Shokichi Teramoto1
(1)
Shinbashi Yume Clinic, Excel Shinbashi Bldg., 2-5-1, Minato-ku, Tokyo 1050004, Japan
Shokichi Teramoto
Email: embryo@yumeclinic.net
Abstract
The term minimal stimulation cycle encompasses a variety of ovulation stimulation methods, from the natural cycle, in which no ovarian stimulation medication is used, to the Letrozole (Femara) cycle, in which a follicular stimulatory effect is thought to take place among some patients, including those with polycystic ovary syndrome (PCOS), although its efficacy with regard to ovarian stimulation is not clear, to the Clomiphene citrate cycle, in which a definite ovarian stimulation efficacy has been demonstrated and the development of more than one follicle is normally observed. Furthermore, cycles where follicle-stimulating hormone (FSH) and human menopausal gonadotropin (hMG) drugs are used in combination at lower-than-usual levels are generally included in the minimal stimulation cycles. This chapter discusses the protocols which maximize the benefit of each method, based on the accurate evaluation of hormonal levels and clinical outcomes.
Keywords
Minimal stimulationLetrozoleClomiphene citrateSmall folliclesIVM
Introduction
In the natural cycle in vitro fertilization (nIVF), normally only one dominant follicle will develop to 16 mm or more in diameter during the final stage of follicular development. Other follicles remain as small follicles, the size of which are 2–8 mm, and their number depends on the patient’s ovarian reserve. This polarization in follicular development is thought to occur because, when the dominant follicle is recruited during the follicular phase, due to the reduction in the levels of follicle-stimulating hormone (FSH), the non-recruited follicles undergo atresia (Ingram 1959). In a cycle, with the aim of in vitro maturation (IVM), therefore, it is thought necessary to retrieve oocytes before the recruitment of the dominant follicle is completed. Oocytes are usually retrieved using a special needle (17-gauge/19-gauge double-lumen needle) when the diameter of the biggest follicle, which has not yet been recruited as the dominant follicle, is less than 13 mm (Son et al. 2008). This is a logical conclusion derived from the follicular atresia theory which suggests that by the final stage of the follicular development, the follicular recruitment has been completed and all the remaining non-recruited follicles are atretic, meaning they have lost their developmental ability. Thus, it has been thought necessary to stimulate the ovary in various ways in order to obtain as many oocytes with a developmental ability as possible. To achieve this, gonadotropin-releasing hormone (GnRH) antagonist protocol has been developed, in addition to the classic long and short protocols. All those protocols are considered to be theoretically correct, but they entail various side effects, such as ovarian hyperstimulation syndrome (OHSS) and the disruption of ovarian functions. Furthermore, the ovaries of older women cannot respond to such potent stimuli as their ovarian function has deteriorated, not only reducing the success rate relative to the applied stimuli but also increasing their adverse effects by expediting functional deterioration. In the face of such a situation, the use of milder ovarian stimulation methods with a lessened potency, which are much gentler on the body, is gradually spreading, although some comment that the success of such methods is limited. The author has been endeavoring to raise the success rate of the natural stimulation and the minimal stimulation methods by improving the oocyte retrieval methods in order to retrieve oocytes from the small follicles (average volume: 0.103 ± 0.041 mL, average diameter: 5.8 mm, N = 3155), something which used to be believed impossible, and also by evaluating the follicular growth backed up by endocrine evidence obtained through accurate analysis of hormonal dynamics. This section provides details of such efforts and a re-evaluation of the efficacy of minimal stimulation methods.
Points to Be Noted
The hormonal levels are measured by AIA 1800, Tosoh, which is based on the principle of fluorescent-enzyme immunoassay (FEIA). The reagent for estradiol (E2) was changed to the new reagent, iE2, and the values of the former and new reagents are slightly different. The ultrasonic device used was HI VISION Avius, Hitachi Medical Corporation. The oocyte retrieval needle used was a proprietary needle which the author commissioned Kitazato Medical Supply to create. All the oocytes were cultured to blastocysts and were cryopreserved using vitrification. They were then thawed and transferred in natural ovulation cycles or hormone replenished cycles. Only one embryo was transferred at a time, without exception.
Oocyte Retrieval in the Natural Cycle
The natural cycle is the method where no drug is administered except gonadotropin-releasing hormone agonist (GnRHa), which is used as an oocyte maturation trigger (Fig. 6.1). As GnRHa facilitates final oocyte maturation and ovulation by inducing endogenous luteinizing hormone (LH) and FSH surges, its administration does not affect the process of the natural cycle as long as it is administered with the right timing. Therefore, its use should be tolerated in order to achieve the minimum required level of convenience for oocyte retrieval.
