that lead to menstruation.
Estrogen and Progesterone Action
The fluctuating levels of ovarian steroids are the direct cause of the endometrial cycle.
Recent advances in the molecular biology of estrogen and progesterone receptors have
greatly improved understanding of their function. The most biologically potent naturally
occurring estrogen—17β-estradiol—is secreted by granulosa cells of the dominant folli-
cle and luteinized granulosa cells of the corpus luteum (see Fig. 5-2). Estrogen is the es-
sen- tial hormonal signal on which most events in the normal men- strual cycle depend.
Estradiol action is complex and appears to involve two classic nuclear hormone recep-
tors designated estrogen receptor α (ERα) and β (ERβ) (Katzenellenbogen, 2001). These
isoforms are the products of separate genes and can exhibit distinct tissue expression.
Both estradiol-receptor complexes act as transcriptional factors that become associ- ated
with the estrogen response element of specific genes. They share a robust activation by
estradiol. However, differences in their binding affinities to other estrogens and their
cell-specific expression patterns suggest that ERα and ERβ receptors may have both dis-
tinct and overlapping function (Saunders, 2005). Both receptors are expressed in the
uterine endometrium (Bombail, 2008; Lecce, 2001). Estrogens function in many cell
types to regulate follicular development, uterine receptivity, or blood flow.

Most progesterone actions on the female reproductive tract are mediated through the nu-
clear hormone receptors, proges- terone receptor type A (PR-A) and B (PR-B). Proges-
terone enters cells by diffusion and in responsive tissues becomes associated with pro-
gesterone receptors (Conneely, 2002). Both progesterone receptor isoforms arise from a
single gene, are members of the steroid receptor superfamily of transcrip- tion factors,
and regulate transcription of target genes. These receptors have unique actions. When
PR-A and PR-B recep- tors are coexpressed, it appears that PR-A can inhibit PR-B gene
regulation. The endometrial glands and stroma appear to have different expression pat-
terns for progesterone recep- tors that vary during the menstrual cycle (Mote, 1999). In
addition, progesterone can evoke rapid responses such as changes in intracellular free
calcium levels that cannot be explained by genomic mechanisms.
The Endometrial Cycle
Proliferative or Preovulatory Endometrial Phase
Fluctuations in estrogen and progesterone levels produce strik- ing effects on the repro-
ductive tract, particularly the endo- metrium. Epithelial—glandular cells; stromal—mes-
enchymal cells; and blood vessels of the endometrium replicate cyclically in reproduc-
tive-aged women at a rapid rate. The endometrium is regenerated during each ovarian–
endometrial cycle. The superficial endometrium, termed the functionalis layer, is shed
and regenerated from the deeper basalis layer almost 400 times during the reproductive
lifetime of most women (Fig. 5-4). There is no other example in humans of such cyclical
shedding and regrowth of an entire tissue.

Follicular-phase estradiol production is the most impor- tant factor in endometrial recov-
ery following menstruation. Although up to two thirds of the functionalis endometrium
is fragmented and shed during menstruation, reepithelializa- tion begins even before
menstrual bleeding has ceased. By the fifth day of the endometrial cycle—fifth day of
menses, the epithelial surface of the endometrium has been restored, and revasculariza-
tion is in progress. The preovulatory endo- metrium is characterized by proliferation of
glandular, stro- mal, and vascular endothelial cells. During the early part of the prolifera-
tive phase, the endometrium is usually less than 2 mm thick. The glands are narrow,
tubular structures that pursue almost a straight and parallel course from the basalis layer
toward the endometrial cavity. Mitotic figures, espe- cially in the glandular epithelium,
are identified by the fifth

cycle day. Mitotic activity in both epithelium and stroma persists until day 16 to 17, or 2
to 3 days after ovulation. Although blood vessels are numerous and prominent, there is
no extravascular blood or leukocyte infiltration in the endo- metrium at this stage.

Clearly, reepithelialization and angiogenesis are important to endometrial bleeding ces-

sation (Chennazhi, 2009; Rogers, 2009). These are dependent on tissue regrowth, which
is estro- gen regulated. Epithelial cell growth also is regulated in part by epidermal
growth factor (EGF) and transforming growth factor α (TGFα). Stromal cell proliferation
appears to increase through paracrine and autocrine actions of estrogen and increased lo-
cal levels of fibroblast growth factor-9 (Tsai, 2002). Estrogens also increase local pro-
duction of VEGF, which causes angiogenesis through vessel elongation in the basalis
(Gargett, 2001; Sugino, 2002).

By the late proliferative phase, the endometrium thick- ens from both glandular hyper-
plasia and increased stromal ground substance, which is edema and proteinaceous mate-
rial. The loose stroma is especially prominent, and the glands in the functionalis layer
are widely separated. This is compared with those of the basalis layer, in which the
glands are more crowded and the stroma is denser. At midcycle, as ovulation nears, glan-
dular epithelium becomes taller and pseudostrati- fied. The surface epithelial cells ac-
quire numerous microvilli, which increase epithelial surface area, and cilia, which aid in
the movement of endometrial secretions during the secretory phase (Ferenczy, 1976).

Dating the menstrual cycle day by endometrial histological criteria is difficult during the
proliferative phase because of con- siderable phase-length variation among women.
Specifically, the follicular phase normally may be as short as 5 to 7 days or as long as 21
to 30 days. In contrast, the luteal or secretory postovulatory phase of the cycle is remark-
ably constant at 12 to 14 days.

