The time-interval of ovarian reserve recovery after laparoscopic unilateral ovarian nonendometriotic cystectomy: a prospective cohort study

Background: Ovarian benign cyst, frequently seen in women of reproductive age, is one of the most important causes of damaging effect of ovarian reserve. Laparoscopic ovarian cystectomy is established as the gold standard surgical approach to the ovarian benign cyst. Studies have shown that potential fertility can be directly impaired by laparoscopic ovarian cystectomy and diminished ovarian reserve. There is little data about the time-interval of ovarian reserve recovery after the laparoscopic unilateral ovarian cystectomy. The objective of this study was to investigate the time-interval of ovarian reserve recovery after laparoscopic unilateral ovarian nonendometriotic cystectomy. Method: In the first part of the study, a total number of 67 patients with unilateral ovarian nonendometriotic cyst who underwent laparoscopic unilateral ovarian cystectomy were recruited as a postoperative observation group (POG). A total number of 69 same-aged healthy women without ovarian cyst who did not undergo surgery were recruited as a referent group (RFG).The serum anti-Müllerian hormone (AMH) levels were measured using a commercially available enzyme-linked immunosorbent assay kit; the Follicle-stimulating hormone (FSH)and E2 levels, measured using a chemiluminescent reagent kit. The ovarian arterial resistance index (OARI) and AFC were measured by transvaginal ultrasonography on the 3rd-5th day of the same menstrual cycle. In the second part of the study, a prospective postoperative 6-month follow-up of cases was performed. Results: When compared with RFG, the AFC of POG’s cyst side showed no difference in the 1 st , 3 rd , 6 th postoperative month (F = 0.03, 0.02, 0.55; P = 0.873, 0.878, 0.460). The OARI of POG’s cyst side presented no difference in the 1 st , 3 rd , 6 th postoperative month ( F = 0.73, 3.57, 1.75; P=0.395, 0.061, 0.188). In the first month, the postoperative AMH levels declined significantly, 1.88 ng/ml (IQR: 1.61-2.16 ng/ml) in POG and 2.57 ng/ml (IQR: 2.32-2.83 ng/ml) in RFG (F = 13.43; P = 0.000). At the time interval, the rate of decline was significantly lower postoperatively than preoperatively in POG (32.75 %),and was also the case in the comparison of POG with RFG (26.67 %). Conclusions: After the laparoscopic unilateral ovarian cystectomy, the optimal time-interval can be the 6th month for ovarian reserve recovery. Semiannually, AMH levels were to be detected to find those whose window time to conceive was likely to be shorter than others of the same age.

Ovarian reserve is defined as a woman's reproductive potential in terms of the number and quality of her remaining oocytes [2]. In 2015, The American College of Obstetricians and Gynecologists recommended that ovarian reserve testing should be performed on those who had ovarian surgery [16]. Ovarian reserve testing should allow individualization of treatment protocols to achieve optimal response while minimizing safety risks [16,17] .
The abnormally elevated follicle-stimulating hormone (FSH) is almost synonymous with late DOR (high positive predictive value), but the majority of women who are tested (including those with DOR) will have a normal test result (low negative predictive value) [18]. The high values of estradiol ( E2 ) have been associated with both poor ovarian response and failure to achieve pregnancy [19]; measurement of both FSH and E 2 on cycle day 3 may therefore help decrease the incidence of false negative testing [20]. AMH levels rise in young women in adolescence and peak at about 25 years of age, then gradually decline until reaching undetectable levels a few years prior to menopause [21]. Therefore, it reflects the size of the primordial oocyte pool [22]. AMH level testing is a useful screening test in women at high risk of diminished ovarian reserve [17], especially in assessing ovarian reserve for young women with cancer [23]. But, the study did not observe a significant decrease in serum AMH levels 3 months after endometriomas cystectomy [24], on the other hand ,the recovery of AMH serum level has been observed 3-6 months after endometriomas cystectomy [25].
Antral follicle count (AFC) is the sum of follicles in both ovaries as observed on 5 ultrasound in the early follicular phase (day 2-4) of the menstrual cycle. A previous study showed that the OARI of patients with hypoestrogenic amenorrhoea were decreased when compared with the eumenorrhoeic subjects [26].
However, there is little data about the time-interval of ovarian reserve recovery after the laparoscopic unilateral ovarian cystectomy. If little is known about the changing curve of ovarian reserve after the surgery, a question still remains how we can accomplish the individualization of treatment protocols. Therefore, we conducted a six-month prospective cohort study to monitor the ovarian reserve in the patients with benign nonendometriotic ovarian cyst who had undergone laparoscopic unilateral ovarian cystectomy. The monitoring took the form of detecting the serum levels of AMH, and FSH and E2, those data acquired using enzyme-linked immunosorbent assay kit and chemiluminescent reagent kit. The data of AFC and OARI were detected using transvaginal ultrasonography.