Fig. 6.1
The procedure of the natural cycle
In the natural cycle method, hormonal measurements of E2, progesterone (P4), LH, FSH, and anti-Mullerian hormone (AMH), as well as measurement of the number and the size of antral follicles are carried out on the third day of the menstrual cycle (d3), in order to decide whether the cycle is viable for treatment. If it is viable, the level of the four factors, namely, E2, P4, LH, and FSH, and the number and the size of the follicles are measured again on d10. As early as this point, the use of an oocyte maturation trigger may be decided, or oocyte retrieval may be carried out on the spot if the ovulation is about to take place due to the advanced state of the LH surge. If natural ovulation is likely to happen immediately, oocyte maturation is not triggered. The rate of increase in the E2 level per day shows a gradual increase as the follicles grow, normally at 1.2–1.4 times per day. However, this varies depending on the individual, and misreading this rate of increase will result in missing the correct timing for oocyte retrieval. When it is necessary to continue monitoring the follicular growth, the next consultation day must be set based on the E2 level. If advanced LH and FSH surges are observed on the final visit, the state of surge progression is assessed based on the rate of increase of the LH and FSH, obtained by comparing their levels to those of the last visit. The author assesses the progression of the LH surge by categorizing the rate of increase into three levels, namely, less than 1.25 times, 1.25 times or more but less than 3 times, and 3 times or more. The progress of the FSH increase is categorized into two levels, namely, less than 2 times and 2 times or more. The FSH surge normally begins later than the LH surge. Therefore, when the FSH surge is considered to have begun, because its level of increase is twice or more, the LH surge tends to be coming to an end. If the LH surge is still at the beginning stage, it can be arrested by administering 0.25 mg of GnRH antagonist. However, if the LH level exceeds 3 times or more since the last visit, arresting the surge is difficult (Table 6.1).
Table 6.1
Measures to be taken at the time of oocyte retrieval based on the LH and FSH levels
|
When LH concentration/LH basal concentration is |
And when |
Measure to take |
|
<1.25 |
Diameter of the dominant follicle |
Administer GnRHa nasal spray at 2200 and retrieve oocytes in 34–35 h (normal oocyte retrieval) |
|
from 1.25_ to <3.0 |
Administer 0.25 mg of GnRH antagonist immediately and GnRH agonist (GnRHa) nasal spray at 2200, then retrieve oocytes in 34 h (suppressed oocyte retrieval) |
|
|
3.0 |
FSH concentrations/FSH basal concentrations <2.0 |
Immediately administer GnRHa nasal spray and retrieve oocytes in 24–28 h (emergency oocyte retrieval). |
|
3.0 |
FSH concentrations/FSH basal concentrations _ 2.0 |
Immediately retrieve oocytes (surge oocyte retrieval) |
Oocyte maturation is triggered when the E2 level and the diameter of the dominant follicle, derived by taking the average length of its long and short axis, have reached their optimum conditions. Basically, such optimum conditions would be when the E2 level has reached 264–268 pg/mL, and the follicular diameter has become 16 mm or more, but the E2 level should take priority over the follicular growth. When these optimum conditions are met, oocyte maturation is triggered by administering 300 μg GnRHa by nasal spray. The timing of the oocyte retrieval should be decided according to the progression of the LH surge but should be carried out immediately without applying artificial oocyte maturation if the LH surge has already been completed (Fig. 6.2). In our clinic, every patient’s first oocyte retrieval is carried out by the natural cycle method without exception. There are two requisite conditions for such an indication: the patient has not received any medication in the cycle immediately prior to the treatment cycle, and the treatment cycle is a natural ovulation cycle.
Fig. 6.2
Hormonal dynamics of the natural cycle
The number of oocyte retrievals in the natural cycle IVF carried out in our clinic for the 2 years from January 1, 2011 to December 31, 2012, was 1979 cases (1979 cycles). The average age of women who underwent the treatment was 37.6 years old (30–45 years old). The level of d3 AMH was 15.6 pM (0.0–108.11 pM), and that of d3 FSH was 8.5 IU/L (1.5–62.3 IU/L). The average number of visits to the clinic was 3.67 ± 0.91 times, and the average number of days from the start of the cycle to the day of the oocyte retrieval was 14.9 ± 2.8 days. The number of cycles where the dominant follicle had ovulated before the day of retrieval was 100 cycles (5.2 %), from which only small follicles were aspirated and retrieved.
