Scholarly article on topic 'Evaluation of ultrasonographic and Anti-Müllerian Hormone (AMH) changes as predictors for ovarian reserve after laparoscopic ovarian drilling for women with polycystic ovarian syndrome'

Evaluation of ultrasonographic and Anti-Müllerian Hormone (AMH) changes as predictors for ovarian reserve after laparoscopic ovarian drilling for women with polycystic ovarian syndrome Academic research paper on "Veterinary science"

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Abstract of research paper on Veterinary science, author of scientific article — Emad M. Seyam, T.G. Mohamed, Momen M. Hasan, Marwa H. Abd Al Mawgood

Abstract Background and aim of the study Laparoscopic ovarian drilling (LOD) is considered one of the effective tools for treatment of women with polycystic ovarian syndrome. The aim of this work was to evaluate the effects of laparoscopic ovarian drilling on ovarian reserve assessed by Anti-Müllerian hormone assay and ultrasonography. Study design The current prospective controlled study included 70 (n =70) primary anovulatory women with PCOS who are allocated to undergo LOD (n =40), who are clomiphene citrate (CC) resistant, and another group receiving incremental doses (50–150mg) of CC (n =30) and 20 healthy age-matched women with a regular menstrual cycle and normal ovaries (confirmed with ultrasound examination) as the control group. Materials and methods For the LOD group, standard laparoscopy ovarian drilling was done just after the end of the menstruation using a monopolar electrocautery needle. For the CC group, clomiphene citrate was given in incremental doses (50–150mg) for up to six cycles from days 2–6 of a menstrual cycle or after a progestin withdrawal bleeding. Evaluation for the ovarian reserve was done using the following tests: (1) AMH level in the third cycle day, (2) antral follicle count (AFC) by TVS, and (3) summed ovarian volume (SOV) by transvaginal ultrasound examination (TVS). Results In the current study, among the (40) PCOS women having LOD, 30/40 (75%) had regular cycles in the 6-month period after LOD, while the ovulation rate was 24/40 (60%) and the pregnancy rate was 11/40 (27.5%) in the 6-month period after LOD. Those rates were not statistically different in the PCO women group treated with CC. There were statistically significant differences between AMH levels and antral follicle count before and after LOD (p =0.001 and 0.009 respectively) and this might indicate a possible diminished ovarian reserve. Conclusion AMH and AFC are reliable markers for assessment of the ovarian reserve and measuring them for women with anovulatory PCOS undergoing LOD may provide a useful tool in evaluating the outcome of LOD.

Academic research paper on topic "Evaluation of ultrasonographic and Anti-Müllerian Hormone (AMH) changes as predictors for ovarian reserve after laparoscopic ovarian drilling for women with polycystic ovarian syndrome"

Middle East Fertility Society Journal (2014) xxx, xxx-xxx

Middle East Fertility Society Middle East Fertility Society Journal

www.mefsjournal.org www.sciencedirect.com

ORIGINAL ARTICLE

Evaluation of ultrasonographic and Anti-Miillerian Hormone (AMH) changes as predictors for ovarian reserve after laparoscopic ovarian drilling for women with polycystic ovarian syndrome

Emad M. Seyam *, T.G. Mohamed, Momen M. Hasan, Marwa H. Abd Al Mawgood

El Minya University Maternity Hospital, College of Medicine, El Minay University, Egypt Received 5 May 2013; revised 16 February 2014; accepted 28 February 2014

KEYWORDS

Polycystic ovarian syndrome; Laparoscopic ovarian drilling;

Ovarian reserve; Anti-Mullerian Hormone; Clomiphene citrate

Abstract Background and aim of the study: Laparoscopic ovarian drilling (LOD) is considered one of the effective tools for treatment of women with polycystic ovarian syndrome. The aim of this work was to evaluate the effects of laparoscopic ovarian drilling on ovarian reserve assessed by Anti-Miillerian hormone assay and ultrasonography.

Study design: The current prospective controlled study included 70 (n = 70) primary anovulatory women with PCOS who are allocated to undergo LOD (n = 40), who are clomiphene citrate (CC) resistant, and another group receiving incremental doses (50-150 mg) of CC (n = 30) and 20 healthy age-matched women with a regular menstrual cycle and normal ovaries (confirmed with ultrasound examination) as the control group.

Materials and methods: For the LOD group, standard laparoscopy ovarian drilling was done just after the end of the menstruation using a monopolar electrocautery needle. For the CC group, clomiphene citrate was given in incremental doses (50-150 mg) for up to six cycles from days 2-6 of a menstrual cycle or after a progestin withdrawal bleeding. Evaluation for the ovarian reserve was done using the following tests: (1) AMH level in the third cycle day, (2) antral follicle count (AFC) by TVS, and (3) summed ovarian volume (SOV) by transvaginal ultrasound examination (TVS).

Results: In the current study, among the (40) PCOS women having LOD, 30/40 (75%) had regular cycles in the 6-month period after LOD, while the ovulation rate was 24/40 (60%) and the pregnancy rate was 11/40 (27.5%) in the 6-month period after LOD. Those rates were not statistically different in the PCO women group treated with CC. There were statistically significant differences between AMH levels and antral follicle count before and after LOD (p = 0.001 and 0.009 respectively) and this might indicate a possible diminished ovarian reserve.

Corresponding author. E-mail address: eemsalah@yahoo.com (E.M. Seyam). Peer review under responsibility of Middle East Fertility Society.

1110-5690 © 2014 Production and hosting by Elsevier B.V. on behalf of Middle East Fertility Society. http://dx.doi.org/10.1016/j.mefs.2014.02.004

Conclusion: AMH and AFC are reliable markers for assessment of the ovarian reserve and measuring them for women with anovulatory PCOS undergoing LOD may provide a useful tool in evaluating the outcome of LOD.

