Preclinical workup using long-read amplicon sequencing provides families with de novo pathogenic variants access to universal preimplantation genetic testing.

STUDY QUESTION: Can long-read amplicon sequencing be beneficial for preclinical preimplantation genetic testing (PGT) workup in couples with a de novo pathogenic variant in one of the prospective parents? SUMMARY ANSWER: Long-read amplicon sequencing represents a simple, rapid and cost-effective preclinical PGT workup strategy that provides couples with de novo pathogenic variants access to universal genome-wide haplotyping-based PGT programs. WHAT IS KNOWN ALREADY: Universal PGT combines genome-wide haplotyping and copy number profiling to select embryos devoid of both familial pathogenic variants and aneuploidies. However, it cannot be directly applied in couples with a de novo pathogenic variant in one of the partners due to the absence of affected family members required for phasing the disease-associated haplotype. STUDY DESIGN, SIZE, DURATION: This is a prospective study, which includes 32 families that were enrolled in the universal PGT program at the University Hospital of Leuven between 2018 and 2022. We implemented long-read amplicon sequencing during the preclinical PGT workup to deduce the parental origin of the disease-associated allele in the affected partner, which can then be traced in embryos during clinical universal PGT cycles. PARTICIPANTS/MATERIALS, SETTING, METHODS: To identify the parental origin of the disease-associated allele, genomic DNA from the carrier of the de novo pathogenic variant and his/her parent(s) was used for preclinical PGT workup. Primers flanking the de novo variant upstream and downstream were designed for each family. Following long-range PCR, amplicons that ranged 5-10 kb in size, were sequenced using Pacific Bioscience and/or Oxford Nanopore platforms. Next, targeted variant calling and haplotyping were performed to identify parental informative single-nucleotide variants (iSNVs) linked to the de novo mutation. Following the preclinical PGT workup, universal PGT via genome-wide haplotyping was performed for couples who proceeded with clinical PGT cycle. In parallel, 13 trophectoderm (TE) biopsies from three families that were analyzed by universal PGT, were also used for long-read amplicon sequencing to explore this approach for embryo direct mutation detection coupled with targeted long-read haplotyping. MAIN RESULTS AND THE ROLE OF CHANCE: The parental origin of the mutant allele was identified in 24/32 affected individuals during the preclinical PGT workup stage, resulting in a 75% success rate. On average, 5.95 iSNVs (SD = 4.5) were detected per locus of interest, and the average distance of closest iSNV to the de novo variant was ∼1750 bp. In 75% of those cases (18/24), the de novo mutation occurred on the paternal allele. In the remaining eight families, the risk haplotype could not be established due to the absence of iSNVs linked to the mutation or inability to successfully target the region of interest. During the time of the study, 12/24 successfully analyzed couples entered the universal PGT program, and three disease-free children have been born. In parallel to universal PGT analysis, long-read amplicon sequencing of 13 TE biopsies was also performed, confirming the segregation of parental alleles in the embryo and the results of the universal PGT. LIMITATIONS, REASONS FOR CAUTION: The main limitation of this approach is that it remains targeted with the need to design locus-specific primers. Because of the restricted size of target amplicons, the region of interest may also remain non-informative in the absence of iSNVs. WIDER IMPLICATIONS OF THE FINDINGS: Targeted haplotyping via long-read amplicon sequencing, particularly using Oxford Nanopore Technologies, provides a valuable alternative for couples with de novo pathogenic variants that allows access to universal PGT. Moreover, the same approach can be used for direct mutation analysis in embryos, as a second line confirmation of the preclinical PGT result or as a potential alternative PGT procedure in couples, where additional family members are not available. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by KU Leuven funding (no. C1/018 to J.R.V.) and Fonds Wetenschappelijk Onderzoek (1241121N to O.T.). J.R.V. is co-inventor of a patent ZL910050-PCT/EP2011/060211-WO/2011/157846 'Methods for haplotyping single-cells' and ZL913096-PCT/EP2014/068315-WO/2015/028576 'Haplotyping and copy number typing using polymorphic variant allelic frequencies' licensed to Agilent Technologies. All other authors have no conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Assessment of sexual function before medically assisted procreation: A mixed-methods study among a sample of infertile women and men cared for in a fertility center.

