The Treatment of Polycystic Ovarian Syndrome

#### **Chapter 6**

## Novel Methods in the Diagnosis of PCOS: The Role of 3D Ultrasonographic Modalities

*Apostolos Ziogas, Emmanouil Xydias and Elias Tsakos*

#### **Abstract**

Polycystic ovary syndrome (PCOS) is a common and complicated endocrine disorder, with its diagnosis based on clinical, laboratory and imaging criteria. The latter is usually assessed via two-dimensional ultrasound; however, the advent of three-dimensional ultrasound, along with three-dimensional power Doppler (3D-PD) could offer more accurate diagnoses and further our understanding of PCOS pathophysiology. Three-dimensional ultrasound (3D-US) has already been used successfully in many fields of gynecology. It offers improved image quality with stored data that can be processed either manually or automatically to assess many parameters useful in PCOS assessment, such as ovarian volume, number of follicles and vascular indices. The examination requires minimal time as data is assessed in post-processing, thus being more tolerable for the patient. 3D-US parameters are generally increased in PCOS patients when compared to controls and 2D measurements, with studies showing improved diagnostic performance, though that remains inconclusive. 3D transrectal ultrasound is more accurate in the diagnosis of virgin PCOS patients than the modalities currently available in that subgroup. Overall, though with some limitations, 3D-US is a promising diagnostic method in the assessment of PCOS which, regardless of diagnostic accuracy, can undoubtedly offer many practical advantages, more objective and reliable measurements, potentially improving PCOS diagnosis standardization.

**Keywords:** polycystic ovary syndrome (PCOS), 3D-transvaginal ultrasonography (3D-TVUS), 3D-power doppler angiography (3D-PDA), 3D-transrectal ultrasonography (3D-TRUS)

#### **1. Introduction**

Polycystic ovary syndrome (PCOS) is a complicated and heterogenous endocrine disorder affecting more than 10% of women worldwide and it is the most common endocrinopathy of women of reproductive age [1]. It is a syndrome with varied clinical manifestations and several degrees of severity. Some characteristics observed in PCOS patients include hyperandrogenemia, accompanied by acne and hirsutism, ovulatory dysfunction such as oligomenorrhea or amenorrhea, obesity, insulin resistance etc [1].

Diagnosis of PCOS was initially based on clinical characteristics alone, with three prevalent clinical features being agreed upon at the first international conference on PCOS [2, 3], namely:


This definition and the diagnostic algorithm were lacking in several ways [3]. The associated clinical features necessary for diagnosis varied considerably in their clinical manifestation among patients, in particular menstrual instability, obesity and hirsutism and acne with the latter two being the manifestation of hyperandrogenism [4, 5]. Furthermore, no ultrasonographic evidence of PCOS was included in the diagnostic guidelines, although such evidence of PCOS was becoming more and more frequently included in the diagnostic workup of PCOS, with several centers, in fact, mandating it [6].

This led to a joint conference of the American Society for Reproductive Medicine and the European Society for Human Reproduction & Embryology in Rotterdam in 2003, where the previous diagnostic guidelines were revised [7]. The new Rotterdam criteria dictated that the diagnosis of PCOS must include at least two of the following:


With the revised criteria both hyperandrogenism and anovulation do not need to be present if ultrasound findings exist for the diagnosis of PCOS, thus including women that would elude diagnosis if the previous criteria were applied. The aforementioned ultrasound features necessary for PCOS diagnosis are the following [8]:


The Rotterdam 2003 revised criteria constitute an important step in the standardization of diagnostic workup in PCOS, however, they do come with certain limitations. One of the most notable ones is the fact that ovarian volume measurements, collected based on data from 2D scans, mandate the use of a mathematical formula and therefore entail certain geometric assumptions and estimates [9]. A formula for a prolate ellipsoid (0.5 × length × width × thickness) is typically used, however, such calculations assume ovarian regularity, whereas PCOS ovaries have been repeatedly shown to be

#### *Novel Methods in the Diagnosis of PCOS: The Role of 3D Ultrasonographic Modalities DOI: http://dx.doi.org/10.5772/intechopen.101995*

more irregular than normal ones [10]. Another limitation of current diagnostic criteria is the lack of ovarian stromal volume and blood flow assessment. It has been shown that PCOS ovaries have increased stromal volume and blood flow [11, 12], two important parameters that could not only assist in the improvement of our understanding of the pathogenesis of the disease, but also serve as possible response predictors in the treatment of PCOS [13, 14]. However, neither of the two parameters are included in the 2003 guidelines, which could be partially attributed to the great degree of observer subjectivity in the description of stromal echogenicity, as well as to the technical difficulties in blood perfusion measurements using conventional Doppler ultrasound.

#### **2. Three-dimensional ultrasound in gynecology**

#### **2.1 Technical aspects**

Three-dimensional ultrasound (3D-US) as imaging technology was first developed in the 1980s and initially applied mostly in obstetrics for more accurate monitoring of fetal in utero development during pregnancy [15]. However, its success in that field led to research and trials for its potential application in gynecology as well.

Mirroring the application of two-dimensional ultrasound (2D-US), its more well-established and clinically applied counterpart, 3D-US is predominantly used transvaginally in the gynecological examination. The transducer is placed in close proximity to the target area and a complete 3D volume is acquired, which can be assessed either in real-time or digitally stored for later analysis. This option of data storage is particularly advantageous, as data acquisition via sweeping can be completed in seconds and thorough assessment at a later time significantly shortens the total examination time. This renders 3D-US a more tolerable and less time-consuming diagnostic modality overall.

The stored data can be displayed in several different ways, including display in three orthogonal planes, surface rendering, individual slice display (similar to conventional tomography) etc. This option for alternative displays can additionally provide detailed information about areas not previously accessible via conventional two-dimensional ultrasonographic display, namely, the coronal plane, which can significantly contribute to the diagnosis of uterine corner and adnexal pathology.

#### **2.2 Application in benign gynecological disorders**

3D-US over the years has been tested and applied in many benign gynecological disorders with varying levels of success, though on average, its performance is at least comparable to the conventional diagnostic methods and yielded results promising for its inclusion in the diagnostic work-up in clinical practice.

3D-US has been successfully applied in the diagnosis of congenital uterine abnormalities, with remarkable results, as studies have shown up to 100% sensitivity and specificity [16, 17]. Furthermore, research has proven the potential contribution of 3D-US to treatment optimization of uterine abnormalities as well, with 3D-US offering auxiliary visual guidance to the surgeon and improving the final surgical outcome [18]. Another application of 3D-US is in the evaluation of leiomyomas, offering the advantage of precise mapping of their location and clearer differentiation between intramural and submucosal variants when compared to the conventional method of assessment, namely 2D-US [19]. The 3D power Doppler modality is

beneficial in leiomyoma assessment as well, via the more precise evaluation of its vascularization. Therefore, more accurate selection and designation of patients as candidates for embolization treatment can be made [20]. The application of 3D-US in adenomyosis has been examined, with research showing encouraging results, as it facilitates superior visualization of the disrupted border between the endometrium and the basal endometrial layer [21, 22]. 3D-US has also been utilized in the assessment of intrauterine contraception device (IUD) malposition. It can clearly depict the device in its entirety and its position relative to the myometrium via the coronal view [23], whereas such images are far more challenging to obtain via conventional 2D-US. 3D-US also seems promising in pre-operational pelvic assessment in cases with deep pelvic endometriosis. Results are comparable to 2D-US and MRI in patients with intestinal loci of endometriosis and superior to the aforementioned imaging techniques in non-intestinal loci [24].

Apart from improving on currently available diagnostic techniques, 3D-US technology provides new, automated modalities as well. Such modalities mainly include being automated volume calculation, antral follicle counting and follicular growth monitoring, mainly utilized during IVF cycles. This technology has been shown to reduce overall cost, examination time and to deliver accurate and reproducible measurements as well [25–27].

#### **2.3 Application in gynecological oncology**

Regarding ovarian malignancies, 3D-US can accurately measure the volume of the mass, as well as visualize its internal structure, including wall irregularities, cystic elements, septae and so on [19], thus more accurately identifying suspicious masses [28]. In addition to 3D-US, 3D Doppler can offer precise information regarding mass vasculature, with increased mass perfusion and highly irregular vessel anatomy being indicative of possible malignancies [29]. In endometrial cancer, 3D-US can accurately measure endometrial volume, which is an important predictor of malignancy, as well as 3D Doppler vascular indices, however, more research is required to establish optimal cut-off values [30].

#### **3. Three-dimensional ultrasound in PCOS**

#### **3.1 Technical aspects**

As has been made evident so far, 3D-US has been successfully applied in the diagnostic work-up of many gynecological pathologies. Therefore, it was inevitable that similar research would be conducted on its application in PCOS assessment, particularly since the currently used technology does come with certain limitations as mentioned above.

Measurements and data acquisition methods vary between referral centers and studies, however, a similar procedure is followed. Measurements usually begin with a brief 2D-US assessment of the pelvis, followed by identification of the ovaries, with follicles larger than 10 mm and ovarian cysts being excluded. Subsequently, 3D mode is entered and the area of interest is defined. Subsequently, slow-sweeping at a 90° sweep angle or 30–45° angles is applied to ensure that the whole ovary is scanned [31, 32]. The resulting volumetric data is then stored for later evaluation. Compatible software, such as 4D view, allows for several calculations and measurements,

*Novel Methods in the Diagnosis of PCOS: The Role of 3D Ultrasonographic Modalities DOI: http://dx.doi.org/10.5772/intechopen.101995*

with techniques for ovarian volume, follicle count and ovarian stromal volume calculations.

Ovarian volume can be calculated by the rotational method, which in brief entails measurements at different rotational angles of the stored volume [31]. Follicle count can be facilitated by inversion mode, which entails setting a specific threshold that dictates which tissues are displayed. Therefore, it could be set to display liquid-filled, hypoechoic formations only, without the surrounding stroma, leading to easier and more accurate follicle counting. The same basic principle can be applied to display ovarian stromal volume or follicular volume and subsequently the voxels above or below the defined threshold can be automatically and accurately calculated to determine the OSV or the total follicular volume. This is known as semi-automatic measurement [31]. More recently, fully automated software such as Sono-AVC can automatically calculate the ovarian volume and follicle count, forgoing the traditional manual methods and providing more objective measurements with remarkable accuracy and reproducibility [32–34]. An example of automated follicle detection and the count is displayed in **Figures 1** and **2**.

#### **3.2 3D-US parameters**

#### *3.2.1 Ovarian volume (OV)*

OV is an important ultrasonographic parameter that has been included in the Rotterdam criteria and is typically increased in polycystic ovaries and PCOS when compared to controls. The same observations are made when OV is measured via 3D-US, however, 3D measurements have been proven more reliable than 2D ones [35],

**Figure 1.** *2D slice of a stored 3D volume of a PCOS ovary.*

#### **Figure 2.**

*Automatic follicle detection and count via post-processing software.*


*ovarian stromal volume (cm3 ), FC: follicle count, N/A: not assessed.*

#### **Table 1.**

*Comparison of three main ultrasonographic parameters assessed by 3D-US in PCOS patients and controls.*

as those necessitate certain mathematical assumptions. OV measurements in PCOS patients and healthy controls via 3D-US are presented in **Table 1**.

#### *3.2.2 Follicle count (FC)*

FC is critical in the ultrasonographic diagnosis of PCOS and is included in the Rotterdam criteria. FC can be referred to as antral follicle count, or follicle number per ovary, or even total follicle count in different studies, but practically they are

#### *Novel Methods in the Diagnosis of PCOS: The Role of 3D Ultrasonographic Modalities DOI: http://dx.doi.org/10.5772/intechopen.101995*

the tertiary follicles adjacent to a fluid-filled cavity, or antrum and can be visualized accurately via ultrasound if they measure more than 2 mm in diameter [33]. Typically and in fact by definition, PCOS patients and patients with polycystic ovarian morphology have increased FC compared to controls. Follicle counting by 3D-US can be more easily conducted compared to conventional 2D-US, like inversion, the model can be applied and seamlessly differentiate between liquid-filled cystic components and the surrounding stroma [3, 31]. Additionally, automated counting software offers a diagnostic alternative to manual counting, which while not conclusively proven to possess superior diagnostic accuracy, is reportedly less time-consuming [36].

Regarding quantitative data, Allemand et al. found that with 3D-US and by application of the subtractive method, mean FC in PCOS patients was 29.8 ± 11.5 and in controls, 9.5 ± 3.1 and the optimal cut-off for PCOS prediction was 20 or more follicles, with 70% sensitivity, 100% specificity and 0.987 AUC resulting in no false-positive diagnoses [40]. Their proposed threshold is higher than what is included in the Rotterdam criteria [7], which can be partially attributed to the use of 3D-US, as it has been shown to measure larger FC than 2D [41]. On that basis, they propose a possible revision of said criteria to include greater thresholds when 3D-US is applied. FC measurements in PCOS patients and healthy controls via 3D-US are presented in **Table 1**.

#### *3.2.3 Ovarian stromal volume (OSV)*

OSV has been considered an important ultrasonographic parameter in PCOS patients and is measured in many studies. Such measurements were traditionally conducted manually and showed that there is a statistically significant increase of stromal volume in PCOS patients compared to controls [11], perhaps indicating that hypertrophy of the thecal cells of the ovarian stromal is the main androgen-producing factor in PCOS, as has been hypothesized [31]. 3D-US allows for the calculation of OSV, that being either manual via the activation of inversion mode or automatic via thresholding, with the latter being less time-consuming than the aforementioned manual methods.

OSV has not been included as a parameter in the Rotterdam criteria, possibly due to concerns of subjectivity during measurements. 3D ultrasonographic calculation of OSV may present an opportunity to re-evaluate that fact, as OSV could prove to be very useful in clinical practice [37]. OSV measurements in PCOS patients and healthy controls via 3D-US are presented in **Table 1**.

#### *3.2.4 Ovarian stromal volume to total ovarian volume ratio (OSV/OV)*

OSV/OV is a proposed diagnostic parameter for the assessment of PCOS patients, based on the increase of stromal volume that has been observed in many studies. Battaglia et al. calculated this parameter in their study and concluded that it was the most accurate predictor of both hyperandrogenemia and hirsutism, with an AUC of 0.915 and 0.891, respectively when compared to every other ultrasonographic parameter assessed, such as OV, FC, 2D and 3D Doppler indices [32]. They also showed that an OSV/OV ratio equal to or greater than 0.84 was the optimal cut-off for the prediction of the aforementioned PCOS manifestations, with a sensitivity of 92% and a specificity of 91%. In addition, this parameter was more accurate if based on 3D-US measurements rather than 2D-US, which can be attributed to the visualization of the stroma of the whole ovary in 3D, compared to measurements conducted on a single 2D slice [32, 42].

#### *3.2.5 Total follicular volume (TFV)*

TFC is a parameter not used as regularly as the others in PCOS assessment, as its contribution is still in debate. Nardo et al. showed that it was in fact better correlated with PCOS laboratory findings compared to stromal volume and proposed that it was the increase of TFV that actually caused the increase in OV in PCOS patients rather than that of stromal volume [43]. This is in disagreement with several other studies showing that there is an increase in stromal volume and that it is an important predictor of PCOS, with TFV being lower in PCOS patients than in controls and being used mainly to calculate the OSV via subtraction from the total OV [32].

#### **3.3 Comparison of 3D and 2D ultrasound**

The comparative studies about 3D-US and 2D-US are not conclusive on one method's superiority over the other with regard to PCOS and polycystic ovarian morphology diagnosis.

On the one hand, some studies showed that the two methods showed no statistically significant difference between them as far as assessing the main ultrasonographic parameters, namely, FC and OV. Battaglia et al. found no significant difference between 3D and 2D-US parameters, however, they do acknowledge that 3D-US is a more appropriate method due to its reproducibility, it is requiring less mathematical assumptions, and its blood flow parameters assessed via 3D-Doppler [32]. As mentioned above, they also proposed the OSV/OV ratio as an important predictor, which was more accurate when based on 3D-US data. Similar conclusions were reached by Sujata et al. [39] as well as far as the comparison of 2D and 3D is concerned, with no significant difference between them is being made apparent.

Studies examining just the differences in FC between the two methods, outside of the PCOS setting also showed that 2D-US produced larger FCs. Deb et al. compared 2D estimations to 3D manual and automated estimations (via the SonoAVC software) of FC, with SonoAVC underestimating FC compared to the two other methods. However, 3D-US images were used for 2D estimates in that study, which might have led to the increase in FC that was observed. Moreover, a lower FC might be indicative of fewer double-counting incidents compared to manual measurements, thus in fact reflecting a more accurate FC. Regardless of FC, automated 3D-US FC was shown to possess greater inter-observer reproducibility than the other two methods [44]. In another comparative study by Deb et al. regarding 2D and 3D-US FC measurements in subfertile women, it was shown that 2D measurements of FC were significantly larger than 3D, but 3D FC semi-automated counting via SonoAVC was significantly faster, averaging approximately 130 s whereas manual counting via 2D-US lasted for an average of 324 s [45].

On the other hand, Nylander et al. concluded that 3D-US was more accurate as far as OV was concerned compared to 2D, as the 3D estimates were in closer agreement with MRI measurements. In their study, 2D measurements of ovarian volume were 14.9% smaller than 3D-US measurements and 11.6% smaller than MRI, which is in agreement with one previous study comparing 2D to MRI and other studies comparing it to volume measurements of anatomical specimens [46]. This observation is attributed to the assumption of a regular ovoid or ellipsoid shape of the ovary and the use of mathematical formulas in the calculation of OV in 2D-US, whereas 3D-US, MRI and anatomical measurements outline the ovary contours and thus result in more

precise measurements [46]. Regarding FC, the research team found that 2D estimates were 18% smaller than those of 3D-US and 16% smaller than those of MRI, suggesting that 3D-US more accurately counts antral follicles than 2D-US.

Overall, the currently available bibliography is still conflicted on which of the two methods provides the most accurate measurements of ultrasonographic parameters, however, what is undisputed is the speed and reproducibility of 3D-US measurements, which is superior to 2D.

#### **4. Three-dimensional power Doppler**

#### **4.1 Technical aspects**

Three-dimensional power Doppler (3D-PD) allows for vascularization and blood perfusion assessment via histogram analysis and has been shown to provide more data than frequency-based Doppler ultrasound, especially in low-velocity flow and when flow alterations take place [47, 48]. It has also been considered a means of objective assessment of vascularization and blood flow, contrary to 2D modalities which examine only specific blood vessels in a single slice and depend on the detection of the most representative image of the examined pathology [48]. With regard to ovarian pathologies, 3D-PD via scanning the organ in its entirety could offer very representative data of the vascularization and perfusion status of the whole ovary and perhaps applied in clinical practice.

The data acquisition procedure closely resembles 3D-US acquisition of 3D volume data, with the notable difference of specific Doppler settings being activated to capture relevant data. Afterward, the acquired information is stored digitally and via post-processing software, such as VOCAL or 4D-view, computer algorithms create a histogram of voxel data and calculate vascular indices. The indices most commonly assessed are the vascularization index (VI), the flow index (FI) and the vascularization and flow index (VFI), as described by Pairleitner et al. [48].

#### **4.2 3D-PD parameters**

#### *4.2.1 Vascularization index (VI)*

VI is the proportion of the scanned volume that emits a flow signal compared to the rest of the organ. In practicality it is the number of colored voxels (representing areas that flow was detected) and expressed as a percentage of the complete volume of the ovary, thus reflecting the blood vessel density in the scanned volume. It could be applied in the diagnosis of pathologies where vascularization either increases or decreases, without changes in the blood flow necessarily, as VI provides no information on the blood flow itself or its intensity [32, 48].

#### *4.2.2 Flow index (FI)*

FI is an average of the signal intensity of the blood flow detected in the scanned volume. In practicality, the software calculates the mean color value of all the colored vessels, representing the average intensity of the blood flow in the scanned volume, which could be used in pathologies where there are changes in blood flow but not in the anatomy of blood vessels or vascularization [32, 48].

#### *4.2.3 Vascularization-flow index (VFI)*

Finally, VFI is the combination of the information provided by the other two indices, as practicality is the product of VI and FI. It could be applied to identify pathologies on the spectrum of low vascularization and decreased blood flow on the one extreme and increased vascularization and blood flow on the other [32, 48].

