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15 Ultrasound inFollicle Monitoring forOvulation Induction/IUI
269
is suggested that hMG/hCG administration should be interrupted in the presence of 11 or more preovulatory follicles, especially if most of them are immature (<9mm).

Conclusion

Ultrasound is the most powerful tool to monitor normal and stimulated cycles; predictions of the assumed time of ovulation allow optimal timing of various procedures such as insemination, ovum aspiration, etc.
In stimulated cycles sonographic detection of too many follicles allows withholding hCG induction thus preventing hyperstimulation.
In the past ovulation function was monitored by estradiol estimation; since the development of sophisticated ultrasonographic techniques, moni­toring of ovarian follicular growth by ultrasound became a routine addition to estradiol measure­ment in most clinics.
Accumulating data based on the Cochrane Database [38] indicate that there is no evidence from randomized trials to support cycle monitoring by ultrasound plus serum estradiol as more efca­cious than cycle monitoring by ultrasound only on outcomes of live birth and pregnancy rates.
As far as OHSS, randomized trial with a suf­ciently large sample is needed. Until such a trial is considered, ultrasound plus serum estradiol may need to be retained as a precautionary good practice point, in patients prone to develop hyperstimulation.

References

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6. Shrestha SM, Costello MF, Sjoblom P, McNally G, Bennett M, Steigrad SJ, et al. Doppler ultrasound assessment of follicular vascularity in the early fol­licular phase and its relationship with outcome of in-vitro fertilization. J Assist Reprod Genet. 2006;23(4):161–9. Epub 2006 Apr 22.
7. Coulam CB, Goodman C, Rinehart JS. Colour Doppler indices of follicular blood ow as predictors of pregnancy after in vitro fertilization and embryo transfer. Hum Reprod. 1999;14(8):1979–82.
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2D Ultrasound inFollicle Monitoring forART
GianlucaGennarelli, TomerTur-Kaspa, AlbertoRevelli, MetteToftager, andDavidP.Cohen
16

