Ovarian reserve markers after discontinuing long-term use of combined oral contraceptives.

RESEARCH QUESTION: How early do the ovarian reserve markers anti-Müllerian hormone (AMH) and antral follicle count (AFC) normalize after discontinuation of the long-term use of combined oral contraceptives (COC). DESIGN: This was a prospective cohort study of 68 women with a history of long-term COC use. Serum AMH concentrations, ovarian volume and AFC were measured during COC use and serially in a 4-month period after discontinuing COC: 1 and 2 weeks after discontinuation, and on cycle day 2-5 during three consecutive menstrual cycles. Changes in AMH and AFC were investigated using linear mixed models of repeated measurements adjusted for relevant covariates. RESULTS: Mean age was 29.4 years and mean duration of COC use 8.0 years. Baseline median AMH concentrations during COC use of 13 pmol/l (interquartile range [IQR] 8.4-22 pmol/l) increased to a median of 22.5 pmol/l (IQR 11-37 mol/l) 3 months after discontinuation. The estimated average increase was 53% (95% confidence interval [CI] 1.40-1.68, P < 0.001). AFC increased from a median value of 17 (IQR 11-25) to 24 (IQR 17-34). The estimated average increase was 41% (95% CI 1.30-1.52, P < 0.001). Ovarian volume increased from 2.4 to 5.8 ml (P < 0.001). The ovarian reserve markers increased continuously from baseline measurements until 2 months after discontinuation. Thereafter a plateau was reached. CONCLUSION: After discontinuation of COC, AMH increased by 53% and AFC by 41%, with values returning to normal within 2 months. This study provides clinicians with the highly relevant knowledge that AMH and AFC can be measured 2 months after discontinuation of COC without having to account for their influence.

Contribution of recruitable follicles to circulating anti-Müllerian hormone levels following maximal gonadotrophin stimulation in patients with limited ovarian reserve.

In women, the majority of anti-Müllerian hormone (AMH) measured in serum originate from small antral follicles measuring 2-10 mm. In gonadotrophin-stimulated cycles prior to assisted reproductive technology (ART), most of the recruitable follicles develop beyond 10 mm in size and thus lose their AMH secretion capacity causing declining serum AMH levels. The aim of this study was to define the residual serum AMH level after elimination of the AMH producing recruitable follicles following maximal gonadotrophin stimulation. We measured serum AMH and number of follicles according to size at several time points during a cycle of maximal gonadotrophin stimulation (fixed dose of 300 IE HP-hMG) in 107 women with low AMH (median AMH 5 pmol/L, interquartile range (IQR) 3.3-8.3). We found that AMH decreased gradually and reached a minimum level of -55.4% (95% CI -59.6; -50.7) of the baseline value four days after ovulation trigger. Our findings suggest that the residual AMH production origins from pre-antral and small antral follicles not visible by sonography and that they account for up to 40% of the circulating AMH.

Ovarian cortical follicle density in infertile women with low anti-Müllerian hormone.

PURPOSE: To evaluate the association between anti-Müllerian hormone (AMH) and follicle density in infertile women with diminished ovarian reserve (DOR) versus women with normal ovarian reserve? METHODS: Case-control study comparing follicle densities in ovarian cortex from 20 infertile women with DOR (AMH ≤ 5 pmol/L) and 100 controls with presumed normal ovarian reserve. RESULTS: For all women > 25 years, the follicle densities correlated positively with AMH levels. For each single picomole per liter increase in AMH the follicle density increased by 6% (95% CI 3.3-8.5%) when adjusted for age. This was similar for women with DOR and controls. The follicle density was 1.8 follicles/mm3 cortical tissue in women with DOR versus 7.0 in age-paired controls (p = 0.04). The women with DOR had a median AMH of 1.8 pmol/L versus 14.4 pmol/L in the age-paired control group (p < 0.001). The ratio of AMH/follicle density was 1:1 (1.8/1.8) in women with DOR and 2:1 (14.4/7.0) in the age-paired controls. Analyses for gonadotropin receptor polymorphisms could not explain the characteristics of women with DOR. The proportion of secondary follicles was higher in women with DOR compared with controls (4.6% versus 1.4%, p = 0.0003). Pooling all patients, the follicle density decreased significantly by 7.7% for every year added (p < 0.0001). The women with DOR had lower follicle densities than the controls, but the slopes were equal in the two cohorts. CONCLUSIONS: Follicle density and AMH concentrations correlate also when AMH is low. However, AMH is only a reliable marker for the true ovarian reserve when age is included in the estimation and women with DOR may have more follicles than their AMH levels imply.

