Laparoscopic surgeries for uterine fibroids and ovarian cysts reduce ovarian reserve via age- and surgical type-manner

Abstract Aim: Diminished ovarian reserve can be characterized by poor response to ovulation and low number of eggs. The risk factors include endometriosis, receiving cancer treatment, luteal phase deficiency and gynecologic surgery. Uterine fibroids and ovarian cysts are the most common benign gynecological diseases in women globally. This prospective study evaluated the influence of laparoscopic surgery treating benign gynecological diseases on ovarian reserve. Methods: A total of 121 patients with uterine fibroids or benign ovarian cysts who received laparoscopic surgeries (41 with hysterectomy, 36 with myomectomy, 18 with adnexectomy, and 26 with ovarian cystectomy) were included. The ovarian reserve status defined as serum anti-Mullerian hormone concentration was assessed at 1 month before and after surgery, respectively. Results: The anti-Mullerian hormone reduction level was 22.34% in cystectomy, 44.1% in adnexectomy, 24.92% in myomectomy, and 47.61% in hysterectomy (p < 0.001). Multivariate analysis showed that adnexectomy had significantly high risk of high serum anti-Mullerian hormone reduction level (adjusted odds ratio (aOR): 14.90, 95% confidence interval (CI): 2.56-86.93, p = 0.003). Conclusions: These 4 laparoscopic surgeries all reduced the AMH levels 1 month after the procedure. Further prospective studies with longer follow-up are necessary to know the real impact on the ovarian reserve. Adnexectomy was an independent factor of reduced ovarian reserve in 1 month after surgery.


Introduction
Ovarian reserve is a concept which describes the storage of woman's oocyte and the ability of producing mature follicle for ovulation [1]. Generally, ovarian reserve will diminish during ovarian aging [2]. However, diminished ovarian reserve could be observed in young women with infertility [3]. Diminished ovarian reserve could be characterized by two conditions: low number of eggs, and poor response to ovulation [2,3]. Recent knowledge reveals the risk factors of diminished ovarian reserve include endometriosis, receiving cancer treatment, luteal phase deficiency, and gynecologic surgery [4][5][6][7].
Uterine fibroids and ovarian cysts are the most common benign gynecological diseases in women with prevalence of 4.5-70% and 7-21% in global, respectively [8,9]. Uterine fibroids are characterized by noncancerous growth in the uterus, which commonly appears during childbearing years; ovarian cysts are a fluid-filled sac or pocket that appears in the ovary or on its surface [10,11]. Laparoscopic surgery is the primary treatment approach for patients with these conditions, especially for symptomatic women. Laparoscopic or robotic surgery is a minimally invasive procedure with a smaller abdominal incision, shorter recovery time, and less discomfort [12,13]. This type of surgery may include hysterectomy, myomectomy, adnexectomy, and ovarian cystectomy. Yet, the effect of laparoscopic surgery on the ovarian reserve remains debatable. A prospective cohort study for investigating the interference of ovarian reserve by applying laparoscopy in endometriosis treatment showed a significant decrease of ovarian reserve [14]. However, recovery of ovarian reserve in 9-12 months after surgery was evident in patients underwent 3-step laparoscopic surgery compared to those underwent one-step surgery [15]. Therefore, knowing the effects of different operations on the ovarian reserve is vital in providing more accurate guidance for patients in selecting individual surgical methods.
Anti-Mullerian hormone (AMH) is a dimer glycoprotein, mainly secreted by follicular granulosa cells [16]. AMH inhibits recruitment of primordial follicles and reduce follicle maturation stimulated by follicular stimulating hormone [17]. Therefore, AMH can keep minimal exhaustion of ovarian pool and is believed as an indicator of ovarian function [18]. Thus, serum AMH concentration is one of the conventional indicators of ovarian reserve [19]. Since serum AMH level changes mildly during different menstrual cycles and is independent to hormonal contraceptives and pregnancy, it can be regarded as a reliable and stable biological marker for evaluating ovarian reserve function [20]. In this study, the pre-and post-operation AMH levels were measured to evaluate the effect of gynecological laparoscopic surgery on ovarian reserve.

Study design
Patients with uterine fibroids or ovarian cysts who underwent laparoscopic surgery at our hospital from January 2017 to December 2019 were enrolled in the study. All cases were diagnosed by postoperative pathology. Inclusion criteria were 1) age between 18 to 50 years with regular menstrual periods (25-35 days), 2) no other endocrine diseases such as thyroid disease, hyperprolactinemia, and Cushing's syndrome, and 3) no hormonal treatment within 3 months before surgery, neither oral contraceptives nor GnRH analogues. The primary outcome was the status of ovarian reserve, indicated by the serum AMH concentration. Relevant clinical data were collected, including age, height, weight, BMI, fertility, and menstruation.
This prospective study complied the Declaration of Helsinki. The protocol was reviewed and approved by Institutional Review Board of South Branch of Fujian Provincial Hospital (K2020-12-026). A signed informed consent was obtained from all included patients.

