Risk Factors for Infertility in Korean Women.

BACKGROUND: Female infertility is a crucial problem with significant implications for individuals and society. In this study, we explore risk factors for infertility in Korean women. METHODS: A total of 986 female patients who visited six major infertility clinics in Korea were recruited from April to December 2014. Fertile age-matched controls were selected from two nationwide survey study participants. Conditional logistic regression after age-matching was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) of each risk factor for infertility. RESULTS: Women with a body mass index (BMI) < 18.5 kg/m² had 1.35 times higher odds of infertility (OR, 1.35; 95% CI, 1.03-1.77), while those with a BMI ≥ 25.0 kg/m² had even higher odds (OR, 2.06; 95% CI, 1.61-2.64) compared to women with a normal BMI (18.5 kg/m² ≤ BMI < 25 kg/m²). Ever-smokers exhibited 4.94 times higher odds of infertility compared to never-smokers (95% CI, 3.45-8.85). Concerning alcohol consumption, women who consumed ≥ 7 glasses at a time showed 3.13 times significantly higher odds of infertility than those who consumed ≤ 4 glasses at a time (95% CI, 1.79-5.48). Lastly, women with thyroid disease demonstrated 1.44 times higher odds of infertility compared to women without thyroid disease (95% CI, 1.00-2.08). CONCLUSION: Female infertility in Korea was associated with underweight, obesity, smoking, alcohol consumption, and thyroid disease.

Comparison of ovarian reserve after cystectomy of ovarian endometrioma by bipolar coagulation, suture method, or hemostatic sealants: An updated meta-analysis.

AIM: The purpose of the study was to compare the ovarian reserve after cystectomy of ovarian endometrioma by bipolar coagulation, suture method, or hemostatic sealants (HSs). METHODS: We performed a meta-analysis of studies in which post-cystectomy serum anti-Müllerian hormone (AMH) values were compared between bipolar coagulation and suture method or between bipolar coagulation and HSs. Through a literature search, we retrieved 14 articles which met inclusion criteria and were eligible for final analysis. The articles included 10 randomized trials, 3 prospective studies, and 1 retrospective study (n = 1435). The primary outcome was post-cystectomy serum AMH values. RESULTS: Both bipolar coagulation and suture methods showed significantly lower post-cystectomy AMH values at 3, 6, and 12 months. However, post-cystectomy serum AMH values at 12 months were significantly higher in the suture method group compared to the bipolar coagulation (weighted mean difference [WMD]: -1.10, 95% confidence interval [CI]: -1.83, -0.38, p = 0.003, I2 = 89, n = 3). The suture method also showed a lower decline rate at 3 months post-cystectomy compared to the bipolar coagulation group (WMD: -25.13%, 95% CI: -49.56 to -0.70, p = 0.04, I2 = 95%, n = 2). Overall, pregnancy rates were similar between the two groups. Between the bipolar coagulation and HSs group, serum AMH values at 3 months post-cystectomy were similar (WMD: -0.46, 95% CI: -1.04 to 0.13, p = 0.13, I2 = 0%, n = 3). However, the HSs group showed a less decline rate at 3 months post-cystectomy compared to the bipolar coagulation group (WMD: -17.02%, 95% CI: -22.81, -11.23, p < 0.00001, I2 = 0%, n = 3). CONCLUSIONS: Both the suture method and HSs may have potential benefits in the preservation of ovarian reserve over the bipolar coagulation method when cystectomy for ovarian endometrioma is performed.

Clinical efficacy of dual trigger with human chorionic gonadotropin and a gonadotropin-releasing hormone agonist for women undergoing fertility preservation.

