Successful live birth in a Chinese woman with P450 oxidoreductase deciency through frozen-thawed embryo transfer and review of the literature

Background: Congenital adrenal hyperplasia (CAH) caused by P450 oxidoreductase deciency (PORD) in 46,XX patients is characterized by genital ambiguity, primary amenorrhea, absent or incomplete sexual maturation, infertility, skeletal malformations and so on. But few pregnancies have been reported from these female patients with PORD. Case Description: A 29-year-old Chinese woman with PORD due to the compound heterozygous mutation (c.1370G>A/c.1196_1204del) in the P450 oxidoreductase( POR ) gene had suffered from primary amenorrhea and infertility. She had one cancelled cycle of ovulation induction due to low serum estradiol(E 2 ), high progesterone(P) levels and thin endometrium,then in vitro fertilization (IVF) was recommended. At the rst IVF cycle, 4 oocytes were retrieved and 4 viable embryos were cryopreserved due to thin endometrium associated with low E 2 and prematurely elevated P after ovarian stimulation, even though oral dexamethasone were used to control adrenal P overproduction at the same time. When basal P fell to <1.5ng/ml, articial endometrial preparation and frozen embryo transfer were performed, resulting in a twin pregnancy. She delivered a healthy boy and a healthy girl by caesarean section at 37 weeks and 2 days of gestation. Conclusions: We report the pregnancy achieved in a CAH woman caused by a compound heterozygous POR mutation, with primary amenorrhea and disorders of steroidogenesis. It seemed that disorders of steroidogenesis caused by PORD didn’t impair the developmental potential of oocytes. IVF and frozen embryo transfer after adequate hormonal control and endometrial preparation should be an effective infertility treatment for PORD women.


years old a
d had suffered from primary infertility for three years, she was referred to treat infertility and she had a cancelled cycle of ovulation induction in the local hospital.The follicle growth and sex hormone changes during the ovulation induction were as the following : human menopausal gonadotropin(HMG)(150IU/d) were administered for 17 days from the cycle 3 of i ducing menstruation after two-month oral contraception pills, and two follicles grew to 18mm and 17mm in size but serum E 2 level remained very low (<5pg/ml) with P level increasing to 25.1ng/ml and a thin endometrium (3mm).Ovulation trigger was cancelled due to the thin endometrium and the abnormal levels of E 2 and P.

Then she was referred to Reproductive Medicine Center of Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University in 2014, willing to have a child.Physical examination revealed the following characteristics: a height of 158cm and weight of 60kg; Tanner scores of four for the breasts and two for axillary and pubic hair; female external genitalia; di culty of bending the metacarpopha-langeal joints from childhood ; no other skeletal malformations were founded.No other infertility factor was identi ed.The evaluations for adrenal, gonadal and pituitary hormones showed that serum levels of P and 17-hydroxyprogesterone(17-OHP) were obvious high, and dehydroepiandrosterone sulfate(DHEA-S), androstenedione, free testosterone were low, as well as the other tests were within the reference ranges.Table 1 summarized the clinical characteristics and hormonal pro les of the patient.A pelvic ultrasonography showed a hypoplastic uterus, t in endometrium and an ovarian cyst(2.9×3.0×2.8cm) in the right ovary.Bilateral integration of the adrenal glands was enlarged, as determin

by a computed tomography scan.


Genetic t
sting

The patient was suspected of having rare forms of CAH according to the clinical manifestations, imaging and laboratory tests.In order to con rm the diagnosis and nd the genetic etiology, a panel of CAH candidate genes by targeted exome next-generation sequencing (NGS) were performed, including CYP21A2,CYP19A1,CYP17A1,CYP11A1, HSD3B2, STAR,AR,EDNRA,NR5A1,PDE8B and POR gene.

Genomic DNA were extracted from the peripheral blood leukocytes using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany).The extracted DNA was segmented by DNA enzyme and puri ed by magnetic bead(Beckman Inc.,USA), followed by PCR ampli cation.DNA library was captured and puri ed twice by a customized Panel probe (Illumina Inc.,USA).The exon, intron-exon boundaries, the 5'and 3' anking regions of the panel genes was sequenced by NextSeq500 (Illumina Inc.,USA).

Raw data was compared with reference sequence retrieved from the University of California at Santa Cruz Genome Browser (http://genome.ucsc.edu)(UCSC,hg19) by the BWA algorithm and annotated using the method reported by Zhang [28].The HGVS (www.hgvs.org/mutnomen/)guidelines for describing sequence variations and numbering were used, with +1 corresponding to the A of the ATG translation initiation codon of the GenBank cDNA sequence and the amino acid sequences.All variants were classi ed according to the American College of Medical Genetics and Genomics (ACMG) 2015 classi cation [23]: pathogenic, likely pathogenic, uncertain signi cance, likely beni n and benign.Sanger sequenced was performed in suspected variations.