Introduction

Ultrasound (US) imaging represents an invaluable tool for the reproductive endocrinologist working with assisted reproductive technologies (ART). It allows for noninvasive monitoring of the ovarian response during controlled ovarian stimulation (COS). Ultrasound imaging of follicle maturation was rst performed in 1978, when a linear corre­lation between follicle size and serum estradiol levels was demonstrated [1]. Later, studies per­formed in the early 1980s conrmed the relation­ship between serum estrogen concentration and both the number and size of growing follicles. An increase in uterine size and endometrial thicken­ing during stimulation was also described [2]. Currently two-dimensional (2D) ultrasound imag­ing is used diagnostically and in treatment, for evaluation of all pelvic organs and for transvagi­nal procedures, respectively.
G. Gennarelli · A. Revelli Sant’Anna Hospital, University of Turin, Department of Obstetrics and Gynecology, Turin, Italy
T. Tur-Kaspa Wesleyan University, Middletown, CT, USA
M. Toftager Fertility Clinic, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark
D. P. Cohen (*) Institute for Human Reproduction, Chicago, IL, USA e-mail: dcohen@infertilityihr.com
Most importantly, the introduction of transvagi­nal ultrasound in 1983 dramatically improved the safety and success of ART. Today, transvaginal ultrasound imaging is imperative when adminis­tering gonadotropins for COS, in order to optimize treatment, reduce the risk of multiple pregnancies, and avoid potentially life- threatening side effects, such as the ovarian hyperstimulation syndrome (OHSS). Indeed, the use of 2D ultrasound for assessing follicular development during gonado­tropin stimulation for ART is virtually universal.
More recently, the addition of power Doppler added to 2D ultrasound has been proposed with the aim of studying ovarian and endometrial blood ow. By monitoring perifollicular blood ow, the physician can identify follicles whose oocytes may result in embryos of better develop­mental competence [3] (see Chaps. 3 and 4).
Why Monitor theFollicular Phase?
During in vitro fertilization (IVF) treatment, exogenous gonadotropins induce the growth of a cohort of ovarian follicles. Monitoring this phe­nomenon is essential in order to optimize and individualize IVF treatments, and ultrasound examination is effective at all stages of the COS.
At a baseline examination, before initiating gonadotropin administration, US monitoring can identify ovarian abnormalities, such as follicular cysts or adnexal masses that need to be addressed
© Springer Nature Switzerland AG 2019 L. A. Stadtmauer, I. Tur-Kaspa (eds.), Ultrasound Imaging in Reproductive Medicine,
https://doi.org/10.1007/978-3-030-16699-1_16
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in advance. Furthermore, a baseline count of small antral follicles (AFC) provides a hint into the probability of success, and it helps identify the optimal starting dose of gonadotropin and the most appropriate type of stimulation protocol. It has been repeatedly shown that the AFC corre­lates positively with the number of oocytes retrieved, the proportion of mature oocytes from the oocytes retrieved, and ultimately the probability of success of the IVF treatment [4, 5], provided a transvaginal US probe with frequency 7MHz is used and the operator is adequately experienced [6].
Furthermore, in cases when GnRH agonists are used to block the endogenous gonadotropin secre­tion from the pituitary, US examination can verify that effective pituitary-ovarian axis suppression has been obtained before starting COS. Additionally, other useful information can be obtained before stimulation, for example, the ease with which the ovaries are accessible to transvaginal needle aspira­tion. Finally, during ovarian stimulation US moni­toring is crucial in order to determine whether the initially suggested gonadotropin stimulation dos­ing protocol is optimal or needs to be adjusted.
As previously mentioned US helps predict the number of oocytes expected to be obtained, but it is also of paramount importance for minimizing the risk of OHSS.When OHSS is incipient, free uid may be identied in the peritoneal cavity. This sign, together with the number and size of the growing follicles and the ovarian volume, is infor­mation currently used to help predict and prevent OHSS.Interestingly, to prevent OHSS in the poly­cystic ovarian syndrome patient resistant to stan­dard ovulation induction, ultrasound has been suggested as a tool to guide ovarian drilling [7].
Finally, ultrasound monitoring is critical in determining the optimal time for induction of the nal maturation of the growing follicles. As a matter of fact, most clinicians agree that ultra­sound examination is the best modality, among those available, to monitor ovulation induction.
which occur during normal folliculogenesis. Follicles grow through two stages, the rst being gonadotropin-independent and the second gonadotropin- dependent. Primordial follicles consist of an oocyte with a thin layer of granulosa and stromal cells, and at this stage, follicles can­not be seen by ultrasound. By the time follicles develop an antral cavity, they become ultrasono­graphically visible, and, importantly, they have reached the gonadotropin-dependent stage. These antral follicles measure between 2 and 10mm in mean diameter and represent the pool of follicles that may be stimulated in the ensuing follicular phase. In a natural cycle, one follicle is ultimately selected for ovulation, and that selection process occurs during the mid-follicular phase of the ovarian cycle, when the endogenous pituitary fol­licle stimulating hormone (FSH) level falls in response to the increasing ovarian estradiol pro­duction. Decreasing FSH levels promote a selec­tion process in which each of the follicular microenvironments competes for the diminishing FSH needed to stimulate granulosa cells to pro­duce aromatase and continue growth. Aromatase, in turn, is necessary to convert testosterone and androstenedione produced in the peripheral theca cells into estradiol and estrone, respectively, in the granulosa cells. Failure of this conversion from androgen to estrogen leads to an elevated androgen-to-estrogen ratio in the microenviron­ment of the follicle and then to follicular atresia. From this cursory review of the anatomy and physiology of oocyte maturation, it is easy to see how ovulation-inducing agents that either indi­rectly increase endogenous FSH (e.g., clomi­phene citrate) or directly add exogenous FSH to the system diminish the competition among fol­licles for FSH and permit the development of multiple dominant follicles [5, 6].

