Endocrinology of Pregnancy

Implantation

The process of embryo implantation was thought to take place between 6 and 7 days after ovulation, ^, but more contemporary studies suggest that in most successful human pregnancies, the embryo implants approximately 8 to 10 days after ovulation. This event involves a series of complex steps: (1) apposition of the blastocyst with respect to the endometrial surface; (2) initial adhesion of the blastocyst to endometrium; (3) meeting of the microvilli on the surface of trophoblast with pinopodes, microprotrusions from the apical end of the uterine epithelium; (4) trophoblastic migration through the endometrial surface epithelium; (5) embryonic invasion with localized disruption of the endometrial capillary beds; and, finally, (6) remodeling of the capillary bed and formation of trophoblastic lacunae. By day 10, the blastocyst is completely encased in the uterine stromal tissue. A diagrammatic representation of this process is shown in Figure 10.1 .

Although recent work with in vitro fertilization (IVF)-related techniques such as embryo donation and frozen embryo transfer has contributed significantly to our understanding of this process, much of our present physiologic information is derived from other mammalian species, because human tissue experiments are limited by ethical constraints. This implantation process has been reviewed by Norwitz and colleagues and Dey and coworkers.

In general, studies of IVF-related techniques suggest a high prevalence of chromosomal errors in the embryos that would result in a high level of pregnancy wastage. Based on studies of pregenetic screening by embryo biopsy, we now recognize that up to 70% of blastomeres in which biopsy is performed have an aneuploidy chromosomal at the cleavage stage in otherwise normal-appearing embryos. Embryo biopsy has yielded controversial and limited data on the use of pregenetic testing to improve embryo implantation success. Some blastomere biopsies result in a mosaic pattern with both normal euploid and aneuploid stem cells present in the blastomere population. Overall, the use of embryo biopsy did not demonstrate improvement in clinical pregnancy or miscarriage rates in patients with recurrent pregnancy loss, although there may be a decreased time to pregnancy. Assisted reproductive technologies suggest a “window” for implantation in which the endometrium is receptive to embryo implantation. In this concept, synchronization between embryonic and uterine endometrial receptivity is required for successful nidation. IVF-generated data suggest that implantation is successful usually after embryo transfer into the uterus, between days 3 and 5 after fertilization (the embryo is at the eight-cell [day 3] to blastocyst stage [day 5] of development). If the embryo is transferred outside this window or is in a different location, embryo demise or the likelihood of ectopic pregnancy increases. Although the process of embryo implantation requires a receptive endometrium, the process is not exclusive to the endometrium, because advanced ectopic (e.g., abdominal) pregnancies have been reported with a viable fetus. During a typical IVF cycle, embryos are transferred to the uterus on day 3 or day 5 after fertilization. By day 3 of embryo culture, embryo development is at the six- to eight-cell stage. Embryos placed back into the uterus at this stage remain unattached to the endometrium and continue developing to the blastocyst stage; the embryo “hatches” or escapes from the zona pellucida and implants by day 6 or 7 of embryo life (i.e., 8 to 9 days following human chorionic gonadotropin [hCG] trigger for ovulation). In IVF programs that transfer on day 3, the chance of each embryo implanting is approximately 17% to 37%. Thus to achieve a reasonable chance of overall pregnancy, most women undergoing IVF will have one to two good-quality embryos placed back into the uterus to achieve implantation rates of 31% to 47% and live birth rates of 26 to 55% per IVF retrieval cycle for women up to 40 years of age based on 2017 US statistics. Because the implantation potential for each embryo is affected by the age of the mother and because embryo morphology alone is imprecise for predicting the likelihood of implantation, transfer of multiple embryos can result in higher-order multiple births, such as twins, triplets, or, rarely, quadruplets.

