Sonography Gynecology Infertility Assessment, Protocols, and Interpretation

Anatomy and Physiology

Understanding the different phases of the endometrium during the menstrual cycle and normal developmental phases in the ovarian follicles is essential for both initial baseline screening assessment and monitoring during natural, IUI, or IVF cycles.

Endometrium

The endometrium goes through consistent changes each month in response to the hormonal milieu. After sloughing of the endometrium and in response to rising estrogen, the endometrium thickens significantly during the follicular phase and shows a trilaminar pattern on ultrasound. After ovulation, in the luteal phase, the lining becomes decidualized, brighter, and is unilaminar until menstruation. The menstrual endometrium can have a heterogeneous appearance with hyper- and hypo- echoic features. Endometrial thickness (ET), endometrial morphological changes, and vascularity are assessed while performing ultrasound during natural or stimulated cycles. 

• Endometrial thickness -  The thickness of the endometrium is thin initially 4.4 +/- 0.2 mm after menses and increases to 10.3 +/- 0.3 mm before ovulation.[2] Raine-Fenning and Bakos also noted that in natural cycles, the mean endometrial thickness ranges from 10 to 12.5 mm before ovulation.[3][4]
• Endometrial morphology can be categorized into three patterns; Trilaminar,  homogenous, and heterogeneous (Figure 1). A trilaminar endometrium is triple-layered and characterized by a hypoechoic endometrium with a central line. This pattern is noted during the follicular phase from 5 to 6 days before ovulation and continues to the day of ovulation.[4] The presence of a trilaminar endometrium on the day of the trigger injection has been associated with higher pregnancy rates in IVF cohorts.[5] Once ovulation has occurred, the organized appearance of the trilaminar lining disappears, and the endometrium becomes more homogenous with poorly defined outer walls and the absence of the central echogenic line. During assisted reproductive technology (ART) cycles, this is often a sonographic clue before laboratory confirmation that the patient may have ovulated (Figure 2). A heterogenous endometrium may be seen while a patient is menstruating due to the presence of blood or clot or when intracavitary pathology (i.e., polyp, myoma, or hyperplastic tissue) is present.[6]  
• Endometrial Vascularity is classified into three grades based on the visual assessment of endometrium on power color flow doppler imaging before ovulation. Grade 1 shows no sub-endometrial blood flow, grade 2 shows sub-endometrial blood flow, and grade 3 shows both endometrial and sub-endometrial blood flow.[7]

Ovary 

Based on the stages of development, the ovaries have primordial, primary, secondary, tertiary, or antral follicles. The number of primordial follicles determines true ovarian reserve. Primordial follicles are small and difficult to be seen by ultrasound. The ovary has 6-7 million primordial follicles at around five months of gestational age, slowly declining to approximately 1000 at menopause.[8][9] 

With the initiation of menarche, a cohort of dormant primordial follicles are recruited during each day of the menstrual cycle, and these follicles grow in size steadily. These primordial follicles develop into primary, secondary, and antral follicles. Development of the fluid-filled cavity, also called the antrum adjacent to the maturing oocyte, results in forming of the antral or tertiary follicle. In normal cycles, one follicle matures and is destined to ovulate while other follicles degenerate and ultimately disappear through a process called apoptosis or programmed cell death.

Antral follicular count (AFC), one of the standard measurements for ovarian reserve, is calculated as the sum of all follicles ranging from 2 to 10 mm contained within both ovaries. The preferred time to measure the AFC is during cycle days 2 through 4 due to the absence of corpus luteum and ovarian cysts. Even though there is a slight variation in the AFC during the menstrual cycle, the AFC can be performed at any point during the menstrual cycle as long as cysts are not obscuring the view of the ovary in its entirety.[10]

Follicle-stimulating hormone (FSH) receptors are developed in the antral follicle once it reaches a minimum diameter of 6 mm, and further growth of this follicle is under control of FSH produced by the anterior pituitary. Dominant follicles are follicles >10 mm. By day 8 to 12, dominant follicles are usually formed, and they grow at an average of 1 to 1.4 mm in diameter per day until they reach a mean diameter of 20 to 24 mm at the time of ovulation in a natural-unstimulated cycle.[11] Other authors have noted a wider range of mean follicular diameter before ovulation.[12] 

For ovulation trigger, most IVF centers usually use a cut-off of 18 to 20 mm before egg retrievals. This differs from patients undergoing clomiphene citrate cycles in which the recent literature suggests follicles closer to 21 to 22 mm have a higher odds of leading to clinical pregnancy.[13] (Figure 3) After ovulation, the follicle collapses to form the corpus luteum, producing progesterone and estradiol to support implantation and nidation. The corpus luteum has a “ring of fire” on color flow doppler due to a large increase in angiogenesis.

