Is large for gestational age in singletons born after frozen embryo transfer associated with freezing technique or endometrial preparation protocol? A longitudinal national French study.

STUDY QUESTION: Is large for gestational age (LGA) observed in babies born after frozen embryo transfer (FET) associated with either the freezing technique or the endometrial preparation protocol? SUMMARY ANSWER: Artificial cycles are associated with a higher risk of LGA, with no difference in rate between the two freezing techniques (vitrification versus slow freezing) or embryo stage (cleaved embryo versus blastocyst). WHAT IS KNOWN ALREADY: Several studies have compared neonatal outcomes after fresh embryo transfer (ET) and FET and shown that FET is associated with improved neonatal outcomes, including reduced risks of preterm birth, low birthweight, and small for gestational age (SGA), when compared with fresh ET. However, these studies also revealed an increased risk of LGA after FET. The underlying pathophysiology of this increased risk remains unclear; parental infertility, laboratory procedures (including embryo culture conditions and freezing-thawing processes), and endometrial preparation treatments might be involved. STUDY DESIGN, SIZE, DURATION: A multicentre epidemiological data study was performed through a retrospective analysis of the standardized individual clinical records of the French national register of IVF from 2014 to 2018, including single deliveries resulting from fresh ET or FET that were prospectively collected in fertility centres. Complementary data were collected from the participating fertility centres and included the vitrification media and devices, and the endometrial preparation protocols. PARTICIPANTS/MATERIALS, SETTING, METHODS: Data were collected from 35 French ART centres, leading to the inclusion of a total of 72 789 fresh ET, 10 602 slow-freezing FET, and 39 062 vitrification FET. Main clinical outcomes were presented according to origin of the transferred embryos (fresh, slow frozen, or vitrified embryos) and endometrial preparations for FET (ovulatory or artificial cycles), comparing five different groups (fresh, slow freezing-ovulatory cycle, slow freezing-artificial cycle, vitrification-ovulatory cycle, and vitrification-artificial cycle). Foetal growth disorders were defined in live-born singletons according to gestational age and sex-specific weight percentile distribution: SGA and LGA if <10th and ≥90th percentiles, respectively. Analyses were performed using linear mixed models with the ART centres as random effect. MAIN RESULTS AND THE ROLE OF CHANCE: Transfers led to, respectively, 19 006, 1798, and 9195 deliveries corresponding to delivery rates per transfer of 26.1%, 17.0%, and 23.5% after fresh ET, slow-freezing FET, and vitrification FET, respectively. FET cycles were performed in either ovulatory cycles (n = 21 704) or artificial cycles (n = 34 237), leading to 5910 and 10 322 pregnancies, respectively, and corresponding to pregnancy rates per transfer of 31.6% and 33.3%. A significantly higher rate of spontaneous miscarriage was observed in artificial cycles when compared with ovulatory cycles (33.3% versus 21.4%, P < 0.001, in slow freezing groups and 31.6% versus 21.8%, P < 0.001 in vitrification groups). Consequently, a lower delivery rate per transfer was observed in artificial cycles compared with ovulatory cycles both in slow freezing and vitrification groups (15.5% versus 18.9%, P < 0.001 and 22.8% versus 24.9%, P < 0.001, respectively). Among a total of 26 585 live-born singletons, 16 413 babies were born from fresh ET, 1644 from slow-freezing FET, and 8528 from vitrification FET. Birthweight was significantly higher in the FET groups than in the fresh ET group, with no difference between the two freezing techniques. Likewise, LGA rates were higher and SGA rates were lower in the FET groups compared with the fresh ET group whatever the method used for embryo freezing. In a multivariable analysis, the risk of LGA following FET was significantly increased in artificial compared with ovulatory cycles. In contrast, the risk of LGA was not associated with either the freezing procedure (vitrification versus slow freezing) or the embryo stage (cleaved embryo versus blastocyst) at freezing. Regarding the vitrification method, the risk of LGA was not associated with either the vitrification medium used or the embryo stage. LIMITATIONS, REASONS FOR CAUTION: No data were available on maternal context, such as parity, BMI, infertility cause, or maternal comorbidities, in the French national database. In particular, we cannot exclude that the increased risk of LGA observed following FET with artificial cycles may, at least partially, be associated with a confounding effect of some maternal factors. No information about embryo culture and incubation conditions was available. Most of the vitrification techniques were performed using the same device and with two main vitrification media, limiting the validity of a comparison of risk for LGA according to the device or vitrification media used. WIDER IMPLICATIONS OF THE FINDINGS: Our results seem reassuring, since no potential foetal growth disorders following embryo vitrification in comparison with slow freezing were observed. Even if other factors are involved, the endometrial preparation treatment seems to have the greatest impact on LGA risk following FET. FET during ovulatory cycles could minimize the risk for foetal growth disorders. STUDY FUNDING/COMPETING INTEREST(S): This work has received funding from the French Biomedicine Agency (Grant number: 19AMP002). None of the authors has any conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Ovarian tissue cryopreservation can be combined simultaneously with oocyte retrieval after controlled ovarian hyperstimulation.

