Anti-Müllerian Hormone in Fertility Preservation: Clinical andTherapeutic Applications

A prospective randomised controlled study of serum anti-mullerian hormone (AMH) as a predictive marker of ovarian hyperstimulation syndrome (OHSS) in IVF-ICSI cycles

The role of anti-müllerian hormone in the classification of anovulation

Objective To explore the effect on ovarian reserve function and the sensitive marker after laparoscopic operation on unilateral ovarian endometrioma. Methods From February 2013 to October 2015, a total of 115 women with ovarian cyst underwent laparoscopic cystectomy were included into this study. They were divided into two groups according to histopathological analysis, group A (n=65, histopathological analysis showed ovarian follicle) and group B (n=50, histopathological analysis showed without ovarian follicle). The serum follicle-stimulating hormone (FSH), estradiol and anti-Mullerian hormone (AMH) levels before operation and at seventh day, third month and sixth month after operation were detected, meanwhile, antral follicle count (AFC) and ovarian volume before operation and at seventh day, third month and sixth month after operation were measured by ultrasonic examination. Serum FSH and estradiol levels were detected by electrochemiluminescence immunoassay, and serum AMH levels were detected by enzyme-linked immunosorbent assay (ELISA). The study protocol was approved by the Ethical Review Board of Investigation in Human Being of Northern Jiangsu People′s Hospital. Informed consent was obtained from the parents of each participating patients. Results ①There were no significant differences between two groups in the aspects of constitution ratio of older than 35 years old, constitution ratio of ovarian endometriotic cyst diameter≥5 cm, operation duration, bleeding volume, et al (P>0.05). ②The level of serum FSH increased at seventh day after the operation than that before the operation, and the levels of serum estradiol and AMH decreased at seventh day after the operation than that before the operation, which both had significant difference (P 0.05). At the sixth month after the operation, the serum AMH of group A still significant lower than that before the operation (P 0.05). As for group B, there were no significant difference in serum FSH, estradiol and AMH levels compared before the operation(P>0.05). ③ There were no significant differences in serum FSH, estradiol and AMH levels between two groups at every different time points (P>0.05). ④ There were no significant differences between two groups in AFC before operation and at sixth month after the operation (P>0.05). The AFC in group A was less than that of group B at the third month after the operations (P 0.05). Conclusions The ovarian reserve function is influenced after laparoscopic operation on unilateral ovarian endometrioma. The patients with ovarian follicle whose ovarian reserve function was still in the recovery phase at the sixth month after the operation. The serum AMH can be used as a more sensitive marker of ovarian reserve function assessment, it could be a follow-up marker of premature ovarian failure. Key words: Ovarian endometriotic cyst; Laparoscopes; Pathology; Sex hormone; Anti-Mullerian hormone; Women

Fertility preservation in girls with Turner syndrome: limitations, current success and future prospects

Biomarkers of ovarian response: current and future applications

Germ cell ovarian tumors (malignant ovarian germ cell tumors – MOGCT) affect young women and are treated by surgery plus chemotherapy. It is well known that cytotoxic treatment may accelerate depletion of the primordial follicle pool leading to impaired fertility and premature menopause. Aim of this study is to identify patient candidates for fertility preservation strategies. We report our experience in preservation of fertility for four patients affected by MOGCT, referred to San Raffaele Hospital Oncofertility Unit. All patients received fertility sparing surgery plus platinum-based chemotherapy. Two patients were affected by mixed germ cell tumors and two by disgerminomas. After 24 months from the end of treatment, serum AMH levels have been measured. We report lower serum anti-Mullerian hormone (AMH) levels in our patients than in healthy general population as serum AMH levels were under the 25th age-specific percentiles. Fertility preservation, in terms of oocytes cryopreservation, was offered to those two patients with serum AMH levels predictive of significantly poor ovarian reserve (1st and 2nd patients). Using the gonadotropin releasing hormone (GnRH) antagonist protocol for ovarian stimulation, we obtained two and six oocytes, respectively.Therefore, serum AMH, as a marker of ovarian function, can improve the identification of patients that need to be referred to fertility preservation strategies.