Materials and Methods
For the current study, which was registered by the Chinese clinical trial Registry  (Table   6 1), a total number of 67 patients with the unilateral ovarian benign nonendometriotic cyst, who had undergone a laparoscopic cystectomy, were invited to participate in the postoperative observation group (POG), and 69 same-aged women formed the referent group (RFG). Because of their pregnancies within six months after the surgery, 11 participants were excluded in POG, and because of their pregnancies and personal reasons, 9 withdrew from RFG ( Figure 1).

Study treatment
The laparoscopic unilateral ovarian cystectomy was performed by the same surgical team. The weak position of the cyst surface was opened with an ultrasound knife upon the visual exploration of pelvic cavity and ovarian cysts, the cyst detached completely from the ovarian cortex while saving the healthy ovarian cortex as much as possible. During the operation, hemostatic method was ensured with bipolar electrocoagulation forceps at the power of 25 watts and for the duration of no more than 5 seconds. A loose knot was made of 2/0 absorbable sutures for controlling bleeding and reshaping ovarian morpholog. The specimens were examined under intraoperative rapid freezing pathology in order to exclude malignancy, which was followed by the pathological routine examination.
After the surgery, the women were monitored and observed in the hospital wards for 48 hours to watch for surgical or anesthesia-associated complications. For all the women, the operative and post-operative course was successful without any specific complication.

Measurements
In POG, the fasting blood of each was collected on the morning of her menstrual cycle's second day to be examined one month prior to the laparoscopic unilateral ovarian cystectomy; the same collecting procedure was performed in RFG, the 7 serum separated from the whole blood and transferred into a sterile polypropylene tube to be stored at -80℃. After the operation, the samples were examined in the 1 st , 3 rd and 6 th month.
In RFG, the serum was collected at the same time point. The serum AMH levels were measured using a commercially available enzyme-linked immunosorbent assay kit (Beckman, Germany); the FSH and E2 levels, measured using a chemiluminescent reagent kit (Siemens, Germany). According to Rosendahl M and J.G. Bentzen1's experimental methods [27,28], OARI and AFC were measured by transvaginal ultrasonography (Philips, Germany) on the 3 rd -5 th day of the same menstrual cycle.

Statistics
SPSS10.0 software package (SPSS Inc., Chicago, IL, USA) was applied to the statistical analysis. In the cases of quantitative variables, after the normality of the data was checked, mean ± SD and median (range) were used to describe normal and non-normal distribution, respectively. Comparisons were made between the two groups based on one-way ANOVA. Statistically, p < 0. 05 was considered as significant.