#### *4.2.4 Mean grayness (MG)*

Mean grayness is not a vascular index, as it assesses the mean signal intensity of the gray voxels, meaning areas without detectable flow. It is a more objective representation of the tissue echogenicity which is traditionally assessed subjectively via 2D-US, as it is calculated by algorithm based on histogram data. Despite it not being a vascularity index, in most studies it is assessed along with the other three 3D-PD parameters, therefore, data on it will be presented along with the other three in this chapter as well [32].

#### **4.3 Study results on 3D-PD parameters**

There have been numerous studies conducted on PCOS patients that used 3D-PD and calculated mean values for both PCOS patients and controls. This data is summarized in **Table 2**. There is significant variation regarding the values acquired among the studies. This could be attributed to differences in study design and protocol (definition of PCOS, time of ultrasonographic data acquisition relative to menstrual cycle), the technology used (different devices, heterogenous settings) and disparities in demographical characteristics of the participants (age, BMI, clinical manifestations etc).

In general, there is still a lack of consensus on whether these parameters can be utilized in PCOS assessment, with many studies showing that some or all indices were significantly increased in PCOS patients, whereas others showed no statistical difference between the values at all. Data on the statistical significance of the differences of several parameters between the PCOS group and the control group are presented in **Table 3**.


*N: number of participants, VI: vascularization index (%), FI: flow index (0–100), VFI: vascularization flow index (0–100), MG: mean grayness (0–100), N/A: not assessed.*

#### **Table 2.**

*3D-PD parameter values in PCOS patients and controls.*

*Novel Methods in the Diagnosis of PCOS: The Role of 3D Ultrasonographic Modalities DOI: http://dx.doi.org/10.5772/intechopen.101995*


*BMI: body mass index, Day: day of the menstrual cycle that 3D-PD measurements were taken (note: in cases with amenorrhea, the days are counted after withdrawal bleeding induced via progesterone administration for a week), OV: ovarian volume (cm3), FC: follicle count, VI: vascularization index (%), FI: flow index (0–100), VFI: vascularization flow index (0–100), MG: mean grayness (0–100), OPI: ovarian pulsatility index, ORI: ovarian resistance index,* ↑*: significant increase in PCOS group,* ↓*: significant decrease in PCOS group, ND: no significant difference between the two groups, N/A: not assessed.\*2D-PD parameters (OPI, ORI) were measured on uterine arteries.*

#### **Table 3.**

*Several parameters of the PCOS group and their difference in comparison to control group measurements.*

From the assessed parameters, VI and VFI appear to be the more reliable of the four parameters, as they are significantly elevated in every study that 3D-PD parameters significantly differ between the PCOS and control groups. FI and MG are not shown to be as reliable relative to the other two, as they do not differ between the two groups in two studies, whereas VI and VFI are increased [31, 47]. No difference or effect on these results was noted based on differences in age, BMI or day of the cycle when the 3D-DP scan was performed.

Some of the included studies compared to the traditional way of ultrasonographic assessment of vascularity and blood flow, namely 2D-PD with the most frequently used parameters being pulsatility and resistance indices of the ovarian vessels to the 3D-PD parameters. Though data on this comparison is only available from four studies, it is inconclusive, as in three of the studies 2D-PD parameters are statistically different between the PCOS and control groups and in one, no statistically significant difference between the two is evident, with the 3D-PD parameters following the same trends in these studies as well. Lam et al. note that is based only on 2D-PD measurements, no difference between the PCOS patients and healthy participants would be noted, whereas that distinction was made apparent when 3D-PD was applied [31].

As far as cut-offs and reference values are concerned, from the studies that did find a significant difference, only Battaglia et al. attempted to create ROC curves and calculate optimal cut-off values, however, since the ROC curve was not statistically significant, no such values were obtained. More research is required, mainly to confirm the significance of 3D-PD measurements, as in half the studies no significant differences were noted and establish optimal cut-off values that could herald the application of 3D-PD in clinical practice as an objective means of vascularization and blood flow assessment in PCOS.

#### **5. Three-dimensional transrectal ultrasound (3D-TRUS)**

PCOS generally manifests during adolescence, in young and usually virgin women. In such patients, the so far described transvaginal ultrasonographic assessment with

its remarkable diagnostic accuracy is not recommended. Therefore, the transabdominal and transrectal approaches are considered viable alternatives, with the latter seeming more promising, as it is frequently difficult to obtain high-quality images via TA-US [51].

The advent of 3D-US technology marks a significant advance in that field, as 3D-TRUS could replace it in the cases that transvaginal cannot be applied, with hopefully similar results. Sun et al. attempted to evaluate 3D-TRUS' diagnostic accuracy in such a population, namely virgin PCOS patients. A total of 45 virgin patients with PCOS, aged 15–25 presenting with the classic PCOS clinical manifestations were enrolled in their study. In addition, 30 patients with only the ultrasonographic findings of polycystic ovarian morphology and no clinical symptoms, along with 25 healthy volunteers were enrolled as well. All patients received 2D-TAUS and 3D-TRUS and several 3D parameters were assessed.

The results were very encouraging, as 3D-TRUS allowed for improved detection of PCOS, in fact even surpassing transvaginal sonography's accuracy, with the most accurate parameter being the stromal area to total area ratio. Though very encouraging for 3D-US application in this specific subgroup of young patients, whose family planning can be severely impacted by PCOS, the results of this study should be verified by future studies on the subject, as the authors stress [51].

#### **6. Limitations of 3D-US**

As with every other diagnostic method, 3D-US is by all means not without some limitations which should be mentioned.

For 3D-US high-quality image acquisition, typically the probes used are larger than the corresponding 2D probes, although not by much. Thus, in theory, this could render the examination less tolerable by the patients, especially in transvaginal or transrectal ultrasounds. However, this in practice is balanced by the shorter examination time, as mentioned above and as 3D technology constantly evolves, it is very likely that such concerns about the transducer size will be eliminated [52].

Another consideration is data storage, as 3D-US stores data regarding the whole volume of the target and not just slice as its 2D counterpart. Therefore more space is required to store patient data, with said requirements likely to further increase, as technology improves and image quality improves exponentially. However, this is offset by the synchronous progress of digital media as well, with digital storage becoming more and more affordable and health centers using servers thus rendering physical storage media, such as DVDs and USBs obsolete [52].

3D-US remains a costly method to this day, with the latest equipment usually being unaffordable by most centers. Apart from the physical devices and peripheral attachments, the cost of software is also a major consideration, as the more advanced modalities that facilitate automatic follicle counting and volume measurement are an additional cost for potential buyers. However, as technology progresses, 3D-US equipment will undoubtedly become more and more affordable, particularly by centers and individuals specialized in PCOS and other fields where it can be applied.

Another easily overlooked limitation is the need for 3D-US operator additional training. Despite the many apparent similarities with the more established 2D-US, special training is required to obtain high-quality images as well as to process the acquired data after the examination. Many inexperienced operators face orientation problems during post-processing and viewing, as the improved space awareness combined with

#### *Novel Methods in the Diagnosis of PCOS: The Role of 3D Ultrasonographic Modalities DOI: http://dx.doi.org/10.5772/intechopen.101995*

an initial lack of correct orientation determination during the examination can lead to the false perception of the stored volume and false assumptions [52].

Finally, like every other imaging technique, 3D-US can produce artifacts, some that are similar to 2D and others limited to themselves due to the acquisition process, the rendering and the post-processing. It is more usual in 3D-US for motion artifacts to be produced, as the whole organ must be scanned and not every patient can stay still throughout the examination. Therefore, training on data acquisition and correct post-processing is required to reduce the number of artifacts that may be introduced to the images, as well as training on artifact recognition, as misinterpretation of them can lead to inaccurate diagnoses.

#### **7. Conclusion**

PCOS is a common endocrine disorder affecting many women worldwide, with varying clinical manifestations. Diagnosis is mainly based on 2D-US, however, the relative advent of 3D-US technology offers a promising alternative. 3D-US entails the acquisition of the complete 3D volume of the region of interest, along with vascularization data if Doppler mode is applied. This process is quick and thus more tolerable for the patients and provides vastly more information than 2D real-time assessment. The stored data can be evaluated at any time by many examiners and the measurements of OV, follicle count and VI, FI, VFI and MG, especially if automatically calculated are more objective and with significantly better inter-observer reproducibility of results.

Data on actual diagnostic performance in comparison to the currently available technology is still lacking and inconclusive, with some studies showing no difference and others indicating that 3D-US more accurately visualizes the underlying ovarian morphology and thus offers a more accurate diagnosis. The bibliography on 3D-PD ultrasound is more conflicted, as many studies did not manage to show statistically significant differences in PCOS patients' Doppler parameters in comparison with the control group. However, some studies actually did show a significant difference and in fact propose that 3D-PD offers a more objective and accurate assessment of the vascularization and blood flow of the ovary, as it visualizes the whole organ and its parameters are calculated based on histogram analysis and not the operator's observations, thus being more objective. 3D-TRUS is shown to be a very promising alternative to the traditional transvaginal approach in virgin patients, with remarkable results.

It is made apparent that more research is required to further assess the diagnostic accuracy and usefulness of 3D-US in PCOS assessment, especially as far as 3D-PD is concerned, as it shows much promise and could potentially lead to the inclusion of objective diagnostic criteria in the guidelines if sufficient evidence is found. In addition, new reference values and cut-offs need to be established, again especially in 3D-PD, as the current bibliography is still lacking in that regard.

#### **Conflict of interest**

The authors have no conflict of interest to declare.

#### **Author details**

Apostolos Ziogas1 \*, Emmanouil Xydias1 and Elias Tsakos2

1 Department of Medicine, School of Health Sciences, University of Thessaly, Larissa, Greece

2 EmbryoClinic, Thessaloniki, Greece

\*Address all correspondence to: ziogasapo@hotmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Novel Methods in the Diagnosis of PCOS: The Role of 3D Ultrasonographic Modalities DOI: http://dx.doi.org/10.5772/intechopen.101995*

#### **References**

[1] Goodarzi MO, Dumesic DA, Chazenbalk G, Azziz R. Polycystic ovary syndrome: Etiology, pathogenesis and diagnosis. Nature Reviews. Endocrinology. 2011;**7**(4):219-231. DOI: 10.1038/nrendo.2010.217

[2] Lujan ME, Chizen DR, Pierson RA. Diagnostic criteria for polycystic ovary syndrome: Pitfalls and controversies. Journal of Obstetrics and Gynaecology Canada. 2008;**30**(8):671-679. DOI: 10.1016/s1701-2163(16)32915-2

[3] Lam PM, Raine-Fenning N. The role of three-dimensional ultrasonography in polycystic ovary syndrome. Human Reproduction. 2006;**21**(9):2209-2215. DOI: 10.1093/humrep/del161

[4] Michelmore KF, Balen AH, Dunger DB, Vessey MP. Polycystic ovaries and associated clinical and biochemical features in young women. Clinical Endocrinology. 1999;**51**(6):779-786. DOI: 10.1046/j.1365-2265.1999.00886.x

[5] Polson DW, Adams J, Wadsworth J, Franks S. Polycystic ovaries—a common finding in normal women. Lancet. 1988;**1**(8590):870-872. DOI: 10.1016/ s0140-6736(88)91612-1

[6] Balen A, Michelmore K. What is polycystic ovary syndrome? Are national views important? Human Reproduction. 2002;**17**(9):2219-2227. DOI: 10.1093/ humrep/17.9.2219

[7] Rotterdam ESHRE/ASRM-Sponsored PCOS consensus workshop group. Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome (PCOS). Human Reproduction. 2004;**19**(1):41-47. DOI: 10.1093/humrep/deh098

[8] Balen AH, Laven JS, Tan SL, Dewailly D. Ultrasound assessment of the polycystic ovary: International consensus definitions. Human Reproduction Update. 2003;**9**(6):505-514. DOI: 10.1093/ humupd/dmg044

[9] Gilja OH, Hausken T, Berstad A, Odegaard S. Measurements of organ volume by ultrasonography. Proceedings of the Institution of Mechanical Engineers. Part H. 1999;**213**(3):247-259. DOI: 10.1243/0954411991534951

[10] DePriest PD, van Nagell JR Jr, Gallion HH, Shenson D, Hunter JE, Andrews SJ, et al. Ovarian cancer screening in asymptomatic postmenopausal women. Gynecologic Oncology. 1993;**51**(2):205-209. DOI: 10.1006/gyno.1993.1273

[11] Kyei-Mensah AA, LinTan S, Zaidi J, Jacobs HS. Relationship of ovarian stromal volume to serum androgen concentrations in patients with polycystic ovary syndrome. Human Reproduction. 1998;**13**(6):1437-1441. DOI: 10.1093/ humrep/13.6.1437

[12] Pan HA, Wu MH, Cheng YC, Li CH, Chang FM. Quantification of Doppler signal in polycystic ovary syndrome using three-dimensional power Doppler ultrasonography: A possible new marker for diagnosis. Human Reproduction. 2002;**17**(1):201-206. DOI: 10.1093/ humrep/17.1.201

[13] Aleem FA, Predanic M. Transvaginal color Doppler determination of the ovarian and uterine blood flow characteristics in polycystic ovary disease. Fertility and Sterility. 1996;**65**(3):510-516. DOI: 10.1016/ s0015-0282(16)58145-x

[14] Agrawal R, Conway G, Sladkevicius P, Tan SL, Engmann L, Payne N, et al. Serum vascular endothelial growth factor and Doppler blood flow velocities in in vitro fertilization: Relevance to ovarian hyperstimulation syndrome and polycystic ovaries. Fertility and Sterility. 1998;**70**(4):651-658. DOI: 10.1016/ s0015-0282(98)00249-0

[15] Turkgeldi E, Urman B, Ata B. Role of three-dimensional ultrasound in gynecology. Journal of Obstetrics and Gynaecology of India. 2015;**65**(3):146- 154. DOI: 10.1007/s13224-014-0635-z

[16] Ghi T, Casadio P, Kuleva M, Perrone AM, Savelli L, Giunchi S, et al. Accuracy of three-dimensional ultrasound in diagnosis and classification of congenital uterine anomalies. Fertility and Sterility. 2009;**92**(2):808-813. DOI: 10.1016/j. fertnstert.2008.05.086

[17] Faivre E, Fernandez H, Deffieux X, Gervaise A, Frydman R, Levaillant JM. Accuracy of three-dimensional ultrasonography in differential diagnosis of septate and bicornuate uterus compared with office hysteroscopy and pelvic magnetic resonance imaging. Journal of Minimally Invasive Gynecology. 2012;**19**(1):101-106. DOI: 10.1016/j.jmig.2011.08.724

[18] Ludwin A, Ludwin I, Pityński K, Banas T, Jach R. Role of morphologic characteristics of the uterine septum in the prediction and prevention of abnormal healing outcomes after hysteroscopic metroplasty. Human Reproduction. 2014;**29**(7):1420-1431. DOI: 10.1093/humrep/deu110

[19] Armstrong L, Fleischer A, Andreotti R. Three-dimensional volumetric sonography in gynecology: An overview of clinical applications. Radiologic Clinics of North America.

2013;**51**(6):1035-1047. DOI: 10.1016/j. rcl.2013.07.005

[20] Bragg AC, Angtuaco TL. Threedimensional gynecologic ultrasound. Ultrasound Clinics. 2010;**5**(2):299-311. DOI: 10.1016/j.cult.2010.03.001

[21] Exacoustos C, Brienza L, Di Giovanni A, Szabolcs B, Romanini ME, Zupi E, et al. Adenomyosis: Threedimensional sonographic findings of the junctional zone and correlation with histology. Ultrasound in Obstetrics & Gynecology. 2011;**37**(4):471-479. DOI: 10.1002/uog.8900

[22] Luciano DE, Exacoustos C, Albrecht L, LaMonica R, Proffer A, Zupi E, et al. Three-dimensional ultrasound in diagnosis of adenomyosis: Histologic correlation with ultrasound targeted biopsies of the uterus. Journal of Minimally Invasive Gynecology. 2013;**20**(6):803-810. DOI: 10.1016/j. jmig.2013.05.002

[23] Benacerraf BR, Shipp TD, Bromley B. Three-dimensional ultrasound detection of abnormally located intrauterine contraceptive devices which are a source of pelvic pain and abnormal bleeding. Ultrasound in Obstetrics & Gynecology. 2009;**34**(1):110-115. DOI: 10.1002/ uog.6421

[24] Guerriero S, Saba L, Ajossa S, Peddes C, Angiolucci M, Perniciano M, et al. Three-dimensional ultrasonography in the diagnosis of deep endometriosis. Human Reproduction. 2014;**29**(6):1189- 1198. DOI: 10.1093/humrep/deu054

[25] Ata B, Tulandi T. Ultrasound automated volume calculation in reproduction and in pregnancy. Fertility and Sterility. 2011;**95**(7):2163-2170. DOI: 10.1016/j.fertnstert.2011.04.007

[26] Ata B, Seyhan A, Reinblatt SL, Shalom-Paz E, Krishnamurthy S, Tan SL. *Novel Methods in the Diagnosis of PCOS: The Role of 3D Ultrasonographic Modalities DOI: http://dx.doi.org/10.5772/intechopen.101995*

Comparison of automated and manual follicle monitoring in an unrestricted population of 100 women undergoing controlled ovarian stimulation for IVF. Human Reproduction. 2011;**26**(1): 127-133. DOI: 10.1093/humrep/deq320

[27] Raine-Fenning N, Jayaprakasan K, Deb S, Clewes J, Joergner I, Dehghani Bonaki S, et al. Automated follicle tracking improves measurement reliability in patients undergoing ovarian stimulation. Reproductive Biomedicine Online. 2009;**18**(5):658-663. DOI: 10.1016/s1472-6483(10)60010-7

[28] Dodge JE, Covens AL, Lacchetti C, Elit LM, Le T, Devries-Aboud M, et al. Preoperative identification of a suspicious adnexal mass: A systematic review and meta-analysis. Gynecologic Oncology. 2012;**126**(1):157-166. DOI: 10.1016/j.ygyno.2012.03.048

[29] Chase DM, Crade M, Basu T, Saffari B, Berman ML. Preoperative diagnosis of ovarian malignancy: Preliminary results of the use of 3-dimensional vascular ultrasound. International Journal of Gynecological Cancer. 2009;**19**(3):354-360. DOI: 10.1111/IGC.0b013e3181a1d73e

[30] Odeh M, Vainerovsky I, Grinin V, Kais M, Ophir E, Bornstein J. Threedimensional endometrial volume and 3-dimensional power Doppler analysis in predicting endometrial carcinoma and hyperplasia. Gynecologic Oncology. 2007;**106**(2):348-353. DOI: 10.1016/j. ygyno.2007.04.021

[31] Lam PM, Johnson IR, Raine-Fenning NJ. Three-dimensional ultrasound features of the polycystic ovary and the effect of different phenotypic expressions on these parameters. Human Reproduction. 2007;**22**(12):3116-3123. DOI: 10.1093/ humrep/dem218

[32] Battaglia C, Battaglia B, Morotti E, Paradisi R, Zanetti I, Meriggiola MC, et al. Two- and three-dimensional sonographic and color Doppler techniques for diagnosis of polycystic ovary syndrome. The stromal/ovarian volume ratio as a new diagnostic criterion. Journal of Ultrasound in Medicine. 2012;**31**(7):1015-1024. DOI: 10.7863/jum.2012.31.7.1015

[33] Coelho Neto MA, Ludwin A, Borrell A, Benacerraf B, Dewailly D, da Silva CF, et al. Counting ovarian antral follicles by ultrasound: A practical guide. Ultrasound in Obstetrics & Gynecology. 2018;**51**(1):10-20. DOI: 10.1002/uog.18945

[34] Froyman W, Van Schoubroeck D, Timmerman D. Automated follicle count using three-dimensional ultrasound in polycystic ovarian morphology. Ultrasound in Obstetrics & Gynecology. 2018;**51**(1):147-149. DOI: 10.1002/ uog.18896

[35] Raine-Fenning NJ, Campbell BK, Clewes JS, Johnson IR. The interobserver reliability of ovarian volume measurement is improved with threedimensional ultrasound, but dependent upon technique. Ultrasound in Medicine & Biology. 2003;**29**(12):1685-1690. DOI: 10.1016/s0301-5629(03)01068-8

[36] Pascual MA, Graupera B, Hereter L, Tresserra F, Rodriguez I, Alcázar JL. Assessment of ovarian vascularization in the polycystic ovary by threedimensional power Doppler ultrasonography. Gynecological Endocrinology. 2008;**24**(11):631-636. DOI: 10.1080/09513590802308099