Indications

Transvaginal Sonogram

• Baseline assessment of the uterus, ovaries, and adnexa to rule out any pathology like fibroids, polyps, uterine anomalies, hydrosalpinx, or PCOS
• Ovarian reserve assessment- antral follicular count
• Monitoring ovulation induction during clomiphene citrate, letrozole, or gonadotropin cycles with or without intrauterine insemination (IUI) and with in-vitro fertilization (IVF) cycles.
• For timing the ovulation trigger during IUI and IVF
• Assessment of endometrial thickness during IUI and IVF
• For performing oocyte retrievals and sometimes for embryo transfer or other uterine procedures, transabdominal scanning is ineffective.

Transabdominal Sonogram

• Women with a large uterus caused by fibroids or adenomyosis, or both.
• When the uterus or ovaries closely adhered to the anterior abdominal wall secondary to scarring from prior surgery, infection or endometriosis.

Saline Infusion Sonogram

• To assess the uterine cavity for pathologies such as polyps, submucosal fibroids, intrauterine synechiae or scarring, and uterine malformations.
• To assess tubal patency (instead of a traditional hysterosalpingogram (HSG)).

Contraindications

Contraindications for an SIS include pregnancy, heavy menstruation or active bleeding, suspicion of infection as noted by patient symptomatology, or a suspicious vaginal discharge upon examination.

Equipment

An ultrasound machine with transabdominal and transvaginal transducers is required. Transducers of 3.5 Mhz or more are used for transabdominal scans, whereas transducers of 5MHz or more are used for transvaginal scans. In recent years, 3D ultrasound has shown value in performing SIS and assessing AFC and ovarian volumes.

Personnel

The required personnel includes a sonographer, a radiologist, an OBGYN trained in ultrasounds, and reproductive endocrinology and infertility specialists.

Preparation

For a transabdominal scan, a distended bladder is recommended to displace the bowel and provide an acoustic window to visualize the pelvic structures. For a transvaginal scan, SIS, or sonohysterosalpingogram, an empty bladder is recommended.

It is ideal for performing SIS or sonohysterosalpingogram during the mid follicular phase when the patient is no longer bleeding to reduce the false-positive rate for detecting polyps or adhesions and better visualization of any pathological conditions. A urine pregnancy test is performed to confirm that the patient is not pregnant before performing SIS or sonohysterosalpingogram.

Technique or Treatment

Transvaginal Ultrasound 

The patient is kept in a dorsal lithotomy position, and the transvaginal probe is introduced into the vagina either by the patient or the sonographer. The transducer is covered with ultrasound gel, and a condom or condom-like cover is placed over the gelled probe. KY jelly or a similar gel is then placed on the covered transducer for patient comfort.

