Oncofertility and Pregnancy in Adolescent and Young Adult Cancers: Physicians' Knowledge and Preferences in India.

PURPOSE: The treatment outcomes of adolescent and young adult (AYA) cancers have improved with advanced oncology care. Hence, fertility preservation (FP) and post-therapy pregnancies (PTPs) become vital issues. MATERIALS AND METHODS: An online survey link with 17 questions regarding oncofertility and PTPs was circulated among oncologists to assess the knowledge, understand the oncofertility care patterns, and seek suggestions to improve oncofertility services. RESULTS: The median age of 179 respondents, predominantly medical oncologists (68.7%), was 37 years (IQR, 10; range, 29-74), working in academic centers (39%) having a median experience of 4 years (IQR, 4; range, 1-42); 23 (12.8%) had dedicated AYA cancer units. Although a quarter (19%-24%) of respondents discussed fertility issues in >90% of AYA patients with cancer, only a tenth (8%-11%) refer >90% for FP, with significantly higher (P < .05) discussions and referrals in males and by more experienced oncologists (P < .05). Forty-six (25.6%) were not well versed with international guidelines for FP. Most (122, 68.1%) oncologists knew about the referral path for semen cryopreservation; however, only 46% were knowledgeable about additional complex procedures. One hundred and ten (61.5%) oncologists never or rarely altered the systemic treatment for FP. Prominent barriers to FP were ignorance, lack of collaboration, and fear of delaying cancer treatment. Lead thrust areas identified to improve FP practices are education, and enhanced and affordable access to FP facilities. Seventy-four (41.3%) respondents knew about international guidelines for PTPs; however, only half (20%) of them often monitored fertility outcomes in survivors. Oncologists have conflicting opinions and uncertainties regarding pregnancy safety, assisted reproductive techniques, breastfeeding, and pregnancy outcomes among survivors. CONCLUSION: Oncologists are uncertain about the guidelines, FP practices, referral pathways, and PTPs. Multipronged approaches to improve awareness and provision for affordable oncofertility facilities are needed to enhance AYA cancer outcomes in India, which will be applicable to other low- and middle-income countries too.

The status of anti-ovarian antibody immunoassays: Valid or invalid, in or out?

There has been a paradign shift in the status of immunoassays. There used to be a time where immunoassays had a very narrow role in clinical medicine, but that is not the case in today's world. Immunoassays have taken a central role in helping us better understand and treat human diseases. The literature around anti-ovarian antibodies (AOA) immunoassay testings have been conflicting. Researchers challenged the specificity of the reported assays, but a systematic study was never elucidated on what/who the trouble maker was in rendering these tests so nonspecific. Attempts were made by our group in Mumbai, India, to throw light on the culprit behind the nonspecificity casative factor in the immunoassays and a method to overcome this was reported and published. This review highlights the stories back five and a half decades to date, to demonstrate where the status of AOA testing was, is and will be.

First Report of an α Chain Variant [Hb Coombe Park (HBA2: c.382A>G)] from India, Coinherited with a Novel SERPINC1 Gene Mutation: A Double Whammy?

Coinheritance of a high oxygen affinity structural hemoglobin (Hb) variant along with a thrombophilia marker is a rare occurrence. This may lead to a multi fold increase in the risk of thrombosis in patients. We report here a first case of Hb Coombe Park (HBA2: c.382A>G; p.Lys128Glu) from India, coinherited with a novel mutation (c.839C>G; p.Ser280Ter) on the SERPINC1 gene. This coinheritance has not been reported before. Though the patient is presently asymptomatic, identification of these variants will help in genetic counseling and to decide the future course of action in case of any clinical complications.

Dysfunctional regulation of pivotal and key inflammatory pathways in infertile Indian women with genital tuberculosis.