The FSH level peaked on d3 and then decreased as the level of E2 increased. It hit the lowest level when the E2 level reached 200 pg/mL on the day before oocyte maturation was triggered but began to rise due to the positive feedback when the E2 level went over 200 pg/mL.
The LH level peaked on d3 and then began to decrease as the level of E2 rose. However, it began to rise due to the positive feedback when the level of E2 went above 150 pg/mL, 2 days before the oocyte maturation was triggered, and increased by 90 % from the day before the oocyte maturation triggering to the day of maturation triggering.
This result shows that the LH surge precedes the FSH surge in the positive feedback.
The increase in rate of the E2 level gradually rose from 6 days before oocyte maturation triggering and showed 35 %, the highest level of increase, 2 days before the maturation triggering to 1 day before the trigger. Therefore, the patient’s next visit to the clinic should be scheduled, based on the increase rate of the E2 level, within 4 days if E2 = 80–100 pg/mL, within 3 days if E2 = 100–120 pg/mL, and within 2 days if E2 = 120–140pg/mL (Fig. 6.2).
The average number of aspirated follicles per patient was 1.13 for dominant follicles developed to a diameter of 11 mm or more and 8.35 for small follicles, the diameters of which were less than 11 mm. The oocyte retrieval rate was 55.6 % and 36.8 %, respectively. The rate of retrieval metaphase II (MII) stage oocytes was 81.0 % and 23.5 %, respectively. The level of maturity was assessed whether the oocyte was denuded immediately after the retrieval or not. All the retrieved oocytes were cultured to blastocysts, and the rate of retrieved oocytes which grew to blastocysts was 38.0 % among oocytes derived from dominant follicles and 6.6 % among oocytes derived from small follicles. The rate of ongoing pregnancies for 22 weeks or longer, including cases where no oocytes were retrieved, was 13.4 %, and 35.7 % of those were achieved from the small follicles (Table 6.2).
Table 6.2
Outcome of the natural cycle
|
Natural cycle |
Follicular size |
Average age (years) |
d3AMH level (PM) |
d3FSH level (IU/L) |
Follicular Aspiration (in total) no. of cycles |
Follicular aspiration |
Oocyte retrieval |
Growth to blastocyst |
No. of cycles embryo was transferred |
No. of cycles pregnancy was achieved |
Rate of ongoing pregnancies b (%) |
||||
|
No. of cyclesa |
Average no. of follicles aspirated |
No. of cycles |
Average no. of oocytes retrieved |
No. of cycles |
Average no. of oocytes grew to blastocysts |
||||||||||
|
Age categories |
30–35 |
Largec |
33.3 |
21.7 |
7.6 |
506 |
468 |
1.14 |
303 |
1.05 |
142 |
1.04 |
137 |
79 |
15.6 |
|
Smalld |
432 |
10.79 |
358 |
4.96 |
99 |
1.32 |
93 |
47 |
9.3 |
||||||
|
36–40 |
Large |
38.1 |
14.4 |
8.6 |
882 |
837 |
1.13 |
498 |
1.06 |
199 |
1.02 |
187 |
67 |
7.6 |
|
|
Small |
767 |
8.36 |
615 |
3.74 |
128 |
1.30 |
113 |
36 |
4.1 |
||||||
|
41–45 |
Large |
42.2 |
7.9 |
10.1 |
474 |
452 |
1.15 |
249 |
1.05 |
71 |
1.00 |
69 |
16 |
3.4 |
|
|
Small |
401 |
5.71 |
298 |
2.85 |
22 |
1.23 |
20 |
7 |
1.5 |
||||||
|
Total |
37.8 |
14.6 |
8.7 |
1862 |
1757 |
1.14 |
1050 |
1.06 |
412 |
1.02 |
393 |
162 |
8.7 |
||
|
1615 |
8.35 |
1271 |
3.87 |
249 |
1.30 |
226 |
90 |
4.8 |
|||||||
aNumbers shown above and below overlap. The total is shown in the left column
bOngoing pregnancies: The denominator is the total number of aspirated cycles
cLarge: Large follicles the size of which is 11 mm or more in diameter
dSmall: Small follicles the size of which is less than 11 mm in diameter
Oocyte Retrieval from Small Follicles
Normally, with the natural cycles or the mild stimulation cycles, only dominant follicles are aspirated. However, in our clinic, we aspirate as many small follicles as possible if their diameter is 3 mm or above. With conventional aspiration needles, such as an 18-gauge needle, the smaller their size, the more difficult it becomes to puncture small follicles of less than 11 mm in diameter, because the needle has a high punctuation resistance and the longitudinal diameter of the opening is as large as 8 mm or longer. However, with the use of the 23-gauge tapered needle, it is possible to learn to puncture quite small follicles with training. This 23-gauge tapered needle was developed by the author in 2007, and it is only sold in Japan presently (Fig. 6.3).