Purpose: To determine the optimal maturation method to increase the yield of mature oocytes, especially for cancer patients with fewer chances of fertility preservation (FP) before gonadotoxic therapy. Methods: A total of 373 cycles in 293 patients undergoing controlled ovarian stimulation (COS) for FP using a gonadotropin-releasing hormone (GnRH) antagonist protocol were enrolled. The control group (n = 225) received 250 µg of recombinant human chorionic gonadotropin (rhCG) while the study group (n = 148) received 250 µg of rhCG and 0.2 mg of triptorelin for triggering. Subgroup analyses were performed for stimulation cycles with diminished ovarian reserve (DOR; anti-Müllerian hormone (AMH) levels <1.1 ng/ml, n = 86), with endometrioma (n = 104), or with breast cancer and endometrial cancer using 5 mg of letrozole during the COS cycles (n = 84). Results: There was no significant difference in the baseline characteristics or the number of total and mature oocytes between the two groups. Subgroup analyses for women with endometrioma or DOR showed similar results. However, the dual trigger group had a significantly higher number of mature oocytes than the rhCG trigger group in breast and endometrial cancer patients using letrozole during the COS cycles (6.9 ± 6.0 vs. 4.6 ± 3.6, p = 0.034). The maturation rate was higher in the dual trigger group, although the difference was not statistically significant (59.3 ± 26.7 vs. 50.0 ± 28.0, p = 0.124). Conclusions: Dual triggering can be an efficient maturation method to maximize the yield of mature oocytes in breast or endometrial cancer patients using letrozole-combined GnRH antagonist protocol for FP.

Injection funnel persistence time and oolemma resistance during intracytoplasmic sperm injection and subsequent embryo development.

OBJECTIVE: This study aimed to investigate whether the injection funnel persistence time and oolemma resistance during the intracytoplasmic sperm injection (ICSI) are associated with subsequent embryo quality. DESIGN: A prospective observational study at a university hospital. METHODS: One hundred and twenty normal-appearing metaphase II oocytes were collected from 54 ICSI cycles. Injection funnel was observed at 0, 30, 60, and 90 s after ICSI, and the injection funnel persistence time was assigned to "no funnel," "0-30," "30-60," "60-90," and ">90 s." The degree of oolemma resistance during ICSI was recorded as "no," "mild," "moderate," and "severe." Subsequent embryos on day 3 after ICSI were evaluated morphologically, and formation of top-quality embryo and embryo score was assessed. We newly developed "oolemma score," based on the injection funnel persistence time and oolemma resistance, and the predictability of top-quality embryo was assessed. RESULTS: Among the five groups by injection funnel persistence time, the proportion of top-quality embryo and embryo score (64.3%, 32) was highest in the "30-60 s," but not significant. Among the four groups by oolemma resistance, the proportion of top-quality embryo and embryo score (53.7%, 32) was highest in "no group." The proportion of top-quality embryo in "no group" was significantly higher than "moderate group" (p = 0.012) and "severe group" (p = 0.043). The median embryo score in "no group" was significantly higher than "severe group" (p = 0.041). Newly developed "oolemma score" could predict well the formation of top-quality embryo with a statistical significance (cutoff >14.5, area under the curve 0.695, p < 0.001). CONCLUSION: Embryo quality or score is more closely associated with oolemma resistance during ICSI. New "oolemma score" would help to identify embryo developmental potential of each mature oocyte in ICSI cycles.

The effects of letrozole on women with endometriosis undergoing ovarian stimulation for in vitro fertilization.

This study aimed to analyze the effects of a new protocol with letrozole on the outcomes of in vitro fertilization (IVF) cycles in women with endometriosis. This retrospective cohort study was conducted for women diagnosed with endometriosis undergoing IVF from an infertility clinic. A new protocol, combination therapy with letrozole and gonadotropin, was used from August 2016 to January 2018 ('protocol 1', n = 38). From March 2014 to July 2016, conventional IVF with gonadotropin was administered ('protocol 2', n = 26). Age and ovarian reserve were comparable between the two groups. The patients who received protocol 1 resulted in a significantly lower peak estradiol level in IVF compared with those received protocol 2 (722 ± 1076 pg/mL versus 2168 ± 1521 pg/mL, p < .001). The length of stimulation, the total dose of gonadotropin, number of oocytes retrieved, fertilization rates, and number of embryos obtained were similar between the two groups. The mean percentage of mature oocytes was lower (69.9 ± 23.7% versus 80.2 ± 21.0%, p = .029) in patients with protocol 1. While maintaining low estrogen levels, the combination therapy with letrozole and gonadotropin produce similar oocyte and embryo yield to the conventional IVF protocol in women with endometriosis.

Erratum: Correction of Author ORCID in the Article: Efficacy of Random-start Controlled Ovarian Stimulation in Cancer Patients.