1. Baseline assessment includes evaluation of uterus, endometrium, ovaries, and cul de sac. 
   • Uterus: The uterus is evaluated in both sagittal and transverse planes (Figure 4). Attention is given to evaluate the size, shape, orientation of the uterus, cervix, myometrium, and endometrium. The length and height of the uterus are measured in the sagittal plane, and width is measured in transverse or coronal planes at the level of the tubal ostia or cornual regions. The myometrium is evaluated for any pathology like fibroids or adenomyosis. The location and character of all pathological lesions are noted and documented in the images and the final report.
   • Endometrium - The endometrium is measured perpendicularly from one echogenic to the other echogenic border at its thickest part in the midsagittal plane (Figure 5). If the fluid is visualized in the cavity, the endometrium is measured by combining the anterior and posterior endometrial thicknesses separately without including the fluid.
   • Adnexa: The adnexa is evaluated for any masses such as hydrosalpinges, para-ovarian cysts, or para-tubal cysts. 
   • Ovaries: The ovaries are identified bilaterally and measured in three dimensions (length, height, and width) (Figure 6). The antral follicles are seen as round or oval anechoic structures. A baseline AFC is performed by counting the number of follicles between 2 to 10 mm and measuring the dimensions of follicles greater than or equal to 10 mm in the early follicular phase in both ovaries.[14] AFC can be measured using three different techniques during the transvaginal scan; a traditional real-time two-dimensional (2D) scan, stored 2D cine loops, or three-dimensional (3D) data sets. Real-time 2D scanning is used to measure the ovary in both the longitudinal and coronal planes. The next step is to optimize the ultrasound settings for better image quality while centering the ovary in its largest axis. The ovary should occupy at least 50% of the screen while counting the follicles ranging from 2 to 10 mm from one ovarian margin to the other. The total follicle number for both ovaries is added to obtain AFC.[10] The 2D cine loop uses the same technique as real-time 2D ultrasound except that the loop involves the continuous sweep of the ovary. The data is saved and assessed at a later time. Even though 3D ultrasound has better reproducibility than 2D methods, it needs additional special training and ultrasound equipment. The technique to perform 3D ultrasound is beyond the scope of this basic topic hence will not be discussed in detail. 
   • Cul de sac: The cul de sac is evaluated for any fluid, and a sliding sign is performed to assess for any adhesions. The examiner's hand is placed on the patient's abdomen to apply pressure on the uterus. The transvaginal probe is placed near the posterior vaginal fornix to assess whether the cervix and the uterus glide over the anterior rectum and sigmoid colon. The lack of sliding of these organs implies adhesive disease of these structures.[15]
2. Monitoring during ovulation induction cycles: A transvaginal ultrasound is performed to assess the follicular growth, endometrium, and uterine arterial blood flow during ovulation induction cycles.
   • Follicular assessment: The measurement of the follicles is usually performed using a transvaginal ultrasound. The follicle size can be done using two different techniques during the transvaginal scan using a traditional real-time 2D scan or sonography-based automated volume calculation (Sono AVC). In traditional real-time 2D scanning, the follicles are measured by calculating the average of two orthogonal diameters. To accommodate the asymmetrical growth in the follicles greater than 15 mm, one can calculate the average of diameters in three orthogonal planes for greater accuracy if clinically warranted.[16]
   • Endometrial assessment includes measurement of endometrial thickness, identifying the endometrial morphological pattern, and grading the endometrial vascularity. To assess endometrial vascularity, the color flow power doppler box is reduced to as small as possible and focused for a few seconds on the endometrial layer, occupying 50% of the screen.

Transabdominal Ultrasound 

A transabdominal ultrasound is performed as needed with infertility. The transducer is placed on the patient's abdomen to assess the uterine size, shape, and orientation. In addition,  the myometrium, endometrium, and ovaries are also assessed. This route of scanning is performed to assess ovarian reserve only in virgin patients, patients with vaginal or pelvic pain, or if the pelvic organs are located cranially and anteriorly due to their enlargement or adhesive disease.

Saline Infusion Sonogram

The patient is kept in a dorsal lithotomy position. Transvaginal ultrasound is performed as described above to assess the uterine size, orientation, adnexa, and ovaries. Any cul de sac or peritoneal fluid is noted and quantified. This detail is important in later considering tubal patency assessment after saline infusion. The transvaginal probe is removed from the vagina. A bivalve speculum is placed in the vagina. Cervix is cleansed with betadine or an alternative anti-septic solution. SIS catheter is introduced in the cervix. There are multiple types of SIS catheters (Figure 7).

When tubal patency needs to be assessed, it is preferable to use a catheter with a balloon that can be wedged in the lower uterine segment to block the backflow of fluid so that the fluid with bubbles can be better directed into the tubal ostia. After the catheter placement, the speculum is removed, and the transvaginal probe is reintroduced into the vagina. Normal saline is introduced through the catheter into the uterine cavity by the assistant while the sonographer or the physician is performing the ultrasound. Instillation of the normal saline into the uterine cavity distends the uterine cavity with the anechoic fluid. Any lesions in the endometrial cavity are visualized and measured in three dimensions and characterized accordingly (Figure 8).[17] 

If tubal patency assessment is planned or required following the intrauterine assessment, a sonohysterosalpingogram (hysterosalpingo contrast sonography -HyCoSy) is performed. During HyCoSy, an agitated mixture of air and normal saline is injected into the uterine cavity through the SIS catheter while performing a real-time ultrasound. Attention is paid to the cornual ends bilaterally to visualize the passage of fluid with air bubbles through the ostia of the fallopian tubes and noting the spilling of the mixed fluid into the adnexa region or elsewhere in the abdominal cavity (Figure 9).

