Human theca arises from ovarian stroma and is comprised of three discrete subtypes.

Theca cells serve multiple essential functions during the growth and maturation of ovarian follicles, providing structural, metabolic, and steroidogenic support. While the function of theca during folliculogenesis is well established, their cellular origins and the differentiation hierarchy that generates distinct theca sub-types, remain unknown. Here, we performed single cell multi-omics analysis of primary cell populations purified from human antral stage follicles (1-3 mm) to define the differentiation trajectory of theca/stroma cells. We then corroborated the temporal emergence and growth kinetics of defined theca/stroma subpopulations using human ovarian tissue samples and xenografts of cryopreserved/thawed ovarian cortex, respectively. We identified three lineage specific derivatives termed structural, androgenic, and perifollicular theca cells, as well as their putative lineage-negative progenitor. These findings provide a framework for understanding the differentiation process that occurs in each primordial follicle and identifies specific cellular/molecular phenotypes that may be relevant to either diagnosis or treatment of ovarian pathologies.

Xenograft model of heterotopic transplantation of human ovarian cortical tissue and its clinical relevance.

In brief: Xenografts of human ovarian cortical tissue provide a tractable model of heterotopic autotransplantation that is used for fertility preservation in patients undergoing ablative chemo/radiotherapy. This study describes the behavior of hundreds of xenografts to establish a framework for the clinical function of ovarian cortex following autotransplantation over short- and long-term intervals. Abstract: More than 200 live births have been achieved using autotransplantation of cryopreserved ovarian cortical fragments, yet challenges remain to be addressed. Ischemia of grafted tissue undermines viability and longevity, typically requiring transplantation of multiple cortical pieces; and the dynamics of recruitment within a graft and the influence of parameters like size and patient age at the time of cryopreservation are not well-defined. Here, we describe results from a series of experiments in which we xenografted frozen/thawed human ovarian tissue (n = 440) from 28 girls and women (age range 32 weeks gestational age to 46 years, median 24.3 ± 4.6). Xenografts were recovered across a broad range of intervals (1-52 weeks post-transplantation) and examined histologically to quantify follicle density and distribution. The number of antral follicles in xenografted cortical fragments correlated positively with the total follicle number and was significantly reduced with increased patient age. Within xenografts, follicles were distributed in focal clusters, similar to the native ovary, but the presence of a leading antral follicle coincided with increased proliferation of surrounding follicles. These results underscore the importance of transplanting ovarian tissue with a high density of follicles and elucidate a potential paracrine influence of leading antral follicles on neighboring follicles of earlier stages. This temporal framework for interpreting the kinetics of follicle growth/mobilization may be useful in setting expectations and guiding the parameters of clinical autotransplantation.

The use of fertility preservation services for cancer patients: a single institution experience.

Objective: To analyze the use of services regarding fertility preservation (FP) in cancer patients at a single institution. Design: A retrospective cohort study. Setting: Academic medical center. Patients: A total of 208 FP referrals. Interventions: None. Main Outcome Measures: Method of FP; time from referral to FP intervention. Results: A total of 553 patients were referred to a reproductive specialist for FP in the setting of a medical diagnosis from 2011 to 2016. Of these, 208 patients satisfied the inclusion criteria and met with a reproductive specialist. Ninety patients underwent FP services. The average age at referral was 30.9 ± 7.9 years. Breast cancer (n=94, 45%) and leukemia/lymphoma (n=62, 30%) were the most prevalent cancer diagnoses. A 68.9% of patients underwent oocyte cryopreservation (n=62), 26.7% underwent embryo cryopreservation (n=24) and 4.4% underwent ovarian tissue preservation (n=4). The time interval from the referral to the FP intervention ranged from 1 to 810 days, with a median of 17 days. Conclusions: In the setting of a cancer diagnosis, most patients undergoing FP intervention underwent oocyte cryopreservation, were <35 years old, and underwent FP intervention in <30 days from referral. Whereas FP should ideally be initiated at the time of cancer diagnosis, all patients with a cancer diagnosis should be referred to a reproductive specialist and counseled on options for FP to preserve the optionality for the reproductive future they desire.

Ovarian Reserve Disorders, Can We Prevent Them? A Review.