[37] Lam P, Raine-Fenning N, Cheung L, Haines C. Three-dimensional ultrasound features of the polycystic ovary in Chinese women. Ultrasound in Obstetrics & Gynecology. 2009;**34**(2): 196-200. DOI: 10.1002/uog.6442

[38] Alcázar JL, Kudla MJ. Ovarian stromal vessels assessed by spatiotemporal image correlation-high definition flow in women with polycystic ovary syndrome: A case-control study. Ultrasound in Obstetrics & Gynecology. 2012;**40**(4):470-475. DOI: 10.1002/ uog.11187

[39] Sujata K, Swoyam S. 2D and 3D trans-vaginal sonography to determine cut-offs for ovarian volume and follicle number per ovary for diagnosis of polycystic ovary syndrome in Indian women. Journal of Reproduction & Infertility. 2018;**19**(3):146-151

[40] Allemand MC, Tummon IS, Phy JL, Foong SC, Dumesic DA, Session DR. Diagnosis of polycystic ovaries by threedimensional transvaginal ultrasound. Fertility and Sterility. 2006;**85**(1): 214-219. DOI: 10.1016/j.fertnstert.2005. 07.1279

[41] Scheffer GJ, Broekmans FJ, Bancsi LF, Habbema JD, Looman CW, Te Velde ER. Quantitative transvaginal two- and three-dimensional sonography of the ovaries: Reproducibility of antral follicle counts. Ultrasound in Obstetrics & Gynecology. 2002;**20**(3):270-275. DOI: 10.1046/j.1469-0705.2002.00787.x

[42] Fulghesu AM, Angioni S, Frau E, Belosi C, Apa R, Mioni R, et al. Ultrasound in polycystic ovary syndrome—the measuring of ovarian stroma and relationship with circulating androgens: Results of a multicentric study. Human Reproduction. 2007;**22**(9): 2501-2508. DOI: 10.1093/humrep/ dem202

[43] Nardo LG, Buckett WM, White D, Digesu AG, Franks S, Khullar V. Threedimensional assessment of ultrasound features in women with clomiphene citrate-resistant polycystic ovarian syndrome (PCOS): Ovarian

stromal volume does not correlate with biochemical indices. Human Reproduction. 2002;**17**(4):1052-1055. DOI: 10.1093/humrep/17.4.1052

[44] Deb S, Jayaprakasan K, Campbell BK, Clewes JS, Johnson IR, Raine-Fenning NJ. Intraobserver and interobserver reliability of automated antral follicle counts made using threedimensional ultrasound and SonoAVC. Ultrasound in Obstetrics & Gynecology. 2009;**33**(4):477-483. DOI: 10.1002/ uog.6310

[45] Deb S, Campbell BK, Clewes JS, Raine-Fenning NJ. Quantitative analysis of antral follicle number and size: A comparison of two-dimensional and automated three-dimensional ultrasound techniques. Ultrasound in Obstetrics & Gynecology. 2010;**35**(3):354-360. DOI: 10.1002/uog.7505

[46] Nylander M, Frøssing S, Bjerre AH, Chabanova E, Clausen HV, Faber J, et al. Ovarian morphology in polycystic ovary syndrome: Estimates from 2D and 3D ultrasound and magnetic resonance imaging and their correlation to anti-Müllerian hormone. Acta Radiologica. 2017;**58**(8):997-1004. DOI: 10.1177/0284185116676656

[47] Mala YM, Ghosh SB, Tripathi R. Three-dimensional power Doppler imaging in the diagnosis of polycystic ovary syndrome. International Journal of Gynaecology and Obstetrics. 2009; **105**(1):36-38. DOI: 10.1016/j. ijgo.2008.11.042

[48] Pairleitner H, Steiner H, Hasenoehrl G, Staudach A. Threedimensional power Doppler sonography: Imaging and quantifying blood flow and vascularization. Ultrasound in Obstetrics & Gynecology. 1999;**14**(2):139-143. DOI: 10.1046/j.1469-0705.1999.14020139.x *Novel Methods in the Diagnosis of PCOS: The Role of 3D Ultrasonographic Modalities DOI: http://dx.doi.org/10.5772/intechopen.101995*

[49] Järvelä IY, Mason HD, Sladkevicius P, Kelly S, Ojha K, Campbell S, et al. Characterization of normal and polycystic ovaries using three-dimensional power Doppler ultrasonography. Journal of Assisted Reproduction and Genetics. 2002;**19**(12):582-590. DOI: 10.1023/a:1021267200316

[50] Garg N, Khaira HK, Kaur M, Sinha S. A comparative study on quantitative assessment of blood flow and vascularization in polycystic ovary syndrome patients and normal women using three-dimensional power Doppler ultrasonography. Journal of Obstetrics and Gynaecology of India. 2018;**68**(2):136-141. DOI: 10.1007/ s13224-017-1082-4

[51] Sun L, Fu Q. Three-dimensional transrectal ultrasonography in adolescent patients with polycystic ovarian syndrome. International Journal of Gynaecology and Obstetrics. 2007;**98**(1):34-38. DOI: 10.1016/j. ijgo.2007.02.024

[52] Bragg AC, Angtuaco TL. Threedimensional gynecologic ultrasound. In: Allison S, Wolfman D, editors. Gynecologic Ultrasound, An Issue of Ultrasound Clinics. 1st ed. Philadelphia: Saunders; 2010. pp. 307-308. DOI: 10.1016/j.cult.2010.03.001

#### **Chapter 7**

## The Novelty of miRNAs as a Clinical Biomarker for the Management of PCOS

*Rana Alhamdan and Juan Hernandez-Medrano*

#### **Abstract**

Polycystic ovary syndrome (PCOS) is a common endocrine disorder that affects around 5–10% of women of reproductive age. The aetiology of PCOS is not fully understood with various genetics, iatrogenic (e.g. chemotherapy) and environmental factors have been proposed. microRNAs (miRNAs) are small non-coding single-stranded RNAs which are known to act as a regulator to gene expression at the post-transcriptional levels. Altered expression of miRNAs has been linked to several disorders including infertility. Recent reports demonstrated the expression of differential levels of miRNAs in the serum, ovarian follicular cells and follicular fluid of PCOS patients when compared with healthy women. Therefore, miRNAs may play important role in the pathogenesis of PCOS. The aim of this chapter is to summarise the current understanding pertaining to miRNAs and PCOS and to expedite its possible role in the diagnosis and management of this disorder.

**Keywords:** PCOS, miRNA, follicular fluid

#### **1. Introduction**

Polycystic ovary syndrome (PCOS) is the most common multifactorial endocrinopathy female disorder. It affects approximately 5–10% of women in their reproductive age [1–3]. It is characterised by oligoanovulatory ovarian dysfunction, polycystic ovarian morphology and clinical and biochemical hyperandrogenism (HR). In addition, other factors such as endocrine dysfunctions, suboptimal follicular environment and oocyte competence make it a leading cause of female infertility. PCOS is also an important risk factor for metabolic disorders such as obesity, hyperlipidaemia and insulin resistance (IR), leading to type-2 diabetes mellitus (T2DM), cardiovascular disease (CVD) and endometrial cancer and other diseases [4, 5]. To date, the aetiology of PCOS remains elusive; however, environmental and genetic variations are proposed as a potential contributing factor. Although recent pedigree and genomewide association studies have revealed apparent interrelation of several genes with PCOS, none have shown direct cause to the occurrence of the syndrome [6]. Thus, it has been postulated that their effect might be apparent in a dose-dependent or synergistic fashion rather than being a one or none effect [6]. Recent reports have suggested miRNAs' involvement in PCOS incidence and development, thanks to the differential expression in patients with this disorder and healthy fertile patients [1, 7].

#### **2. miRNAs as a potential novel clinical biomarker for PCOS**

**miRNAs** are a new class of endogenously produced short non-coding single-strand RNAs with 20–25 nucleotides [1, 4, 8]. miRNAs are first transcribed from the genome as a primary miRNA (pri-miRNA) by RNA polymerases to form a hairpin like structure. For the miRNA to exert its biological function, it cleaves into a precursor miRNA (pre-miRNA) by a nuclear protein complex containing a Dorsha enzyme. These small transcripts (60–110 nt) can then leave the nucleus to the cytoplasm and subsequently processed further by the Dicer enzyme and another protein complex into the mature form of the miRNA. The mature form of the miRNA can regulate gene expression post-transcriptionally via binding to the 3'untranslated region (UTR) of the target mRNA, thus preventing their translation and/or causing their destabilisation of molecule [1, 9]. miRNAs have been shown to be widely expressed throughout the body, including organs such as muscles, adipose tissue and ovaries, at the intra- and extracellular levels [1, 4]. Recently, miRNAs have been isolated from extracellular fluids with variable expression profile depending on the fluid [1]; urine [10], saliva, plasma/ serum [9] and follicular fluid [11]. Furthermore, miRNAs can be encapsulated in microvesicles [12] which are extracellular hetergeneous membraneous vesicles (EVs) originating from the cells. The smallest subpopulation of which are called exosomes, and they are known to act as a mediator to transport and release miRNAs between target cells [12]. It has been reported that miRNAs are more stable in exosomes with greater gene regulatory activity [13]. miRNA's functions become feasible through participation in processes involved in biological growth, development and disease state. The main impact of miRNAs has been demonstrated during cellular proliferation, differentiation, metabolism, and apoptosis with regulatory roles on RNA processing and transcription, chromatin structure and chromosome segregation [14]. In humans, it has been reported that around 60% of proteins-coding genes serve as target sites of miRNA [14, 15]. miRNAs can also act in an epigenetic manner to regulate the amplification and inhibition of miRNA signals through the feedback mechanism which may lead to a significant modification expression that contributes to different pathological conditions [4]. Moreover, miRNAs have been shown to play an important role in ovarian physiology and pathology such as primordial follicle activation and development, oocyte maturation, ovulation, ovarian cancer and endometriosis [4, 14]. Growing evidence demonstrates a differential expression of miRNAs in patients with and without PCOS [1], potential diagnostic and therapeutic markers for PCOS. Nevertheless, considering the large degree of heterogeneity of PCOS and the complexity of miRNAs regulatory actions, to our knowledge to date is still preliminary. Understanding the role of miRNAs in PCOS pathogenesis is crucial for possible alternative management and treatment approaches to this syndrome.

To this end, the aim of this chapter is to summarise and discuss the current knowledge regarding the possible interplay between miRNAs and PCOS and to establish the potential clinical role of some miRNAs that may offer a novel insight for the management and treatment of the syndrome.

#### **2.1 Possible role of miRNAs in ovarian dysfunction in PCOS**

Several studies and theories have been proposed in an endeavour to explore the possible cause of the altered follicle growth and development, large number of small and generally immature follicles as well as anovulation associated with PCOS. Hormonal-induced alterations to granulosa cells (GCs) appearance and function, as

*The Novelty of miRNAs as a Clinical Biomarker for the Management of PCOS DOI: http://dx.doi.org/10.5772/intechopen.104386*

well as steroidogenesis abnormalities by the theca cells (TC), have also been reported (**Figure 1**). However, the mechanisms, chronology and the relative criticality in the cascade of events leading to PCOS are still unestablished. Since PCOS is largely influenced by environmental and genetic modifications including miRNAs [16], and their epigenetics alteration can modulate gene expression at the cellular levels, thus miRNAs' profiles in the ovarian compartments in PCOS have been studied in several animal and human models and are discussed further in the section below.

#### *2.1.1 miRNAs in granulosa cells (GCs) and PCOS*

During follicle development, GCs govern oocyte growth by modulating nutrient availability and activity of regulatory molecules. GCs have been identified as the primary site of endocrine signalling and oestrogen synthesis [17]. It has been demonstrated that GC may contribute to the abnormal folliculogenesis in PCOS patients [18]. These abnormalities have been further defined by the proliferation inhibition and increased rate of GCs death, thus supporting the link between the functional disorder of GCs and the disease nature of the PCOS [18].

A vast array of miRNAs has been shown to be differentially expressed among various size follicles. They have been also indicated to modulate GCs apoptosis and proliferation [19]. miR-1275, a regulator of GC death, has been reported to upregulate during follicular atresia and induce early porcine GCs apoptosis [20]. miR-23a and miR-27a have been shown to stimulate GC apoptosis, whereas miR-93 and Let-7 family of miRNAs act to promote proliferation [4, 17]. Moreover, Let-7 family of miRNAs has also been reported to induce GC apoptosis via the inhibition of mitogenic activated protein kinase 1 (MAP3K1). Interestingly, let-7c, 23a and 27a were all shown

**Figure 1.** *The complexity of miRNAs interactions with multiple organs leading to PCOS.*

to be highly expressed in patients with premature ovarian failure when compared with a healthy control patient [21]. Furthermore, a high expression of miR-93 [22] and lower miR-23a have been identified in the GCs from PCOS patients. They indicated that miR-23a induces cell cycle arrest via the inhibition of FGD4 signalling [23]. Another group has also demonstrated a significantly lower expression of miR-126-5p and miR-29a in the GCs of PCOS patients when compared with healthy individuals, which were indicated to induce GC apoptosis in PCOS [24]. A recent study proposed that the high expression profile of miR-141 and miR-200 in PCOS may target the Wnt and PI3K signalling pathway to inhibit GC proliferation [25]. Moreover, miR-3940-5P, miR-486-5P, miR-206 and miR-204 are known to modulate ovarian GC proliferation and apoptosis [26, 27]. In PCOs, miR-3940-5p was reported to be markedly upregulated; however, low expression of miR-206, miR-204 and miR-486-5p has been indicated in the GC of PCOS when compared with normal controls [19, 26]. One questionable miRNA, which has been known to be involved in the proliferation and apoptosis of GC, is miR-485-5P. This miRNA has been reported contradictory in PCOS, with one report demonstrating its upregulation [18], and the other showed a lower expression of miR-485-5P in the GC of PCOS [28].

Furthermore, the dysregulation of miRNAs in GC can be a cause of oestrogen difeiciency which is known as a main characteristic of PCOS. Zhang et al. [29] reported a significantly lower expression of miR-320a in the cumulus cells (CCs) of PCOS, which served via 320a/RUNX2/CYP11A1 (CYP19A1) cascade to induce oestrogen deficiency. miR-182 and miR-15a are known as an essential regulator of GC proliferation and apoptosis as well as steroidogenesis. These miRNAs were found to markedly decreased in the GC of PCOS patients [4].

Studies on miRNAs expression in GC and its relation to PCOS are intensive and generally linked to the increased GC proliferation and apoptosis rates in GCs. Although it may sound contradicting, but it does make sense when thinking about the progression features of the disease in relation to follicular development. The increased proliferation rate of GCs can contribute to a more follicles progressing to the primary stage, which is reflected in the polycystic countenance of PCOS [30]. Whereas the increased apoptosis rate is indicated by the anovulatory feature of the disease [4, 31].

Overall, to date, all reports indicate that GCs miRNAs are clearly involved in the regulation of folliculogenesis and steroidogenesis, and any dysregulation or alteration may subsequently contribute to the pathogenicity of PCOS.

#### *2.1.2 miRNAs in theca cells (TCs) and PCOS*

Theca cells (TCs) are the primary site of androgen synthesis in the ovaries. Under the influence of LH, theca cells express the mRNA of the three important steroidogenic enzymes CYP11A, CYP17 and 3ß-HSD, which are involved in the androgen *de novo* synthesis pathway [32, 33]. Androgen excess is one of the most important key features that is used to diagnose PCOS [14, 34]. It can negatively impact ovarian follicle growth and maturation and therefore female infertility. It has been reported that the increased expression or activity of CYP17 and P450scc enzyme is associated with the abnormal high androgen production in the theca cells [2, 35, 36]. Another key androgen producing-related gene GATA6 and an insulin receptor gene IRS-2 were demonstrated to be markedly upregulated in the theca cells of PCOS patients. Lin et al. [2] documented that miR-92a and miR-92b are involved in the regulation of GATA6 and IRS-2, and their expressions were significantly lower in theca tissues of women with PCOS. The results of miRNA microarray analysis have demonstrated that

miR-200a, miR-200c, miR-141 and miR-502-3p were significantly increased in the theca interna in women with PCOS [14].

Limited data are available on the theca cells miRNAs in PCOS; however, it indicates that it may play a pivotal role in the dysregulation of androgen synthesis pathways in women with PCOS.

#### *2.1.3 miRNA in follicular fluid and PCOS*

Follicular fluid (FF) provides a perfect microenvironment for oocyte development and maturation. It allows for an efficient cross talk between the blood, granulosa and TCs [9]. FF comprises various hormones, multiple proteins, metabolites, antiapoptotic factors and regulatory nucleotides including miRNAs [13]. Therefore, FF composition reflects the secretory activity of oocyte, GC and TCs. In addition, it may serve as a relatively easy and less invasive method for the collection and the analysis of miRNA during oocytes retrieval for assisted reproduction [4].

miRNAs have been reported abundant in FF, and some researchers focused on investigating their association to PCOS [1, 13]. A recent study found 29 differentially expressed miRNAs in the FF of subjects with and without PCOS, with 12 involved in reproductive pathways [37]. Of these, miR-382-5p was found to positively associate with free androgen index (FAI) and age, miR-199b-5p allied with AMH, and miR-127-3p was linked to insulin resistance [37]. A study by Roth et al. [38] reported a significantly increased expression of miR-9, miR-18b, miR-32, miR-34c and miR-135a in the FF of women with PCOS. In addition, the study demonstrated that synaptogamin 1, insulin receptor 2 and interleukin 8 were the target genes for these miRNAs [38]. The study by Scalici et al. showed an increased expression of miR-30a and a significantly decreased levels of let7b and miR-140 in the FF of PCOS individuals. FOXL-2, essential gene for ovarian development, was identified as the target gene for miR-30a, and the inhibition of this miRNA in mouse resulted into disrupted GC morphology and androgen production by the TCs [39]. Furthermore, two members of the TGF-ß family, Smad 2/3 and activin receptor I, were possible target genes for let-7b. The dysregulation of TGF-ß was reported to be a potential cause of PCOS [1, 40]. Scalici et al. [39] also concluded that the combination of Let-7b, miR-30a and miR-140 can be used as a possible novel biomarker, with a sensitivity of 70% and specificity of 80%, to distinguish between normal controls and PCOS. miR-93 has been described as a novel diagnostic marker for PCOS due to the significantly consistent increased expression when compared with healthy individuals [41]. Moreover, a study by Lin et al. reported a significant downregulation of miR-92a and miR-92b in PCOS [2].

Studies focusing on miRNAs' involvement in androgen metabolism and PCOS have identified a specific group of miRNAs in the FF of PCOS patients; however, there is no specific consensus on the effect of these miRNAs in steroidogenesis in PCOS patients. Of these miRNAs, miR-151 was shown to negatively associate with free testosterone; while miR-29a and miR-518 were positively correlated to testosterone; miR-155, miR-9 and miR-18b inhibited testosterone and miR-146a, miR-132 and miR-135 inhibited both testosterone and progesterone secretion [42]. Other contradicting miRNAs, miR-132 and miR-320, which are known to be involved in oestradiol release, showed significant lower expression in PCOS patients [43], while another author reported increased expression of miR-320 in PCOS individuals [39, 44]. A third report demonstrated no change in 320a expression in PCOS subjects when compared with healthy women [3, 38]. miR-518-3p was shown to highly expressed in androgenic

PCOS phenotype. Further analysis demonstrated the reduction in miR-24-3p, miR-29a, miR-151-3p and miR-574-3p levels in PCOS subjects when compared with healthy controls [3].

All above, data indicates that miRNAs in PCOS can serve as a potential novel biomarker for the diagnosis and maybe further for the classification of different PCOS phenotypes. Furthermore, it may provide insights into the molecular changes related to the cells from which the fluid is derived from and thus support therapeutic decisions. Ovarian cells and circulating miRNAs that have been proposed to be dysregulated and involved in the pathogenicity of PCOS are summarised in the **Table 1**.

#### **2.2 miRNAs altered androgenic and metabolic consequences of the PCOS**

HA and metabolic disorders are inevitably associated in women with PCOS. It is always manifested by hyperandrogenism (HA), insulin resistance (IR) and compensatory hyperinsulinemia, which is known as an essential contributor to the pathogenicity of PCOS. Therefore, the mechanism behind this connection could shed lights on key markers in the diagnosis of PCOS.