Results
Between POG and RFG, no significant differences were found in age, BMI, OARI (the cyst-side ovary), AFC (the cyst-side ovary), median baseline levels of antimüllerian hormone, estradiol (E2), FSH and CA125; such data were detected one month prior to the laparoscopic unilateral ovarian cystectomy (P > 0.05; Table 2).
In the POG, the cyst size was 4.67± 3.12cm; pathological types were teratoma ( Figure 2). At the time interval, the rate of decline was significantly lower (Figure 3) postoperatively than preoperatively in POG (32.75 %), and was also the case in the comparison of POG with RFG (26.67 %). In the 3 rd and 6 th month, however, the postoperative AMH levels were found to be similar between POG and RFG (F=1.42, 0.75; P=0.784, 0.102) ( Table 5).
These evidences showed the postoperative AMH levels restored gradually to the preoperative in the 6 th month, showing a statistical significance between three 9 detecting time intervals (F=14.21; P= 0.000). As indicated by Table 4, the interaction between the detecting time and grouping was statistically significant (F=111.89; P=0.000). The postoperative E2 and FSH levels were similar in the 1 st , 3 rd , and 6 th month between POG and RFG (P > 0.05). There was no statistical significance between the three detecting time intervals (P > 0.05), between the detecting time point and treatment factors (P > 0.05), and between the treatment factors (P > 0.05).

