Diminished Ovarian Reserve: A Narrative Review of Etiologies and Possible Therapeutic Approaches

Study question Is whole exome sequencing (WES) a potential routine diagnostic tool to assess genetic causes in patients with diminished ovarian reserve (DOR), together with FMR1 analysis? Summary answer WES is a promising diagnostic tool in patients with DOR, together with FMR1 screening. In addition, WES could predict progression to premature ovarian insufficiency (POI). What is known already DOR and primary ovarian insufficiency (POI) are among the leading causes of female infertility related to ovarian dysfunction. We very recently conducted the first NGS study investigating a custom-made NGS-POI panel comprising all known genes causing POI (n = 88) in a large cohort of 120 patients with unexplained DOR (Lafraoui et al. IJMS 2024, PMID: 39595984). We highlighted a monogenic etiology in 24 % of cases leading to personalized medicine. This preliminary study showed a genetic link between POI and DOR. Study design, size, duration A multicenter prospective cohort study was conducted in 50 supplementary patients with unexplained DOR. Participants/materials, setting, methods All patients included were under 35 years with AMH plasma levels <1.2ng/ml. All patients signed informed consent with the study. Examination of FMR1 premutation was performed together with WES using Twist exome V2.0 on NovaSeqX. Copy Number Variations (CNV) were studied. In silico analysis was focused on gene panel known to cause POI (Lafraoui et al, IJMS 2024 PMID 39595984). Variants were assessed according to the ACMG guidelines using Franklin, Varsome, Mobidetail and Intervar softwares. Main results and the role of chance The mean age of our cohort was 30.5 years (24-35 years), the mean plasma AMH level was 0.76 ng/ml (0.02-1.2) and the mean FSH level was 9.5 IU/l (2-111). All positive patients carry one or two class 4 or 5 variants depending on the known mode of transmission of the gene. The positive genes identified were FOXL2 (2 patients), BNC1, AR, GATA4 (2 patients), BMPR2 and ESR2, mainly belonging to two pathways: ovarian development and follicular growth. One patient had two large CNV of X chromosome. One patient had a premutation of FMR1 gene. We thus report a genetic diagnosis in 10 patients of our DOR cohort, i.e. 20% of patients. Eight percent of patients are heterozygous carriers for a single pathogenic variant in a known recessive POI gene. Our study confirms the genetic heterogenity of DOR as for POI. Moreover, it confirms the genetic link between POI and DOR and underscores the importance of routine genetic diagnosis for all patients with unexplained DOR. Limitations, reasons for caution The size of the cohort needs to be increased to confirm these results. The pathogenicity of the identified variants was assessed by in silico analyses according to American College of Medical Genetics guidelines. Wider implications of the findings Study highlights the importance of routine genetic studies in all unexplained DOR, including FMR1 analysis together with WES and CNV studies. Whole exome sequencing allows to implement genetic diagnosis as soon as new genes are identified. Genetics could be a predictive biomarker of progression to POI in patients with DOR. Trial registration number Yes

Anti-Müllerian hormone (AMH) defines, independent of age, low versus good live-birth chances in women with severely diminished ovarian reserve