The ovarian reserve is finite and begins declining from its peak at mid-gestation until only residual follicles remain as women approach menopause. Reduced ovarian reserve, or its extreme form, premature ovarian insufficiency, stems from multiple factors, including developmental, genetic, environmental exposures, autoimmune disease, or medical/surgical treatment. In many cases, the cause remains unknown and resulting infertility is not ultimately addressed by assisted reproductive technologies. Deciphering the mechanisms that underlie disorders of ovarian reserve could improve the outcomes for patients struggling with infertility, but these disorders are diverse and can be categorized in multiple ways. In this review, we will explore the topic from a perspective that emphasizes the prevention or mitigation of ovarian damage. The most desirable mode of fertoprotection is primary prevention (intervening before ablative influence occurs), as identifying toxic influences and deciphering the mechanisms by which they exert their effect can reduce or eliminate exposure and damage. Secondary prevention in the form of screening is not recommended broadly. Nevertheless, in some instances where a known genetic background exists in discrete families, screening is advised. As part of prenatal care, screening panels include some genetic diseases that can lead to infertility or subfertility. In these patients, early diagnosis could enable fertility preservation or changes in family-building plans. Finally, Tertiary Prevention (managing disease post-diagnosis) is critical. Reduced ovarian reserve has a major influence on physiology beyond fertility, including delayed/absent puberty or premature menopause. In these instances, proper diagnosis and medical therapy can reduce adverse effects. Here, we elaborate on these modes of prevention as well as proposed mechanisms that underlie ovarian reserve disorders.

A synopsis of the 2021 International Society of Fertility Preservation bi-annual meeting.

On November 19, 2021, the first virtual meeting of the International Society for Fertility Preservation (ISFP) took place. Eight experts in the field of reproductive medicine presented important updates on their research in the field of fertility preservation and reproductive surgery for absolute uterine factor infertility. Presentations included talks on ovarian stem cell therapy for premature ovarian insufficiency, practical aspects of oocyte vitrification, ovarian stimulation for patients with breast cancer, in vitro maturation of oocytes at the time of ovarian tissue harvesting, male fertility preservation, and uterine transplantation. These presentations are summarized below and can be viewed in their entirety at www.isfp-fertility.org.

Chronic superphysiologic AMH promotes premature luteinization of antral follicles in human ovarian xenografts.

Anti-Müllerian hormone (AMH) is produced by growing ovarian follicles and provides a diagnostic measure of reproductive reserve in women; however, the impact of AMH on folliculogenesis is poorly understood. We cotransplanted human ovarian cortex with control or AMH-expressing endothelial cells in immunocompromised mice and recovered antral follicles for purification and downstream single-cell RNA sequencing of granulosa and theca/stroma cell fractions. A total of 38 antral follicles were observed (19 control and 19 AMH) at long-term intervals (>10 weeks). In the context of exogenous AMH, follicles exhibited a decreased ratio of primordial to growing follicles and antral follicles of increased diameter. Transcriptomic analysis and immunolabeling revealed a marked increase in factors typically noted at more advanced stages of follicle maturation, with granulosa and theca/stroma cells also displaying molecular hallmarks of luteinization. These results suggest that superphysiologic AMH alone may contribute to ovulatory dysfunction by accelerating maturation and/or luteinization of antral-stage follicles.

Psychosocial response of infertile patients to COVID-19-related delays in care at the epicenter of the global pandemic.

BACKGROUND: The aim of this study was to describe the psychosocial response of the infertile population whose care was curtailed due to the COVID-19 pandemic. METHODS: A web-based cross-sectional survey was administered to 117 infertile patients at our Center who had their infertility treatment delayed due to suspension of care at our hospital during the COVID-19 pandemic. The survey consisted of 52-question multiple-choice questions including the Life Orientation Test-Revised (LOT-R) and the Hospital Anxiety and Depression Scale (HADS) instruments. Characteristics of respondents who "agreed" (strongly agree and agree) that "delaying treatment has permanently impacted my chances at future conception" were compared with participants who "disagreed" (neutral, disagree, and strongly disagree) using Fischer's Exact Test. RESULTS: In total, 79.5% agreed that delaying treatment has permanently impacted their chances at future conception. There were no discernible demographic differences between patients who "agreed" versus "disagreed" with the above statement. The mean LOT-R score was 14.1 (5.1) with an optimism score of 6.8 (2.6) and a pessimism score of 7.3 (2.9). The mean HADS depression score was 5.4 (3.4) with 28.2% reporting scores in the borderline-abnormal to abnormal range. The mean HADS anxiety score was 9.0 (3.9) with 64.6% reporting scores in the borderline-abnormal to abnormal range. Nearly one third of respondents (36.8%) reported wanting to "expedite/be more aggressive with treatment," whereas only 5.1% wanted to postpone treatment. CONCLUSIONS: Women undergoing ART during the COVID-19 pandemic express significant concern and signs of distress about how delays in care affect their future reproductive potential.

