Although the first successful human IVF cycle utilized a natural menstrual cycle, subsequently higher pregnancy rates were achieved when ovarian stimulation with gonadotropins was utilized. Exogenous follicle stimulating hormone (FSH) causes growth of follicles, upregulates aromatase, and prevents the physiologic decrease in FSH in a natural cycle when the dominant follicle is selected. This allows for multi-follicular development during controlled ovarian hyperstimulation. Luteinizing hormone (LH) acts on the theca cells to increase testosterone production by the ovary, which becomes the substrate for estradiol by the granulosa cells in the developing follicles. LH is also the signal by the pituitary to cause luteinization with large amounts of progesterone from the corpus luteum. Recent data have shown that variations in the relative proportions of FSH and LH may have a substantial impact on the outcomes of ovarian stimulation, with an optimal LH: FSH ratio of 0.30: 0.60 [25]. Monitoring the response to ovarian stimulation is accomplished with a combination of transvaginal ultrasonography, serum estradiol levels, and occasionally progesterone levels.

There are various ways to begin an IVF cycle, and these will be described. Oral contraceptive pills (OCPs) are often used prior to stimulation to attenuate the FSH rise and induce a more homogenous follicular cohort, as well as prolong the FSH-responsive window and prevent the occurrence of spontaneous LH surges [26]. In addition, in low responder patients, suppression with OCPs has been shown to improve the response [27], however, patients over the age of 35 who undergo ovarian suppression with OCPs may require a longer duration of stimulation with gonadotropins [26]. OCPs may also provide more flexibility with the timing of appointments for both patients and providers. In addition, some clinics batch cycles, or plan the stimulation start so procedures are likely to occur when staffing is optimal. If women are unable to take OCPs due to a preexisting contraindication (such as migraine headache with aura) or intolerance related to side effects, an IVF cycle may be started with menses. If a woman is amenorrheic or oligomenorrheic, menses may be induced with progesterone withdrawal. In addition, fertility preservation patients often have cancer treatments scheduled and need to start stimulation as soon as possible. There are data that the timing of cycle start does not affect outcomes, even when gonadotropins are started in the luteal phase [4, 5].

The goal of ovarian stimulation for IVF is eventually to harvest a cohort of mature oocytes before the patient ovulates. If ovulation occurs, the oocytes cannot be retrieved, and the cycle must be cancelled. When a patient undergoes controlled ovarian hyperstimulation, the estradiol levels usually far surpass the estradiol threshold that would cause an LH surge under physiologic conditions. Prior to the use of gonadotropin releasing hormone (GnRH) agonists to combat this issue, more than one-quarter of stimulation cycles were cancelled because of a premature LH surge and ovulation [28]. There are three standard IVF protocols to physiologically prevent or delay an LH surge.

Once a cohort of mature follicles is present, urinary human chorionic gonadotropin (hCG, 5000–10,000 international units), or recombinant hCG (250–500 μg), is typically administered to mimic the LH surge and complete oocyte maturation. Oocyte retrieval is performed prior to ovulation, 34–36 h after the hCG injection. Urinary and recombinant hCG products are equivalent [32]. HCG, however, has a relatively long half-life and remains elevated in the serum for 6 days [33]; for this reason, it may exacerbate symptoms of ovarian hyperstimulation syndrome in patients at risk. Alternately, a GnRH agonist trigger may be used in patients on an antagonist protocol to cause an endogenous LH surge. In patients on a GnRH agonist protocol, there are limited data to suggest the use of recombinant LH to trigger ovulation in patients at risk of ovarian hyperstimulation syndrome (OHSS). This is appealing due to the shorter duration (approximately 34 h) [33], although more data are needed to determine the optimal dose of recombinant LH [34].

Embryo transfer is the final, and arguably the most important, step in IVF. The basic steps of an embryo transfer include placing a speculum in the vagina, inserting a catheter into the uterus, and injecting the embryo near the uterine fundus. The optimal technique for embryo transfer has been studied, as there is large variation in techniques. Cervical flushing has not been shown to improve pregnancy rates [45]. By contrast, ultrasound guidance seems to maximize the chance for a successful embryo transfer, likely by allowing for an atraumatic transfer 1.5–2.0 cm from the fundus. Soft catheters seem to improve outcomes compared to stiff catheters [46]. There is controversy about whether a single lumen catheter (embryo pre-loaded into the catheter) or a double lumen catheter (embryo loaded after correct placement of catheter) is superior, although there are no strong data to suggest the superiority of either [47].

An embryo transfer cycle of previously frozen embryos has significant advantages compared to a controlled ovarian hyperstimulation cycle, including decreased risk of OHSS, ability to have genetic information for the embryos, and some data suggest better pregnancy outcomes. Because the ovaries are not hyperstimulated, the risk of ovarian hyperstimulation syndrome is essentially negligible, which is especially important for patients with polycystic ovarian syndrome or patients who have a robust response to controlled ovarian hyperstimulation. In addition, couples that elect PGS or PGD on embryos will be able to have this information prior to a transfer if embryos are frozen. Although some clinics are able to offer trophectoderm biopsy and transfer during the same (fresh) cycle, this requires proximity to a genetics lab and is not widely available.