STUDY QUESTION: Can ovarian tissue cryopreservation (OTC) be performed after controlled ovarian hyperstimulation (COH)? SUMMARY ANSWER: Unilateral oophorectomy after transvaginal oocyte retrieval is feasible on stimulated ovaries during one surgical step. WHAT IS KNOWN ALREADY: In the fertility preservation (FP) field, the timeframe between patient referral and start of curative treatment is limited. Combining oocyte pick-up with ovarian tissue (OT) extraction has been reported to improve FP but COH applied before OT extraction is not currently recommended. STUDY DESIGN, SIZE, DURATION: This retrospective cohort-controlled study involved 58 patients who underwent oocyte cryopreservation immediately followed by OTC between September 2009 and November 2021. The exclusion criteria were a delay between oocyte retrieval and OTC of >24 h (n = 5) and IVM of oocytes obtained ex vivo in the ovarian cortex (n = 2). This FP strategy was performed either after COH (stimulated group, n = 18) or after IVM (unstimulated group, n = 33). PARTICIPANTS/MATERIALS, SETTING, METHODS: Oocyte retrieval followed by OT extraction on the same day was performed either without previous stimulation or after COH. Adverse effects of surgery and ovarian stimulation, mature oocyte yield and pathology findings of fresh OT were retrospectively analysed. Thawed OTs were analysed prospectively, for vascularization and apoptosis using immunohistochemistry, when patient consent was obtained. MAIN RESULTS AND THE ROLE OF CHANCE: No surgical complication occurred after OTC surgery in either group. In particular, no severe bleeding was associated with COH. The number of mature oocytes obtained increased after COH (median = 8.5 (25% = 5.3-75% = 12.0)) compared to the unstimulated group (2.0 (1.0-5.3), P < 0.001). Neither ovarian follicle density nor cell integrity was affected by COH. Fresh OT analysis showed congestion in half of the stimulated OT which was higher than in the unstimulated OT (3.1%, P < 0.001). COH also increased haemorrhagic suffusion (COH + OTC: 66.7%; IVM + OTC: 18.8%, P = 0.002) and oedema (COH + OTC: 55.6%; IVM + OTC: 9.4%, P < 0.001). After thawing, the pathological findings were similar between both groups. No statistical difference in the number of blood vessels was observed between the groups. The oocyte apoptotic rate in thawed OT was not statistically different between the groups (ratio of positive cleaved caspase-3 staining oocytes/total number of oocytes equal to median 0.50 (0.33-0.85) and 0.45 (0.23-0.58) in unstimulated and stimulated groups respectively, P = 0.720). LIMITATIONS, REASONS FOR CAUTION: The study reports FP from a small number of women following OTC. Follicle density and other pathology findings are an estimate only. WIDER IMPLICATIONS OF THE FINDINGS: Unilateral oophorectomy can be successfully performed after COH with limited bleeding risk and an absence of impact on thawed OT. This approach could be proposed to post pubertal patients when the number of mature oocytes expected is low or when the risk of residual pathology is high. The reduction of surgical steps for cancer patients also has positive implications for introducing this approach into clinical practice. STUDY FUNDING/COMPETING INTEREST(S): This work was made possible through the support of the reproductive department of Antoine-Béclère Hospital and of the pathological department of Bicêtre Hospital (Assistance Publique Hôpitaux de Paris, France). The authors have no conflict of interest to disclose in this study. TRIAL REGISTRATION NUMBER: N/A.

Preimplantation genetic testing for mitochondrial DNA mutation: ovarian response to stimulation, outcomes and follow-up.

RESEARCH QUESTION: How do carriers of pathogenic mitochondrial DNA (mtDNA) respond to ovarian stimulation? DESIGN: A single-centre, retrospective study conducted between January 2006 and July 2021 in France. Ovarian reserve markers and ovarian stimulation cycle outcomes were compared for couples undergoing preimplantation genetic testing (PGT) for maternally inherited mtDNA disease (n = 18) (mtDNA-PGT group) with a matched-control group of patients undergoing PGT for male indications (n = 96). The PGT outcomes for the mtDNA-PGT group and the follow-up of these patients in case of unsuccessful PGT was also reported. RESULTS: For carriers of pathogenic mtDNA, parameters of ovarian response to FSH and ovarian stimulation cycle outcomes were not different from those of matched-control ovarian stimulation cycles. The carriers of pathogenic mtDNA needed a longer ovarian stimulation and higher dose of gonadotrophins. Three patients (16.7%) obtained a live birth after the PGT process, and eight patients (44.4%) achieved parenthood through alternative methods: oocyte donation (n = 4), natural conception with prenatal diagnosis (n = 2) and adoption (n = 2). CONCLUSION: To the best of our knowledge, this is the first study of women carrying a mtDNA variant who have undergone a PGT for monogenic (single gene defects) procedure. It is one of the possible options to obtain a healthy baby without observing an impairment in ovarian response to stimulation.

Fetal growth disorders following medically assisted reproduction: due to maternal context or techniques? A national French cohort study.