PROBLEM: Diagnosis of female genital tuberculosis (FGTB) remains elusive due to the paucibacillary nature of the disease. We evaluated if analysis of inflammatory pathways of endometrial tissue could establish a better diagnosis of FGTB. METHOD OF STUDY: One hundred and four infertile women suspected of having GTB or having been treated for GTB in the past, underwent endometrial biopsies for diagnosis and Gene Inflammatory Pathways analysis at our center between 2018-2020. Diagnosis of FGTB was based on acid-fast bacilli culture, immunocytochemistry, nested-polymerase chain reaction, histopathological examination, TB GeneXpert, or combinations thereof. Gene expression profiles were also analyzed. RESULTS: Based on diagnostic tests of 104 women, 44 (42%) were considered TB-positive, 35 (34%) TB-negative, and 25 (24%) TB-negative after TB treatment in the past. Inflammatory pathways were significantly upregulated in TB-positive women versus TB-negative (41% vs. 6%; p = .0005), and in women who were TB-negative after TB treatment in the past versus TB-negative (never treated for TB in the past) (38% vs. 6%; p = .0037). Two-hundred seventy-one genes were upregulated, and 61 genes were downregulated in TB-positive women versus those who were TB-negative. Differentially expressed genes were mapped to various interlinked inflammatory signaling pathways, including mitogen-activated protein kinase (MAPK), Natural Killer (NK) cells, nuclear factor kappa-B (NF-kB), tumor necrosis factor (TNF), and Toll-like receptors (TLR) signaling. CONCLUSIONS: Inflammatory pathways and gene expression profiles add to the diagnostic tools to identify TB-positive women at an early stage. The results from this study are still experimental and large multi-centric studies are suggested before their recommendation in routine clinical practice.

Evolution and Utility of Preimplantation Genetic Testing for Monogenic Disorders in Assisted Reproduction - A Narrative Review.

Preimplantation genetic testing (PGT) for monogenic disorders and assisted reproductive technology have evolved and progressed in tandem. PGT started with single-cell polymerase chain reaction (PCR) followed by fluorescent in situ hybridisation for a limited number of chromosomes, later called 'preimplantation genetic diagnosis (PGD) version 1'. This review highlights the various molecular genetic techniques that have evolved to detect specific inherited monogenic disorders in the preimplantation embryo. Literature review in English was performed in PubMed from 1990 to 2021, using the term 'preimplantation genetic diagnosis'. With whole-genome amplification, multiple copies of embryonic DNA were created. This helped in avoiding misdiagnosis caused by allele dropout. Multiplex fluorescent PCR analysed informative short tandem repeats (STR) and detected mutations simultaneously on automated capillary electrophoresis sequencers by mini-sequencing. Comparative genomic hybridisation (CGH) and array CGH were used for 24 chromosome aneuploidy screening. Subsequently, aneuploidies were detected by next-generation sequencing using single-nucleotide polymorphism arrays, while STR markers were used for haplotyping. 'PGD version 2' included accurate marker-based diagnosis of most monogenic disorders and detection of aneuploidy of all chromosomes. Human leukocyte antigen matching of embryos has important implications in diagnosis and cure of haemoglobinopathies and immunodeficiencies in children by means of matched related haematopoietic stem cell transplantation from an unaffected 'saviour sibling' obtained by PGT.

Anti-Mullerian Hormone Levels in Indian Women Seeking Infertility Treatment: Are Indian Women Facing Early Ovarian Senescence?

BACKGROUND: Antimullerian hormone (AMH) is a key marker of ovarian reserve and predictor of response to fertility treatment. AIM: To understand the prevalence of low ovarian reserve in Indian women seeking infertility treatment, compare their AMH with age-matched fertile Indian controls and understand ethnic differences with Caucasian women. SETTING AND DESIGN: Retrospective observational study done as collaboration between our in vitro fertilization centre and a laboratory with Pan-India presence. MATERIALS AND METHODS: Women aged 20-44 years were selected as Group A (seeking infertility treatment n = 54,473), Group B (conceived naturally in the past; n = 283) and Group C (data of Caucasian women; n = 718). Serum AMH levels were measured and descriptive analysis done. STATISTICAL ANALYSIS: Descriptive statistics and Chi-square test. RESULTS: In Group A, 28.7%, 48.7% and 70.6% of women aged <30 years, 30-34 years and 35-39 years had serum AMH levels ≤2 ng/mL and the proportions were higher than Group B. The rate at which median AMH decreased was 1.1-2 times faster in Group B as compared to Group C. The decrease in median AMH across age groups in Group A was similar to Group B. CONCLUSIONS: Indian women in their late twenties and early thirties visiting fertility centers showed a worrisome trend of low AMH. Our study can be used as a reference for those women considering postponing pregnancy. It may be time to look at intangible cultural factors linked to social habits, ethnicity, diet, genetic predispositions, and environmental factors like endocrine disrupting chemicals contributing to premature ovarian senescence.