Study question Identify the genetic cause of a cohort of patients with unexplained Diminished ovarian reserve (DOR) by next-generation sequencing and compare with pregnancy outcome. Summary answer A high-yield positive genetic diagnosis was obtained: 27% of cases. Defects of genes involved in DNA repair/meiosis appeared to have an unfavorable prognosis. What is known already Ten percent of women undergoing Medically Assisted Procreation-MAP have a DOR defined by an AMH level <1.2 and an antral follicle count (AFC) <5. However, most causes of DOR are unknown. There is no known criteria of success in MAP. Primary ovarian insufficiency (POI) corresponds to a complete cessation of ovarian function in 1-4% of women under 40 years. We have very recently shown in a large cohort of POI that a custom-made target next-generation sequencing (NGS) POI panel allowed a genetic diagnosis in 30% of unexplained POI and leads to personalized medicine (Heddar et al., EBioMedicine. 2022 doi: 10.1016/j.ebiom.2022.104246). Study design, size, duration Prospective genetic study of a cohort of 100 patients with undergoing MAP using a custom-made NGS-POI panel comprising 88 validated POI-causing genes. These patients were for 77% European, 20% North African and 3% Asian. The classification of the variants detected were based on the American College of Medical Genetic criteria 2015. Only pathogenic and likely pathogenic variants were used for diagnosis. Candidate gene studies were performed in negative patients. Participants/materials, setting, methods One hundred patients aged 16-35 with DOR, normal karyotype and FMR1 gene, were studied with the NGS POI panel. The segregation of the variants in the available families was performed by Sanger sequencing. Cytogenetic studies of chromosomal fragility was performed if necessary. The outcome of the pregnancy (spontaneous or induced) was recorded. A correlation between the result of the genetic study and the outcome of the pregnancy has been performed. Main results and the role of chance One family included two sisters, one with POI and the other with DOR, highlighting the proximity of the two syndromes. A high positive genetic yield was found: 27% of cases. Genes were involved 1) in follicular growth and gonadal development (SOX8, AR, NOBOX, BMPR1A, BMPR1B, GNAS) (35%) 2) DNA repair (BNC1, ERCC6, BRCA1, ATM) (31%) 3) metabolism and mitochondrial functions (POLG, STAR) (12%), 4) cellular aging (24%), in particular the LMNA gene. In a patient with isolated DOR, we identified for the first time, bi-allelic truncating variants of BRCA1, a major breast/ovarian cancer susceptibility gene. Cytogenetic studies revealed increased chromosomal breaks with radial figures typical of Fanconi Anemia (FA). However there was no sign of FA or cancer in the patient and family. This observation is reminiscent of the family with isolated POI and biallelic mutations of BRCA2 (Heddar et al, J Med Genet 2021; DOI: 10.1136/jmedgenet-2019-106672). Among the 27 patients with an established genetic diagnosis, six were able to obtain a pregnancy. However, no pregnancies were achieved in the DNA repair gene suggesting an unfavorable prognosis for this gene family. On the other hand, patients with mutations of other gene families seem to have a better prognosis of fertility. Limitations, reasons for caution The size of the cohort should be implemented to confirm these results. The issues of pregnancy are not available for the whole cohort. Wider implications of the findings This is the first genetic study of a cohort of DOR and the first implication of BRCA1 in isolated DOR. It shows i) the need for genetic studies of DOR ii) the genetic link between POI and DOR iii) NGS study could give information on the fertility prognosis of DOR. Trial registration number not applicable

Distribution of the FMR1 gene in females by race-ethnicity: women with diminished ovarian reserve versus women with normal fertility (SWAN study)

