Variation in follicle health and development in cultured cryopreserved ovarian cortical tissue: a study of ovarian tissue from patients undergoing fertility preservation.

This study investigated how follicle health and development in human ovarian tissue cryopreserved for fertility preservation varied between patients before and after 6 d of in vitro culture. Ovarian tissue from 12 patients (9-25 years) was used. In 3 patients, a 1hr neutral red (NR) incubation was used to identify tissues with viable follicles. Tissues were fixed, sectioned and follicles staged and graded for health. Inter-patient differences were observed in the non-cultured tissue in the number of both healthy follicles (p = 0.005) and growing follicles (p = 0.005). After culture there was significant variation in the number of transitional, primary and secondary follicles between patients (p < 0.001). Asymmetric primary follicles with a single complete layer of granulosa cells plus two or more additional partial layers were 5.5 times more likely to be observed in cultured compared to non-cultured tissue (p = 0.0063). Non-cultured (p = 0.0125) and cultured (p < 0.001) tissue selected using NR had more healthy follicles compared to tissue not selected using NR. Non-cultured and cultured tissue selected using NR had more healthy follicles compared to tissue not selected using NR (p = 0.0125; p < 0.001). We demonstrate that inter-patient variation exists in the health and development of follicles before and after culture. Culture systems need to be optimized to support cryopreserved ovarian tissue and these findings should prompt researchers to consider patient variation when evaluating culture systems.

Analysing culture methods of frozen human ovarian tissue to improve follicle survival.

In vitro follicle growth is a potential fertility preservation method for patients for whom current methods are contraindicated. Currently, this method has only been successful using fresh ovarian tissue. Since many patients who may benefit from this treatment currently have cryopreserved ovarian tissue in storage, optimising in vitro follicle growth (IVG) for cryopreserved-thawed tissue is critical. This study sought to improve the first step of IVG by comparing different short-term culture systems for cryopreserved-thawed human ovarian tissue, in order to yield a higher number of healthy multilayer follicles. We compared two commonly used culture media (αMEM and McCoy's 5A), and three plate conditions (300 µL, 1 mL on a polycarbonate membrane and 1 mL in a gas-permeable plate) on the health and development of follicles after 6 days of culture. A total of 5797 follicles from three post-pubertal patients (aged 21.3 ± 2.3 years) were analysed across six different culture conditions and non-cultured control. All culture systems supported follicle development and there was no difference in developmental progression between the different conditions tested. Differences in follicle morphology were evident with follicles cultured in low volume conditions having significantly greater odds of being graded as morphologically normal compared to other conditions. Furthermore, culture in a low volume of αMEM resulted in the highest proportion of morphologically normal primary and multilayer follicles (23.8% compared to 6.3-19.9% depending on condition). We, therefore, recommend culturing cryopreserved human ovarian tissue in a low volume of αMEM to support follicle health and development. LAY SUMMARY: Ovaries contain a large number of follicles, each containing an immature egg and other important cells. Cancer treatments can lead to long-lasting negative side effects to the ovaries including the destruction of follicles, resulting in infertility. One strategy to preserve fertility is freezing of ovaries or ovarian tissue in girls and women undergoing cancer treatment. The long-term aim is to thaw and grow their ovarian tissue in the laboratory to obtain mature eggs, which can then be fertilised. In this study, we compared six different methods of growing previously frozen human ovarian tissue in order to best support follicle growth and health. We found that using the lowest amount of αMEM medium (a specific type of nutrient-rich growth solution) resulted in the highest proportion of healthy follicles. Improving the methods used to grow ovarian tissue, particularly frozen tissue, is important for future fertility preservation.

Rescue In Vitro Maturation in Polycystic Ovarian Syndrome Patients Undergoing In Vitro Fertilization Treatment who Overrespond or Underrespond to Ovarian Stimulation: Is It A Viable Option? A Case Series Study.