A mixed-methods study was conducted to investigate sexual function among infertile patients undergoing medically assisted procreation for the first time. The study employed an interview and content analysis approach, involving 45 infertile patients prior to their medically assisted procreation procedures. The findings revealed that infertile patients are a group at risk for sexual distress. Furthermore, patients with sexual dysfunctions exhibited lower levels of sexual activity, potentially diminishing their chances of achieving pregnancy. Participants faced challenges in openly discussing their sexual problems and demonstrated limited knowledge of sexual functioning. Among infertile women with sexual dysfunctions, the most frequently reported issues were sexual interest/arousal disorders, with a majority also experiencing pain during sexual activity and associated genital-pelvic pain disorders. In contrast, delayed ejaculation and erectile disorder seem to be more common in infertile men, while sexual desire and excitement disorders and premature ejaculation disorders appeared to be as common as in the general population. While the relationship between infertility and sexuality is complex, our study suggests that sexual dysfunctions or the absence of sexual activity may explain infertility. Therefore, it is imperative for clinicians to evaluate the sexual functioning of both men and women undergoing medically assisted procreation treatment, to increase their chances of procreation and offer them sexological support if needed. Future studies should expand their scope to include a larger sample size and delve into the potential etiological factors associated with sexual dysfunctions.

Transplantation of Refrozen Ovarian Cortical Strips Retrieved from a Cryopreserved Whole Ovary: Proof of Feasibility.

We report successful clinical outcomes after transplantation of refrozen-rethawed cortical strips from a cryopreserved whole ovary in a patient diagnosed with stage IIIb rectal adenocarcinoma. Whole ovary cryopreservation was proposed as a fertility preservation strategy in 2006 prior to radiotherapy, chemotherapy and oncological surgery. To allow for minimal residual disease screening before ovarian reimplantation, the whole ovary was thawed and dissected into cortical strips. While awaiting the results, the majority of the cortical strips were refrozen. These refrozen-rethawed cortical strips were laparoscopically grafted to 2 sites: the previously irradiated pelvic cavity and the non-irradiated extrapelvic cavity. Ovarian function resumption was assessed by recovery of menses, hormone levels, ultrasound and oocyte pick-up following controlled ovarian stimulation (COS). Restoration of ovarian function occurred 6 months after reimplantation, with recovery of menses and estradiol secretion. A total of 12 cycles were followed by the IVF department. A second reimplantation was performed 1.5 years later, since the grafts were found to have stopped functioning for >3 consecutive months. Overall, 3 fertilizable oocytes were retrieved transabdominally from the extrapelvic graft following COS, yielding 2 embryos and culminating in one fresh embryo transfer, but no pregnancy. Concerning the reimplantation site, no ovarian activity was detected in the graft placed in the previously irradiated pelvic cavity. Indeed, only fibrotic-looking tissue was observed in the pelvic site at second laparoscopy 1.5 years later, while ovarian activity was noted in the extrapelvic graft, showing a large antral follicle. All in all, transplantation of refrozen-rethawed cortical strips from a cryopreserved whole ovary can lead to ovarian function resumption and embryo development if grafted to a non-irradiated field.

Live birth after allografting of ovarian cortex between genetically non-identical sisters.

Aggressive chemotherapy generally results in the loss of both endocrine and reproductive functions. If the patient has not undergone previous oocyte, embryo or ovarian tissue cryopreservation, orthotopic allotransplantation of fresh ovarian tissue from a genetically non-identical sister may be considered. Here, we describe a case report. The patient, aged 15 years and presenting with homozygous sickle cell anemia, underwent chemotherapy (busulfan, cyclophosphamide) and total body irradiation before bone marrow transplantation, the donor being her HLA-compatible sister. HLA group analysis later revealed complete chimerism. When the patient was 32 years old, ovarian allografting was performed, with the ovarian tissue donor being the same sister who had already donated bone marrow. The goal was to restore ovarian activity and natural fertility. No immunosuppressive therapy was administered. No sign of rejection was observed. Restoration of ovarian function was achieved 3.5 months after transplantation, as proved by the first estradiol peak and follicular development detected by ultrasound. After 9 months of regular ovulatory cycles, IVF was attempted because proximal tubal stenosis (unknown at the time of grafting) could not be repaired by tubal reanastomosis. After stimulation, three oocytes were retrieved. Two embryos were obtained. One embryo was frozen and the other was transferred, resulting in an ongoing pregnancy. The patient delivered a healthy baby girl weighing 3.150 g at 37 2/7 weeks of gestation.

Haplotyping-based preimplantation genetic testing reveals parent-of-origin specific mechanisms of aneuploidy formation.