© 2014 Production and hosting by Elsevier B.V. on behalf of Middle East Fertility Society.

1. Introduction

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder affecting 5-10% of women of reproductive age. It is the major cause of ovulation-related infertilities, accounting for at least 75% of cases with anovulatory infertility. In women with polycystic ovary syndrome (PCOS) who do not ovulate when treated with clomiphene citrate (CC), ovulation induction with gonadotrophins and laparoscopic electrocau-tery of the ovaries are established second line treatments with comparable success rates (1-3).

Surgical therapy with laparoscopic ovarian 'drilling' may avoid or reduce the need for gonadotrophins or may facilitate their use. However, the procedure, though effective, can be traumatic on the ovaries, which may cause postoperative adhesions and/or diminished ovarian reserve (DOR). Although the available data in the literature are limited, there was no concrete evidence of a decreased ovarian reserve (DOR) or premature ovarian failure (POF) associated with LOD in women with PCOS (4-6).

The ovarian reserve is related to the size, number, and quality of oocytes within follicles. To assess the ovarian reserve, measuring serum level of FSH, Inhibin B and E2 in the follic-ular phase is a useful tool. One of the best ovarian reserve determining factors is antral follicle count which is assessed by transvaginal ultrasound tests during the follicular phase. Within the last years, serum AMH measurement has been introduced as one of the best and easiest markers of the ovarian reserve (7-10).

Serum AMH evaluation is a practical marker in infertility as it assesses the ovarian reserve and recognizes women with poor fertility. Evaluation of AMH serum levels can be used as a predictor of laparoscopic possible damage to the ovarian reserve. AMH levels disappear in cycle bleeding and the whole cycle has minimum intra-cycle changes. Women with PCOS display elevated circulating AMH levels compared with age and body mass index (BMI)-matched controls with normal menstrual regularity. Furthermore, AMH levels are higher in amenorrheic women with PCOS compared with oligomenor-rhea women with PCOS in conjunction with significantly elevated 2- to 9-mm follicle number per ovary, and AMH has been proposed as a surrogate marker for antral follicle count (AFC) in PCOS (2,4,7,8,11-13).

It has been accepted that AFC may be a good quantitative predictor of the ovarian reserve. The number of antral follicles, which is closely related to reproductive age, could substantially reflect the number of remaining primordial follicles. Therefore, on the one hand, LOD should have some diathermal effect on the ovarian tissue leading to a decrease in AFC and the ovarian reserve. Importantly, on the other hand, the ovarian reserve in PCOS women undergoing LOD remains high (1,3,7,10).

The aim of the current study was to evaluate the subsequent effects of laparoscopic ovarian drilling on the ovarian reserve,

using the ultrasonographic and AMH changes developing after the procedure, in addition to studying some pre-operative relevant factors, which might be used as predictors for LOD prognosis.

2. Materials and methods

The current prospective controlled study included 70 (n = 70) primary anovulatory women, diagnosed as PCOD, where 40 (n = 40) are clomiphene-resistant PCOS (who had failed to ovulate before and after maximum CC dose for at least 3 cycles), allocated to undergo LOD, and another group receiving incremental doses of 150 mg of CC (n = 30). It was conducted between May 2011 and November 2013 and all enrolled women were recruited from the attendants of the Out patient Clinic and Endoscopy Unit in a tertiary referral. The study was approved by the Local Ethics Committee for Research. All women were informed about the study and a detailed written informed consent was taken from all participants before being included in the study.

The age of the included subjects was between 25 and 40 years with primary anovulatory infertility of one year duration or more, with a BMI between 20 and 30 kg/m2. PCOS women were diagnosed according to the 2003 ESHRE/ASRM (Rotterdam) criteria. All women included in this study should have normal HSG and their partners had normal semen analysis according to WHO criteria. The Rotterdam consensus expanded the diagnostic criteria to include at least two of the following three features: (a) oligo- or anovulation, (b) clinical and/or biochemical signs of hyperandrogenism, or (c) polycys-tic ovaries by sonographic features (4,5).

Irregular menstruation was defined as oligomenorrhea (cycle lasting longer than 35 days) or amenorrhea (absence of menstrual cycle for 3 cycles or more). The most common clinical manifestation of hyperandrogenism in women was hir-sutism (assessed with hirsutism score), followed by ovulatory and menstrual irregularity, acne and male-pattern alopecia. The biochemical evidences of hyperandrogenism include elevated serum LH (p10IU/l), elevated LH/FSH ratio (P2) and/or raised serum concentrations of androgens (testosterone P 2.6 nmol/l) or free androgen index (FAI) > 5.

ASRM/ESHRE consensus defines sonographic features of polycystic ovaries if at least one of the ovaries has at least one of the following: either 12 or more subcortical follicles (2-9 mm in diameter) with dense stroma, or increased ovarian volume (>10 cm3). If there is a follicle >10 mm in diameter, the scan should be repeated at a time of ovarian quiescence in order to calculate volume and area.

The subjects were composed of three groups as follows: (1) LOD group (PCOS women who underwent LOD): Forty (40) primary anovulatory women with clomiphene-resistant PCOS undergoing laparoscopic ovarian drilling were enrolled in this group. (2) CC group (PCOS women who received CC): Thirty (30) primary anovulatory women with PCOS who received

clomiphene citrate in incremental doses (50-150 mg). (3) Control group (non-PCOS women with normal menstruation): Twenty (20) healthy women with a regular menstrual cycle and normal ovaries (by ultrasound examination) were enrolled as controls. These women were age-matched with other study groups.