BACKGROUND: This study intends to present the role of rescue in vitro maturation (IVM) in polycystic ovarian syndrome (PCOS) patients undergoing in vitro fertilization (IVF) treatment who have inappropriate responses to ovarian stimulation. MATERIALS AND METHODS: This was a retrospective case series study of five PCOS patients undergoing IVF treatment considered for cycle cancellation due to increased risk of ovarian hyperstimulation syndrome (OHSS) as group A or poor response to ovarian stimulation as group B. Patients in group A had high oestradiol levels and recruitment of high numbers of small/intermediate sized follicles that did not meet the criteria for human chorionic gonadotropin (hCG) triggering. Patients in group B responded inadequately to hormonal stimulation despite high gonadotropin dosage. Treatment was changed to rescue IVM cycles after the patients provided consent. RESULTS: In group A, three IVF patients deemed to have high chances of developing OHSS as evidenced by high oestradiol levels were converted to IVM. A total of the 58/68 oocytes retrieved were mature or matured in vitro. There were 26 cleaving embryos obtained. Two patients had live births and one patient suffered a miscarriage. In group B, rescue IVM was implemented in two patients due to poor ovarian response (POR). A total of 22/26 oocytes retrieved were mature or matured in vitro. There were 13 cleaving embryos obtained. One patient had a live birth, whilst the other suffered a miscarriage. CONCLUSION: Rescue IVM could be a viable option in PCOS patients undergoing IVF treatment who are unable to safely meet the criteria for hCG triggering due to overresponse to ovarian stimulation or ovarian resistance to high doses of stimulation. Conversion to IVM can still result in reasonable oocyte retrieval and lead to clinical pregnancy and live births without the risks of OHSS.

Time to consider ovarian tissue cryopreservation for girls with Turner's syndrome: an opinion paper.

Turner's syndrome (TS) is the most common sex chromosome abnormality in women. In addition to short stature and gonadal dysgenesis, it is associated with cardiac and renal anomalies. Due to rapid follicular atresia, the majority of women with TS suffer from primary ovarian insufficiency around puberty. Thus far, donor oocyte conception has been the key fertility option for these women. With advancing technology, ovarian tissue cryopreservation (OTCP) has emerged as a clinically justifiable option especially for pre-pubertal girls with cancer. Recently published results following the use of cryopreserved ovarian tissue are reassuring. It would be prudent to consider the extension of these technological and scientific advances to other conditions, such as TS, where accelerated follicular atresia is suspected. It is possible to obtain competent oocytes from cryopreserved ovaries of girls with TS provided the ovaries were preserved before ovarian failure. However, it is a complex decision whether and when to offer OTCP as a fertility preservation (FP) option for girls with TS. The rate of decline in fertility is variable in girls with TS and can be more complex in cases with mosaicism. On the other hand, OTCP has shown some promising results in patients with cancer, which can potentially be replicated in TS and other benign indications of patients at risk of premature ovarian failure. There are proven psychological and clinical benefits of FP. Thus, an argument could be made for offering OTCP to these patients to endow these girls with the option of having biological fertility using this innovative technology. Ethical, clinical and psychological dilemmas should be considered, discussed and addressed before considering such a novel approach. We believe that the time has come to start this discussion and open this avenue of FP for girls with TS.

Ovarian response and follow-up outcomes in women diagnosed with cancer having fertility preservation: Comparison of random start and early follicular phase stimulation - cohort study.

