9.2.1 Timing of FET in a Natural Cycle

In contrast to a HRT cycle where follicle growth and spontaneous ovulation result in cycle cancellation, in a natural cycle estrogen and progesterone production from a dominant follicle and subsequently from the corpus luteum are crucial for endometrial preparation. In a natural cycle, estradiol synthesis increases progressively from the dominant follicle and initiates the LH surge. Prior to the LH surge, a small increase in progesterone level is seen, which reflects the increasing LH pulse amplitude and frequency leading up to the surge. An LH surge of 24–36 hours is sufficient to initiate the resumption of oocyte meiosis, luteinization of granulosa cells, ovulation, and the initial phase of corpora lutea development. Progesterone and 17α-hydroxyprogesterone (17α-OHP) plasma concentrations increase rapidly after the LH surge indicating the beginning of granulosa and theca cell luteinization (13). The corpus luteum produces up to 40 mg of progesterone per day in addition to a significant quantity of androgens and estradiol (14).

Following the pathophysiology of natural conception, the embryo is deemed to arrive in the uterine cavity approximately five days after fertilization of the oocyte by the sperm in the fallopian tube while the aforementioned hormonal changes prepare the receptive endometrium for implantation. The process of implantation, which involves embryo apposition, attachment to the maternal endometrial epithelium and invasion into the endometrial stroma can only occur when the endometrium is receptive (Figure 9.1) (15). This phase is commonly referred to as the “window of implantation” (WOI) (15–17).

FET can be planned in a pure natural cycle (NC) in patients with regular spontaneous ovulatory cycles or in a modified natural cycle (mNC), using hCG (Human Chorionic Gonadotropin) for ovulation induction. In a natural cycle, follicle growth and spontaneous ovulation are crucial for endometrial preparation. The key to success of embryo transfer in a natural cycle is the accurate detection of the LH surge and subsequent ovulation in order to determine the timing of the embryo transfer accurately. As described above, the LH surge in a natural cycle is initiated through increasing estradiol levels, produced by the growing dominant follicle. When the LH concentration rises by 180 percent above the most recent serum values and continues to rise thereafter, the LH surge is considered to have begun (18). As a consequence of rising LH levels, luteinization of granulosa cells, and synthesis of progesterone is stimulated and a drop in estradiol levels follows the initiation of the LH surge. When LH surge, ovulation, and the rise of progesterone levels are confirmed by blood tests, subsequently the embryo transfer has to be planned according to the embryo development stage at the time of embryo freezing/vitrification. Figure 9.2 depicts the hormonal constellation of estradiol (E2), progesterone (P4) and LH to identify the LH surge and the day of ovulation.

Figure 9.2 Hormonal constellation to identify the LH surge and the day of ovulation.

Detection of the LH surge and the estradiol decrease in blood requires frequent patient visits to the clinic, therefore the use of urinary LH kits to detect ovulation may seem to be a more patient-friendly and convenient approach in a natural cycle. However, the use of urinary LH kits have several disadvantages which may result in incorrect identification of the LH surge. LH levels in the urine follow the serum peak by nearly 24 hours because of the required time for urinary LH clearance (19). In addition, urinary LH kits may demonstrate LH surges in the absence of ovulation (20) and finally a high percentage of normally cycling women may demonstrate premature LH surges which do not trigger ovulation and therefore if a clinician depends solely on urinary LH kits to identify the LH surge it is very likely that the timing of embryo transfer will be inaccurate and result in an unsuccessful cycle (20, 21). It is worth bearing in mind that the conclusions of studies using LH urinary kits to determine timing of the LH surge have to be evaluated critically and may not be scientifically accurate.

9.2.2 Timing of FET in Hormonal Replacement Cycle

The advantages of a HRT-FET cycle are obvious: first, disturbances due to cycle variation can be avoided, second, the number of patient visits to the clinic for ultrasound scans and hormonal monitoring can be reduced and third, this approach facilitates flexibility with scheduling of the embryo transfer procedure which is an advantage for both the clinic and patient.

In a hormonal replacement therapy (HRT) cycle for FET, the hormonal changes of a natural cycle are simulated by commencing estradiol administration on the second or third day of the cycle and continuing throughout the cycle to promote endometrial development. When a sufficient endometrial thickness is confirmed by ultrasound examination, progesterone is introduced to induce secretory transformation of the endometrium. The critical step in a HRT-FET cycle is to adjust the duration of progesterone exposure to the developmental stage of the embryo following estradiol administration to prepare the endometrium. Theoretically, progesterone can be administered orally, rectally, subcutaneously, vaginally, and via the intramuscular route with the two latter modes of administration being the most commonly applied in clinical practice (22). The serum progesterone’s rise and it´s subsequent effect on the endometrium vary depending on the route of progesterone administration.