Hematological and Molecular Findings in the First Case of Hb J-Norfolk [HBA2: c.173G>A (or HBA1] in an Indian Patient.

We here report a case of a 23-year-old female from Mumbai, Maharashtra, India who was detected to carry the α chain variant Hb J-Norfolk [HBA2: c.173G>A (or HBA1]. She had no clinical symptoms and was referred to us for routine investigations and screening. An abnormal peak was detected on both high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) with a fast-moving band on cellulose acetate electrophoresis. There is no detailed study on the HPLC and CE pattern of this hemoglobin (Hb) variant, and therefore, this study will help in detecting and avoiding missing these variants during routine investigations and population screening. This is the first report of this variant in the Indian population.

Detection of t(14;16)(q32;q22) and Monosomy 13 by FISH Analysis in a Patient with Multiple Myeloma Associated with Sjögren's Syndrome: The First Case Report from India.

Sjögren's syndrome (SS) is a chronic slowly progressive autoimmune disorder characterized by symptoms of oral and ocular dryness, exocrine dysfunction, and lymphocytic infiltration of exocrine glands. Multiple myeloma (MM) is a bone-marrow-based malignant neoplasm of plasma cells associated with serum/urine monoclonal paraproteins and lytic skeletal lesions. There have been very few reported cases of MM, who had SS as the first presentation. We report a case of a woman diagnosed with Sjögren's syndrome, who was later suspected to have multiple myeloma on serum protein electrophoresis. Fluorescence in situ hybridization (FISH) was carried out to check for deletions of loci 13q14.3, ATM, p53, and IGH (14q32) rearrangements on a bone marrow aspirate. Monosomy 13 was observed in 49% of cells, and a rearrangement at the IGH locus was seen in 42% of cells. To determine the partner chromosome associated with the IGH rearrangement, further FISH tests were set up for t(4;14)(p16;q32) followed by t(14;16)(q32;q22) on fresh slides. The test was negative for t(4;14) but positive for t(14;16) in 27% of cells. This confirmed the diagnosis of MM. We report the first case from India, having an association of Sjögren's syndrome with multiple myeloma, which showed t(14;16) and monosomy 13 by FISH analysis.

Identification and validation of candidate biomarkers involved in human ovarian autoimmunity.

Antibodies to multiple ovarian antigens have been proposed as markers of ovarian autoimmunity. The role of ovarian autoantibodies has been widely discussed in the pathophysiology of premature ovarian failure and unexplained infertility, but the autoantigens are yet to be identified. Three immunodominant ovarian autoantigens, α-actinin 4 (αACTN4), heat shock 70 protein 5 (HSPA5) and ß-actin (ACTB), have been identified using anti-ovarian antibody-positive sera from women with idiopathic premature ovarian failure (n=50) and women undergoing IVF (n=695), using mass spectrometry. These autoantigens were subsequently validated using Western blot, immunohistochemistry and enzyme-linked immunosorbent assay. These autoantigens are localized to different components of the ovary such as the ooplasm of the oocyte, theca, granulosa, corpus luteum and zona pellucida. All the above antigens were found to be expressed in the ooplasm throughout follicular development. All the autoantigens are expressed specifically in the oocyte except αACTN4. The three autoantigens could contribute to the array of biomarkers to be used for developing specific and sensitive tests for diagnosis of women at risk of premature ovarian failure and IVF failure due to ovarian autoimmunity and could give an insight into the molecular mechanisms involved in the pathophysiology of these conditions.

Can anti-ovarian antibody testing be useful in an IVF-ET clinic?