OBJECTIVES: To determine response to controlled ovarian stimulation in a random start cycle and utilisation of cryopreserved oocytes and embryos in cancer patients. STUDY DESIGN: A retrospective cohort study was carried out in an assisted reproductive treatment centre. Participants included 137 cancer patients who underwent controlled ovarian stimulation for fertility preservation between 1 Feb 2003 and 30 June 2016. The primary outcome variable was number of oocytes retrieved. Multivariable logistic regression analysis was performed, and differences compared using Chi squared test and student t-test as appropriate. P < 0.05 was considered statistically significant. RESULTS: Using the antagonist protocol, there was no difference in number of oocytes retrieved between the early follicular phase or at random start stimulation; 11.9 (95% CI 10.3-13.5) and 12.9 (95% CI 9.6-16.2), P = 0.602, respectively. Similarly, the number of embryos frozen was comparable between those starting stimulation in early follicular and random phase, 6.7 (95% CI 5.7-7.7) and 5.1 (95% CI 3.6-6.5), P= 0.1508 respectively. Among patients undergoing fertility preservation, those who returned to attempt a pregnancy had an ongoing pregnancy rate of 24.3%. Overall, 65% of oocytes and embryos were still in storage, however, 16 (11.7%) had elected to have their oocytes or embryos disposed of. CONCLUSION(S): For women faced with potential gonadotoxic treatment and requiring urgent fertility preservation, ovarian stimulation with the antagonist protocol can be started at random without compromising ovarian response. Pregnancy rates following utilisation of frozen-thawed oocytes and embryos are promising, however, more research is needed to understand reasons underlying disposition of oocytes and embryos especially when survival following cancer treatment has improved significantly.

Grade of the inner cell mass, but not trophectoderm, predicts live birth in fresh blastocyst single transfers.

Debate continues over which morphological parameter is most important in selecting blastocysts for transfer. We aimed to investigate which parameter more accurately predicts the occurrence of a live birth by designing a retrospective cohort study of 1084 fresh elective single blastocyst transfers. Primary outcome was live birth rate (LBR) and secondary outcomes were implantation, clinical pregnancy and early pregnancy loss rates. Blastocyst expansion and inner cell mass (ICM), but not trophoectoderm, were associated with LBR in the definitive multivariable regression analysis. When ICM grade dropped from A to C the likelihood of achieving a live birth was reduced by 55% (OR= 0.45, 95% CI 0.26-0.79, p = .005). These results were similar for clinical pregnancy rates. Early pregnancy loss rates of embryos with ICM grade C were more than double (38.0%) compared to those of grades A (15.95%) and B (17.17%, p = .002). The transfer of an embryo with an optimal inner cell mass reduces early pregnancy loss and increases the likelihood of a live birth. We did not find any significant association between trophectoderm and LBR in the multivariable analysis in contrast with recent studies.

Deleterious effects of obesity upon the hormonal and molecular mechanisms controlling spermatogenesis and male fertility.

Worldwide obesity rates have nearly doubled since 1980 and currently over 10% of the population is obese. In 2008, over 1.4 billion adults aged 20 years and older had a body mass index or BMI above a healthy weight and of these, over 200 million men and nearly 300 million women were obese. While obesity can have many ramifications upon adult life, one growing area of concern is that of reproductive capacity. Obesity affects male infertility by influencing the hypothalamic-pituitary-gonadal axis, thus causing detrimental effects upon spermatogenesis and subsequent fertility. In particular, evidence indicates that excess adipose tissue can alter the relative ratio of testosterone and oestrogen. Additional effects involve the homeostatic disruption of insulin, sex-hormone-binding-globulin, leptin and inhibin B, leading to diminished testosterone production and impairment to spermatogenesis. Aberrant spermatogenesis arising from obesity is associated with downstream changes in key semen parameters, defective sperm capacitation and binding, and deleterious effects on sperm chromatin structure. More recent investigations into trans-generational epigenetic inheritance further suggest that molecular changes in sperm that arise from obesity-related impaired spermatogenesis, such as modified sperm RNA levels, DNA methylation, protamination and histone acetylation, can impact upon the development of offspring. Here, we summarise our current understanding of how obesity exerts influence over spermatogenesis and subsequent fertility status, and make recommendations for future investigative research.

In vitro maturation and surrogacy in patients with vascular-type Ehlers-Danlos syndrome--a safe assisted reproductive technology approach.