Monitoring Follicular Maturation

Normal Folliculogenesis

Before reviewing the parameters of follicular growth studied with 2D ultrasound during ART treatment, it is helpful to summarize the events
Methods forMonitoring
It is difcult to predict the optimal number of growing ovarian follicles in an IVF cycle, since there is considerable variation in ovarian response among patients. The ovarian response depends on
16 2D Ultrasound inFollicle Monitoring forART
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age, ovarian reserve, the type of stimulation pro­tocol, the FSH dose, FSH receptor polymor­phisms, etc.
Follicular maturation in IVF cycles can be monitored clinically by:
• Serum estradiol value alone
• 2D ultrasound alone
• 3D ultrasound alone
• Serum estradiol and ultrasound combined
• Supplemental power Doppler imaging
There are numerous studies on the use of these different methods for monitoring follicular maturation. Traditional monitoring of an IVF treatment cycle includes a combination of 2D ultrasonography and serum estradiol concentra­tions and has long been accepted as the gold standard. However, whether estradiol monitor­ing adds any advantage to US monitoring remains controversial. Obviously, US examina­tion provides more accurate measurement of fol­licle number and size than can be obtained by serum estradiol alone. Whereas available data are not conclusive, the most recent literature does not support the notion that measuring estra­diol serum concentrations during COS is of sig­nicant advantage in terms of both results and safety [8].
The frequency of US check and the time points at which the dose of exogenous gonadotro­pins could be changed show wide variations, depending on the experience of the clinician and the routine applied at each IVF clinic. Some monitoring methods are very complex, whereas other methods are simpler. However, whatever the complexity of COS monitoring, the outcomes of IVF cycles does not differ signicantly [9].
When viewed on ultrasound, follicles appear as echo-free structures within the more echo­genic ovarian tissue. By convention, follicle size in 2D is estimated by calculating the mean of the maximum follicular internal diameter in two perpendicular planes [10]. Alternatively, the fol­licle size can be estimated in three dimensions, the x, y, and z planes, and using this technique, it is possible to calculate the volume of each fol­licle. Most recently, three-dimensional (3D) software programs that distinguish the echo-
genicity of the well-circumscribed, sharp-edged, echolucent follicular uid from the surrounding greater echogenicity of the ovarian cortical parenchyma have automated this process and allow follicular volume calculations from data derived from 2D images. The clinician can sim­ply sweep through the ovarian tissue, and the stored image data is analyzed, reducing the time usually needed when multiple diameters are measured for each follicle, separately. This option is particularly useful in busy practices, when a large number of follicular monitoring scans are performed every day.
Standard Ultrasound Monitoring Program
Follicular growth can be directly monitored with 2D ultrasound, since the follicular diameter increases during development in the follicular phase. Most clinicians measure the follicles at baseline (Fig.16.1), prior to initiating gonadotro­pin stimulation, and then again after approxi­mately 5days of gonadotropin stimulation, and then every 24–48hours depending on the rate of development (Figs.16.2, 16.3, 16.4, and 16.5). Once the mature follicle measures 17–21 mm, the physician can trigger ovulation with human chorionic gonadotropin (hCG, a luteinizing hor­mone (LH) surrogate) (Fig. 16.6) or a gonadotropin- releasing hormone analog. In sim­ple ovulation induction, conrmation of ovula­tion can be demonstrated by ultrasound as well, observing the sudden change of the intact folli­cle, made up of concentric layers of theca cells surrounding granulosa cells enclosing the follicu­lar uid and the oocyte that is suddenly changed after follicular rupture and ovulation. Physiologically, it is at this moment that both testosterone-secreting theca cells and estradiol- secreting granulosa cells convert intra­cellular steroid production to preferentially favor progesterone production. Thus, ovulation is accompanied by a dramatic loss of the concentric architecture of the preovulatory follicle and an increase in blood supply, presumably aimed at increasing dramatically the output of progester­one into the bloodstream. A very specic, nearly
276
Fig. 16.1 Baseline,
prior to initiating gonadotropin stimulation. Ovary with antral follicles
Fig. 16.2 Stimulation
day 5, showing recruited follicles measuring 10–12mm
G. Gennarelli et al.
Fig. 16.3 Stimulation
day 7, showing ovary with leading follicle >12mm
16 2D Ultrasound inFollicle Monitoring forART
Fig. 16.4 Stimulation
day 9, showing ovary with growing follicles
Fig. 16.5 Stimulation
day 11, 2–3 follicles measuring 17–18mm
277
Fig. 16.6 Day of
ovulation induction. Leading follicles measuring more than 18mm
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Fig. 16.7 Corpus
luteum cyst– “ring of re”
G. Gennarelli et al.
pathognomonic “ring of re” ultrasound nding is easily discerned around each corpus luteum at this time (Fig.16.7). Doppler technology added to the 2D image enables observation of this typi­cal ow pattern which, in combination with the classic echogenicity of the corpus luteum, makes the diagnosis of ovulation quite simple [11].
Follicular Size andVolume
During COS for IVF, it is realistic to recruit 5–10 ovarian follicles in each ovary; however the num­ber, the rate of growth of each follicle, and the number of stimulation days can vary greatly between patients.
After 6–7 days of gonadotropin stimulation, follicles measuring more than 10mm are expected.
Once a dominant follicle measures more than 12mm in mean diameter, a growth rate of 2mm (1–3mm) per day is expected [12]. Growth con­tinues until follicular maturation at 17–21mm, and at that point, the oocyte is ready to ovulate, complete meiosis in response to the LH trigger, and be released into the peritoneal cavity.
