Fig. 3.1
Differences in the endocrine milieu in follicles of NC-IVF (NC) and cIVF. Concentrations of different hormones in the follicular fluid at the time of follicle aspiration. All concentrations are significantly different in both therapies (n = 76) (Reprinted from von Wolff et al. 2014a)
The marked difference in the concentration of the putative implantation marker AMH in NC-IVF follicles raised the question, if the concentration of AMH correlates with other follicular fluid and serum parameters, suggesting a metabolic link. Such a direct or indirect link was analyzed by a regression analysis of AMH and testosterone. Testosterone concentrations are positively correlated (r = 0.35, p = 0.0002) with AMH concentrations, suggesting that the higher AMH concentrations in NC-IVF than in cIVF follicles are due to higher testosterone concentration in NC-IVF follicles (von Wolff et al. 2014a).
The significantly higher testosterone concentration in NC-IVF follicles might be either due to increased testosterone production due to increased LH activity in NC-IVF or due to an inhibition of the follicular aromatase, inhibiting the conversion of T in E2. A positive correlation of testosterone and LH (r = 0.48, p < 0.0001) (von Wolff et al. 2014a) suggests first a metabolic link between LH and testosterone and second that high testosterone concentration in NC-IVF follicles is probably due to much LH activity in NC-IVF than in cIVF. An inhibition of the aromatase activity with a reduced conversion of T into E2 seems to be unlikely as such an effect would result in an accumulation of testosterone with a non-linear correlation of T and E2, which could not be demonstrated by von Wolff et al. (2014a).
Two essential questions have derived from these results: (1) What significance do increased AMH concentrations have? (2) Which regulatory mechanisms lead to increased AMH concentrations?
The first question, the significance of follicular AMH on oocyte quality, cannot currently be answered easily, as the significance of AMH is unclear. However, it is proven that AMH is produced by granulosa cells (Vigier et al. 1984) and that atretic granulosa cells do not produce AMH (De Vet et al. 2002). The degree of apoptosis on the granulosa cells correlated with the developmental competence of the oocyte (Nakahara et al. 1997). These relationships lead to the hypothesis that a high AMH concentration may have no direct effect on the oocyte but is only a marker for the granulosa cell function and as such, is of relevance for the function of the oocyte. Several studies hypothesize that there is a direct link between the oocyte function and the AMH production of the granulosa cells. The oocyte seems to activate various physiological processes in the surrounding granulosa cells. In a mouse model, it was shown that the oocyte influenced the AMH expression by this mechanism (Salmon et al. 2004).
The second question, which regulatory mechanisms lead to AMH production, can only be answered indirectly at the moment. Andersen and Lossl (2008) indirectly proved that during IVF treatment, induction with human chorionic gonadotropin (hCG before gonadotropin stimulation leads to higher intrafollicular androgen concentrations as well as increased follicular AMH concentrations, i.e., that intrafollicular testosterone possibly stimulated the AMH production. Based on our study results, this means that the increased androgen concentrations in the naturally matured follicles are the reason for the increased AMH concentrations. The precise mechanisms for the stimulation of AMH production are, however, still unknown. Androgens may induce FSH receptor expression in the granulosa cells (Weil et al. 1999). A direct stimulatory effect of LH is also possible. A stimulatory effect of hCG/LH on the AMH production in granulosa cells of polycystic ovaries (PCO) patients but not in the granulosa cells of healthy women was found by Phy et al. (2004). In our study, we detected a strong correlation between the follicular testosterone concentration and the AMH concentration, which supports the supposition of a dependency of the AMH concentration on the testosterone concentration.
This in turn raises the question of which mechanisms lead to an increased follicular testosterone concentration. The increased androgen concentrations in naturally matured follicles can be based upon two different mechanisms. Either AMH inhibits the aromatase, as a result of which androgens accumulate or androgenesis in the theca cells is (mediated by LH) increased.
The second hypothesis implies that androgen synthesis is increased in naturally matured follicles in the theca cells. Thecal androgen synthesis is stimulated by LH. This appears to confirm the hypothesis that in NC-IVF, the LH concentrations in the serum as well as in the follicular fluid are significantly higher, because LH suppression using gonadotropin-releasing hormone (GnRH) analogs or GnRH antagonists, as in cIVF, is not performed.
The unphysiological suppression of LH in cIVF seems to affect a cascade of metabolic changes within the follicle (Fig. 3.2). This possibly leads to lower implantation potential of the oocytes in cIVF and is probably one reason for the higher implantation potential in NC-IVF.
Fig. 3.2
Model for the effect of LH on the follicular endocrine milieu. The model is based on the data presented in Fig. 3.1 and on the study by von Wolff et al. (2013a) and Vaucher et al. (2013). Suppressed LH, as found in cIVF, due to GnRH agonists or GnRH antagonists result in lower follicular testosterone concentrations and thereby, in lower AMH concentration, leading possibly to a lower implantation potential of the oocyte in cIVF. AMH has been shown to be a marker for the implantation potential of the oocyte (Fanchin et al. 2007) (dark blue: theca cells, light blue: granulosa cells, green: oocyte)
Treatment Protocol in NC-IVF
Theoretically, NC-IVF is performed without any hormones. Even hCG is not used to induce follicle maturation. Practically, this approach is not useful, as the efficacy of the treatment would be far too low to be able to compete with cIVF.