Ehlers-Danlos syndrome (EDS) is an autosomal dominant connective tissue disorder with one of the highest maternal mortality rates of any condition. Patients with the vascular type of EDS are prone to spontaneous arterial and visceral ruptures. The occurrence of these severe and life-threatening complications is increased in pregnancy. Moreover, these patients carry a 50% risk of having an affected child. However, little is known about the risks of assisted conception treatments on these patients. We present the case of a 33-year-old woman suffering from EDS with a history of repeated ruptures of arterial aneurysms and a recently ruptured aneurysm of the splenic artery during her first intracytoplasmic sperm injection (ICSI) cycle who was then advised to undergo only unstimulated cycles. After a few natural ICSI cycles, the patient safely underwent two in vitro maturation cycles with pre-implantation genetic diagnosis in our unit. An unaffected blastocyst was transferred into a surrogate host. To our knowledge, this is the first case of EDS in assisted reproduction technologies including pre-implantation genetic diagnosis to be reported in the medical literature. This case has shown that unstimulated in vitro maturation and pre-implantation genetic diagnosis can safely be offered for vascular-type Ehlers-Danlos patients.

The case for aromatase inhibitors use in oncofertility patients. Should aromatase inhibitors be combined with gonadotropin treatment in breast cancer patients undergoing ovarian stimulation for fertility preservation prior to chemotherapy? A debate.

Breast cancer is one of the hormone-dependent cancers that may be adversely affected by elevated oestrogen or progesterone concentrations, particularly the endocrine active (hormone receptor positive) breast cancers. Treatment for breast cancer patients aimed at fertility preservation, includes ovarian hyperstimulation, the harvest of oocytes, and subsequent cryopreservation of oocytes or embryos. Classically, gonadotrophins have been used effectively for ovulation induction, a treatment often accompanied by high blood oestrogen concentrations produced by the hyperstimulated granulosa cells. Despite the uncertainty which surrounds this issue and the lack of clear-cut clinical evidence, it is still of major concern that these ensuing high hormone levels might be associated with a high risk of recurrence of the cancer. A growing number of clinical studies have strongly suggested the benefits of using aromatase inhibitors in infertility treatment, both as single agents or as adjuncts to FSH-containing ovulation induction regimes in reproductive medicine. Combining gonadotrophins with aromatase inhibitors would augment the stimulation effect, with a reduced increase in serum concentrations of estradiol. We propose to open a debate over the use of aromatase inhibitors in combination with FSH in ovulation induction treatment of breast cancer oncofertility patients. As the safety of aromatase inhibitors such as letrozole has recently been demonstrated in several studies, and there is growing concern over the possible detrimental effects of high estradiol levels on breast cancer cells (at least in mouse models), the co-administration of letrozole in these patients would reduce both the high supraphysiologic serum levels of estradiol and the intratumoral in situ production of oestrogen. However, since it is unlikely that a well-founded evidence-based justification of this treatment will be formulated in the near future, based on well-designed prospective randomised controlled trials, we advocate a wider use of aromatase inhibitors in combination with gonadotrophins in breast cancer patients, especially those with hormone-receptor-positive tumours.

In vitro maturation or in vitro fertilization for women with polycystic ovaries? A case-control study of 194 treatment cycles.