Fig. 6.3
23-gauge tapered needle
Oocyte Retrieval in the Femara Cycle
Femara (Novartis Pharma K.K.) is an aromatase inhibitor (Letrozole), which reduces the estrogen actions in the hypothalamus by inhibiting estrogen synthesis at the granulosa cell layer. As a result, it is generally understood that the negative feedback of the GnRH is blocked, the FSH action on the ovary increase, and the number of dominant follicles increase. Furthermore, it is understood that the presence of androgen in the follicular microenvironment will be excessive, causing the sensitivity of the granulosa cell layer to FSH to increase, in turn facilitating follicular growth. Although Femara is used for assisted reproductive technology (ART) based on this assumption, continued use of Femara makes E2 stop reflecting follicular growth, thereby increasing the risk of missing the timing to trigger oocyte maturation. As the biggest worry in ART is missing oocytes because they have already been ovulated by the time of retrieval, it is necessary to consider carefully the dosing duration in order to maximize the efficacy of Femara on follicular growth as well as to avoid unscheduled ovulation. In order to achieve this delicate balance, the author has devised a protocol to administer Femara (2.5 mg) for the 3 days from d3 to d5 (Fig. 6.4).
Fig. 6.4
The procedure of the Femara cycle
The FSH level rose from d3 and peaked on d6 which was immediately after the cessation of Femara administration. Then, it continued to decrease, reaching the lowest level on the day before triggering oocyte maturation but rising slightly on the maturation triggering day due to the positive feedback.
The LH level continued its decrease from its peak on d3 but it turned to rise 2 days before triggering oocyte maturation and increased by 130 % from the day before to the day of maturation triggering. Compared to the natural cycle, the LH surge tends to advance more rapidly and at a lower E2 level in the Femara cycle.
E2 decreased once from d3 and hit the lowest level on d6 but then began to rise. From 4 days before oocyte maturation was triggered, it showed a rapid increase of 20 % or more, and it reached 38 % on the last day. The shift in the level of E2 is important in setting the next consultation day, but the increase in the E2 level only becomes stable 3 days prior to oocyte maturation triggering. Therefore, during the Femara cycle, the next consultation day must be set within 3 days, even when the E2 level remains low (Fig. 6.5).
Fig. 6.5
Hormonal dynamics of the Femara cycle
The half-life of Femara (68.6 ± 36.7 h) is involved in this shift in the E2 level, showing that a total of only 7.5 mg Femara administered during 3 days affects hormonal dynamics and follicular growth in no small measure. Therefore, a long period of Femara administration will make prediction of the dynamics of E2, LH, and FSH more difficult, and thus, we are limiting the use of Femara to 3 days from d3.
The number of oocyte retrievals in the Femara cycle IVF carried out in our clinic for the 2 years from January 1, 2011 to December 31, 2012, was 1260 cases (1260 cycles). The average age of women who underwent the treatment was 38.2 years old (30–45 years old). The level of d3 AMH was 13.3 pM (0.0–136.0 pM), and that of d3 FSH was 8.2 IU/L (0.5–40.6 IU/L). The average number of visits to the clinic was 3.66 ± 0.92 times, and the average number of days from the start of the cycle to the day of the oocyte retrieval was 13.8 ± 2.4 days. The number of cycles where the dominant follicle had ovulated before the day of retrieval was 65 cycles (6.9 %), from which only small follicles were aspirated and retrieved.
The average number of aspirated follicles per patient was 1.31 for dominant follicles developed to a diameter of 11 mm or more and 5.36 for small follicles, the diameter of which were less than 11 mm. The oocyte retrieval rate was 55.8 % and 41.8 %, respectively. The rate of retrieving MII-stage oocytes was 77.8 % and 29.1 %, respectively. The rate of retrieved oocytes growing to blastocysts was 36.2 % among oocytes derived from dominant follicles and 12.5 % among oocytes derived from small follicles. The rate of ongoing pregnancies, including cases where no oocytes were retrieved, was 16.2 %, and 40.4 % of those were achieved from the small follicles (Table 6.3).