RESEARCH QUESTION: What part do maternal context and medically assisted reproduction (MAR) techniques play in the risk of fetal growth disorders? DESIGN: This retrospective nationwide cohort study uses data available in the French National Health System database and focuses on the period from 2013 to 2017. Fetal growth disorders were divided into four groups according to the origin of pregnancy: fresh embryo transfer (n = 45,201), frozen embryo transfer (FET, n = 18,845), intrauterine insemination (IUI, n = 20,179) and natural conceptions (n = 3,412,868). Fetal growth disorders were defined from the percentiles of the weight distribution according to gestational age and sex: small and large for gestational age (SGA and LGA) if <10th and >90th percentiles, respectively. Analyses were performed using univariate and multivariate logistic models. RESULTS: Compared with births following natural conception, multivariate analysis showed that the risk of SGA was higher for births following fresh embryo transfer and IUI (adjusted odds ratio [aOR] 1.26 [1.22-1.29] and 1.08 [1.03-1.12], respectively) and significantly lower following FET (aOR 0.79 [0.75-0.83]). The risk of LGA was higher for births following FET (aOR 1.32 [1.27-1.38]), especially in artificial cycles when compared with ovulatory cycles (aOR 1.25 [1.15-1.36]). In the subgroup of births without any obstetrical or neonatal morbidity, the same increased risk of SGA and LGA were observed following fresh embryo transfer or IUI and FET (aOR 1.23 [1.19-1.27] or 1.06 [1.01-1.11] and aOR 1.36 [1.30-1.43], respectively). CONCLUSIONS: An effect of MAR techniques on the risks for SGA and LGA is suggested independently from maternal context and obstetrical or neonatal morbidities. Pathophysiological mechanisms remain poorly understood and should be further evaluated, as well as the influence of embryonic stage and freezing techniques.

Obstetrical outcomes and maternal morbidities associated with COVID-19 in pregnant women in France: A national retrospective cohort study.

BACKGROUND: To the best of our knowledge, no study has exhaustively evaluated the association between maternal morbidities and Coronavirus Disease 2019 (COVID-19) during the first wave of the pandemic in pregnant women. We investigated, in natural conceptions and assisted reproductive technique (ART) pregnancies, whether maternal morbidities were more frequent in pregnant women with COVID-19 diagnosis compared to pregnant women without COVID-19 diagnosis during the first wave of the COVID-19 pandemic. METHODS AND FINDINGS: We conducted a retrospective analysis of prospectively collected data in a national cohort of all hospitalizations for births ≥22 weeks of gestation in France from January to June 2020 using the French national hospitalization database (PMSI). Pregnant women with COVID-19 were identified if they had been recorded in the database using the ICD-10 (International Classification of Disease) code for presence of a hospitalization for COVID-19. A total of 244,645 births were included, of which 874 (0.36%) in the COVID-19 group. Maternal morbidities and adverse obstetrical outcomes among those with or without COVID-19 were analyzed with a multivariable logistic regression model adjusted on patient characteristics. Among pregnant women, older age (31.1 (±5.9) years old versus 30.5 (±5.4) years old, respectively, p < 0.001), obesity (0.7% versus 0.3%, respectively, p < 0.001), multiple pregnancy (0.7% versus 0.4%, respectively, p < 0.001), and history of hypertension (0.9% versus 0.3%, respectively, p < 0.001) were more frequent with COVID-19 diagnosis. Active smoking (0.2% versus 0.4%, respectively, p < 0.001) and primiparity (0.3% versus 0.4%, respectively, p < 0.03) were less frequent with COVID-19 diagnosis. Frequency of ART conception was not different between those with and without COVID-19 diagnosis (p = 0.28). When compared to the non-COVID-19 group, women in the COVID-19 group had a higher frequency of admission to ICU (5.9% versus 0.1%, p < 0.001), mortality (0.2% versus 0.005%, p < 0.001), preeclampsia/eclampsia (4.8% versus 2.2%, p < 0.001), gestational hypertension (2.3% versus 1.3%, p < 0.03), postpartum hemorrhage (10.0% versus 5.7%, p < 0.001), preterm birth at <37 weeks of gestation (16.7% versus 7.1%, p < 0.001), <32 weeks of gestation (2.2% versus 0.8%, p < 0.001), <28 weeks of gestation (2.4% versus 0.8%, p < 0.001), induced preterm birth (5.4% versus 1.4%, p < 0.001), spontaneous preterm birth (11.3% versus 5.7%, p < 0.001), fetal distress (33.0% versus 26.0%, p < 0.001), and cesarean section (33.0% versus 20.2%, p < 0.001). Rates of pregnancy terminations ≥22 weeks of gestation, stillbirths, gestational diabetes, placenta praevia, and placenta abruption were not significantly different between the COVID-19 and non-COVID-19 groups. The number of venous thromboembolic events was too low to perform statistical analysis. A limitation of this study relies in the possibility that asymptomatic infected women were not systematically detected. CONCLUSIONS: We observed an increased frequency of pregnant women with maternal morbidities and diagnosis of COVID-19 compared to pregnant women without COVID-19. It appears essential to be aware of this, notably in populations at known risk of developing a more severe form of infection or obstetrical morbidities and in order for obstetrical units to better inform pregnant women and provide the best care. Although causality cannot be determined from these associations, these results may be in line with recent recommendations in favor of vaccination for pregnant women.

Live birth rate after use of cryopreserved oocytes or embryos at the time of cancer diagnosis in female survivors: a retrospective study of ten years of experience.