The results showed that no mutation and copy number variation were found in CYP21A2,CYP19A1,CYP17A1,CYP11B1,HSD3B2,AR,EDNRA,NR5A1,PDE8B and STAR, but a compound heterozygous mutation was found in POR gene(NM_000941.2):c.1370G>A (p.Arg457His,rs28931608,) and c.1196_1204del (p.Pro399_Glu401del ) [Figure 1].The sequencing results of her parents showed that her father was a heterozygous carrier for c.1370G>A and her mother was a heterozygous carrier for c.1196_1204del.The c.1370G>A had been found in some PORD patients(HGMD:CM040474), which are common in Japanese and Chinese patients [2,7,8,11,12].The mutation of c.1370G>A in POR gene leads to a conversion of arginine at amino acid position 457 to histidine (R457H) which supports only 3% of 17-hydroxylase activity, no detectable 17,20 lyase activity [11,14], and only 1% of aromatase activity [21].The c.1196_1204del mutation in POR gene was rstly reported in two unrelated Turkish PORD patients(HGMD ID:CD117091) and cause a loss of three amino acid p.Pro399_Glu401del (P399_E401del) [

] .In comparison
to wild-type POR, this P399_E401del mutation was found to decrease catalytic e ciency of 21hydroxylase by 68%, 17α-hydroxylase and 17,20 lyase by 76% and 69%, and aromatase by 85% [5,10].The variants c.1370G>A and c.1196_1204del were classi ed by pathogenic and likely pathogenic respectively according to ACMG.


Fertility treatment

According the clinical presentation, the r sults of biochemical tests and genetic testing the patient was diagnosed as PORD.Then she was given HRT composed of estradiol and progesterone (Femoston, Abbott Biologicals B.V, Netherland), combined with oral dexamethasone (0.375mg/d) for two months.After the therapy her basal serum P and 17-OHP levels fell to normal levels(0.35ng/mland 0.23ng/ml respectively) with disappearance of the ovarian cyst.

According to our several successful cases with atypical CAH caused b 17α-hydroxylase de ciency through frozen embryo transfer after IVF and the pregnant case with 17α-hydroxylase de ciency published in 2016 [4] who showed similar changes of sex hormones and inadequate endometrial development during ovarian stimulation, IVF management was recommended.Oral dexamethasone(0.375mg/d) was maintained during all treatment phases.


Ovarian stimulation for IVF

We performed a long gonadotropin releasing hormone agonist (GnRHa) protocol with down regulation using a single dose of 1.3mg long-acting triptorelin and ovarian stimulation with 225IU/d of recombinant FSHα(rFSH) and 75IU/d HMG.When four follicles reached to 20 mm, 19mm,16mm and 15mm in diameter, 10,000IU of human chorionic gonadotropin(HC ) was administered for triggering the maturation of oocytes on Day 21 of stimulation.On the triggering day serum levels of E 2 and P were 33pg/ml and 2.3ng/ml respectively with a thin endometrium (4mm), which showed less disorder comparing with them in the cycle of ovulation induction without oral dexamethasone and GnRH agonist down regulation.Then 4 oocytes were retrieved 36 hours after HCG triggering and 4 cleavage embryos were available and cryopreserved.The details were shown in Table 2.


Pregnancy after frozen-thawed embryo transfer with arti cial endometrial preparation

The patient's menses came 17 days after oocyte retrieval.On cycle 3 serum P level was 0.6 ng/ml and arti cial endometrial preparation was started with oral estradiol valerate (4mg/d).When endometrial thickness reached 10.4mm, progesterone in oil(60mg/d) was administered by intramuscular injection, and 3 days later, two frozen-thawed embryos were transferred.After embryo transfer, oral dexamethasone wasn't given any longer considering the patient had never presented with adrenal insu ciency before.A twin pregnancy was attained and estradiol and progesterone was maintained during the rst trimester of pregnancy.The pregnancy proceeded uneventfully, with the regular monitor in the department of Endocrinology and Obstetrics.A healthy boy and a healthy girl were delivered by caesarean sectio

after 37 weeks and 2
days of gestation, weighing 2.5kg and 2.3kg respectively.No perinatal problems were observed, and the puerperium was uneventful.During the pregnancy and post-partum period, she had not presented with adrenal insu ciency and no need for glucocorticoids replacement.She remained amenorrhea one year after delivery and has been accepting HRT until now.