Practically speaking, this type of cycle may be considered a uterine preparation cycle. Once the embryos have been created, they remain in cryopreservation until the patient desires to use them. Typically, the patient’s hypothalamic–pituitary–ovarian axis is suppressed with oral contraceptive pills (OCPs) and/or leuprolide acetate. As with fresh IVF cycles, non-OCP start cycles are possible as well. Estrogen is given to grow the uterine lining. When the lining is adequately thick (studies support >6 mm [48]), progesterone is administered, and the number of days corresponds to the stage at which the embryos were cryopreserved, as this is when the endometrium and embryo will be chronologically in sync. Then, the embryo(s) is(are) transferred. A pregnancy test is performed 7–11 days later, corresponding to the time of a missed period. Approximately 97% of embryos survive the thawing process, and the pregnancy rates are not worse compared to fresh IVF cycles [49].

In fact, recent data have suggested that vitrified-warmed embryo transfer cycles may have better outcomes compared to fresh embryo transfer cycles, such as higher implantation rates, higher day 14 beta hCG levels per implantation, lower ectopic pregnancy rates, higher live birth rates per transfer, and higher infant birth weight [50]. Other studies have shown significantly higher ongoing pregnancy rates and clinical pregnancy rates compared to fresh cycles [51]. These findings may be due to better endometrial receptivity and placentation in vitrified-warmed cycles.

There are also disadvantages of transferring previously frozen embryos, not the least of which is timing. Most patients who have suffered from infertility want to become pregnant as soon as possible, and choosing to freeze all embryos may result in a delay of several weeks. Another disadvantage is the small chance that the embryo may not survive the thaw, although survival rates have been improved with vitrification protocols [51].

A decision must be made whether to fertilize the oocytes with conventional IVF (putting sperm and oocytes in close proximity in the laboratory) or intracytoplasmic sperm injection (ICSI, which is a micromanipulation procedure in which a single sperm is injected directly into an oocyte to attempt fertilization). ICSI is the treatment of choice for male factor infertility, as it may overcome the negative effects of abnormal semen characteristics and sperm quality on fertilization. For couples that have conceived together previously, conventional in vitro fertilization is usually appropriate. For individuals and couples who do not fit into these categories, the IVF lab may provide guidance .

Reports on the risk of birth defects associated with ICSI, compared to conventional insemination, have yielded conflicting results. The largest study to date has suggested that ICSI is associated with an increased risk of congenital anomalies [52]. Whether the association is due to the ICSI procedure itself, or to inherent sperm defects, has yet to be determined. Although the relative risk of ICSI is increased compared to conventional IVF (1.57, 95% CI 1.3–1.9, vs. 1.07, 95% CI 0.90–1.26), the absolute risk is low. The estimated risk for congenital anomalies in all ART pregnancies is estimated to be 4.3% [53], compared to 3.0% in the general population. Notably, studies have shown that couples with infertility have a higher risk of birth defects, even in the absence of ART [52].

ICSI has also been implicated in other effects on the offspring. In particular, an early report suggested that children conceived with ICSI had an increased risk of developmental delay, however, more recent data have not detected any differences in the development of children born after ICSI, conventional IVF, or natural conception [54]. The prevalence of sex chromosome abnormalities in children conceived via ICSI is higher than observed the general IVF population, but the absolute difference between the two groups is small (0.8–1.0% in ICSI offspring vs. 0.2% in the general IVF population) [55]. The reason for this is unclear—it may result from the ICSI procedure itself, or it may reflect a direct paternal effect. Men with sperm problems are more likely themselves to have genetic abnormalities and often produce sperm with abnormal chromosomes. More studies are needed to clarify these areas of ambiguity, although given the low absolute risk of these issues, most patients are willing to accept the risk.

With the advent of new technologies in the laboratory, genetic testing of the embryos is becoming more common. It is often used for aneuploidy screening, single gene disorders, chromosomal abnormalities such as translocations, mitochondrial disorders, and gender selection in sex-chromosome linked disorders [56]. If patients desire preimplantation genetic testing, the outer layer of a mammalian blastocyst, also called the trophectoderm, may be biopsied on day 5 or 6 following the oocyte retrieval. Although a day 3 biopsy can technically be performed, day 5 or day 6 biopsy is the standard of care due to better sensitivity and specificity [57].

Following embryo biopsy, these cells can be sent for Preimplantation Genetic Screening (PGS) testing, which is the process of removing one or more cells from an embryo to test for a normal number of chromosomes, or Preimplantation Genetic Diagnosis (PGD) testing, which is a similar process but testing for an allele that is associated with a particular disease. PGS provides chromosomal information about the embryos: whether all are present, and whether there are deletions, duplications, or translocations. Pregnancy rates are higher when a euploid embryo is transferred [58, 59]. PGD provides information regarding the presence or absence of a particular disease-associated allele in each embryo that was biopsied. The specific mutation must be known ahead of time, and the testing platform must be built prior to proceeding with controlled ovarian hyperstimulation. There are long-term data that these technologies are safe [60], and they are helping patients to make informed choices prior to the embryo transfer. A day 5 or 6 biopsy usually necessitates cryopreservation of embryos following the biopsy, and waiting for the results before proceeding with a vitrified-warmed blastocyst transfer cycle.

Third Party Reproduction, which is the use of oocytes, sperm, or embryos that have been donated by a third person (donor) to enable an infertile individual or couple (intended recipient) to become a parent (or parents), is another area that has grown considerably in the recent years. Third party reproduction also includes the use of a gestational carrier. Patients who are entering into third party reproduction need additional testing and counseling, including a psychological evaluation and a consultation with a lawyer.