Azoospermia factor c microdeletions and outcomes of assisted reproductive technology: a systematic review and meta-analysis.

OBJECTIVE: To investigate whether Azoospermia Factor c (AZFc) microdeletions affect Assisted Reproductive Technology (ART) outcomes. DESIGN: Systematic review and meta-analysis. SETTING: Not applicable. PATIENTS: Infertile men with and without AZFc microdeletions. INTERVENTION(S): Electronic databases were searched for case-control studies reporting sperm retrieval rates and outcomes of ART in infertile men with and without AZFc microdeletions from inception to April 2023. Study quality was assessed using the Newcastle-Ottawa Scale. Summary effect sizes (odds ratio [OR] with 95% confidence interval [CI]) were calculated for both categories of infertile men. MAIN OUTCOME MEASURES: The primary outcome was successful sperm retrieval and the secondary outcomes were outcomes of ART. RESULTS: Case-control studies reporting sperm retrieval rates and ART outcomes in men with AZFa and AZFb deletions were unavailable. On the basis of the data from 3,807 men, sperm retrieval rates were found to be higher in men with AZFc microdeletions compared to their non-deleted counterparts [OR = 1.82, 95% CI 0.97, 3.41], but the difference was not statistically significant. A significantly lower fertilization rate (OR = 0.61, 95% CI [0.50, 0.74]), clinical pregnancy rate (OR = 0.61, 95% CI [0.42, 0.89]), and live birth rate (OR = 0.54, 95% CI [0.40, 0.72]) were observed in men with AZFc deletions compared with men without deletions. There was no statistically significant difference in rates of embryo cleavage, blastocyst formation, good-quality embryos, implantation, and miscarriage between the two groups. On correcting for female factors, the fertilization rate (OR = 0.76, 95% CI [0.71, 0.82]), cleavage rate (OR = 0.54, 95% CI [0.41, 0.72]), clinical pregnancy rate (OR = 0.39, 95% CI [0.30, 0.52]), and live birth rate (OR = 0.48, 95% CI [0.35, 0.65]) were significantly lower in men with AZFc deletions compared with controls. CONCLUSIONS: Presence of AZFc microdeletions adversely affects outcomes of ART in infertile men. Further in-depth studies delineating the role of the AZF genes in embryonic development are necessary to understand the full-impact of this finding. CLINICAL TRIAL REGISTRATION NUMBER: CRD42022311738.

False-positive detection of Group B Streptococcus (GBS) in chromogenic media (Strep B Carrot Broth) due to presence of Enterococcus faecalis in High Vaginal swabs.

Introduction. Vaginal colonization of Group B Streptococcus (GBS) is associated with preterm births and neonatal sepsis. Thus routine screening of GBS in prenatal care is recommended.Hypothesis. Chromogenic media (carrot broth) aids in specific and rapid detection of GBS.Aim. To investigate the efficiency of Strep B Carrot Broth for detection of GBS in high vaginal swabs from pregnant women.Methods. In this study 201 vaginal swab samples were collected from pregnant women. Swabs were inoculated in chromogenic media (Strep B Carrot Broth). The positive and negative cultures were inoculated on blood agar and crome agar plates. The colonies were subjected to 16S rRNA sequencing and gene-specific PCR for confirmation. The Christie Atkins Munch Peterson (CAMP) and bile esculin agar (BEA) tests were used for biochemical confirmation. PCR was performed on genomic DNA isolated from uncultured vaginal swabs.Results. It was found that 20/201 (9.9 %) vaginal swab samples were positive in the Strep B Carrot Broth and 17/20 (85 %) and 19/20 (95 %) of these samples yielded colonies on blood agar and crome agar, respectively. Of the 181 carrot broth-negative samples, 1 (0.5 %) and 38 (20.9 %) yielded colonies on blood agar and crome agar plates, respectively. However, 16 s rRNA sequencing revealed that none of the 20 carrot broth-positive cultures were GBS and had sequence similarities to Enterococcus faecalis. This was also confirmed by using gene-specific PCR and BEA positivity. Furthermore, E. faecalis was detected by PCR in DNA isolated from 57 uncultured vaginal swabs samples, GBS could only be detected by PCR in four samples.Conclusion. Carrot broth-based culture can lead to false-positive detection due to the presence of E. faecalis. Thus GBS-positive results in carrot broth must be confirmed by the other molecular and biochemical tests before making a final diagnosis.