PURPOSE: The aim of this study was to evaluate the outcomes of frozen oocytes or embryos cryopreserved after controlled ovarian stimulation (COS) or in vitro maturation (IVM) for female cancer patients who underwent a fertility preservation (FP) prior to gonadotoxic therapy. METHODS: A retrospective cohort study from 2009 to December 2017 was conducted. Among the 667 female cancer patients who underwent oocytes or embryos cryopreservation for FP, 40 (6%) have returned to the fertility clinic between 2011 and 2019 to use their frozen material after being cured. We compared these thaw cycles outcomes according to the techniques used at the time of cryopreservation. RESULTS: Among the 40 women cancer survivors who used their cryopreserved material, thirty patients have benefited from at least one embryo transfer. Ten patients did not have an embryo transfer since the oocytes did not survive after the thawing process or because no embryo was obtained after fertilization. We related three live births following FP using IVM (two from frozen oocytes and one after embryo cryopreservation). Five live births were obtained when COS was performed at the time of FP (one from frozen oocytes and four after embryo cryopreservation). CONCLUSIONS: Our preliminary results, although they are obtained in a small sample, are encouraging and show that different FP techniques can be used in female cancer patients and lead to live births. IVM is one of the options available that does not delay the start of chemotherapy or if ovarian stimulation using gonadotropins is contraindicated.

Does the prognosis after PGT for structural rearrangement differ between female and male translocation carriers?

RESEARCH QUESTION: Chromosomal translocations are known genetic causes of premature ovarian insufficiency syndrome. Are certain translocations associated with decreased capacity of small antral follicles to respond to exogenous FSH? Does the prognosis after preimplantation genetic testing for structural rearrangements differ in couples with female or male translocation carriers and according to the type of translocation? DESIGN: A single-centre, retrospective, observational study covering a 10-year period. One hundred and thirty-nine females carrying a translocation were compared with 192 partners of male translocation carriers. To evaluate ovarian response to FSH, the follicular output rate was used, defined by ratio between the pre-ovulatory follicle count on day of HCG x 100/antral follicle count (AFC). To determine a cut-off of metaphase II oocytes and biopsied embryos as predictor of obtaining a balanced embryo transfer, receiver operator characteristic curves were plotted. RESULT: A decreased capacity of small antral follicles to respond to exogenous FSH in female translocation carriers was found. The number of metaphase II oocytes in both groups was weakly informative as a predictor of obtaining an embryo transfer. The number of biopsied embryos had some clinical value, however, and allowed a cut-off of 6.5 to be determined for female translocation carriers versus 5.5 for the partners of male translocation carriers. Live birth rates, however, were not different between female and male translocations carriers. CONCLUSIONS: Female translocation carriers may respond poorly to ovarian stimulation, and present a higher rate of unbalanced embryos, which means that higher gonadotrophin doses may be required to increase the number of biopsied embryos.

Chromosomal translocations and semen quality: A study on 144 male translocation carriers.

RESEARCH QUESTION: Chromosomal translocations are known genetic causes of male infertility. Are certain translocations or chromosomal regions more directly associated with sperm defects? Is there a threshold of sperm impairment that can be relevant for detection of translocations? DESIGN: This is a monocentric retrospective observational study covering a 10-year period. Eighty-one patients carrying a reciprocal translocation (RCT) and 63 carrying a Robertsonian translocation (ROBT) were compared with 105 fertile patients. Semen quality before and after sperm migration was compared. The aims were to define whether a threshold based on sperm analysis could be proposed for detection of translocations and to identify whether some redundant chromosomal regions might be associated with sperm quality defects. RESULTS: The number of progressive spermatozoa retrieved after sperm preparation (NPS-ASP) was altered in both RCT and ROBT carriers compared with controls, with a stronger alteration in ROBT. Based on the NPS-ASP results in this large group of translocation carriers, a relatively robust threshold, fixed at less than 5 million, may be proposed for detection of translocations. The alteration of NPS-ASP was independent of the chromosome involved in ROBT, while in RCT, four redundant chromosomal regions (1q21, 6p21, 16q21, 17q11.2) were associated with poor or very poor NPS-ASP. CONCLUSIONS: The NPS-ASP appears to be a good parameter to assess sperm function and would be a useful tool to detect chromosomal translocations. Four redundant regions have been identified on four chromosomes, suggesting that they may contain genes of interest to study sperm functions.

Mutation dependance of the mitochondrial DNA copy number in the first stages of human embryogenesis.

Mitochondrial DNA (mtDNA) content is thought to remain stable over the preimplantation period of human embryogenesis that is, therefore, suggested to be entirely dependent on ooplasm mtDNA capital. We have explored the impact of two disease-causing mutations [m.3243A>G myopathy, encephalopathy, lactic acidosis and stroke-like syndrome (MELAS) and m.8344A>G myoclonic epilepsy associated with ragged-red fibers (MERRF)] on mtDNA amounts in human oocytes and day 4-5 preimplantation embryos. The mtDNA amount was stable in MERRF and control materials, whereas gradually increasing from the germinal vesicle of oogenesis to the blastocyst stage of embryogenesis in MELAS cells, MELAS embryos carrying ∼3-fold higher mtDNA amount than control embryos (P = 0.0003). A correlation between mtDNA copy numbers and mutant loads was observed in MELAS embryos (R(2) = 0.42, P < 0.0013), suggestive of a compensation for the respiratory chain defect resulting from high mutation levels. These results suggest that mtDNA can replicate in early embryos and emphasize the need for sufficient amount of wild-type mtDNA to sustain embryonic development in humans.