This corrects the article on p. 290 in vol. 30, PMID: 25729252.

Predictors of blastocyst formation rate in elective day 5 transfer cycle.

This study aimed to investigate the factors affecting blastocyst formation rate. One hundred and seven fresh in vitro fertilisation (IVF) and elective day 5 blastocyst transfer cycles were selected. Univariate and multivariate analyses revealed that intracytoplasmic sperm injection (ICSI) (r = -.236, p = .014 vs. p = .005) was advantageous for blastocyst formation. In addition, the number of mature oocytes (r = -.274, p = .004 vs. p = .002) was a significant factor associated with blastocyst and good-quality blastocyst formation rates (p = .021, r = -.389). Both blastocyst and good-quality blastocyst formation rates were significantly higher with ICSI than with conventional insemination (65.0 ± 24.5% vs. 50.0 ± 21.2%, p = .012; 43.1 ± 22.8% vs. 30.9 ± 19.8%, p = .038, respectively). The number of mature oocytes appears to be the most important predictor of blastocyst formation rate. Additionally, ICSI fertilisation is superior to conventional insemination in terms of blastocyst formation rate.IMPACT STATEMENTWhat is already known on this subject? There are many advantages of blastocyst transfer cycle over cleavage transfer cycle, but there are no known routine selection criteria for the timing of embryo transfer. To date, the number of blastomeres, number of retrieved oocytes, quality of embryos and fertilisation method have been suggested as the important factors involved in blastocyst formation. However, the number of studies on this issue is limited, and some studies have shown conflicting results.What do the results of this study add? This study showed that the number of mature oocytes and ICSI fertilisation are the significant factors associated with blastocyst formation rate in elective day 5 transfer cycle.What are the implications of these findings for clinical practice and/or further research? This paper demonstrated that the number of mature oocytes and the fertilisation method should be considered before embryo transfer. Consideration of these factors would be meaningful in selecting patients who will be suitable for extended culture up to day 5.

Adverse symptoms during short-term use of ulipristal acetate in women with uterine myomas and/or adenomyosis.

AIM: To evaluate various adverse symptoms during short-term use of ulipristal acetate in women with uterine myomas (n = 90), adenomyosis (n = 3) or both (n = 7). METHODS: One hundred premenopausal women who received ulipristal acetate for 4-12 weeks during 2016 to 2017 were selected. The medical records were reviewed and the following information was collected; adverse symptoms during medication, presence of menorrhagia or menstrual cramps, blood hemoglobin and liver function test. Adverse symptoms were recorded in the medical records as a checklist form including 76 specific progestin-related symptoms. RESULTS: Overall, the most frequent adverse symptom was amenorrhea (43%), followed by weight gain (29%), fatigue (27%), abdominal discomfort (21%), decreased menstrual flow (19%) and dizziness (18%). In 89 symptomatic women (with heavy menstrual bleeding and/or menstrual cramping pain and/or anemia), the most frequent adverse symptom was weight gain (27%) and fatigue (27%), followed by abdominal discomfort (21%), dry eye (18%), facial flushing (17%), dizziness (17%), headache (17%) and increased vaginal discharge (15%). Fourteen women stopped the medication due to unwanted adverse symptoms. Of this discontinuation group, major complaint was fatigue (50%), followed by weight gain (36%) and breast discomfort (35.7%). CONCLUSION: Adverse symptoms were common and discontinuation rate was somewhat higher during short-term course of ulipristal acetate. Information about incidence of various adverse symptoms should be given to women who willing to take ulipristal acetate.

Symptom-alleviating effect and adverse effect of dienogest in Korean women with endometriosis.