Those with other aetiologies of hyperandrogenism (congenital adrenal hyperplasia, androgen secreting tumours and Cushing's syndrome) had been excluded, patients with infertility due to any factor other than anovulation (e.g. tubal or male factors), and with no history of ovarian surgery or pathology detected by TVS, or any organic pelvic diseases at laparoscopy or diseases potentially affecting the ovarian environment and/ or function (including endometriosis and leiomyomas) or women with a single ovary, or women with any factor affecting OR (like smokers, current metformin users) had been excluded.

All enrolled women were subjected to full detailed history either medical, surgical, past history, obstetric history or sexual history with a special stress on menstrual pattern, fertility status, any previous investigations or treatment given to the patient for PCOS. Thorough general examination was done with special stress on anthropometric measurements and clinical manifestations of hyperandrogenism. On day three of the natural cycle or after progestin induced menstruation, anthropometric measurements were obtained from all women of all three groups.

Transvaginal ultrasound examination (TVS) was done using a 7.5-mHz transducer (TOSHIBA SSA 270 AUS machine, Toshiba Co., Tokyo, Japan) for all women of all three study groups. Transvaginal ultrasound examination was done to confirm the presence of the diagnostic ultrasound criteria of PCOS, to evaluate endometrial thickness, ovarian volume, to check the antral follicle count, to exclude other pelvic pathology and to confirm the diagnosis of pregnancy. The TVS was performed by the same person for all enrolled women of the three groups in the early follicular. TVS was done before LOD then 3 and 6 months after LOD.

2.1. Procedure for measuring the AFC

Antral follicles were defined as all echo lucent rounded structures measuring 2-10 mm seen within the ovarian substance. Serial scans were obtained by making a slow sweep with the transvaginal probe from the medial toward the lateral border of the ovary in two perpendicular planes. The procedure was repeated on the contra lateral ovary to obtain the total Antral follicle count (AFC) defined as the count of all antral follicles measuring 2-10 mm in both ovaries at the baseline examination session. Ovarian volume = length x width x thickness x 0.523. The procedure was repeated on the contra lateral ovary and the sum of volumes of both ovaries was calculated giving the total summed ovarian volume (SOV).

2.2. Blood sample collection

Blood samples were collected before and one week after LOD to measure plasma concentrations of AMH, LH, FSH, testosterone, sex hormone binding globulin (SHBG) and other hormones. Further blood samples were collected 3 and 6 months after LOD for the hormonal assays. Similarly in

women receiving clomiphene citrate, hormonal assays were measured before treatment, on cycle Day 2 of the following menstrual cycle, and at 3- and 6-month follow-up. The presence of hyperprolactinemia was excluded with a single assay of plasma prolactin (PRL) levels (normal values < 25 ng/ ml). Non classical congenital adrenal hyperplasia was excluded with a single measurement of serum 17-hydroxyprogesterone (17-OHP) levels (normal value < 1.98 ug/l).

Plasma samples were assayed for AMH in duplicate using a commercial enzyme-linked immunosorbent assay kit (Immunotech, Beckman-Coulter UK Ltd, High Wycombe, Buckinghamshire, UK) according to the manufacturer's protocol. The sensitivity of the assay was 0.24 ng/ml. The intra- and inter-assay variabilities were <5% and 8%, respectively. In each woman, the free androgen index (FAI) was calculated using the following formula:

FAI = 100 x

Total Testosterone SHBG

2.3. Ovulation induction by CC and LOD

For CC group, clomiphene citrate was given in 150 mg dose per day for up to six cycles starting in the second day and for 5 days of a menstrual cycle or after a progestogen withdrawal bleeding. Laparoscopic ovarian electrocautery was done just after the end of the menstruation. LOD was carried out by a conventional 10 mm Storz laparoscopic equipment using a monopolar electrocautery needle. Four (4-6) punctures were made per ovary at a power setting of 30 W applied for 5 s per puncture.

2.4. Detailed surgical procedure of laparoscopic electrocautery

The same experienced operator performed the laparoscopic procedures during the early post-menstrual phase (spontaneous or withdrawn). The routine tubal patency testing with methylene blue was followed for all patients. The ovarian ligament is grasped with a traumatic grasping forceps introduced through second puncture; the ovary is moved to the front of the body of the uterus or toward the anterior abdominal wall to prevent injury of the pelvic organs.

The ovary was fixed away from intestine by grasping the ovarian ligament with a traumatic grasper introduced through one of the 5 mm ports. The drilling needle was introduced through the other 5 mm port connected to at monopolar current. The needle was held against the ovarian surface with gentle pressure for approximately 4-5 s using a power of 30 W; the number of drills for each ovary ranged from 4 to 6 according to the size of the ovary. The number of drills was the same for right and left ovaries in each patient. The ovary was cooled after each drill by lactated Ringer's solution.

At the end of the procedure, repeated suction irrigation of the pelvis with lactated Ringer's solution was done and 500 cc was left intraperitoneally. Duration of surgical procedure, blood loss, and hospital stay were recorded. All intra- and postoperative complications were carefully recorded for each group. Based on clinical conditions, patients were discharged either on the same day of the surgical procedure or one day postoperatively.

2.5. Clinical and reproductive outcome

All Patients were followed up until they successfully conceived or for up to 6 month period. The main outcome measures included menstrual pattern, ovulation rate, pregnancy rate and the ovarian reserve. Evaluation of those parameters was done at the beginning of the study, one week after treatment and at 3 and 6 month follow up periods.

Follicular monitoring was evaluated by transvaginal U/S measurements. The scans were performed by the same experienced examiner at three day interval, starting on day 7 (during the first month after surgery), and subsequently on the second day after the onset of menses. When the follicular dimensions achieved at least 16 mm, the TVS started to be performed daily. The day of ovulation had been defined retrospectively with the observation of follicular collapse and of appearance of free fluid in the cul-de-sac.