#### *2.2.1 miRNA and androgens dysregulation in PCOS*

HA is a common characteristic of PCOS that is detected and used for diagnosis in both serum and ovarian compartment. The source of androgen excess is not exclusively resolved, it might be due to increased steroidogenic enzyme activity, increased androgen synthesis by the TCs as a response to LH overstimulation, androgen receptor (AR) defects at the target organs level, cortisol metabolism defects and/or increased adrenal gland androgen production [3, 4, 45]. Evidence indicates that testosterone, androstenedione (A4) and dihydrotestosterone (DHT) are all involved in the pathogenicity of PCOS. In normal subjects, a major fraction of free testosterone is bound to sex hormone binding globulin (SHBG) and albumin. In women with PCOS, SHBG levels are decreased resulting into an increased level of bioavailable testosterone [4]. Furthermore, increased expression of AR has been reported in patients with PCOS [46].

The complexity of abnormal sex hormone production makes it difficult to define the specific miRNAs involved in this process. Several studies have highlighted the role played by miRNAs in androgens and steroid synthesis in the ovarian cells and body fluids. miR-592 was found to positively correlate with LH/chorionic gonadotropin receptor (LH/CGH), a key factor in the mechanism involved in HA in PCOS [47]. A recent study showed a negative association between serum testosterone and miR-146a, an miRNA that has been found to inhibit the secretory activity of steroid hormones [1, 48]. Oestradiol was noted to positively linked with miR-222, miR-132, miR-320 and miR-520-3p, whereas a negative relationship has been observed between progesterone concentration and miR-193b, miR-24 and miR483-5p [43, 49]. Furthermore, miR-320, miR-518, miR107 and miR-29a were also found to positively correlated with high levels of serum testosterone [4, 9]. On the other hand, expression profile of miR-151 was found to negatively relate to serum testosterone [9]. Another study demonstrated a strong positive association between levels of free testosterone and miR-155, miR-27b, miR-103 and miR-21 [1]. Interestingly, Xiong et al. reported that the chance of PCOS development has been reduced by 0.01-fold for every elevated fold expression of miR-23a [50]. Lower oestrogen synthesis has been linked to the over-expression of miR-181a and miR-378 via the downregulation of aromatase

*The Novelty of miRNAs as a Clinical Biomarker for the Management of PCOS DOI: http://dx.doi.org/10.5772/intechopen.104386*



#### **Table 1.**

*Circulating and ovarian miRNAs that been proposed altered in PCOS.*

enzyme [51, 52]. It is intriguing to note that miR-200b is involved in ovulation at the hypothalamo-pituitary ovarian axis level and is a downstream target for AR; such information may indicate that miR-200 plays a role in HA and PCOS [14].

HA is associated with an array of pathological changes that interact to promote the development of PCOS. Of these changes, hyperinsulinemia, IR and dyslipidaemia are well characterised.

#### *2.2.2 miRNAs and insulin resistance (IR) and/or hyperinsulinemia in PCOS*

IR plays a major role in the pathogenicity of PCOS with a pervasiveness of 70% among patients. It is known to be associated with impaired glucose metabolism, T2DM, metabolic syndrome, dyslipidaemia and increased risk of CVD. Hyperinsulinemia contributes to the increased ovarian steroidogenesis and androgen production via insulin growth factor-1 (IGF-1) receptor to initiate LH-induced TCs excess androgen secretion [53]. IR synergistically stimulated androgen via increasing CYP17 enzyme activity leading to lower SHBG levels and thus increase free testosterone levels [4, 54]. IGF-1 signalling, peroxidase proliferator receptor (PPAR) and angiopoietin, have been associated with PCOS and are potentially regulated by miR-223 [41]. miR-483-5p has been shown to lower IRs in women with PCOS [28]. It has been suggested that miR-320 and miR-132 can play a role in modulating insulin

resistance [43]. The author predicted that RABSB and HMGA2 are the target genes for miR-320 and miR-132, respectively, and that both gene expressions are altered by the reduced levels of miR-320 and miR-132 in the FF of PCOS individuals [43]. Increased expressions of miR-194, miR-193b and miR-122 have been noted in PCOS patients when compared with controls. The upregulation of these three miRNAs was found to target several pathways including insulin signalling pathway [22]. A strong association has been reported between the expression of miR-93 and miR-33b-5p and the development of IR in women with PCOS. They have been shown to exert their effect through the downregulation of glucose transporter 4 (GLUT4) expression [10, 55]. A significant positive correlation has been found between miR-222 expression and serum insulin [48]. Lin et al. [2] reported that 200a expression is associated with IR and T2DM. miR-1, miR-29, miR126 and miR-19a have been postulated to regulate glucose uptake via modulating PI3K [56].

All these findings indicate a cross talk between HA and hyperinsulinemia in PCOS. miRNAs involved in the regulation of these two dominant features are key for the development of specific markers for the diagnosis and treatment of the syndrome.

#### *2.2.3 miRNA and dyslipidaemia in PCOS*

The prevalence of hyperlipidaemia is 70% in PCOS. It is generally manifested by lipid profile dysfunctions, including increased levels of low-density lipoprotein cholesterol (LDL-C), reduced levels of high-density lipoproteins cholesterol (HDL-C) and high triglycerides [57]. Even though obesity is not indicative of PCOS, however, visceral adipose tissues (VATs) were found higher in patients with PCOS, whether they are obese or not, when compared with a control group [33]. To date, studies have found a close correlation between androgen excess, obesity and PCOS [5]. Androgen excess promotes the deposition of abdominal visceral fat, which in turn drives the secretion of adrenal and ovarian androgens via several pathways induced by adipose tissue dysfunction [58].

Current data on the interaction between miRNAs, dyslipidaemia and PCOS is sparse. In animal models, miR-33 has been shown to exert some control on LDL-C secretion and modulation of cholesterol biosynthesis [4, 59]. In addition, miR-128-1, miR-185 and miR-148a were reported to significantly decrease LDL-C levels, whereas miR-148a expression increased HDL-C [60]. The target genes for these miRNAs were adenosine triphosphate (ATP) binding cascade transporter A1 (ABCA1) and ABCG1 [60]. Furthermore, miR-34c-5p, miR-760, miR-597 and miR-1468 were found to be positively linked to hirsutism score. miR-597 and miR-1468 were shown to target the androgen receptor pathway [58]. Murri et al. [58] identified a group of miRNAs that are differentially expressed among PCOS patients. Of which, miR-30c-5p, miR-34c5p, miR-151a-5p, miR-193a-5p, miR-199a-3p, miR-1539, miR-26a-5p, miR-107, miR-142-3p, miR-126-5p and miR-598 were shown to correlate either positively or negatively with abdominal adiposity, obesity and metabolic dysfunction. Among all, miR-107, miR-30c-5p, miR-199a-3p and miR-26a-5p were noted to significantly associate with fatty acid biosynthesis and metabolism, and their expressions were found reduced in PCOS. The same group demonstrated further an increased expression of miR-338-3p, miR-365, miR-223-3p and miR-197-3 in obese PCOS patients when compared with the control subjects. All the reported miRNAs were correlated with androgen excess, glucose metabolic index and BMI [58]. Furthermore, miR-548-3p and miR-34c-5p expressions were also found to increase in PCOS individuals when compared with controls. Both miR-548-3p and miR-34c-5p markedly correlate with fatty acid biosynthesis and metabolisms [58].

Taken together, miRNAs play a pivotal role in the interaction of HA, hyperinsulinemia, dyslipidaemia and PCOS. Therefore, targeting miRNAs involved in the regulation of these features can shed light on the clinically relevant diagnostics and therapeutics markers of PCOS.

#### **3. Considering miRNAs as a potential therapeutic target in PCOS**

Based on the current knowledge presented in previous sections, miRNAs seem to be associated to the development of PCOS. However, no reports are available postulating the use of drugs to target miRNAs as a potential treatment option. Current guidance on disease management options is only focusing on improving prognosis and mitigating the symptoms. Of interest, PCOS patients are mostly undergoing in vitro fertilisation and thus involved in pharmacological protocols to support their fertility treatment. Available treatment options include the combined Letrozole and clomiphene citrate treatment, which is typically used for ovulation induction. Oral contraceptives (OCPs) control ovulation and prevent cyst formation. Anti-androgen drugs such as flutamide and spironolactone are used to manage increased androgens-related problems. Metformin is an insulin sensitivity agent that can improve the life quality for these patients by reducing serum insulin and androgen levels and elevating SHBG [4, 9, 61]. Polyphenols are a natural compound that has been used traditionally as a treatment option for several conditions; however, their limited bioavailability restricted their use. It has recently dragged some attention as a therapeutic target to ameliorate PCOS symptoms and reverse expression of crucial genes; nevertheless, more data are required before their administration into clinical use [61].

Studies focusing on therapeutics that may target small molecules such as miRNAs are rarely reported. Recently, metformin administration has been found to either positively or negatively target some miRNAs. Bao et al. demonstrated that metformin can inhibit markers for pancreatic cancer stem cells via the upregulation of miR-26a [62]. In addition, another study reported that metformin decreased the expression of miR-221 and miR-222 in T2DM patients [4, 9, 63]. Even more, metformin with sitagliptin combined therapy has been proposed as a treatment strategy to ameliorate PCOS in patients with IR [64]. This strategy was found to reduce GC apoptosis via mediating IncRNA H19 expression [33, 64].

Incretins-based therapies have been recently discovered to influence miRNAs expression and its potential for the management of PCOS. Glucagon-like peptide 1 agonist receptor agonist (GLP-1RA) decreased blood sugars via the downregulation of miR-375 and miR-23 and the increased expression of miR-192, miR-132 and miR-27a [65].

Treatment approaches targeting miRNAs associated with the metabolic feature of the disease may improve the clinical outcomes. miRNAs regulating androgen hormones are worthy at the top of the list. However, it is still a rich field of scientific research and validation.

#### **4. Clinical application of miRNAs is still limited**

Despite the hope, great effort and the scientific work done to clarify the role of miRNAs in PCOS, it is still a long way to go before it is possible to utilise miRNAs as a *The Novelty of miRNAs as a Clinical Biomarker for the Management of PCOS DOI: http://dx.doi.org/10.5772/intechopen.104386*

diagnostic and therapeutic tool. It is perhaps due to the complexity of both miRNAs and PCOS.

The pathogenicity of PCOS involves several phenotypes described by the Rotterdam criteria and other less sever criterion, thus providing difficult and ununited diagnosis. In addition, the fact that one mRNA maybe regulated by multiple miRNAs and that miRNAs can modulate each other complicates the matter even further. Moreover, most of the studies performed are limited by the sample size and sometimes with contradicting results. Furthermore, metformin is mostly taken by PCOS individuals during their infertility treatment and has been shown to influence miRNA expression, thus may false mask facts and lead to contradicting results. Therefore, it is imperative to deepen the current research to develop consistent and reliable miRNA-based diagnostics and therapeutics.

#### **5. Conclusion and future direction**

Taken together, miRNAs play a key role to fine-tune events leading to ovarian cell apoptosis, proliferation, follicular development, HA, altered insulin and dyslipidaemia in PCOS. Yet, it is not possible to determine whether miRNAs-altered expression is a cause or is an aftereffect event of the syndrome. Furthermore, the dynamic expression and action of miRNAs complicate facts further. Thus, further functional studies on mRNA-miRNA and the interplay between epigenetic regulation and altered miRNA expression are required to highlight the pathogenicity of the disease. It is, however, possible to use miRNAs as biomarker to categorise and identify PCOS subphenotypes. miRNAs are a promising candidate to modulate pathways involved in the pathogenicity of this syndrome.

#### **Author details**

Rana Alhamdan1,2\* and Juan Hernandez-Medrano3

1 Department of Pathology and Laboratory Medicine (DPLM), King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia

2 Division of Child Health, Obstetrics and Gynaecology, School of Medicine, Queen's Medical Centre, University of Nottingham, Nottingham, UK

3 Department of Production Animal Health, Alberta, Canada

\*Address all correspondence to: amsf199705@hotmail.co.uk

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Chen B, Xu P, Wang J, Zhang C. The role of MiRNA in polycystic ovary syndrome (PCOS). Gene. 2019;**706**: 91-96

[2] Lin L, Du T, Huang J, Huang L-L, Yang D-Z. Identification of differentially expressed microRNAs in the ovary of polycystic ovary syndrome with hyperandrogenism and insulin resistance. Chinese Medical Journal. 2015;**128**:169

[3] Sørensen AE, Wissing ML, Englund ALM, Dalgaard LT. MicroRNA species in follicular fluid associating with polycystic ovary syndrome and related intermediary phenotypes. The Journal of Clinical Endocrinology & Metabolism. 2016b;**101**:1579-1589

[4] Abdalla M, Deshmukh H, Atkin SL, Sathyapalan T. miRNAs as a novel clinical biomarker and therapeutic targets in polycystic ovary syndrome (PCOS): A review. Life Sciences. 2020; **259**:118174

[5] Zeng X, Xie Y-J, Liu Y-T, Long S-L, Mo Z-C. Polycystic ovarian syndrome: Correlation between hyperandrogenism, insulin resistance and obesity. Clinica Chimica Acta. 2020a;**502**:214-221

[6] Jiang X, Li J, Zhang B, Hu J, Ma J, Cui L, et al. Differential expression profile of plasma exosomal microRNAs in women with polycystic ovary syndrome. Fertility and Sterility. 2021; **115**:782-792

[7] Cai G, Ma X, Chen B, Huang Y, Liu S, Yang H, et al. MicroRNA-145 negatively regulates cell proliferation through targeting IRS1 in isolated ovarian granulosa cells from patients with polycystic ovary syndrome. Reproductive Sciences. 2017;**24**:902-910

[8] He L, Hannon GJ. MicroRNAs: Small RNAs with a big role in gene regulation. Nature Reviews Genetics. 2004;**5**: 522-531

[9] Sørensen AE, Wissing ML, Salö S, Englund ALM, Dalgaard LT. MicroRNAs related to polycystic ovary syndrome (PCOS). Genes. 2014;**5**:684-708

[10] Chen Y-H, Heneidi S, Lee J-M, Layman LC, Stepp DW, Gamboa GM, et al. miRNA-93 inhibits GLUT4 and is overexpressed in adipose tissue of polycystic ovary syndrome patients and women with insulin resistance. Diabetes. 2013;**62**:2278-2286

[11] Wu W, Duan C, Lv H, Song J, Cai W, Fu K, et al. MiR-let-7d-3p inhibits granulosa cell proliferation by targeting TLR4 in polycystic ovary syndrome. Reproductive Toxicology. 2021;**106**: 61-68

[12] Jiang L, Huang H, Qian Y, Li Y, Chen X, Di N, et al. miR-130b regulates gap junctional intercellular communication through connexin 43 in granulosa cells from patients with polycystic ovary syndrome. Molecular Human Reproduction. 2020;**26**:576-584

[13] Cui C, Wang J, Han X, Wang Q, Zhang S, Liang S, et al. Identification of small extracellular vesicle-linked miRNA specifically derived from intrafollicular cells in women with polycystic ovary syndrome. Reprod Biomed Online. 2021; **42**:870-880

[14] Xue Y, Lv J, Xu P, Gu L, Cao J, Xu L, et al. Identification of microRNAs and genes associated with hyperandrogenism in the follicular fluid of women with polycystic ovary syndrome. Journal of Cellular Biochemistry. 2018;**119**: 3913-3921

*The Novelty of miRNAs as a Clinical Biomarker for the Management of PCOS DOI: http://dx.doi.org/10.5772/intechopen.104386*

[15] Suzuki H, Maruyama R, Yamamoto E, Kai M. Epigenetic alteration and microRNA dysregulation in cancer. Frontiers in Genetics. 2013;**4**:258

[16] Fenichel P, Rougier C, Hieronimus S, Chevalier N. Which Origin for Polycystic Ovaries Syndrome: Genetic, Environmental or both? Annales D'endocrinologie. Amsterdam, Netherlands: Elsevier; 2017. pp. 176-185

[17] Hou Y, Wang Y, Xu S, Qi G, Wu X. Bioinformatics identification of microRNAs involved in polycystic ovary syndrome based on microarray data. Molecular Medicine Reports. 2019;**20**: 281-291

[18] Xu B, Zhang Y-W, Tong X-H, Liu Y-S. Characterization of microRNA profile in human cumulus granulosa cells: Identification of microRNAs that regulate notch signaling and are associated with PCOS. Molecular and Cellular Endocrinology. 2015;**404**:26-36

[19] Zhou J, Jin X, Sheng Z, Zhang Z. miR-206 serves an important role in polycystic ovary syndrome through modulating ovarian granulosa cell proliferation and apoptosis. Experimental and Therapeutic Medicine. 2021;**21**:1-1

[20] LIu J, Li X, Yao Y, Li Q, Pan Z, Li Q. miR-1275 controls granulosa cell apoptosis and estradiol synthesis by impairing LRH-1/CYP19A1 axis. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms. 2018; **1861**:246-257

[21] Cao, R., Wu, W., Zhou, X., Liu, K., Li, B., Huang, X., Zhang, Y. & Liu, H. 2015. Let-7g induces granulosa cell apoptosis by targeting MAP3K1 in the porcine ovary. The International Journal of Biochemistry & Cell Biology*,* 68**,** 148-157.

[22] Jiang L, Huang J, Chen Y, Yang Y, Li R, Li Y, et al. Identification of several circulating microRNAs from a genomewide circulating microRNA expression profile as potential biomarkers for impaired glucose metabolism in polycystic ovarian syndrome. Endocrine. 2016;**53**:280-290

[23] Lin J, Huang H, Lin L, Li W, Huang J. MiR-23a induced the activation of CDC42/PAK1 pathway and cell cycle arrest in human cov434 cells by targeting FGD4. Journal of Ovarian Research. 2020;**13**:1-10

[24] Mao Z, Fan L, Yu Q, Luo S, Wu X, Tang J, et al. Abnormality of klotho signaling is involved in polycystic ovary syndrome. Reproductive Sciences. 2017; **25**:372-383

[25] He T, Liu Y, Jia Y, Wang H, Yang X, Lu G, et al. MicroRNA-141 and MicroRNA-200c are overexpressed in granulosa cells of polycystic ovary syndrome patients. Frontiers in Medicine. 2018;**5**:299

[26] GAO L, Wu D, Wu Y, Yang Z, Sheng J, Lin X, et al. MiR-3940-5p promotes granulosa cell proliferation through targeting KCNA5 in polycystic ovarian syndrome. Biochemical and Biophysical Research Communications. 2020;**524**: 791-797

[27] Pei C-Z, Jin L, Baek K-H. Pathogenetic analysis of polycystic ovary syndrome from the perspective of omics. Biomedicine & Pharmacotherapy. 2021; **142**:112031

[28] Shi L, Liu S, Zhao W, Shi J. miR-483- 5p and miR-486-5p are down-regulated in cumulus cells of metaphase II oocytes from women with polycystic ovary syndrome. Reproductive Biomedicine Online. 2015;**31**:565-572

[29] Zhang C-L, Wang H, Yan C-Y, Gao X-F, Ling X-J. Deregulation of RUNX2 by miR-320a deficiency impairs steroidogenesis in cumulus granulosa cells from polycystic ovary syndrome (PCOS) patients. Biochemical and Biophysical Research Communications. 2017;**482**:1469-1476

[30] Das M, Djahanbakhch O, Hacihanefioglu B, Saridogan E, Ikram M, Ghali L, et al. Granulosa cell survival and proliferation are altered in polycystic ovary syndrome. The Journal of Clinical Endocrinology & Metabolism. 2008;**93**: 881-887

[31] Worku T, Rehman ZU, Talpur HS, Bhattarai D, Ullah F, Malobi N, et al. MicroRNAs: New insight in modulating follicular atresia: A review. International Journal of Molecular Sciences. 2017;**18**: 333

[32] Mlynarcikova A, Fickova M, Scsukova S. Impact of endocrine disruptors on ovarian steroidogenesis. Endocrine Regulations. 2014;**48**:201-224

[33] Zeng Z, Lin X, Xia T, Liu W, Tian X, Li M. Identification of crucial lncRNAs, miRNAs, mRNAs, and potential therapeutic compounds for polycystic ovary syndrome by bioinformatics analysis. BioMed Research International. 2020b, 2020;**2020**:1817094

[34] Nandi A, Chen Z, Patel R, Poretsky L. Polycystic ovary syndrome. Endocrinology and Metabolism Clinics of North America. 2014;**43**: 123-147