OBJECTIVE: To compare the outcome of unstimulated in vitro maturation (IVM) and routine IVF/intracytoplasmic sperm injection (ICSI) for women with polycystic ovaries (PCO). DESIGN: Retrospective case-control study. SETTING: Fertility unit. PATIENT(S): Ninety-seven patients undergoing IVM were compared with 97 patients undergoing IVF. All had PCO and matched for age, infertility diagnosis, and ovulatory status. INTERVENTION(S): In vitro maturation cycles were unstimulated and hCG was administered 35-40 hours before oocyte retrieval. Oocytes were matured in vitro for 24-48 hours before insemination by ICSI. Endometrial priming with E(2) and P was commenced from the day of egg retrieval and one to two embryos were transferred on days 2-5 of development. Standard long protocol IVF/ICSI was used in the control group. MAIN OUTCOME MEASURE(S): Live birth rate per cycle and ovarian hyperstimulation syndrome (OHSS) rate. RESULT(S): Overall, 65% of IVM eggs matured in vitro in the IVM group. Implantation rates were significantly higher in the IVF group (19.4% vs. 12.9%) as clinical pregnancy rates (50.5% vs. 19.6%) and live birth rates (44.3% vs. 16.5%) than in the IVM group. The OHSS rate was significantly higher in the IVF group (8.2% vs. 0%). CONCLUSION(S): In vitro maturation is a safer and simpler alternative to conventional IVF for women with PCO. It avoids difficulties of gonadotropin stimulation and the risk of OHSS but has a significantly lower live birth rate. Current research projects aim to close the success gap between IVM and IVF.

Thin unresponsive endometrium--a possible complication of surgical curettage compromising ART outcome.

PURPOSE: Endometrial thickness is important for implantation. Little data addresses the etiology of persistently thin endometrium. We present a patient cohort in order to define common features and draw conclusions. METHODS: Thirteen out of 1,405 IVF patients repeatedly had thin unresponsive endometrium (<7 mm). Age, history, uterine cavity status, treatment type and outcome were examined. RESULTS: Patient age was 35.9 +/- 5.7 years. Ten patients had a curettage performed previously. Nine patients had normal cavity and endometrium, and in four adhesions were diagnosed and removed. Out of 99 cycles performed afterwards, endometrial thickness increased in 22. ETs were performed in 49 cycles resulting in 11 pregnancies. Their outcome was eight miscarriages, two terminations due to malformations, and one live birth. CONCLUSIONS: Thin unresponsive endometrium was associated with curettage, not necessarily with intrauterine adhesions. Even if adequate thickening eventually occurred, the reproductive outcome was still very poor. Therefore other alternatives should be sought for these patients.

Is estradiol mandatory for an adequate follicular and embryo development? A mouse model using aromatase inhibitor (anastrozole).

BACKGROUND: Although high levels of estradiol are found in the follicular fluid, little is known about its necessity for adequate follicular growth, oocyte maturation and embryo development. Arimidex (anastrozole) is a potent aromatase inhibitor capable to induce an in-vivo milieu deprived of estradiol. This study uses a mouse model applying Arimidex to create an in-vivo system lacking of estradiol, in order to explore whether this gonadal steroid hormone is mandatory for folliculogenesis followed by normal fertilization and embryo development. METHODS: Experiment 1: Immature C57 Black female mice, aged 3-4 weeks were superovulated by 5 IU PMSG given intraperitoneally. A study group (9 mice) was concomitantly injected with 0.1 mg of Arimidex intraperitoneally given the morning day before PMSG, the morning day of PMSG injection and the following two days. The control group (8 mice) was similarly injected with normal saline. Estradiol (E2) and progesterone (P) serum levels were tested 48 hours after PMSG and the ovaries of each mouse blindly examined by a pathologist to evaluate follicular development. Experiment 2: 48 h after PMSG superovulation, hCG (7.5 IU) was injected intraperitoneally, followed by mating. The study group was treated with Arimidex 0.1 mg intraperitoneally daily from a day prior to PMSG injection to the day of sacrifice. The control group was treated similarly by normal saline. Forty-two hours after mating blood was withdrawn for E2 and P levels followed by tubal dissection. Embryos of 2-4 cells were cultured in-vitro and the development to the morula, blastocyst and hatching blastocyst stages were examined 24, 42, and 48 h later. RESULTS: Experiment 1: A significant reduction of E2 levels was achieved in the Arimidex group in comparison to control group (126.3+/-104.8 and 1910+/-960 pmol/L, respectively; p < 0.0001). Nevertheless, the two groups did not differ by the mean number of follicles (27+/-9.5 and 30.4+/-13.0) or the distribution for antral (65% and 68.4%) and pre-antral (35% and 31.6%) follicles, respectively. Experiment 2: The reduction of estradiol during follicular phase did not hamper follicular development, in-vivo fertilization and in-vitro embryo development. Similar rates of embryo development to the morula stage (90.6% and 86%), blastocyst stage (86% and 89%) and hatching blastocyst (81% and 78%) were achieved in the Arimidex group and the control group, respectively. CONCLUSIONS: Adequate folliculogenesis is independent of estrogen but is conditioned on gonadotropin stimulation. Moreover, depletion of estradiol in the vicinity of the oocyte did not impair its developmental potential, including its fertilization and development into morulae, blastocysts and hatching blastocysts.