Pregnancy was diagnosed by positive quantitative of b-hCG serum level (more than 50 IU/ml or a rising titer is diagnostic as well) and the appearance of a gestational sac by transvaginal ultrasound examination. The ovarian reserve was evaluated using the following markers: (1) AMH in the third cycle day (2) antral follicle count (AFC) by TVS, summed ovarian volume (SOV) by TVS.

2.6. Statistical analysis

Statistical analysis was done using Statistical Package for Social Sciences Software (for Windows 17.0, SPSS Inc. Chicago, IL, USA). Data were presented as numbers and percent for non-parametric variables and as mean ± SEM (standard error of the mean) for parametric variables. For comparison between parametric data, unpaired t-test was used to compare between two independent study groups and ANOVA was used to compare between more than two groups. For non-parametric data, Mann-Whitney U-test test was used to compare between two independent study groups and Wilcoxon signed-ranks test was used to compare between more than two groups. Comparisons of categorical data were carried out using v2 (Chi-square test). Spearman's rank correlation was used to test correlations between plasma AMH and clinical, hormonal and sonographic variables. p value <0.05 was considered statistically significant.

3. Results

Table 1 summarizes the baseline clinical characteristics the 70 enrolled women with anovulatory PCOS (LOD and CC groups) included in this study compared with another 20 age-matched healthy women with a regular menstrual cycle as a control group. There were no statistical significant differences in age, body mass index, waist/hip ratio, age at menarche and pattern of menstruation among the two study groups. Acne, seborrhea and hirsutism were more present in PCOS groups than the control group (p = 0.008, p = 0.04 and p = 0.007, respectively).

Table 2 summarizes the baseline biochemical assays of the study groups. Baseline total testosterone levels were significantly higher in the LOD and CC groups than in the control group (p = 0.0004, p = 0.01, respectively) without a statistically significant difference found between the LOD and CC groups (p = 0.542). Baseline androstenedione levels were significantly higher in the LOD and CC groups than in the control group (p = 0.0001, p = 0.0001, respectively) but no statistically significant difference was found between LOD and CC groups (p = 0.108). No statistically significant differences were found between groups as regards baseline DHEAS or SHBG levels. Baseline free androgen index (FAI) was significantly higher in the LOD and CC groups than in the control group (p = 0.0001, p = 0.0001, respectively) but without a statistically significant difference found between LOD and CC groups (p = 0.287).

No statistically significant differences were found between all included groups regarding the baseline estradiol (E2) levels. Baseline FSH, LH, and the LH:FSH ratio were significantly higher in the LOD and CC groups than in the control group but no statistically significant difference was found between LOD and CC groups. Baseline AMH levels were significantly higher in the LOD and CC groups than in the control group (p = 0.0001, p = 0.0001, respectively) but without a statistically significant difference found between LOD and CC groups (p = 0.527).

Table 3 summarizes the baseline sonographic findings of the study groups. Mean AFC was significantly higher in the LOD and CC groups than in the control group (p = 0.014, p = 0.009, respectively) but no statistically significant difference was found between LOD and CC groups (p = 0.724).

Table 1 Baseline clinical characteristics of patients.

Baseline patients' characteristics LOD group (n = 40) CC group (n = 30) Control group (n = 20) p value

Age (years) 31.6 ± 4.5 30.9 ± 4.1 32.8 ± 4.2 0.315

BMI (kg/m2) 27.1 ± 5.2 26.8 ± 6.7 24.6 ± 5.4 0.271

WHR 0.86 ± 0.06 0.87 ± 0.1 0.84 ± 0.05 0.375

Age at menarche (years) 14.1 ± 2.3 13.9 ± 2.7 13.6 ± 2.3 0.755

Menstrual pattern

Oligomenorrhoea 32 (80%) 25 (83.3%) 0 (0%)

Amenorrhea 8 (20%) 5 (16.7%) 0 (0%) 0.723

Acne 16 (40%) 14 (46.7%) 1 (5%) 0.008

Seborrhea 15 (37.5%) 15 (50%) 3 (15%) 0.04

Hirsutism 14 (35%) 11 (36.7%) 0 (0%) 0.007

Data are shown as mean ± SEM (standard error of the mean) or as number (%).

Table 2 Baseline biochemical assays of patients.

Biochemical assays LOD group (n = 40) CC group (n = 30) Control group (n = 20) p value

LOD Vs CC LOD vs controls CC vs controls

Androgens

T (nmol/l) 2.8 ± 0.2 2.7 ± 1.01 1.21 ± 0.4 0.542 0.0004 0.01

A (nmol/L) 11.11 ± 0.26 11.2 ± 0.18 9.1 ± 0.24 0.108 0.0001 0.0001

DHEAS (ig/dl) 134.5 ± 12.2 132.4 ± 11.8 131.1 ± 9.5 0.472 0.279 0.683

SHBG (nmol/l) 42 ± 2.9 43 ± 2.4 44 ± 1.7 0.129 0.07 0.113

FAI 6.6 ± 1.2 6.3 ± 1.1 2.75 ± 0.8 0.287 0.0001 0.0001

E2 (pg/ml) 54.8 ± 10.2 56.5 ± 9.7 56.6 ± 9.4 0.483 0.511 0.971

FSH (IU/ml) 5.4 ± 2.7 5.2 ± 2.1 3.9 ± 2.3 0.737 0.037 0.044

LH (IU/l) 12.6 ± 1.2 12.2 ± 1.5 4.9 ± 1.2 0.219 0.0001 0.0001

LH/FSH ratio 2.3 ± 0.2 2.35 ± 0.4 1.2 ± 0.2 0.495 0.0001 0.0006

AMH (ng/ml) 5.99 + 2.3 5.65 + 2.1 2.52 + 2.1 0.527 0.0001 0.0001

Data are shown as mean ± SEM.