Objective: To evaluate the possible effect of methotrexate (MTX) on rat ovaries by measuring serum antimullerian hormone (AMH), the novel marker of the ovarian reserve. Methods: Pretreatment serum AMH levels were measured in 15 Wistar albino rats. MTX was given in 1 mg/kg dose in days 1, 3, 5, and 7. Serum AMH levels were measured twenty-four hours after each MTX administration. Pre- and post-treatment serum AMH levels were compared. Results: Pretreatment median serum AMH was 102.4 ng/mL (25%: 41.9; 75%: 179.8). The median serum AMH levels were 70.6 ng/mL (25%: 54.08; 75%: 125.5); 136.1 ng/mL (25%: 57.3; 75%: 223.09); 121.2 ng/mL (25%: 52.5; 75%: 151.5); and 104.7 ng/mL (25%: 65.8; 75%: 265.5) after the first, second, third, and fourth methotrexate administrations, respectively. The ratio of the final (eighth day) median serum AMH level to the pretreatment median AMH level was 1.27 (25%: 0.84 and 75%: 2.57). Wilcoxon related samples test showed that final AMH was significantly higher as compared to the second day AMH measurement (p = 0.041). Conclusion: MTX administration did not cause a statistically significant change between pretreatment and final serum AMH levels in rats. There was no decrease in AMH levels indicating a decrease in ovarian reserve.

ObjectiveThe value of serum AMH, INHB, and bFSH levels in assessing postoperative ovarian reserve function was analyzed by measuring serum anti-Mullerian hormone (AMH), inhibin B (INHB), and basal follicle-stimulating hormone (bFSH) levels in patients after laparoscopic cystectomy for endometrioma.MethodsFrom June 2019 to December 2021, 124 patients underwent laparoscopic cystectomy for endometrioma in our hospital were selected, and the serum AMH, INHB, bFSH level, antral follicle count (AFC) of all patients before and after operation were detected and compared. According to the results of postoperative testing, all the patients were divided into normal group (n = 86), diminished ovarian reserve (DOR) group (n = 27), and premature ovarian failure (POF) group (n = 11). Pearson correlation model and subject operating characteristic curve (ROC) were used to analyze the correlation and diagnostic value of serum AMH, INHB and bFSH levels with postoperative ovarian reserve function, respectively.ResultsAfter operation, the levels of serum AMH, INHB and AFC in the DOR group and POF group decreased compared with those before the operation, and the serum bFSH levels increased (p < 0.05). After operation, the levels of serum AMH, INHB and AFC in DOR group and POF group were lower than those in normal group,and the serum bFSH levels were higher than the normal group; the levels of serum AMH, INHB and AFC in POF group were lower than those in DOR group, and the serum bFSH levels were higher than the DOR group (p < 0.05). Pearson analysis showed that serum AMH and INHB levels were negatively correlated with bFSH, and positively correlated with the number of AFC, the serum bFSH level was negatively correlated with the number of AFC (p < 0.05). The diagnostic values of serum AMH, bFSH, INHB and the combination of the three tests for postoperative abnormal ovarian reserve function were 0.866 (95% CI, 0.801–0.923), 0.810 (95% CI, 0.730–0.890), 0.774 (95% CI, 0.687–0.860) and 0.940 (95% CI, 0.900–0.981), respectively.ConclusionSerum AMH and INHB levels decreased and bFSH levels increased in patients after laparoscopic cystectomy for endometrioma, both of which were closely related to postoperative ovarian reserve function, and both could evaluate ovarian reserve function after ovarian cyst debulking, and the combined test could significantly improve the detection rate.

To explore the serum anti-Mullerian hormone (AMH) level in women of childbearing age with normal menstrual cycles. A total of 1 423 women with regular menstrual cycles were selected and divided into 5 groups according to their ages, i.e. ≤ 25, 26-30, 31-35, 36-40, ≥ 41 years. Their serum levels of AMH were measured, and the relationship between AMH and age was analyzed. The serum AMH levels of 5 groups according to ages (≤ 25, 26-30, 31-35, 36-40, ≥ 41 years) were 3.62, 3.10, 2.27, 1.07, 0.45 µg/L, respectively. The comparison of serum AMH levels in different age groups had significant difference (P < 0.01). Serum AMH level declined with increasing age, and dropped significantly after 36. The serum AMH level and age showed a negative correlation with significant difference (r = -0.374, P < 0.01). Quadratic regression of logAMH proximally reflected the relationship between AMH and age. AMH determination for women of childbearing age could provide reference for the evaluation of ovarian function.