Table 6.3
Outcome of the Femara cycle
|
Femara cycle |
Follicular size |
Average age (years) |
d3AMH level (PM) |
d3FSH level (IU/L) |
Total follicular aspiration no. of Cycles |
Follicular aspiration |
Oocyte retrieval |
Growth to blastocyst |
No. of cycles embryo was transferred |
No. of cycles pregnancy was achieved |
Rate of ongoing pregnancies (%) |
||||
|
No. of cycles |
Average no. of follicles aspirated |
No. of cycles |
Avenge no. of oocytes retrieved |
No. of cycles |
Average no. of oocytes grew to blastocysts |
||||||||||
|
Age categories |
30–35 |
Large |
33.1 |
18.1 |
7.9 |
168 |
154 |
1.40 |
101 |
1.21 |
53 |
1.09 |
49 |
34 |
20.2 |
|
Small |
136 |
6.61 |
110 |
3.03 |
45 |
1.29 |
33 |
19 |
11.3 |
||||||
|
36–40 |
Large |
38.3 |
12.2 |
8.0 |
356 |
328 |
1.28 |
201 |
1.11 |
84 |
1.06 |
74 |
26 |
7.3 |
|
|
Small |
289 |
5.62 |
228 |
3.02 |
68 |
1.29 |
68 |
26 |
7.3 |
||||||
|
41–45 |
Large |
42.1 |
9.6 |
8.5 |
209 |
199 |
1.30 |
124 |
1.19 |
37 |
1.11 |
37 |
11 |
5.3 |
|
|
Small |
166 |
3.91 |
129 |
2.32 |
14 |
1.07 |
16 |
3 |
1.4 |
||||||
|
Total |
38.2 |
12.8 |
8.1 |
733 |
681 |
1.31 |
426 |
1.15 |
174 |
1.08 |
160 |
71 |
9.7 |
||
|
612 |
5.37 |
467 |
2.83 |
127 |
1.27 |
117 |
48 |
6.5 |
|||||||
Oocyte Retrieval in the Femara-Clomiphene Citrate Cycle
Femara and Clomiphene citrate are used in combination with this stimulation method. Femara’s efficacy in ART is not significant when compared to the natural cycle, and ovulation caused by the onset of the LH surge is always a concern, placing a great stress on medical practitioners. Meanwhile, Clomiphene citrate is a leading competitive inhibitor of estrogen, which works as a potent estrogen antagonist in the hypothalamus. Under the influence of Clomiphene citrate, the positive feedback is interrupted, increasing the GnRH and delaying the LH surge. Therefore, using Clomiphene citrate as a measure to delay the LH surge in addition to Femara can be expected to improve ART performance and at the same time, contribute to reducing the stress placed on medical practitioners.
The agent for the antagonistic action of Clomiphene citrate is thought to be enclomiphene, also known as trans-isomer, one of the two stereoisomeric forms of Clomiphene citrate. Its half-life is considered to be less than 1 day. This means its effect fades out within a few days after the end of administration, and thus, continuous use is necessary in order to delay the LH surge and to avoid ovulation. We administer Femara for 3 days from d3 to d5 and 12.5 mg of Clomiphene citrate on d6 onward until the day oocyte maturation is triggered (Fig. 6.6).
Fig. 6.6
The procedure of the Femara-Clomiphene cycle
As is the case with the Femara cycle, the FSH level in the Femara-Clomiphene citrate cycle peaked on d6 immediately after the end of Femara administration and then decreased gradually. However, it began to rise gradually from 2 days before the oocyte maturation was triggered.
The LH level hardly changed after d3 and its rate of increase remained at 43 % even on the last day. This result shows that the Clomiphene citrate’s competitive inhibition action on E2 was at work, blocking the positive feedback and inhibiting FSH and LH surges.
The rate of increase in E2 level in this cycle is always higher and more stable than those of the natural cycle and the Femara cycle, making the prediction of the correct timing of oocyte retrieval possible from 5 days before the oocyte maturation triggering. Therefore, in this cycle, it is possible to plan the patients’ visiting schedule confidently which significantly alleviates physicians’ psychological burden, compared to the natural cycle and the Femara cycle (Fig. 6.7).