Chromosome instability is inherent to human IVF embryos, but the full spectrum and developmental fate of chromosome anomalies remain uncharacterized. Using haplotyping-based preimplantation genetic testing for monogenic diseases (PGT-M), we mapped the parental and mechanistic origin of common and rare genomic abnormalities in 2300 cleavage stage and 361 trophectoderm biopsies. We show that while single whole chromosome aneuploidy arises due to chromosome-specific meiotic errors in the oocyte, segmental imbalances predominantly affect paternal chromosomes, implicating sperm DNA damage in segmental aneuploidy formation. We also show that postzygotic aneuploidy affects multiple chromosomes across the genome and does not discriminate between parental homologs. In addition, 6% of cleavage stage embryos demonstrated signatures of tripolar cell division with excessive chromosome loss, however hypodiploid blastomeres can be excluded from further embryo development. This observation supports the selective-pressure hypothesis in embryos. Finally, considering that ploidy violations may constitute a significant proportion of non-viable embryos, using haplotyping-based approach to map these events might further improve IVF success rate.

Restoration of ovarian function after allografting of ovarian cortex between genetically non-identical sisters.

BACKGROUND: Aggressive chemotherapy generally results in the loss of both endocrine and reproductive functions. For some women, however, oocyte, embryo or ovarian tissue cryopreservation were not proposed at the time. For three such women, orthotopic allotransplantation of fresh ovarian tissue from their genetically non-identical sister was performed. METHODS: Three women, aged 20, 15 and 12 years, respectively, underwent chemotherapy and total body irradiation before bone marrow transplantation (BMT), the donor in each case being their HLA-compatible sister. Years later, HLA group analysis revealed complete chimerism, and ovarian allografting was performed, with the ovarian tissue donor being the sister who had already donated bone marrow. No immunosuppressive therapy was administered. No sign of rejection was observed. RESULTS: Restoration of ovarian function occurred in all three cases, respectively, 6, 3.5 and 3.5 months after transplantation. The timing of the first estradiol peaks and the persistence of ovarian function were probably related to the primordial follicle density of donor ovarian tissue. CONCLUSIONS: Even in the absence of immunosuppressive therapy, ovarian allografting between genetically non-identical sisters allowed restoration of ovarian function in cases where previous BMT from the HLA-compatible sister resulted in full chimerism, avoiding the threat of rejection.

Experience With Medical Treatment of Cesarean Scar Ectopic Pregnancy (CSEP) With Local Ultrasound-Guided Injection of Methotrexate.

Objective: Ectopic pregnancy within Cesarean section scars is a rare condition. Late diagnosis carries significant risk of bleeding with poor prognosis for survival. There is no consensus on the management of this type of pregnancy. Historically, our facility offered an intra-muscular injection of methotrexate that resulted in a significant failure rate and later need for surgery. We hypothesized that injecting methotrexate directly into the gestational sac would improve the success rate of the treatment. Patients and Methods: This retrospective, uni-centric study examined nine patients aged between 33 and 42 years (mean age = 36.5 years) with Cesarean scar ectopic pregnancy (CSEP) between 2010 and 2018. CSEP was diagnosed by transvaginal ultrasound at a mean gestational age of 8w0/7. CSEP was treated under general anesthetic by ultrasound-guided methotrexate injection directly into the gestational sac. HCG levels and subsequent childbearing were monitored post-treatment. Results: Half of the patients were asymptomatic at the time of diagnosis. All patients tolerated treatment well and all ectopic pregnancies were successfully removed. HCG levels returned to negative within 3 months without additional medical or surgical intervention. The post-treatment pregnancy rate was 50%. Discussions/Conclusions: Our findings indicate that local ultrasound-guided injection of methotrexate into the gestational sac is a safe and effective therapeutic approach when performed by a trained team on a hemodynamically stable patient in the early stages of CSEP.

IVF outcome in patients with orthotopically transplanted ovarian tissue.

BACKGROUND: Chemo- or radiotherapy can induce premature ovarian failure (POF), and ovarian tissue cryopreservation and transplantation may be proposed to restore ovarian function. Our aim was to evaluate the quality of oocytes and embryos derived from frozen-thawed transplanted ovarian tissue. MATERIALS AND METHODS: Women were 21-28 years old at tissue cryopreservation. Nine women suffering POF following chemotherapy with or without radiotherapy underwent orthotopic ovarian tissue transplantation. After 12 months of spontaneous cycles without pregnancy, oocyte retrieval was performed in four patients during mildly stimulated or spontaneous cycles. ICSI was performed in all cases, with embryo transfer on day 3. Light and electron microscopy was used to study oocytes and embryos. RESULTS: Signs of ovarian function restoration (estradiol peak, decreased FSH, follicular development) began 16-26 weeks after reimplantation. Twenty-one oocyte retrieval attempts were made. At least one oocyte was collected in 15 cases, giving an empty follicle rate per retrieval of 29% (6/21). Sixteen oocytes were recovered, of which 6 were abnormal or immature (38%) and 10 (62%) were in metaphase II (MII). Three MII oocytes failed to fertilize, two showed abnormal fertilization and five normal MII oocytes successfully fertilized with subsequent normal embryo development (Grade 2), yielding an embryo transfer rate of 24% per retrieval. No pregnancy occurred. CONCLUSIONS: IVF in women with orthotopically grafted frozen-thawed ovarian tissue involves a higher risk of empty follicles, abnormal or immature oocytes, and low embryo transfer rates.