Most IVF programs have the capability to culture embryos for up to 5 to 6 days. Embryos at this stage are at the blastocyst stage. The overall implantation rate for each good-quality embryo at this stage is between 30% and 50% per embryo. Thus to achieve a reasonable chance of pregnancy, most women have only one or two good-quality blastocyst-stage embryos transferred to the uterus, reducing the chances of higher-order multiple births. The American Society for Reproductive Medicine has recommended that only one good-quality embryo be placed for women younger than 38 years of age. A study from population-based control data indicates that the use of assisted reproductive technology accounts for a disproportionate number of low-birth-weight and very-low-birth-weight infants, in part because of multiple births and in part because of higher rates of low birth weight among singleton infants conceived with assisted reproductive technologies. Therefore the practice of elective single embryo transfer, defined by the transfer of one embryo when more than one are available for transfer, should even be considered in older women aged 35–40 with top-quality embryos to avoid multiple gestations, especially in the age of improving IVF technologies.

Concept of Immunotolerance in the Uterus

The embryo and resultant fetus can be viewed as a successful allograft, because the embryo is genetically different from the mother. There is growing evidence that a variety of mechanisms are in play to provide an immunologic barrier between the mother and the fetus.

One mechanism that has been described in the mouse model is “chemokine gene silencing in decidual stromal cells that limits T-cell access to the maternal-fetal interface.” These experiments show that in the specialized stromal tissue surrounding the fetus and placenta, effector cells cannot penetrate or accumulate within the decidua. These researchers were able to identify that this was due in part to pregnancy-induced epigenetic changes in the inflammatory chemokine genes in decidual stromal cells.

Clinically, patients may experience recurrent pregnancy loss in the first trimester (two or more pregnancy losses), and some patients undergoing IVF experience repeated implantation failure, defined as three consecutive cycles with transfer of good-quality embryos without conception. These may be due to failure to form an adequate functional immunologic barrier between the mother and fetus. In a recent study of patients with recurrent implantation failure, midluteal phase endometrial biopsies of 17 of 32 patients with this diagnosis revealed an increase in natural killer cells in the endometrium. These findings suggest one possible defect in the immunologic barrier between the maternal-fetal interface.

Concept of MicroRNA in Implantation Regulation

Emerging studies have proposed that posttranscriptional regulation of gene expression through microRNAs may play a role in embryo implantation and endometrial receptivity. Studies in mouse models have shown that miRNA expression regulates embryonic angiogenesis, development, and overall survival. Abnormal embryo development through abnormal miRNA expression may play a role in recurrent implantation failure.

Additionally, miRNAs have been suggested to have a differential role in endometrial receptivity. Genomic profiling through endometrial receptivity array (ERA), a commercially available test, is used to detect alterations in biomarkers that affect implantation. These alterations are proposed to be due to endometrial miRNA regulation in both animal and human studies. ^,

Human Chorionic Gonadotropin Production

hCG is one of the earliest products of the cells forming the embryo and should be viewed as one of the first embryonic signals elaborated by the embryo even before implantation. This glycoprotein is a heterodimer (36 to 40 kDa). It is composed of a 92–amino acid α subunit that is homologous to thyroid-stimulating hormone, luteinizing hormone (LH), and follicle-stimulating hormone and a 145–amino acid β subunit that is similar to LH. The α subunit gene for hCG has been localized to chromosome 6; the β subunit gene is located on chromosome 19, fairly close to the LH-β gene.

In contrast to LH, the presence of sialic acid residues on hCG-β accounts for its prolonged half-life in the circulation. After implantation, hCG is produced principally by the syncytiotrophoblast layer of the chorionic villus and is secreted into the intervillous space. Cytotrophoblasts are also able to produce hCG.

Clinically, hCG can be detected in either the serum or urine 7 to 8 days before expected menses and is the earliest biochemical marker for pregnancy ( Fig. 10.2 ). In studies during IVF cycles in which embryos were transferred 2 days after fertilization, β-hCG was detected as early as the eight-cell stage, whereas intact hCG was not detectable until 8 days after egg retrieval. The increase in hCG levels between days 5 and 9 after ovum collection is principally the result of the production of free β-hCG, whereas by day 22 most of the circulating hCG is in the dimeric form.

These observations correspond to in vitro studies that indicate a two-phase control of dimer hCG synthesis mediated principally through a supply of subunits. In contrast to LH secretion in the pituitary gland, hCG is secreted constitutively as subunits are available and is not stored in secretory granules. Initially, immature syncytiotrophoblast produces free β-hCG subunits, whereas the cytotrophoblast’s ability to produce the α subunit appears to lag by several days. As the trophoblast matures, the ratio of α subunits to β subunits reaches 1:1, and a peak of approximately 100,000 mU/mL is reached by the 9th or 10th week of gestation ( Fig. 10.3 ). By 22 weeks’ gestation, the placenta produces more of the α subunit than β-hCG. At term gestation, the ratio of α subunit to hCG release is approximately 10:1.

