Decrease in serum anti‐Müllerian hormone level per puncture with laparoscopic ovarian drilling using ultrasonically activated device

Abstract Purpose To determine the contributing factor in infertility treatment with laparoscopic ovarian drilling (LOD) to the decrease in serum anti‐Müllerian hormone (AMH) levels in patients with polycystic ovarian syndrome using an ultrasonically activated device. Methods A retrospective analysis was performed in 60 patients (aged 23–36 years) who received 25–120 punctures in each ovary with LOD treatment from January 2014 to December 2018. Results The mean decrease in serum AMH level per puncture with LOD was 0.07 ± 0.04 ng/ml in all 60 patients and 0.08 ± 0.04 ng/ml in patients with ≥10 ng/ml preoperative serum AMH level, which was significantly higher than in those with <10 ng/ml (0.05 ± 0.02 ng/ml). The mean decrease in serum AMH level per puncture in patients with body mass index (BMI) < 18.5 kg/m2 (0.10 ± 0.03 ng/ml) was significantly higher than in those with BMI 18.5–25 kg/m2 (0.07 ± 0.04 ng/ml) and >25 kg/m2 (0.06 ± 0.02 ng/ml). Conclusions The mean decrease in serum AMH levels per puncture with LOD using an ultrasonically activated device depends on the preoperative serum AMH level and BMI of patients.


| INTRODUC TI ON
Laparoscopic ovarian drilling (LOD) has been widely used to treat polycystic ovary syndrome (PCOS). [1][2][3][4][5][6] In general, the clinical results indicate that LOD restores menstrual cyclicity and ovulation in approximately 70-80% of patients, and pregnancy rate following LOD is approximately 40-60%. 2,3,6,7 There was no statistically significant difference in pregnancy or miscarriage rates between LOD and gonadotropin therapy. 3,7 LOD is performed through various methods: electrocautery; lasers including CO 2 , argon, KTP, and Nd-YAG; and ultrasonically activated devices. 2,[8][9][10] In LOD using electrocautery, power setting, duration of penetration, depth of penetration, and the number of punctures to be applied vary in each case because it is impossible to objectively measure the cauterized area of the ovarian cortex. [1][2][3]5,6 In addition, LOD with lasers, laser machines, power settings, and the number of punctures were different. 7,8,10,11 LOD using electrocautery or laser is influenced by the subjectivity and experience of the operator.
Serum anti-Müllerian hormone (AMH) is useful in diagnosing ovarian function, and LOD is expected to normalize the high preoperative serum AMH levels in patients with PCOS. A decrease in serum AMH level after LOD compared to the preoperative value appears to be a good predictor of the ovarian response to LOD 12 ; however, this has not been clarified yet.
In LOD using an ultrasonically activated device, the effect of treatment can be assessed only by the number of applied punctures. 9 In this study, we retrospectively determined the decrease in serum AMH level per puncture with LOD in 60 patients with PCOS who received LOD treatment at our clinic. The ultimate goal of this study was to establish a formula for the number of punctures with LOD required to achieve the target serum AMH level. However, the decrease in serum AMH level per puncture would depend on patient background. Therefore, we conducted an exploratory analysis of potential confounding factors such as preoperative serum AMH levels.

| Subjects
A retrospective analysis was performed in 60 patients (aged 23-36 years) with PCOS who received 25-120 punctures in each ovary with LOD treatment from January 2014 to December 2018 at the Institution of Central Clinic. LOD was indicated in patients who met any of the following criteria: (I) had PCOS with clomiphene citrate (CC) and/or letrozole resistance and no desire for gonadotropin therapy; (II) had PCOS with failed infertility treatment with CC and/or letrozole and no desire for gonadotropin therapy; and (III) had PCOS with failed infertility treatment with CC and/or letrozole at high risk of ovarian hyperstimulation syndrome (OHSS) by gonadotropin therapy and no desire for in vitro fertilization, and had desired for LOD in any case.
PCOS was diagnosed when at least two of the following three criteria were satisfied, as proposed by the Rotterdam Consensus Meeting 13 : oligomenorrhea or amenorrhea, clinical hyperandrogenism and/or hyperandrogenemia, and polycystic ovaries.

| Laparoscopic ovarian drilling
The LOD was performed under general anesthesia using a threepuncture laparoscopy method. A 5-mm laparoscope was inserted via the sub-umbilical route, and two 5-mm manipulating trocars were to make a single puncture; therefore, the total duration of drilling was <10 min, and the total operation time was approximately 30-40 min.

| Follow-up data
The serum AMH level was determined using the original protocol of the AMH electrochemiluminescence immunoassay (SRL Inc., Japan). Serum AMH level was measured within 2 months before and 1 month after LOD to retrospectively calculate the decrease in serum AMH level per puncture using the following formula: The results were also categorized by two age groups: <32 years and ≥32 years, based on a study reporting that the fecundity of women decreases significantly around 32 years of age and decreases more rapidly after 37 years of age, 14 by body mass index (BMI): <18.5 kg/m 2 (underweight), 18.5-25 kg/m 2 (normal weight), or >25 kg/m 2 (overweight and obesity) according to the BMI classification by WHO, 15 and by preoperative serum AMH level <10 ng/ml or ≥10 ng/ml based on a study reporting that >97% of women with highly elevated AMH levels (>10 ng/ml) had PCOS. 16 All data were analyzed using the IBM Statistical Package for Social Sciences for Windows (version 11.0; Armonk, NY, USA).
Welch's t-test was used to compare the results. Statistical significance was set at p < .05.