Contribution to More Patient-Friendly ART Treatment: Efficacy of Continuous Low-Dose GnRH Agonist as the Only Luteal Support-Results of a Prospective, Randomized, Comparative Study.

Background. The aim of this pilot study was to evaluate intranasal buserelin for luteal phase support and compare its efficacy with standard vaginal progesterone in IVF/ICSI antagonist cycles. Methods. This is a prospective, randomized, open, parallel group study. Forty patients underwent ovarian hyperstimulation with human menopausal gonadotropin under pituitary inhibition with gonadotropin-releasing hormone antagonist, while ovulation trigger and luteal support were achieved using intranasal GnRH agonist (group A). Twenty patients had their cycle downregulated with buserelin and stimulated with hMG, while ovulation trigger was achieved using 10,000 IU human chorionic gonadotropin with luteal support by intravaginal progesterone (group B). Results. No difference was observed in estradiol levels. Progesterone levels on day 5 were significantly lower in group A. However, significantly higher levels of luteinizing hormone were observed in group A during the entire luteal phase. Pregnancy rates (31.4% versus 22.2%), implantation rates (22% versus 15.4%), and clinical pregnancy rates (25.7% versus 16.7%) were not statistically different between groups, although a trend towards higher rates was observed in group A. No luteal phase lasting less than 10 days was recorded in either group. Conclusion. Intranasal administration of buserelin is effective for providing luteal phase support in IVF/ICSI antagonist protocols.

Laparoscopic management of peritoneal endometriosis, endometriotic cysts, and rectovaginal adenomyosis.

Peritoneal endometriosis is probably caused by the implantation of regurgitated menstrual cells. The ovarian endometrioma is the consequence of non-hormone-regulated bleeding from intraovarian epithelial inclusions after they have undergone metaplasia into endometrial-like tissue. Rectovaginal adenomyosis is, in fact, an adenomyotic lesion and can develop from Müllerian rests. In conclusion, peritoneal endometriosis, ovarian endometriosis, and rectovaginal adenomyotic nodules must be considered as three separate entities with different pathogeneses that require a different therapeutic approach.

Pre- and post-surgical management of endometriosis.

The efficacy of medical and surgical treatment of endometriosis-associated infertility and pelvic pain is a source of questions and controversies. Complete resolution of endometriosis is not yet possible, but therapy has essentially three main objectives: (1) to reduce pain, (2) to increase the possibility of pregnancy, and (3) to delay recurrence for as long as possible. It could be concluded that a consensus will probably never be reached on minimal and mild endometriosis. In cases of moderate and severe endometriosis-associated infertility, the combined approach (operative laparoscopy with gonadotropin-releasing hormone agonist) must be considered as first-line treatment. The mean pregnancy rate of 50% reported in the literature following surgery provides scientific proof that operative treatment should first be undertaken to give our patients the best chance of conceiving naturally. In cases of rectovaginal adenomyotic nodule, surgery must be considered as first-line therapy, medical therapy being relatively inefficacious.

Pelvic mass in a woman with Mayer-Rokitansky-Kuster-Hauser syndrome.

Magnetic resonance imaging and laparoscopy demonstrated the presence of a myoma in a woman with Rokitansky syndrome.

Safety of conservative management and fertility outcome in women with borderline tumors of the ovary.