Exogenous insulin-like growth factor 1 accelerates growth and maturation of follicles in human cortical xenografts and increases ovarian output in mice.

OBJECTIVE: To measure the influence of exogenous insulin-like growth factor 1 (IGF1) on follicle growth and maturation in human ovarian cortical xenografts. DESIGN: Xenotransplantation model. SETTING: University-based research laboratory. PATIENTS/ANIMALS: Ovarian tissue was donated with consent and institutional review board approval by brain-dead organ donors or patients undergoing ovarian tissue cryopreservation for fertility preservation. Cortical fragments were transplanted into immunocompromised mice. INTERVENTIONS: Cryopreserved ovarian cortical fragments from four women (aged 19, 25, 33, and 46 years) were transplanted into the gluteus muscle of immunocompromised mice in a fibrin matrix containing endothelial cells that were transduced with lentiviral particles encoding secreted IGF1. Xenografts were recovered after 3, 8, and 14 weeks. In addition, C57/Bl6 mice underwent intraovarian injection of saline or recombinant IGF1 (60 µg), followed by superovulation, analysis of ethynyl-deoxyuridine incorporation, and ribonucleic acid sequencing of the whole ovaries. MAIN OUTCOME MEASURES: For xenografts: follicle count and distribution; antral follicle count; and corpora lutea/albicans count. For mice: follicle count and distribution; oocyte yield, ethynyl-deoxyuridine incorporation (granulosa cell proliferation); and ovarian transcriptomic signature. RESULTS: At 3 weeks, xenografts in the IGF1 condition revealed a decreased percentage of primary follicles and increased percentage of secondary follicles that were concentrated in the preantral subtype; at 8 weeks, an increase in secondary follicles was concentrated in the simple subtype; after 14 weeks, primordial follicles were reduced, and while the number of advanced follicles did not power the experiment to demonstrate significance, antral follicles reduced and corpora lutea increased. Supporting experiments in mice revealed an increase in normal oocytes following intraovarian injection of recombinant IGF1 (60 µg) as well as increased proliferative index among follicles of secondary and preantral stages. Ribonucleic acid sequencing analysis of the whole ovaries following injection of recombinant IGF1 (25 µg) revealed an acute (24 hours) upregulation of transcripts related to steroidogenesis and luteinization. CONCLUSIONS: Exogenous IGF1 advances the pace of growth among primordial, primary, and secondary stage follicles but results in near absence of antral stage follicles in long-term (14 weeks) xenografts. In mice, acute administration of IGF1 promotes follicle advance and increased oocyte yield. The results suggest that while superphysiological IGF1 alone advances the pace of growth among early/preantral follicles, a sustained and/or later-stage influence undermines antral follicle growth/survival or promotes premature luteinization. These findings provide a temporal framework for interpreting follicle growth/mobilization and may be useful in understanding the clinical application of human growth hormone in the context of assisted reproduction.

Comparison of Human Antral Follicles of Xenograft versus Ovarian Origin Reveals Disparate Molecular Signatures.

The activation, growth, and maturation of oocytes to an ovulatory phase, termed folliculogenesis, is governed by the orchestrated activity of multiple specialized cell types within the ovary; yet, the mechanisms governing diversification and behavior of discrete cellular sub-populations within follicles are poorly understood. We use bulk and single-cell RNA sequencing to distinguish the transcriptional signature of prospectively isolated granulosa and theca/stroma cell subsets within human antral follicles derived from xenografts or ovaries. The analysis deconstructs phenotypic diversification within small (<4 mm) antral follicles, identifying secreted factors that are differentially enriched between mural and oophorus granulosa cells, and segregating stromal/support and steroidal activity between theca externa and interna, respectively. Multiple factors are differentially expressed in follicles of xenograft versus ovarian origin. These data capture a high-resolution transcriptional signature of granulosa and theca subpopulations and provide a systems-level portrait of cellular diversification in early antral human follicles.

