Valeria Stella Vanni

Is the oocyte quality affected by endometriosis? A review of the literature

Endometriosis is an estrogen-dependent chronic inflammatory condition that affects women in their reproductive period causing infertility and pelvic pain. The disease, especially at the ovarian site has been shown to have a detrimental impact on ovarian physiology. Indeed, sonographic and histologic data tend to support the idea that ovarian follicles of endometriosis patients are decreased in number and more atretic. Moreover, the local intrafollicular environment of patients affected is characterized by alterations of the granulosa cell compartment including reduced P450 aromatase expression and increased intracellular reactive oxygen species generation. However, no comprehensive evaluation of the literature addressing the effect of endometriosis on oocyte quality from both a clinical and a biological perspective has so far been conducted. Based on this systematic review of the literature, oocytes retrieved from women affected by endometriosis are more likely to fail in vitro maturation and to show altered morphology and lower cytoplasmic mitochondrial content compared to women with other causes of infertility. Results from meta-analyses addressing IVF outcomes in women affected would indicate that a reduction in the number of mature oocytes retrieved is associated with endometriosis while a reduction in fertilization rates is more likely to be associated with minimal/mild rather than with moderate/severe disease. However, evidence in this field is still far to be conclusive, especially with regards to the effects of different stages of the disease and to the impact of patients’ previous medical/surgical treatment(s).

Top quality blastocyst formation rates in relation to progesterone levels on the day of oocyte maturation in GnRH antagonist IVF/ ICSI cycles

Cycles with progesterone elevation during controlled ovarian stimulation (COS) for IVF/ICSI are commonly managed with a “freeze-all” strategy, due to a well-recognized detrimental effect of high progesterone levels on endometrial receptivity. However, also a detrimental effect of elevated progesterone on day-3 embryo quality has recently been found with regards to top quality embryo formation rate. Because blastocyst culture and cryopreservation are largely adopted, we deemed relevant to determine whether this detrimental effect is also seen on blastocyst quality on day 5–6. This issue was investigated through a large two-center retrospective study including 986 GnRH antagonist IVF/ICSI cycles and using top quality blastocyst formation rate as the main outcome. Results showed that on multivariate analysis sperm motility (p<0.01) and progesterone levels at ovulation triggering (p = 0.01) were the only two variables that significantly predicted top quality blastocyst formation rate after adjusting for relevant factors including female age, BMI, basal AMH and total dose of FSH used for COS. More specifically, progesterone levels at induction showed an inverse relation with top quality blastocyst formation (correlation coefficient B = -1.08, 95% CI -1.9 to -0.02) and ROC curve analysis identified P level >1.49 ng/ml as the best cut-off for identification of patients at risk for the absence of top quality blastocysts (AUC 0.55, p<0.01). Our study is the first to investigate the top quality blastocyst formation rate in relation to progesterone levels in IVF/ICSI cycles, showing that increasing progesterone is associated with lower rates of top quality blastocyst. Hence, the advantages of prolonging COS to maximize the number of collected oocytes might eventually be hindered by a decrease in top quality blastocysts available for transfer, if increasing progesterone levels are observed. This observation extends the results of two recent studies focused on day-3 embryos and deserves further research.

Recombinant LH administration in subsequent cycle after "unexpected" poor response to recombinant FSH monotherapy.

Abstract Poor ovarian response (POR) is most frequently linked to the condition known as diminished ovarian reserve, but it can also occur in the absence of pathological ovarian reserve tests (“unexpected” POR). Because possible explanations include theca cells function deficiency, our aim was to evaluate the effect of r-LH administration in “unexpected” poor responders. A retrospective, single-centre, cohort study was conducted on 65 patients with AMH >0.5 ng/ml and/or AFC >5 with POR in their first cycle. Patients underwent a second IVF cycle with same protocol (long- or antagonist) and same starting dose of r-FSH used in the first cycle, plus daily addiction of 150 IU of r-LH from day 1. Compared to the first cycle, r-LH addition in the second cycle determined an increase in number of oocytes retrieved (p < 0.001), number of metaphase II oocytes (p < 0.05), E2 levels at hCG triggering (p < 0.001) and number of embryos transferred (p = 0.002). A 15% clinical pregnancy rate was also observed in the second cycle. Our results suggest that patients with non-pathological ovarian reserve tests and previous “unexpected” POR seem to benefit from r-LH addition in subsequent cycles without the need to increase the r-FSH starting dose, possibly due to an underlying occult theca cells deficiency. Chinese abstract 卵巢低反应常常与卵巢储备功能下降的情况相关，但是它也会发生在卵巢储备正常的情况下（即所谓的“非预料”卵巢低反应POR）。由于有人认为这种卵巢低反应与卵泡膜细胞的功能缺陷有关，因此，我们的研究是评估在IVF中给予这部分患者重组LH的治疗效果。 这是一项回顾性、单中心的队列研究， 65名抗苗勒氏管激素AMH>0.5ng/ml和/或窦卵泡数AFC>5患者.，在IVF的第一个周期呈现卵巢低反应。在随后的第二个周期采用与第一个周期相同的方案（GnRH拮抗剂长方案）以及相同的FSH的起始应用剂量，同时从第一天开始每天加用150IU的重组LH。与第一个周期不同的是，第二个周期重组LH的给予增加了获卵数(p<0.001)，卵母细胞成熟率(p<0.05)，HCG诱发排卵时的雌二醇的水平(p<0.001)以及移植的胚胎数量(p=0.002)，在第二个周期获得了15%的临床妊娠率。 我们的研究结果提示，在IVF的治疗中，对于那些可能存在潜在的卵泡膜细胞功能缺陷的患者，其卵巢储备功能正常但是却出现卵巢低反应，在随后的治疗周期中加用重组LH，而不需要增加重组FSH的初始剂量，看起来是有效果的。