The purpose was to evaluate the effects of dienogest on Korean women with endometriosis. A cross-sectional questionnaire-based survey was conducted for 100 premenopausal women. They had taken or were taking 2 mg of dienogest daily. We assessed the pelvic pain score and quality-of-life (QOL) score before and after taking dienogest as well as the prevalence of short-term (≤12 weeks) and long-term adverse effects (>52 weeks). Patients were classified into three groups: dienogest treatment immediately following surgery (A), dienogest treatment for a recurrence of endometriosis after surgery (B), or dienogest treatment without any surgery (C). In groups A and C, the median pain score (from 5 to 0, p <.001; from 7 to 1.5, p <.001) and median QOL score (from 10 to 5, p = .002; from 7.5 to 6.5, p = .008) were significantly decreased. Irregular bleeding and decreased menstrual flow were more prevalent in patients with dienogest intake of fewer than 12 weeks, while amenorrhea, weight gain, hair loss, and dorsal pain were more prevalent in patients with dienogest treatment of over 52 weeks. Accordingly, proper counseling is necessary when prescribing dienogest.

Successful in Vitro Fertilization and Embryo Transfer after Transplantation of Cryopreserved Ovarian Tissue: Report of the First Korean Case.

For patients at risk of premature ovarian failure with cancer treatment, it is an important option to re-implant the ovarian tissue (OT) after cryopreservation to preserve endocrine function and fertility. With this technique, about 30% of pregnancy success rate and about 90 live births have been reported to date. However, there has been no case report of successful in vitro fertilization (IVF) and embryo transfer (ET) with oocytes collected from transplanted cryopreserved OT in Korea. We report a 30-year old woman with rectal cancer who underwent IVF and ET after cryopreserved OT thawing and re-implantation. She has been diagnosed with stage IIIC rectal cancer after surgery, and right ovary was removed and cryopreserved between cycles of chemotherapy. After completion of chemotherapy and radiotherapy, the patient underwent orthotopic transplantation of cryopreserved OTs. Three months after transplantation, the serum follicle-stimulating hormone level decreased from 91.11 mIU/mL to 43.69 mIU/mL. Thereafter, the patient underwent 11 ovarian stimulation cycles, and in 7 cycles, follicle growth was observed at the OT graft site. In one of these cycles, the oocyte was successfully retrieved and one embryo was transplanted after IVF. The patient was not pregnant, but the cryopreservation of OT can save the fertility after anticancer chemotherapy.

Major Disease Prevalence and Menstrual Characteristics in Infertile Female Korean Smokers.

We investigated the prevalence of smoking and factors associated with smoking in infertile Korean women. Smoking status, education, occupation, personal habits, past medical history, current illness, stress level, and menstrual characteristics were collected from self-report questionnaires. The Beck Depression Inventory (BDI) was used to assess the degree of depression. Data on the causes of infertility and levels of six reproductive hormones were collected from medical records. Among 785 women less than 42 years of age, the prevalence of current, secondhand, past, and never smokers were 12.7%, 45.7%, 0.9%, and 40.6%, respectively. Primary infertility was more frequent in secondhand smokers. Causes of infertility were similar among current, secondhand, and never smokers. Current smokers were less educated (P < 0.001) and more likely to consume alcohol than secondhand or never smokers (P < 0.001). Secondhand smokers slept less than current smokers (P = 0.041). Among several major diseases, only the prevalence of diabetes mellitus (4.0%) was significantly higher in current smokers than in secondhand smokers (0.0%, P = 0.002) or never smokers (0.6%, P = 0.031). The self-reported prevalence of depression, and the degree of depression were similar among women with different smoking statuses. There were no differences in menstrual characteristics or serum levels of six reproductive hormones between current, secondhand, and never smokers, even after excluding women with polycystic ovary syndrome. In conclusion, education/employment status, alcohol drinking, and the prevalence of primary infertility and diabetes mellitus were significantly different according to smoking status among infertile women.

Reference Values for the Revised Anti-Müllerian Hormone Generation II Assay: Infertile Population-based Study.

Anti-Müllerian hormone (AMH) is now accepted as an important clinical marker of ovarian reserve and is increasingly measured as an initial evaluation at infertility clinics. The aim of this study was to establish reference values for the revised second generation (Gen II) assay using population-based data. In this population-based cohort study, AMH data from unselected infertile women aged 25-45 years from June 2013 to June 2014 (n = 15,801) were collected. The AMH values were measured using the revised Gen II assay. We established and validated 5 AMH-age regression models. Based on the optimal AMH-age model, reference values and centile charts were obtained. The quadratic model (log AMH = 0.410 × age -0.008 × age² -3.791) was the most appropriate for describing the age-dependent decrease in AMH measured using the revised Gen II assay. This is the largest population-based study to establish age-specific reference values of AMH using the revised Gen II assay. These reference values may provide more specific information regarding the ovarian reserve estimation of infertile women.