T = testosterone, A = androstenedione, DHEAS = dehydroepiandrosterone sulfate, SHBG = sex hormone binding globulin, FAI = free androgen index, E2 = Estradiol, FSH = follicle-stimulating hormone, LH = lutenizing hormone, AMH = Anti-Müllerian Hormone.

Table 3 Baseline sonographic findings of patients.

Sonographic findings LOD group (n = 40) CC group (n = 30) Control group (n = 20) p value

LOD vs CC LOD vs controls CC vs controls

AFC Summed ovarian volume (cm3) 16.75 ± 3.2 12.2 ± 2.5 17 ± 2.5 12.9 ± 1.7 13 ± 1.4 8.5 ± 1.2 0.724 0.191 0.014 0.007 0.009 0.0001

Data are shown as mean ± SEM (standard error of the mean). AFC = antral follicles count.

There were also differences in the mean summed ovarian volume (cm3) between groups.

Mean summed ovarian volume was significantly higher in the LOD and CC groups than in the control group (p = 0.007, p = 0.0001, respectively) but no statistically significant difference was found between LOD and CC groups (p = 0.191).

Table 4 summarizes the comparison of the ovarian reserve hormonal markers before and after LOD and CC groups. Among the 40 PCOS women having LOD, 30/40 (75%) had regular cycles after treatment, while in the CC group 19/30 (63.33%) women had regular cycles after treatment. These results were not statistically different between the two LOD and CC groups (p = 0.292). The ovulation rate was 24/40 (60%) for women after LOD and 19/30 (63.33%) for women after CC treatment, without a statistically significant difference between both groups (p = 0.777). The pregnancy rate was 11/ 40 (27.5%) for women after LOD and 12/30 (40%) for women after CC treatment, without a statistically significant difference between both groups (p = 0.271).

There were no significant changes in the FSH throughout the follow-up periods after LOD or CC treatment. FSH increased shortly after LOD, and then gradually returned to baseline values but without significant changes. In the CC group, FSH levels slightly increased a week and 3-months after treatment, and then gradually returned to baseline values at 6-months but without significant changes. LH and the LH:FSH ratio decreased significantly a week after LOD and remained low at 3- and 6-month follow-up periods. However, in the

CC group, LH and the LH:FSH ratio did not show significant changes during the same time periods. In the LOD group, estradiol levels (E2) increased slightly during the first week after LOD and then increased at 3- and 6-months of follow-up but without statistical significance. In the CC group, there were no significant changes in the E2 levels between the baseline and 3-months or 6-months periods after treatment.

Following LOD, AMH level significantly decreased after one week and remained low at 3- and 6-month follow-up. In the CC group, there were no significant changes in the AMH levels between the baseline and at 3-month or 6-month periods after treatment (Table 5). There were significant positive correlations between plasma AMH levels and mean summed ovarian volume (r = 0.826; p = 0.001), the mean antral follicles count (r = 0.817; p = 0.001) and the plasma testosterone concentrations (r = 0.856; p = 0.001). No significant correlation was found between plasma AMH and age, BMI, FAI, LH, or FSH and other clinical and hormonal variables (Table 6).

The mean (SOV) was significantly higher in responders than in non-responders before LOD. However, after LOD no significant differences were found. However, after LOD responders had a more significant decrease in mean AFC. Responders had significantly lower testosterone levels in baseline and after LOD in comparison to non-responders. No significant differences were found in DHEAS or SHBG levels before or after surgery in responders compared with non-responders. Responders had significantly lower FAI levels in baseline and after LOD in comparison to non-responders (Table 5).

Table 4 Pre versus post treatment ovarian reserve markers and other reliable hormones in the LOD and CC groups.

Ovarian reserve markers Pre treatment 1-Week post treatment 3-Months post treatment 6-Months post treatment

and other hormones

Mean ± SEM Mean ± SEM p value* Mean ± SEM p value** Mean ± SEM p value***

Ovarian reserve

FSH (IU/ml)

LOD (n = 40) 5.4 ± 2.7 5.7 ± 2.3 0.594 5.5 ± 2.1 0.854 5.45 ± 2.4 0.93

CC (n = 30) 5.2 ± 2.1 5.3 ± 2.2 0.858 5.4 ± 2.3 0.726 5.18 ± 2.2 0.971

LH/FSH ratio

LOD (n = 40) 2.3 ± 0.2 1.44 ± 0.21 0.0001 1.48 ± 0.21 0.0001 1.49 ± 0.33 0.0001

CC (n = 30) 2.35 ± 0.4 2.23 ± 0.33 0.209 2.2 ± 0.16 0.0611 2.34 ± 0.22 0.905

E2 (pg/ml)

LOD (n = 40) 54.8 ± 10.2 58.6 ± 8.5 0.074 63.2 ± 9.1 0.321 64.6 ± 9.7 0.523

CC (n = 30) 56.5 ± 9.7 58.1 ± 9.4 0.519 58.6 ± 9.5 0.4 59.1 ± 10.1 0.313

AMH (ng/ml)

LOD (n = 40) 5.99 + 2.3 3.4 + 1.7 0.0001 3.2 + 1.7 0.0001 3.1 + 1.5 0.0001

CC (n = 30) 5.65 + 2.1 5.5 + 1.9 0.773 5.4 + 1.6 0.606 5.1 + 1.7 0.269

LOD (n = 40) 16.75 ± 3.2 14.2 ± 2.8 0.0002 12.5 ± 2.6 0.0001 12.2 ± 1.6 0.0001

CC (n = 30) 17 ± 2.5 16.8 ± 2.4 0.753 16.6 ± 2.2 0.513 16.5 ± 2.3 0.423

Summed ovarian volume (cm3)