The final step is to evaluate the area where the balloon is located. The balloon is deflated, and the catheter is removed under ultrasound guidance. This part of the procedure is imperative to fully evaluate the previously occupied area by the balloon. Labeling of all pathological structures should also be noted on the ultrasound pictures while scanning.

Complications

The complications for SIS and sonohysterosalpingogram include pain, bleeding, and infection. Patients should be advised to take pain medication generally one hour before the procedure. Most advocate the use of ibuprofen 400 to 600 mg orally one hour before the procedure. Patients should be advised to look for signs of infections, including fever, abnormal vaginal discharge, pelvic pain, or tenderness up to one month after the procedure. A mild amount of bleeding is common after the procedure, which usually resolves within 24 hours. Additional bleeding may warrant evaluation.

Clinical Significance

Importance of Ovarian Assessment by Ultrasound

1. Role of AFC: Evaluation of AFC has similar accuracy as Anti-Mullerian Hormone levels in predicting the ovarian reserve and the response to ovarian stimulation to gonadotropins and pregnancy success rates with IVF.[18]  AFC less than 5 is considered low.[1] AFC more than or equal to 20 is associated with a high ovarian response and a higher risk for ovarian hyperstimulation syndrome.[10] Hence AFC can be used to adjust gonadotropin dosage. AFC less than or equal to 4 have an increased risk of menopause within 7 years.[19][20] 
2. Follicle assessment
   • Gonadotropin stimulated cycles - The use of gonadotropins leads to the recruitment of multiple follicles compared with the typical single follicle in a natural, unstimulated cycle. Ultrasonographic assessment of follicular growth is an important step for clinical decision-making and early detection of complications such as ovarian hyperstimulation.[5] Under the influence of exogenous gonadotropins, the follicles grow at an average of 1.7 to 2 mm per day instead of 1 to 1.4 mm per day. Follicular size is directly related to oocyte recovery rates. The highest oocyte recovery rate ( 83.5%) is noted when the follicle diameter is between 18 to 20 mm and significantly lower when it is less than 12 mm or higher than 24 mm.[21] Therefore ultrasounds are performed to assess ovarian response to stimulation and determine the timing of oocyte maturity and timing for hCG or GnRH agonist triggers. Gonadotropin stimulation can also increase the risk of forming multiple follicles instead of a single follicle and result in ovarian hyperstimulation and /or multiple births. Ultrasound is an easy, reliable imaging technique to identify the formation of maturing follicles in which cycles can be modified or canceled as needed.
   • Oral ovulation-inducing medications- Follicular assessment using ultrasound in this population is controversial as there is less chance of ovarian hyperstimulation and multiple births. Additionally, the increase of cycle monitoring with ultrasound may be cost-prohibitive for many patients without providing a significant difference in pregnancy rates. Although ultrasound is not routinely used in all patients, it is utilized in patients using an Hcg trigger for ovulation to time intercourse or IUI and also when there is difficulty predicting ovulation with ovulation predictor kits. In addition, a recent retrospective study including approximately 50,000 IUI cycles showed a higher risk of multifetal pregnancy in patients less than 40 years of age and with >2 mature ovarian follicles. Hence, it may be beneficial to monitor follicular growth in patients using oral ovulation induction medications to reduce multifetal gestations.[22]
3. Diagnosis of Polycystic ovarian syndrome: PCOS contributes to female infertility secondary to anovulation. The ultrasound criteria to diagnose PCOS include assessing follicle number per ovary (FNPO) and ovarian volume. (Figure 10). An ovarian volume of greater than 10 ml and an AFC of greater than 12 per ovary on ultrasound is consistent with PCOS based on the Rotterdam Consensus conference.  However,  greater than 60% of women aged 25 to 30 were found to have ovaries with PCOS based on this criteria. Therefore, this criteria is being changed to FNPO greater than 20 and/ or ovarian volume greater than 10 ml without corpus luteum or other dominant follicles or cysts.[23] 
4. Diagnosis of other ovarian cysts like cystic teratomas (Figure 11), borderline or malignant ovarian tumors, and endometriomas (Figure 12). Cystic teratomas have hyperechoic areas, sometimes with calcifications. Endometriomas are notable for homogeneous low-level internal echoes or ground glass appearance.