Fig. 6.7
Hormonal dynamics of the Femara-Clomiphene citrate cycle
The number of oocyte retrievals in Femara-Clomiphene citrate cycle IVF carried out in our clinic for the 2 years from January 1, 2011, to December 31, 2012, was 1456 cases (1456 cycles). The average age of women who underwent the treatment was 38.4 years old (30–45 years old). The level of d3 AMH was 15.3 pM (0.0–126.5 pM), and that of d3 FSH was 7.6 IU/L (1.0–20.3 IU/L). The average number of visits to the clinic was 3.86 ± 1.10 times, and the average number of days from the start of the cycle to the day of the oocyte retrieval was 13.9 ± 2.7 days. The number of cycles where the dominant follicle had ovulated before the day of retrieval was 54 cycles (3.7 %), from which only small follicles were aspirated and retrieved.
The average number of aspirated follicles per patient was 1.69 for dominant follicles developed to a diameter of 11 mm or more and 9.54 for small follicles, the diameter of which was less than 11 mm. The oocyte retrieval rate was 64.4 % and 37.2 %, respectively. The rate of retrieving MII-stage oocytes was 88.9 % and 34.2 %, respectively. The rate of retrieved oocytes growing to blastocysts was 34.4 % among oocytes derived from dominant follicles and 9.5 % among oocytes derived from small follicles. The rate of ongoing pregnancies, including cases where no oocytes were retrieved, was 19.2 %, and 38.3 % of those were achieved from the small follicles (Table 6.4).
Table 6.4
Outcome of Femara-Clomiphene citrate cycle
|
Femara-CC cycle |
Follicular size |
Average age (years) |
d3AMH level (PM) |
d3FSH level (IU/L) |
Total follicular aspiration no. of cycles |
Folicular aspiration |
Oocyte retrieval |
Growth to blastocyst |
No. of cycles embryo was transferred |
No. of cycles pregnancy was achieved |
Rate of ongoing pregnancies (%) |
||||
|
No. of cycles |
Average no. of follicles aspirated |
No. of cycles |
Average no. of oocytes retrieved |
No. of cycles |
Average no. of oocytes grew to blastocysts |
||||||||||
|
Age categories |
30–35 |
Large |
33.3 |
24.3 |
6.9 |
306 |
297 |
1.89 |
221 |
1.51 |
131 |
1.24 |
120 |
67 |
21.9 |
|
Small |
303 |
13.87 |
270 |
5.31 |
95 |
1.79 |
89 |
44 |
14.4 |
||||||
|
36–40 |
Large |
38.2 |
15.0 |
7.6 |
724 |
696 |
1.63 |
539 |
1.39 |
226 |
1.17 |
203 |
87 |
12.0 |
|
|
Small |
690 |
9.59 |
602 |
4.11 |
175 |
1.45 |
170 |
60 |
8.3 |
||||||
|
41–45 |
Large |
42.3 |
9.4 |
8.3 |
426 |
409 |
1.65 |
321 |
1.38 |
92 |
1.08 |
87 |
19 |
4.5 |
|
|
Small |
402 |
6.17 |
336 |
3.09 |
40 |
1.18 |
33 |
3 |
0.7 |
||||||
|
Total |
38.4 |
15.3 |
7.6 |
1456 |
1402 |
1.69 |
1081 |
1.41 |
449 |
1.17 |
410 |
173 |
11.9 |
||
|
1395 |
9.54 |
1208 |
4.09 |
310 |
1.52 |
292 |
107 |
7.3 |
|||||||
Oocyte Retrieval in the Clomiphene-FSH Cycle
Because Clomiphene citrate’s action in retaining the endogenous FSH level is limited, the FSH level decreases as the E2 level increases. Therefore, this method was devised in an effort to retain the FSH level at 7.5 IU/L or above, which is equivalent to the d3 FSH level in the natural cycle, by replenishing exogenous FSH. Exogenous FSH basically has an accumulative effect. By administering 75 IU of FSH daily, the FSH level reaches 5 IU/L on the third day of administration and keeps a plateau level of 6–7 IU/L. As this level is excessive when taking the presence of endogenous FSH into consideration, it is expected that a sufficient dose should be around 37.5–50 IU a day. With this method, we measure the FSH level on d3, d6, d9, and d12. On d6 and subsequent visits, we decide the necessity for and the dose of the exogenous FSH based on the serum FSH concentration measured. The basic dose is 37.5 IU. However, as the endogenous FSH level decreases against the increase in E2, the dose should be decided to some extent in such a way as to compensate for the decline.