Surgical approach
All surgeries were performed by Dr. Xiao-Long Shi from South Branch of Fujian Provincial Hospital (Fujian, China) using laparoscope. Briefly, pneumoperitoneum needle was injected into the umbilical hole, after which CO 2 gas was injected into the abdominal cavity (the pressure was maintained at 12 mmHg). One 1.0 cm incision was made to place puncture devices at the umbilical hole, and three 0.5-1.2 cm puncture holes were made in the left and right lower abdomen, respectively. When necessary, simple uterine lifting devices were used to help adjust the uterine direction.

Uterine fibroids
Patients with uterine fibroids underwent hysterectomy or myomectomy according to the indications and personal willingness. No ovaries were removed; no ovarian cystectomy was performed during hysterectomy and myomectomy.
Hysterectomy: hysterectomy was performed in a conventional manner. Bilateral fallopian tubes were removed. Bilateral tubes, pelvic ligaments, and ovarian ligaments were bilaterally transected, and the uterus was vaginally removed. Cutting and haemostasis were achieved with an ultrasonic scalpel and bipolar coagulation.
Myomectomy: a vasopressin solution was injected into the serosa and myometrium overlying the fibroid to decrease bleeding. Suitable myometrial incisions were performed with unipolar cautery. Fibroids were enucleated along the cleavage plane with great care to avoid the endometrial cavity and to avoid manipulation of the corner or fallopian tubes. The myometrium was closed in layers using 0 or 2-0 absorbable suture. The excised fibroids were removed with an electrical morcellator.

Ovarian cysts
Patients with ovarian cysts underwent adnexectomy or ovarian cystectomy according to the indications and personal willingness. All adnexectomies were unilateral, while ovarian cystectomy has no restrictions.
Adnexectomy: ultrasonic scalpel (ACE/HAR, Johnson, USA) and bipolar combination (RoBi38151, Karl Storz, Germany) were used to cut off the ovarian ligaments and fallopian tubes. The excised ovarian cyst was placed in the specimen bag and removed from the puncture hole.
Ovarian cystectomy: two noninvasive forceps were used to blunt exfoliate the cyst or cystic wall through traction and back-traction. Scissors were used to cut off the lesion tissue when the cyst and ovarian tissue were dense adherents, after which the oophoroplasty was performed. Bipolar electrocoagulation was occasionally applied for hemostasis.

Serum AMH test
Blood samples were collected before and at 1 month after surgery. Serum from each blood sample was isolated by centrifugation after coagulation and stored at −80 °C before use. Analysis of serum AMH level was measured by automatic electrochemical luminescence analyzer (Roche Cobas 8000, Hoffmann-La Roche Ltd., Basel, Basel-Stadt Switzerland) coupled with specific AMH analysis kit (Elecsys® AMH, Hoffman-La Roche).

Statistical analysis
Continuous data from each treatment are firstly tested with normality by Shapiro-Wilk test followed by presented as the median (Q1, Q3) and were analyzed by Kruskal-Wallis test and the pairwise comparison are performed by Dunn's test. Categorical data were analyzed with the chi-square test or Fisher's exact test and presented as n (%). Logistic regression models were used to estimate the odds ratio (OR) and 95% CIs for percentage (relative) of △AMH ≥30%. The multivariate logistic regression included the variable that P-value less than 0.2 in univariate logistic regression. All statistical analyses were carried out with SAS 9.4. A two-tailed P value < 0.05 is considered significant.