Association of AMH and AMHR2 gene polymorphisms with ovarian response and pregnancy outcomes in Indian women.

PURPOSE: To evaluate the association of single-nucleotide polymorphisms (SNPs) in the anti-Müllerian hormone (AMH) and AMH type II receptor (AMHR2) genes with ovarian response and clinical pregnancy outcomes in women undergoing controlled ovarian hyperstimulation. METHODS: In this prospective study, we genotyped AMH polymorphisms (c. -649 T > C, c. 146 T > G, c. 252 G > A, and c. 303 G > A) in 365 women and AMHR2 polymorphisms (c. -482 A > G, c. 622-6 C > T, c. 4952 G > A, c. 10 A > G) in 80 women undergoing controlled ovarian hyperstimulation for IVF. RESULTS: Higher doses of exogenous FSH and lower numbers of preovulatory follicles were noted in women having AMH c. -649 T > C and AMH c. -146 T > G polymorphisms, respectively. Overall, we found that the presence of a polymorphic genotype (homozygous or heterozygous) at positions c. -649 T > C, c. 146 T > G, c. 252 G > A, and c. 303 G > A in the AMH gene was associated with higher doses of FSH for ovulation induction (p < 0.001). Interestingly, a higher live birth rate was noted in women with a homozygous polymorphic genotype for all four AMH SNPs investigated while none of the women showing a homozygous polymorphic genotype at all AMHR2 SNPs investigated in this study had a live birth. CONCLUSION: Our results show that presence of AMHR2 SNPs (c. 482 A > G, c. 622-6 C > T, c. 4952 G > A, and c. 10 A > G) negatively correlate with live birth rate. However, these findings need to be validated by using larger sample size.

Mitochondrial DNA Levels in Trophectodermal Cells Show No Association with Blastocyst Development and Pregnancy Outcomes.

Background: In patients undergoing assisted reproduction, levels of mitochondrial DNA (mtDNA) in the trophectodermal cells of the developing blastocyst are suggested to be associated with its ability to implant. However, discrepancies exist regarding the use of mtDNA levels as a reliable biomarker to predict outcomes of assisted reproduction. Aims: The aim of the study is to explore the association of trophectodermal mtDNA levels to determine blastocyst quality, implantation potential of blastocyst and clinical outcomes in couples who have undergone pre-implantation genetic testing for aneuploidy (PGT-A). Study Setting: Private fertility centre. Study Design: Retrospective analysis. Materials and Methods: We analysed mtDNA levels in the trophectodermal cells of 287 blastocysts from 61 couples undergoing PGT-A. The levels of mtDNA were estimated by next-generation sequencing method. mtDNA levels were correlated with maternal age, blastocyst morphology, ploidy status, implantation rates, miscarriage rate and live birth rate. Statistical Analysis Used: Linear regression and one-way ANOVA with Tukey's all column comparison test. Results: The trophectodermal mtDNA levels did not correlate with maternal age. There were no significant differences in their levels in grade 1 and grade 2 blastocysts. No significant differences were seen between mtDNA levels of implanted and non-implanted blastocysts or those blastocysts that resulted in miscarriage or live birth. However, significantly lower amounts of mtDNA were seen in euploid blastocysts as compared to that in aneuploid blastocysts. Conclusion: mtDNA levels in the trophectodermal cells of the blastocyst do not associate with blastocyst quality (grade 1 and grade 2), implantation potential and clinical outcomes but can differentiate between aneuploid and euploid blastocysts. Our study does not support the use of trophectodermal mtDNA levels as a biomarker for blastocyst quality and predictor of clinical outcomes.

Significance of borderline HbA2 levels in ß thalassemia carrier screening.

Increased HbA2 levels are the characteristic feature of ß-thalassemia carriers. A subset of carriers however do not show HbA2 levels in the typical carrier range (≥ 4.0%) but show borderline HbA2 levels. As a result, these carriers escape diagnosis and carry the risk of having ß-thalassemia major offspring. Borderline HbA2 values may occur as a consequence of mild ß-thalassemia mutations, co-inherited ß-thalassemia and α- or δ- thalassemia or iron deficiency anemia. However, there is insufficient knowledge regarding the cause of borderline HbA2 levels in specific populations. This study aimed to identify the determinants of borderline HbA2 levels (which we have considered as HbA2 3.0-3.9%) in 205 individuals. Primary screening involved detecting the presence of iron deficiency anemia followed by molecular analysis of α, ß and δ globin genes. Remarkably, 168 of 205 individuals were positive for a defect. 87% (149/168) of positive individuals were heterozygous for ß thalassemia with (59/149) or without (90/149) the presence of co-existing IDA, α or δ gene defects. Notably, 20 of 149 ß thalassemia carriers showed HbA2 < 3.5% and MCV > 80fL. 7 of these 20 carriers were married to carriers of hemoglobinopathies. Our findings describe the genetic basis of borderline HbA2 levels and emphasize the necessity of a molecular diagnosis in these individuals in the routine clinical setting.