Importance of Endometrial Assessment by Ultrasound

Ultrasound plays a key role in identifying different endometrial and uterine factors that affect embryo implantation. 

1. Exponential endometrial growth is observed in women undergoing gonadotropin stimulated cycles, whereas reduction in the endometrial lining was noted with increasing age and use of clomiphene citrate (CC).[24][25]  Clomiphene citrate, a selective estrogen receptor modulator, affects endometrial development. On ultrasound, a thin endometrial stripe is sometimes developed due to CC's anti-estrogenic effect. (Figure 13). This negative effect on endometrial thickness correlates with the increasing dosage of CC. This effect of clomiphene citrate is noted to last until ovulation. A preovulatory endometrial lining of 6.5 to 7 mm or higher is associated with a better pregnancy rate than an endometrial lining of 6 mm or less.[26][27][28] Oral and/or vaginal Ethinyl estradiol or transdermal estradiol can be given during the CC cycle to increase the endometrial thickness. However, many studies have not shown that supplementation with estrogen for thin linings increases pregnancy rates. In patients with a thin endometrium, while taking CC, alternative ovulation-inducing agents in subsequent cycles may improve endometrial thickness, such as Tamoxifen. Tamoxifen is a selective estrogen receptor modulator, which has a stimulatory effect on estrogen receptors at the level of the uterus compared to the inhibitory effect of CC, thus promoting endometrial growth and increased endometrial thickness.  Aromatase inhibitors like letrozole can also be tried in patients who have side effects with CC.
2. Higher clinical pregnancy rates are noted when grade 3 endometrial vascularity was observed on color flow power doppler.[29][30]
3. Despite other confounding factors affecting successful implantation, a few studies noted a positive correlation between higher clinical pregnancy rates and both triple-layer endometrium and endometrial thickness of 7 mm or higher.[31][24] Endometrial echogenic patterns alone do not alter clinical decision-making during infertility treatments but are important to consider when considering alternative treatments.
4. The presence of polyps, endometrial fluid, adenomyosis, and leiomyomas is associated with abnormal endometrial molecular expressions, in turn altering endometrial receptivity and negatively impacting implantation and early embryo development.[32] On many occasions, a careful look at the endometrial lining can reveal endometrial polyps, submucosal fibroids, intrauterine fluid accumulation, or a congenital uterine anomaly. Even though endometrium can be assessed by transvaginal scan, SIS showed 93% specificity and 75 % sensitivity in detecting intrauterine adhesions compared to HSG, TVUS, SIS with hysteroscopy.[33] MRI, SIS, hysteroscopy were equally effective and superior to TVUS in detecting intracavitary lesions.[34] 

• Endometrial polyps are either sessile or pedunculated localized tumors with endometrial glands, stroma, and blood vessels. Polyps are seen on ultrasound as hyperechoic masses with a feeding vessel on color flow Doppler (Figure 14). Endometrial polyps have a prevalence of 32% and are common in patients with infertility.[35] AAGL recommends surgical removal of polyps in infertile patients to improve their pregnancy success rates.[36]
• The most common causes for endometrial fluid in reproductive age women include intrauterine adhesions (Asherman’s syndrome), hydrosalpinx, cesarean scar niche, and subclinical intrauterine infection. Ultrasound features of intrauterine adhesions include disruption to the endometrial- myometrial junction, hypoechoic bands traversing the endometrial cavity, presence of skip lesions, and areas of normal functional endometrium (Figure 15).[37] 

 Importance of Uterine Assessment by Ultrasound

• Submucosal fibroids: On ultrasound, submucosal fibroids appear as hypoechoic structures in the endometrium with a circular pattern on color flow doppler. Submucosal fibroids can reduce implantation, live birth rates, and their surgical resection has improved clinical pregnancy rates, live birth rates in some studies.[38][39][40] (Figure 16). Intramural fibroids distorting the cavity can also decrease live birth rates by 21% and clinical pregnancy rates by 15% per IVF cycle.[41] In contrast, subserosal fibroids do not affect implantation but can obstruct the visualization of ovaries and oocyte collection. After a systematic review, ASRM proposed that myomectomy for cavity distorting myomas improves pregnancy rates and reduces the risk of early pregnancy loss.[42]