oocytes to be fertilized; mature oocytes are dened as those that have completed meiosis I, extruded the rst polar body, and rearrested in metaphase of meiosis II.Most commonly hCG is administered to mimic the endogenous LH surge, which in turn triggers meiotic reinitiation from the oocyte’s pro­phase I resting state. As noted previously, stimula­tion protocols vary and are often modied during the stimulation. However, in most cases ovulation is triggered when 3 follicles 17mm in diameter are identied on ultrasound. Alternatively, the trig­ger is administered either when 3 follicles, each with a maximum diameter of 18mm, are identied or when 1 follicle of 18mm and 3 follicles of 15mm are observed. Other criteria have taken into consideration serum estradiol levels as well; in these criteria, hCG is usually administered when the leading follicle reaches 18–20 mm and the coincident serum estradiol level suggests “satis­factory” follicular development. More in detail, it is suggested that induction of the nal oocyte mat­uration be performed in the presence of at least one follicle 20mm and a serum estradiol level 1200pg/mL [13]. So far, there are no conclusive data in favor of any specic criteria.
Criteria Used forTriggering Ovulation
Whereas the criteria used for triggering ovulation and inducing the nal oocyte maturation vary between protocols, all aim to produce mature
How toPredict Retrieval ofMature Oocytes?
Follicular size and the volume of follicular uid have traditionally been recognized as possible
16 2D Ultrasound inFollicle Monitoring forART
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predictors of oocyte quality, i.e., oocytes that will be fertilized and result in embryos that implant and progress into a viable pregnancy [14]. However, attempts to nd a universally accepted threshold of a worth-to-be-punctured follicle size have been disappointing, due to conicting results. What is commonly accepted is that a large follicle is more likely to lead to the retrieval of a mature oocyte than a smaller follicle. The correlation between the follicle size and the like­lihood of retrieving a mature oocyte may reect the notion that larger follicles have completed the maturation process and released the oocyte­cumulus cell mass as a free-oating structure in the antral uid just before follicle rupture. According to early data from Teissier etal. [15], 14 mm in diameter should be considered the threshold follicle size to get an acceptable chance of nding meiotically competent oocytes at retrieval, both in normal and polycystic ovaries. However, follicles measuring as small as 11–15mm have been reported to have as much as a 50% chance of yielding a mature oocyte [16].
Conversely, follicles with a mean diameter above 22mm result in lower recovery of mature, fertilizable oocytes as they often contain postma­ture eggs, probably the result of intrafollicular atresia and degenerative phenomena [17].
More recent studies have observed a correla­tion between oocyte competence and ultrasound­measured follicular size prior to trigger and oocyte retrieval. A higher proportion of immature oocytes (germinal vesicle-GV-stage) are indeed found in smaller follicles, particularly those below 12mm in mean diameter. It is important to remember, however, that this is not a universal nding since even small follicles can generate mature MII oocytes [18]. Wittmaack et al. [19] found the optimal follicle volume to be between 1ml, which corresponds to 12mm in mean diam­eter, and 6–7 ml, corresponding to 24 mm in mean diameter. Higher oocyte recovery rates, higher fertilization rates, and higher cleavage rates for follicles in this interval were reported. However, within this range, the higher the follic­ular volume, the higher the fertilization rate and the better the embryo quality. Furthermore, in sync with those observations, a recent study
showed that delaying the oocyte maturation trig­ger by 1 day, once the leading follicles have reached 18 mm in diameter, would allow the retrieval of signicantly more mature oocytes, provided no progesterone rise is detected [20].
Most compelling, the results of a prospective study, including 9933 follicles from 535 IVF cycles, conrmed that oocytes from follicles with a volume <1ml (<12mm in mean diameter) had a signicantly lower fertilization rate than oocytes from larger follicles. However, the same oocytes, once fertilized, yielded embryos of com­parable quality; no signicant differences in the implantation rate, clinical pregnancy rate, or live birth rate were detectable from embryos derived from oocytes recovered from either small or large follicles [21].
It is also true that in general, it is not possible to identify a clear relationship between follicle size and the morphological quality of the embryos. This may be due to the fact that follicles with a volume within a certain interval contain oocytes that lead to embryos of comparable morphologic scores and/or to the fact that the male gamete con­tributes to embryo quality as well and should be considered along with the oocyte.
Likewise, follicular size, volume, morphol­ogy, and vascularity do not provide any predic­tive information on the chances of conception of a euploid fetus.
The Uterine Cavity andMonitoring ofEndometrial Proliferation
During the diagnostic evaluation of the infertile couple, assessment of the uterine cavity and the patency of the fallopian tubes is essential. The standard means of assessment of these two struc­tures is hysterosalpingography or more invasive laparoscopy and hysteroscopy. In the last decade, the use of saline infusion into the cavity to assess both the cavities in several planes using 2D and 3D (more recently) transvaginal imaging has made assessment of the uterine cavity and the tubes an ofce procedure that is less painful, involves no radiation, and is less expensive and more accessible.