Poor correlations in the levels of pathogenic mitochondrial DNA mutations in polar bodies versus oocytes and blastomeres in humans.

Because the mtDNA amount remains stable in the early embryo until uterine implantation, early human development is completely dependent on the mtDNA pool of the mature oocyte. Both quantitative and qualitative mtDNA defects therefore may negatively impact oocyte competence or early embryonic development. However, nothing is known about segregation of mutant and wild-type mtDNA molecules during human meiosis. To investigate this point, we compared the mutant levels in 51 first polar bodies (PBs) and their counterpart (oocytes, blastomeres, or whole embryos), at risk of having (1) the "MELAS" m.3243A>G mutation in MT-TL1 (n = 30), (2) the "MERRF" m.8344A>G mutation in MT-TK (n = 15), and (3) the m.9185T>G mutation located in MT-ATP6 (n = 6). Seven out of 51 of the PBs were mutation free and had homoplasmic wild-type counterparts. In the heteroplasmic PBs, measurement of the mutant load was a rough estimate of the counterpart mutation level (R(2) = 0.52), and high mutant-load differentials between the two populations were occasionally observed (ranging from -34% to +34%). The mutant-load differentials between the PB and its counterpart were higher in highly mutated PBs, suggestive of a selection process acting against highly mutated cells during gametogenesis or early embryonic development. Finally, individual discrepancies in mutant loads between PBs and their counterparts make PB-based preconception diagnosis unreliable for the prevention of mtDNA disorder transmission. Such differences were not observed in animal models, and they emphasize the need to conduct thorough studies on mtDNA segregation in humans.

Is it acceptable to destroy or include human embryos before day 5 in research programmes?

Day-3 poor-quality embryos (PQE) from IVF-embryo transfer cycles are usually destroyed or are included in research programmes. Knowing that these embryos have the ability to evolve to the blastocyst stage and yield embryonic stem cell lines, this study postulated that they could also give rise to live births. This is a prospective study including 186 IVF-embryo transfer candidates who had obtained at least one supernumerary PQE on day 3. PQE were kept for extended culture and high-quality blastocysts were frozen. A total of 620 PQE were eligible for the study, 217 (35.0%) reached the blastocyst stage and 73 (33.6%) were frozen. Blastulation rates were 7-fold higher (OR 7.29, 95% CI 5.01-10.61) in embryos compacted on day 4. Of the frozen blastocysts, 40 were thawed during 33 thawed blastocyst transfer cycles, which led to 10 clinical pregnancies. These pregnancies resulted in five miscarriages and five healthy live births at full term. PQE may achieve their development to the blastocyst stage, be frozen-thawed and harbour reasonable implantation potential. These results, thereby, raise an ethical issue regarding the fate reserved to PQE.

PGT and deferred embryo transfer: Is blastocyst biopsy more effective than cleaved embryo biopsy?

OBJECTIVE: Blastocyst biopsy has recently been implemented in our laboratory for PGT with a "freeze all" indication. The aim of this study is to compare PGT results between embryos biopsied at the cleaved and embryos biopsied at the blastocyst stage. STUDY DESIGN: This is a retrospective cohort study conducted from January 2017 to December 2022 in France. All couples with a "freeze all" indication the day of hCG trigerring during the study period were included in the study. Patients were retrospectively assigned in one group of two groups based on the day of embryo biopsy: the cleavage group if a blastomere biopsy was performed on day 3/4 or the blastocyst group if a trophectoderm biopsy was performed on day 5/6. We evaluated and compared the results between the two groups for biological parameters and clinical outcomes. RESULTS: In total, 325 PGT cycles (291 patients) were included in our study. Frozen-thawed embryo transfer was performed for 285 cycles, 122 in the blastocyst group and 163 in the cleavage group. The number of biopsied embryos per cycle is significantly higher in the cleavage group with a mean of 7.2 ± 4.1 embryos biopsied per cycle vs. 2.9 ± 2.8 embryos in the blastocyst group (p < 0.001). The rate of the useful embryos was similar between the two groups with 14.6 % of frozen healthy embryos among the 1352 cleaved embryos obtained in blastocyst group, compared to 17.1 % in the cleavage group. No significant differences in clinical pregnancy rate per transfer and implantation rate were observed between the blastocyst and cleavage groups (36.4% vs. 40.4 % and 33.1% vs. 33.2 % respectively). CONCLUSIONS: For "freeze all" PGT cycles, the day of embryo biopsy (cleaved vs blastocyst biopsy) does not impact pregnancy outcomes. Knowing how to perform embryo biopsy at different stages helps to better organize daily laboratory activity and to rescue some undiagnosed embryos after day 3 biopsy.

Loss of LH-induced down-regulation of anti-Müllerian hormone receptor expression may contribute to anovulation in women with polycystic ovary syndrome.