Delay in IVF treatment up to 180 days does not affect pregnancy outcomes in women with diminished ovarian reserve.

STUDY QUESTION: Will a delay in initiating IVF treatment affect pregnancy outcomes in infertile women with diminished ovarian reserve? SUMMARY ANSWER: A delay in IVF treatment up to 180 days does not affect the live birth rate for women with diminished ovarian reserve when compared to women who initiate IVF treatment within 90 days of presentation. WHAT IS KNOWN ALREADY: In clinical practice, treatment delays can occur due to medical, logistical or financial reasons. Over a period of years, a gradual decline in ovarian reserve occurs which can result in declining outcomes in response to IVF treatment over time. There is disagreement among reproductive endocrinologists about whether delaying IVF treatment for a few months can negatively affect patient outcomes. STUDY DESIGN, SIZE, DURATION: A retrospective cohort study of infertile patients in an academic hospital setting with diminished ovarian reserve who started an IVF cycle within 180 days of their initial consultation and underwent an oocyte retrieval with planned fresh embryo transfer between 1 January 2012 and 31 December 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: Diminished ovarian reserve was defined as an anti-Müllerian hormone (AMH) <1.1 ng/ml. In total, 1790 patients met inclusion criteria (1115 immediate and 675 delayed treatment). Each patient had one included cycle and no subsequent data from additional frozen embryo transfer cycles were included. Since all cycle outcomes evaluated were from fresh embryo transfers, no genetically tested embryos were included. Patients were grouped by whether their cycle started 1-90 days after presentation (immediate) or 91-180 days (delayed). The primary outcome was live birth (≥24 weeks of gestation). A subgroup analysis of more severe forms of diminished ovarian reserve was performed to evaluate outcomes for patients with an AMH <0.5 and for patients >40 years old with an AMH <1.1 ng/ml (Bologna criteria for diminished ovarian reserve). Logistic regression analysis, adjusted a priori for patient age, was used to estimate the odds ratio (OR) with a 95% CI. All pregnancy outcomes were additionally adjusted for the number of embryos transferred. MAIN RESULTS AND THE ROLE OF CHANCE: The mean ± SD number of days from presentation to IVF start was 50.5 ± 21.9 (immediate) and 128.8 ± 25.9 (delayed). After embryo transfer, the live birth rate was similar between groups (immediate: 23.9%; delayed: 25.6%; OR 1.08, 95% CI 0.85-1.38). Additionally, a similar live birth rate was observed in a subgroup analysis of patients with an AMH <0.5 ng/ml (immediate: 18.8%; delayed: 19.1%; OR 0.99, 95% CI 0.65-1.51) and in patients >40 years old with an AMH <1.1 ng/ml (immediate: 12.3%; delayed: 14.7%; OR 1.21, 95% CI 0.77-1.91). LIMITATIONS, REASONS FOR CAUTION: There is the potential for selection bias with regard to the patients who started their IVF cycle within 90 days compared to 91-180 days after initial consultation. In addition, we did not include patients who were seen for initial evaluation but did not progress to IVF treatment with oocyte retrieval; therefore, our results should only be applied to patients with diminished ovarian reserve who complete an IVF cycle. Finally, since we excluded patients who started their IVF cycle greater than 180 days from their first visit, it is not known how such a delay in treatment affects pregnancy outcomes in IVF cycles. WIDER IMPLICATIONS OF THE FINDINGS: A delay in initiating IVF treatment in patients with diminished ovarian reserve up to 180 days from the initial visit does not affect pregnancy outcomes. This observation remains true for patients who are in the high-risk categories for poor response to ovarian stimulation. Providers and patients should be reassured that when a short-term treatment delay is deemed necessary for medical, logistic or financial reasons, treatment outcomes will not be affected. STUDY FUNDING/COMPETING INTEREST(S): No financial support, funding or services were obtained for this study. The authors do not report any potential conflicts of interest. TRIAL REGISTRATION NUMBER: Not applicable.