Expressions of aquaporin family in human luteinized granulosa cells and their correlations with IVF outcomes.

STUDY QUESTION: Are mRNAs for specific aquaporins (AQPs) expressed in human luteinized granulosa cells (GCs) and are their expression levels correlated with in vitro fertilization (IVF) outcomes? SUMMARY ANSWER: The mRNAs of AQP1-7, 9, 11, and 12 were expressed in human luteinized GCs; the level of AQP1 mRNA was negatively associated with retrieved oocyte number and the level of AQP7 mRNA was positively associated with fertilization rate. WHAT IS KNOWN ALREADY: mRNAs of AQP1-4 and AQP9 have been detected in human GCs. STUDY DESIGN, SIZE, DURATION: Prospective observational study involving 111 women undergoing a stimulated IVF cycle. PARTICIPANTS/MATERIALS, SETTING, METHODS: In a preliminary experiment on 27 women, the mRNA expression of AQP0-12 in GCs was explored by RT-PCR. In the main experiment, luteinized GCs were obtained from 111 women at the time of oocyte retrieval of whom 102 had an embryo replacement. Real-time quantitative RT-PCR (qRT-PCR) was performed to quantify the mRNA level of AQP1-7, 9, 11, and 12. The mRNA for luteinizing hormone receptor (LHR) and steroidogenic acute regulatory protein (StAR) were also quantified by qRT-PCR. MAIN RESULTS AND THE ROLE OF CHANCE: mRNAs for AQP0, 8 and 10 were not detected in the preliminary experiment. In samples from 111 women, retrieved oocyte number was negatively associated with the mRNA levels of AQP1, 4, 6, and 11 and LHR (r = -0.311, r = -0.233, r = -0.203, r = -0.194, and r = -0.202, respectively, P < 0.05 for each), however, after adjustment for woman's age and serum anti-Müllerian hormone (AMH) levels, only correlation with AQP1 was found (r = -0.299, P < 0.05). BMI was negatively associated (after adjustment for age) with the mRNA level of AQP7 (r = -0.259, P < 0.05). Fertilization rate was positively associated with the mRNA level of AQP7 (r = 0.269, P < 0.05). The number or quality of embryos or clinical pregnancy was not associated with the mRNA levels of any of ten AQP subtypes. The mRNA levels for the ten AQP subtypes were correlated positively with LHR expression but negatively with StAR expression. Amongst high responders (oocyte number ≥14), the mRNA levels of AQP11 (1.4 ± 0.7 versus 1.7 ± 0.6) and LHR (1.3 ± 0.7 versus 1.7 ± 0.7) were significantly lower in the group with PCOS than in the non-PCOS group. LIMITATIONS, REASONS FOR CAUTION: A relative small number of subjects in PCOS group is the main limitation of our study. P-values were not corrected for multiple comparisons. WIDER IMPLICATION OF THE FINDINGS: AQP1 may be one of the factors that modulate individual ovarian response to exogenous gonadotrophin. The mRNA level of AQP7 was positively associated with fertilization rate, which is a surrogate marker of oocyte competence, thus expression of AQP7 could be a marker for adequate folliculogenesis and healthy oocytes. STUDY FUNDING/COMPETING INTERESTS: This work was supported by grant no. A120043 from the Korea Health Care Technology R&D Project, Ministry of Health and Welfare, Korea. None of the authors has any conflict of interest to declare.

Comparison of normal and abnormal fertilization of in vitro-matured human oocyte according to insemination method.