Borderline HbA2 levels: Dilemma in diagnosis of beta-thalassemia carriers.

There is inconsistency in the exact definition of diagnostic levels of HbA2 for ß thalassemia trait. While many laboratories consider HbA2 ≥4.0 % diagnostic, still others consider HbA2 ≥3.3 % or HbA2 ≥3.5 % as the cut-off for establishing ß thalassemia carrier diagnosis. This is because, over the years, studies have described ß thalassemia carriers showing HbA2 levels that lie above the normal range of HbA2 but below the typical carrier range of ß thalassemia. These, "borderline HbA2 levels", though not detrimental to health, are significant in ß thalassemia carrier diagnosis because they can lead to misinterpretation of results. In this review, we have evaluated the prevalence of borderline HbA2 levels and discussed the causes of borderline HbA2 values. We have also compiled an extensive catalogue of ß globin gene defects associated with borderline HbA2 levels and have discussed strategies to avoid misdiagnosing borderline HbA2 ß thalassemia carriers. Our analysis of studies that have delineated the cause of borderline HbA2 levels in different populations shows that 35.4 % [626/1766] of all individuals with borderline HbA2 levels carry a molecular defect. Among the positive samples, 17 % [299/1766] show ß globin gene defects, 7.7 % [137/1766] show α thalassemia defects, 2.7 % [49/1766] show KLF1 gene mutations, 2.3 % [41/1766] show the co-inheritance of ß and α thalassemia, 2.0 % [37/1766] show the co-inheritance of ß and δ thalassemia and 1.8 % [32/1766] show α globin gene triplication. It appears that a comprehensive molecular work up of the ß globin gene is the only definite method to detect borderline HbA2 ß thalassemia carriers, especially in populations with a high prevalence of the disease. The presence of associated genetic or acquired determinants may subsequently be assessed to identify the cause of borderline HbA2.

Single-Cell RNA-seq Identifies Cell Subsets in Human Placenta That Highly Expresses Factors Driving Pathogenesis of SARS-CoV-2.

Infection by the Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) results in the novel coronavirus disease COVID-19, which has posed a serious threat globally. Infection of SARS-CoV-2 during pregnancy is associated with complications such as preterm labor and premature rupture of membranes, and a proportion of neonates born to infected mothers are also positive for the virus. During pregnancy, the placental barrier protects the fetus from pathogens and ensures healthy development. To predict if the placenta is permissive to SARS-CoV-2, we utilized publicly available single-cell RNA-seq data to identify if the placental cells express the necessary factors required for infection. SARS-CoV-2 binding receptor ACE2 and the S protein priming protease TMPRSS2 are co-expressed by a subset of syncytiotrophoblasts (STB) in the first trimester and extravillous trophoblasts (EVT) in the second trimester human placenta. In addition, the non-canonical receptor BSG/CD147 and other proteases (CTSL, CTSB, and FURIN) are detected in most of the placental cells. Other coronavirus family receptors (ANPEP and DPP4) were also expressed in the first and second trimester placental cells. Additionally, the term placenta of multiple species including humans expressed ACE2, DPP4, and ANPEP along with the viral S protein proteases. The ACE2- and TMPRSS2-positive (ACE2 + TMPRSS2 +) placental subsets expressed mRNA for proteins involved in viral budding and replication. These cells also had the mRNA for proteins that physically interact with SARS-CoV-2 in host cells. Further, we discovered unique signatures of genes in ACE2 + TMPRSS2 + STBs and EVTs. The ACE2 + TMPRSS2 + STBs are highly differentiated cells and express genes involving mitochondrial metabolism and glucose transport. The second trimester ACE2 + TMPRSS2 + EVTs are enriched for markers of endovascular trophoblasts. Both these subtypes abundantly expressed genes in the Toll-like receptor pathway. The second trimester EVTs are also enriched for components of the JAK-STAT pathway that drives inflammation. We carried out a systematic review and identified that in 12% of pregnant women with COVID-19, the placenta was infected with SARS-CoV-2, and the virus was detected in STBs. To conclude, herein we have uncovered the cellular targets for SARS-CoV-2 entry and have shown that these cells can potentially drive viremia in the developing human placenta. Our results provide a basic framework toward understanding the paraphernalia involved in SARS-CoV-2 infections in pregnancy.