OBJECTIVE: To assess the safety of fertility-sparing treatment and the remaining chance of childbearing after surgery. DESIGN: Retrospective clinical study. DESIGN: Gynecology department of a university teaching hospital. PATIENT(S): Seventy-five women underwent surgical management in our institution between 1986 and 2001 for borderline tumors of the ovary. INTERVENTION(S): Fifty-nine patients were treated by radical, fertility-compromising surgery. The remaining 16 patients underwent conservative surgery, preserving the uterus and at least some functional ovarian tissue. Seven unilateral adnexectomies, one simple cystectomy, and two adnexectomies associated with contralateral cystectomy were performed. MAIN OUTCOME MEASURE(S): Recurrence, survival, and pregnancy rates. RESULT(S): The observed recurrence rates after radical and conservative surgery were 0.0% and 18.7%, respectively. No disease-related deaths occurred in any group; there is no significant difference in survival rates. We can report 12 pregnancies in 7 of 11 women who underwent fertility-sparing management and who wished to become pregnant. CONCLUSION(S): In certain circumstances, conservative management offers a safe solution for borderline tumors of the ovary. Recurrence is noted significantly more often after this type of treatment, but all cases of recurrent disease can be detected with close follow-up and can be treated accordingly. No significant change in survival rates was found. Moreover, the pregnancy rate in women desiring pregnancy, those treated conservatively, was as high as 63.6%.

Surgical management of endometriosis.

The efficacy of medical and surgical treatment of endometriosis-associated infertility and pelvic pain is a source of ongoing controversy. Complete resolution of endometriosis is not yet possible and current therapy has three main objectives: (1) to reduce pain; (2) to increase the possibility of pregnancy; and (3) to delay recurrence for as long as possible. It is possible that a consensus will never be reached on the optimal treatment of minimal and mild endometriosis. In case of moderate and severe endometriosis-associated infertility, the combined approach (operative laparoscopy with a gonadotropin-releasing hormone (GnRH) agonist) should be considered as 'first-line' treatment. The mean pregnancy rate of 50% reported in the literature following surgery provides scientific proof that operative treatment should first be undertaken to give our patients the best chance of conceiving naturally. In case of rectovaginal adenomyotic nodules, surgery must be considered as first-line therapy, medical therapy being relatively in-efficacious.

Typical and subtle atypical presentations of endometriosis.

The diagnosis of peritoneal endometriosis at the time of laparoscopy is often made by the observation of typically puckered black or bluish lesions. There are also numerous subtle appearances of peritoneal endometriosis. The lesions are frequently non-pigmented. Red flame-like lesions, glandular excrescences, and subovarian adhesions must be considered as the most active lesions. Sometimes, however, subtle endometriotic lesions can be the only lesions seen at laparoscopy.

Ovarian tissue cryopreservation followed by controlled ovarian stimulation and pick-up of mature oocytes does not impair the number or quality of retrieved oocytes.

BACKGROUND: The objective of this study was to evaluate the feasibility of fertility preservation in cancer patients by combined bilateral ovarian cortex cryopreservation and embryo freezing. METHODS: This was a cohort-controlled study in a university hospital center. Sixteen patients with a recent cancer diagnosis were included in the study. They all consented to fertility preservation by a combined technique: ovarian tissue cryopreservation (OTC) followed by ovarian stimulation for in vitro fertilization (IVF) and embryo freezing. The control group included 100 women of the same age undergoing IVF for male factor infertility. RESULTS: The mean number of metaphase II oocytes was 8.3 per patient (±7.7) and was not statistically different from the control group (8.1 ± 5.6). The mean number of good quality embryos obtained was not statistically different in the 2 groups (4.2 versus 4.4). CONCLUSION: OTC before embryo freezing does not impair the number or quality of cryopreserved embryos, but increases fertility preservation potential.

Live birth after transplantation of frozen-thawed ovarian tissue after bilateral oophorectomy for benign disease.

OBJECTIVE: To report the restoration of ovarian function and pregnancy in a woman after bilateral oophorectomy for benign disease after autotransplantation of cryopreserved ovarian cortex. DESIGN: Case report. SETTING: Gynecology research unit in a university hospital. PATIENT(S): A 28-year-old woman who underwent bilateral adnexectomy for ovarian abscesses at the age of 18 years. INTERVENTION(S): We performed ovarian cortex autotransplantation to a peritoneal pocket in the broad ligament. MAIN OUTCOME MEASURE(S): Restoration of ovarian activity and pregnancy. RESULT(S): Restoration of ovarian function began at 20 weeks and was achieved 24 weeks after transplantation. After the fifth stimulation attempt, two mature oocytes were obtained and microinjected. One embryo (seven cells) was obtained and transferred, leading to a normal pregnancy. The patient delivered a healthy baby boy weighing 2,370 g at 38 weeks of gestation. CONCLUSION(S): Ovarian cortex cryopreservation can be performed at the time of surgery for benign diseases when fertility is impaired. We report the first pregnancy to occur after ovarian tissue cryopreservation for benign ovarian pathology after bilateral oophorectomy.