Secretory or Postovulatory Endometrial Phase

During the early secretory phase, endometrial dating is based on glandular epithelium
histology. After ovulation, the estrogen- primed endometrium responds to rising proges-
terone levels in a highly predictable manner. By day 17, glycogen accumulates in the
basal portion of glandular epithelium, creating subnuclear vacuoles and pseudostratifica-
tion. This is the first sign of ovula- tion that is histologically evident. It is likely the re-
sult of direct progesterone action through receptors expressed in glandular cells (Mote,
2000). On day 18, vacuoles move to the apical portion of the secretory nonciliated cells.
By day 19, these cells begin to secrete glycoprotein and mucopolysaccharide contents
into the lumen (Hafez, 1975). Glandular cell mitosis ceases with secretory activity on
day 19 due to rising progesterone lev- els, which antagonize the mitotic effects of estro-
gen. Estradiol action is also decreased because of glandular expression of the type 2 iso-
form of 17β-hydroxysteroid dehydrogenase. This con- verts estradiol to the less active
estrone (Casey, 1996).

Dating in the mid- to late-secretory phase relies on endome- trial stromal changes. On
days 21 to 24, the stroma becomes edematous. On days 22 to 25, stromal cells surround-
ing the spiral arterioles begin to enlarge, and stromal mitosis becomes apparent. Days 23
to 28 are characterized by predecidual cells, which surround spiral arterioles.

An important feature of secretory-phase endometrium between days 22 and 25 is striking

changes associated with pre- decidual transformation of the upper two thirds of the func-
tionalis layer. The glands exhibit extensive coiling, and luminal secretions become visi-
ble. Changes within the endometrium also can mark the so-called window of implanta-
tion seen on days 20 to 24. Epithelial surface cells show decreased microvilli and cilia
but appearance of luminal protrusions on the apical cell surface (Nikas, 2003). These
pinopodes are important in preparation for blastocyst implantation. They also coincide
with changes in the surface glycocalyx that allow acceptance of a blastocyst (Aplin,
2003).

The secretory phase is also highlighted by the continu- ing growth and development of
the spiral arteries. Boyd and Hamilton (1970) emphasized the extraordinary importance
of the endometrial spiral or coiled arteries. They arise from the radial arteries, which are
myometrial branches of the arcuate and ultimately, uterine vessels (see Fig. 5-4). The
morphologi- cal and functional properties of spiral arteries are unique and essential for
establishing blood flow changes to permit either menstruation or implantation. During
endometrial growth, spiral arteries lengthen at a rate appreciably greater than the rate of
endometrial tissue thickening (Fig. 5-5). This growth discordance obliges even greater
coiling of the already spiral- ing vessels. Spiral artery development reflects a marked in-
duc- tion of angiogenesis, consisting of widespread vessel sprouting and extension. Per-
rot-Applanat and associates (1988) described progesterone and estrogen receptors in the
smooth muscle cells of the uterus and spiral arteries. Such rapid angiogenesis is reg-
ulated, in part, through estrogen- and progesterone-regulated synthesis of VEGF (An-
celin, 2002; Chennazhi, 2009).

Menstruation
The midluteal–secretory phase of the endometrial cycle is a critical branch point in en-
dometrial development and differen- tiation. With corpus luteum rescue and continued
progesterone secretion, the decidualization process continues. If luteal pro- gesterone
production decreases with luteolysis, events leading to menstruation are initiated (Critch-
ley, 2006; Thiruchelvam, 2013).

A notable histological characteristic of late premenstrual- phase endometrium is stromal

infiltration by neutrophils, giv- ing a pseudoinflammatory appearance to the tissue.
These cells infiltrate primarily on the day or two immediately preceding menses onset.
The endometrial stromal and epithelial cells pro- duce interleukin-8 (IL-8), a chemotac-
tic–activating factor for neutrophils (Arici, 1993). Similarly, monocyte chemotactic pro-
tein-1 (MCP-1) is synthesized by endometrium and pro- motes monocyte recruitment
(Arici, 1995).

Leukocyte infiltration is considered key to both endometrial extracellular matrix break-

down and repair of the functionalis layer. The term “inflammatory tightrope” refers to
the ability of macrophages to assume phenotypes that vary from proin- flammatory and
phagocytic to immunosuppressive and repara- tive. These are likely relevant to menstru-
ation, in which tissue breakdown and restoration occur simultaneously (Evans, 2012).
Invading leukocytes secrete enzymes that are members of the matrix metalloprotease
(MMP) family. These add to the pro- teases already produced by endometrial stromal
cells and effec- tively initiate matrix degradation. This phenomenon has been proposed
to initiate the events leading to menstruation (Dong, 2002). As tissue shedding is com-
pleted, microenvironment- regulated changes in macrophage phenotype promote repair
and resolution Anatomical Events During Menstruation. The classic study by Markee
(1940) described tissue and vascular changes in endometrium before menstruation. First,
there are marked changes in endometrial blood flow essential for menstruation. With en-
dometrial regression, spiral artery coiling becomes suf- ficiently severe that resistance to
blood flow increases strikingly, causing endometrial hypoxia. Resultant stasis is the pri-
mary cause of endometrial ischemia and tissue degeneration (see Fig. 5-5). Vasocon-
striction precedes menstruation and is the most striking and constant event observed in
the cycle. Intense spiral artery vasoconstriction also serves to limit menstrual blood loss.
Blood flow appears to be regulated in an endocrine manner by sex steroid hormone–in-
duced modifications of a paracrine- mediated vasoactive peptide system as described
subsequently.