[35] Strauss JF, Wood JR, Christenson LK, Mcallister JM. Strategies to elucidate the mechanism of excessive theca cell androgen production in PCOS. Molecular and Cellular Endocrinology. 2002;**186**:183-188

[36] Wood JR, Ho CK, Nelson-Degrave VL, Mcallister JM, Strauss JF. The molecular signature of polycystic ovary syndrome (PCOS) theca cells defined by gene expression profiling. Journal of Reproductive Immunology. 2004;**63**: 51-60

[37] Butler AE, Ramachandran V, Hayat S, Dargham SR, Cunningham TK, Benurwar M, et al. Expression of microRNA in follicular fluid in women with and without PCOS. Scientific Reports. 2019;**9**:16306

[38] Roth LW, Mccallie B, Alvero R, Schoolcraft WB, Minjarez D, Katz-Jaffe MG. Altered microRNA and gene expression in the follicular fluid of women with polycystic ovary syndrome. Journal of Assisted Reproduction and Genetics. 2014;**31**:355-362

[39] Scalici E, Traver S, Mullet T, Molinari N, Ferrieres A, Brunet C, et al. Circulating microRNAs in follicular fluid, powerful tools to explore in vitro fertilization process. Scientific Reports. 2016;**6**:1-10

[40] Raja-Khan N, Urbanek M, Rodgers RJ, Legro RS. The role of TGF-β in polycystic ovary syndrome. Reproductive Sciences. 2014;**21**:20-31

[41] Sathyapalan T, David R, Gooderham NJ, Atkin SL. Increased expression of circulating miRNA-93 in women with polycystic ovary syndrome may represent a novel, non-invasive biomarker for diagnosis. Scientific Reports. 2015;**5**: 16890

[42] Sørensen AE, Udesen PB, Wissing ML, Englund ALM, Dalgaard LT. MicroRNAs related to androgen metabolism and polycystic ovary syndrome. Chemico-Biological Interactions. 2016a;**259**:8-16

*The Novelty of miRNAs as a Clinical Biomarker for the Management of PCOS DOI: http://dx.doi.org/10.5772/intechopen.104386*

[43] Sang Q, Yao Z, Wang H, Feng R, Wang H, Zhao X, et al. Identification of microRNAs in human follicular fluid: Characterization of microRNAs that govern steroidogenesis in vitro and are associated with polycystic ovary syndrome in vivo. The Journal of Clinical Endocrinology & Metabolism. 2013;**98**: 3068-3079

[44] Yin M, Wang X, Yao G, Lü M, Liang M, Sun Y, et al. Transactivation of micrornA-320 by microRNA-383 regulates granulosa cell functions by targeting E2F1 and SF-1 proteins. The Journal of Biological Chemistry. 2014; **289**:18239-18257

[45] Kempná P, Marti N, Udhane S, Flück CE. Regulation of androgen biosynthesis – A short review and preliminary results from the hyperandrogenic starvation NCI-H295R cell model. Molecular and Cellular Endocrinology. 2015;**408**: 124-132

[46] Apparao K, Lovely LP, Gui Y, Lininger RA, Lessey BA. Elevated endometrial androgen receptor expression in women with polycystic ovarian syndrome. Biology of Reproduction. 2002;**66**:297-304

[47] Song J, Luo S, Li SW. miRNA-592 is downregulated and may target LHCGR in polycystic ovary syndrome patients. Reproductive Biology. 2015;**15**:229-237

[48] Long W, Zhao C, Ji C, Ding H, Cui Y, Guo X, et al. Characterization of serum microRNAs profile of PCOS and identification of novel non-invasive biomarkers. Cellular Physiology and Biochemistry. 2014;**33**:1304-1315

[49] Eiras MC, Pinheiro DP, Romcy KAM, Ferriani RA, Dos Reis RM, Furtado CLM. Polycystic ovary syndrome: The epigenetics behind the disease. Reproductive Sciences. 2021:1-15 [50] Xiong W, Lin Y, Xu L, Tamadon A, Zou S, Tian F, et al. Circulatory microRNA 23a and microRNA 23b and polycystic ovary syndrome (PCOS): The effects of body mass index and sex hormones in an eastern Han Chinese population. Journal of Ovarian Research. 2017;**10**:1-11

[51] Xu S, Linher-Melville K, Yang BB, Wu D, Li J. Micro-RNA378 (miR-378) regulates ovarian estradiol production by targeting aromatase. Endocrinology. 2011;**152**:3941-3951

[52] Zhang Q, Sun H, Jiang Y, Ding L, Wu S, Fang T, et al. MicroRNA-181a suppresses mouse granulosa cell proliferation by targeting activin receptor IIA. PLoS ONE. 2013;**8**: e59667

[53] Diamanti-Kandarakis E, Christakou CD. Insulin resistance in PCOS. Diagnosis and Management of Polycystic Ovary Syndrome. 2009:35-61

[54] Zhang G, Garmey JC, Veldhuis JD. Interactive stimulation by luteinizing hormone and insulin of the steroidogenic acute regulatory (StAR) protein and 17αhydroxylase/17, 20-lyase (CYP17) genes in porcine theca Cells1. Endocrinology. 2000;**141**:2735-2742

[55] Yang Y, Jiang H, Xiao L, Yang X. MicroRNA-33b-5p is overexpressed and inhibits GLUT4 by targeting HMGA2 in polycystic ovarian syndrome: An in vivo and in vitro study. Oncology Reports. 2018;**39**(6):3073-3085

[56] Chakraborty C, Doss CGP, Bandyopadhyay S, Agoramoorthy G. Influence of miRNA in insulin signaling pathway and insulin resistance: Micromolecules with a major role in type-2 diabetes. Wiley Interdisciplinary Reviews: RNA. 2014;**5**:697-712

[57] Legro RS, Kunselman AR, Dunaif A. Prevalence and predictors of dyslipidemia in women with polycystic ovary syndrome. The American Journal of Medicine. 2001;**111**:607-613

[58] Murri M, Insenser M, Fernández-Durán E, San-Millán JL, Luque-Ramírez M, Escobar-Morreale HF. Non-targeted profiling of circulating microRNAs in women with polycystic ovary syndrome (PCOS): Effects of obesity and sex hormones. Metabolism. 2018;**86**:49-60

[59] Soh J, Iqbal J, Queiroz J, Fernandez-Hernando C, Hussain MM. MicroRNA-30c reduces hyperlipidemia and atherosclerosis in mice by decreasing lipid synthesis and lipoprotein secretion. Nature Medicine. 2013;**19**:892-900

[60] Wagschal A, Najafi-Shoushtari SH, Wang L, Goedeke L, Sinha S, Delemos AS, et al. Genome-wide identification of microRNAs regulating cholesterol and triglyceride homeostasis. Nature Medicine. 2015;**21**:1290-1297

[61] Mihanfar A, Nouri M, Roshangar L, Khadem-Ansari MH. Polyphenols: Natural compounds with promising potential in treating polycystic ovary syndrome. Reproductive Biology. 2021; **21**:100500

[62] Bao B, Wang Z, Ali S, Ahmad A, Azmi AS, Sarkar SH, et al. Metformin inhibits cell proliferation, migration and invasion by attenuating CSC function mediated by deregulating miRNAs in pancreatic cancer cells. Cancer Prevention Research. 2012;**5**:355-364

[63] Coleman CB, Lightell DJ Jr, Moss SC, Bates M, Parrino PE, Woods TC. Elevation of miR-221 and-222 in the internal mammary arteries of diabetic subjects and normalization with metformin. Molecular and Cellular Endocrinology. 2013;**374**:125-129

[64] Wang Q, Shang J, Zhang Y, Zhou W. Metformin and sitagliptin combination therapy ameliorates polycystic ovary syndrome with insulin resistance through upregulation of lncRNA H19. Cell Cycle. 2019;**18**: 2538-2549

[65] Radbakhsh S, Sathyapalan T, Banach M, Sahebkar A. Incretins and microRNAs: Interactions and physiological relevance. Pharmacological Research. 2020;**153**:104662

#### **Chapter 8**

## Role of Oxidative Stress and Carnitine in PCOS Patients

*Bassim Alsadi*

#### **Abstract**

Polycystic ovary syndrome (PCOS) is a common female endocrine and reproductive system disorder which is found in 6–10% of the female population. PCOS is considered a multifactorial metabolic disease characterized by several clinical manifestations, such as hyperandrogenism, polycystic ovaries and ovulatory dysfunctions. PCOS patients have an increase in the oxidative stress with generation of excessive amounts of reactive oxygen species (ROS) and reduction of antioxidant capacity. Oxidative stress is defined as the imbalance between the production of free radicals and the ability of the organism to defend itself from their harmful effects damaging the plasma membrane, DNA and other cell organelles, inducing apoptosis. Oxidative stress markers are circulating significantly higher in PCOS patients than in healthy women, so these can be considered as potential inducers of the PCOS pathology. Therefore, the central role of the oxidative stress may be involved in the pathophysiology of various clinical disorders including the PCOS. This chapter reviewed the role of oxidative stress and carnitine in PCOS patients, indicating the beneficial action of the carnitine pool, and L-carnitine contributes to restore the energy balance to the oocyte during folliculogenesis and maturation, which represent an important strategy to improve the intraovarian environment and increase the probability of pregnancy.

**Keywords:** polycystic ovary syndrome, ultrasound, anovolution, infertility, hyperandrogenism, oxidative stress, carnitine pool, insulin resistance, advanced glycation end products, RAGE (receptor for AGEs), hyperinsulinemia, reactive oxygen species (ROS)

#### **1. Introduction**

Polycystic ovary syndrome (PCOS) is a common female endocrine and reproductive system disorder which is found in 6–10% of the female population [1].

In general, it is considered a multifactorial metabolic disease characterized by several clinical manifestations such as hyperandrogenism, polycystic ovaries aspects on ultrasound and ovulatory dysfunctions which makes it the most common cause of anovulation infertility in women, but also from metabolic problems such as obesity, insulin resistance, hyperinsulinemia and type II diabetes which may enhance cardiovascular complications and other neurological and psychological implication such as anxiety and depression [2, 3].

The carnitines are essential in the metabolism of fatty acids and can act to protect from mitochondrial damage and altered energy balance conditions such as those present in polycystic ovary syndrome (PCOS) as also highlighted by the reduced levels of L-carnitine in the serum of patients with this disease.

Restoring the energy balance and adequate energy reserves to the oocyte during folliculogenesis and maturation can represent an important strategy to improve the intraovarian environment and increase the probability of pregnancy. In this context, metabolic compounds, such as carnitines, with positive effects on mitochondrial activity and free radical scavenging, can contribute to mitigate the effects of PCOS.

#### **2. Clinical remarks of PCOS**

In the last decade there has been a plenty of discussion regarding the pathogenesis of PCOS, the causes are not yet known, but environmental and genetic factors may be involved [4, 5].

In particular, the genetic abnormalities appear to play a key role in the metabolic complications with a high rate of hyperandrogenism and type II diabetes in first degree relatives of women with PCOS [6, 7].

Recently, some studies have indicated that a defect in insulin action could be the primary cause of PCOS [8, 9].

Other studies have instead observed how important the role of socio-economic status and unhealthy life style, which includes smoking, poor diet, poor exercise and obesity [10, 11]. Furthermore, other studies have suggested that ethnic background may also be associated PCOS probably due to the increased number of insulin resistance and type II diabetes in this population [12, 13]. Polycystic ovary syndrome is the most common cause of menstrual irregularity leads to infertility and it is estimated that 90% of anovolution cases are caused by PCOS [14].

In addition to endocrine and reproductive clinical findings, PCOS also leads to consequences on mental health. Studies showing the correlation between PCOS and reduced quality of life [15, 16] with the increase in anxiety and depression [17]. This is not surprising, since the main phenotypes of this syndrome (obesity, infertility and hirsutism) are major problems that can cause psychological stress. Neuroendocrine dysfunction in gamma-aminobutyric acid (GABA) signaling and neuronal androgen receptors that might alter hypothalamic sensitivity and lead to an impairment of estradiol and progesterone feedback. Elevated concentrations of GABA in the cerebrospinal fluid of women with PCOS, GABA seems to exert an excitatory effect on GnRH neurons and this leads to greater secretion of LH by the pituitary gland, as occurs in PCOS [18, 19].

The metabolic implications of PCOS increase the risk of cardiovascular complications in PCOS patients [20]. Chronic anovulation in PCOS patient may lead to endometrial hyperplasia increasing the risk of endometrial cancer. Obesity, insulin resistance and type 2 diabetes associated to PCOS will enhance the risk of endometrial cancer in PCOS patients [21, 22].

PCOS patients have an increased risk of type II mellitus [23], in addition, insulin resistance plays a central role in the pathogenesis of PCOS [24] as it provokes hyper-insulinemia and accelerates the over-production of androgens in the ovary. Hyper-insulinemia which, in turn, contributes to the development of diabetes and dyslipidemia [25].

#### **3. Oxidative stress in PCOS**

Central role of the oxidative stress may be involved in the pathophysiology of various clinical disorders including the PCOS.

PCOS patients have an increase in the oxidative stress with generation of excessive amounts of reactive oxygen species (ROS) and reduction of antioxidant capacity [26].

Oxidative stress is defined as the imbalance between the production of free radicals and the ability of the organism to defend itself from their harmful effects damaging the plasma membrane, DNA and other cell organelles, inducing apoptosis [27]

Oxidative stress markers are circulating significantly higher in PCOS patients than in healthy women, so these can be considered as potential inducers of the PCOS pathology [28].

#### **4. Advanced glycation end products (AGEs)**

Among the most important post-translational modifications is the non-enzymatic modification of proteins, lipids and nucleic acids with glucose and their consequent conversion into AGEs.

AGEs (advanced glycation end products) therefore represent the final products of a chemical process known as the Maillard reaction, in which carbonyls of glucose or other reactive sugars react non-enzymatically with amino groups of proteins. The further reorganization of which leads to the formation of the Amadori product: the proteins containing this product are known as glycated proteins and the process of formation is known as glycation. Depending on the nature of these glycation products, protein adducts or protein cross-linking are formed, giving rise to the AGEs [29].

The end product of this reaction (AGE), in turn, induce oxidative stress and accelerate the Maillard reactions ultimately leading to inflammation and the propagation of tissue damage [30–32].

Advanced glycation end-products (AGEs) such as glycated hemoglobin commonly used in clinical practice as a marker of hyperglycemia is an Amadori product implicated in the development diabetes mellitus [32].

AGEs can be taken exogenously, through the consumption of food and smoke, or produced endogenously. In fact, in physiological conditions, AGEs are formed very slowly while, in particular conditions like hyperglycemia, insulin resistance, obesity, aging, oxidative stress and hypoxia, their formation process is accelerated [33].

Any accumulation of AGE is associated with various diseases, such as diabetes mellitus type 2, metabolic syndrome, cardiovascular diseases, ovarian aging, neurodegenerative disorders, obesity and PCOS [33–35].

Once formed, AGEs can damage cellular structures through a number of mechanisms, including the formation of cross-links between key molecules of the basement membrane of the extracellular matrix and interaction with receptors on cell surfaces, leading to this way to alteration of cellular function [36, 37].

However, the AGE content in the body is not defined only by their rate of formation, but also from rate of removal. The body cells in fact have developed pathways of detoxification against the accumulation of AGE [38].

The interaction between circulating AGEs and RAGE (receptor for AGEs) will trigger and enhance the pro-inflammatory state, cell toxicity cell and damage **Figure 1** [40].

RAGE is a transmembrane receptor and is expressed in numerous tissues including ovaries, heart, lung and skeletal muscle, but also in monocytes, macrophages and lymphocytes [41]. In physiological conditions this receptor is down-regulated while with aging its expression increases, probably due to the accumulation of ligands which, through positive feedback, regulate the expression of receptor itself [40–42].

In conditions like diabetes, inflammation, atherosclerosis and PCOS, there is a marked induction of RAGE due to the action of ligands and the numerous mediators activated by inflammatory cells **Figure 2** [44].

#### **5. Factors that induce the production of advanced glycation end products (AGEs)**

AGE levels in blood and tissues depend on endogenous sources (chemical reactions) and exogenous sources (diet and smoking). In particular foods rich in protein and fat, like meat, cheese and egg yolk, they are in fact rich in AGE, moreover, cooking methods (such as high temperatures) also increase their concentration drastically [45].

Smoke is another exogenous source of AGE and it has in fact been seen that the serum levels of AGE in smokers are significantly higher compared to non-smokers [46].

The presence of AGE in ovarian tissue, together with an altered metabolic profile and elevated testosterone levels therefore provides evidence for a double effect of the AGE taken with the diet on reproductive and metabolic function [39].

*Advanced glycation end products and their relevance in female reproduction [39].*

**Figure 2.** *The role of advanced glycation end products in human infertility [43].*

#### **6. Correlation between AGEs and feature of PCOS**

PCOS has been defined as a disorder due to an excess of androgens and insulin resistance, about 50–70% of women with PCOS have a certain degree of resistance to insulin, which is defined as a state in which more insulin is required than the normal to obtain an appropriate response [47]. Besides contributing to PCOS-associated hyperandrogenism, insulin resistance is also linked the development of impaired glucose tolerance and type 2 diabetes mellitus [48], in both obese and non-obese women with PCOS [49]. It is unclear whether hyperandrogenism is the result of hyperinsulinemia or vice versa [50]. Both insulin-like growth factor 1 (IGF-1) and insulin are potent stimulators of production of ovarian androgens, an action probably mediated by the insulin receptor [50, 51], furthermore, it is possible that the increase of circulating insulin levels potentiate the effect of luteinizing hormone (LH) on cells of the ovarian theca. Another mechanism of possible hyperandrogenism observed in the PCOS is the insulin-mediated inhibition of sex hormone binding globulin, which results in an increase of free androgens [52].

Since oxidative stress and inflammation are closely associated with insulin resistance, it is conceivable that the AGE-RAGE system may play a role in pathogenesis of insulin resistance observed in PCOS [30], regardless of circulating glucose levels, weight and obesity. A study conducted by Cai et al. has in fact identified the AGEs as a risk factor for insulin resistance independent of over-nutrition in non-obese mice [53], with such insulin resistance that occurred before changes in blood glucose levels. Additionally, recent work on overweight women reported that a low AGE diet improves insulin sensitivity [54].

About 30–75% of women with PCOS are obese [55] and such patients are likely at more risk to suffer from severe consequences than PCOS, such as hyperandrogenism and metabolic syndrome, compared to patients with a normal BMI [1, 56]. Moreover, it has been shown that modest weight loss regulates menstruation, improves reproductive performance and hirsutism, reduces serum androgen and insulin levels and improves the index of insulin sensitivity in women with PCOS [1].

In addition, the distribution and morphology of adipose tissue appear to contribute significant to the pathophysiology underlying PCOS: most affected women in fact, it presents an abdominal distribution of adipose tissue (central obesity) independent of BMI, which is an effect probably associated with the high amount of circulating androgens [56].

Circulating AGEs correlate with indicators inflammation, such as C reactive protein (CRP), and with oxidative stress [57]. In addition, the accumulation of AGE in the tissues induces cellular oxidative stress and promotes inflammation, thus increasing the vulnerability of the target tissues [58]. The dietary restriction of AGE, in fact, is associated with a significant reduction in inflammatory markers, such as plasma CRP, TNF-α (tumor necrosis factor-α) and VCAM-1 (vascular cell adhesion molecule-1) [59]. Furthermore, AGEs are directly correlated with the physiology of adipocytes as AGEs may also stimulate adipogenesis [60].

Patients with classic PCOS phenotype show alterations in the follicular fluid intermediate metabolites and the cumulus cells have an increase in oxidative stress, which causes the alteration of processes of follicular growth and oocyte development, causing the reduction in the pregnancy rate [61].

#### **7. AGE-RAGE system in serum and ovarian PCOS**

Insulin resistant women with PCOS without hyperglycemia have elevated levels serum of AGE and the expression of RAGE in circulating monocytes [44]. Furthermore, serum AGE levels are positively correlated with levels of testosterone, free androgens and insulin [50]. An increase in the serum AGE levels suggesting that serum AGEs are high in PCOS regardless of the presence of insulin resistance [62]. Recent studies have also shown that RAGE and AGE-modified proteins are expressed in human ovarian tissue [35, 63]. Specifically, women with PCOS have an increase in the expression of AGE and RAGE in the theca and granulosa cell layers, compared to healthy women [34, 64].