Progesterone is rapidly absorbed after intramuscular (IM) injection and higher doses will result in earlier and higher peak serum levels: following injection of 25 mg, 50 mg, and 100 mg of progesterone, peak concentrations were 16.9 ng/mL, 36.5 ng/mL, and 83.8 ng/mL, respectively, and these peak levels were attained 5 hours after injection of the 25 mg and 50 mg dosage and 3.5 hours following the injection of the 100 mg dose. Thirteen hours after the injection, progesterone plasma levels decreased to 10.9 ng/mL (25 mg), 19.8 ng/mL (50 mg), and 40.7 ng/mL (100 mg), respectively (23).

In contrast to the rapidly increasing serum progesterone levels following IM injection, serum progesterone concentrations reach maximal levels two to four hours after vaginal application of a 400 mg suppository with a maximum peak of 35 ng/mL (24). It is well recognized that vaginal absorption of progesterone is enhanced following previous estrogenization (25).

Despite low serum progesterone levels, adequate secretory endometrial transformation is achieved by vaginally administered progesterone. This suggests a direct local effect on the endometrium before entering the systemic circulation, the so-called first uterine pass effect.

HRT-FET can be performed with or without co-treatment with a GnRH (Gonadotropin-Releasing-Hormone) analog. Incorporating a GnRH analog into the treatment protocol reduces the risk of cycle cancellation due to spontaneous ovulation which may occur despite the administration of exogenous hormones. The advantages of employing this approach include a reduction in the number of patient visits to the clinic for ultrasound and hormonal monitoring, cycle cancellations due to cycle variation can be avoided, and this approach facilitates embryo transfer planning.

9.2.3 Is the “True NC-FET” in the Literature Really “True”?

Data on the efficacy of a “true“ NC-FET cycle compared to a HRT-FET are limited. Previous studies have focused for the most part on modified natural cycles using human chorionic gonadotrophin to induce ovulation of the dominant follicle which has been shown to be inferior to a spontaneous natural cycle for planning of frozen-thawed embryo transfer (26). Administration of hCG in the late follicular phase induces changes in the endometrium, which would have occurred several days later in a natural cycle (26). In addition, human chorionic gonadotrophin (hCG) and LH act on the endometrium through the same receptor and it has been suggested that their simultaneous presence may have an adverse effect on pregnancy rates (26, 27). Cochrane meta-analyzes, comparing different FET approaches, were published in 2008 and 2017 both concluding that there is insufficient evidence to support the use of one cycle regimen in preference to another in preparation for FET in subfertile women with regular ovulatory cycles (28, 29). The overall quality of the evidence was very poor in both meta-analyses. The main limitation was poor reporting of study methods. In addition, the studies referenced as studies of the natural cycle in the 2017 analysis were essentially abstracts and never published as full papers (30, 31). It also has to be noted that the study by Karimzadeh et al., did not meet the criteria for a “true” NC-FET as hCG was used for ovulation induction which has been clearly shown to be significantly less effective as compared to a true natural cycle (31).

A recent study comparing artificial and natural cycles concluded that the optimal means of endometrial preparation for frozen-thawed cycle remains unclear and both options may be offered to women with regular ovulatory cycles in line with the previous Cochrane meta-analyses (32). However this study identified ovulation based on ultrasound findings alone which means that a premature LH or progesterone rise would be missed leading to inaccurate timing of ovulation. In addition, the natural cycle was modified by the use of hCG to induce ovulation which has been shown to be inferior to a spontaneous natural cycle (26). A further recent systematic review and meta-analysis of the available evidence concluded that no significant difference with regard to clinical, ongoing pregnancy rates and live birth rates were found comparing true NC-FET to HRT-FET (33). However the conclusion of this meta-analysis should not be universally accepted without determining the methods adopted in each study to determine the timing of ovulation which we know is of critical importance. Only two studies included in this meta-analysis used serum samples to identify the LH surge (34, 35) and the study by Xiao et al., used urinary LH tests for LH surge identification (36). Due to the aforementioned possible inaccuracies of urinary LH detection kits, the findings of this study are questionable.

Recently published studies comparing NC-FET to other approaches for endometrial preparation used solely ultrasonographic disappearance of the dominant follicle in combination with urinary LH tests for detection of ovulation (37, 38). The outcomes are contradictory as the study by Cardenas et al. (37) favored NC-FET whereas Madani et al. (38) did not find one approach superior to the other.