LOD (n = 40) 12.2 ± 2.5 11.5 ± 2.1 0.741 11.2 ± 1.8 0.755 11.2 ± 1.6 0.781

CC (n = 30) 12.9 ± 1.7 12.8 ± 1.5 0.811 12.6 ± 1.6 0.484 12.4 ± 1.5 0.232

Other hormones

T (nmol/l)

LOD (n = 40) 2.8 ± 0.2 1.5 ± 0.1 0.0001 1.56 ± 0.2 0.0001 1.58 ± 0.1 0.0001

CC (n = 30) 2.7 ± 1.01 2.65 ± 1.1 0.858 2.62 ± 1.1 0.77 2.67 ± 0.8 0.672

A (nmol/l)

LOD (n = 40) 11.11 ± 0.26 9.2 ± 0.3 0.0001 9.1 ± 0.2 0.0001 8.5 ± 0.25 0.0001

CC (n = 30) 11.2 ± 0.18 11.1 ± 0.3 0.123 10.88 ± 1.1 0.121 11 ± 1.3 0.408

DHEAS (ig/dl)

LOD (n = 40) 134.5 ± 12.2 112.5 ± 10.2 0.0001 114.1 ± 11.5 0.0001 115.2 ± 10.3 0.0001

CC (n = 30) 132.4 ± 11.8 134 ± 12.1 0.606 133.6 ± 11.4 0.69 133.1 ± 11.2 0.814

SHBG (nmol/l)

LOD (n = 40) 42 ± 2.9 33.33 ± 3.1 0.0001 37.1 ± 2.6 0.0001 33.2 ± 4.5 0.0001

CC (n = 30) 43 ± 2.4 42.7 ± 2.5 0.637 42.6 ± 2.4 0.521 44.8 ± 2.6 0.007

LOD (n = 40) 6.6 ± 1.2 4.5 ± 0.8 0.0001 4.2 ± 0.4 0.0001 2.5 ± 0.2 0.0001

CC (n = 30) 6.3 ± 1.1 6.2 ± 1.2 0.738 6.15 ± 1.1 0.599 5.8 ± 1.1 0.083

LH (IU/ml)

LOD (n = 40) 12.6 ± 1.2 8.2 ± 1.3 0.0001 8.13 ± 1.4 0.0001 8.12 ± 1.4 0.0001

CC (n = 30) 12.2 ± 1.5 11.8 ± 1.4 0.289 11.9 ± 1.6 0.457 12.1 ± 1.3 0.783

p value is for degree of significance between groups..

Data are shown as mean ± SEM. FSH = follicle-stimulating hormone, E2 = estradiol, AMH = Anti-Müllerian Hormone, AFC = antral

follicles count, T = testosterone, A = = androstenedione, DHEAS = dehydroepiandrosterone sulfate, SHBG = Sex Hormone Binding Glob-

ulin, FAI = free androgen index, LH [ = lutenizing hormone,

Pre treatment vs 1 week post treatment.

Pre treatment vs 3-months post treatment.

Pre treatment vs 6-months post treatment.

4. Discussion

The aim of the present study was to evaluate the effects of laparoscopic ovarian drilling on the ovarian reserve assessed by Anti-MUllerian Hormone (AMH) assay and ultrasonography changes in women with polycystic ovary syndrome. All patients were followed up after treatments until they conceived or for up to a period of 6 months. Main outcome measures

included menstrual pattern, ovulation rate, pregnancy rate and the ovarian reserve. Evaluation was done at the beginning of the study, 1 week after treatment and at 3 and 6 month follow up periods.

In the current work, among the (40) PCOS women having LOD, 30/40 (75%) had regular cycles in the 6-month period after LOD. The ovulation rate was 24/40 (60%) and the pregnancy rate was 11/40 (27.5%) in the 6-month period after

Table 5 Comparison between responders and non-responders to LOD regarding the pre and post treatment ovarian reserve markers and other reliable hormones.

Pre-LOD Post-LOD p value

Ovarian reserve markers and other hormones FSH (IU/ml) Responders (n = 24) 5.7 ± 2.6 Non-responders (n = 16) 5.1 ± 1.7 p value 0.421 5.8 ± 2.8 5.5 ± 2.3 0.724 0.898 0.579

LH/FSH ratio Responders (n = 24) Non-responders (n = 16) p value 3.03 ± 0.6 1.92 ± 0.3 0.009 1.95 ± 0.4 2.67 ± 0.5 0.0001 0.0001 0.0001

E2 (pg/ml) Responders (n = 24) Non-responders (n = 16) p value 60.7 ± 12.7 58.4 ± 14.6 0.56 64.7 ± 14.9 67.5 ± 13.4 0.127 0.219 0.075

AMH (ng/ml) Responders (n = 24) Non-responders (n = 16) p value 5.5 ± 2.4 8.7 ± 3.2 0.0001 4.2 ± 1.8 9.4 ± 3.3 0.025 0.04 0.546

AFC Responders (n = 24) Non-responders (n = 16) p value 17.4 ± 3.5 12.4 ± 2.1 0.02 10.1 ± 1.8 12.2 ± 2.7 0.005 0.005 0.816

Summed ovarian volume (cm3) Responders (n = 24) Non-responders (n = 16) p value 12.6 ± 2.6 9.5 ± 2.4 0.004 10.5 ± 1.4 8.9 ± 2.1 0.115 0.346 0.456

Other hormones T (nmol/l) Responders (n = 24) Non-responders (n = 16) p value 2.4 ± 1.6 3.6 ± 1.8 0.03 1.5 ± 1.2 2.8 ± 1.5 0.004 0.0.03 0.181