Discussion
In the current study, we undertook a prospective cohort study to test our hypothesis that the optimal time-interval of ovarian reserve recovery can be in the 6 th month after the laparoscopic unilateral ovarian nonendometriotic cystectomy. We found that the ovarian reserve decreased after the surgery, and that in comparison with E 2 , FSH levels and OARI and AFC, the serum AMH levels could be a convenient and reliable marker for testing ovarian reserve in the short-term. Intriguingly, AMH levels in POG showed a significant decline in the 1 st post-operative surgery ( The best surrogate marker for oocyte quality is age [16]. Actually, age is a rough 10 indicator; therefore we need more useful and accurate indicators for discussing prognosis and recommending a treatment plan in short-term clinical practice, especially for those younger women with decreased or diminished ovarian reserve. It is reasonable to inform the woman that her window of pregnancy possibility may be shorter than anticipated, before formulating an individualized treatment protocol. Over the years, various tests and markers of ovarian reserve have been reported such as the basal FSH in 1988, the antral follicle count (AFC) in 1997 and AMH in 2002 [29][30][31]. The basal FSH plus E 2 levels, AMH levels, AFC as appropriate ovarian reserve screening tests should be used in clinic practice [16,18].
With the increase of reproductive age, the basal FSH level is various because of the inherent variability of each reproductive cycle, FSH multiple cut-off points >10 IU/L (10-20 IU/L) associated with diminished ovarian reserve, but its sensitivity is generally poor (11-86%) [19,20] . In terms of predicting failure to evaluate ovarian reserve at a short time, therefore, a single FSH value has limited reliability [30]. The basal E 2 has low predictive accuracy for poor ovarian response and failure to conceive; therefore, this test should not be used in isolation to assess ovarian reserve [19]. In the current study, the E 2 , FSH levels were similar between POG and RFG in the 1st, 3rd and 6th postoperative month, respectively (P>0.05); thus E2 and FSH levels was not an effective and sensitive indicator of ovarian reserve changes in the short term.
AFC correlates with the quantity of remaining follicles, and good intercycle and interobserver reliability has been demonstrated [32,33]. However, since AFC has inherent variability related to technology and inter-observer variability [18], it is difficult to assess the exact number of antral follicles of the cystic ovary before cystectomy [34]. In the current study, AFC presented similarity between POG and RFG in the 1 st , 3 rd and 6 th month postoperatively (P>0.05). Our findings suggest that AFC cannot evaluate ovarian reserve in the short term, especially after laparoscopic ovarian nonendometriotic cystectomy.
AMH has been reported to relatively independent of gonadotropins circulating at physiologic levels, allowing for testing anytime throughout the menstrual cycle [35][36][37][38]. The level of AMH declines by 5.6% per year [39], which reflects the size of the primordial oocyte pool [40]. An undetectable AMH level suggests diminished ovarian reserve and may allow a treatment to be tailored to each individual [17,41]. The recent studies have shown that serum AMH level has been accepted as the most reliable and easily measurable marker for post-operative assessment of ovarian reserve, for it can show a postoperative decline [5,[42][43][44][45][46][47][48][49][50][51].
In recent years, many doctors have paid growing attention to the ovarian reserve, their concerns ranging from the decline of ovarian reserve in cancer patients undergoing chemotherapy or radiotherapy to the impact of various gynecological operations on ovarian reserve.
Recent studies have suggested that AMH could be used to predict ovarian follicle loss and follow the evolution of ovarian reserve during chemotherapy. Pretreatment serum AMH concentrations could predict long-term ovarian function after chemotherapy for the patients with early breast cancer; this marker was the only significant predictor of menses compared with age, Inhibin B and FSH, a 55% decrease of AMH levels after one chemotherapy cycle [52]. In 2017, a study analyzed a large prospective multicentric cohort of 249 breast cancer patients [53], in which with the mean basal AMH levels of 4.19 ng/ml (median 2.95 ng/ml), AMH levels were of 0.78 ± 1.40 ng/ml four months after chemotherapy cycle completion. The breast cancer patients were reported to have AMH levels above 0.7 ng/ml before chemotherapy, and those aged under 40 were overweight or obese (BMI > 25) were more likely to regain ovarian function [54]. Radiation therapy was also recognized as highly ovario-toxic even at low doses, associated to 149 extremely low or undetectable AMH dosages in the post-treatment patients [55]. In 2018, a prospective pilot study showed decreased ovarian reserve of thyroid carcinoma patients after radioiodine therapy, the median AMH levels being 3.25 (0.32-17.42), 1 (0.01-3.93), 1.13 (0.08-6.12), and 1.37 (0.09-6.1) ng/ml before and in the 3rd, 6th, and 12th month after therapy [56]. A prospective study showed that AMH levels of 42 pediatric females with cancer who had underwent fertility preservation procedures dropped by approximately 40-50%, and their AMH levels after anticancer and ovarian folliculus preservation therapy increased until approximatly10 years later and then decreased [57].