Consequences of Y chromosome microdeletions beyond male infertility.

PURPOSE: The human Y chromosome plays a central role in sex determination and spermatogenesis. The azoospermia factor (AZF) loci on the Y chromosome contain genes that were thought to be testis specific with their deletions leading to spermatogenic failure. However, beyond the testis, the AZF genes (mainly those in AZFa and AZFb loci) are widely expressed in multiple tissues. Further, these genes are predicted to play roles in processes such as gene regulation and protein synthesis. These observations suggest that the AZF genes may have functions beyond regulation of fertility. RESULTS: Three major areas have emerged where alternations in AZF genes have effects beyond infertility. (1) Poor-quality embryos are generated in assisted reproduction when sperm from men harboring Y chromosome microdeletions are used, (2) a higher preponderance of neuropsychiatry disorders is observed in men with deletions in AZF genes, and (3) copy number variations and altered expression of AZF genes are found in several cancers. CONCLUSION: While our data is preliminary and observational in nature, systematic studies are required to address how genetic alterations in the Y chromosome can affect the health of men beyond infertility. This information will provide a different perspective in the area of androgenetics and have implications in devising strategies for maintaining the overall well-being of infertile males.

Paternal factors contributing to embryo quality.

PURPOSE: Advancing maternal and paternal age leads to a decrease in fertility, and hence, many infertile couples opt for assisted reproductive technologies [ART] to achieve biological parenthood. One of the key determinants of achieving a live outcome of ART, embryo quality, depends on both the quality of the oocyte and sperm that have created the embryo. Several studies have explored the effect of oocyte parameters on embryo quality, but the effects of sperm quality on the embryo have not been comprehensively evaluated. METHOD: In this review, we assess the effect of various genetic factors of paternal origin on the quality and development of the embryo. RESULTS: The effects of sperm aneuploidy, sperm chromatin structure, deoxyribonucleic acid [DNA] fragmentation, role of protamines and histones, sperm epigenetic profile, and Y chromosome microdeletions were explored and found to negatively affect embryo quality. CONCLUSION: We propose that careful assessment of spermatozoal parameters is essential to achieve embryo development and a healthy live birth. However, the heterogeneity in test results and the different approaches of assessing a single sperm parameter highlight the need for more research and the development of standardized protocols to assess the role of sperm factors affecting embryo quality.

Genetics of the human Y chromosome and its association with male infertility.

The human Y chromosome harbors genes that are responsible for testis development and also for initiation and maintenance of spermatogenesis in adulthood. The long arm of the Y chromosome (Yq) contains many ampliconic and palindromic sequences making it predisposed to self-recombination during spermatogenesis and hence susceptible to intra-chromosomal deletions. Such deletions lead to copy number variation in genes of the Y chromosome resulting in male infertility. Three common Yq deletions that recur in infertile males are termed as AZF (Azoospermia Factor) microdeletions viz. AZFa, AZFb and AZFc. As estimated from data of nearly 40,000 Y chromosomes, the global prevalence of Yq microdeletions is 7.5% in infertile males; however the European infertile men are less susceptible to Yq microdeletions, the highest prevalence is in Americans and East Asian infertile men. In addition, partial deletions of the AZFc locus have been associated with infertility but the effect seems to be ethnicity dependent. Analysis of > 17,000 Y chromosomes from fertile and infertile men has revealed an association of gr/gr deletion with male infertility in Caucasians and Mongolian men, while the b2/b3 deletion is associated with male infertility in African and Dravidian men. Clinically, the screening for Yq microdeletions would aid the clinician in determining the cause of male infertility and decide a rational management strategy for the patient. As these deletions are transmitted to 100% of male offspring born through assisted reproduction, testing of Yq deletions will allow the couples to make an informed choice regarding the perpetuation of male infertility in future generations. With the emerging data on association of Yq deletions with testicular cancers and neuropsychiatric conditions long term follow-up data is urgently needed for infertile men harboring Yq deletions. If found so, the information will change the current the perspective of androgenetics from infertility and might have broad implication in men health.