Clinical cases have been reported with women who got pregnant with confirmed low serum anti-Müllerian hormone (AMH) concentrations, thus demonstrating that low serum AMH concentration cut-points could be fairly specific for poor ovarian response (POR) to gonadotrophin stimulation, but not for pregnancy. That observation prompted the question whether serum AMH concentration accurately corresponded to the whole amount of AMH secreted by granulosa cells. To measure AMH levels in peritoneal fluid and their correlations with serum AMH concentrations. The reported study involved 48 female patients, aged 18-40 years, diagnosed with benign ovarian cysts and qualified for a laparoscopic cystectomy. Prior to surgery, the ovarian reserve was assessed using serum AMH concentration assay. The peritoneal fluid was also collected during the laparoscopy and AMH concentrations in peritoneal fluid were measured. The AMH present in the peritoneal fluid strongly correlated with AMH levels in blood serum (r = 0.54; p < 0.001) and higher serum AMH concentrations corresponded to higher AMH concentrations in the peritoneal fluid. There was also a significant correlation between AMH levels in serum and in peritoneal fluid, collected from patients with endometrioma and other benign cysts (r = 0.61; p = 0.001 vs r = 0.43; p = 0.03). The AMH is present in the peritoneal fluid and its concentrations significantly correlate with AMH levels in serum. The assessment of AMH concentration in the peritoneal fluid may be a valuable complement to the evaluation of ovarian reserve and the diagnosis of infertility after adnexal surgery.

Study question Our goal was to determine whether the administration of temsirolimus and recombinant AMH, concomitant with chemotherapy, is effective in protecting fertility from chemotherapy-induced damage. Summary answer The administration of temsirolimus and recombinant AMH concomitant with chemotherapy proved effective in protecting the fertility, as assessed five weeks after treatment. What is known already Alkylating agents cause apoptosis of growing follicles which produce anti-Müllerian hormone (AMH), a key factor controlling primordial follicle dormancy. Their loss lowers AMH levels, leading to indirect overactivation and extensive recruitment of primordial follicles. Additionally, alkylating agents directly overactivate primordial follicles through the PI3K–mTOR pathway, ultimately depleting the ovarian reserve. Given chemotherapy’s role in activating primordial follicles, recent studies have shown that blocking both indirect and direct activation pathways by administering AMH and an mTOR inhibitor effectively protects the ovary from chemotherapy-induced damage. However, no data is available on residual fertility and longer-term effectiveness. Study design, size, duration Thirty-eight female NMRI mice were randomly assigned to one of three treatment groups: (i) control group; (ii) chemotherapy group; and (iii) gonadoprotection group. Five weeks after treatment, all mice underwent ovarian stimulation and were mated with males to evaluate residual fertility. After mating, the animals were euthanized for embryo collection and ovarian recovery. The ovarian reserve was assessed by follicle count, while follicle damage was evaluated through analyses of apoptosis, activation and proliferation. Participants/materials, setting, methods The control group (n = 10) received a 100µL intraperitoneal injection of PBS. The chemotherapy group (n = 13) received a single 100µL intraperitoneal injection of chemotherapy (120mg/kg cyclophosphamide + 12 mg/kg busulfan). The gonadoprotection group (n = 15) received five injections of 5 mg/kg temsirolimus over one week prior to chemotherapy, followed by a chemotherapy injection, then five injections of 5µg rAMH over the 24 hours following chemotherapy. All mice were stimulated five weeks later with Menopur 3.75IU. Main results and the role of chance In terms of residual fertility, the control group showed the highest mean number of retrieved embryos (41.40 ± 14.74), closely followed by the gonadoprotection group (36.27 ± 17.22), with comparable fertility outcomes (p = 0.4449). In contrast, the chemotherapy group exhibited a significant reduction (20.63 ± 12.12) compared to both the control group (p = 0.0046) and the gonadoprotection group (p = 0.0349). Regarding ovarian reserve, the control group showed the highest total follicle count (897.4 ± 392.8), with no significant difference compared to the gonadoprotection group (714.4 ± 283.9). In contrast, the chemotherapy group demonstrated a significant decline in total follicle count per ovary (320.7 ± 145.5) compared to both the control group (p &amp;lt; 0.0001) and the gonadoprotection group (p = 0.0008). Follicle damage was assessed by immunohistochemistry analyses of caspase-3 for apoptosis, phospho-Akt for activation and Ki67 for proliferation. Follicle proliferation rates were significantly higher in the chemotherapy group (59.38% ± 14.67%) than in the control group (20.72% ± 10.52%) (p &amp;lt; 0.0001). Gonadoprotective treatment led to a decrease in follicle proliferation (42.23% ± 18.37%) compared to the chemotherapy group (p = 0.0083), but the proliferation rate remained significantly higher than in the control group (p = 0.0020). No significant difference was observed between groups for follicle apoptosis and activation. Limitations, reasons for caution Temsirolimus and AMH show gonadoprotective potential in murine models but their effects on human ovarian tissue remain unclear. Studies on AMH in xenografts report conflicting results: some indicating inhibition of follicle activation and others suggesting promotion of follicle growth. Further research is needed to clarify their applicability in human models. Wider implications of the findings Our findings suggest that temsirolimus and AMH show promise as gonadoprotective agents, potentially preserving fertility and the ovarian reserve against chemotherapy. This could provide new fertility preservation options for patients who cannot benefit from current available techniques, such as oocyte and embryo cryopreservation or ovarian tissue cryopreservation and transplantation. Trial registration number No