Literature search

We searched the PubMed database using search terms for Medical Subject Headings and/or text words relating to PORD and pregnancy.The retrieved papers were hand-searched for additional relevant articles using our inclusion criteria (papers reporting POR gene mutations in 46, XX females).Only two papers and three female cases with homozygous or compound heterozygous mutations in POR gene were reported to be successful pregnant (22,25).Including our case, all four successful pregnant cases were obtained by

F and no spontaneous pregnan
y has been reported (Table 3).


Discussion

A recently published review showed that PORD is a complex disorder with many possible mutations affecting a large number of enzymes and the most common mutations were R457H(25%) and A287P(24%) in 180 individual POR mutations from 90 patients [7].Several phenotypic features were very common in PORD women but occurred across a range of mutations, including of high serum concentrations of P (100%), pregnenolone (100%), 17OHP (96%), corticosterone (83%) and deoxycorticosterone (70%), DSD(78%), ovarian cysts(39%), skeletal malformations(84%), and adrenal insu ciency(78%) with most of mild cases [7].For late-onset PORD primary amenorrhea/oligomenorrhea or infertility could be the main clinical manifestation [2,22,24], but little is known about the optimal way to investigate and treat patients ith adult-onset PORD.Our case was a late-onset PORD woman presented with features of high P and 17OHP, primary amenorrhea, ovarian cyst, minor skeletal malformation and no obvious sign of adrenal insu ciency.A compound heterozygotes for c.1370G>A (R457H) and c.1196_1204del (P339_E401del) were found which had been con rmed to reduce activities of P450c17, P450c21, and P450aro [5,10,11,14,21].

As so far the clinical course and of PORD in adulthood and the long-term consequence for fertility remain unknown and until now no spontaneous pregnancy in PORD female patients has been reported.In 2018, Song et al. reported that a patient with a history of vaginal atresia, PORD was suspected during ovarian stimulation, after the patient exhibited low serum E 2 levels and high P levels.After controlling P level by GnRHa and dexamethasone, a successful IVF frozen embryo transfer was achieved [25].In a recent published paper from France, PORD was biologically and genetically con rmed in ve adult women with chronically elevated serum P who were referred for oligo-/amenorrhea and/or infertility, and two of these ve PORD women were reported to obtain successful live births by assisted reproductive treatment [22].However comparing with our case, the reported th

e patients had mil
er clinical phenotypes with oligomenorrhea and infertility, and different homozygous and compound heterozygous mutations in POR gene [22,25].Our case provided additional information of effective infertility treatment in PORD women with different ethnicity, clinical phenotype and POR gene mutation.The impairment of reproductive capacity in PORD women may be mainly explained by the effects of estradiol de ciency and progesterone excess from both adrenal and gonad, accentuated by ovarian stimulation, on endometrial development.These conditions occurred in our case.During the ovulation induction with HMG she presented with normal follicular growth but higher serum P and thin endometrium with undetectable serum E 2 , which means that ovarian stimulation and follicular development increase the P overproduction from ovary.The similar serum steroid pro le have been shown in the other reports (Table 3), the consisting high basal P and the discordance between the normal number of follicles and the low E 2 levels but high P levels during ovarian stimulation should be considered the possibility of PORD.

IVF can be used to segment ovarian stimulation and embryo transfer to avoid the negative effect of high P and low E 2 on endometrial receptivity by freezing all available embryos.Freeze-all policy have been successfully used in women undergoing IVF under various conditions such as the premature elevation of serum P after conventional ovarian simulation [15,18], luteal phase stimulation [18,26] and progestin-primed ovarian stimulation protocol and so on [15,27].Therefore, it suggests that high P level during the period of follicular growth may not impair the developmental capacity of the oocyte and the effect of high P on endometrium can be overcome with c yopreservation and frozen-thawed embryo transfer(FET).As for low estradiol, previous reports showed estrogen may not play a key role in folliculogenesis and follicular development in vivo and in vitro [13,19], but gonadotrophins play a vital role in the growth and maturation of follicles [9].In the successful pregnancies in CAH women caused by 17-hydroxylase de ciency and steroidogenic acute regulatory protein mutations, the patients presented with primary amenorrhea and absent or incomplete sexual maturation [1,4].The authors both reported that during their IVF treatment, endogenous estrogen level was very low but follicles grew normally after ovarian stimulation and normal embryos and pregnancies were obtained [1,4], just as our case reported.So we suggest that the disorders of gonadal steroidogenesis caused by rare forms of CAH may have little effect on the follicular growth and the developmental capacity of the oocytes.Although only few cases have been reported to be successful pregnancies, it may be an effective way to help them have their own children, through IVF and FET after using exogenous estrogen for endometrial preparation and corticoids or combined with GnRH agonist when necessary to suppress the overproduction of progesterone from adrenal and gonad.Of course, a multidisciplinary team including reproductive endocrinologist, internal endocrinologist, obstetrician and geneticist is needed for these women to get through the pregnancy and delivery [6].