Furthermore, the damage of gynecological surgeries on ovarian function can be evaluated by comparing pre-and post-operative AMH levels. A pilot randomized controlled trial in 2013 suggested that AMH levels were not significantly different at the baseline in the 4-6 postoperative weeks, in the 3rd postoperative month among women with salpingectomy during laparoscopic hysterectomy versus no salpingectomy [58]. The previous studies showed that serum AMH level is a statistically significant decline after ovarian nonendometriotic cystectomy [42,43,45,47]. It was reported that ovarian reserve evaluated with AMH was reduced in patients with ovarian endometriomas when compared with those with other benign ovarian cysts, and with those with healthy ovaries [59].The current study indicated that the ovarian reserve decreased after the surgery, and that in comparison with E2, FSH levels and OARI and AFC, the serum AMH levels could be a convenient and 13 reliable marker for testing ovarian reserve in the short-term. AMH levels showed a significant decline in POG when compared with those in RFG (1.88 ng/ml vs. 2.57 ng/ml; P=0.000) in the 1st post-operative month, the declining rate significantly lower than the preoperative in POG (32.75 %) as well as than that in RFG (26.67 %).Our finding was similar with those previously reported. AMH can be used to evaluate ovarian reserve after chemotherapy and gynecological operation. However, some researchers have pointed out that AMH is not a hormone but a paracrine factor. The mechanism of AMH secretion into the peripheral circulation is unknown; however, there has been sufficient evidence to suggest that the reduced serum AMH in women with endometriomas can indicate a real and definitive damage to the ovarian reserve rather than only a transient and potentially reversible interference with ovarian physiology [60].
The mechanism of the decline of ovarian reserve following ovarian cystectomy remains largely unknown. It is partially because of the removal of some healthy ovarian tissue in laparoscopic ovarian nonendometriotic cystectomy, which exist a certain number of oocytes; it is possibly related to the use of electrocoagulation for hemostasis in the operation, which can cause damages to the healthy ovarian tissue [5,6,61]. A previous study showed that the number of acquired eggs was significantly decreased in IVF after surgery [62]. Therefore, the hemostatic method in the current study was ensured with bipolar electrocoagulation forceps at the power of 25 watts and for the duration of no more than 5 seconds, then with a loose knot made of 2/0 absorbable sutures for controlling bleeding . Thus it is imperative that the ovarian tissue be protected as much as possible during the operation.
In 2018, a small randomized clinical trial suggested that the AFC of the operated ovary was significantly increased in the patients with endometriomas who had 14 undergone CO2 fiber laser vaporization when compared with those with endometriomas after laparoscopic cystectomy, AMH levels found to be significantly reduced at the time interval of 3 months in those after laparoscopic cystectomy when compared with those who had undergone CO2 fiber laser vaporization [63]. The CO2 fiber laser vaporization treatment may mislead the treatment of potential malignant patients due to the lack of pathological results. However, the previous studies provided an alternative treatment to endometriomas and nonendometriomas with minimal damage to the adjacent healthy ovarian tissue. The current study also provided a clinical strategy to find the women with decreased or diminished ovarian reserve by detecting AMH every six months after laparoscopic unilateral ovarian nonendometriotic cystectomy.
However, the current study has several weaknesses. The major limitation was the recruitment of patients with unilateral ovarian nonendometriotic cyst and the healthy women without ovarian cyst, excluding bilateral nonendometriotic cysts and endometrial cyst. The impact on ovarian reserve could be limited by ovarian unilateral cysts, and the mechanism of the postoperative endocrine and paracrine changes in the healthy ovaries is still unknown. Consequently, AFC and OARI were detected at the pre-and postoperative intervals, the AFC of POG's cyst side showing no difference in the 1st, 3rd, 6th postoperative month and the OARI of POG's cyst side presenting no difference in the 1st, 3rd, 6th postoperative month. Therefore, the optimal time-interval can be the 6th month for ovarian reserve recovery after the laparoscopic ovarian unilateral cystectomy.
Another limitation was the relatively short follow-up. The postoperative measuring of ovarian reserve was performed every six months for two years, which can depict a change curve of ovarian reserve. We will continue our efforts to disentangle the intricate relations between ovarian cysts, ovarian reserve and the impact of surgery. Future studies should aim to reveal the optimal interval of ovarian reserve recovery of the patients with unilateral ovarian nonendometriotic cyst and bilateral nonendometriotic cysts and endometrial cyst after surgery. Moreover, it is necessary that we have a better understanding of the mechanism of causing the decreased ovarian reserve to develop a clinical strategy and ameliorate the surgical techniques in use.
Since there is a dearth of literature on the changing curve of ovarian reserve, we cannot determine an optimal time-interval of ovarian reserve recovery after laparoscopic unilateral ovarian nonendometriotic cystectomy. The current study suggested that the optimal interval of ovarian reserve recovery could be in the 6 th month following the laparoscopic ovarian nonendometriotic cystectomy. Based on our findings, it can serve as a clinical strategy to detect AMH levels at the timeinterval of the 6th month after the surgery, which is to be semiannually performed.
If the test results suggest decreased or diminished ovarian reserve, it is reasonable for the gynecologist and reproductive endocrinology physician to inform the woman that her window of opportunity to conceive may be shorter than that of those women at the same age, encouraging her to conceive sooner rather than later.

Authors' contributions
Li HuaPing designed and performed the research, analyzed the data, and drafted the manuscript; Liu Zhou guided the research, and Yan Bing, Wang YanLi, Shu ZhiMing, Liu YaHong collected and analyzed the data. All of the authors read and approved the final manuscript.

Ethics approval and consent to participate
Ethics approval (NO.2014031) was received from the Committee of Medical Ethics, Shanghai Punan Hospital and written informed consent was obtained from all participants.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Consent for publication
Not applicable. The serum AMH levels of POG and RFG in the 1st postoperative month. The serum AMH levels The decline rate of serum AMH levels of POG and RFG The rate of decline was 32.75%, the co

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