Distribution and responsiveness of rat anti-Müllerian hormone during ovarian development and VCD-induced ovotoxicity

STUDY QUESTIONDo polymorphisms in the anti-Müllerian hormone (AMH) promoter have an effect on AMH levels in patients with polycystic ovary syndrome (PCOS)?SUMMARY ANSWERWe have identified a novel AMH promoter polymorphism rs10406324 that is associated with lower serum AMH levels and is suggested to play a role in the mechanism of regulation of AMH gene expression in women.WHAT IS KNOWN ALREADYFollicle number is positively correlated with serum AMH levels, reflected by elevated AMH levels in women with PCOS. In addition, it is suggested that AMH production per follicle is higher in women with PCOS than in normo-ovulatory women, implying an altered regulation of AMH in PCOS.STUDY DESIGN, SIZE, DURATIONA discovery cohort of 655 PCOS women of Northern European ancestry and both an internal and external validation PCOS cohort (n = 458 and n = 321, respectively) were included in this study. Summary-level data of an AMH genome-wide association study meta-analysis including 7049 normo-ovulatory women was included as a control cohort. A genetic approach was taken through association analysis and in silico analysis of the associated variants in the AMH promoter. In vitro analysis was performed to investigate the functional mechanisms.PARTICIPANTS/MATERIALS, SETTING, METHODSAll common two-allelic single-nucleotide polymorphisms (SNPs) in the region Chr19:2 245 353–2 250 827 bp (Build 37) were selected for the analysis. Linear regression analyses were performed to determine the association between SNPs in the AMH promoter region and serum AMH levels. For the in silico analysis, the webtools ‘HaploReg’ v4.1 for ENCODE prediction weight matrices and ‘atSNP’ were used. In vitro analysis was performed using KK1 cells, a mouse granulosa cell line and COV434 cells, a human granulosa tumor cell line. Cells were transfected with the reference or the variant human AMH promoter reporter construct together with several transcription factors (TFs). Dual-Glo® Luciferase Assay was performed to measure the luciferase activity.MAIN RESULTS AND THE ROLE OF CHANCEPolymorphism rs10406324 was significantly associated with serum AMH levels in all three PCOS cohorts. Carriers of the minor allele G had significantly lower log-transformed serum AMH levels compared to non-carriers (P = 8.58 × 10−8, P = 1.35 × 10−3 and P = 1.24 × 10−3, respectively). This result was validated in a subsequent meta-analysis (P = 3.24 × 10−12). Interestingly, rs10406324 was not associated with follicle count, nor with other clinical traits. Also, in normo-ovulatory women, the minor allele of this variant was associated with lower serum AMH levels (P = 1.04 × 10−5). These findings suggest that polymorphism rs10406324 plays a role in the regulation of AMH expression, irrespective of clinical background. In silico analysis suggested a decreased binding affinity of the TFs steroidogenenic factor 1, estrogen-related receptor alpha and glucocorticoid receptor to the minor allele G variant, however in vitro analysis did not show a difference in promoter activity between the A and G allele.LIMITATIONS, REASONS FOR CAUTIONFunctional analyses were performed in a mouse and a human granulosa cell line using an AMH promoter reporter construct. This may have limited assessment of the impact of the polymorphism on higher order chromatin structures. Human granulosa cells generated from induced pluripotent stem cells, combined with gene editing, may provide a method to elucidate the exact mechanism behind the decrease in serum AMH levels in carriers of the −210 G allele. We acknowledge that the lack of follicle number in the external validation and the control cohort is a limitation of the paper. Although we observed that the association between rs10406324 and AMH levels was independent of follicle number in our discovery and internal validation PCOS cohorts, we cannot fully rule out that the observed effects on serum AMH levels are, in part, caused by differences in follicle number.WIDER IMPLICATIONS OF THE FINDINGSThese results suggest that variations in serum AMH levels are not only caused by differences in follicle number but also by genetic factors. Therefore, the genetic context should be taken into consideration when assessing serum AMH levels in women. This may have clinical consequences when serum AMH levels are used as a marker for the polycystic ovarian morphology phenotype.STUDY FUNDING/COMPETING INTEREST(S)No external funding was used. J.S.E.L. has received consultancy fees from the following companies: Ferring, Roche Diagnostics and Ansh Labs and has received travel reimbursement from Ferring. J.A.V. has received royalties from AMH assays, paid to the institute/lab with no personal financial gain. The other authors declare no competing interests.TRIAL REGISTRATION NUMBERN/A.