Biopsying, fragmentation and autotransplantation of fresh ovarian cortical tissue in infertile women with diminished ovarian reserve.

STUDY QUESTION: Can ovarian biopsying per se and/or autotransplantation of fragmented ovarian cortical tissue activate dormant follicles and increase the number of recruitable follicles for IVF/ICSI in women with diminished ovarian reserve (DOR)? SUMMARY ANSWER: Ovarian biopsying followed by immediate autotransplantation of fragmented cortical tissue failed to increase the number of recruitable follicles for IVF/ICSI 10 weeks after the procedure either at the graft site or in the biopsied ovary, but 12 of the 20 women subsequently had a clinical pregnancy during the 1-year follow-up. WHAT IS KNOWN ALREADY: Infertile women with DOR constitute a group of patients with poor reproductive outcome mainly due to the low number of mature oocytes available for IVF/ICSI. Recent studies have shown that in vitro activation of residual dormant follicles by both chemical treatment and tissue fragmentation has resulted in return of menstrual cycles and pregnancies in a fraction of amenorrhoeic women with premature ovarian insufficiency. STUDY DESIGN, SIZE, DURATION: This is a prospective clinical cohort study including 20 women with DOR treated at the fertility clinic, Rigshospitalet, Denmark, during April 2016-December 2017. Non-pregnant patients were on average followed for 280 days (range 118-408), while women who conceived were followed until delivery. Study follow-up of non-pregnant patients ended in September 2018. PARTICIPANTS, MATERIALS, SETTING, METHODS: The study included infertile women aged 30-39 years with preserved menstrual cycles, indication for IVF/ICSI and repeated serum measurements of anti-Müllerian hormone (AMH) ≤ 5 pmol/L. Patients were randomized to have four biopsies taken from either the left or the right ovary by laparoscopy followed by fragmentation of the cortical tissue to an approximate size of 1 mm3 and autotransplanted to a peritoneal pocket. The other ovary served as a control. Patients were followed weekly for 10 weeks with recording of hormone profile, antral follicle count (AFC), ovarian volume and assessment for ectopic follicle growth. After 10 weeks, an IVF/ICSI-cycle with maximal ovarian stimulation was initiated. MAIN RESULTS AND THE ROLE OF CHANCE: No difference in the number of mature follicles after ovarian stimulation 10 weeks after the procedure in the biopsied versus the control ovaries was observed (1.0 vs. 0.7 follicles, P = 0.35). In only three patients, growth of four follicles was detected at the graft site 24-268 days after the procedure. From one of these follicles, a metaphase II (MII) oocyte was retrieved and fertilized, but embryonic development failed. Overall AMH levels did not change significantly after the procedure (P = 0.2). The AFC increased by 0.14 (95% CI: 0.06;0.21) per week (P < 0.005), and the biopsied ovary had on average 0.6 (95% CI: 0.3;-0.88) follicles fewer than the control ovary (P = 0.01). Serum levels of androstenedione and testosterone increased significantly by 0.63 nmol/L (95% CI: 0.21;1.04) and 0.11 nmol/L (95% CI: 0.01;0.21) 1 week after the procedure, respectively, and testosterone increased consecutively over the 10 weeks by 0.0095 nmol/L (95% CI: 0.0002;0.0188) per week (P = 0.045). In 7 of the 20 patients, there was a serum AMH elevation 5 to 8 weeks after the procedure. In this group, mean AMH increased from 2.08 pmol/L (range 1.74-2.34) to 3.94 pmol/L (range 3.66-4.29) from Weeks 1-4 to Weeks 5-8. A clinical pregnancy was obtained in 12 of the 20 (60%) patients with and without medically assisted reproduction (MAR) treatments. We report a cumulated live birth rate per started IVF/ICSI cycle of 18.4%. LIMITATIONS, REASON FOR CAUTION: Limitations of the study were the number of patients included and the lack of a non-operated control group. Moreover, 9 of the 20 women had no male partner at inclusion and were treated with donor sperm, but each of these women had an average of 6.8 (range 4-9) unsuccessful MAR treatments with donor sperm prior to inclusion. WIDER IMPLICATIONS OF THE FINDINGS: Although 12 out of 20 patients became pregnant during the follow-up period, the current study does not indicate that biopsying, fragmenting and autotransplanting of ovarian cortical tissue increase the number of recruitable follicles for IVF/ICSI after 10 weeks. However, a proportion of the patients may have a follicular response in Weeks 5-8 after the procedure. It could therefore be relevant to perform a future study on the possible effects of biopsying per se that includes stimulation for IVF/ICSI earlier than week 10. STUDY FUNDING/COMPETING INTEREST(S): This study is part of the ReproUnion collaborative study, co-financed by the European Union, Interreg V ÖKS. The funders had no role in the study design, data collection and interpretation, or decision to submit the work for publication. None of the authors have a conflict of interest. TRIAL REGISTRATION NUMBER: NCT02792569.