Unravelling the ovarian endometrioma pathogenesis: “The long and winding road”* across the various theories

Controversy exists regarding the pathogenesis of endometriotic ovarian cysts. Different and complex theories have been proposed over the years since the description of chocolate cysts by Sampson in 1921. We have herein reviewed findings in support and against the most widely accepted theories. According to the theory of Hughesdson and Brosens, a prerequisite for endometrioma formation seems to be the inversion and progressive invagination of the ovarian cortex after the accumulation of menstrual debris derived from bleeding of superficial endometriotic implants, which are located on the ovarian surface and adherent to the peritoneum. Disproving the metaplasia hypothesis put forward by Donnez and coworkers and supporting the involvement of the ovulation process in the development of ovarian endometriosis, Vercellini and colleagues have recently demonstrated that a cystic corpus luteum may be a transitory step toward endometrioma formation. As these theories are not able to explain the various aspects of endometrioma formation fully, the possibility that the coelomic metaplasia of the ovarian mesothelium with changes into typical endometrial glands and stroma might be responsible for the endometrioma formation cannot be totally ruled out. Further research is needed to clearly elucidate the pathogenetic aspects of endometriotic ovarian cysts.

A direct healthcare cost analysis of the cryopreserved versus fresh transfer policy at the blastocyst stage

A cost analysis covering direct healthcare costs relating to IVF freeze-all policy was conducted. Normal- and high- responder patients treated with a freeze-all policy (n = 63) compared with fresh transfer IVF (n = 189) matched by age, body mass index, duration and cause of infertility, predictive factors for IVF (number of oocytes used for fertilization) and study period, according to a 1:3 ratio were included. Total costs per patient (€6952 versus €6863) and mean costs per live birth were similar between the freeze-all strategy (€13,101, 95% CI 10,686 to 17,041) and fresh transfer IVF (€15,279, 95% CI 13,212 to 18,030). A mean per live birth cost-saving of €2178 (95% CI -1810 to 6165) resulted in a freeze-all strategy owing to fewer embryo transfer procedures (1.29 ± 0.5 versus 1.41 ± 0.7); differences were not significant. Sensitivity analysis revealed that the freeze-all strategy remained cost-effective until the live birth rate is either higher or only slightly lower (≥-0.59%) in the freeze-all group compared with fresh cycles. A freeze-all policy does not increase costs compared with fresh transfer, owing to negligible additional expenses, i.e. vitrification, endometrial priming and monitoring, against fewer embryo transfer procedures required to achieve pregnancy.

Advances in improving fertility in women through stem cell-based clinical platforms

ABSTRACT Introduction: Due to their regenerative ability, stem cells are looked at as a promising tool for improving infertility treatments in women. As the main limiting factor in female fertility is represented by the decrease of ovarian reserve, the main goals of stem cell-based clinical platforms would be to obtain in vitro or in vivo neo-oogenesis. Refractory endometrial factor infertility also represents an obstacle for female reproduction for which stem cells might provide novel treatment strategies. Areas covered: A systematic search of the literature was performed on MEDLINE/PubMed database to identify relevant articles using stem-cell based clinical or research platforms in the field of female infertility. Expert opinion: In vitro oogenesis has not so far developed beyond the stage of oocyte-like cells whose normal progression to mature oocytes and ability to be fertilized was not proved. Extensive epigenetic programming of gamete precursors and the complex interactions between somatic and germ cells required for human oogenesis likely represent the main obstacles in stem-cell-based neo-oogenesis. Also resuming oogenesis in vivo in adulthood still appears a distant hypothesis, as there is still a lack of consensus about the existence and functionality of adult ovarian stem cells.