What is the influence of age and chemotherapy regimen on the longitudinal blood anti-Müllerian hormone (AMH) variations in a large series of adolescents and young adult (AYA) (15-24 years old) and non-AYA (25-35 years old) lymphoma patients? In case of alkylating regimen treatment, there was a deep and sustained follicular depletion in AYA as well as non-AYA patients; however in both groups, the ovarian toxicity was extremely low in cases of non-alkylating treatments. AMH is now well-recognised to be a real-time indicator of ovarian follicular depletion and recovery in women treated by chemotherapy. Its longitudinal variations may discriminate between highly and minimally toxic protocols regarding ovarian function. It has been shown, in different cancer types, that age, type of chemotherapy regimen and pre-treatment AMH levels are the main predictors of ovarian recovery. Large studies on longitudinal AMH variations under chemotherapy in lymphoma patients are few but can provide the opportunity to assess the degree of follicle loss at a young age. This prospective cohort study was conducted in the Fertility Observatory of the Lille University Hospital. Data were collected between 2007 and 2016. Non-Hodgkin or Hodgkin lymphoma patients (n = 122) between 15 and 35 years old were prospectively recruited before commencing chemotherapy. Patients were treated either by a non-alkylating protocol (ABVD group; n = 67) or by an alkylating regimen (alkylating group; n = 55). Serial AMH measurements were performed at baseline (AMH0), 15 days after the start of chemotherapy (AMH1), 15 days before the last chemotherapy cycle (AMH2), and at time 3, 6, 9, 12, 18 and 24 months from the end of chemotherapy. The whole study population was divided into two groups according to age: AYA (15-24; n = 65) and non-AYA (25-35; n = 57). All patients received a once monthly GnRH agonist injection during the whole treatment period. A linear mixed model was used to account for the repeated measures of single patients. At baseline, non-AYA patients had higher BMI and lower AMH levels than AYA patients. All AYA and non-AYA patients having received ABVD protocols had regular cycles at 12 months of follow-up. In case of alkylating regimens, amenorrhoea was more frequent in non-AYA patients than in AYA patients at 12 months (37% vs 4%, P = 0.011) and at 24 months (24% vs 4%, P = 0.045). We distinguished a similar depletion phase from AMH0 to AMH2 between ABVD and alkylating groups but significantly different recovery phases from AMH2 to AMH + 24 months. AMH recovery was fast and complete in case of ABVD protocols whatever the age: AMH reached pre-treatment values as soon as the 6th month of follow-up in the AYA group (mean (95% CI) in log AMH M0 vs M6: 3.07 (2.86 to 3.27) vs 3.05 (2.78 to 3.31), P = 1.00) and in the non-AYA group (mean (95% CI) in log AMH M0 vs M6: 2.73 (2.40 to 3.05) vs 2.47 (2.21 to 2.74), P = 1.00). In contrast, no patients from the alkylating group returned to pre-treatment AMH values whatever the age of patients (AYA or non-AYA). Moreover, none of the AMH values post-chemotherapy in the non-AYA group were significantly different from AMH2. Conversely in the AYA group, AMH levels from 6 months (mean (95% CI) in log AMH: 1.79 (1.47 to 2.11), P < 0.001) to 24 months (mean (95% CI) in log AMH: 2.16 (1.80 to 2.52), P ≤ 0.001) were significantly higher than AMH2 (mean (95% CI) in log AMH: 1.13 (0.89 to 1.38)). Considering the whole study population (AYA and non-AYA), pre-treatment AMH levels influenced the pattern of the AMH variation both in alkylating and ABVD protocols (interaction P-value = 0.005 and 0.043, respectively). Likewise, age was significantly associated with the pattern of the recovery phase but only in the alkylating group (interaction P-value =0.001). BMI had no influence on the AMH recovery phase whatever the protocol (interaction P-value = 0.98 in alkylating group, 0.72 in ABVD group). There was a large disparity in subtypes of protocols in the alkylating group. The average duration of chemotherapy for patients treated with alkylating protocols was longer than that for patients treated with ABVD. These results make it possible to develop strategies for fertility preservation according to age and type of protocol in a large series of young lymphoma patients. In addition, it was confirmed that young age does not protect against ovarian damage caused by alkylating agents. This work was supported by Agence Régionale de Santé Hauts de France and Agence Onco Hauts-de-France who provided finances for AMH dosages (n° DOS/SDES/AR/FIR/2019/282). There are no competing interests. DC-2008-642 and CNIL DEC2015-112.