A (nmol/L) Responders (n = 24) Non-responders (n = 16) p value 10.12 ± 1.7 11.9 ± 1.9 0.003 9.1 ± 1.4 11.1 ± 1.6 0.0001 0.03 0.206

DHEAS (ig/dl) Responders (n = 24) Non-responders (n = 16) p value 135.1 ± 13.2 130.2 ± 12.3 0.241 137.73 ± 16.3 135.4 ± 14.6 0.651 0.546 0.283

SHBG (nmol/l) Responders (n = 24) Non-responders (n = 16) p value 41.5 ± 2.8 42.2 ± 3.1 0.462 40.8 ± 2.2 41.3 ± 2.7 0.524 0.34 0.387

FAI Responders (n = 24) Non-responders (n = 16) p value 5.8 ± 1.4 8.5 ± 1.7 0.0001 3.7 ± 1.1 6.8 ± 1.6 0.0001 0.0001 0.006

LH (IU/ml) Responders (n = 24) Non-responders (n = 16) p value 17.3 ± 5.1 9.8 ± 2.3 0.02 11.3 ± 3.5 14.7 ± 3.2 0.003 0.0001 0.0001

Data are shown as mean ± SEM. FSH = follicle-stimulating hormone, E2 = estradiol, AMH = Anti-Müllerian Hormone, AFC = antral follicles count, T = testosterone, A = androstenedione, DHEAS = dehydroepiandrosterone sulfate, SHBG = Sex Hormone Binding Globulin, FAI = free androgen index, LH = lutenizing hormone. * Pre LOD vs post LOD. Responders vs non-responders.

Table 6 Spearman's rank correlations between plasma AMH and other clinical, hormonal and sonographic variables in women with PCOS.

Correlation coefficient (r) p value

Age 0.154 0.921

BMI -0.101 0.895

FSH 0.188 0.940

LH/FSH ratio 0.124 0.756

E2 0.241 0.689

AFC 0.817 0.001

Summed ovarian volume 0.826 0.001

T 0.856 0.001

A 0.423 0.083

DHEAS 0.102 0.866

SHBG 0.111 0.874

FAI 442 0.075

LH 0.261 0.785

FSH = follicle-stimulating hormone, E2 = estradiol,

AMH = Anti-Mullerian Hormone, AFC = antral follicles count, T = testosterone, A = androstenedione, DHEAS = dehydroepi-androsterone sulfate, SHBG = sex hormone binding globulin, FAI = free androgen index, LH = lutenizing hormone.

LOD. These results are in agreement with previous studies (15-20). Several studies described the success and utility of this procedure, with ovulation rates ranging from 64% to 92% and pregnancy rates from 41% to 80%21 (5-9,14-17).

In the present study, 29/40 (72.5%) women with PCOS were still resistant to LOD and did not conceive despite high ovulation rate being observed. A possible explanation is that the amount of ovarian tissue destroyed during LOD was not enough to induce favorable changes on reproductive parameters in some patients such as intra-ovarian AMH levels. Another cause may be hyperprolactinemia observed in some (8 women) patients after LOD, which had been significantly improved 6 months after LOD. Therefore it is important to monitor the patients for prolactin levels after LOD.

Our study confirmed no significant changes in the serum concentration of FSH throughout the follow-up periods after. FSH concentrations increased shortly after LOD, and then gradually returned to baseline values but without significant changes. In addition, no significant differences in FSH levels were found between pre and post LOD either in responders or non-responders. These findings are in agreement with previous reports. Estradiol levels (E2) increased slightly during the first week after LOD and then increased at 3- and 6-months of follow-up but without statistical significance, without a significant difference in E2 levels between pre and post LOD either in responders or non-responders. These findings are in agreement with many previous studies (8-12,18-21).

Our study results demonstrated a mean summed ovarian volume in cm3 was decreased after one week and remained low for 3-6-months follow-up after LOD. Those findings are in agreement with many previous studies. Another study reported on the short-term effect of ovarian drilling on the ovarian volume as measured by three-dimensional (3D) ultrasound. They found that ovarian drilling resulted in a transient increase followed by a significant reduction in ovarian volume from a pre-operative mean value of 12.2-6.9 ml 3 weeks after surgery (11-15,19-23).

Our study outcomes showed that no significant differences in mean summed ovarian volume (cm3) were found between responders and non-responders after LOD. These findings are consistent with many studies that did not detect a significant difference in ovarian volume between normal and poor responders in women aged less than 37 years and when evaluating women at high risk for cancellation of assisted reproduction cycles. A recent systematic review concluded that ovarian volume has little clinical application in prediction of poor pregnancy response.

However, another recent review commented on the value of ovarian volume with regard to its easy execution, and therefore could be included in preparatory protocols providing data for continuity of research (9-15,24-26).

The relative contribution of each individual measure of the ovarian reserve is clearer and most authors agree that antral follicle counts and serum Anti-Mullerian Hormone levels have the most discriminative. Antral follicle counts are easy to perform and cheap in comparison as all units have access to ultrasound facilities. Follicle counts, as a quantitative measure of the ovarian reserve, are also subject to 'assay' variation due to intra- and inter-observer differences and require additional time and manpower to perform.

In the current work, the mean AFC was significantly decreased after one week and remained low at 3- and 6-month follow-up after LOD. However, after LOD responders had a more significant decrease in mean AFC. These findings were similar to those of other previous studies (7-10,24-27).

It has been accepted that AFC may be a good quantitative predictor of the ovarian reserve. The number of antral follicles, which is closely related to reproductive age, could substantially reflect the number of remaining primordial follicles. Therefore, on the one hand, LOD should have some diathermal effect on the ovarian tissue leading to a decrease in AFC and the ovarian reserve. Importantly, on the other hand, the ovarian reserve in PCOS women undergoing LOD remains high.