STUDY QUESTION: Are anti-Müllerian hormone (AMH) and AMH type II receptor (AMHR-II) mRNAs similarly regulated by gonadotrophins in lutein granulosa cells (GCs) from control, normo-ovulatory and oligo/anovulatory women with polycystic ovary syndrome (PCOS)? SUMMARY ANSWER: AMH mRNA expression was induced by LH only in lutein GC of oligo/anovulatory PCOS women; down-regulation of AMHR-II, induced by LH in control and normo-ovulatory PCOS women, was absent in oligo/anovulatory women. WHAT IS KNOWN ALREADY: It was suggested that AMH could be responsible for the blockade of follicles at the small antral stage in PCOS women. In keeping with this hypothesis, both AMH and AMHR-II are overexpressed in lutein GCs from oligo/anovulatory PCOS women. STUDY DESIGN, SIZE, DURATION: Women undergoing IVF were included in this prospective study, either in the control group (30 women) or in the PCOS group (21 normo-ovulatory and 19 oligo/anovulatory patients) between January 2010 and July 2012. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human lutein GCs were isolated from follicular fluid during IVF protocols. Twenty-four hours after seeding, lutein GCs from each woman were serum starved and cultured for 48 h ± FSH, LH or cAMP. Then AMH and AMHR-II mRNAs were quantified by quantitative RT-PCR and AMH protein concentration was measured in the culture medium by ELISA. Experimental results were analyzed, within each group of women, by the non-parametric Wilcoxon test for paired comparisons between cells cultured in control medium and FSH, LH or cAMP treated cells. Clinical comparisons between the three groups of women were performed on log values using the ANOVA test with Bonferroni correction. MAIN RESULTS AND THE ROLE OF CHANCE: FSH up-regulated both AMH expression and secretion by lutein GCs from the three groups of women (P < 0.05). LH had no effect on AMH mRNAs levels in lutein GCs from controls and normo-ovulatory PCOS women, but increased AMH expression in oligo/anovulatory PCOS women (P < 0.05). Interestingly, LH and cAMP treatments reduced AMHR-II expression by lutein GCs from controls and normo-ovulatory PCOS women (P < 0.05), but had no effect on AMHR-II mRNA levels in oligo/anovulatory PCOS women. LIMITATIONS, REASONS FOR CAUTION: The lutein GCs are not the best model to study AMH and AMHR-II regulation by gonadotrophins. Indeed, AMH and AMHR-II are down-regulated in luteinized cells. Furthermore, these cells have been exposed to non-physiological levels of gonadotrophins and hCG. However, AMH and AMHR-II mRNAs are quantifiable by real-time RT-PCR, and the cells are still responsive to FSH and LH. The age of patients is significantly different between control and oligo/anovulatory PCOS women: this may be a bias in the interpretation of results but older women in the control group had a good ovarian reserve. WIDER IMPLICATIONS OF THE FINDINGS: The overexpression of AMH and AMHR-II in oligo/anovulatory PCOS women could be due to increased LH levels and/or inhibition of its repressive action. The fact that this dysregulation is observed in oligo/anovulatory, but not in normo-ovulatory, PCOS women emphasizes the role of LH in the follicular arrest of PCOS women and suggests that this involves the AMH/AMHR-II system. STUDY FUNDING/COMPETING INTEREST(S): The Assistance-Publique Hôpitaux de Paris provided a Contrat d'Interface and the Agence de Biomédecine provided a grant to Nathalie di Clemente. Schering-Plough provided an FARO grant to Alice Pierre. The authors have nothing to disclose.

Higher risk of pre-eclampsia and other vascular disorders with artificial cycle for frozen-thawed embryo transfer compared to ovulatory cycle or to fresh embryo transfer following in vitro fertilization.

Background: Risks of maternal morbidity are known to be reduced in pregnancies resulting from frozen embryo transfer (FET) compared to fresh-embryo transfer (fresh-ET), except for the risk of pre-eclampsia, reported to be higher in FET pregnancies compared to fresh-ET or natural conception. Few studies have compared the risk of maternal vascular morbidities according to endometrial preparation for FET, either with ovulatory cycle (OC-FET) or artificial cycle (AC-FET). Furthermore, maternal pre-eclampsia could be associated with subsequent vascular disorders in the offspring. Methods: A 2013-2018 French nationwide cohort study comparing maternal vascular morbidities in 3 groups of single pregnancies was conducted: FET with either OC or AC preparation, and fresh-ET. Data were extracted from the French National Health System database. Results were adjusted for maternal characteristics and infertility (age, parity, smoking, obesity, history of diabetes or hypertension, endometriosis, polycystic ovary syndrome and premature ovarian insufficiency). Results: A total of 68025 single deliveries were included: fresh-ET (n=48152), OC-FET (n=9500), AC-FET (n=10373). The risk of pre-eclampsia was higher in AC-FET compared to OC-FET and fresh-ET groups in univariate analysis (5.3% vs. 2.3% and 2.4%, respectively, P<0.0001). In multivariate analysis the risk was significantly higher in AC-FET compared to fresh-ET: aOR=2.43 [2.18-2.70], P<0.0001). Similar results were observed for the risk of other vascular disorders in univariate analysis (4.7% vs. 3.4% and 3.3%, respectively, P=0.0002) and in multivariate analysis (AC-FET compared to fresh-ET: aOR=1.50 [1.36-1.67], P<0.0001). In multivariate analysis, the risk of pre-eclampsia and other vascular disorders were comparable in OC-FET and fresh-ET: aOR=1.01 [0.87-1.17, P= 0.91 and aOR=1.00 [0.89-1.13], P=0.97, respectively).Within the group of FET, the risks of pre-eclampsia and other vascular disorders in multivariate analysis were higher in AC-FET compared to OC-FET (aOR=2.43 [2.18-2.70], P<0.0001 and aOR=1.5 [1.36-1.67], P<0.0001, respectively). Conclusion: This nationwide register-based cohort study highlights the possibly deleterious role of prolonged doses of exogenous estrogen-progesterone supplementation on gestational vascular pathologies and the protective role of the corpus luteum present in OC-FET for their prevention. Since OC-FET has been demonstrated not to strain the chances of pregnancy, OC preparation should be advocated as first-line preparation in FET as often as possible in ovulatory women.