Study question Investigate whether screening for autoimmune etiologies is necessary in women with diminished ovarian reserve (DOR) as recommended in the evaluation of premature ovarian insufficiency (POI). Summary answer Adrenocortical antibodies (ACA) screening can be performed in the evaluation of women with idiopathic DOR, especially those with a family history of autoimmune disease. What is known already Autoimmune disorders are more common in POI than in the general population. The most important association is with autoimmune Addison’s disease. Measurement of ACA and / or 21 OH-A is recommended in every POI patients as they appear to be the marker with the highest diagnostic sensitivity for autoimmune POI. Also thyroid peroxidase autoantibodies (TPO-Ab) should be assayed due to the common association between thyroid disease and POI. The underlying etiologies of DOR in young women can be expected to be similar to the etiology of POI since they represent a continuum in the phenotypic expression of premature ovarian aging. Study design, size, duration This pilot case-control study was conducted between January 2019 and April 2020. The study group consisted of patients under the age of 35, who was diagnosed with idiopathic DOR by ovarian reserve tests during infertility work up. Controls were patients of the same age range who diagnosed with isolated tubal factor or male infertility and had normal ovarian reserve test results during infertility work up. Participants/materials, setting, methods Patients with a history of ovarian surgery, cancer, genetic or autoimmune disease were excluded. Abnormal ovarian reserve tests are defined as antral follicle count < 5 and AMH < 1.2 ng/dl corresponding to group 3 according to POSEIDON criteria. In total, 35 DOR patients and 35 controls were included in the study. ACA and TPO-Ab screening were performed in serum samples using indirect immunofluorescence method. Demographics and family history of autoimmune diseases were also evaluated. Main results and the role of chance A higher rate of ACA positivity was detected in the DOR group (34.3%) compare to controls (17.1%), although it was not found to be statistically significant (p = 0.101). The incidence of family history of autoimmune diseases in first degree relatives was positively correlated with ACA positivity (p = 0.006). In DOR group, autoimmune disease history in the family was significantly higher in ACA (+) patients compared to ACA (-) individuals (p = 0.03). TPO-Ab positivity rates were similar between 2 groups (17.1% vs%20, p = 0.75). Limitations, reasons for caution Since this is an observational study and also due to the small sample size, a causal conclusion cannot be reached. Wider implications of the findings: Even if there is no specific treatment option yet for autoimmune ovarian damage, screening for ACA or 21 OH-A may be considered in young women with idiopathic DOR based on knowledge that identification of women with autoimmune POI is clinically important for the identification of subclinical autoimmune Addison’s cases. Trial registration number non applicable

Study question Are young infertile patients with diminished ovarian reserve (DOR) elegible to perform the FMR1 premutation study? Summary answer Study of the FMR1 premutation should be considered in infertile young patients with DOR in order to give them an adequate genetic counselling. What is known already FMR1 gene may have some reproductive implications. Most notable is that FMR1 premutation expansions are associated with premature ovarian insufficiency (POI), diagnosed by amenorrhea or oligomenorrhea and FSH hormonal levels >25U/L before 40 years old. Presence of FMR1 premutation implies a risk of develop POI up to 24% and having an offspring with fragile X syndrome. The frequency of FMR1 premutation in general population is estimated in 0.3-0.7%. The role of FMR1 premutation expansions in diminished ovarian reserve (DOR) patients is not clearly established and could be considered as a previous step to POI that may be related to sterility. Study design, size, duration Retrospective review of the FMR1 gene study requested in patients of an Assisted Reproduction Unit of a tertiary Hospital in Barcelone from January-2016 to December-2019. A total of 307 cases were evaluated to determine the number of CGG repeat and AGG interruptions to assess the FMR1 gene status. Participants/materials, setting, methods A total of 307 samples were assessed. Clinical and reproductive data were collected. The FMR1 status was requested on patients who present: a) POI (n = 60); b) Family history of the FMR1 mutation (n = 11); c) Infertile normo-ovulatory and young (≤35 years old) women with DOR defined as antral follicle count (AFC) < 7 and antimüllerian hormone <0.8ng/ml (n = 71); d) Miscellaneous (n = 29) FMR1 was studied in 136 oocyte donors (screened by protocol), this was considered control group. Main results and the role of chance Mean age (±SD) of infertile DOR group was 32.7 +/- 2.1 years old (range 26-35) and showed altered ovarian reserve markers: AMH 0.43 ng/ml (SD ± 0.28) and AFC 4.27 (SD ± 2.1) follicles. In this group, 4 FMR1 premutation cases were found. Mean age (±SD) in control group was 26.28 +/- 5.2 years old and presented normal AMH and AFC values. One FRM1 premutation carrier was detected among 136 patients, prevalence comparable to the non-sterile population. The prevalence of FRM1 premutation was significantly higher in the DOR infertile group 5,6% vs 0,73% in the donors’ group (p = 0.02). Significant differences were observed also in terms of age and ovarian reserve markers between both groups. Very few cases of POI patients or family history of Fragile X Syndrome have been evaluated, due to the fact we are not a reference of these kind of patients. Among patients with a family history, 1 case from 11 (9.1%) was detected. In the POI group, three cases of premutation out of 60 (5%) were found. Limitations, reasons for caution This is a retrospective study with limited determinations of FMR1 studies. Donor screening and young infertile patients with significant low ovarian reserve are the main indications to request FMR1 status gene, so may lead to a selection bias. Wider implications of the findings These results should be confirmed prospectively in a higher population of infertile young patients with DOR, in order to identify the profile of infertile patient with diminished ovarian reserve who are elegible to perfom FMR1 gene premutation to give them an adequate clinical and genetic counselling. Trial registration number not apllicable