AMH is considered to be a marker that can estimate the quantity and activity of recruitable follicles in early stages of growth, thus being more reliable for the prediction of the ovarian reserve. Studies have shown reduced variability in the levels of AMH as compared to other endocrine markers of the ovarian reserve (15-17,22-26).

The current research showed that the baseline AMH levels were not statistically significant difference was found between LOD and CC groups. These results of significant high levels of plasma AMH in women with anovulatory PCOS compared with healthy controls were confirmed by previous reports. Other previous studies showed that women with PCOS have 2-3 times increased level of the serum AMH concentration which was related to increment in the number of small follicles (13-18,25-27).

The developed results showed that, responders (ovulating women) to LOD had a significantly lower pre-operative AMH levels compared with the non-responders. After LOD, a significant reduction in AMH levels occurred in responders to LOD. These findings are in agreement with previous studies. Several previous studies on the ovarian reserve have shown low AMH levels to be associated with poor ovarian responsiveness to ovarian hyperstimulation in IVF programmes in women without PCOS (10-16,22-26).

In the current work, there was a significant positive correlation between plasma AMH levels and both mean summed

ovarian volume and mean antral follicle count. These results are in agreement with previous studies. These findings may be explained by the fact that ovarian volume is a reflection of the number of small antral follicles present in PCOS, which are the only source of AMH.

On the other hand, there were no significant correlations between plasma AMH and age, BMI, FAI, LH, or FSH and other clinical and hormonal variables in the present study. This may be possibly attributed to the small age range included in the study, the small study samples and the strict inclusion criteria (Figs. 1 and 2).

The present research work confirmed a significant positive correlation between plasma AMH levels and plasma testosterone concentrations. This is in agreement with many previous studies. Another study also showed that this correlation is specific to PCOS because it was not observed in a control group of women without PCO. This positive association between testosterone and AMH could be explained by the stimulatory effect of androgens on primordial follicular growth and granulose cell proliferation, which in turn could increase AMH secretion, or the inhibitory effect of AMH on aromatase activity, resulting in an increase in androgens (12-18,25-27).

In the present study, mean levels of androgens (especially serum testosterone, androstenedione, and FAI) and LH levels had a significant high decrease after one week and remained low at 3- and 6-month follow-up after LOD. Also, the serum levels of LH and the LH:FSH ratio decreased significantly 1-week after LOD and remained low during the 3- and 6-month follow-up periods. The reduction of androgens and LH levels after LOD has been reported by other investigators.

Our study outcome showed that, responders had significantly lower androgen level (except DHEA S) than non-responders in baseline and after LOD. These findings are in agreement with many other articles. Whereas many studies found no difference in baseline and post-treatment androgen levels between responders and non-responders, or between obese and lean patients. Only one report noted higher testosterone values post-operatively (21-25).

The aforementioned results of the present study showed that there were statistically significant differences between AMH levels and AFC before and after LOD. Although the after LOD values were found to be lower than the before

AMH (ng/iri)

Figure 1 Correlations between plasma concentrations of AMH and summed ovarian volume in women with PCOS show that there was a positive correlation between the level of AMH in (ng/ ml) and the summed ovarian volume (SOV).

AMH (ng/ml)

Figure 2 Correlations between plasma concentrations of AMH and mean antral follicle count in women with PCOS show that there was a positive correlation between the AMH in (ng/ml) and the AFC in the study.

LOD values by means of ovarian reserve markers, the after values stayed higher than normal when compared with normal women without PCOS. Moreover, the baseline of the ovarian reserve, depicted by histological and hormonal evidence, in PCOS women is much higher than that of women with normal menstruation, as found in other studies (23-26).

Based on previous studies it can be deduced that the LOD had normalized ovarian function which is a significant factor in the follicular recruitment and their maturation. Therefore, LOD has no negative effect on ovarian supply. It seems that damage to the ovarian tissue occurs during and may extend for only a short period of time after the LOD procedure as evidenced by the fact that AMH levels and AFC, which are important parameters, did not correlate with the time since LOD (26,27).

Reduction in the AMH levels may be due to the use of the bilateral diathermy technique. Previous reports showed that the ovarian reserve of patients with PCOS does not change significantly after LOD; therefore, reduction of AMH after LOD might be referred to the normalization of PCO women after the laparoscopic procedure. Therefore, the likeliness of traumatic injury in this form of intervention is zero. Overall, it seems that although LOD leads to the decreased levels of AMH in patients with PCOS, these changes are not statistically significant and only indicate the patient's normality and has no negative impact on ovarian supply (11-22).

As a conclusion for the current study, the aforementioned results of the present study showed that there were statistically significant differences between AMH levels and antral follicle count before and after LOD and it may indicate a possible diminished ovarian reserve. Although the after LOD values were found to be lower than the before LOD values by means of ovarian reserve markers, the after values stayed higher than normal when compared with normal women without PCOS''.

LOD had appeared after the study not to be associated with an increased risk of diminished ovarian reserve. Most of the changes in the ovarian reserve markers raised with the current work after LOD could be interpreted with the normalization of ovarian function in the enrolled PCO women rather than the reduction of the ovarian reserve. The authors of the study could also recommend using the AMH and AFC as reliable markers of the ovarian reserve and measuring them for women

with anovulatory PCOS undergoing LOD may provide a useful tool in evaluating the outcome of LOD as the gold standard treatment for CC resistant PCO women.

Conflict of interest

None declared. Acknowledgment

All authors also are quite thankful and grateful for all colleagues in the University Obstetrics and Gynecology department in El Minya University Hospital for their kind support for this work.

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