Differences in transcriptomic profiles of human cumulus cells isolated from oocytes at GV, MI and MII stages after in vivo and in vitro oocyte maturation.

BACKGROUND: Oocyte maturation and competence to development depends on its close relationship with cumulus cells (CCs). However, the maturation conditions of human cumulus-oocyte complexes (COCs) might affect gene expression in both oocyte and CCs. We thus compared the transcriptome profiles of CCs isolated from in vivo and in vitro matured COCs at different nuclear maturation stages. METHODS: Three groups of CCs from patients who underwent ICSI were included: CCs of patients with polycystic ovary syndrome (PCOS) referred for in vitro maturation (IVM), CCs from patients with PCOS for in vivo maturation (used as controls) and CCs from normal responders referred for in vivo maturation. CCs were isolated from COCs at the germinal vesicle, metaphase I and metaphase II stages. Microarray technology was used to analyse the global gene expression and significance analysis of microarray to compare the expression profiles of CCs from COCs at different nuclear maturation stages following IVM or in vivo maturation. Selected genes were validated by RT-qPCR. RESULTS: In CCs isolated after IVM, genes related to cumulus expansion and oocyte maturation, such as EREG, AREG and PTX3, were down-regulated, while cell cycle-related genes were up-regulated in comparison with CCs from in vivo matured COCs from PCOS and normal responder patients. Moreover, irrespective of the stage of oocyte maturation, genes involved in DNA replication, recombination and repair were up-regulated in CCs after IVM. CONCLUSIONS: The CC transcriptomic signature varies according to both the oocyte maturation stage and the maturation conditions. Our findings suggest a delay in the acquisition of the mature CC phenotype following IVM, opening a new perspective for the improvement in IVM conditions.

MLPA and sequence analysis of DPY19L2 reveals point mutations causing globozoospermia.

STUDY QUESTION: Do DPY19L2 heterozygous deletions and point mutations account for some cases of globozoospermia? SUMMARY ANSWER: Two DPY19L2 heterozygous deletions and three point mutations were identified, thus further confirming that genetic alterations of the DPY19L2 gene are the main cause of globozoospermia and indicating that DPY19L2 molecular diagnostics should not be stopped in the absence of a homozygous gene deletion. WHAT IS KNOWN ALREADY: Globozoospermia is a rare phenotype of primary male infertility characterized by the production of a majority of round-headed spermatozoa without acrosome. We demonstrated previously that most cases in man were caused by a recurrent homozygous deletion of the totality of the DPY19L2 gene, preventing sperm head elongation and acrosome formation. In mammals, DPY19L2 has three paralogs of yet unknown function and one highly homologous pseudogene showing >95% sequence identity with DPY19L2. Specific amplification and sequencing of DPY19L2 have so far been hampered by the presence of this pseudogene which has greatly complicated specific amplification and sequencing. STUDY DESIGN, SIZE, DURATION: In this cohort study, 34 patients presenting with globozoospermia were recruited during routine infertility treatment in infertility centers in France and Tunisia between January 2008 and December 2011. The molecular variants identified in patients were screened in 200 individuals from the general population to exclude frequent non-pathological polymorphisms. PARTICIPANTS/MATERIALS, SETTING, METHODS: We developed a Multiplex Ligation-dependent Probe Amplification test to detect the presence of heterozygous deletions and identified the conditions to specifically amplify and sequence the 22 exons and intronic boundaries of the DPY19L2 gene. The pathogenicity of the identified mutations and their action on the protein were evaluated in silico. MAIN RESULTS AND THE ROLE OF CHANCE: There were 23 patients who were homozygous for the DPY19L2 deletion (67.6%). Only eight of the eleven non-homozygously deleted patients could be sequenced due to poor DNA quality of three patients. Two patients were compound heterozygous carrying one DPY19L2 deleted allele associated respectively with a nonsense (p.Q342*) and a missense mutation (p.R290H). One patient was homozygous for p.M358K, another missense mutation affecting a highly conserved amino acid. Due to the localization of this mutation and the physicochemical properties of the substituted amino acids, we believe that this variant is likely to disrupt one of the protein transmembrane domains and destabilize the protein. Overall, 84% of the fully analysed patients (n = 31) had a molecular alteration of DPY19L2. There was no clear phenotypic difference between the homozygous deleted individual, patients carrying a point mutation and undiagnosed patients. LIMITATIONS, REASONS FOR CAUTION: Globally poor fertilization rates are observed after intracytoplasmic sperm injection of round spermatozoa. Further work is needed to assess whether DPY19L2 mutated patients present a better or worse prognostic than the non-diagnosed patients. Evaluation of the potential benefit of treatment with a calcium ionophore, described to improve fertilization, should be evaluated in these two groups. WIDER IMPLICATIONS OF THE FINDINGS: In previous work, deletions of DPY19L2 had only been identified in North African patients. Here we have identified DPY19L2 deletions and point mutations in European patients, indicating that globozoospemia caused by a molecular defect of DPY19L2 can be expected in individuals from any ethnic background. STUDY FUNDING/COMPETING INTEREST(S): None of the authors have any competing interest. This work is part of the project 'Identification and Characterization of Genes Involved in Infertility (ICG2I)' funded by the program GENOPAT 2009 from the French Research Agency (ANR).