| Baseline characteristics of patients
The baseline characteristics of the 60 patients enrolled in the study are presented in Table 1. The mean age (± SD) was 29.8 ± 3.2 years, and the mean BMI (± SD) was 23.0 ± 4.8 kg/m 2 . All patients were diagnosed with PCOS according to the Rotterdam criteria and underwent LOD. Of the 60 patients, 20 were classified into PCOS criterion I, 19 in criterion II, and 21 in criterion III.

| Decrease in serum AMH level after LOD
Serum AMH levels were measured within 2 months before and 1 month after the LOD. The number of ovarian punctures with LOD based on preoperative serum AMH levels is shown in Figure 2. One hundred and more punctures were performed in 91.9% (34/37) and 52.3% (12/23) of patients with preoperative serum AMH levels ≥10 ng/ml and <10 ng/ml, respectively. One hundred and fifty or more punctures were performed in 45.9% (17/37) of patients with preoperative serum AMH levels ≥10 ng/ml because their ovaries were 1.5-to 2-fold larger than normal. None of the patients with preoperative serum AMH level <10 ng/ml had ovaries 1.5-fold larger than normal. The mean (± SD) preoperative and postoperative serum AMH levels were 12.70 ± 6.50 and 3.96 ± 2.41 ng/ml, respectively.
Age-stratified analysis showed that the mean (± SD) preoperative and postoperative serum AMH levels were 12.20 ± 3.01 ng/ ml and 4.21 ± 2.94 ng/ml, respectively, in patients aged ≥32 years,
The mean decrease in serum AMH level per puncture in patients with BMI 18.5-25 kg/m 2 was significantly higher than in those with BMI > 25 kg/m 2 (p = .046) ( Figure 5). resulting in normal ovulation and pregnancy. 18 The procedure has been effective for patients with CC-resistant PCOS, but there is a risk of postoperative adhesion formation leading to mechanical infertility.

| DISCUSS ION
Laparoscopic ovarian electrocautery and ovarian drilling for the treatment of PCOS have been reported since the 1980s. [1][2][3][4][5][6] It is now recognized that LOD is an effective second-line treatment for anovulatory and oligo-ovulatory infertility associated with PCOS.
In the LOD using an ultrasonically activated device, a blade is placed onto the surface of the ovarian capsule to induce a shock wave called cavitation, which breaks tissues. 9 Bleeding is rare, and there is no ex- The effects of LOD were objectively evaluated by analyzing ovulation, serum LH/FSH ratio, number of antral follicles, and free androgen index. 20 Recently, serum AMH levels have been recognized as a more objective index for the assessment of LOD treatment. 12 Postoperative reduction mechanism in AMH levels could involve the atresia of several small follicles as part of normal follicular development leading to ovulation. However, there seems to be no consensus regarding the appropriate timing to measure postoperative AMH levels in assessing the clinical efficacy of LOD. Therefore, this study utilized data from postoperative checkups conducted 1 month after LOD.
We started LOD treatment in 2010 according to Aoki's report stating that the appropriate number of punctures with LOD is approximately 40-50 per ovary. 21 However, no clinical efficacy was observed in patients with large ovaries (1.5-fold larger than normal), requiring a second LOD. Therefore, we increased the number of punctures per ovary to approximately 100 in patients with large ovaries, after which clinical efficacy was observed in patients even with a single LOD. Therefore, we decided to perform LOD with 25-120 punctures per ovary in this study.
The biggest concern in treating PCOS patients with LOD is determining the exact number of punctures required to impart maximum clinical effect while preventing substantial decrease in ovarian functions caused by LOD. Therefore, we explored the possibility of serum AMH levels before LOD as an index for deciding the number of punctures.
The target AMH level decrease after LOD was set at 1.0-2.0 ng/ ml plus age-specific mean AMH values. 22 Although LOD is a recognized method to lower AMH levels in PCOS patients, a formula for calculating the adequate number of punctures has not been established. Therefore, it would be clinically useful to predict serum AMH levels after LOD using a simple index.
In this study, we found that the mean decrease in serum AMH level per puncture applied with LOD was 0.07 ± 0.04 ng/ml in 60 patients, and it was significantly larger in patients with preopera- utes to a decrease in testosterone, androstenedione, AMH, and insulin growth factor-1. 11 Considering these facts, it is suggested that a study with a large sample is necessary to verify the results of this exploratory study.
In conclusion, 0.07 ng/ml is the standard value of the decrease in serum AMH level per puncture with LOD using an ultrasonically activated device in patients with PCOS, and it fluctuates depending on preoperative serum AMH level and BMI. We expect that LOD will be more widely recognized as one of the options for reducing AMH levels to accelerate the accumulation of data to clarify the correlation between the number of punctures and AMH levels and between AMH reduction and fertility improvement and will further contribute to the improvement of fertility in patients with PCOS.

ACK N OWLED G M ENTS
The authors are greatly thankful to Dr. Shigeo Araki for providing advices on this manuscript.

CO N FLI C T S O F I NTE R E S T
The authors have no conflicts of interest to declare.

H U M A N R I G HT S S TATE M E NT S A N D I N FO R M E D CO N S E NT
All the procedures accorded with the ethical standards of the relevant committees on human experimentation (institutional and national) and with the Helsinki Declaration of 1964 and its later amendments. The study design was approved by the ethics committee of Institutional Review Board of Institute of Central Clinic. This is a retrospective study in patients who submitted informed consent for undergoing fertility treatment at our clinic.

A N I M A L S TU D I E S
This article does not contain any study with animal participants that have been performed by any of the authors.