AIM: Our purpose was to compare the normal fertilization rate, multi-pronuclei (PN) formation rate, and embryonic development of in vitro-matured oocytes between conventional insemination and intracytoplasmic sperm injection (ICSI). METHODS: A total of 213 stimulated in vitro fertilization (IVF) cycles were selected, in which at least one immature oocyte was obtained (from 2010 to 2014). Immature oocytes were assigned to germinal vesicle (GV)-stage or metaphase I (MI)-stage oocyte groups. Cycles with obligatory ICSI due to male-factor infertility were excluded. Cycles were divided into two groups according to fertilization method: there were 97 cycles with conventional insemination and 116 cycles with ICSI. After in vitro maturation of 324 GV-stage oocytes and 341 MI-stage oocytes, the fertilization rate, multi-PN formation rate, and embryonic development were compared according to the fertilization method. RESULTS: The normal fertilization rate was similar in the conventional insemination and the ICSI both in GV-derived and MI-derived oocytes. Both fertilization methods resulted in a similar multi-PN formation rate in GV-derived oocytes; however, in MI-derived oocytes, the multi-PN formation rate was zero with ICSI and this was significantly lower than that with conventional insemination (9.6%, P = 0.001). CONCLUSION: In non-male-factor infertility, ICSI should be considered when MI oocytes are matured.

Efficacy of random-start controlled ovarian stimulation in cancer patients.

This study aimed to evaluate the efficacy of random-start controlled ovarian stimulation (COS) in cancer patients for emergency fertility preservation. In this retrospective comparative study, 22 patients diagnosed with cancer and 44 infertile women undergoing conventional in vitro fertilization (IVF) were included. In cancer patients, ovarian stimulation was started on the day of referral, irrespective of their menstrual cycle date. The control group was selected by age matching among women undergoing conventional IVF. COS outcomes were compared between groups. The number of total and mature oocytes retrieved and the oocyte maturity rate were higher in the random-start group than in the conventional-start group. However, duration of ovarian stimulation was longer in the random-start group (11.4 vs. 10.3 days, P = 0.004). The addition of letrozole to lower the estradiol level during COS did not adversely affect total oocytes retrieved. However, oocyte maturity rate was lower in cycles with letrozole than in cycles without letrozole (71.6% vs. 58.2%, P = 0.019). Our study confirms the feasibility and effectiveness of random-start COS in cancer patients.

Factors affecting occurrence of twin pregnancy after double embryo transfer on day 3.

AIM: The aim of this study was to identify the risk factors of twin gestation in fresh double day-3 embryo transfer cycles. MATERIAL AND METHODS: Out of a total of 386 cycles of fresh double embryo transfers on day 3 between 2006 and 2013 at a single center, 72 women with single intrauterine gestational sac (GS) (single-GS group) and 32 women with double GS (double-GS group) were identified. The patients' clinical characteristics and laboratory results were extracted from electronic medical records and were compared. RESULTS: The double-GS group had a significantly younger age (P = 0.012), and higher bodyweight (P = 0.033) compared with the single-GS group. The total number of blastomeres (P = 0.013) and cumulative embryo score (P = 0.012) were higher in the double-GS group. By receiver-operator curve analysis, maternal age ≤ 35 years, maternal bodyweight > 60 kg, total number of blastomeres > 14, and cumulative embryo score > 49 were the cut-off values to predict twin pregnancy. Among eight women with all four risk factors, the twin pregnancy rate was 87.5%. There was no twin pregnancy among seven women with no risk factors. CONCLUSION: Younger age, higher bodyweight, and better embryo quality are all associated with a higher incidence of twin pregnancy after double embryo transfer on day 3. Single embryo transfer might be advisable for couples with risk factors for twin pregnancy.

Addition of lysophosphatidic acid to mouse oocyte maturation media can enhance fertilization and developmental competence.