• Adenomyosis -ultrasound features of adenomyosis include globular uterine enlargement without any leiomyomas, cystic anechoic spaces in the myometrium, asymmetric uterine wall thickening, subendometrial echogenic linear striations, heterogeneous echotexture of myometrium, obscure endometrial/ myometrial border, thickening of the transition zone.[43] (Figure 17).  Ultrasound has a sensitivity of 82.5% and specificity of 84.6% in diagnosing adenomyosis and is comparable to MRI.[44][45] Despite the conflicting evidence, adenomyosis has an overall negative impact on fertility due to its effect on endometrial receptivity and decidualization.[32]

• Congenital anomalies of the uterus or Mullerian duct anomalies significantly affect preterm delivery, pregnancy losses, malpresentation, and possibly infertility. While there are many different variants of Mullerian anomalies, septate, arcuate, and bicornuate uteri are the most common types seen in clinical practice. (Figure 18) Bicornuate uterus increases the risk for preterm birth and malpresentation at delivery, whereas septate uterus increases the risk for early miscarriage.[46] When a Mullerian anomaly is suspected on a 2D ultrasound, the American Society for Reproductive Medicine (ASRM) recommends further investigation by performing a saline infusion sonogram with or without 3D reconstruction or hysteroscopy to characterize the anatomy better. Using 3D ultrasound, one can measure the degree of indentation of the protruding medial portion to distinguish between arcuate and septate uteri, which has a reported cut-off ranging between 1.0-1.5cm.[47] 3D ultrasound has similar efficacy as MRI for the diagnosis of congenital anomalies of the uterus.[48] 

Importance of tubal Assessment 

• The presence of hydrosalpinges reduced live birth rates by 30% due to the reflux of toxic fluid into the endometrium.[49][50] Ultrasound findings of hydrosalpinx include thick or thin-walled, tubular, or contorted S-shaped anechoic fluid-filled structure with partial septa.  (Figure 19) Sometimes the hydrosalpinx looks like “beads on a string.” ASRM recommends laparoscopic salpingectomy or proximal tubal occlusion with cautery or partial salpingectomy to improve subsequent pregnancy and live birth rates through IVF  in patients with hydrosalpinges.[51]
• Evaluation of tubal patency plays a key role in counseling infertility patients considering IUI or IVF therapies.  Sonohysterosalpingogram (HycoSY) is a quick, simple technique to confirm tubal patency rapidly. A combined SIS with 2D or 3D ultrasound can be performed around day 5 through 9 as a “one-stop-shop” infertility evaluation.[52] HyCoSy is comparable to HSG for assessment of tubal patency when compared with the laparoscopic chromopertubation.[53]

Role of Ultrasound in Embryo Transfer

Transabdominal ultrasound is usually used to guide the embryo transfer catheter during embryo transfer. An increase in pregnancy rate from 30.1% to 36.8% was noted when embryo transfer was performed using ultrasound versus clinical touch method.[54][55]

Enhancing Healthcare Team Outcomes

Ultrasound is a safe, reliable, cost-effective, and easily available imaging modality to assess the anatomy and morphology of the female reproductive organs. Performing 2D ultrasound along with SIS, HyCoSy, and 3D ultrasound in one setting can curtail the significant time delay in performing infertility workup. Performing a 2D ultrasound is part of general ob-gyn training, but performing SIS, HyCoSy, 3D ultrasound generally needs additional training. All general obstetricians and gynecologists are not equipped with this special skill. Therefore collaborating with an REI provider or physicians who provide fertility services is important to provide care in a timely and efficient manner.

Patient care coordinators and nurses also play a crucial role in managing infertility patients. The timing of ultrasound is typically precise to the patient’s menstrual cycle. Therefore, scheduling patients for ultrasounds expeditiously is essential to an infertility evaluation. Involving an interprofessional team of providers who can prioritize the patient's needs, provide education and support while navigating their journey to a successful pregnancy can increase patient satisfaction and alleviate burnout for care providers. This interprofessional care coordination involving nurses, physician assistants, nurse practitioners, and physicians can improve patient outcomes.