The requirements of NC-IVF are therefore:
1.
As few consultations as possible before follicle aspirations to reduce the patient’s effort to a minimum
2.
High yield of oocytes and the highest possible transfer rate per treatment cycle
3.
Simple and almost painless follicle aspiration, as described in the chapter below
4.
Lowest possible treatment costs combined with the highest possible pregnancy chance per treatment time, as described in the chapter below
To fulfill the first two requirements, the physician can either monitor the follicles every 1–2 days to identify the ideal day for ovulation induction, which requires many expensive and time-consuming consultations. Or, the physician uses a treatment concept that allows reduction of consultations without reducing pregnancy rate.
As gonadotropins and GnRH antagonists are expensive and as Clomiphene citrate at the common dosage of 50 mg/day has negative effects on endometrial function and can induce cyst formations resulting in cycle cancelations in the following cycle, we have introduced a treatment protocol with very low dosages of Clomiphene citrate (von Wolff et al. 2014b).
Patients received 25 mg Clomiphene citrate per day, started on day 6 or 7 until 24 h before ovulation induction with hCG is given (Fig. 3.3). The first consultation takes place on cycle day 10 ± 2. Follicular diameter and endometrial thickness are analyzed by ultrasound and concentrations of estradiol (E2) and LH by serum analysis. The results are used to calculate the expected time of ovulation. In a few cases, a second consultation is required. 5,000 IU of human chorionic gonadotropin is given 36 h before follicle aspiration when the follicle is ≥15 mm and estradiol concentration is ≥700 pmol/L.
Fig. 3.3
Treatment protocol of NC-IVF as performed in Berne (clomiphene citrate 25 mg/day = CC25)
The results of this treatment protocol are shown in Fig. 3.4. Less than 5 % of patients described mild side effects such as hot flushes or headache. These results clearly indicate the superiority of the treatment protocol in respect to the transfer/cycle ratio. They also demonstrate that Clomiphene citrate did not reduce pregnancy rate.
Fig. 3.4
Outcome of the 108 NC-IVF and the 103 ccNC-IVF (with clomiphene citrate) therapies. Significant differences of the treatment steps between the treatment groups are marked by an asterisk. Patients underwent both kind of therapies without randomization. One hundred three patients started with an NC-IVF cycle and, if not pregnant after the first cycle, followed by a ccNC-IVF cycle. Fourteen patients started with a ccNV-IVF cycle (Modified according to von Wolff et al. 2014b)
Therefore, to perform treatment cycles with the lowest possible number of consultations, lowest possible treatment costs and highest possible pregnancy chances per cycle, the use of Clomiphene citrate at low dosages should be considered. Accordingly, the calculation of treatment costs and pregnancy rate per treatment time in the chapter below is based on the treatment results achieved by the use of low dosages of Clomiphene citrate.
Follicle Aspiration in NC-IVF
The technique of follicle aspiration plays an important role in NC-IVF as first, it needs to be highly efficient to get the highest possible number of oocytes and second, it should be simple and not very painful in order to allow monthly treatment cycles. To improve the efficacy of follicle aspiration, we have reintroduced follicle flushing in monofollicular NC-IVF.
This change in our treatment protocol is in contrast to the common scientific evidence. A Cochrane analysis even concluded that “there is no evidence that follicular aspiration and flushing is associated with an increase in oocyte yield.” Follicular flushing even seemed to be disadvantageous as “the operative time is significantly longer and more opiate analgesia is required for pain relief during oocyte retrieval” (Wongtra-Ngan et al. 2010). However, this statement was based on different studies in which follicle aspiration was performed in polyfollicular IVF (normal responders) and oligofollicular IVF (low responders) following controlled ovarian hyperstimulation.
We therefore performed a clinical study to analyze if this statement also applies to NC-IVF (von Wolff et al. 2013a): 164 aspirations were performed in monofollicular IVF cycles. Follicles were aspirated without any anesthesia, using 19G single-lumen needles (250 mmHg). Pain intensity during follicle aspiration was low (Fig. 3.5). After initial aspiration, follicles were flushed and aspirated three times each with 2 mL flushing medium with heparin. Total oocyte yield/aspiration was 44.5 % in the aspirate, 20.7 % in the first flush, 10.4 % in the second flush, and 4.3 % in the third flush (Table 3.1). By flushing, the total oocyte yield increased significantly (p < 0.01) by 80.9 % from 44.5 to 80.5 %.
Fig. 3.5
Pain intensity in NC-IVF aspiration without analgetics/anesthesia. Pain intensity of follicle aspiration using a 19G aspiration needle in relation to venous blood sampling according to a survey performed in Berne
Table 3.1
Effect of follicle flushing on oocyte yield in NC-IVF
|
NC-IVF | ||||
|---|---|---|---|---|
|
Aspirations (n) |
164 | |||
|
Mean age (y) ± SD |
37.0 ± 3.8 (28–45)
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