Study question Is there any association between diminished ovarian reserve (DOR) and sub-clinical hypothyroidism (SCH) in lower age group (<35 years) women undergoing IVF? Summary answer In women <35 years with DOR, the odds of associated SCH is over 5 times compared to women with normal ovarian reserve What is known already Known causes of DOR include advancing age, poor primordial follicular pool, endometriosis, ovarian surgery, and other environmental or lifestyle factors. Although the exact mechanisms for DOR are poorly understood, age is considered to be the most important factor. Younger women are expected to have better ovarian reserve and response to IVF stimulation. In our clinical practise, we noticed that many women with DOR also presented with SCH. Recent reports have indicated an association between SCH and DOR, although, to the best of our knowledge, there are no reports yet exploring this association in lower-age patients undergoing IVF. Study design, size, duration We retrospectively analysed last 3 years' IVF data to investigate whether women < 35 years old who presented with DOR (AMH less than or equal to 1.2 ng/ml) had greater odds of also showing evidence of SCH (TSH: 2.5-4.5 mIU/L) compared to women with normal ovarian reserve (AMH> 1.2 ng/ml). 608 cycles were initially included in the study. Women with overt clinical hypothyroidism were excluded and data from 573 cycles was analysed. Participants/materials, setting, methods The study was conducted in a teaching hospital affiliated private IVF institute. Following institutional ethical committee clearance, data was analysed from cycles of women < 35 years age who were enlisted for IVF. Statistical analysis was done using SPSS V.6 to investigate whether patients with DOR (AMH = < 1.2 ng/ml) showed greater odds (primary outcome measure) of SCH (TSH: 2.5-4.5 mIU/L), compared to women with normal ovarian reserve (AMH> 1.2 ng/ml). Main results and the role of chance Data analysis revealed that women < 35 years of age who presented with DOR (AMH = < 1.2 ng/ml) were 5 times as likely to also have SCH (TSH: 2.5-4.5 mIU/L), in comparison to women who had normal ovarian reserve (Odds Ratio: 5.2, p < 0.01). Moreover, mean AMH level was significantly lower in patients with SCH (n = 409) when compared to patients without SCH (n = 164), across the entire cohort of normo-responders and DOR patients under the age of 35 years (AMH in SCH vs Euthyroid: 1.65± 1.46 ng/ml vs 3.32± 2.22 ng/ml, p < 0.01, Student’s t test). However, when we performed a sub-group analysis of DOR patients, no significant differences were found in oocyte yield, fertilisation rate (2PN rate) or top quality blastocyst rate between euthyroid and SCH groups, although top quality blastocyst rate was higher in euthyroid patients compared to SCH patients (41% vs 27.9%). Limitations, reasons for caution This retrospective study reports an association of SCH with DOR in women <35 years undergoing IVF. However, we are unable to explain the possible underlying mechanisms. Controlled prospective studies are required to elucidate the molecular underpinnings of these findings, and investigate possible association with laboratory and clinical IVF outcome parameters. Wider implications of the findings Screening and treatment for SCH could be an option in managing DOR patients <35 years. Further studies are required to confirm our observation of a remarkably high incidence of SCH amongst younger DOR patients in the Indian population (88.2%), as well as to elucidate the underlying mechanisms behind this association. Trial registration number No