Conclusions

In conclusion, It is a report of successful pregnancy in a PORD patient who had primary amenorrhea and different POR mutation with the published three cases who had been reported to obtain successful pregnancies after IVF.For this rare form of CAH, it seemed that disorders of steroidogenesis caused by PORD didn't impair the developmental potential of oocytes.The successful pregnancy could be obtained through IVF and FET after adequate hormonal control and endometrial preparation.Our report will hope ully improve the effective treatment of infertility in PORD women.Twins live birth.

Abbreviation: E 2 : estradiol; P: progesterone 17-OHP: 17α-hydroxyprogesterone ACTH: adrenocorticotropic hormone; IVF: in vitro fertilization; GnRHa: gonadotrophin releasing hormone agonist.



Abbreviationsadrenal hyperplasia; PORD: P450 oxidoreductase de ciency; POR: P450 oxidoreductase; E 2 : estradiol; P: progesterone; IVF: in vitro fertilization; NADPH: nicotinamide adenine dinucleotide phosphate; DSD: sexual development; HRT: hormone replacement therapy; HMG: human menopausal gonadotropin; 17-OHP: 17-hydroxyprogesterone; DHEA-S: dehydroepiandrosterone sulfate; NGS: targeted exome next-generation sequencing; ACMG: the American College of Medical Genetics and Genomics; HCG: human chorionic gonadotropin; GnRHa: gonadotropin releasing hormone agonist; FET: frozenthawed embryo transfer


Table 1 and
1
Hormonal Profiles of patient
Case(years)29MenstruationPrimary amenorrheaHeight (cm)158Weight (kg)60BMI (kg/m 2 )24.03Blood pressure (mmHg)118/79Karyotype46XX, 1qh+Antral follicle count (n)6AMH (ng/ml)2.53FSH (IU/L)14.80LH (IU/L)8.84E 2 (pg/ml)21Prolactin (IU/L)20.9Testosterone (nmol/L)1.21Progesterone (ng/ml)>40.117-OHP (ng/ml)>20Free testosterone (pg/ml)0.19DHEA-S (ng/ml)223.99Androstenedione (ng/ml)0.82SHBG (nmol/L)43.39TSH(mIU/L)2.61ACTH (pg/ml) 8:00 a.m140.00ACTH (pg/ml) 4:00 p.m21.00Cortisol (nmol/L) 8:00 a.m474.39Cortisol (nmol/L) 4:00 p.m271.82Aldosterone (ng/L)115.51Serum potassium (mmol/L)4.23Serum sodium (mmol/L)141.9Renin concentration (ng/ml/h) 2.95
Abbreviation: BMI: body mass index; AMH: anti-Müllerian hormone; FSH: follicle-stimulating hormone; LH: luteinizing hormone; E 2 : estradiol; 17-OHP: 17αhydroxyprogesterone; DHEA-S: dehydroepiandrosterone sulfate; SHBG: sex hormone-binding globulin; TSH: thyroid stimulating hormone; ACTH: adrenocorticotropic hormone.


Table 2
2
Ovarian stimulation for IVF and the changes of hormones Abbreviation: rFSH: recombinant FSHα; HMG: human menopausal gonadotropin; HCG: human chorionic gonadotrophin; FSH: follicle-stimulating hormone;
Cycle day-210611172326dexamethasone0.375mg/dlong-acting tr

visions,
collected the clinical and laboratory data, performed the genetic analysis and took part in discussions regarding he results.XC and JH-participated i managing the whole infertility treatment of the case, retrieved the data, proof read the paper and took part in discussio

regarding the results.DY-proof read the pa
er and took part in discussions regarding the results.YL-designed and performed the study, oversaw the data interpretation and cr

ically revised the manus
ript.All authors read and approved t

nal ma
uscript.Ethics approval and consent to participateThe study was approved by the ethical committee of Sun Yat-sen Memorial Hospital, Sun Yat-sen Univer ity (SYSEC-KY-KS-2019-052).Consent for pu