The exponential rise of hCG after implantation is characterized by a doubling time of 30.9 ± 3.7 hours. The hCG doubling time has been used as a characteristic marker by clinicians to differentiate normal from abnormal gestations (i.e., ectopic pregnancy). Most recent studies suggest that the rate of rise of hCG is more variable. An hCG rise of 35% over a 2-day interval will achieve the optimal sensitivity and specificity for differentiation of a normal pregnancy from a pregnancy of unknown location. The inability to detect an intrauterine pregnancy (gestational sac) by endovaginal ultrasound when serum hCG levels reach 1100 to 1500 mU/mL suggests an abnormal gestation or ectopic pregnancy. Higher than normal hCG levels may indicate a molar pregnancy or multiple-gestational pregnancies. Levels of hCG in combination with maternal α-fetoprotein and unconjugated estriol have been used as a screening test for detection of fetal anomalies (see Chapter 30 ).

Maintenance of Early Pregnancy: Human Chorionic Gonadotropin and Corpus Luteum of Pregnancy

The major biologic role of hCG during early pregnancy is to rescue the corpus luteum from premature demise while maintaining progesterone production. Although the secretory pattern of hCG is not well characterized, hCG is required for rescue and maintenance of the corpus luteum until the luteal-placental shift in progesterone synthesis occurs. This concept is supported by observations that immunoneutralization of hCG results in early pregnancy loss. ^,

Studies in early pregnancy show that secretion of hCG and progesterone from the corpus luteum appears to be irregularly episodic with varying frequencies and peaks. ^, In first-trimester explant experiments, intermittent gonadotropin-releasing hormone administration enhances the pulse-like secretion of hCG from these explants, indirectly implicating placental gonadotropin-releasing hormone as a paracrine regulator of hCG secretion. In nonconception cycles, the corpus luteum is preprogrammed to undergo luteolysis, which is regulated through apoptotic mechanisms. Acting through the LH receptor, hCG is also able to stimulate parallel production of estradiol, 17-hydroxyprogesterone, and other peptides, such as relaxin and inhibin, from the corpus luteum.

Timing of the Luteal-Placental Shift

Ovarian progesterone production is essential for maintenance of early pregnancy. If progesterone action is blocked by a competitive progesterone antagonist, such as mifepristone (RU-486), pregnancy termination results. During later gestation, placental production of progesterone is sufficient to maintain pregnancy. To uncover the timing of this luteal-placental shift, Csapo and colleagues ^, performed corpus luteum ablation experiments. They demonstrated that removal of the corpus luteum before, but not after, the seventh week of gestation usually resulted in subsequent abortion. Removal of the corpus luteum after the ninth week appears to have little or no influence on gestation ( Fig. 10.4 ). Thus progesterone supplementation is required if corpus luteum function is compromised before 9 to 10 weeks of gestation.

Fetoplacental Unit as an Endocrine Organ

The fetus and placenta must function together in an integrated fashion to control the growth and development of the unit and subsequent expulsion of the fetus from the uterus. The changes occurring in the maternal endocrine milieu contribute to fetal and placental activity. Estrogens, androgens, and progestins are involved in pregnancy from before implantation to parturition. They are synthesized and metabolized in complex pathways involving the fetus, the placenta, and the mother.

The fetal ovary is not active and does not secrete estrogens until puberty. In contrast, the Leydig cells of the fetal testes are capable of producing large amounts of testosterone, so the circulating testosterone concentration in the first-trimester male fetus is similar to that in the adult man. hCG provides the initial stimulus of the testes. Fetal testosterone is required to promote differentiation and masculinization of the male external and internal genitalia. In addition, local conversion of testosterone to dihydrotestosterone by 5α-reductase localized in situ at the genital target tissues ensures final maturation of the external male genital structures. The maternal environment is protected from the testosterone produced by the male fetus because of the abundance of placental aromatase, which can convert testosterone to estradiol.