Hydroxyurea Is a Potential Risk Factor for Diminished Ovarian Reserve in Young Adults with Sickle Cell Anemia

Diminished ovarian reserve (DOR) and primary ovarian insufficiency (POI) are primary factors leading to infertility. However, there is a lack of appropriate animal models of DOR usable for assessing new therapeutic strategies. In this study, we aimed to evaluate whether chemotherapy treatment in mice could reproduce features similar of that observed in women with DOR. Twenty-one Nonobese diabetic/severe combined immunodeficiency (NOD/SCID) female mice were allocated to 3 groups (n = 7/group): control, single dose of vehicle (Dimethyl Sulfoxide [DMSO]); DOR, single reduced chemotherapy dose; and POI, single standard chemotherapy dose. After 21 days, mice underwent ovarian hyperstimulation and mating. Part of the animals were harvested to analyze ovarian reserve, ovulation and fertilization rates, and morphology, apoptosis, and vascularization of the ovarian stroma. The remaining mice underwent multiple matings to assess pregnancy rates and litter sizes. The DOR and POI mice showed an impaired estrous cyclicity and a decrease in ovarian mass, number of follicles, Metaphase II (MII) oocytes, and embryos as well as in ovarian stroma vascularization. Mice in both models showed also an increase in the percentage of morphologically abnormal follicles, stromal degeneration, and apoptosis. Similar to that observed in DOR and POI patients, these impairments were less severe in DOR than in POI mice. None of the POI females were able to achieve a pregnancy. Meanwhile, DOR females achieved several consecutive pregnancies, although litter size was decreased when compared to controls. In conclusion, a mouse model which displayed most of the ovarian characteristics and fertility outcomes of women with DOR has been established using a single dose of chemotherapy.

Diminished ovarian reserve (DOR) and primary ovarian insufficiency (POI) are primary factors leading to infertility. However, there is a lack of appropriate animal models of DOR usable for assessing new therapeutic strategies. In this study, we aimed to evaluate whether chemotherapy treatment in mice could reproduce features similar of that observed in women with DOR. Twenty-one Nonobese diabetic/severe combined immunodeficiency (NOD/SCID) female mice were allocated to 3 groups (n = 7/group): control, single dose of vehicle (Dimethyl Sulfoxide [DMSO]); DOR, single reduced chemotherapy dose; and POI, single standard chemotherapy dose. After 21 days, mice underwent ovarian hyperstimulation and mating. Part of the animals were harvested to analyze ovarian reserve, ovulation and fertilization rates, and morphology, apoptosis, and vascularization of the ovarian stroma. The remaining mice underwent multiple matings to assess pregnancy rates and litter sizes. The DOR and POI mice showed an impaired estrous cyclicity and a decrease in ovarian mass, number of follicles, Metaphase II (MII) oocytes, and embryos as well as in ovarian stroma vascularization. Mice in both models showed also an increase in the percentage of morphologically abnormal follicles, stromal degeneration, and apoptosis. Similar to that observed in DOR and POI patients, these impairments were less severe in DOR than in POI mice. None of the POI females were able to achieve a pregnancy. Meanwhile, DOR females achieved several consecutive pregnancies, although litter size was decreased when compared to controls. In conclusion, a mouse model which displayed most of the ovarian characteristics and fertility outcomes of women with DOR has been established using a single dose of chemotherapy.