OBJECTIVE: To establish importance of anti-ovarian antibodies (AOA) testing in infertile women. DESIGN: A clinical reproductive outcome comparative study between two groups of women undergoing IVF-ET. Group 1 consists of women tested positive for AOA, put on corticosteroid therapy, reverted to AOA negative and then taken up for IVF-ET. Group 2 were seronegative for AOA. SETTING: Major urban infertility reference centre and National research institute. PATIENT(S): Five hundred seventy infertile women enrolled for IVF-ET. INTERVENTION(S): AOA testing, corticosteroid therapy and IVF-ET/ICSI. MAIN OUTCOME MEASURE(S): Comparable clinical outcome and significance of AOA testing established. RESULTS: AOA positive serum samples were sent periodically to re-investigate presence of AOA after corticosteroid therapy and women turned AOA negative were taken up for IVF-ET. Of the 70/138 women in group 1 who were treated with corticosteroids and turned seronegative for AOA, 22/70 were poor responders and needed donor oocyte-recipient cycles. Results demonstrated that fertilization and clinical pregnancy rates between both groups are comparable. Nevertheless, it is also observed that there is poor response to stimulation protocol, smaller number of oocytes retrieved and more spontaneous abortions in group 1 women. Hence not all outcomes following the treatment are comparable between the two groups. Usefulness of the test was established in two case studies. CONCLUSIONS: AOA testing could be included in the battery of tests investigating and treating infertility.

Epigenetic regulatory mechanisms associated with infertility.

Infertility is a complex human condition and is known to be caused by numerous factors including genetic alterations and abnormalities. Increasing evidence from studies has associated perturbed epigenetic mechanisms with spermatogenesis and infertility. However, there has been no consensus on whether one or a collective of these altered states is responsible for the onset of infertility. Epigenetic alterations involve changes in factors that regulate gene expression without altering the physical sequence of DNA. Understanding these altered epigenetic states at the genomic level along with higher order organisation of chromatin in genes associated with infertility and pericentromeric regions of chromosomes, particularly 9 and Y, could further identify causes of idiopathic infertility. Determining the association between DNA methylation, chromatin state, and noncoding RNAs with the phenotype could further determine what possible mechanisms are involved. This paper reviews certain mechanisms of epigenetic regulation with particular emphasis on their possible role in infertility.

A case-control study identifying chromosomal polymorphic variations as forms of epigenetic alterations associated with the infertility phenotype.

OBJECTIVE: To study the association of chromosomal polymorphic variations with infertility and subfertility. DESIGN: A comparative case-controlled association study using cytogenetic techniques to compare the frequency of chromosomal variations in infertile individuals versus fertile controls. SETTING: Department of Infertility Management and Assisted Reproduction, Jaslok Hospital and Research Centre, Mumbai, India. PATIENT(S): 760 infertile individuals and 555 fertile controls. INTERVENTION(S): ICSI, IUI, karyotyping, inverted 4',6-diamidino-2-phenylindole (DAPI), CBG banding. MAIN OUTCOME MEASURE(S): Frequency of chromosomal polymorphic variations in infertile individuals undergoing infertility treatment versus fertile individuals. RESULT(S): A highly statistically significant increase in the frequency of total chromosomal variants in infertile women (28.31% vs. 15.16%) and infertile men (58.68% vs. 32.55%) was observed. The frequency of 9qh+ was statistically significantly increased in women with primary infertility (16.22% vs. 6.41%) and in men with severe male factor infertility (14.69% vs. 4.25%). A highly statistically significant increase in the frequency of Yqh+ was observed in men whose wives had a bad obstetric history (30.20% vs. 12.74%). CONCLUSION(S): The statistically significantly higher incidence of heterochromatic variations found in infertile individuals stresses on the need to evaluate their role in infertility and subfertility. Potential epigenetic, genetic, and chromosomal modifications could be associated with certain complex disorders such as infertility and bad obstetric history.

Specific and sensitive immunoassays detect multiple anti-ovarian antibodies in women with infertility.

Serum anti-ovarian antibodies (AOAs) have been shown in autoimmune premature ovarian failure and in vitro fertilization-embryo transfer (IVF-ET) cases. The specificity of assays detecting these antibodies has been questioned. Researchers have used several techniques (e.g., ELISA and indirect immunofluorescence). Few have reported on the non-specificity and the type of molecular and cellular targets. We reported earlier on the presence of naturally occurring anti-albumin antibodies as the likely factor for non-specificity. Having developed a novel blocking recipe, we show substantial elimination of this non-specificity. With these standardized tests, we hereby report multiple targets at protein and histological levels. In our study group, 15 of 50 (30%) patients with premature ovarian failure and 13 of 50 (26%) IVF-ET patients showed the presence of AOAs. Western blotting showed a large number of patients making AOAs to a 90-kDa protein, followed by 97- and 120-kDa proteins. Histochemically, it was evident that the sera of these patients predominantly react with the oocyte; other somatic cellular targets are also involved. The specific non-invasive test developed by us was found to be useful because it could carry out a reliable diagnosis of an autoimmune etiology that would be very helpful to select patients in whom immune-modulating therapy could be recommended, which in turn may restore ovarian function and fertility.