STUDY QUESTION: Does exposure to lysophosphatidic acid (LPA) during in vitro maturation (IVM) enhance the maturation and developmental competence of mouse oocytes? SUMMARY ANSWER: Supplementation of IVM medium with 30 µM LPA enhanced the developmental competence of in vitro matured oocytes and so made them more comparable to in vivo matured control oocytes. WHAT IS KNOWN ALREADY: LPA is a small phospholipid that acts as an extracellular signaling molecule by binding to and activating at least five G protein-coupled receptors. LPA has various biological actions, with both developmental and physiological effects. STUDY DESIGN, SIZE, DURATION: During IVM, LPA at six different doses (0, 1, 10, 30, 50 or 100 µM) was added into the TCM-199 medium. After maturation, the developmental competence and other parameters of the oocytes were assessed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Immature GV stage oocytes from 5- to 6-week-old female BDF-1 mice were incubated for 17-18 h in IVM medium containing 0, 1, 10 or 30 µM LPA and then either fertilized in vitro with epididymal sperm, or assessed for spindle morphology, mitochondrial membrane potential (ΔΨm) or the mRNA expression of a meiotic checkpoint gene (Mad2), a microtubule structure gene (Hook1), two maternally derived genes (Mater and Hsf1) and an apoptosis-related gene (Caspase6). The fertilized embryos were grown in vitro to assess blastocyst-formation rates, differential cell counts and apoptosis. MAIN RESULTS AND THE ROLE OF CHANCE: Rates of maturation, fertilization and blastocyst formation and hatching were significantly higher in the 30 µM LPA-supplemented group (94.3, 96.3, 79.1 and 51.3%, respectively) than in the unsupplemented control (0 µM) group (80.5, 87.5, 61.3 and 37.8%, respectively) and more comparable to that of the in vivo matured oocytes (100, 96.5, 95.3 and 92.9%, respectively). LPA did not adversely affect mitochondrial activity, spindle integrity, blastocyst cell number, caspase positivity or Mad2 expression. Oocytes matured in 30 µM LPA had reduced Caspase6 expression, but Hook1, Mater and Hsf1 were up-regulated in all of the LPA-supplemented groups. LIMITATIONS, REASONS FOR CAUTION: Chromosomal aneuploidy in the resultant blastocysts and the production of normal pups were not assessed. Only mouse oocytes were assessed. WIDER IMPLICATIONS OF THE FINDINGS: Supplementation of IVM medium with 30 µM LPA may enhance the developmental competence of mouse oocytes without affecting apoptosis, spindle normalcy or mitochondrial integrity. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by a research grant (02-2012-021) from the Seoul National University Bundang Hospital. The authors declare that they have no competing interests.

Optimal vitrification protocol for mouse ovarian tissue cryopreservation: effect of cryoprotective agents and in vitro culture on vitrified-warmed ovarian tissue survival.

STUDY QUESTION: What is the optimal vitrification protocol according to the cryoprotective agent (CPA) for ovarian tissue (OT) cryopreservation? SUMMARY ANSWER: The two-step protocol with 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 10 min then 20% EG, 20% DMSO and 0.5 M sucrose for 5 min showed the best results in mouse OT vitrification. WHAT IS KNOWN ALREADY: Establishing the optimal cryopreservation protocol is one of the most important steps to improve OT survival. However, only a few studies have compared vitrification protocols with different CPAs and investigated the effect of in vitro culture (IVC) on vitrified-warmed OT survival. Some recent papers proposed that a combination of CPAs has less toxicity than one type of CPA. However, the efficacy of different types and concentrations of CPA are not yet well documented. STUDY DESIGN, SIZE, DURATION: A total of 644 ovaries were collected from 4-week-old BDF1 mice, of which 571 ovaries were randomly assigned to 8 groups and vitrified using different protocols according to CPA composition and the remaining 73 ovaries were used as controls. After warming, each of the eight groups of ovaries was further randomly divided into four subgroups and in vitro cultured for 0, 0.5, 2 and 4 h, respectively. Ovaries of the best two groups among the eight groups were autotransplanted after IVC. PARTICIPANTS/MATERIALS, SETTING, METHODS: The CPA solutions for the eight groups were composed of EDS, ES, ED, EPS, EF, EFS, E and EP, respectively (E, EG; D, DMSO; P, propanediol; S, sucrose; F, Ficoll). The IVC medium was composed of α-minimal essential medium, 10% fetal bovine serum and 10 mIU/ml follicle-stimulating hormone (FSH). Autotransplantation of vitrified-warmed OTs after IVC (0 to 4 h) using the EDS or ES protocol was performed, and the grafts were recovered after 3 weeks. Ovarian follicles were assessed for morphology, apoptosis, proliferation and FSH level. MAIN RESULTS AND THE ROLE OF CHANCE: The percentages of the morphologically intact (G1) and apoptotic follicles in each group at 0, 0.5, 2 and 4 h of IVC were compared. For G1 follicles at 0 and 4 h of IVC, the EDS group showed the best results at 63.8 and 46.6%, respectively, whereas the EP group showed the worst results at 42.2 and 12.8%, respectively. The apoptotic follicle ratio was lowest in the EDS group at 0 h (8.1%) and 0.5 h (12.7%) of IVC. All of the eight groups showed significant decreases in G1 follicles and increases in apoptotic follicles as IVC duration progressed. After autotransplantation, the EDS 0 h group showed a significantly higher G1 percentage (84.9%) than did the other groups (42.4-58.8%), while only the ES 4 h group showed a significant decrease in the number of proliferative cells (80.6%, 87.6-92.9%). However, no significant differences in apoptotic rates and FSH levels were observed between the groups after autotransplantation. LIMITATIONS, REASONS FOR CAUTION: The limitation of this study was the absence of in vitro fertilization using oocytes obtained from OT grafts, which should be performed to confirm the outcomes of ovarian cryopreservation and transplantation. WIDER IMPLICATIONS OF THE FINDINGS: We compared eight vitrification protocols according to CPA composition and found the EDS protocol to be the optimal method among them. The data presented herein will help improve OT cryopreservation protocols for humans or other animals.