Diminished Ovarian Reserve (DOR) is one of the main causes of female infertility and is common in women under of 40s. The definition and diagnosis are pragmatic and the treatment in mainstream medicine is not promising as many of the sufferers have to choose IVF as the last resort. DOR could be the reduced ability of producing quality egg or the poor quality of eggs produced. Premature Ovarian Insufficiency (POI) belongs to its aggravate stage. Traditional Chinese medicine has long been employed in treating female infertility, and a large proportion of the cases in the records now could be classified as DOR or POI. Some clinical reports have demonstrated great potential in treating DOR & POI. The authors have treated many cases of DOR and POI, and great achievements have been recorded. The protocol used in the treatment is a whole system approach which combines acupuncture, Chinese herbal medicine and lifestyle advice into an organic composition to maximize the effects. The cases reported here are of good details and could demonstrate the application of the TCM diagnosis of syndrome pattern which logically leads to the whole-system treatment implementation.

Study question Does ferroptosis, through its regulation of Hippo signaling pathway, contribute to follicular atresia, thereby leading to diminished ovarian reserve (DOR) and associated infertility? Summary answer Ferroptosis in cumulus cells may induce follicular atresia and causing DOR through Hippo signaling pathway. What is known already Ferroptosis is a novel form of regulated cell death associated with oxidative stress and lipid peroxidation. Its activation has been implicated in various pathologies, including ovarian cancer, endometriosis, polycystic ovary syndrome and primary ovarian insufficiency. The Hippo signaling pathway regulates cellular growth and apoptosis, and recent evidence suggests a potential interaction between ferroptosis and this pathway in ovarian cells. Oocytes are dependent on cumulus cells for their energy supply, with the energy necessary for oocyte maturation, fertilization and early embryonic development. However, the relationship between ferroptosis and Hippo signaling in the context of DOR has not been fully elucidated. Study design, size, duration This cross-sectional study was conducted in an IVF clinic between July 2023 and December 2024. 81 women aged between 18 and 39 years undergoing IVF treatment were included, divided into two groups: 46 patients diagnosed with DOR with a serum AMH level of < 1.1 ng/ml and/or ≤3 oocytes retrieved in prior IVF cycles. 35 with normal ovarian reserve (NOR) defined by serum AMH levels between 1.1 and 3.5 ng/ml,and 4-15 oocytes retrieved during egg collection. Participants/materials, setting, methods Cumulus-oocyte complexes were retrieved after ovarian stimulation with recombinant FSH and GnRH antagonist protocol and ovulation trigger with recombinant HCG. Cumulus cells were collected during egg retrieval, mechanically using a 31G insulin syringe. Ferroptosis-related (GPX4, EMP1) and Hippo pathway-related genes (MST, YAP, LATS) were analyzed by qRT-PCR. Gene expression levels were compared between DOR and NOR groups by using delta-CT values. Additionally, iron and reactive oxygen species levels in follicular fluid were measured. Main results and the role of chance Significantly higher expression levels of EMP1 and GPX4 were observed in the DOR group compared to the NOR group (p = 0.024 and p = 0.015, respectively), while no significant difference was found in the expression of other genes. The increase in both EMP1 and GPX4 in the DOR group suggest the activation of ferroptosis pathway in cumulus cells, which, in turn, may reflect a compensatory activation of the antioxidant system. No difference was observed in follicular fluid iron levels between the DOR and NOR groups; however, reactive oxygen species (ROS) levels were significantly elevated in the DOR group (p = 0.008). The increase in ROS levels may serve as an indirect indicator of the ferroptosis activation triggered by oxidative stress. Although these results support a relationship between ferroptosis and DOR, the influence of confounding factors such as age and ovarian stimulation protocols cannot be entirely excluded. Limitations, reasons for caution The study’s limitations include its cross-sectional design and the small sample size. The findings, while promising, may not be fully generalizable due to the limited number of cases. Additionally, the complexity of cellular interactions and the lack of longitudinal follow-up limits the ability to establish causality. Wider implications of the findings These findings highlight the potential role of the relationship between ferroptosis and the Hippo signaling pathway in the pathophysiology of DOR. Understanding these mechanisms could open new avenues for therapeutic strategies aimed at preserving ovarian reserve and improving fertility outcomes in women with DOR. Trial registration number No