Rare ß- and δ-Globin Gene Mutations in the Pathare Prabhus: Original Inhabitants of Mumbai, India.

Genetic structure of the Indian population is influenced by waves of several immigrants from West Eurasia. Therefore, genetic information of various ethnic groups is valuable to understand their origins, the pattern of migration as well as the genetic relationship between them. No genetic data is available on Pathare Prabhu, which is a small indigenous Hindu community from Mumbai, Maharashtra State, India. The aim of this study was to screen the Pathare Prabhus for hemoglobinopathies, which is a major public health problem in India. Two hundred and fifty-seven unrelated Pathare Prabhus subjects were screened for various hemoglobinopathies. Complete blood counts (CBC) were done on an automated hematology counter. High performance liquid chromatography (HPLC) was used to identify ß-thalassemia (ß-thal) carriers. Molecular characterization of the ß gene defects was done by reverse dot-blot hybridization, amplification refractory mutation system (ARMS) and DNA sequencing. Deletional α-thalassemia (α-thal) was detected by multiplex polymerase chain reaction (PCR). Hb A2-Saurashtra (HBD: c.301C>T) was identified by DNA sequencing; its modeling was also done. The prevalence of ß-thal was 3.89%, while deletional α-thal was 5.4%. The initiation codon (ATG>ACG) (HBB: c.2T>C) was seen in eight individuals (80.0%), Hb D-Punjab (HBB: c.364G>C) and Hb A2-Saurashtra, was found in two and one individual, respectively. A community-specific ß-thal mutation was found in Pathare Prabhus in significant proportions. This information is useful in developing an algorithm for a prenatal diagnosis (PND) program.

Effect of the Hemochromatosis Mutations on Iron Overload among the Indian ß Thalassemia Carriers.

BACKGROUND: Hereditary hemochromatosis is a disorder of iron metabolism characterized by increased iron absorption.HFE gene mutations C282Y and H63D are responsible for the majority of hereditary hemochromatosis cases. METHODS: We tried to look at the effect of HFE mutations on the iron status. A total of 100 ß thalassemia traits (BTT) with 100 normal individuals were screened for the C282Y and H63D mutations using PCR-RFLP. The serum ferritin levels were determined using ELISA kit. RESULTS: We did not find the C282Y mutation in our study group. The allelic frequencies for H63D mutation did not differ significantly between ß-thalassemia traits (8.5%) and normal controls (9%). ΒΤΤ with H63D genotype of H/D (143.16 ± 80.3 ng/ml) and D/D (504 ng/ml) showed higher ferritin levels as against H/H genotype (88.64 ± 92.43 ng/ml). The statistically significant difference was observed in the mean serum ferritin levels among the individuals showing H/H and D/D genotypes (P < 0.002) and H/D and D/D genotype (P < 0.01) in both the groups. CONCLUSION: This suggests that iron load in BTT tends to aggravated with the co-inheritance of the H63D mutation. The mutant H63D gene showed the presence of haplotype 6 which is reported in the European population suggesting a common origin.

HbD Punjab/HbQ India Compound Heterozygosity: An Unusual Association.

BACKGROUND: Haemoglobinopathies are the commonest hereditary disorders in India and pose a major health problem. Both beta thalassaemia and structural haemoglobin variants are relatively common in northwestern India. Here we report a 29-year-old Sindhi female who was referred to us for a haemoglobinopathy work up and genetic counseling since her spouse was a classical beta thalassaemia carrier. METHOD: A complete blood count was done on an automated cell counter. Haemoglobin analysis was carried out using HPLC Variant Haemoglobin Testing System. The cellulose acetate electrophoresis was carried out [pH 8.9]. Confirmation of mutations was done by automated DNA sequencing. RESULTS: HPLC analysis showed four major peaks, HbA0, a peak in the HbD window, an unknown peak [retention time 4.74 minutes] and a peak in the HbC window. The HbA2 level was 2.2%, and the HbF level was 0.7%. Cellulose acetate electrophoresis at alkaline pH, a slow moving band was seen at the HbS/D position along with a prominent band at the HbA2 position. DNA sequencing of the ß and α genes showed presence of the two hemoglobin variants: Hb D [ß 121GAA → CAA] and Hb Q [α 64 AAG → GAG]. The δ globin gene was normal. The additional peak in the HbC window was due to the formation of a heterodimer hybrid. CONCLUSION: Both HbD Punjab and HbQ India are relatively common in India, but their co-inheritance has not been described in the country. This case is the third report of compound heterozygosity for HbQ India/HbD Punjab haemoglobinopathy globally and the second one from India.