Results
A total of 121 patients were included in this study, including 26 patients undergoing cystectomy (group 1), 18 patients undergoing adnexectomy (group 2), 36 patients with myomectomy (group 3), and 41 patients with hysterectomy (group 4). Patient characteristics are listed in Table 1. There are significant different of age, gravidity, parity, BMI, hospital stay, histological type in OVC patients, surgery time, blood loss, and FT4 in the four groups. The hysterectomy group had highest median age (47 vs 29.5 ~ 40.5 years, p < 0.001; difference: group 4 vs 1 ~ 3), gravidity 2) of all groups. The myomectomy group had significantly higher age, surgery time, and blood loss than cystectomy group (difference: group 3 vs 1). The two OVC groups had higher FT4 than the two UTF groups (p = 0.022). In the two OVC groups, adnexectomy group had significantly higher proportion of serous cystadenoma than cystectomy group (38.89% vs 15.38%, p = 0.031). No significant difference in subject demographics between two OVC groups. Table 2 shows the outcome in the 4 groups. There were significant differences in preoperative AMH level (p < 0.001), postoperative AMH level (p < 0.001), △AMH level (p = 0.001), percentage (relative) of △AMH (p < 0.001), and patient number of percentage (relative) of △AMH ≥30% (p = 0.001) among the four surgery groups. The hysterectomy had lowest median preoperative AMH level (0. 36 1, 3). The four surgery groups had significant different proportion of percentage (relative) of △AMH ≥30% (p = 0.001), adnexectomy group had the highest rate (72.22%), and the cystectomy group had the lowest rate (26.92%).

Discussion
This is the first study to evaluate the influence on the postoperative serum AMH levels of different operations performed in benign gynecological diseases. Our results suggest that the four different laparoscopic surgeries for UTF and OVC all impaired ovarian reserve. Adnexectomy is associated with a significantly higher risk in ovarian reserve reduction more than 30%.
In the current study, serum AMH reduction level was up to 45-50% in adnexectomy and hysterectomy; univariate analysis also showed significantly high risks in these two surgeries for serum AMH reduction more than 30% after surgery. Interestingly, after adjusting for age, only adnexectomy still had significantly high risk. Adnexectomy is a conventional surgery for treating benign  or malignant disease on ovary or fallopian tubes [21], especially for patients without pregnancy plans. Because a removal of ovary adjacent to uterus by adnexectomy may damage the ovary reserve. The influence of hysterectomy in ovarian reserve is contradicted. A prospective study showed that both laparoscopic and non-laparoscopic hysterectomy caused permanent serum AMH reduction [22], whereas other studies showed no significant difference in ovarian reserve in 2-3 months after laparoscopic hysterectomy or myomectomy [23,24]. Fluctuation of ovarian artery by hysterectomy was considered as the cause of serum AMH reduction [24,25]. However, Abdelazim et al. showed no significant difference in the ovary volume and ovarian-related hormone content between pre-and post-hysterectomy [26]. Accordingly, the reduction of serum AMH concentration after hysterectomy might be also transient. Considering that patients received hysterectomy had higher healthy-related quality of life than those received myomectomy [27], hysterectomy is more recommended than myomectomy if the patient has no plan to get pregnancy.
Our results showed no significant difference was found in AMH reduction level between cystectomy and myomectomy; myomectomy does not elevate the risk of diminished ovarian reserve compared to cystectomy. It has been reported that serum AMH level was reduced after myomectomy and recovered in 3 months [28]. Laparoscopic cystectomy is believed in preserving ovarian reserve by minimal removal of normal ovarian tissue [29]. Although the permeant harm of ovarian reserve after laparoscopic cystectomy has been reported, the reduced ovarian reserve could recover to 65% in 3 months after surgery [30]. Meanwhile, it is worth noting that an excess removal of normal ovarian tissue my happen during cystectomy. A study showed that 6% patients who received cystectomy underwent an excessive removal of ovarian tissues [31]. Although an excessive removal of ovarian tissue could effectively diminish the recurrent rate, it may also accelerate the reduction of ovarian reserve [32]. The damage level in ovarian reserve by laparoscopic cystectomy depends on the surgeon's skill [33]. Therefore, improvements of surgeons' skill and novel surgical techniques such as robotic assisted cystectomy or single-port cystectomy could effectively preserve normal ovarian tissues [34,35]. In addition, the influence of cystectomy on ovarian function is reported to correlate to basal AMH level and bilateral operation, not size of cyst [36].
This study has some limitations. Firstly, the sample size is small. Secondly, it lacked a long-time follow-up in ovarian reserve. Studies have shown a recovery of ovarian reserve in 2 or 3 months after hysterectomy, myomectomy, or cystectomy surgery [23,28,30]. Hence, it's foreseeable to follow monitor serum AMH concentration in at least 3 months after surgery to evaluate the long-term effect on ovarian reserve of the four surgeries.

Conclusion
All four laparoscopic surgeries impair ovarian function in one month after surgery. Adnexectomy is an independent factor of reduced ovarian reserve in 1 month after surgery. It is necessary to make a prudent decision when selecting surgical treatment for perimenopausal women with uterine fibroids or ovarian cysts.

Disclosure statement
No potential conflict of interest was reported by the authors.