BRCA carriers have similar reproductive potential at baseline to noncarriers: comparisons in cancer and cancer-free cohorts undergoing fertility preservation.

OBJECTIVE: To investigate whether BRCA carriers with and without malignancy have decreased ovarian reserve at baseline compared with BRCA noncarriers. DESIGN: Retrospective cohort study. SETTING: Academic medical center. PATIENT(S): Seven-hundred and ninety-five oocyte cryopreservation patients, comprising BRCA carriers with and without malignancy (n = 57) and BRCA noncarriers (n = 738). INTERVENTION(S): Fertility preservation with oocyte cryopreservation. MAIN OUTCOME MEASURE(S): Antral follicle count (AFC), antimüllerian hormone (AMH) concentration, day-3 follicle-stimulating hormone (FSH) level, number of harvested oocytes, and number of mature/cryopreserved oocytes. RESULT(S): In the cancer cohort we compared BRCA-positive breast cancer (n = 38) with BRCA-negative breast cancer (n = 53) and with non-breast-cancer malignancies (n = 85). In the cancer-free cohort we compared BRCA carriers (n = 19) with women undergoing elective egg freezing (n = 600). We also compared the BRCA1 (n = 31) versus the BRCA2 carriers (n = 18). The patients' mean ages were 32.4 ± 3.6 years and 35.5 ± 4.3 years in the BRCA carrier and noncarrier cohorts, respectively. BRCA status was associated with a higher day-3 FSH level in the cancer cohort, but we found no changes in the other outcomes compared with the BRCA-negative cancer groups. BRCA carriers without cancer exhibited a higher AFC and number of mature oocytes compared with the patients undergoing planned egg freezing. Overall (cancer and cancer-free cohorts), the BRCA carriers had an increased AFC (15.5 ± 4.6 vs. 12.6 ± 5.7) and number of mature/cryopreserved oocytes (14.0 ± 7.9 vs. 10.4 ± 6.9) compared with the BRCA noncarriers but had no differences in other outcomes. CONCLUSION(S): BRCA carriers with and without malignancy exhibit comparable ovarian reserve and responses to ovarian stimulation compared with women with BRCA-negative cancers and cancer-free controls.

Co-transplantation of Human Ovarian Tissue with Engineered Endothelial Cells: A Cell-based Strategy Combining Accelerated Perfusion with Direct Paracrine Delivery.

Infertility is a frequent side effect of chemotherapy and/or radiotherapy and for some patients, cryopreservation of oocytes or embryos is not an option. As an alternative, an increasing number of these patients are choosing to cryopreserve ovarian tissue for autograft following recovery and remission. Despite improvements in outcomes among patients undergoing auto-transplantation of cryopreserved ovarian tissue, efficient revascularization of grafted tissue remains a major obstacle. To mitigate ischemia and thus improve outcomes in patients undergoing auto-transplantation, we developed a vascular cell-based strategy for accelerating perfusion of ovarian tissue. We describe a method for co-transplantation of exogenous endothelial cells (ExECs) with cryopreserved ovarian tissue in a mouse xenograft model. We extend this approach to employ ExECs that have been engineered to constitutively express Anti-Mullerian hormone (AMH), thus enabling sustained paracrine signaling input to ovarian grafts. Co-transplantation with ExECs increased follicular volume and improved antral follicle development, and AMH-expressing ExECs promoted retention of quiescent primordial follicles. This combined strategy may be a useful tool for mitigating ischemia and modulating follicular activation in the context of fertility preservation and/or infertility at large.

Gonadotropin-releasing hormone agonist trigger increases the number of oocytes and embryos available for cryopreservation in cancer patients undergoing ovarian stimulation for fertility preservation.