To determine whether recombinant AMH (rAMH) could prevent post-transplant follicular depletion by acting on the stemness markers Oct-4, Sox2, and NANOG. This was an experimental study where 12 ovariectomized nude mice were xenotransplanted with vitrified/warmed ovarian cortex obtained from a pre-pubertal girl and Alzet pumps delivering rAMH, or placebo (control), were inserted intra-abdominally. Previously vitrified/warmed ovarian cortex fragments were transplanted after 7days and then harvested after 14days from pump placement. We performed real-time RT-PCR analyses, ELISA for AMH, FSH, and estradiol, histologic measurement of ovarian follicles, and immunohistochemistry for Ki67 and TUNEL. The main outcome measures were serum levels and tissue expression of the parameters under investigation and follicle count. Serum AMH, FSH, and estradiol reflected post-ovariectomy profiles and were mildly influenced by rAMH administration. Ovarian cortex expression of AMH, AMH-R2, VEGF, GDF9, Oct-4, and Sox2 was lower in rAMH mice than in controls, while NANOG was upregulated. There was a non-significant decrease in primordial follicles after vitrification-warming, and xenotransplantation further decreased this number. There were lower cell replication and depressed apoptosis in the rAMH group. Administration of recombinant AMH in the peri-transplant period did not protect the initial follicular depletion but decreased apoptosis and cellular activation and regulated stem cell markers' tissue expression. These results aid our understanding of the inhibitory effects of AMH on follicular development and show the benefit of administering exogenous AMH at the time of pre-pubertal ovarian cortex transplant to protect the follicles from pre-activation and premature depletion.