Diminished ovarian reserve (DOR) is defined as reduced capacity of the ovaries to produce oocytes; the oocytes produced are of poorer quality leading to the formation of poor quality embryos. The most severe form of DOR can be represented as premature ovarian failure (POF). There are various reasons leading to DOR, the most important factor being increasing age, others being endometriosis and surgeries on the ovary. POF can be due to chromosomal aberrations or secondary to chemotherapy, radiotherapy, infections, or surgeries involving the ovaries. Patients with DOR may present with infertility and menstrual cycle abnormalities; patients with sudden onset POF may also present with hot flushes and vaginal dryness. There are various tests for finding out ones's ovarian reserve, the most widely used being follicle-stimulating hormone (FSH), anti-Mullerian hormone, and antral follicle count. It is important to know a patient's ovarian reserve before recruiting her for in vitro fertilization. Various modalities have been tried to improve the outcome in candidates with DOR undergoing assisted reproductive technology. This includes high-dose FSH treatment, luteinizing hormone supplementation, GnRH antagonist cycle, and use of adjuvant treatments such as estrogen priming, growth hormone, L-arginine, and dehydroepiandrosterone. Patients who are planned for chemotherapy or radiotherapy may undergo oocyte or embryo cryopreservation before the cancer treatment. To conclude, patients with DOR and POF should be provided with good counseling and emotional support.

Study question Does autologous bone marrow derived stem cell (BMDSC) ovarian transplantation optimize ovarian reserve parameters in young infertile women with diminished ovarian reserve (DOR) ? Summary answer The autologous stem cell ovarian transplantation (ASCOT) improves AFC and AMH by facilitating the recruitment of existing dormant follicles in young women with DOR. What is known already Oocyte donation is the practical therapeutic option when patients with premature ovarian ageing desire pregnancy. It involves significant psychological burden in terms of not able to have their own biological child. ASCOT has opened new doors in poor responders and premature ovarian insufficiency through its beneficial effects on ovarian reserve and IVF outcomes. However recent studies have shown contradictory results in terms of its efficacy. No prior study has been contemplated in DOR group Study design, size, duration An open label non randomized controlled trial was conducted at Division of Reproductive Medicine in collaboration with stem cell facility at tertiary care institute. Forty two infertile women less than 35 years age with DOR (AFC<5, AMH<1.2ng/ml and /or high FSH>8IU/l) were enrolled in the study during a period from January 2020 to December 2020. 20 women who did not opt for the intervention were treated as control group whereas 22 women received the intervention. Participants/materials, setting, methods Baseline hormonal profile ( Day 2 FSH, estradiol, AMH and AFC) was done in all patients. Women with abnormal uterine cavity, endometriosis, prior ovarian surgery, abnormal karyotype were excluded. Bone marrow aspiration followed by mesenchymal stem cells isolation was performed. The stem cells were transplanted in both the ovaries through transvaginal route on the same day. Follow up visits were planned at one and six months to assess ovarian reserve parameters. Main results and the role of chance The mean age, BMI and duration of infertility were comparable between the control and study group (29.5±3.34vs29.36±2.95years, 21.51±1.40vs21.87±1.93kg/m2, 6.9±1.94vs7. 04±3.67 years) . The positive response in terms of improved AMH and AFC was seen in 68% (15/22) patients. The mean number of stem cells injected in these women were 77.71±25.33 million. At first follow up, there was no significant difference between mean FSH, estradiol levels and mean right and left ovarian volume (9.23±3.95 vs 9.02±3.92mIU/l, 61.46±29.25 vs 68.12±62.52 pg/ml, 2.82±2.18 vs 2.44±1.25 cc, 2.02±1.54 vs 2.72±1.06 cc, p < 0.05). There was significant increase in AMH and AFC values as compared to baseline (0.79±0.43 vs 1.26±0.82ng/ml, p = 0.03; 3.47±1.30 vs 6.40±2.23, p < 0.001). At second follow up visit, the significant increase in ovarian reserve persisted for AMH and AFC (0.79±0.43 vs 1.22±0.76 ng/ml, p = 0.02; 3.47±1.30 vs 6.93±1.71,p<0.001). There was no significant difference between serum FSH , Estradiol and ovarian volume. None of the patients developed any complication and the improvement in AFC and AMH persisted during 10 month follow up period. Limitations, reasons for caution The limitation of present study is small sample size and non randomization. However, time period for which positive effect lasts has not been documented in earlier studies. This study is currently being endeavored, and women with improved ovarian reserve are followed up for any spontaneous conception or following assisted reproduction. Wider implications of the findings: The present study demonstrates beneficial role of stem cells in improving ovarian reserve parameters in women with DOR with no acquired cause. If supported by future randomized clinical studies, it could represent a paradigm shift for fertility treatment in these women providing an opportunity to have their own biological child. Trial registration number CTRI/2020/01/022726