Cumulus coculture and cumulus-aided embryo transfer increases pregnancy rates in patients undergoing in vitro fertilization.

OBJECTIVE: To evaluate the effect of the use of cumulus-aided embryo transfer on pregnancy rates. To study the proximity of expanded cumulus cells to the developing embryo. To document by light microscopy the anchoring of day 3 to day 4 embryos by the expanded cumulus cells. To demonstrate by transmission electron microscopy the cellular activity of the expanded cumulus cells. To evaluate the expression of growth factors (vascular endothelial growth factor, interleukin-6, insulin-like growth factor I) that are secreted by the cumulus cells. DESIGN: A comparative study of a group of women undergoing cumulus coculture and cumulus-aided embryo transfer, with those who underwent cumulus coculture but did not undergo cumulus-aided embryo transfer. The endpoint was the achievement of pregnancy. SETTING: Department of Infertility Management and Assisted Reproduction, Jaslok Hospital and Research Centre, Mumbai, India. PATIENT(S): Five hundred seventeen women undergoing treatment for infertility using intracytoplasmic sperm injection and embryo transfer and fulfilling set criteria. To validate our initial results, we conducted a similar study on 208 women where randomization was performed. INTERVENTION(S): Embryos were cocultured with the patient's own cumulus cells and were transferred into the uterus with approximately 30 microL of the expanded cumulus cells. MAIN OUTCOME MEASURE(S): Pregnancy, implantation, and multiple gestation rates. RESULT(S): Our study demonstrated a significant increase in the implantation rate in the study group (group A) of 25.6% versus 14.5% in the control group (group B) and a significant increase in the pregnancy rate in the study group (group A) of 47.6% versus 34% achieved in the control group (group B). Although the incidence of multiple gestation was similar (38.6% in the study group and 32.9% in the control group), the higher-order multiple gestation rate was significantly more in the study group as compared with the control group (18.1% vs. 2.4%). Similar pregnancy and implantation rates were observed in the randomized study. CONCLUSION(S): This study demonstrates the efficacy of cumulus-aided embryo transfer, using autologous cumulus cells. It indicates a significant increase in implantation and pregnancy rates. The results suggest that cumulus cells play an important role in embryonic development, and that they may provide a mechanism to improve embryo-uterine adhesion by physical proximity, and by secreting cytokines and growth factors required to aid the implantation process.

Polymorphic variants on chromosomes probably play a significant role in infertility.

Polymorphic variants on chromosomes are considered 'normal', as heterochromatin has no coding potential and nucleolar organizing regions (NOR) contain genes coding for rRNA. Variants have been reported in infertility and recurrent abortions. With refined molecular techniques, genes for fertility and viability are now thought to reside in heterochromatin. DNA sequence analysis of human chromosome 9 has shown that it is highly structurally polymorphic, with many intrachromosomal and interchromosomal duplications, and contains the largest autosomal block of heterochromatin. Transcriptional activation of constitutive heterochromatic domains of the human genome in response to environmental stress was reported recently. Heat shock triggers the assembly of nuclear stress bodies on the pericentromeric heterochromatin of human chromosomes including chromosome 9. These are characterized by an epigenetic status typical of euchromatic regions. On acrocentric chromosomes, NOR-associated protein count and morphology was reported to separate benign and malignant melanocytic lesions. Hence all variants may not be 'normal'. The present study of karyotyping 842 individuals attending an IVF clinic with primary infertility or repeated miscarriages, showed polymorphic variants in 28.82% of males and 17.19% of females, which was quite high. It is suggested that variants should not be ignored by cytogeneticists. Screening prospective gamete donors for chromosome variants may help enhance the success of IVF.