Autotransplantation of fragmented ovarian cortical tissue: a laparoscopic demonstration.

OBJECTIVE: To describe and demonstrate a simple and secure procedure for laparoscopic autotransplantation of fragmented ovarian cortical tissue in women with diminished ovarian reserve as part of in vitro activation (IVA) of ovarian follicles. DESIGN: Step-by-step video explanation of the surgical procedure with still pictures and surgical video clips to demonstrate the detailed technique. SETTING: Fertility clinic and obstetrics and gynecology department at a university hospital. PATIENT(S): Women with idiopathic diminished ovarian reserve and indication for in vitro fertilization (IVF), aged 25 to 39 years with an antral follicle count bilaterally of ≤5, antimüllerian hormone level of ≤5 pmol/L, and two ovaries. INTERVENTION(S): The laparoscopic autotransplantation consists of six steps: [1] obtaining ovarian cortical biopsy samples, [2] preparing the peritoneal pocket, [3] fragmenting the ovarian cortical tissue into pieces of approximately 1 mm3, [4] installing the tissue fragments into a catheter, [5] transplanting the tissue fragments into the peritoneal pocket, and [6] closing the peritoneal pocket with a surgical clip. After the procedure, the patients are evaluated with blood samples and ultrasound scans followed by controlled ovarian stimulation. Ethics committee approval was obtained. MAIN OUTCOME MEASURE(S): Feasibility of a six-step laparoscopic autotransplantation procedure using fragmented ovarian cortical tissue. RESULT(S): A simple, fast laparoscopic procedure for taking biopsy samples and autotransplanting cortical tissue fragments in an all-in-one procedure ensures the rapid handling and correct placement of the small tissue fragments. The procedure is performed in an outpatient setting with an operation time of 1 hour. We have performed this procedure on 20 patients with no complications. CONCLUSION(S): In vitro activation is a new, developing option for women in fertility treatment who have diminished ovarian reserve. Fragmentation of murine ovarian tissue has shown to suppress the Hippo pathway, thereby initiating proliferation and growth. This surgical procedure resembles that used when transplanting pieces of frozen-thawed ovarian tissue for fertility restoration, but the fragmented ovarian tissue is only 1 mm3, which makes it difficult to transplant. Until now no surgical procedures for transplanting small IVA fragments of cortical tissue has been published. With this video we report in detail a simple way of autotransplanting small fragments of IVA cortical tissue using what is already accessible in the operating theater. Among the many advantages of this procedure are its short duration (1 hour) and outpatient setting, which enable fast recovery and minimal postoperative pain. The procedure also allows fast handling and minimal manipulation of the tissue (limited to the fragmentation). The effect of autotransplantation of fragmented tissue in women with diminished ovarian reserve is currently being studied in ongoing trials. If the technique is combined with chemical IVA, a better outcome may be seen.