Oocyte cryopreservation following failed testicular sperm extraction: a French case report with implications for the management of non-obstructive azoospermia.

The French law regulating assisted reproductive technologies forbids donor spermatozoa to be available in case of failed testicular sperm extraction (TESE) performed on the day of oocyte retrieval. This article reports the first French live birth after intracytoplasmic sperm injection of donated spermatozoa into frozen-thawed oocytes cryopreserved following failure of TESE. By reinforcing the relevance of TESE performed on the day of oocyte retrieval, oocyte cryopreservation in couples having beforehand consented to go to sperm donation will avoid cycle cancellation and potentially lead to successful live birth. Therefore, it could modify the French policy of management of patients suffering from non-obstructive azoospermia.

New trends in female fertility preservation: in vitro maturation of oocytes.

As antineoplastic treatments have become more successful, an increasing number of women with cancer survive to endure the long-term consequences of chemotherapy. One of the most important of these consequences in young females is premature ovarian failure and infertility. Owing to increasing survival rates, many of these young women are seeking methods to preserve their fertility. Currently, embryo/oocyte cryopreservation obtained after controlled ovarian stimulation appears to provide the best fertility preservation option. However, patients may not have enough time to undergo ovarian stimulation prior to chemotherapy and/or have contraindications to exogenous gonadotropin administration owing to estrogen-dependant tumors. In vitro maturation of oocytes is an attractive alternative for fertility preservation in cancer patients because it does not require ovarian stimulation and it can be performed at any time of the menstrual cycle. In addition, this technique can be combined with ovarian tissue cryobanking. In this review, we discuss the position of in vitro maturation of oocytes in the fertility preservation strategy in young women.

Second biopsy for embryos with inconclusive results after preimplantation genetic testing: Impact on pregnancy outcomes.

In this study, we aimed to evaluate the pregnancy outcomes for embryos biopsied twice at cleavage and blastocyst stage for preimplantation genetic testing (PGT). This retrospective monocentric study, conducted between January 2016 and March 2021, described all PGT results on one hand and the PGT results for undiagnosed embryos submitted to a second biopsy on the other hand. Among the 5865 embryos biopsied during the study period, 510 embryos were genetic undiagnosed after the first embryo biopsy at cleavage stage (8.7%). The rate of undiagnosed embryos was significantly higher for PGT for structural rearrangement (PGT-SR) than PGT for monogenic disease (PGT-M) (10.2% vs 7.4% respectively, p < 0.001). Thirty-three embryos were compatible with a second biopsy at blastocyst stage before being directly frozen. Among them 17 were diagnosed as healthy for the researched pathology (51.5%). At the time of our study, 11 of the 17 preserved embryos were thawed and transferred. Embryo survival at thawing was 100% and 5 pregnancies were obtained (clinical pregnancy rate of 45.5% per transfer), including 3 live births. A second biopsy for inconclusive embryos after PGT does not seem to have an impact on thaw survival and implantation rate. For couple, this strategy avoids to discard transferable embryos.

Segregation of mtDNA throughout human embryofetal development: m.3243A>G as a model system.

Mitochondrial DNA (mtDNA) mutations cause a wide range of serious diseases with high transmission risk and maternal inheritance. Tissue heterogeneity of the heteroplasmy rate ("mutant load") accounts for the wide phenotypic spectrum observed in carriers. Owing to the absence of therapy, couples at risk to transmit such disorders commonly ask for prenatal (PND) or preimplantation diagnosis (PGD). The lack of data regarding heteroplasmy distribution throughout intrauterine development, however, hampers the implementation of such procedures. We tracked the segregation of the m.3243A>G mutation (MT-TL1 gene) responsible for the MELAS syndrome in the developing embryo/fetus, using tissues and cells from eight carrier females, their 38 embryos and 12 fetuses. Mutant mtDNA segregation was found to be governed by random genetic drift, during oogenesis and somatic tissue development. The size of the bottleneck operating for m.3243A>G during oogenesis was shown to be individual-dependent. Comparison with data we achieved for the m.8993T>G mutation (MT-ATP6 gene), responsible for the NARP/Leigh syndrome, indicates that these mutations differentially influence mtDNA segregation during oogenesis, while their impact is similar in developing somatic tissues. These data have major consequences for PND and PGD procedures in mtDNA inherited disorders.