OBJECTIVE: To compare the oocyte and embryo yield associated with GnRH-agonist triggers vs. hCG triggers in cancer patients undergoing controlled ovarian stimulation (COS) for fertilization preservation. DESIGN: Retrospective cohort study. SETTING: Academic center. PATIENT(S): Cancer patients undergoing COS with letrozole and gonadotropins or gonadotropin-only protocols for oocyte or embryo cryopreservation. INTERVENTION(S): Gonadotropin-releasing hormone agonist or hCG trigger. MAIN OUTCOME MEASURE(S): Number of metaphase II (MII) oocytes or two-pronuclei (2PN) embryos available for cryopreservation were primary outcomes. Separate multivariate linear regression models were used to assess the effect of trigger type on the primary outcomes, after controlling for confounders of interest. RESULT(S): A total of 341 patients were included, 99 (29.0%) in the GnRH-agonist group and 242 (71%) in the hCG group. There was no difference in the baseline demographics of patients receiving GnRH-agonist or hCG triggers. Within the letrozole and gonadotropins group (n = 269), the number (mean ± SD, 11.8 ± 5.8 vs. 9.9 ± 6.0) and percentage of MII oocytes (89.6% vs. 73.0%) available for cryopreservation was higher with GnRH-agonist triggers compared with hCG triggers. Similar results were noted with GnRH-agonist triggers in the gonadotropin-only group (n = 72) (i.e., a higher number [13.3 ± 7.9 vs. 9.3 ± 6.0] and percentage of MII oocytes [85.7% vs. 72.8%] available for cryopreservation). Multivariate linear regression demonstrated approximately three more MII oocytes and 2PN embryos available for cryopreservation in the GnRH-agonist trigger group, irrespective of cancer and COS protocol type. CONCLUSION(S): Utilization of a GnRH-agonist trigger increases the number of MII oocytes and 2PN embryos available for cryopreservation in cancer patients undergoing COS for fertility preservation.

Fertility Preservation and Sexual Health After Cancer Therapy.

Recent developments in cancer diagnostics and treatments have considerably improved long-term survival rates. Despite improvements in chemotherapy regimens, more focused radiotherapy and diverse surgical options, cancer treatments often have gonadotoxic side-effects that can manifest as loss of fertility or sexual dysfunction, particularly in young cancer survivors. In this review, we focus on two pertinent quality-of-life issues in female cancer survivors of reproductive age-fertility preservation and sexual function. Fertility preservation encompasses all clinical and laboratory efforts to preserve a woman's chance to achieve future genetic motherhood. These efforts range from well-established protocols such as ovarian stimulation with cryopreservation of embryos or oocytes, to nascent clinical trials involving cryopreservation and re-implantation of ovarian tissue. Therefore, fertility preservation strategies are individualized to the cancer diagnosis, time interval until initiation of treatments must begin, prognosis, pubertal status, and maturity level of patient. Some patients choose not to pursue fertility preservation, and the conversation then centers around other quality of life issues. Not all cancer treatments cause loss of fertility; however, most treatments can directly impact the physical and psychosocial aspects of sexual function. Cancer treatment is also associated with fear, anxiety, and depression, which can further decrease sexual desire, function, and frequency. Sexual dysfunction after cancer treatment is generally ascertained by compassionate inquiry. Strategies to promote sexual function after cancer treatment include pelvic floor exercises, clitoral therapy devices, pharmacologic agents, as well as couples-based psychotherapeutic and psycho-educational interventions. Quality-of-life issues in young cancer survivors are often best addressed by utilizing a multidisciplinary team consisting of physicians, nurses, social workers, psychiatrists, sex educators, counselors, or therapists.

Engineered endothelium provides angiogenic and paracrine stimulus to grafted human ovarian tissue.

Despite major advances in tissue cryopreservation and auto-transplantation, reperfusion ischemia and hypoxia have been reported as major obstacles to successful recovery of the follicular pool within grafted ovarian tissue. We demonstrate a benefit to follicular survival and function in human ovarian tissue that is co-transplanted with exogenous endothelial cells (ExEC). ExECs were capable of forming functionally perfused vessels at the host/graft interface and increased both viability and follicular volume in ExEC-assisted grafts with resumption of antral follicle development in long-term grafts. ExECs that were engineered to constitutively express anti-mullerian hormone (AMH) induced a greater proportion of quiescent primordial follicles than control ExECs, indicating suppression of premature mobilization that has been noted in the context of ovarian tissue transplantation. These findings present a cell-based strategy that combines accelerated perfusion with direct paracrine delivery of a bioactive payload to transplanted ovarian tissue.