Does postmenopausal hormone replacement therapy affect intraocular pressure?

PURPOSE: To assess the effects of postmenopausal hormone replacement therapy (HRT) on intraocular pressure (IOP). PATIENTS AND METHODS: This was a cross-sectional controlled study, including 107 women aged 60 to 80 years receiving HRT and 107 controls who have never received HRT. All subjects underwent IOP assessment and funduscopic photography for cup-to-disc (C/D) ratios, and completed questionnaires regarding personal and family history of glaucoma, hormone replacement therapy, lifetime estrogen and progesterone exposure, and cardiovascular risk factors. Main Outcome Measures included IOP, prevalence of increased IOP, and C/D ratios. RESULTS: The groups did not differ in mean IOP (15.3 versus 15.3 mm Hg), mean vertical (0.18 versus 0.21) and horizontal (0.17 versus 0.14) C/D ratios, and in prevalence of increased IOP (15% versus 14%), C/D ratio (7% versus 7%), or glaucoma (9% versus 11%). A personal history of ischemic heart disease was the only risk factor associated with increased IOP (O.R. = 4.63, P = 0.003). Lifetime estrogen and progesterone exposure, including pregnancies, deliveries, menstruation years, and the use of oral contraceptives did not significantly affect the risk for increased IOP. CONCLUSION: Hormone replacement therapy and lifetime estrogen and progesterone exposure do not seem to affect IOP or the risk for increased IOP. A personal history of ischemic heart disease may be associated with a higher risk for this disorder.

The effect of hormone therapy on the risk for age-related maculopathy in postmenopausal women.

OBJECTIVE: To evaluate the effect of postmenopausal hormone therapy (HT) as well as the use of oral contraceptives and lifetime endogenous hormone exposure on the risk for age-related maculopathy (ARM) in postmenopausal women. DESIGN: This was a cross-sectional, controlled study. A total of 102 women from 60 to 80 years of age who were receiving HT and 100 controls underwent a detailed clinical funduscopic evaluation and stereoscopic fundus photography for the presence and grading of ARM. All participants completed a standardized questionnaire regarding vascular risk factors, HT, and lifetime exogenous and endogenous estrogen and progesterone exposure. Statistical analysis was performed using Student's t test, chi2 test, and a multivariate logistic regression model. RESULTS: The HT and the non-HT groups did not differ in terms of early (11% v 15%), late (6% v 6%), or wet (2% v 2%) ARM prevalence rates. Women with ARM were significantly older than controls (69 v 66 years; P = 0.001, 95% CI = 0.008 - 0.027) and were more likely to have ischemic heart disease (21% v 9%; OR = 2.86, P = 0.03, 95% CI = 0.020 - 0.360). Lifetime exogenous and endogenous hormone exposures and other cardiovascular risk factors were not significantly different among women with ARM as compared with controls. CONCLUSION: Postmenopausal HT may not affect the risk for either early or late ARM in women aged 60 to 80 years. The risk for both entities is not necessarily affected by either exogenous or endogenous lifetime hormone exposure. A history of ischemic heart disease may be associated with an increased risk for ARM.