The AGE-RAGE system may be responsible for the failure of ovulation characteristic of PCOS: in a model of human cell lines of granulosa, observed that the AGE interfere in vitro with the action of LH leading to altered follicular development and therefore the dysfunction ovulatory associated with PCOS [65]. The AGEs within the ovary alter glucose metabolism and the folliculogenesis, the AGE could be responsible for the reduction of glucose uptake by granulosa cells, with consequent alteration of follicular growth [66].

The relationship between the AGE-RAGE system and infertility was also documented: AGEs have a negative effects on the reproductive outcome in women undergoing ART (assisted reproduction technology), AGE high levels in NON PCOS women appear to be related to the decrease in ovarian reserve and abnormal folliculogenesis. The pathological significance of these inflammatory AGE molecules, which are harmful to the follicles, clearly requires further investigation, but the identification of specific AGEs could offer potential therapeutic options for treating the decreased response ovarian **Figure 3**.

Intra ovarian dyslipidemia is probably a consequence of the changes associated with the metabolism in the follicles [67]. In addition, the exposure of cumulus-oocyte complex to a high lipid concentrations are known to have negative influences on oocyte maturation [68].

#### **Figure 3.**

*Relationship of the AGE-RAGE system with PCOS and infertility [39].*

#### **8. Role of Carnitine in PCOS and female infertility**

Levocarnitine (L-carnitine) plays a central role in the cellular energy metabolism as it is an essential molecule for the transport of long-chain fatty acids across the internal mitochondrial membrane. It was first isolated in 1905 in bovine muscle [69] and only the L isomer is bioactive. The carnitines as a whole they belong to a special class of nutrients called "quasi-vitamins" or "Conditionally essential" nutrients [70]. L-carnitine can be synthesized endogenously or taken with the diet, in particularly through meat and dairy products [71], hence its homeostasis reflects the balance between endogenous biosynthesis, absorption from the diet and renal reabsorption [72].

Numerous clinical studies have reported that the administration of L-carnitine (LC) and/or acetyl-L-carnitine (ALC) alleviates some effects of PCOS resulting in an increase reproductive outcome [73–77].

Both LC and ALC are commonly used in reproductive biology to improve mitochondrial function in the treatment of female infertility [78, 79]. Specifically, ALC is predominantly used for its antioxidant and anti-aging effect, while the use of LC to promote capacity of the body to oxidize fat cells to produce energy and burning fat [80]. LC also prevents DNA fragmentation induced by the harmful actions of free radicals [81].

Numerous studies have indicated that administration of L-carnitine (LC) and its acetylated form, acetyl L-carnitine (ALC) improves conditions such as PCOS [73], endometriosis [82] and amenorrhea [83]. In addition, carnitines increase levels of gonadotropins and sex hormones, as well as improve oocyte health **Figure 4** [83].

The administration of ALC instead increases the serum levels of other reproductive hormones such as estradiol, progesterone and luteinizing hormone (LH) and decreases prolactin [83, 85]. Hence, through their indirect endocrine effect, carnitines can prevent PCOS, amenorrhea and other pathological conditions related to the reproductive cycle female.

#### **Figure 4.**

*(a) Molecular structures of L-carnitine and acetyl-L-carnitine, (b) systemic and reproductive functions of L-carnitine. CoA, coenzyme A; ER, endoplasmic reticulum; FFA, free fatty acid; IFN, interferon; IL, interleukin; TNF, tumor necrosis factor [84].*

LC and ALC also affect the hypothalamic-pituitary-gonadal (HPG) axis, inducing secretion of reproductive hormones [83, 85, 86]. Among the neural centers, the concentration of LC is higher in the hypothalamus [87]. LC reduces the death rate of nerve cells and the damage associated with aging [88], thanks to its cholinomimetic activity [89]. It also increases the secretion of gonadotropin-releasing hormone (GnRH) from part of the hypothalamus, inducing the depolarization of hypothalamic nerve cells to increase its secretory activity **Figure 5** [90, 91].

Regarding PCOS, Samimi et al. observed that supplementation with LC (250 mg oral L-carnitine supplementation for 12 weeks) leads to a significant reduction in body weight, body mass index and waist and hip circumference decreasing blood glucose levels and favors the contrast of insulin resistance [73], which may be attributed to the increase in β-oxidation of fatty acids and the metabolic rate base line induced by LC [74].

As women with PCOS present also an imbalance between male and female hormones as their ovaries tend to produce androgens in excessive quantities, such phenomena of hyperandrogenism and/or insulin resistance in non-obese women with PCOS may be associated with the lowering of serum levels of LC [75]. Recent studies based on mass spectrometry confirmed altered fatty acid levels and carnitine in the serum of PCOS patients [92].

*Role of Oxidative Stress and Carnitine in PCOS Patients DOI: http://dx.doi.org/10.5772/intechopen.104327*

**Figure 5.**

*Mechanism of L-carnitine action on female fertility [84].*

#### **9. Carnitine and human assisted reproduction**

Due to their beneficial effects on female fertility, carnitines have been used in numerous in vitro studies focused on improving the health and maturation of oocytes, embryonic development in assisted reproduction, in fact they allow to reduce the delay in embryonic development due to ROS, the fragmentation of the DNA and the development of an abnormal blastocyst due to prolonged culture [93, 94].

It has been observed that during oocyte development the cumulus-oocyte complex (COC) plays an essential role in lipid metabolism and therefore in energy production: therefore, in the oocyte, the maintenance of a correct lipid metabolism without or with the minimum generation of free radicals is necessary to preserve its quality [95].

LC is essential for maintaining cellular energy balance and to reduce oxidative stress [96] and to minimize cell death by apoptosis [97], which is necessary for adequate growth of the oocyte and for the maturation of the blastocyst. LC promotes the lipid metabolism of the cumulus-oocyte complex (COC), which is one of the main regulators of oocyte maturation, by transferring the fatty acids in the mitochondria and facilitating their β-oxidation [95].

Carnitine also has an anti-inflammatory effect as the integration of diet with LC decreases the anti-proliferative effect induced by the presence of interleukins such as TNF-α and its detrimental action reducing the consumption of glucose of the embryo in its early development [98] and reducing growth of the inner cell mass and trophoectoderm in the blastocyst, which leads to delayed embryonic development and reduction of vitality of the embryo [99, 100].

It has been observed that the integration of the culture medium with ALC stabilized the mitochondrial membrane, increased the energy supply to the organelles and protected the developing embryo preventing its fragmentation [101]. Furthermore, the integration of the culture medium with LC in addition to showing anti-apoptotic effects, increases the rate of development of blastocysts [97].

Supplementation of the culture medium with LC during the in vitro maturation of the oocytes favored the acquisition of competence for development, as it improved cytoplasmic and nuclear maturation and reduced ROS levels in the culture medium, showing an antioxidant effect [102].

Furthermore, women with endometriosis have a marked increase in TNF-α concentration in the granulosa cells [103–106], which leads to the reduction in the size of the inner cell mass and in the proliferation of the trophoectoderm in the blastocyst, it was observed that the integration of the culture medium with LC allowed to neutralize the antiproliferative effect of TNF-α and to limit DNA damage during embryo development [93]. LC also had a protective effect against oocytes and embryos against the toxic effects of peritoneal fluid in women with endometriosis, reducing apoptosis levels in embryos and enhancing the microtubular structure [107].

Another typical feature of PCOS is chronic anovulation and the standard approach for the treatment of women with anovulation infertility consists of administration of clomiphene citrate to induce ovulation, however, some women fail to ovulate despite taking increasing doses of clomiphene citrate and, therefore, defined as clomiphene citrate-resistant PCOS. The administration of clomiphene citrate together with LC increases both the ovulation and pregnancy rate in women with clomiphene citrateresistant PCOS. In addition, the integration with L-carnitine increases the number of follicles capable of ovulating (diameter ≥17 mm), and oocyte maturation, as well as serum levels of estradiol and progesterone [76].

An alternative treatment to induce ovulation in patients with citrate-resistant clomiphene PCOS consists of therapeutic treatment with gonadotropins; however, some of these women do not respond to both treatments, the addition of LC to therapy stimulates the growth of dominant follicles, favoring the pregnancy rate, it also increases the average thickness of the endometrium and the size of ovarian follicles [77].

#### **10. Conclusion**

The beneficial effects of carnitines on the reproductive system and ovarian function as well the differential action of the carnitine pool. The carnitines are essential in the metabolism of fatty acids and can act to protect from mitochondrial damage and altered energy balance conditions such as those present in polycystic ovary syndrome (PCOS). The L-carnitine contributes to restore the energy balance and provide

*Role of Oxidative Stress and Carnitine in PCOS Patients DOI: http://dx.doi.org/10.5772/intechopen.104327*

adequate energy reserves to the oocyte during folliculogenesis and maturation and can represent an important strategy to improve the intraovarian environment and increase the probability of pregnancy. In this context carnitines, with positive effects on mitochondrial activity and free radical scavenging, can contribute to mitigate the effects of PCOS.

#### **Author details**

Bassim Alsadi Rome University 'La Sapienza', Rome, Italy

\*Address all correspondence to: balsadi@hotmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Norman RJ, Dewailly D, Legro RS, Hickey TE. Polycystic ovary syndrome. Lancet. 2007;**370**:685-697

[2] Barthelmess EK, Naz RK. Polycystic ovary syndrome: Current status and future perspective. Frontiers in Bioscience. 2014;**6**:104-119

[3] Carmina E, Oberfield SE, Lobo RA. The diagnosis of polycystic ovary syndrome in adolescents. American Journal of Obstetrics and Gynecology. 2010;**203-204**:e1-e5

[4] Franks S, McCarthy M. Genetics of ovarian disorders: Polycystic ovary syndrome. Reviews in Endocrine & Metabolic Disorders. 2004;**5**:69-76

[5] Vink JM, Sadrzadeh S, Lambalk CB, Boomsma DI. Heritability of polycystic ovary syndrome in a Dutch twinfamily study. The Journal of Clinical Endocrinology and Metabolism. 2006;**91**:2100-2104

[6] Ehrmann DA, Kasza K, Azziz R, Legro RS, Ghazzi MN. Effects of race and family history of type 2 diabetes on metabolic status of women with polycystic ovary syndrome. The Journal of Clinical Endocrinology and Metabolism. 2005;**90**:66-71

[7] Legro RS, Driscoll D, Strauss JF, Fox J, Dunaif A. Evidence for a genetic basis for hyperandrogenemia in polycystic ovary syndrome. Proceedings of the National Academy of Science USA. 1998;**95**:14956-14960

[8] Dunaif A, Finegood DT. Beta-cell dysfunction independent of obesity and glucose intolerance in the polycystic ovary syndrome. The Journal of Clinical Endocrinology and Metabolism. 1996;**81**:942-947

[9] Svendsen PF, Nilas L, Nørgaard K, Jensen JE, Madsbad S. Obesity, body composition and metabolic disturbances in polycystic ovary syndrome. Human Reproduction. 2008;**23**(9):2113-2121

[10] Thurston RC, Kubzansky LD, Kawachi I, Berkman LF. Is the association between socioeconomic position and coronary heart disease stronger in women than in men? American Journal of Epidemiology. 2005;**162**:57-65

[11] Martorell R, Khan LK, Hughes ML, Grummer-Strawn LM. Obesity in women from developing countries. European Journal of Clinical Nutrition. 2000;**54**:247-252

[12] Azziz R, Woods KS, Reyna R, Key TJ, Knochenhauer ES, Yildiz BO. The prevalence and features of the polycystic ovary syndrome in an unselected population. The Journal of Clinical Endocrinology and Metabolism. 2004;**89**:2745-2749

[13] Park YW, Zhu S, Palaniappan L, Heshka S, Carnethon MR, Heymsfield SB. Themetabolic syndrome: Prevalence and associated risk factor findings in the US population from the Third National Health and Nutrition Examination Survey, 1988- 1994. Archives of Internal Medicine. 2003;**163**:427-436

[14] Balen AB, Rutherford AJ. Managing anovulatory infertility and polycystic ovary syndrome. BMJ. 2007;**335**:663-666

[15] Coffey S, Bano G, Mason HD. Health-related quality of life in women with polycystic ovary syndrome: A comparison with the general population using the Polycystic Ovary Syndrome Questionnaire (PCOSQ ) and the

*Role of Oxidative Stress and Carnitine in PCOS Patients DOI: http://dx.doi.org/10.5772/intechopen.104327*

Short Form-36 (SF-36). Gynecological Endocrinology. 2006;**22**:80-86

[16] Barnard L, Ferriday D, Guenther N, Strauss B, Balen AH, Dye L. Quality of life and psychological well being in polycystic ovary syndrome. Human Reproduction. 2007;**22**:2279-2286

[17] Deeks AA, Gibson-Helm ME, Paul E, Teede HJ. Is having polycystic ovary syndrome a predictor of poor psychological function including anxiety and depression? Human Reproduction. 2011;**26**:1399-1407

[18] Ruddenklau A, Campbell RE. Neuroendocrine Impairments of Polycystic Ovary Syndrome Endocrinology. Vol. 160. Oxford: Oxford University Press; 2019. pp. 2230-2242

[19] Tata B, Mimouni NEH, Barbotin AL, Malone SA, Loyens A, Pigny P, et al. Elevated prenatal anti-Müllerian hormone reprograms the fetus and inducespolycystic ovary syndrome in adulthood. Nature Medicine. 2018;**24**(6):834-846

[20] Orio F Jr, Palomba S, et al. The cardiovascular risk of young women with polycystic ovary syndrome: An observational, analytical, prospective case-control study. The Journal of Clinical Endocrinology and Metabolism. 2004;**89**:3696-3701

[21] Balen A. Polycystic ovary syndrome and cancer. Human Reproduction Update. 2001;**7**:522-525

[22] Chittenden BG, Fullerton G, Maheshwari A, Bhattacharya S. Polycystic ovary syndrome and the risk of gynaecological cancer: A systematic review. Reproductive Biomedicine Online. 2009;**19**:398-405

[23] Victor VM, Rocha M, Banuls C, Alvarez A, de Pablo C, Sanchez-Serrano M, et al. Induction of oxidative stress and human leukocyte/ endothelial cell interactions in polycystic ovary syndrome patients with insulin resistance. The Journal of Clinical Endocrinology and Metabolism. 2011;**96**:3115-3122

[24] Arslanian SA, Lewy VD, Danadian K. Glucose intolerance in obese adolescents with polycystic ovary syndrome: Roles of insulin resistance and beta-cell dysfunction and risk of cardiovascular disease. The Journal of Clinical Endocrinology and Metabolism. 2001;**86**:66-71

[25] Dunaif A. Insulin resistance and the polycystic ovary syndrome: Mechanism and implications for pathogenesis. Endocrine Reviews. 1997;**18**:774-800

[26] Fenkci V, Fenkci S, Yilmazer M, Serteser M. Decreased total antioxidant status and increased oxidative stress in women with polycystic ovary syndrome may contribute to the risk of cardiovascular disease. Fertility and Sterility. 2003;**80**:123-127

[27] Wang X, Martindale JL, Liu Y, HolbrookNJ. The cellular response to oxidative stress: Influences of mitogenactivated protein kinase signalling pathways on cell survival. The Biochemical Journal. 1998;**333**:291-300

[28] Murri M, Luque-Ramírez M, Insenser M, Ojeda-Ojeda M, Escobar-Morreale HF. Circulating markers of oxidative stress and polycystic ovary syndrome (PCOS): A systematic review and metaanalysis. Human Reproduction Update. 2013;**19**:268-288

[29] Bucala R, Cerami A. Advanced glycosylation: Chemistry, biology, and implications for diabetes and aging. Advances in Pharmacology. 1992;**23**:1-34 [30] Unoki H, Yamagishi S. Advanced glycation end products and insulin resistance. Current Pharmaceutical Design. 2008;**14**:987-9

[31] Stadtman ER, Berlett BS. Reactive oxygen-mediated protein oxidation in aging and disease. Chemical Research in Toxicology. 1997;**10**:485-494

[32] Thorpe SR, Baynes JW. Role of the Maillard reaction in diabetes mellitus and diseases of aging. Drugs & Aging. 1996;**9**:69-77

[33] Yamagishi S, Nakamura K, Imaizumi T. Advanced glycation end products (AGEs) and diabetic vascular complications. Current Diabetes Reviews. 2005;**1**:93-106

[34] Diamanti-Kandarakis E, Piperi C, Patsouris E, Korkolopoulou P, Panidis D, Pawelczyk L, et al. Immunohistochemical localization of advanced glycation end-products (AGEs) and their receptor (RAGE) in polycystic and normal ovaries. Histochemistry and Cell Biology. 2007;**127**:581-589

[35] Tatone C, Amicarelli F. The aging ovary-the poor granulosa cells. Fertility and Sterility. 2013;**99**:12-17

[36] Inagi R. Inhibitors of advanced glycation and endoplasmic reticulum stress. Methods Enzymology. 2011;**491**:361-380

[37] Piperi C, Adamopoulos C, Dalagiorgou G, Diamanti-Kandarakis E, Papavassiliou AG. Crosstalk between advanced glycation and endoplasmic reticulum stress: Emerging therapeutic targeting for metabolic diseases. The Journal of Clinical Endocrinology and Metabolism. 2012;**97**:2231-2242

[38] Thornalley PJ. The enzymatic defence against glycation in health, disease and therapeutics: A symposium to examine the concept. Biochemical Society Transactions. 2003;**31**:1341-1342

[39] Merhi Z. Advanced glycation end products and their relevance in female reproduction. Human Reproduction. 2014;**29**:135-145

[40] Kalea AZ, Schmidt AM, Hudson BI. RAGE: A novel biological and genetic marker for vascular disease. Clinical Science (London, England). 2009;**116**:621-637

[41] Basta G. Receptor for advanced glycation endproducts and atherosclerosis: From basic mechanisms to clinical implications. Atherosclerosis. 2008;**196**:9-21

[42] Yan SF, D'Agati V, Schmidt AM, Ramasamy R. Receptor for advanced glycation endproducts (RAGE): A formidable force in the pathogenesis of the cardiovascular complications of diabetes & aging. Current Molecular Medicine. 2007;**7**:699-710

[43] Zhu J-L, Cai Y-Q, Long S-L, Chen Z, Mo Z-C. The role of advanced glycation end products in human infertility. Life Sciences. 2020;**255**:117830

[44] Diamanti-Kandarakis E, Piperi C, Kalofoutis A, Creatsas G. Increased levels of serum advanced glycation end-products in women with polycystic ovary syndrome. Clinical Endocrinology. 2005;**62**:37-43

[45] Goldberg T, Caiw M, Dardaine V, Baliga BS, Uribarri J, Vlassara H. Advanced glycoxidation end products in commonly consumed foods. Journal of the American Dietetic Association. 2004;**104**:1287-1291

[46] Cerami C, Founds H, Nicholl I, Mitsuhashi T, Giordano D, Vanpatten S, *Role of Oxidative Stress and Carnitine in PCOS Patients DOI: http://dx.doi.org/10.5772/intechopen.104327*

et al. Tobacco smoke is a source of toxic reactive glycation products. Proceedings of the National Academy of Science USA. 1997;**94**:13915-13920

[47] Diamanti-Kandarakis E. Insulin resistance in PCOS. Endocrine. 2006;**30**:13-17

[48] Legro RS, Kunselman AR, Dodson WC, Dunaif A. Prevalence and predictors of risk for type 2 diabetes mellitus and impaired glucose tolerance in polycystic ovary syndrome: A prospective, controlled study in 254 affected women. The Journal of Clinical Endocrinology and Metabolism. 1999;**84**:165-169

[49] Dunaif A, Segal KR, Futterweitw DA. Profound peripheral insulin resistance, independent of obesity, in polycystic ovary syndrome. Diabetes. 1989;**38**:1165-1174

[50] Burghen GA, Givens JR, Kitabchi AE. Correlation of hyperandrogenism with hyperinsulinism in polycystic ovarian disease. The Journal of Clinical Endocrinology and Metabolism. 1980;**50**:113-116

[51] Barbieri RL, Smith S, Ryan KJ. The role of hyperinsulinemia in the pathogenesis of ovarian hyperandrogenism. Fertility and Sterility. 1988;**50**:197-212

[52] Lindstedt G, Lundberg PA, Lapidus L, Lundgren H, Bengtsson C, Bjorntorp P. Low sex-hormone-binding globulin concentration as independent risk factor for development of NIDDM. 12-yr follow-up of population study of women in Gothenburg, Sweden. Diabetes. 1991;**40**:123-128