Anti-Müllerian hormone levels in the follicular fluid of the preovulatory follicle: a predictor for oocyte fertilization and quality of embryo.

This prospective study investigated the relationship between anti-Müllerian hormone (AMH) level in the follicular fluid (FF) and the quality of the oocyte and embryo. A total of 65 FF samples from 54 women were included in this study. FF was collected from the largest preovulatory follicle sized≥20 mm of mean diameter from each ovary. Samples were divided into 3 groups according to the FF AMH levels: below the 33th percentile (low group, FF AMH<2.1 ng/mL, n=21), between the 33th and the 67th percentile (intermediate group, FF AMH=2.1-3.6 ng/mL, n=22), and above the 67th percentile (high group, FF AMH>3.6 ng/mL, n=22). The quality of the ensuing oocytes and embryos was evaluated by fertilization rate and embryo score. FF AMH levels correlated positively with the matched embryo score on day 3 after fertilization (r=0.331, P=0.015). The normal fertilization rate was significantly lower in the low group than in the intermediate group (61.9% vs. 95.5% vs. 77.3%, respectively, P=0.028). Our results suggest that the FF AMH level could be a predictor of the ensuing oocyte and embryo quality.

Impact of Ablation Versus Cystectomy for Endometrioma on Ovarian Reserve, Recurrence, and Pregnancy: An Updated Meta-Analysis.

This study aimed to compare the impact of ablation and cystectomy for ovarian endometrioma on ovarian reserve, recurrence rates, and pregnancy rates. Databases were searched for studies reporting ovarian reserve (measurement of anti-müllerian hormone [AMH] only), recurrence rates, and/or pregnancy rates after cystectomy and ablation for ovarian endometrioma. Six randomized clinical trials and five prospective cohort studies were included in this meta-analysis. All included studies had a low risk of bias. After ablation, weighted mean difference (WMD) between post- and pre-operative AMH values was - 0.40 (95% confidence interval [CI]: -0.61 to -0.19, p = 0.0002, I2 = 0%). After cystectomy, the WMD of AMH was - 1.08 (95% CI: -1.34 to -0.82, p < 0.00001, I2 = 48%). The intergroup comparison revealed that pre-operative AMH values were similar between the two groups, but post-operative AMH was significantly higher in the ablation group (WMD: 0.38, 95% CI: 0.13 to 0.63, p = 0.003, I2 = 3%). Subgroup analysis showed that the favorable effects of ablation compared to cystectomy are evident after three months of operation, in a bilateral setting, or with the laser ablation method. Overall recurrence rate (risk ratio [RR]: 1.51, 95% CI: 1.08 to 2.12, p = 0.02, I2 = 0%) and one-year recurrence rate (RR: 2.36, 95% CI: 1.30 to 4.31, p = 0.005, I2 = 0%) were significantly higher in the ablation group than in the cystectomy group. Pregnancy rates were similar between the two groups (odds ratio: 1.18, 95% CI: 0.92 to 1.52, p = 0.20, I2 = 72%). These results demonstrate that ablation could result in a smaller serum AMH decrement than cystectomy, but the recurrence rate could be higher after ablation.