With this method, where the E2 level reaches as high as 1000 pg/mL, the LH surge needs to be potently inhibited. Therefore, Clomiphene citrate administration begins from d3 at a daily dose of 25 mg and continues until the day before oocyte maturation is triggered (Teramoto and Kato 2007) (Fig. 6.8).
Fig. 6.8
The procedure of the Clomiphene-FSH cycle
In our study, the FSH level continued to keep a level of 8 IU/L or more until the middle of the cycle and remained steady until the last day.
The LH level also continued to be steady at lower level compared to other stimulation cycles.
The increase in rate of E2 was already high even at 6 days before triggering oocyte maturation and showed a steady increase. The author decided to trigger oocyte maturation using GnRHa when the E2 level per dominant follicle reached 200 pg/mL or more (Fig. 6.9).
Fig. 6.9
Hormonal dynamics of the Clomiphene citrate-FSH cycle
The number of oocyte retrievals in Clomiphene citrate-FSH cycle IVF carried out in our clinic for the 2 years from January 1, 2011, to December 31, 2012, was 1705 cases (1705 cycles). The average age of women who underwent the treatment was 36.3 years old (30–45 years old). The level of d3 AMH was 26.8 pM (1.6–113.8 pM), and that of d3 FSH was 8.2 IU/L (0.1–23.2 IU/L). The average number of visits to the clinic was 4.67 ± 1.04 times, and the average number of days from the start of the cycle to the day of the oocyte retrieval was 15.9 ± 2.2 days. The average total dosage of exogenous FSH was 472 ± 192 IU. The average number of aspirated follicles per patient was 8.68, the average number of oocytes retrieved was 4.55, and the rate of MII-stage oocytes retrieved was 77.1 %. The number of cycles where the dominant follicle had ovulated before the day of retrieval was 3 cycles (0.18 %).
The rate of retrieved oocytes growing to blastocysts was 72.3 %, calculated on the basis of cycles, and 35.7 % on the basis of oocytes. The rate of ongoing pregnancies, including cases where no oocytes were retrieved, was 37.5 % (Table 6.5).
Table 6.5
Outcome of the Clomiphene-FSH cycle
|
CC-FSH cycle |
Average age (years) |
d3AMH level (pM) |
d3FSH level (IU/L) |
Follicular aspiration |
Oocyte retrieval |
Growth to blastocyst |
No. of cycles embryo was transferred |
No. of cycles pregnancy was achieved |
Rate of ongoing pregnancies (%) |
||||
|
No. of cycles |
Average no. of follicles aspirated |
No. of cycles |
Average no. of oocytes retrieved |
No. of cycles |
Average no. of oocytes grew to blastocysts |
||||||||
|
Age categories |
30–35 |
33.2 |
29.3 |
7.8 |
686 |
10.17 |
658 |
5.10 |
514 |
2.49 |
706 |
402 |
47.2 |
|
36–40 |
37.9 |
24.5 |
8.5 |
866 |
7.89 |
815 |
4.23 |
581 |
2.12 |
797 |
363 |
34.4 |
|
|
41–45 |
41.7 |
21.7 |
8.4 |
153 |
6.41 |
135 |
3.82 |
67 |
1.58 |
84 |
18 |
11.8 |
|
|
Total |
36.3 |
26.8 |
8.2 |
1705 |
8.68 |
1608 |
4.55 |
1162 |
2.25 |
1587 |
783 |
37.5 |
|
As the Clomiphene-FSH Cycles included many oocyte retrieval cycles in which aspiration of small follicles was not intended, it was impossible to show the results by the size of the follicles in this table
With the Clomiphene citrate-FSH cycle, oocytes are mainly retrieved from the developed follicles, and therefore, uniformity of follicular growth becomes an issue. In order to achieve this, it is necessary to equalize the size of the antral follicles by administering birth control pills in the previous cycle. What matters then is the fact that due to the FSH inhibition by the birth control pills, the number of follicles decreases, and AMH level is reduced by around 30 %. Therefore, this method is not applicable to women with a low AMH in the first place, and the prerequisite condition for indication to this method is to have an AMH level of at least 15 pM. Because of those limitations and the fact that the improvement in the success rate of the Femara-Clomiphene citrate protocol is so significant, the author has ceased performing this Clomiphene-FSH cycle since May 2012.