[53] Cai W, Ramdas M, Zhu L, Chen X, Striker GE, Vlassara H. Oral advanced glycation endproducts (AGEs) promote insulin resistance and diabetes by depleting the antioxidant defenses AGE receptor-1 and sirtuin 1. Proceedings of the National Academy of Sciences of the United States of America. 2012;**109**(39):15888-15893

[54] Mark AB, Poulsen MW, Andersen S, Andersen JM, Bak MJ, Ritz C, et al. Consumption of a diet low in advanced glycation end products for 4 weeks improves insulin sensitivity in overweight women. Diabetes Care. 2014;**37**(1):88-95

[55] Ehrmann DA. Polycystic ovary syndrome. The New England Journal of Medicine. 2005;**352**:1223-1236

[56] Kirchengast S, Huber J. Body composition characteristics and body fat distribution in lean women with polycystic ovary syndrome. Human Reproduction. 2001;**16**:1255-1260

[57] Uribarri J, Cai W, Peppa M, Goodman S, Ferrucci L, Striker G, et al. Circulating glycotoxins and dietary advanced glycation endproducts: Two links to inflammatory response, oxidative stress, and aging. The Journals of Gerontology. Series A, Biological Sciences and Medical Sciences. 2007;**62**:427-433

[58] Vlassara H, Palace MR. Diabetes and advanced glycation endproducts. Journal of Internal Medicine. 2002;**251**:87-101

[59] Uribarri J, Ramdas M, Goodmans C, Pyzik R, Chen X, Zhu L, et al. Restriction of advanced glycation end products improves insulin resistance in human type 2 diabetes: Potential role of AGER1 and SIRT1. Diabetes Care. 2011;**34**:1610-1616

[60] Jia X, Chang T, Wilson TW, Wu L. Methylglyoxal mediates

adipocyte proliferation by increasing phosphorylation of Akt1. PLoS One. 2012;**7**:e36610

[61] Zhao H, Zhao Y, Li T, Li M, Li J, Li R, et al. Metabolism alteration in follicular niche: The nexus among intermediary metabolism, mitochondrial function, and classic polycystic ovary syndrome. Free Radical Biology & Medicine. 2015;**86**:295-307

[62] Diamanti-Kandarakis E, Katsikis I, Piperi C, Kandaraki E, Piouka A, Papavassiliou AG, et al. Increased serum advanced glycation end products is a distinct finding in lean women with polycystic ovary syndrome (PCOS). Clinical Endocrinology. 2008;**69**:634-641

[63] Fujii EY, Nakayama M. The measurements of RAGE, VEGF, and AGEs in the plasma and follicular fluid of reproductive women: The influence of aging. Fertility and Sterility. 2010;**94**:694-700

[64] Azhary JMK, Harada M, Kunitomi C, Kusamoto A, Takahashi N, Nose E, et al. Androgens increase accumulation of advanced glycation end products in granulosa cells by activating ER stress in PCOS. Endocrinology. 2020;**161**(2)

[65] Diamanti-Kandarakis E, Piperi C, Livadas S, Kandaraki E, Papageorgiou E, Koutsilieris M. Interference of AGE-RAGE Signaling with Steroidogenic Enzyme Action in Human Ovarian Cells. San Francisco, CA: Endocrine Society; 2013

[66] Piperi C, Koutsilieris M, Diamanti-Kandarakis E. Advanced Glycation End- Products Inhibit Insulin Signaling in Human Granulosa Cells: A Causative Link to PCOS Pathogenesis. San Francisco, CA: Endocrine Society; 2013

[67] Bousmpoula A, Benidis E, Demeridou S, Kapeta-Kourkouli R, Chasiakou A, Kouskouni E, et al. Serum and follicular fluid irisin levels in women with polycystic ovaries undergoing ovarian stimulation: Correlation with insulin resistance and lipoprotein lipid profiles. Gynecological Endocrinology. 2019;**35**:803-806

[68] Yang X, Wu LL, Chura LR, Liang X, Lane M, Norman RJ, et al. Exposure to lipid-rich follicular fluid is associated with endoplasmic reticulum stress and impaired oocyte maturation in cumulusoocyte complexes. Fertility and Sterility. 2012;**97**:1438-1443

[69] Gulewitch VS, Krimberg R. Zur Kenntnis der Extraktivstoffe der Muskeln. II. Mitteilung: Über das Carnitin. Zeitschrift für Physiologische Chemie. 1905;**45**:326-330

[70] Vassiliadis S, Athanassakis I. A "conditionally essential" nutrient, L-carnitine, as a primary suspect in endometriosis. Fertility and Sterility. 2011;**95**:2759-2760

[71] Longo N, Frigeni M, Pasquali M. Carnitine transport and fatty acid oxidation. Biochimica et Biophysica Acta. 2016;**1863**(10):2422-2435

[72] Rebouche CJ. Kinetics, pharmacokinetics, and regulation of L-carnitine and acetyl-L- carnitine metabolism. Annals of the New York Academy of Sciences. 2004;**1033**:30-41

[73] Samimi M, Jamilian M, Ebrahimi FA, Rahimi M, Tajbakhsh B, Asemi Z. Oral carnitine supplementation reduces body weight and insulin resistance in women with polycystic ovary syndrome: A randomized, double-blind, placebocontrolled trial. Clinical Endocrinology. 2016;**84**:851-857

[74] Center SA, Warner KL, Randolph JF, Sunvold GD, Vickers JR.

#### *Role of Oxidative Stress and Carnitine in PCOS Patients DOI: http://dx.doi.org/10.5772/intechopen.104327*

Influence of dietary supplementation with (L)-carnitine on metabolic rate, fatty acid oxidation, body condition, and weight loss in overweight cats. American Journal of Veterinary Research. 2012;**73**:1002-1015

[75] Fenkci SM, Fenkci V, Oztekin O, Rota S, Karagenc N. Serum total L-carnitine levels in non obese women with polycystic ovary syndrome. Human Reproduction. 2008;**23**:1602-1606

[76] Ismail AM, Hamed AH, Saso S, Thabet HH. Adding L-carnitine to clomiphene resistant PCOS women improves the quality of ovulation and the pregnancy rate. A randomized clinical trial. European Journal of Obstetrics, Gynecology, and Reproductive Biology. 2014;**180**:148-152

[77] Latifian S, Hamdi K, Totakneh R. Effect of addition of l-carnitine in polycystic ovary syndrome (PCOS) patients with clomiphene citrate and gonadotropin resistant. International Journal of Current Research and Academic Review. 2015;**3**:469-476

[78] Dunning KR, Robker RL. Promoting lipid utilization with L-carnitine to improve oocyte quality. Animal Reproduction Science. 2012;**134**:69-75

[79] Cheng HJ, Chen T. Clinical efficacy of combined L-carnitine and acetyl-Lcarnitine on idiopathic asthenospermia. Zhonghua Nan Ke Xue. 2008;**14**:149-151

[80] Rebouche CJ. Carnitine function and requirements during the life cycle. The FASEB Journal. 1992;**6**:3379-3386

[81] Abdelrazik H, Sharma R, Mahfouz R, Agarwal A. L-carnitine decreases DNA damage and improves the in vitro blastocyst development rate in mouse embryos. Fertility and Sterility. 2009;**91**:589-596

[82] Dionyssopoulou E, Vassiliadis S, Evangeliou A, Koumantakis EE, Athanassakis I. Constitutive or induced elevated levels of L-carnitine correlate with the cytokine and cellular profile of endometriosis. Journal of Reproductive Immunology. 2005;**65**:159-170

[83] Genazzani AD, Lanzoni C, Ricchieri F, Santagni S, Rattighieri E, Chierchia E, et al. Acetyl-L-carnitine (ALC) administration positively affects reproductive axis in hypogonadotropic women with functional hypothalamic amenorrhea. Journal of Endocrinological Investigation. 2011;**34**:287-291

[84] Agarwal A, Sengupta P, Durairajanayagam D. Role of L-carnitine in female infertility. Reproductive Biology and Endocrinology. 2018;**16**(1):5

[85] Krsmanovic LZ, Virmani MA, Stojilkovic SS, Catt KJ. Actions of acetyl-Lcarnitine on the hypothalamopituitary-gonadal system in female rats. The Journal of Steroid Biochemistry and Molecular Biology. 1992;**43**:351-358

[86] Virmani MA, Krsmanovic LZ, Stojilkovic SS, Catt KJ. Stimulatory effects of Lacetyl carnitine on the pituitary-gonadal axis in female rats. In: Advances in Human Female Reproduction. New York: Raven Press; 1991. pp. 291-296

[87] Bresolin N, Freddo L, Vergani L, Angelini C. Carnitine, carnitine acyltransferases, and rat brain function. Experimental Neurology. 1982;**78**:285-292

[88] Amenta F, Cavallotti C, de Rossi M, Bossoni G, Carpi C. Effect of acetylLcarnitine treatment on some behavioural, histochemical and histological parameters of methylazoxymethanol microencephalic rats. International Journal of Tissue Reactions. 1986;**8**:513-526

[89] Bodis-Wollner I. Physiological effects of acetyl-levo-carnitine in the central nervous system. International Journal of Clinical Pharmacology Research. 1990;**10**:109-114

[90] Bigdeli H, Snyder PJ. Gonadotropin releasing hormone release from the rat hypothalamus: Dependence on membrane depolarization and calcium influx. Endocrinology. 1978;**103**:281-286

[91] Krsmanovic LZ, Virmani MA, Stojilkovic SS, Catt KJ. Stimulation of gonadotropin releasing hormone secretion by acetyl-L-carnitine in hypothalamic neurons and GT1 neuronal cells. Neuroscience Letters. 1994;**165**:33-36

[92] Chen X, Lu T, Wang X, Sun X, Zhang J, Zhou K, et al. Metabolic alterations associated with polycystic ovary syndrome: A UPLC Q-exactive based metabolomic study. Clinica Chimica Acta. 2019

[93] Abdelrazik H, Agarwal A. L-carnitine and assisted reproduction. Archives of Medical Science. 2009;**1A**:S43-S47

[94] Mishra A, Reddy IJ, Gupta PS, Mondal S. L-carnitine mediated reduction in oxidative stress and alteration in transcript level of antioxidant enzymes in sheep embryos produced in vitro. Reproduction in Domestic Animals. 2016;**51**:311-321

[95] Dunning KR, Cashman K, Russell DL, Thompson JG, Norman RJ, Robker RL. Beta oxidation is essential for mouse oocyte developmental competence and early embryo development. Biology of Reproduction. 2010;**83**:909-918

[96] Vanella A, Russo A, Acquaviva R, Campisi A, di Giacomo C, Sorrenti V, et al. L-propionyl-carnitine as superoxide scavenger, antioxidant, and DNA

cleavage protector. Cell Biology and Toxicology. 2000;**16**:99-104

[97] Abdelrazik H, Sharma R, Mahfouz R, Agarwal A. L-carnitine decrease DNA damage and improves the in vitro blastocyst development rate in mouse embryos. Fertility and Sterility. 2009;**91**:589-596

[98] Pampfer S, Moulaert B, Vanderheyden I, Wuu YD, de Hertogh R. Effect of tumor necrosis factor alpha on rat blastocyst growth and glucose metabolism. Journal of Reproduction and Fertility. 1994;**101**:199-206

[99] Glabowski W, Kurzawa R, Wiszniewska B, Baczkowski T, Marchlewicz M, Brelik P. Growth factors effects on preimplantation development of muose embryos exposed to tumor necrosis factor alpha. Reproductive Biology. 2005;**5**:83-99

[100] Whiteside EJ, Boucaut KJ, Teh A, Garcia-Aragon J, Harvey MB, Herington AC. Elevated concentration of TNF-alpha induces trophoblast differentiation in mouse blastocyst outgrowths. Cell and Tissue Research. 2003;**14**:275-280

[101] Pillich RT, Scarsella G, Risuleo G. Reduction of apoptosis through the mitochondrial pathway by the administration of acetyl-Lcarnitine to mouse fibroblasts in culture. Experimental Cell Research. 2005;**306**:1-8

[102] Zare Z, Masteri Farahani R, Salehi M, Piryaei A, Ghaffari Novin M, Fadaei Fathabadi F, et al. Effect of L-carnitine supplementation on maturation and early embryo development of immature mouse oocytes selected by brilliant cresyle blue staining. Journal of Assisted Reproduction and Genetics. 2015;**32**:635-643

*Role of Oxidative Stress and Carnitine in PCOS Patients DOI: http://dx.doi.org/10.5772/intechopen.104327*

[103] Agarwal A, Gupta S, Sharma RK. Role of oxidative stress in female reproduction. Reproductive Biology and Endocrinology. 2005;**3**:28

[104] Carlberg M, Nejaty J, Froysa B, Guan Y, Soder O, Bergqvist A. Elevated expression of tumour necrosis factor alpha in cultured granulosa cells from women with endometriosis. Human Reproduction. 2000;**15**:1250-1255

[105] Bedaiwy MA, Falcone T. Peritoneal fluid environment in endometriosis. Minerva Ginecology. 2003;**55**:333-345

[106] Gupta S, Goldberg JM, Aziz N, Goldberg E, Krajcir N, Agarwal A. Pathogenic mechanisms in endometriosisassociated infertility. Fertility and Sterility. 2008;**90**:247-257

[107] Mansour G, Abdelrazik H, Sherma RK, Radwan E, Falcone T, Agarwal A. L-carnitine supplementation reduces oocyte cytoskeleton damage and embryo apoptosis induced by incubation in peritoneal fluid from patients with endometriosis. Fertility and Sterility. 2009;**91**:2079-2086

#### **Chapter 9**

## Polycystic Ovary Syndrome Phenotypes and Infertility Treatment

*Anđelka Radojčić Badovinac and Neda Smiljan Severinski*

#### **Abstract**

The polycystic ovary syndrome (PCOS) includes different clinical, endocrine, metabolic, and morphological criteria in women of reproductive age and consequently different health risks in later life of a woman. Controversy and debates related to diagnostic criteria are constant and current worldwide. As a result of many proposals for PCOS diagnostic criteria, clinicians recognize four phenotypes of PCOS. PCOS is a frequent cause of infertility with an overall prevalence of 5–15% and counts for approximately 70% of all cases of ovulation disorders. There are many aspects of studying differences between PCO phenotypes and problems in infertility treatments. Ovulation induction is often used to treat anovulatory patients with PCOS, but many of these women fail to conceive and the next step in the treatment is assisted reproduction. The contribution of oocyte health to reproductive potential varies and largely depends on the PCOS phenotype and comorbidities associated with PCOS. Contrary to the previous one, PCOS phenotype is not significantly associated with the morphological quality of oocytes. It seems that a combination of hyperandrogenism and chronic anovulation is associated with a negative impact on the cumulative pregnancy rate in medically assisted reproduction.

**Keywords:** PCOS, PCOS phenotype, ART, ovulatory failure, reproductive hormone, *in vitro* maturation

#### **1. Introduction**

Management of PCOS (polycystic ovary syndrome) related to infertility, includes lifestyle changes, ovulation induction by pharmaceuticals, or assisted reproductive technology (ART) as an *in vitro* fertilization (IVF) with or without intracytoplasmic sperm injection (ICSI) and *in vitro* maturation (IVM) of the oocyte. It can be followed by a "freeze-all" procedure. PCOS patients have a higher risk of developing ovarian hyperstimulation syndrome (OHSS), a life-threatening condition, therefore ART is no favored procedure in current international guidelines.

Hyperandrogenism, anovulation, and ovarian morphology are the basic determinants in the diagnosis of the polycystic ovarian syndrome (PCOS) according to international guidelines. Given the different clinical presentations in patients, the criteria for the diagnosis of this condition are still discussed, as well as whether the syndrome involves several different diseases with the same clinical picture, as well as discussions about what is really a clinical picture of the polycystic ovary. Therefore, different approaches in the diagnosis and treatment of patients, have been proposed for different phenotypes of PCOS. The criteria for pre-recognition of this condition have been adopted for years by various authoritative bodies at international meetings, such as the National Institute for Health (NIH), Rotterdam consensus, Androgen Excess, and PCO Society, but there has been a constant difference over the mandatory criteria for PCOS [1]. An important starting point in the diagnosis was to exclude diseases of other endocrine glands (pituitary gland, thyroid, and adrenal gland), which give a similar clinical picture and can be confused with PCOS.

Ovulation disorder in the general population of women is estimated at 15% (12–18%) [2]. Regular menstrual cycles are not the exclusive evidence of ovulation, since in some women there is a "subclinical disorder" of ovulation that is proven only by serum values of progesterone in the middle lutein phase of the cycle (21–24.d.c. which must be >5 ng/mL). In the case of PCOS, almost 80% of patients have ovulation disorder [3].

Hyperandrogenism (hyperandrogenemia) implies clinical and/or biochemical evidence of elevated serum androgens, but the incidence in the general population of women is unknown. Hirsutism, androgenic alopecia, and acne are clinical manifestations of hyperandrogenism. The intensity of hirsutism differs ethnically and geographically, and it is desirable to develop population-specific criteria for hirsutism. Almost 70% of women with hirsutism have PCOS, 40% have severely expressed acne, and only 22% have androgenic alopecia [4]. Hyperandogenemia (biochemical hyperandrogenism) is determined by free testosterone and free androgen index (FAI—free androgen index) [5]. A total of 78% of patients with PCOS have hyperandrogenism and 40% in an unselected population of patients with BMI >25 [6].

Polycystic ovary morphology (PCOM) is evaluated by ultrasound examination based on the number of antral follicles (> of 20 per ovary) and/or on the basis of total ovarian volume (> 10 mL), where the frequency of the ultrasonic probe is an extremely important parameter. Based on these international criteria, the prevalence of PCOM in the population is 12.5% [7, 8]. Ultrasonic examination of nonselective population, based only on PCOM, significantly increase the incidence of PCOS and vice versa.

Thus, on the basis of the described criteria, four PCOS phenotypes with different prevalence in the general and separate population are defined, which are as follows [5]:


Compared to phenotype C and D, patients with phenotype A and B (classical phenotype) are more often obese, with hirsutism, more likely to have insulin resistance, dyslipidemia, fatty liver, and metabolic syndrome in later life. The frequency of individual phenotype differs significantly in different populations with symptoms of PCOS and also in the general population [9]. Each of the PCOS phenotypes has its own specifics in the treatment of impaired fertility.

#### **2. PCOS phenotype and complications of treatment with medically assisted reproduction procedures**

The first line of treatment of patients with PCOS is the induction of ovulation with clomiphene citrate or letrozole. *In vitro* fertilization (IVF) procedures are indicated when this initial treatment fails or in cases where the patient's partner has severe male infertility. Patients with PCOS phenotype A have significantly more frequent resistance to clomiphene despite increasing the dose of the drug through three consecutive stimulation cycles, compared to phenotype D (non-androgenic phenotype) [10].

Gonadotropin stimulation in patients with PCOS is associated with the development of a significantly higher number of follicles in the ovaries, as well as oocytes, a significantly higher number of developed embryos and embryos in excess for cryopreservation. Ovarian stimulation in these patients lasts longer and higher doses of gonadotropin are often required, which is associated with disorders of folliculogenesis caused by hyperandrogenism. Estimating the right dose of gonadotropin is the biggest challenge in the phase of ovarian stimulation and is often insufficient. The follicles do not grow, due to hyperandrogenism, and by increasing the dose, the ovary enters in hyperstimulation, which is an extreme of the ovarian response. A newer approach to ovarian stimulation with follitropin delta, based on the patient's body mass and AMH value, proved to be the best, especially in the PCOS patient population and has a significant reduction in the risk of ovary hyperstimulation. Patients with hyperandrogenism and polycystic ovarian morphology (phenotype A and C) have the highest risk of ovary hyperstimulation [11].

Ovarian hyperstimulation syndrome (OHSS) is an iatrogenic complication of ovarian stimulation, and PCOS patients have the highest risk for complications during the IVF (*in vitro* fertilization) procedure. The frequency of OHSS is from 3 to 6% of IVF cycles. Patients with antral follicles count >24, AMH concentration > 3.5 ng/mL, or estradiol concentration > 3500 pg., have a risk of developing OHSS. Clinical OHSS is recognized in three stages, and depending on the severity of symptoms, we distinguish between mild, medium severe, and severe types of hyperstimulation. Severe ovarian hyperstimulation can be a life-threatening condition, requiring hospitalization and treatment to maintain vital circulatory and pulmonary functions, and can also end with the death of a patient. Identification of patients at risk for OHSS is the basis of the strategy for the prevention of this serious iatrogenic condition and the safety of IVF procedures.