Reproductive function assessment after surgery plus chemotherapy for germ cell ovarian tumors (MOGCT): novel clues deriving from the field of fertility preservation

Abstract 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. Chinese abstract 卵巢恶性生殖细胞肿瘤（MOGCT）好发于年轻女性，常用手术加化疗进行治疗。众所周知，化疗的细胞毒性作用会加速始基卵泡的消耗，导致生殖功能受损及过早绝经。本研究的目的是找出保留患者生育能力的策略。我们报道了对San Raffaele医院肿瘤生殖科的四位MOGCT患者保留其生育功能方面的体会。所有患者接受保留生育功能手术加以铂为基础的化疗方案。两名患者罹患混合性生殖细胞肿瘤，另两名为无性细胞瘤患者。治疗结束24个月后，测其血清AMH水平。我们发现患者的血清抗苗勒氏管激素（AMH）水平较健康人群低，其血清AMH水平低于不同年龄层的第25百分位数。对于血清AMH水平极低并因此预测到其卵巢功能显著受损的第一、二名患者，保存其生殖能力如卵母细胞冷冻保存。用促性腺激素释放激素（GnRH）拮抗剂方案促排卵，我们分别获得2个和6个卵母细胞。因此，血清AMH可作为监测卵巢功能的一项指标，提高我们对需要施行生育能力保存方案的患者的鉴别能力。

Clinical application of a nomogram based on age, serum FSH and AMH to select the FSH starting dose in IVF/ICSI cycles: a retrospective two-centres study

OBJECTIVE To externally validate a nomogram based on ovarian reserve markers as a tool to optimize the FSH starting dose in IVF/ICSI cycles. STUDY DESIGN A two-centres retrospective study including 398 infertile women undergoing their first IVF/ICSI cycle (June 2013-June 2014). IVF data were retrieved from two independent IVF centres in Italy (San Raffaele Hospital, Centre 1; Verona Hospital, Centre 2). A central lab for the routine measurement of AMH and FSH was used for both centres. All women were treated based on physical and hormonal characteristics according to locally adopted protocols. The nomogram was then retrospectively applied to the patients comparing the calculated starting dose to the one actually given. RESULTS In Centre 1, 64/131 women (48.8%) had an ovarian response below the target. While 45 of these patients were treated with a maximal FSH starting dose (≥225 IU), n=19/131 (14.5%) were treated with a submaximal dose. The vast majority of them (n=17/19) would have received a higher FSH starting dose by using the nomogram. Seventeen patients (n=17/131) had hyper response and about half of them would have been treated with a reduced FSH starting dose according to the nomogram. In Centre 2, 142/267 patients (53.2%) had an ovarian response below the target. While 136 of these were treated with a maximal FSH starting dose (≥225 IU), n=6/267 were treated with a submaximal dose. The majority of them (n=5/6) would have received a higher FSH starting dose. Thirty-two (n=32/267) patients had hyper response and more than half of them would have been treated with a reduced FSH dose. CONCLUSION In both Centres, applying the nomogram would have resulted in more appropriate FSH starting doses compared to the the ones actually given based on clinicians choices. The use of an objective algorithm based on patients age, serum FSH and AMH levels may thus be an effective advice on the selection of the tailored FSH starting dose. Hence, the use of this easily available nomogram could increase the proportion of patients achieving the optimal ovarian response.

Association between let-7 microRNA-binding-site polymorphism in the KRAS 3’UTR and endometriosis: a replication study and meta-analysis

Background Endometriosis is inherited as a complex genetic trait, and the single nucleotide polymorphism (SNP) rs61764370 within the KRAS gene on chromosome subband 12p12.1 has been proposed as a potential candidate gene. By disrupting a binding site for microinterference RNA (miRNA) let-7, the rs61764370 SNP variation site may increase activation of KRAS and favor disease development. Conflicting evidence, however, has emerged on the association between the rs61764370 SNP and endometriosis. Due to the potential implications of this issue for the diagnosis and treatment of endometriosis, we sought to replicate the sequencing of the KRAS rs61764370 SNP in a population of cases and controls and to perform a meta-analysis encompassing all currently available studies as well as our novel replication. Methods We sought to replicate for the first time the sequencing of KRAS rs61764370 SNP in a highly selected population of 86 cases of women with laparoscopically proven endometriosis and 72 healthy controls, and to perform a meta-analysis encompassing currently available studies and including affected subjects (n = 2,225) and controls (n = 1,923). Results The rs61764370 minor allele was observed in 12 of 86 women with endometriosis (14.0%) and in 15 of 72 controls (20.8%) (odds ratio [OR] = 0.76, 95% confidence interval [95% CI], 0.36-1.60, p = 0.48). The meta-analysis failed to identify any significant association (OR = 1.03, 95% CI, 0.89-1.20, p = 0.67, phet = 0.46). Conclusions KRAS variation site rs61764370 is unlikely related to disease development, and other loci with potential implications for diagnosis or treatment for endometriosis should be identified.