Conclusions
In natural cycle IVF/IVM, improvement of the success rate was sought by concurrently performing IVF to the MII-stage oocytes derived from the dominant follicle, the diameters, of which were 12–14 mm, and IVM to the immature oocytes derived from the small follicles (Chian et al. 2004). However, it has been concluded that the developmental ability of the oocytes from the small follicles is reduced when the diameter of the dominant follicle goes above 14 mm (Son et al. 2008). In another report on IVF, it was reported with surprise that MII-stage oocytes were retrieved from the small follicles, the size of which were less than 11 mm, in response to hCG (Yang et al. 2010). However, this surprise concerned the fact that the oocytes from the small follicles were able to mature in response to hCG. This surprise was about something quite different from what the author is discussing in this section, the fact that the oocytes from the small follicles could be matured even after the dominant follicle, both in terms of E2 and its size, had been definitely recruited. Traditionally, it has been thought that the LH receptor only developed after the dominant follicle had been recruited and that the small follicles have a low LH receptivity and therefore, could not luteinize nor resume meiosis (Richards 1994). However, the author has retrieved oocytes from small follicles at the rate of 40 %, the size of which was only 2–6 mm in diameter, at the very timing in which the dominant follicle is ovulating. Moreover, more than 30 % of those oocytes were MII-stage oocytes; their development rate to blastocysts was equivalent to that of the dominant follicle, and more than 2000 cases have resulted in either live births or ongoing pregnancies. It is true that 60 % of the oocytes retrieved from the small follicles are at germinal vesicle (GV) stage and that the developmental rate of those oocytes to blastocysts is as low as 2.9 %. However, the rate is not different from those of the GV-stage oocytes retrieved in the normal IVF, and it is thought that the reason for the low rate was due to the involvement of a high rate of chromosomal defects (Nugeira et al. 2000).
But, even at the time when the dominant follicle is ready to ovulate and the LH surge has begun, 40 % of the oocytes in the small follicles are MII-stage oocytes, and 29 % of those can develop into blastocysts, which are worth transferring. Those numbers are not so small that they can be ignored from the point of view of controlled stimulation cycles and the culturing results of IVM (Lim et al. 2009).
Techniques of retrieving matured oocytes from small follicles will transform the landscape of ART. Furthermore, a tiny amount of medication, namely, the combined use of Femara and Clomiphene citrate, not only increases the number of oocytes retrieved from the small follicles and improves the maturity rate but also relieves medical practitioners of the necessity for frequent hormonal measurement. These protocols do not require a cumbersome culturing process, such as that for IVM, are extremely economical, place few burdens on the body and have a high success rate. In other words, these methods will make an ideal ART possible. What is required in order to achieve this is the skill of the physician only – not the culturing technique, nor new drugs. I believe that a new door to ART can be opened by respecting and becoming versed in the hormonal dynamics of the natural cycle and, most of all, by becoming thoroughly adept at oocyte retrieval techniques.
References
Chian RC, Lim JH, Tan SL. State of the art in in-vitro oocyte maturation. Curr Opin Obstet Gynecol. 2004;16:211–9.CrossRefPubMed
Ingram DL. The effect of gonadotrophins and oestrogen on ovarian atresia in the immature rat. J Endocrinol. 1959;19:117–22.CrossRefPubMed
Lim JH, Yang SH, Xu Y, Yoon SH, Chian RC. Selection of patients for natural cycle in vitro fertilization combined with in vitro maturation of immature oocytes. Fertil Stertil. 2009;91:1050–5.CrossRef
Nugeira D, Staessen C, Van de Velde H, et al. Nuclear status and cytogenesis of embryos derived from in vitro matured oocytes. Fertil Stertil. 2000;74:295–8.CrossRef
Richards JS. Hormonal control of gene expression in the ovary. Endocr Rev. 1994;15:725–51.CrossRefPubMed
Son WY, Chung JT, Herrero B, et al. Selection of the optimal day for oocyte retrieval based on the diameter of the dominant follicle in hCG-primed in vitro maturation cycles. Hum Reprod. 2008;23:2680–5.CrossRefPubMed
Teramoto S, Kato O. Minimal ovarian stimulation with clomiphene citrate: a large-scale retrospective study. RBM Online. 2007;15:134–48.PubMed
Yang SH, Qin SL, Xu Y, Yoon SH, et al. Healthy live birth from vitrified blastocysts produced from natural cycle IVF-IVM. Reprod BioMed Online. 2010;20:656–9.CrossRefPubMed
16 mm; estradiol concentration per follicle of 16 mm in diameter