The protocol of choice for ovarian stimulation in patients with PCOS and risk for OHSS is an antagonistic protocol that can be fixed or flexible. In this stimulation, it is possible to achieve the final maturation of oocyte with GnRH (gonadotropinreleasing hormone) agonists, thereby avoiding the administration of hCG (human chorionic gonadotropin) injection, which is the basic molecule in the mechanism of development of OHSS in at-risk patients. In this way, the basic mechanism of vascular permeability and compromising circulation by leaking plasma from the vascular system into extracellular spaces are avoided. Those are signs of a more severe form of OHSS. Likewise, the stimulation cycle is abruptly "extinguished." Menstrual bleeding occurs within a few days after the application of the GnRH agonist. Harvested oocytes are fertilized by IVF/ICSI procedure and developed embryos are cryopreserved, most

often in the blastocyst stage, which represents the so-called "freeze-all" strategy that gives safety to the treatment of patients with PCOS. Embryo transfer is planned in the next cycle in which signs of hyperstimulation do not exist. Hormonal preparation of the endometrium, and ovarian stimulation, in this case, is not required.

Additional treatment of PCOS patients involves the use of various medications that have metabolic effects and that could significantly improve the treatment of these patients in IVF procedures by individualizing therapy. The fact is that within the PCOS population with the same PCOS phenotype, an individual woman may have a significantly different response to different types of treatments with respect to the unique hormonal/metabolic status associated with the PCOS phenotype as well. There is a large gap in the literature that indicates the need for new research and the need for an individual approach in the treatment of infertility of these patients.

Spontaneous abortions in patients with PCOS are more common compared to the general population and they are associated with insulin resistance, hyperandrogenism, and obesity. These conditions are very often associated with PCOS, but they are also separate risks for the spontaneous loss of pregnancy. Studies link spontaneous abortion to impaired endometrial receptivity and to more frequent embryo aneuploidy of patients with PCOS. In the Asian population of women with PCOS phenotypes who have hyperandrogenism (A, B, C types), a higher risk for spontaneous miscarriage after IVF procedures was observed than in phenotype D [12]. Impaired glucose and insulin metabolism at the endometrial level and excessive expression of androgen receptors in the endometrium are associated with a signal transduction disorder during the implantation process in patients with PCOS [13]. The causes of more frequent embryo aneuploidy in PCOS patients have not yet been clarified. There are assumptions that impaired glucose metabolism and steroidogenesis lead to DNA molecule instability [14].

#### **3. PCOS phenotypes and the outcome of medically assisted reproduction procedures**

During the stimulated IVF cycle, various indicators of quality and success of treatment are monitored. Among other things, these are the total dose of gonadotropin used for stimulation, the number of aspirated oocytes, the number of oocytes in metaphase II, the percentage of fertilization, the number of developed embryos on the 3rd day, the number of developed blastocysts on the 5th day, the number of cryopreserved embryos, the proportion of conceived pregnancies, the number of born children, etc. Since PCOS phenotypes imply hormonal and metabolic differences, the question arises whether the indicators of the course of treatment are different in patients with different PCOS phenotypes.

The results of the studies so far indicate significant differences in treatment between PCOS patients and women who do not have this syndrome and who in studies represent the usual control group. Studies most often follow PCOS patients as a single group. Different criteria for defining PCOS phenotype are associated with problems of analysis and comparison of parameters that monitor the course and outcome of the IVF procedures in different studies [15]. There are two fundamental factors that are most often analyzed and compared in patients with PCOS—hyperandrogenism and PCO morphology of the ovaries, which are clinically very important factors in decision-making during the treatment of infertility by medically assisted fertilization procedures. The role of androgens in folliculogenesis is still unclear and

#### *Polycystic Ovary Syndrome Phenotypes and Infertility Treatment DOI: http://dx.doi.org/10.5772/intechopen.101994*

there are conflicting results of studies dealing with this problem. The results of studies analyzing differences in treatment outcomes among defined PCOS phenotypes indicate a negative effect of hyperandrogenism in IVF procedures, and a higher incidence of complications later in pregnancy [16]. In patients with phenotype A and B, for every 1 pg./ml increase in free testosterone concentration, the proportion of clinically confirmed pregnancies decreases by 50–60% as well as the proportion of live births [17]. According to recent findings, the differences between PCOS phenotypes refer only to the number of good embryos for transfer, which is significantly higher in patients with hyperandrogenism and ovulation disorder, but without the typical PCO morphology of the ovaries (phenotype B). The proportion of biochemical and clinically confirmed pregnancies, as well as the number of couples with born children, do not differ significantly among phenotypically different PCOS patients [17, 18]. In addition, studies indicate that the proportion of clinically confirmed pregnancies, is significantly lower in women with PCOS phenotypes A, B and C compared to control patients [17]. The number of children born does not differ in different PCOS phenotypes. In some areas of the world, certain PCOS phenotypes have not been found at all, for example, there are no phenotypes B and C among Vietnamese women with PCOS [19]. Since the anti-Müller hormone (AMH) is often elevated in patients with PCOS, it has become a powerful factor that should have prognostic value in clinically assessing the outcome of treatment with medically assisted fertilization, however, it has been proven useful only in the group of patients with phenotype B. The proportion of clinically confirmed pregnancies and the proportion of babies born increases by 1.3 times for each 1 ng/ml serum AMH concentration increase [17].

#### **4. PCOS phenotypes and the impact on oocytes and embryos quality**

PCOS patients' oocytes quality can be associated with the hormonal and metabolic conditions, and therefore, consequently with the quality of the embryo. Poorer oocyte quality is part of the problem of subfertility in patients with PCOS. There is evidence that oocyte quality depends on PCOS phenotype and accompanying diseases and conditions that are more common in PCOS patients. Oocyte quality is defined by the morphology and morphology of associated structures, such as zona pellucida, cumulus oophorus, and corona radiata. An ovarian microenvironment in which follicles and oocytes grow and mature is exposed to multiple hormonal abnormalities in patients with PCOS. Well-known disruptive mechanisms include elevated concentrations of LH (luteinizing hormone) and FSH (follicle-stimulating hormone), impaired ratio of these hormones, elevated AMH values, impaired insulin-like growth factor secretion, and enzymes involved in the conversion of androgens to estrogens.

Hyperandrogenism interferes with the normal feedback loop between the ovaries, pituitary gland, and hypothalamus, which leads to an increased frequency of excretion of the releasing hormone for gonadotropins, and consecutively results in premature luteinization of granulose cells and abnormal maturation of the oocytes. There is also a direct effect of hyperandrogenism on the oocyte by activating its proapoptotic mechanism [20]. Hyperandogenic ovarium microenvironment interferes with the oocyte in the continuation of meiosis, promotes mitochondrial abnormalities and oxidative stress, and interferes with lipid metabolism in the oocyte [21].

High concentrations of AMH synthesized by granulosa cells, inhibit the recruitment of follicles, and therefore, the selection of follicles that will ovulate, leading to a vicious cycle of anovulation and hyperandrogenism. In addition, by blocking

the action of FSH on follicle growth and blocking the action of aromatase in charge of converting androgens synthesized in theca cells to estrogens in granulosa cells, the chronic state of hyperandrogenism is again supported. There is evidence that in patients with PCOS an increased concentration of AMH in follicular fluid exists along with oocytes of low quality. Molecular mechanisms that lead to disruption in the growth and maturation of oocytes are not known [22]. Significantly lower follicular fluid AMH levels were observed in follicles of fertilized MII oocytes than in nonfertilized non-PCOS patients [23]. Also in our non-PCOS patients with sterility and impaired fertility, gene for the AMH and androgen receptor in human cumulus cells surrounding morphologically highly graded oocytes are underexpressed [24].

Hyperinsulinemia, insulin resistance, and obesity are metabolic disorders associated with PCOS that intertwine with hormonal disorders and further worsen the conditions of oocyte microenvironments. Hyperinsulinemia reduces the synthesis of binding globulin for sex hormones (SHBG), and insulin also competes with androgens for binding sites on this carrier, which means that it promotes hyperandrogenism and all its negative effects. The direct effect of hyperinsulinemia on oocytes has been proven to disrupt the expression of genes associated with the dynamics of the division spindle and the function of centrosomes. In the case of insulin resistance, there is a change in gene expression for glucose carriers in granulose cells, and therefore, a possible decrease in energy sources for the metabolism of the oocyte itself and the processes of meiosis [25].

Based on PCOS phenotype in the population of women being treated with medically assisted reproduction procedures, no difference has been found so far in the proportion of oocytes in metaphase II, percentage of fertilization, or the evaluation of quality embryos for transfer [17, 26]. According to available data to date, patients who have a classic PCOS phenotype (A and B) associated with insulin resistance and obesity also have the highest risk for low-quality oocytes [27].

Besides poor quality oocytes, PCOS patients can have larger numbers of germinal vesicle stages – metaphase I oocyte collected from IVF, due to their elevated antral follicles count. Those are commonly maturated with unsatisfactory results. When optimized maturation procedure will serve, not only for PCOS and infertile patients but also in cancer patients for the preservation of fertility and as a more patientfriendly alternative than standard controlled ovarian stimulation. PCOS patients are not the only ones that could benefit from *in vitro* maturation (IVM) technology. IVM has numerous clinical applications. Under proper culture media additives, immature oocytes in the stage of metaphase I go to the final stage of maturation [28]. Although the IVM seems to have improved lately [29], still a success rate remains lower than traditional IVF [30]. International guidelines do not favor IVM over the other options due to lack of evidence [5] but conceived children are not endangered after IVM procedure [31]. Improving the IVM techniques can definitely increase the success of IVF/ICSI procedures in PCOS patients and lower the risk of OHSS.

#### **5. Conclusion**

The definition of phenotypes of polycystic ovarian syndrome stemmed from a diverse and complex clinical picture of this endocrine disorder. Diagnostic criteria of individual phenotype, contribute to new concepts of research into the effects of obesity, hyperandrogenism, and metabolic disorders on reproduction in humans. According to the outcomes of the treatment of infertility of patients with this disorder, *Polycystic Ovary Syndrome Phenotypes and Infertility Treatment DOI: http://dx.doi.org/10.5772/intechopen.101994*

significant differences in the chances of conception compared to the population of infertile women who do not have polycystic ovary syndrome have been clearly proven. Less clear is the difference in infertility treatment outcomes between women with a defined polycystic ovarian syndrome phenotype, which is the area of new research. In cases of classical phenotype polycystic ovarian syndrome (A and B) associated with obesity and insulin resistance, negative effects of this disease on gametes and embryos are possible due to cellular process disorders related to glucose and androgen metabolism.

#### **Acknowledgements**

The publication is supported by H2020: MESOC – measuring the social dimension of culture; under Grant agreement no. 870935. Uniri-biomed-18-161 project: Extracellular vesicles in human follicular fluid: content and role in oocyte maturation and embryo quality.

### **Conflict of interest**

Authors have no conflict of interest.

#### **Author details**

Anđelka Radojčić Badovinac1 \* and Neda Smiljan Severinski2

1 Department of Biotechnology University of Rijeka and Department of Medical Biology and Genetics, Faculty of Medicine, University of Rijeka, Rijeka, Croatia

2 Department of Gynecology and Obstetrics, Faculty of Medicine, University of Rijeka, Rijeka, Croatia

\*Address all correspondence to: andjelka@biotech.uniri.ri

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Mumusoglu S, Yildiz BO. Polycystic ovary syndrome phenotypes and prevalence: Differential impact of diagnostic criteria and clinical versus unselected population. Current Opinion in Endocrine and Metabolic Research. 2020;**12**:66-71

[2] Bozdag G, Mumusoglu S, Zengin D, Karabulut E, Yildiz BO. The prevalence and phenotypic features of polycystic ovary syndrome: A systematic review and meta-analysis. Human Reproduction. 2016;**31**:2841-2855

[3] Azziz R, Carmina E, Dewailly D, Diamanti-Kandarakis E, Escobar-Morreale HF, Futterweit W, et al. The androgen excess and PCOS society criteria for the polycystic ovary syndrome: The complete task force report. Fertility and Sterility. 2009;**91**:456-488

[4] Lauritsen MP, Bentzen JG, Pinborg A, Loft A, Forman JL, Thuesen LL, et al. The prevalence of polycystic ovary syndrome in a normal population according to the Rotterdam criteria versus revised criteria including anti-Mullerian hormone. Human Reproduction. 2014;**29**:791-801

[5] Teede HJ, Misso ML, Costello MF, Dokras A, Laven J, Moran L. Recommendations from the international evidence-based guideline for the assessment and management of polycystic ovary syndrome. Fertility and Sterility. 2018;**110**:364-379

[6] Alexiou E, Hatziagelaki E, Pergialiotis V, Chrelias C, Kassanos D, Siristatidis C, et al. Hyperandrogenemia in women with polycystic ovary syndrome: Prevalence, characteristics and association with body mass index. Hormone Molecular Biology and Clinical Investigation. 2017;**29**:105-111

[7] Dewailly D, Lujan ME, Carmina E, Cedars MI, Laven J, Norman RJ, et al. Definition and significance of polycystic ovarian morphology: A task force report from the Androgen excess and polycystic ovary syndrome society. Human Reproduction Update. 2014;**20**:334-352

[8] Bozdag G, Mumusoglu S, Coskun ZY, Yarali H, Yildiz BO. Anti-Mullerian hormone as a diagnostic tool for PCOS under different diagnostic criteria in an unselected population. Reproductive Biomedicine Online. 2019;**39**:522-529

[9] Lizneva D, Kirubakaran R, Mykhalchenko K, Suturina L, Chernukha G, Diamond MP, et al. Phenotypes and body mass in women with polycystic ovary syndrome identified in referral versus unselected populations: Systematic review and meta-analysis. Fertility and Sterility. 2016;**106**:1510-15120 e2

[10] Sachdeva G, Gainder S, Suri V, Sachdeva N, Chopra S. Comparison of the different PCOS phenotypes based on clinical metabolic, and hormonal profile, and their response to clomiphene. Indian Journal of Endocrinology and Metabolism. 2019;**23**:326-331

[11] Fauser BC, Diedrich K, Devroey P. Predictors of ovarian response: Progress towards individualized treatment in ovulation induction and ovarian stimulation. Human Reproduction Update. 2008;**14**:1-14

[12] Ma L, Cao Y, Ma Y, Zhai J. Association between hyperandrogenism and adverse pregnancy outcomes in patients with different polycystic ovary syndrome phenotypes undergoing *in vitro* fertilization/intracytoplasmic sperm injection: A systematic review and meta-analysis. Human Reproduction. 2020;**35**(10):2272-2279

*Polycystic Ovary Syndrome Phenotypes and Infertility Treatment DOI: http://dx.doi.org/10.5772/intechopen.101994*

[13] Schulte MM, Tsai JH, Moley KH. Obesity and PCOS: The effect of metabolic derangements on endometrial receptivity at the time of implantation. Reproductive Sciences. 2015;**22**:6-14

[14] Li Y, Wang L, Xu J, Niu W, Shi H, Hu L, et al. Higher chromosomal aberration rate in miscarried conceptus form polycystic ovary syndrome women undergoing assisted reproductive treatment. Fertility and Sterility. 2019;**111**:936-943

[15] Jamil AS, Alalaf SK, Al-Tawil NG, Al-Shawaf T. Comparison of clinical and hormonal characteristics among four phenotypes of polycystic ovary syndrome based on the Rotterdam criteria. Archives of Gynecology and Obstetrics. 2016;**293**:447-456

[16] De Vos M, Pareyn S, Drakopoulos P, Raimundo JM, Anckaert E, Santos-Ribeiro S, et al. Cumulative live birth rates after IVF in patients with polycystic ovaries: Phenotype matters. Reproductive Biomedicine Online. 2018;**37**(2):163-171

[17] Ramezanali F, Ashrafi M, Hemat M, Arabipoor A, Jalali S, Moini A. Assisted reproductive outcomes in women with different polycystic ovary syndrome phenotypes: The predictive value of anti-Müllerian hormone. Reproductive Biomedicine Online. 2016;**32**:503-512

[18] Selçuk S, Özkaya E, Eser A, Kuyucu M, Kutlu HT, Devranoğlu B, et al. Characteristics and outcomes of in vitro fertilization in different phenotypes of polycystic ovary syndrome. Turk Journal of Obstetrics and Gynecology. 2016;**13**:1-6

[19] Ho VNA, Pham TD, Hoang HLT, Vuong LN. Impact of polycystic ovary syndrome phenotypes on in vitro

fertilization outcomes in Vietnamese women: A secondary analysis of a randomized controlled trial. Fertility & Reproduction. 2021;**3**(3):78-83

[20] Qiao J, Feng HL. Extra- and intraovarian factors in polycystic ovary syndrome: Impact on oocyte maturation and embryo developmental competence. Human Reproduction Update. 2011;**17**(1):17-33

[21] Thompson JG, Brown HM, Kind KL, Russell DL. The ovarian antral follicle: Living on the edge of hypoxia or not? Biology of Reproduction. 2015;**92**(6):153, 1-6. DOI: 10.1095/biolreprod.115.128660

[22] Dewailly D, Robin G, Peigne M, Decanter C, Pigny P, Catteau-Jonard S. Interactions between androgens, FSH, anti-Müllerian hormone and estradiol during folliculogenesis in the human normal and polycystic ovary. Human Reproduction Update. 2016;**22**(6):709-724

[23] Tramišak Milaković T, Panić Horvat L, Čavlović K, Smiljan Severinski N, Vlašić H, Vlastelić I, et al. Follicular fluid anti-Müllerian hormone: A predictive marker of fertilization capacity of MII oocytes. Arch Gynecol Obstet. 2015;**291**:681-687. DOI: 10.1007/ s00404-014-3460-9

[24] Dević PS, Tramišak MT, Panić HL, Čavlović K, Vlašić H, Manestar M, et al. Genes for anti-Müllerian formone and androgen receptor are underexpressed in human cumulus cells surrounding morphologically highly graded oocytes. SAGE Open Medicine. 2019;**7**:1-8. DOI: 10.1177/2050312119865137

[25] Chen YH, Heneidi S, Lee JM, Layman LC, Stepp DW, Gamboa GM, et al. Azziz R: miRNA-93 Inhibits GLUT4 and is overexpressed in adipose tissue of polycystic ovary syndrome patients and

women with insulin resistance. Diabetes. 2013;**62**(7):2278-2286

[26] Sigala J, Sifer C, Dewailly D, Robin G, Bruyneel A, Ramdane N, et al. Is polycystic ovarian morphology related to a poor oocyte quality after controlled ovarian hyperstimulation for intracytoplasmic sperm injection? Results from a prospective, comparative study. Fertility and Sterility. 2015;**103**(1):112-118

[27] Palomba S, Daolio J, La Sala JB. Oocyte competence in women with polycystic ovary syndrome. Trends in Endocrinology & Metabolism. 2017;**28**(3):186-198

[28] Yang ZY, Chian RC. Development of in vitro maturation techniques for clinical applications. Fertility and Sterility. 2017;**108**:577-584

[29] Walls ML. Hart RJ In vitro maturation. Best Practice & Research. Clinical Obstetrics & Gynaecology. 2018;**53**:60-72

[30] Walls ML, Hunter T, Ryan JP, Keelan JA, Nathan E, Hart RJ. In vitro maturation as an alternative to standard in vitro fertilization for patients diagnosed with polycystic ovaries: A comparative analysis of fresh, frozen and cumulative cycle outcomes. Human Reproduction. 2017;**32**:1341-1350

[31] Roesner S, von Wolff M, Elsaesser M, et al. Two-year development of children conceived by IVM: A prospective controlled single-blinded study. Human Reproduction. 2017;**32**:1341-1350

### *Edited by Zhengchao Wang*

Polycystic ovary syndrome (PCOS) is a heterogeneous hormone-imbalance disorder that occurs in reproductive-aged women worldwide and is characterized by hyperandrogenism, ovulatory process dysfunction and polycystic ovaries. This book includes two sections that cover the pathogenesis and treatment of PCOS. It provides a comprehensive overview of the latest PCOS research to benefit the population of women with this disorder.

Published in London, UK © 2022 IntechOpen © 7activestudio / iStock

Polycystic Ovary Syndrome - Functional Investigation and Clinical Application

Polycystic Ovary Syndrome

Functional Investigation

and Clinical Application

*Edited by Zhengchao Wang*