Study question Does diminished ovarian reserve (DOR) and its etiology impact the AMH/AFC ratio? Summary answer AMH/AFC ratio varies according to the etiology of DOR in young women, suggesting different impact on the follicular health, and further oocyte quality. What is known already Anti-Müllerian hormone and antral follicle count currently represent the two most accurate markers of the follicular ovarian status. Even though they may diagnose a reduction in the follicular stockpile, low values remain inefficient for predicting poor oocyte quality, in particular in young women. Since AMH is produced by the granulosa cells of follicles ranging from primary to small antral follicles, we hypothesized that the etiology of diminished ovarian reserve might differently impact the follicular health and their capacity of producing this peptide. Study design, size, duration From November 2018 to December 2021, we conducted a monocentric, retrospective study including a total of 484 infertile patients < 37 years with DOR. Participants/materials, setting, methods All patients underwent measurement of AMH levels and AFC. DOR was diagnosed according to the Bologna criteria (AMH < 1.1 ng/mL and AFC < 7). AMH/AFC ratio was compared to values obtained in 154 tubal or male infertility patients matched for age and BMI, with AMH and AFC in the normal ranges. This ratio was studied according to the etiology of DOR: genetic (n = 26), post-chemotherapy (n = 102), idiopathic (n = 215) or ovarian diseases (ovarian cyst or history of ovarian surgery, n = 141). Main results and the role of chance Overall, median age of women with DOR was 30 (18-37) years. As expected, age and BMI were comparable in women with DOR and those having normal ovarian reserve tests. In addition, the AMH/AFC ratio failed to show any difference between these 2 groups (0.143 ± 0.22 vs. 0.166 ± 0.11, NS, respectively). Among women with DOR, the etiology was significantly associated with different AMH/AFC ratio. Indeed, patient with DOR of surgical origin (ovarian diseases group) displayed higher mean values (0.283 ± 0.32 ng/mL/ Foll) when compared with those included in genetic (0.079 ± 0.15 ng/mL/ Foll, p < 0.01), idiopathic (0.103 ± 0.16 ng/mL/ Foll, p < 0.03) or post-chemotherapy (0.084 ± 0.20 ng/mL/ Foll, p < 0.01) groups. Moreover, genetic and post-chemotherapy DOR was also associated with lower AMH/AFC ratio in comparison with idiopathic DOR. Limitations, reasons for caution Despite interesting results, the retrospective nature of the present study may represent a limitation. Moreover, AMH/AFC ratio constitute an indirect method for assessing per follicle AMH production. We hypothesized that this ratio might reflect the follicular health. Its impact on natural conception and assisted reproductive technologies outcome is not known. Wider implications of the findings AMH/AFC ratio may represent an innovative tool aiming to indirectly assess follicular health and possibly oocyte quality in young women with DOR. The etiology of DOR differently impacts the follicular function as reflected by AMH/AFC ratio. Further data on live birth rates following natural or medically assisted pregnancies is needed. Trial registration number N/A