Clinical and hematological presentation among Indian patients with common hemoglobin variants.

BACKGROUND: Co-inheritance of structural hemoglobin variants like HbS, HbD(Punjab) and HbE can lead to a variable clinical presentation and only few cases have been described so far in the Indian population. METHODS: We present the varied clinical and hematological presentation of 22 cases (HbSD(Punjab) disease-15, HbSE disease-4, HbD(Punjab)E disease-3) referred to us for diagnosis. RESULTS: Two of the 15 HbSD(Punjab) disease patients had moderate crisis, one presented with mild hemolytic anemia; however, the other 12 patients had a severe clinical presentation with frequent blood transfusion requirements, vaso occlusive crisis, avascular necrosis of the femur and febrile illness. The 4 HbSE disease patients had a mild to moderate presentation. Two of the 3 HbD(Punjab)E patients were asymptomatic with one patient's sibling having a mild presentation. The hemoglobin levels of the HbSD(Punjab) disease patients ranged from 2.3 to 8.5 g/dl and MCV from 76.3 to 111.6 fl. The hemoglobin levels of the HbD(Punjab)E and HbSE patients ranged from 10.8 to 11.9 and 9.8 to 10.0 g/dl whereas MCV ranged from 67.1 to 78.2 and 74.5 to 76.0 fl respectively. CONCLUSIONS: HbSD(Punjab) disease patients should be identified during newborn screening programmes and managed in a way similar to sickle cell disease. Couple at risk of having HbSD(Punjab) disease children may be given the option of prenatal diagnosis in subsequent pregnancies.

Masking of a ß-thalassemia determinant by a novel δ-globin gene defect [Hb A2-Saurashtra or δ100(G2)Pro→Ser; HBD: c.301C>T] in Cis.

Abstract The molecular basis of ß-thalassemia (ß-thal) syndromes have been well documented, while the spectrum of mutations causing δ-thalassemia (δ-thal) has not been well characterized. δ-Thalassemia has no clinical symptoms but its coinheritance with heterozygous ß-thal may cause misdiagnosis, especially in countries with a high prevalence of ß-thal where prevention programs have been implemented. The coinheritance of ß- and δ-globin mutations in India is not common. This association may interfere with correct diagnosis and genetic counseling of ß-thal in screening programs. Here we report two families showing borderline Hb A2 levels belonging to the Koli Community, indigenous to the Saurashtra Province of Gujarat, India. They were referred to us for thalassemia molecular screening as they had children clinically presenting before 2 years of age and requiring regular blood transfusions. Interestingly, both families carried a novel δ-globin gene mutation at codon 100 (C > T) linked to a polyadenylation (polyA) site [AATAAA > A(-AATAA)] 5 bp deletional ß-thal mutation, never before reported in the Indian population. This report highlights the importance of considering δ-globin gene analysis during ß-thal screening to avoid false-negative results in the detection of at-risk couples. It also highlights how incomplete diagnosis of a borderline or normal Hb A2 level may lead to the probable birth of a ß-thal major (ß-TM) child. This has important implications in prenatal diagnosis.

Compromising for carrier detection of beta thalassemia based on measurement of HbA2 levels in unusual cases.

BACKGROUND: An increased HbA2 level is the hallmark for identification of ß thalassemia carriers. However, in some carriers the level of HbA2 is not typically elevated creating difficulties in making a diagnosis. METHODS: We describe a family having an affected child referred to us for confirmation of diagnosis of ß thalassemia. RESULTS: The father has a classical ß thalassemia trait and the mother showed typical reduced red cell indices with a high RBC count but the HbA2 level was normal (2.4%). On molecular analysis she was a heterozygous carrier having IVS1 nt 5 (G→C) ß thalassemia mutation. Further analysis of δ globin gene showed that the reduction in HbA2 was due to the presence of the δ mutation HbA2 Pelendri [CD 141(Leu→Pro, CTG→CCG)]. CONCLUSIONS: The diagnosis of a ß thalassemia carrier could have been compromised, and states the importance of comprehensive molecular analysis for accurate diagnosis in couples where one partner has ß thalassemia trait.